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PrefaceⅰContents

ⅲ

Introduction

ⅵ

Experimental information

ⅷ

Chapter 1

Discovery of the tube radial distribution phenomenon (TRDP) and development oftube radial distribution chromatography (TRDC)

1

1.1 Novel capillary chromatography based on tube radial distribution of aqueousorganic mixture carrier solvents
Chapter 2

Observation and characterization of tube radial distribution phenomenon (TRDP)

11

2.1 Observation and characterization of TRDP with ﬂuorescence microscope-CCD
camera system
2.2 Observation and characterization of TRDP with various ﬂow rates andcapillary tube lengths
2.3 Observation and characterization of TRDP with various inner diameters ofcapillary tubes
Chapter 3

Consideration of tube radial distribution phenomenon (TRDP)
3.1 Experimental consideration through the phase diagram in TRDC and TRDP
3.2 Experimental consideration through the phase diagram including solubility
curves at different temperatures in TRDC and TRDP
3.3 Experimental consideration through the phase diagram including tie lines andsolubility curves in TRDC and TRDP
3.4 Experimental consideration through the estimation of the dimensionless Weber
number in TRDP
3.5 Experimental consideration through the inner and outer phase formation in
TRDP using various types of mixed solvent solutions

46

Investigation of Tube Radial Distribution Phenomenon (TRDP) and Its Function Appearance

Contents

Development of tube radial distribution chromatography (TRDC)

89

4.1 Analytical conditions and separation performance in TRDC
4.2 Influences of analyte injection volumes and concentrations in TRDC
4.3 Investigation of the composition for a ternary mixed solvent system in TRDC
using PTFE capillary tube
4.4 Investigation of the composition for a ternary mixed solvent system in TRDC
using fused-silica capillary tube
4.5 Fundamental experiments for tentative comparison of TRDC and capillaryzone electrophoresis
Chapter 5

Effects of inner wall characteristics of capillary tubes on tube radial distribution
chromatography (TRDC)

138

5.1 Effects of the inner-wall characteristics of the fused-silica capillary tube on TRDC
5.2 Effects of inner-wall-modified capillary tubes on TRDC
5.3 Effects of the inner wall materials of capillary tubes on TRDC
Chapter 6

Detection methods in tube radial distribution chromatography (TRDC)

158

6.1 Introduction of fluorescence and chemiluminescence detection to TRDC
Chapter 7

Microchip-tube radial distribution chromatography (microchip-TRDC)

168

7.1 Michrochip-TRDC with chemiluminescence detection
Chapter 8

Examination of various types of analytes with tube radial distribution
chromatography (TRDC)

179

8.1 Examination of carboxylated polymer particles as analytes with TRDC using apolyethylene capillary tube
8.2 Examination of polymer compounds as analytes with TRDC using a fusedsilica capillary tube
8.3 Examination of metal compounds as analytes with TRDC

Investigation of Tube Radial Distribution Phenomenon (TRDP) and Its Function Appearance

Chapter 4

8.5 Examination of lambda-DNA molecules as analytes with TRDC
8.6 Examination of metal ions as analytes with TRDC using an absorption reagent
8.7 Examination of dansyl-DL-amino acids as analytes with TRDC using
cyclodextrin
Chapter 9

Consideration of tube radial distribution chromatography (TRDC)

229

9.1 Experiments and consideration through temperature effect in TRDC
9.2 Experiments and consideration through adding surfactants to an analyte
solution in TRDC
9.3 Theoretical consideration through outer phase formation in TRDC
9.4 Considerations through computer simulation in TRDC
Chapter 10

Development of tube radial distribution extraction (TRDE)

258

10.1 TRDE using a microchip that has triple-branched microchannels
10.2 TRDE using a device having double tubes of different inner diameters
Chapter 11

Development of tube radial distribution mixing (TRDM)

272

11.1 TRDM using a microchip having three-to-one line microchannels
Chapter 12

Development of tube radial distribution reaction (TRDR)

284

12.1 TRDR featuring the liquid-liquid interface created with TRDP
Chapter 13

Two-phase separation systems of mixed solutions and TRDP
13.1 Two-phase extraction of metal ions using a ternary water-acetonitrile-ethyl
acetate mixed solution
13.2 TRDP created with an aqueous ionic liquid mixed solution
13.3 TRDP created with an aqueous non-ionic surfactant mixed solution
13.4 TRDP created with a fluorocarbon and hydrocarbon organic solvent mixed
solution

296

Investigation of Tube Radial Distribution Phenomenon (TRDP) and Its Function Appearance

8.4 Examination of biomolecules as analytes with TRDC

solution
Chapter 14

Applications and related techniques with microfluidic behavior of mixed solvent
solutions337

14.1 TRDP in bent and wound microchannels on microchips
14.2 Introduction of double tubes having different inner diameters to TRDC system
14.3 Capillary chromatography using an annular and sluggish flow in the ternarywater–acetonitrile–ethyl acetate mixed solution
14.4 Microfluidic inverted flow of aqueous and organic solvent mixed solution in a
microchannel
Conclusions

366

A list of the TRDP-related manuscripts reported by the author’s groupin chronological order

366

Investigation of Tube Radial Distribution Phenomenon (TRDP) and Its Function Appearance

13.5 TRDP created with a water-acetonitrile containing sodium chloride mixed

Investigation ofTube Radial Distribution Phenomenon(TRDP)and Its Function Appearance

Kazuhiko TsukagoshiPreface
My laboratory has mainly investigated microflow analysis systems, such as microflow
injection analysis, capillary chromatography using coated-capillary tubes, capillaryelectrophoresis, microchip capillary electrophoresis, and microchip flow analysis
systems since 1995 in Doshisha University. I have been especially interested inchemiluminescence detection that are introduced into microflow analysis systems. I
have done extensive research in this area especially for 20 years with my students in mylaboratory. First of all, looking back upon very happy days in my laboratory, I greatly
appreciate all my students; bachelor, master, and doctoral course students, as well as myco-workers in Doshisha University and in other universities, research institute centers,
and companies.
Herein I describe a specific microfluidic behavior exhibited by mixed-solvent
solutions in a microspace, coined as the tube radial distribution phenomenon (TRDP).
The related manuscript was first reported in 2009. The specific fluidic behavior wasobserved in the following solution systems: ternary water–hydrophilic/hydrophobicorganic solvents, water–surfactant, water–ionic liquid, water-hydrophilic solvents
containing salts, and fluorous/organic solvents. When the mixed homogeneous solutions
were delivered into a microspace under certain conditions, the solvent molecules wereradially distributed in the microspace, generating inner and outer phases with a kinetic
liquid-liquid interface. The TRDP was fundamentally evaluated by fluorescencemicroscopy, phase diagram construction, and the elution behaviors of solutes in acapillary tube. A TRDP-based capillary chromatography, referred to as tube radial
distribution chromatography (TRDC); where the outer phase serves as a
pseudo-stationary phase under laminar flow conditions, has been developed as one of
the applications of TRDP. We have also investigated TRDP-based extraction, chemical
reaction, and mixing processes, coined as tube radial distribution extraction (TRDE),
tube radial distribution reaction (TRDR), and tube radial distribution mixing (TRDM),
respectively. The concept and experimental findings regarding TRDP, TRDC, TRDE,TRDR, and TRDM are described in this book.
In 2011 the author established “tube radial distribution phenomenon (TRDP)
research center” in Doshisha University together with co-workers. In 2012, themanuscript (Y. Masuhara, et al., Analytical Sciences, 2012, 28, 439) with the title “TheMicro-Flow Reaction System Featured the Liquid-Liquid Interface Created withTernary Mixed Carrier Solvents in a Capillary Tube”, was given an award of Hot Article
Award Analytical Sciences. The review of the TRDP and the TRDP-related technologies
that was written in Japanese and was published in 2013 (K. Tsukagoshi, Bunseki
Kagaku, 2013, 29, 393). Also, in 2013, the manuscript (M. Murakami, et al., Analytical
Sciences, 2011, 27, 793) of the title, Tube Radial Distribution Phenomenon of Ternary
Mixed Solvents in a Microspace under Laminar Flow Conditions, was given the awardiPrefaceiTube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesof Most Cited Paper Award of Analytical Sciences-2012. In January 2014, an English
review of each of paper was invited to be included in the 30th anniversary publication of
Analytical Sciences (K. Tsukagoshi, Analytical Sciences, 2014, 30, 65). My students, 10bachelors, 10 masters, and 2 doctorates, as well as my co-workers in and outside
Doshisha University have begun this academic year`s, 2014, research into, TRDP andother related studies.
Once again, I would like to offer my heartfelt thanks to all my students andco-workers for their experimental support and data, as well as the useful and helpful
discussions regarding TRDP and it’s related studies.

Professor Kazuhiko Tsukagoshi
October 20, 2014

Professor Kazuhiko Tsukagoshi
Department of Chemical Engineering and Materials Science, Doshisha University,
Kyotanabe, Kyoto 610-0321, Japan
Tube Radial Distribution Phenomenon Research Center, Doshisha University,
Kyotanabe, Kyoto 610-0321, Japan
E-mail: ktsukago@mail.doshisha.ac.jp

Tel & Fax: +81-75-462-4636
ii

ii

Contents
PrefaceⅰContents

ⅲ

Introduction

ⅵ

Experimental information

ⅷ

Chapter 1

Discovery of the tube radial distribution phenomenon (TRDP) and development oftube radial distribution chromatography (TRDC)

1

1.1 Novel capillary chromatography based on tube radial distribution of aqueousorganic mixture carrier solvents
Chapter 2

Observation and characterization of tube radial distribution phenomenon (TRDP)

11

2.1 Observation and characterization of TRDP with fluorescence microscope-CCD
camera system
2.2 Observation and characterization of TRDP with various flow rates andcapillary tube lengths
2.3 Observation and characterization of TRDP with various inner diameters ofcapillary tubes
Chapter 3

Consideration of tube radial distribution phenomenon (TRDP)

46

3.1 Experimental consideration through the phase diagram in TRDC and TRDP
3.2 Experimental consideration through the phase diagram including solubility
curves at different temperatures in TRDC and TRDP
3.3 Experimental consideration through the phase diagram including tie lines andsolubility curves in TRDC and TRDP
3.4 Experimental consideration through the estimation of the dimensionless Weber
number in TRDP
3.5 Experimental consideration through the inner and outer phase formation in
TRDP using various types of mixed solvent solutions
Chapter 4

Development of tube radial distribution chromatography (TRDC)

89

4.1 Analytical conditions and separation performance in TRDC
4.2 Influences of analyte injection volumes and concentrations in TRDC
4.3 Investigation of the composition for a ternary mixed solvent system in TRDC
using PTFE capillary tube

Contentsiii

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

4.4 Investigation of the composition for a ternary mixed solvent system in TRDC
using fused-silica capillary tube
4.5 Fundamental experiments for tentative comparison of TRDC and capillaryzone electrophoresis
Chapter 5

Effects of inner wall characteristics of capillary tubes on tube radial distribution
chromatography (TRDC)

138

5.1 Effects of the inner-wall characteristics of the fused-silica capillary tube on TRDC
5.2 Effects of inner-wall-modified capillary tubes on TRDC
5.3 Effects of the inner wall materials of capillary tubes on TRDC
Chapter 6

Detection methods in tube radial distribution chromatography (TRDC)

158

6.1 Introduction of fluorescence and chemiluminescence detection to TRDC
Chapter 7

Microchip-tube radial distribution chromatography (microchip-TRDC)

168

7.1 Michrochip-TRDC with chemiluminescence detection
Chapter 8

Examination of various types of analytes with tube radial distribution
chromatography (TRDC)

179

8.1 Examination of carboxylated polymer particles as analytes with TRDC using apolyethylene capillary tube
8.2 Examination of polymer compounds as analytes with TRDC using a fusedsilica capillary tube
8.3 Examination of metal compounds as analytes with TRDC
8.4 Examination of biomolecules as analytes with TRDC
8.5 Examination of lambda-DNA molecules as analytes with TRDC
8.6 Examination of metal ions as analytes with TRDC using an absorption reagent
8.7 Examination of dansyl-DL-amino acids as analytes with TRDC using
cyclodextrin
Chapter 9

Consideration of tube radial distribution chromatography (TRDC)
9.1 Experiments and consideration through temperature effect in TRDC
9.2 Experiments and consideration through adding surfactants to an analyte
solution in TRDC
9.3 Theoretical consideration through outer phase formation in TRDC
9.4 Considerations through computer simulation in TRDC

iv

229

Chapter 10

Development of tube radial distribution extraction (TRDE)

258

10.1 TRDE using a microchip that has triple-branched microchannels
10.2 TRDE using a device having double tubes of different inner diameters
Chapter 11

Development of tube radial distribution mixing (TRDM)

272

11.1 TRDM using a microchip having three-to-one line microchannels
Chapter 12

Development of tube radial distribution reaction (TRDR)

284

12.1 TRDR featuring the liquid-liquid interface created with TRDP
Chapter 13

Two-phase separation systems of mixed solutions and TRDP

296

13.1 Two-phase extraction of metal ions using a ternary water-acetonitrile-ethyl
acetate mixed solution
13.2 TRDP created with an aqueous ionic liquid mixed solution
13.3 TRDP created with an aqueous non-ionic surfactant mixed solution
13.4 TRDP created with a fluorocarbon and hydrocarbon organic solvent mixed
solution13.5 TRDP created with a water-acetonitrile containing sodium chloride mixed
solution
Chapter 14

Applications and related techniques with microfluidic behavior of mixed solvent
solutions337

14.1 TRDP in bent and wound microchannels on microchips
14.2 Introduction of double tubes having different inner diameters to TRDC system
14.3 Capillary chromatography using an annular and sluggish flow in the ternarywater–acetonitrile–ethyl acetate mixed solution
14.4 Microfluidic inverted flow of aqueous and organic solvent mixed solution in a
microchannel
Conclusions

366

A list of the TRDP-related manuscripts reported by the author’s groupin chronological order

366

Contentsv

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Introduction
Microfluidic
behaviorssolvents,
confineda microspace,e.g.,
microchannels
Microfluidic
behaviorsof ofsolvents,
conf
ined in ina microspace,e.g.,
microchannels
in inaamicrochip
microchipororcapillary
capillarytubes,
tubes,have
havebeen
beeninvestigatedinvestigated since
since thethe nineteenth century, andare known to generate interesting and useful physical or hydrodynamic phenomena,such as electroosmotic and laminar flows. Electroosmotic flow was first reported in1809 by Reuss along with electrophoresis [1]. In the last century, capillaryelectrophoresis [2], micellar electrokinetic chromatography [3], and capillaryelectrochromatography [4] were developed on the basis of electroosmotic flow. Suchflow is instigated under high-applied voltages. Laminar flow was reported by Hagen in1839 [5] and Poiseuille in 1841 [6]. Reynolds later studied turbulent and laminar flows
and formulated a dimensionless parameter, known as the Reynolds number, in fluid
mechanics [7]. In the last century, the application of laminar flow led to thedevelopment of hydrodynamic chromatography [8], wide-bore hydrodynamic
chromatography [9], and field flow fraction chromatography [10].
Recently, the microfluidic behavior of solvents, based on electroosmotic and laminarflows, has been examined through the use of varied channel configurations, solvent
flow rates, aqueous–organic solvent mixtures, and by introducing specific obstacles into
the microchannel [11-13]. The fluidic behavior of solvents in the microchannel isrelated to the separation, diffusion, and reaction of the solutes. The development ofnovel uses for the microfluidic behaviors of solvents is both important and useful for
designing microreactors or micro-total analysis systems [14,15]. We have reported aninteresting, and unique, microfluidic behavior based on a mixed-solvents system
confined in a microspace, which we named: the tube radial distribution phenomenon(TRDP). TRDP was observed in various types of mixed solutions, namely, ternarywater–hydrophilic/hydrophobic organic solvents, water–surfactant, water–ionic liquid,water-hydrophilic solvents containing salts, and fluorous/organic solvents. TRDP
induces the formation of a kinetic liquid–liquid interface in a microfluidic flow, leading
to the development of inner and outer phases.
A capillary chromatography system, operating under TRDP, where the outer phasefunctions as a pseudo-stationary phase under laminar flow conditions, has beendeveloped. Such a system is referred to as tube radial distribution chromatography(TRDC). We have also applied TRDP to the extraction, chemical reaction, and mixing
processes. The respective TRDP-based processes are coined as tube radial distribution
extraction (TRDE), tube radial distribution reaction (TRDR), and tube radial
distribution mixing (TRDM). Since the development of TRDP in 2009, the latter has
been widely investigated in several areas pertaining to microfluidics. This book mainly
reviews the development and application of TRDP, TRDC, TRDE, TRDR, and TRDM
in a specific water–acetonitrile–ethyl acetate ternary system.
References[1] F. F. Reuss, Proceedings of the Imperial Society of Naturalists of Moscow, 1809, 2,
vi
vi

327.
[2] J. W. Jorgenson and K. D. Lukacs, Anal. Chem., 1981, 53, 1298.
[3] S. Terabe, K. Otsuka, K. Ichikawa, A. Tsuchiya, and T. Ando, Anal. Chem., 1984,
56, 111.
[4] T. Tsuda, Anal. Chem., 1987, 59, 521.
[5] G. Hagen, Ann. Phys. Chem., 1839, 46, 423.
[6] R. B. Bird, W. E. Stewart, and E. N. Lightfoot, “Transport Phenomena", 2nd ed.,
2002, Chapter. 2, (Wiley, Toronto, Canada).
[7] O. Reynolds, Phil. Trans. R. Soc., 1883, 174, 935.
[8] H. Small, J. Colloid Interface Sci., 1974, 48, 147.
[9] M. Harada, T. Kido, T. Matsudo, and T. Okada, Anal. Sci., 2005, 21, 491.
[10] J. C. Giddings, M. N. Myers, G.-C. Lin, and M. Martin, J. Chromatogr., A, 1977,
142, 23.
[11] Y. Kikutani, H. Hisamoto, M. Tokeshi, and T. Kitamori, Lab Chip, 2004, 4, 328.
[12] A. Hibara, M. Tokeshi, K. Uchiyama, H. Hisamoto, and T. Kitamori, Anal. Sci.,
2001, 17, 89.
[13] N. Kaji, Y. Okamoto, M. Tokeshi, and Y. Baba, Chem. Soc. Rev., 2010, 39, 948.
[14] H. Nakamura, Y. Yamaguchi, M. Miyazaki, H. Maeda, and M. Uehara, Chem.
Commun., 2002, 2844.
[15] H. Kawazumi, A. Tashiro, K. Ogino, and H. Maeda, Lab Chip, 2002, 2, 8.
[16] N. Jinno, M. Hashimoto, and K. Tsukagoshi, Anal. Sci., 2009, 25, 145.

vii
Introductionvii

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Experimental information
Water and reagents: Water was purified with an Elix 3 UV (Millipore Co., Billerica,
MA). All reagents used were commercially available and of analytical grade.
Capillary tubes: A fused-silica capillary tube, a high-density polyethylene (PE) capillarytube, a polytetrafluoroethylene (PTFE) capillary tube, and a copolymer of(tetrafluoroethylene-perfluoroalcoxyethylene) (PTFE-PFAE) capillary tube werepurchased from GL Science (Tokyo, Japan), Natume Co. (Tokyo, Japan), Yasaka
Industries, Inc. (Tokyo, Japan), and Iwase Co. (Tokyo, Japan), respectively.
Microchips: A microchip made of glass and incorporating microchannel lines was
acquired from Microchemical Technology (Kanagawa, Japan).
Tube radial distribution chromatography (TRDC) system: The system comprised a
microsyringe pump (MF-9090; Bioanalytical Systems, Inc., West Lafayette, IN, USA)and an absorption detector (modified SPD-10AV spectrophotometric detector;
Shimadzu Co., Kyoto, Japan), a fluorescence detector (modified RF-535 fluorescence
detector; Shimadzu Co.), or a chemiluminescence (CL) detection (Model EN-21,
Kimoto Electric Co., Ltd.) as main devices. The following figure shows a schematic
diagram of typical TRDC system.

Fluorescence microscope-CCD camera system: A fluorescence microscope (BX51;
Olympus, Tokyo, Japan) equipped with an Hg lamp and a filter (U-MWU2, ex 330–385
nm, em. >420 nm) and CCD camera (JK-TU53H; Toshiba, Tokyo, Japan) was operated
in the system.
Fluorescence profiles: The obtained fluorescence images were transformed into line
drawings in order to evaluate the color depth using a computer. The observed colors
included red, green, and blue (RGB). The images principally consisted of blue andgreen because perylene and Eosin Y emit light at approximately 470 nm and 550 nm
viii
viii

respectively. The blue and green color depths were numerically expressed as digital dataon a computer (with BMP2CSV free-software), afterwhich the numbers werestandardized to line drawing data to give fluorescence profiles (with Microsoft Excel).
The following figure shows a schematic diagram of fluorescence microscope-CCD
camera and the transformation of fluorescence photograph to fluorescence profile
through the computer.

ix
Experimental informationix

Chapter 1

Chapter 1 Discovery of the tube radial distribution
phenomenon (TRDP) and development of tube radial
distribution chromatography (TRDC)
The discovery of the tube radial distribution phenomenon (TRDP) and the developmentof tube radial distribution chromatography (TRDC), that is, TRDP-based capillarychromatography, were first reported in 2009. As this discovery was not confirmed byvisual data, such as a fluorescence photograph through a fluorescencemicroscope-CCD camera system, strictly speaking, the hypothesis of TRDP might be amore suitable expression than the discovery, at least for the moment. The part of thischapter is reconstructed and rewritten based on the related manuscripts that have been
published.1,2)

1.1 Novel capillary chromatography based on tube radial distributionof aqueous-organic mixture carrier solvents
A capillary chromatography system was newly developed using open capillary tubes
made of fused-silica, polyethylene, or poly(tetrafluoroethylene), and an aqueous-organic
mixture (water-acetonitrile-ethyl acetate mixture) as a carrier solution. Model analyte
mixture solutions, such as 2,6-naphthalenedisulfonic acid and 1-naphthol, Eosin Y andperylene, bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein and 1,1-bi-2-naphthol,and 2,7-naphthalenedisulfonic acid and p-nitroaniline, were introduced into the capillary
tube by gravity. The analyte solutions were subsequently delivered through the capillary
tube with the carrier solution by a micro-syringe pump. The system worked underlaminar flow conditions. The analytes were separated through the capillary tube anddetected on-capillary by an absorption detector. For example, 2,6-naphthalenedisulfonicacid and 1-naphthol were detected in this order with a carrier solution ofwater-acetonitrile-ethyl acetate (volume ratio 15:3:2), but this order was reversed whena carrier solution of water-acetonitrile-ethyl acetate (volume ratio 2:9:4) was used. The
other analyte solutions were similarly separated by the system and their elution times
could be easily reversed by changing the component ratio of the solvents in the carrier
solution.
Introduction
Capillary chromatography including capillary electrochromatography [1,2], micellar
electrokinetic capillary chromatography [3,4], and capillary high-performance liquid
chromatography using packed and monolithic capillary columns [5,6] have attracted a
lot of attention in the analytical chemistry field as well as in separation science ever
1

Discovery of TRDP and development of TRDC

1

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculessince the last century. Most capillary chromatography systems feature rapid
measurements, easy procedures, inexpensive and small apparatus, small sample
volumes, and low costs. However, only a few new concepts concerned with capillarychromatography have been proposed in the last decade. In one example, wide-borehydrodynamic chromatography [7,8] was carried out using an open capillary tube offused-silica. Diffusive and non-diffusive analytes showed quite different elution
behavior in the capillary tube under laminar flow conditions. However, these analytes
were not completely separated using the principle of the method, i.e., the diffusive andnon-diffusive analytes were overlapped on the chromatogram. In a second example,
Tabata et al. have recently reported a capillary chromatography system based on the
microphase separation of mixed solvents [9]. They described that when an
acetonitrile-water mixture with suitable salt concentration was pumped into afused-silica capillary with a negatively charged inner wall due to the dissociation of its
silanol group, microphase separation occurred near the capillary wall, resulting in awater-enriched aqueous phase attached to the capillary inner wall. The mixture ofhydrophilic and hydrophobic analytes was separated in this order using
chromatography.
Here, a capillary chromatography system using open capillary tubes made of either
fused-silica, polyethylene, or poly(tetrafluoroethylene) (PTFE), and a salt-freewater-hydrophilic-hydrophobic organic solvent mixture carrier solution is described. In
our previous communication,1) we briefly described the results of a fused-silica or
polyethylene capillary tube that was applied to the system, and here we mainly report on
the results concerning a PTFE capillary tube. Four analyte solution mixtures ofhydrophilic and hydrophobic molecules (2,6-naphthalenedisulfonic acid and 1-naphthol,
Eosin Y and perylene, bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein and1,1-bi-2-naphthol, and 2,7-naphthalenedisulfonic acid and p-nitroaniline) were
examined in the present system and were found to separate well. Separation in thecapillary chromatography system was performed without the use of any specific packed
capillary tubes, additions of gels; surfactants; or salts, or high-voltage supply devices.
Furthermore, the elution order of the analytes could be changed easily by altering thecomponent ratio of the solvents in the carrier solution. The separation performance ofthe system is discussed based on the results obtained using fused-silica, polyethylene,
and PTFE capillary tubes. The elution behavior of the analytes in the system provided
some novel information that will be useful in separation science.
Experimental
A fused-silica capillary tube (50 µm i.d., 150 µm o.d.), a high-density polyethylene
capillary tube (200 µm i.d., 500 µm o.d.), and a PTFE capillary tube (100 µm i.d., 200µm o.d.) were used. The present capillary chromatography system was comprised of anopen capillary tube, a micro-syringe pump, and an absorption detector. A fused-silica or

2

Chapter 12polyethylene capillary tube, 120 cm in length (effective length: 100 cm), and a PTFE
capillary tube, 90 cm in length (effective length: 70 cm), were placed at room
temperature in the system. Water-acetonitrile-ethyl acetate mixtures with volume ratiosof 15:3:2, 4:7:9, 2:5:9, 2:7:4, and 2:9:4 were used as carrier solutions. Analyte solutions
were prepared with the carrier solutions.
The analyte solution was introduced directly into the capillary inlet side for 20 s froma height of 20 cm for the fused-silica tube, for 5 s from a height of 20 cm for thepolyethylene tube, or for 5 s from a height of 20 cm for the PTFE tube by the gravity
method. After analyte injection, the capillary inlet was connected through a joint to a
micro-syringe. The syringe was set on a micro-syringe pump. The carrier solution was
fed in the capillary tube at a flow rate of 0.2 µL min-1 for fused-silica, 8.0 µL min-1 forpolyethylene, or 0.8 µL min-1 for PTFE under laminar flow conditions. On-capillary
absorption detection (232, 254, or 280 nm) was performed with the detector.
Results and discussion
Properties of PTFE capillary tube
Although a PTFE capillary tube is useful andavailable for micro-flow separation systems because of its inert inner surface andflexibility [10,11], it should be noted that the crystallinity of the PTFE causes lightscattering. Therefore, we first examined the optical properties of the PTFE capillary
tube in the manner that follows. The PTFE capillary was fixed to the modified
absorption detector. The capillary was filled with water, and absorption in the capillary
tube was examined at 200-650 nm. The obtained results are shown in Fig. 1. The
capillary showed maximum absorption at 200 nm which decreased with increasing
wavelength. Absorption detection in the capillary chromatography system, particularlyin the ultraviolet region, was disturbed by the innate light scattering properties of PTFE.
However, by adjusting the apparent absorption due to the scattering of the tube to abaseline for measuring the absorption profile, compounds with absorption behavior suchas the analytes used here were detected without any problems at wavelengths of 232,
254, or 280 nm.
Effects of the component ratio of the carrier on separation In our communication, 1)
a solution of 2,6-naphthalenedisulfonic acid (hydrophilic) and 1-naphthol (hydrophobic)
was analyzed using the capillary chromatography system with open capillary tubes
made of fused-silica or polyethylene. When using a water-rich carrier solution ofwater-acetonitrile-ethyl acetate with a volume ratio of 15:3:2 for both capillary tubes,
the mixture of 2,6-naphthalenedisulfonic acid and 1-naphthol was separated through theopen capillary tube and were detected in this order. On the other hand, using an organic
solvent rich-carrier solution of water-acetonitrile-ethyl acetate with a volume ratio of
2:7:4 or 2:5:9 for fused-silica or polyethylene, respectively, they were detected withinverse elution times, i.e., 1-naphthol and 2,6-naphthalenedisulonic were detected in this3

Discovery of TRDP and development of TRDC

3

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

order (data not shown). A solution of 2,6-naphthalenedisulfonic acid and 1-naphthol
was also analyzed using the present system with an open PTFE capillary tube and an
aqueous-organic carrier solution. The obtained absorption profiles are shown in Fig. 2
using three carrier solutions of water-acetonitrile-ethyl acetate with volume ratios of
15:3:2, 4:7:9, and 2:9:4. When using a water-rich carrier solution ofwater-acetonitrile-ethyl acetate with a volume ratio of 15:3:2, 2,6-naphthalenedisulfonicacid and 1-naphthol were separated through the open capillary tube and detected at ca.
6.8 and 8.5 min, respectively (Fig. 2 a). The components of the analytes,
2,6-naphthalenedisulfonic acid and 1-naphthol, in the profile were confirmed withindividual absorption signals. Confirmation was performed for all experimental data
shown in Figs. 2-4. On the other hand, using the organic solvent-rich carrier solution ofwater-acetonitrile-ethyl acetate with volume ratios of 4:7:9 and 2:9:4, the analytes weredetected with inverse elution times. 1-Naphthol and 2,6-naphthalenedisulfonic acid
were detected in this order at ca. 7.0 and 8.3 min for the carrier with a volume ratio of
4:7:9 (Fig. 2 b) and at ca. 7.1 and 8.9 min for the carrier with a volume ratio of 2:9:4(Fig. 2 c). The separation behavior of the mixture of 2,6-naphthalenedisulfonic acid and1-naphthol, including the reversibility of the elution times by changing the component
ratio of the solvents in the carrier solution, was observed for fused-silica, polyethylene,
and PTFE capillary tubes. Based on the obtained results, the properties of the capillary
tube material, i.e., fused-silica, polyethylene, and PTFE, seemed to have little influenceon the separation behavior of the analytes in the current capillary chromatographysystem.
Separation of the other analyte mixtures using a PTFE capillary tube
Other mixturesof Eosin Y and perylene, bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein and1,1-bi-2-naphthol, as well as 2,7-naphthalenedisulfonic acid and p-nitroaniline were
subjected to separation with the present system using the PTFE capillary tube using awater-rich carrier solution of water-acetonitrile-ethyl acetate (volume ratio 15:3:2). The
obtained absorption profiles are shown in Fig. 3. In each profile, the hydrophilic Eosin
Y, bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, or 2,7-naphthalenedisulfonicacid was eluted first, followed by the hydrophobic perylene, 1,1-bi-2-naphthol, or
p-nitroaniline, with good separation. In addition, these mixtures were subjected toseparation using the present system with an organic solvent-rich carrier solution ofwater-acetonitrile-ethyl acetate (volume ratio 2:9:4). The obtained absorption profiles
are shown in Fig. 4. In each profile, the hydrophobic perylene, 1,1-bi-2-naphthol, or
p-nitroaniline was eluted first, followed by the hydrophilic Eosin Y,
bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, or 2,7-naphthalenedisulfonic acid,with good separation.

4

Chapter 1

4Elution times of the two analytes in the mixture
A similar separation behavior for a
mixture of 2,6-naphthalenedisulfonic acid and 1-naphthol was observed when open
capillary tubes made of fused-silica, polyethylene, or PTFE were used. In addition, thefour mixed analyte solutions, i.e., 2,6-naphthalenedisulfonic acid and 1-naphthol, EosinYandperylene,bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein
and1,1-bi-2-naphthol, and 2,7-naphthalenedisulfonic acid and p-nitroaniline, wereconfirmed to be similarly separated with the capillary chromatography system using thePTFE capillary tube. That is, in all of the experiments using the three different types ofcapillary tubes and four different analyte mixtures, when a water-rich carrier solution,such as water-acetonitrile-ethyl acetate (volume ratio 15:3:2) was used, the hydrophiliccompound was eluted earlier than the hydrophobic compound. Conversely, when acarrier solution with a large organic solvent component, such as water-acetonitrile-ethyl
acetate (volume ratio 2:9:4) was used, the hydrophobic compound was eluted earlier
than the hydrophilic compound. Thus, the elution order can be reversed easily bychanging the component ratio of the solvents in the carrier solution in the three capillarytubes. The Reynolds number was estimated to be roughly < 1 under the present
analytical conditions, confirming that the system worked under laminar flow conditions.
Under these conditions, the linear velocity of the fluid in the capillary tube showed aparabolic curve; the linear velocity showed a maximum value around the middle of thecapillary tube and decreased approaching the inner wall of the tube. Solutes that are
diffusive or of low molecular weight are eluted from the tube with an average linear
velocity and a Gaussian distribution [7,8]. The average linear velocities using the
fused-silica, polyethylene, and PTFE capillary tubes were estimated to be 1.7, 4.2, and
1.7 mm s-1, respectively, under the corresponding analytical conditions. As shown in
Figs. 2-4 and discussed in our communication1), the elution times for the first peaks inthe fused-silica, polyethylene, and PTFE capillary tubes were ca. 9.6, 4.0, and 6.9 min,respectively, showing no change in the carrier solution for the same capillary tube.
Moreover, the elution times of the first peaks nearly corresponded to those calculatedwith the average linear velocities, and the second peaks were eluted with smaller
velocities than the average linear velocities.
Consideration of separation performance
Tabata et al. [9] reported a capillarychromatography system using an open fused-silica capillary tube, in which the innerwall was negatively charged due to the dissociation of silanol groups, and awater-acetonitrile mixture including sodium chloride with high concentration as thecarrier. Although they separated the hydrophilic and hydrophobic molecules in thisorder, but could not change the order of elution, we have demonstrated the separation ofa mixture of hydrophilic and hydrophobic molecules by capillary chromatography using
open capillary tubes made of fused-silica, polyethylene, or PTFE, and a salt-freewater-hydrophilic-hydrophobic organic solvent mixture carrier solution. Thus, we
5Discovery of TRDP and development of TRDC

5

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

determined that the negative charge due to the silanol groups on the inner wall plays nomajor role in creating a water-rich phase near the inner wall, at least in our present
system. Furthermore, the elution times of the analytes in the present system can beeasily reversed by changing the component ratio of the carrier solvents in all types ofcapillary tubes. In addition such a salt-free solvent mixture is easy to use in a capillary
tube without any problems of salt deposition. Based on our results, we proposed that
separation in the present capillary chromatography system was performed using the tube
radial distribution of the aqueous-organic mixture solvents under laminar flow
conditions, refered to here as a tube radial distribution chromatography (TRDC) system.
Possible separation performance in the TRDC system using an open capillary tube andan aqueous-organic solvent mixture carrier solution is described as follows andillustrated in Fig. 5 (water-rich case in 5a and organic solvent-rich case in 5b). The
linear velocity of the fluid in the capillary tube under laminar flow conditions isexpressed as a parabolic curve in Fig. 5; however, the results in a water-organic solvent
mixture carrier solution may deviate from an ideal parabolic curve. Water and organic
solvents in the carrier solution are not dispersed uniformly in the capillary tube. Relative
to the internal volume, the inner wall of the tube features an extremely large specificsurface area. This leads to the generation of a water-rich phase and an organic
solvent-rich phase based on a tube radial distribution of the aqueous-organic solvent
mixture under laminar flow conditions. A major solvent phase forms around the middleof the tube, while a minor solvent phase forms near the inner wall. When a water-richcarrier solution is used, a major solvent phase or a water-rich phase forms around the
middle of the tube (Fig. 5a). However, when an organic solvent-rich carrier solution isused, a major solvent phase or an organic solvent-rich phase forms around the middle ofthe tube (Fig. 5b). Such tube radial distribution of the solvent molecules in the carrier
solution must be caused through a specific flow in the capillary tube under the laminarflow conditions. The specific microfluidic behavior of the solvents is called tube radial
distribution phenomenon (TRDP). Subsequently, as the analyte mixtures consisted oftypical hydrophilic and hydrophobic molecules, the former (2,6-naphthalenedisulfonic
acid,
Eosin
Y,
bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein,
and2,7-naphthalenedisulfonic acid) is mainly dissolved or dispersed in the water-rich phase,
while the latter (1-naphthol, perylene, 1,1-bi-2-naphthol, and p-nitroaniline) is dissolvedin the organic solvent-rich phase, leading to tube radial distribution of analyte moleculesin the capillary tube. In the case of a water-rich carrier solution, the hydrophilic analyte,
which is dispersed in the water-rich phase of the major solvent phase (around the middleof the capillary tube), is eluted at a nearly average linear velocity. The hydrophobic
analyte, which is dispersed in the organic solvent-rich phase of the minor solvent phasenear the inner wall of the tube (pseudo-stationary phase), is eluted at a lower than
average linear velocity (Fig. 5a). In the case of an organic solvent-rich carrier solution,
the hydrophobic analyte dispersed in the organic solvent-rich phase of the major solvent

6

Chapter 16phase (around the middle of the capillary tube) is eluted with nearly average linear
velocity. The hydrophilic analyte dispersed in the water-rich phase of the minor solvent
phase near the inner wall of the tube (pseudo-stationary phase) is eluted with a lower
than average linear velocity (Fig. 5 b). Therefore, the elution times of the analytes canbe easily reversed by altering the component ratio of the solvents in the carrier solution.
The separation in the TRDC was performed without using any special materials such as
packed and monolithic capillary tubes or applying a high voltage for electrophoresis, as
it is done in conventional capillary chromatography.
In conclusion, we developed a capillary chromatography system using an open
capillary tube and an aqueous-organic mixture carrier solution that worked underlaminar flow conditions. In contrast to conventional capillary electrochromatography,
capillary electrophoresis, and capillary liquid chromatography, separation in this system
was performed without the use of any specific materials such as packed capillary tubes,
additives such as gels, surfactants, host molecules or salts, or high-voltage supply
devices. We examined various mixtures consisting of hydrophilic and hydrophobic
molecules in this chromatography system using fused-silica, polyethylene, or PTFE
capillary tubes and a water-acetonitrile-ethyl acetate mixture carrier solution. The
analytes were well separated through the capillary tubes and detected on-capillary by anabsorption detector, and the elution times of the components could be reversed easily bychanging the component ratio of the solvents in the carrier solution. The separation
performance in the system was explained based on the tube radical distribution of theaqueous-organic mixture carrier solvents under laminar flow conditions.

Figure 1. Relationship between detection wavelength and absorption in the PTFE
capillary tube.
7

Discovery of TRDP and development of TRDC

7

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2. Absorption profiles of a mixture of 2,6-naphthalenedisulfonic acid and1-naphthol by the current system using a PTFE capillary tube. Conditions: Capillarytube, 90 cm (effective length: 70 cm) of 100 µm i.d. PTFE; carrier, a)water-acetonitrile-ethyl acetate (15:3:2) mixture solution, b) water-acetonitrile-ethyl
acetate (4:7:9) mixture solution, and c) water-acetonitrile-ethyl acetate (2:9:4) mixture
solution; sample injection, 20 cm height (gravity)×5 s; flow rate, 0.8 µL min-1; andanalyte concentration, 1 mM each.

Figure 3. Absorption profiles of the mixtures using the current system with a water-richcarriersolution.
a)
Eosin
Y
and
perylene,b)bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein and 1,1-bi-2-naphthol, and c)
2,7-naphthalenedisulfonic acid and p-nitroaniline. Conditions: Capillary tube, 90 cm(effective length: 70 cm) of 100 µm i.d. PTFE; carrier, water-acetonitrile-ethyl acetate(15:3:2) mixture solution; sample injection, 20 cm height (gravity)×5 s; flow rate, 0.8µL min-1; and analyte concentration, 1 mM each.

8

Chapter 18

Figure 4. Absorption profiles of the mixtures using the current system with an organic
solvent-richcarriersolution.
a)
Eosin
Y
and
perylene,b)bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein and 1,1-bi-2-naphthol, and c)
2,7-naphthalenedisulfonic acid and p-nitroaniline. Conditions: Capillary tube, 90 cm(effective length: 70 cm) of 100 µm i.d. PTFE; carrier, water-acetonitrile-ethyl acetate(2:9:4) mixture solution; sample injection, 20 cm height (gravity)×5 s; flow rate, 0.8 µL
min-1; and analyte concentration, 1 mM each.

9

Discovery of TRDP and development of TRDC

9

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 5. Illustration of separation performance in the capillary chromatography system.
a) Water-rich carrier solution and b) organic solvent-rich carrier solution.

References[1] M. C. Breadmore, J. R. E. Thabano, M. Dawod, A. A. Kazarian, J. P. Quirino, and R.
M. Guijt, Electrophoresis, 2009, 30, 230.
[2] I. Miksik and P. Sedlakova. J. Sep. Sci., 2007, 30, 1686.
[3] M. Silva, Electrophoresis, 2009, 30, 50.
[4] S. K. Poole and C. F. Poole, J. Chromatogr. A, 2008, 1182, 1.
[5] M. C. Jung, N. Munro, G. Shi, A. C. Michael, and S. G. Weber,. Anal. Chem., 2006,
78, 1761.
[6] J. Urban and P. Jandera, J. Sep. Sci., 2008, 31, 2521.
[7] M. Harada, T. Kido, T. Masudo, and T. Okada, Anal. Sci., 2005, 21, 491.
[8] K. Tsukagoshi, S. Ishida, and R. Nakajima, J. Chem. Eng. Jpn., 2008, 41, 130.
[9] M. Tabata, Y. G. Wu, T. Charoenraks, and S. S. Samaratunga, Bull. Chem. Soc. Jpn.,
2006, 79, 1742.
[10] M. Macka, W-C. Yang, P. Zakaria, A. Shitangkoon, E. F. Hilder, P. Andersson, P.
Nesterenko, and P. R. Haddad, J. Chromatogr. A, 2004, 1039, 193.
[11] K. Tsukagoshi, S. Ishida, Y. Oda, K. Noda, and R. Nakajima, J. Chromatogr. A,
2006, 1125, 144.

10

Chapter 1

10Chapter 2

Chapter 2 Observation and characterization of tube radial
distribution phenomenon (TRDP)
Tube radial distribution phenomenon (TRDP), including inner and outer phaseformation, was created in various types of microspaces, such as microchannels on a
microchip and capillary tubes, under laminar flow conditions. The TRDP was observedwith fluorescence photographs through a fluorescence microscope-CCD camera system,
and mainly characterized with the elution behavior of analytes in the TRDP-based
chromatographic separation system, that is, tube radial distribution chromatography(TRDC). The part of this chapter is reconstructed and rewritten based on the related
manuscripts that have been published.6,14,17,38)

2.1 Observation and characterization of TRDP with fluorescencemicroscope-CCD camera system
We developed a capillary chromatography system by using an open capillary tube
made of fused silica, polyethylene, or polytetrafluoroethylene, and awater-hydrophilic/hydrophobic organic mixture carrier solution, called tube radial
distribution chromatography (TRDC) system. Comparing with chromatograms
obtained via the TRDC system, fluorescence photographs and profiles of thefluorescent dyes dissolved in the carrier solvents in capillary tubes were observed
under laminar flow conditions. The chromatograms were obtained for a model
mixture analyte consisting of 1-naphthol and 2,6-naphthalenedisulfonic acid with the
TRDC system, by using a fused-silica capillary tube and a water–acetonitrile–ethyl
acetate carrier solution. By altering the carrier flow rates, we were able to examine
the fluorescence both the photographs as well as the profiles of the dyes, perylene
and Eosin Y, which were dissolved in the carrier solvents in the capillary tube withthe use of a fluorescence microscope equipped with a CCD camera. As confirmed byfluorescence observations, the major inner and minor outer phases generated in thecapillary tube were based on the tube’s radial distribution of the carrier solvents. We
designed and manufactured a microchip incorporating microchannels in which three
narrow channels combined to form one wide channel. When the carrier solvents
containing the dyes were fed into the channels, the inner and outer phase generations
were also observed in the narrow and wide channels, supporting our previous
conclusions concerning the tube radial distribution phenomenon (TRDP) of thesolvents.
Introduction
11

Observation and characterization of TRDP

11

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Analytical methods using capillary tubes, usually composed of fused silica, haveattracted great interest over the last century. Well-known methods include capillaryelectrophoresis [1,2], capillary electrochromatography [1,3], micellar electrokinetic
capillary chromatography [4,5], and high-performance liquid capillarychromatography using packed and monolithic columns[3,6]. However, only a few
new concepts concerning capillary chromatography have been reported in the last
decade [7,8]. Related research has been discussed in detail in our papers.2,10) We havedeveloped a capillary chromatography system using an open capillary tube and awater-hydrophilic/hydrophobic organic mixture carrier solution. This system works
under laminar flow conditions and requires no packed reagents in the capillary tube,
nor does it require the application of high voltage to the ends of the tube. Such asystem is called a tube radial distribution chromatography (TRDC) system.
The experimental device and data in the TRDC system have the following
features: (1) various usable types of capillary tubes, including fused-silica,
polyethylene, and polytetrafluoroethylene; (2) an aqueous-organic (homogeneous)
carrier solution in which water, hydrophilic, and hydrophobic organic solvents must
be delivered into the capillary tube under laminar flow conditions; (3) easily
reversible elution times of hydrophobic and hydrophilic analytes, changed by thecomponent ratios of the carrier solvents, e.g., an organic solvent-rich carrier solution(water–acetonitrile–ethyl acetate, 3:8:4 volume ratio) and a water-rich carrier
solution (water–acetonitrile–ethyl acetate, 15:3:2 volume ratio); and (4) a first peakwith nearly the average linear velocity and with subsequent peaks eluted withvelocities at lower than the average linear velocity under laminar flow conditions inboth the organic solvent-rich and the water-rich carrier solutions.
We delivered carrier solvents containing fluorescent dyes into a microchannel in a
microchip, and observed the fluorescence photographs of the dyes using afluorescence microscope equipped with a CCD camera.6) We found that the dyes
dissolved in the carrier solution were distributed radially, based on their hydrophilicor hydrophobic natures in the microchannel in the microchip. However, fluorescencephotographs in capillary tubes that were actually used in the TRDC system have yet
to be examined. As more information concerning the TRDC system is necessary toconfirm and further discuss the TRDP of the carrier solvents in a capillary tube,
direct fluorescence observations in the capillary tube in which the TRDC mode isbeing undertaken would provide visible information, so together withchromatographic data, it would be very benefitial to the TRDC investigation.
Here, we examined separation performances in the TRDC system from thechromatograms obtained for model mixture analytes, and the fluorescencephotographs and profiles obtained through a fluorescence microscope equipped witha CCD camera in a fused-silica capillary tube that was used under the TRDC
separation mode. Furthermore, we redesigned the microreactor, incorporating

12

Chapter 2

12microchannels. The distribution phenomena of the carrier solvents that were fed into
the channels under laminar flow conditions were examined with a fluorescencemicroscope equipped with a CCD camera.
Experimental
Materials Fused-silica capillary tubes (75 µm i.d.) were used. Fig. 1 illustrates the
microchip that we designed. The three narrow channels (each 100 µm-wide × 40
µm-deep) combined to form one wide channel (300 µm-wide × 40 µm-deep) in the
microchip. Herein, we designate these channels as channels N1–N3 and channel W,
respectively. Also, the combining points are designated as points 1 and 2,
respectively, as shown in Fig. 1.
TRDC system with absorption detection
An open, fused-silica capillary tube (75μm i.d., 120 cm length; 100 cm effective length), a microsyringe pump, and anabsorption detector made up the TRDC system with absorption detection.2,10) The
tube temperature was controlled by immersing the capillary tube in water maintained
at 15 °C in a stirred beaker. We used water–acetonitrile–ethyl acetate mixtures involume ratios of 3:8:4 and 15:3:2 as carrier solutions. We prepared analyte solutions,
including 1-naphthol and 2,6-naphthalenedisulfonic acid, with the carrier solutions.
The analyte solution was introduced directly into the capillary inlet side via thegravity method (20 cm height for 30 s). After analyte injection, the capillary inlet
was connected through a joint to a microsyringe. The syringe was set on the
microsyringe pump. The carrier solution was fed into the capillary tube at acontrolled flow rate (0.8 µL min 1) under laminar flow conditions. We performed
on-capillary absorption detection (254 nm) with the detector.
−

Fluorescence microscope equipped with a CCD camera system
We set up a
fused-silica capillary tube the same size (75 µm i.d., 120 cm length) as that of the
TRDC system with absorption detection, and a microchip incorporating the
microchannels for the fluorescence microscope-CCD camera system. The
fluorescence in the capillary tube was mainly monitored at approximately 100 cm
from the capillary inlet (the focus depth, ca. 6.1 µm, under the present conditions in
the microscope device was adjusted to the center of the capillary tube), and thefluorescence in the microchannel was monitored around the combining points using
a fluorescence microscope equipped with an Hg lamp and a filter and CCD camera.
The organic solvent-rich carrier solution (water–acetonitrile–ethyl acetate; 3:8:4
volume ratio) contained 0.1 mM perylene and 1 mM Eosin Y, while the water-richcarrier solution (water–acetonitrile–ethyl acetate; 15:3:2 volume ratio) contained 1
mM Eosin Y (perylene was little dissolved in this solution). We delivered the carrier
13

Observation and characterization of TRDP

13

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

solution into the capillary tube, or into the microchannel at a definite flow rate using
the microsyringe pump. The fluorescence photographs thus obtained were alsotransformed into line drawings to assess color depth using a computer. The colors
observed on the photographs were divided into red, green, and blue (RGB). The
photographs mainly consisted of blue and green because perylene and Eosin Y emit
light at approximately 470 and 550 nm, respectively. Blue and green color depths
were expressed numerically as digital data on a computer, and finally, the numbers
were standardized to the line drawing data to give the fluorescence profiles.
Results and discussion
Chromatograms obtained with the TRDC system with absorption detection
We analyzed a model mixture solution of 1-naphthol and 2,6-naphthalenedisulfonicacid by using the present TRDC system with absorption detection. Fig. 2 shows theobtained chromatograms. Fluorescence photographs and profiles in the capillarytubes are examined under the same analytical conditions as those acquired using the
TRDC system in the following sections. By using the organic solvent-rich carrier
solution of water–acetonitrile–ethyl acetate in a volume ratio of 3:8:4, the mixture of
1-naphthol and 2,6-naphthalenedisulfonic acid was separated through the open
capillary tube and detected in the stated order (Fig. 2a). In contrast, using thewater-rich carrier solution of water–acetonitrile–ethyl acetate in a volume ratio of
15:3:2, the mixture solution was separated and detected with inverse elution times,
i.e., 2,6-naphthalenedisulfonic acid and 1-naphthol were detected in this order (Fig.
2b). We confirmed the components of the analytes on the chromatograms from theirindividual absorption signals. In both chromatograms, we detected the first peaks
with elution times roughly corresponding to the average linear velocities underlaminar flow conditions, and the second peaks were eluted with velocities below theaverage linear velocities. When we used the organic solvent-rich carrier solution, thecomparatively hydrophobic analyte was eluted first. In contrast, when we used thewater-rich carrier solution as the carrier solution, the comparatively hydrophilic
analyte was eluted first. That is, the chromatograms clearly indicated reversible
elution times, by altering the component ratio of the solvents in the carrier solution.
The results shown in Fig. 2 are consistent with the tube radial distribution behaviorof the carrier solvents in the TRDC system that we proposed in our reports earlier. In
addition, as mentioned in the Introduction, the outer phase in the TRDC system
works as a stationary phase in the usual chromatography. But the outer phase in the
TRDC is very specific as a stationary phase; it comprises the carrier solvents,organic solvent-rich or water-rich. So, the outer phase in the system features large
diffusion coefficient and large thickness of µm order, compared to the usual
stationary phase in chromatography. The large diffusion coefficient and thickness ofthe outer phase that works as a stationary phase in the TRDC system might lead to

14

Chapter 2

14the broadening in the second peaks as shown in Fig. 2.
Fluorescence photographs and profiles of the dyes in a capillary tube
We
examined the fluorescence photographs and profiles of the fluorescent dyes
dissolved in the aqueous-organic solvent mixture carrier solution in the
microchannels (the cross-sectional view was a rectangle, 100 µm-wide × 40
µm-deep) in a microchip through the fluorescence microscope equipped with a CCD
camera. We reported the preliminary data in our communication.6) Here, we
attempted to examine the fluorescence photographs and profiles in the fused-silica
capillary tube, although the cross section of the tube (circular) made it difficult toobserve them compared to the microchannels (rectangular). The experiment using
the fluorescence-CCD camera system with the fused-silica capillary tube was
performed under the same analytical conditions, including the capillary tube length,
the tube inner diameter, and the flow rate, as the TRDC system with absorption
detection to give the chromatograms shown in Fig. 2. Fig. 3 depicts the fluorescencephotographs and profiles observed for the fused-silica capillary tube in which thedye-containing aqueous-organic solvent mixture carrier solutions were delivered.
These solutions were observed in the capillary tube using the TRDC analytical mode.
Fluorescence photographs and profiles for the organic solvent-rich carrier solution
showed that the hydrophobic perylene molecule (blue) was distributed around thecenter of the capillary tube and away from the inner wall of the tube, while thehydrophilic Eosin Y molecule (green) was distributed near the tube’s inner wall (Fig.
3a). In contrast, fluorescence photographs and profiles for the water-rich carrier
solution showed that Eosin Y was distributed around the center of the capillary tube
(Fig. 3b). Due to the low solubilty of perylene, we were unable to obtain any
information regarding it’s behavior in a water-rich carrier solution.We could not
obtain any information regarding the behavior of perylene in the water-rich carrier
solution because of its very low solubility in that solution. The tube radial
distribution of perylene and Eosin Y present in the carrier solution thus confirmed
the partition reversal between the organic solvent-rich and water-rich carrier
solutions in the fused-silica capillary tube (Fig. 3). Furthermore, using the same
analytical condiitons, we tried to observe the fluorescence photographs and profiles
from other parts of the capillary tube (effective lengths of 30 and 60 cm). under thesame analytical conditions. The obtained fluorescence photographs and profiles
obtained from this further inquiry are shown in Fig. 4. These data allowed us toconfirm the tube radial distribution of the fluorescence dyes in the capillary tube at
the effective lengths of 30, 60 and even 100 cm. The distribution behavior observed
for the fluorescent dyes dissolved in the carrier solution in the capillary tube wasconsistent with the tube radial distribution of the solvent molecules proposed for the
TRDC system. That is to say, the distribution of perylene and Eosin Y at the center
15

Observation and characterization of TRDP

15

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesof the capillary tube, or near its inner wall supports the non-uniform dispersion of theofthe capillarytube, ornear its inner
wallcarrier
supports
the non-uniform
dispersionof theaqueousand organic
componentsof thesolutionsin the capillary
tube in
TRDC
Theycomponents
each generatea majorphasein (organic
solvent-richoraqueoussystem.
and organicof thecarrierinner
solutionsthe capillary
tube in the
TRDC system.
each outer,
generatemajor innersolvent-richorwater-rich)and Theya minoror acapillarywall phase
phase (organic(water-richor organic
solvent-rich)andin thecapillarytube.orAscapillarya result, thedistributed
water-rich)a minorouter,
wallanalytes
phase are(water-richor between
organic
theinner andin outerphases tube.
dependingon theirnatures are
and distributed
are separable
via
solvent-rich)the capillary
As a result,the analytes
betweentheinner and outerphases dependingon their naturesand inare Fig.
separablevia
chromatography,
corresponding
to the chromatogramsshown
2. In our11)
the mixtureof perylene and
Eosin
as an
paper, we examinedchromatography,
corresponding
to solution
the chromatogramsshown
in YFig.
2. analyte
In our11)
examined
mixturesystemsolutionequippedof perylenewith
and Eosin
Y as an analyte
paper,
solution weusing
the theTRDC
a fluorescenceorchemiluminescence
detector.
Peryleneand Eosin
Y wereseparatedand detected orinsolutionusing theTRDC
systemequippedwitha fluorescence
this order with the detector.
organic solvent-richsolution,
and wereandelutedin thechemiluminescence
Perylene andcarrier
Eosin Y
were separated
detected
in
this
order
withwiththe theorganic
solvent-richsolution,
and were eluted
thereverseorder
water-richcarrier carrier
solution.
The chromatograms
datain also
verified orderthat perylene
Eosin Y actedhydrophobichydrophilic molecules,
reversewith theandwater-richcarrieras solution.
Theandchromatograms
data also
verifiedthat perylene
and Eosin
acted solutions
as hydrophobic
and hydrophilic
respectively,in the ternary
mixedYcarrierin the TRDC
system. molecules,
respectively, in the ternary mixed carrier solutions in the TRDC system.
Effects of flow rates on the tube radial distribution of the dyes in the capillary tube
Effectsof flowthe
rates
on thetube rate
radialof thedyes in thecapillary
We examined
effectsof flow
on distribution
the tube radial
distributionof thedyes intube
thecapillarytube theusing
an organic
carriersolutioncontaining
peryleneWe
examined
effectsof flowsolvent-richrate on the tube
radialdistributionof thedyes in andthecapillaryusing an organic
solvent-richcontaining
and
Eosin
Y. tubeThe fluorescencephotographs
and carrier
profilessolution
were observedfor perylene
the capillary
tube
thefluorescencedye-containing
organic solvent-richsolution wasdeliveredat
Eosinwhere
Y. Thephotographs
and profilescarrier
were observed
for thecapillary1
tube where
the dye-containing
solutionthesewas images.
deliveredatflowrates varying
from 0.5 to organic
3.0 μL solvent-rich
min . Fig. 5carriersummarizes
The
1
flow rates fluorescencevarying from photographs
0.5 to 3.0 μLand
minprofiles. Fig. 5 showedsummarizes
images. The
observedthat these
the hydrophobic
observed
fluorescenceand aroundprofilestheshowed
thatthethehydrophobic
perylene molecule(blue)photographs
was distributed
center of
tube,
while thehydrophilic
Eosin Y(blue)
molecule
was distributed
theof
tube’sinner while
wall overperylenemoleculewas (green)
distributed
around the near
centerthe tube,the1hydrophilic
Eosin
Y molecule
nearwethedidtube’s
wall
overa
the
flow rates
between
0.5 and(green)
2.0 μLwas
mindistributed. In contrast,
not innerobservesuch
1
tubeflowradialdistributionof thedissolved
in contrast,
the carrierwesolution
flow rates
the
rates
between 0.5
anddyes
2.0 μLmin . In
did not atobserve
suchofa
1
tube
radialof thedyes dissolvedin the carrier
solutionat flow
rates of
2.5 and
3.0 distribution
μL min . The
fluorescencephotographs
and profiles
clearly
confirmed
1
2.5 and3.0 μL solution
min . The
fluorescencephotographs
and profilesforclearly
confirmed
that
the carrierin thecapillary tube
was heterogeneous
flow rates
0.5 –
1
1
that μL
themincarrierin the capillaryheterogeneousfor flow
0.5of–
andsolution
homogeneousfor flow tube
rateswasof 2.5and 3.0 μL min. Therates
degree
2.0
1
1
2.0
and
homogeneousfor flow
ratesunderof 2.5laminarand 3.0flow
μL min. The degreeofthe μL
flowminrate
or pressure
in capillary
tubes
conditions
togethertheor pressure
in ofcapillarytubes solvents
under laminar
withflow
the ratecomponent
ratiosthe carrier
must flow
have conditions
important together
roles indetermining
the tube radialof thesolvents
carrier solventsin the
TRDC system.
withthe component
ratiosdistributionof the carrier
must haveimportantroles in
Fluorescencethe
photographs
profiles ofdissolved
thethewater–acetonitrile
determining
tube radial anddistributionof thethedyescarriersolventsinin
TRDC system.
Fluorescencephotographs
and profiles
the dyes dissolvedin the water–acetonitrile
mixture carrier
solution without
ethyl ofacetate
We confirmedthat the analytes
were notcarrier
separatedon thechromatograms
usingWe
anyconfirmed
water–acetonitrile
mixturemixturesolution
without
ethyl acetatethat the analytes
wereseparatedchromatograms
usinginany
water–acetonitrile
mixturecarriernot
solutionsthat ondid thenot include
ethyl acetate
all typesof capillary tube,i.e.,
fused solutions
silica, polyethylene,or polytetrafluoroethylene.
fluorescencecarrierthat did not include
ethyl acetate in all types ofHere,capillarytube, i.e.,
photographs
profiles of theor dyes
dissolved in the water–acetonitrile
carrierfusedsilica,andpolyethylene,
polytetrafluoroethylene.
Here, fluorescencephotographs
and profilesof the weredyes dissolvedin the throughwater–acetonitrile
carriersolution without
ethyl acetate
also examined
the fluorescencesolution without ethyl acetate were also examined through the fluorescence
−

−

−

−

−

−

16

Chapter 2

−

−

−

−

16
16

mixture carrier solution without ethyl acetate
We confirmed that the analytes
were not separated on the chromatograms using any water–acetonitrile mixture
carrier solutions that did not include ethyl acetate in all types of capillary tube, i.e.,
fused silica, polyethylene, or polytetrafluoroethylene. Here, fluorescencephotographs and profiles of the dyes dissolved in the water–acetonitrile carrier
microscope
equipped
withacetate
a CCDwerecamerathe sameanalyticalassolution without
ethyl
alsounder
examinedthroughthe conditions
fluorescencemicroscope
equippeda CCDcamera theundertheradial
samedistribution
analytical conditionsas
those in Figs.
2 and 3.with
We didnot observe
tube
of the carrier
solvents
for thewater–acetonitrile
ethyl acetateofatthe
anycarrier
flow
those in Figs.
2 and
3. We did not carrier
observesolution
the tubewithout
radial distribution
solvents
the water–acetonitrile
carrier16solution
without
ethyl acetate
at any flow
rate. Fig.for6 shows
a typical fluorescencephotographand profile.
The hydrophobicorganic
acetatewas necessary
preparation
of an
rate.
Fig. solvent
6 shows ethyl
a typical
fluorescencephotographfor
and theprofile.
The hydrophobic
aqueous-organic
the TRDCforsystem.
organic
solvent mixture
ethyl carrier
acetatesolution
was innecessary
the preparation of anaqueous-organic mixture carrier solution in the TRDC system.
Fluorescence photographs of the dyes in the microchip
Fluorescencephotographs ofthe dyesin the (water–acetonitrile–ethyl
microchip
The organic solvent-richcarriersolutionacetate, 3:8:4
volumeratio) solvent-richcontaining Eosin
Y andperylene
was delivered into a newly
designed
The organic
carriersolution(water–acetonitrile–ethyl
acetate,3:8:4
microchip,fromthe channels(N1-N3)to the onewas
wide
channel,into
channelW, designed
at a flow
volumeratio)
containing
Eosin
Y and perylenedelivereda newly1
microchip,
the channels
the one We
wideobserved
channel, thechannelW, at a dyes
flow
rate
of 5.0 fromμL minfor each(N1-N3)
channeltoN1–N3.
fluorescent1
rate
of 5.0 inμLthe
minaqueous-organicfor each channel
N1–N3.
We observed
fluorescent dyes
dissolved
solvent
mixturein the themicrochannels,
andmonitored inthemthe microscope-CCD
camera system.
7a depicts andthedissolved
the withaqueous-organic
solvent mixture
in the Fig.
microchannels,
monitored
them with themicroscope-CCD
camerasystem.
Fig.together
7a depicts
obtained fluorescencephotographof the dyesin thechannelwith theits
analytical fluorescenceconditions. The
photographthatinin theeachchannel
narrow together
channel, withchannelobtained
photographof provesthe dyes
its
N1–N3, perylene
molecule(blue) was proves
distributed
centerof the channel
analyticalconditions.
The photographthat aroundin each thenarrowchannel,N1–N3,
perylenemolecule(blue)
waswall,
distributed
center of (green)
the channel
and awayfrom thechannelinner
while around
Eosin Ythemolecule
wasdistributed
distribution
the carrier
andaway nearfrom thethechannel’schannel inner
innerwall.
wall,The
while
Eosin Yphenomena
molecule of(green)wasdistributed
inner clearlywall. The
distribution
phenomena
the carrier
solvents fednear
intothethechannel’s
microchannels
existedunder laminarflow ofconditionsin1
solvents
fed into
the microchannels
clearly
existedineach narrow
channel,channels N1–N3.
The
flow underrate oflaminar
5.0 μLflow
minconditions(the linear
1-1lineareach narrow
channel,The flowof 5.0velocityof 125cm minchannels) in the N1–N3.
microchannels
wereratelargerthanμLtheminflow (therate of2.5-1
1-1
velocity
125
cm min) in ofthe57microchannels
werelarger than
the flow
rateprovideof 2.5μL
min of(thelinearvelocitycm min ) in thecapillarytube that
did not
1
μL(the linear
ofouter
57 cmphases
min -1)ininthe
thetube.
capillarytube that
did not must
providethe minformationof the velocity
inner andThe phaseformationbetheformation
the innerand view,
outer phasesin thephase formation
mustinbea
influenced
by of
cross
sectional
flow rate(or tube.
linearThevelocity),and pressure
influenced
by cross
sectionalview, flowthat
rateflow(or rates
linearlarger
velocity),
andµLpressurechannel or tube.
Actually
we confirmed
than 50
min-1 ininthea-1channel or tube.could
Actually
we confirmedthat formations
flow rates larger
thanthe50 innerµL mininthemicrochannels
not form
stable phaseincludingandouterphasesin the microchannels.
We observed
an interesting
behavior
for themicrochannels
could not form stablephase formations
includingthe innerandminorouterphasesin theinmicrochannels.
interesting
behavior
forN1
theandminor
outer phases
channel N2 andWe
forobserved
the minoranouterphases inchannels
N3,
whichphases
were close
to channel
N2.forTheouterin channel
N2 andtheminorminorouter
outerphases
phasesininchannel
channelsN2N1combinedand N3,
which
were
close
to channel
The minorouter
in channel
combinedwith the
minor
outer
phases inN2.
channels
N1 andN3phasesat points
1 and 2,N2
respectively.
That theis, minor
the twoouterminor
outerin phases,
N11 andN2, orwithphaseschannelscombining
N1 and N3channel
at points
and channel
2, respectively.
channelN3, combinedat respective
combining
points,pointN2,
1 and
Thatis, N2the andtwochannel
minor outerphases, combining
channelN1 andchannelorchannelandcombined
channel N3,
combined
at respective
combining points,
point
1 and
point 2. N2
These
minor
outer phases
were consecutively
deliveredintothewide channel
laminarflow outerconditions,they consecutively
were diffused deliveredin channelinto
W steppoint
2. Theseunder
combinedminor
phasesaswere
thewidechannelunderflowminor
conditions,as theydisappeared
were diffusedchannel aroundW stepby step.
Finally,thelaminarcombinedouter phases
at ina distance
ca.
10 mm
from thethe combined
points 1 and2 inouterchannelW. disappeared
The organicatsolvent-richcarrierby step.
Finally,minor
phasesa distance aroundsolutioncontaining
dyes 1wasthe microchip
at a low flowrate
ca. 10 mm
from thethe
pointsandalso
2 indelivered
channel into
W. Theorganic solvent-richcarrier1solution
the dyes
wasN1–N3.
also delivered
into thethe
microchipat adyes
low dissolved
flow rateof
2.0 μLcontaining
min for eachchannel
We observed
fluorescentof 2.0 μL min 1 for each channel N1–N3. We observed the fluorescent dyes dissolved
−

−

−

−

−

−

−

−

17
17

Observation and characterization of TRDP

17

point 2. These combined minor outer phases were consecutively delivered into thewide channel under laminar flow conditions, as they were diffused in channel W step
Tube
Distribution
Phenomenon(TRDP)disappearedof Solvent Molecules
by step. Finally,
theRadial
combinedminor
outer phases
at a distance aroundca. 10 mm from the points 1 and 2 in channel W. The organic solvent-rich carrier
solution containing the dyes was also delivered into the microchip at a low flow rate
in 2.0the μLaqueous-organic
microchannels,
and monitored
them
channel mixture
N1–N3. in
Wethe
observed
the fluorescentdyes dissolvedofmin 1 for each solvent
inthetheaqueous-organic
solvent
mixturein Fig.
the microchannels,
and monitored
them
with
microscope-CCD
camerasystem.
7b shows the obtained
fluorescencephotographof the dyes incamera
the channels.
The 7bobserved
fluorescencephotographwith the microscope-CCD
system. Fig.
shows theobtained fluorescencephotographthe rate
dyesofin2.0theobserved
obtained at aofflow
μLchannels.
min 1 wassimilarto that fluorescenceat a flow ratephotographof 5.0 μL
17 The11obtained
at a forflowtherateof 2.0 μL min
similarthat at arounda flow therate combiningof 5.0 μL
min, except
combinationof thewasminoroutertophasespoints.
Both offorthetheminor
outer phases
in channel
N2 werecombined
the minor
min 1, except
combinationof theminor outerphasesaround withthe combining
points.phases
Both ofminorN1
outer
in werechannelN2towerecombinedwith the
minor
outer
in thechannelsandphases
N3 thatclose
channelN2 at points
1 and2,
respectively.
After
the twoouter
were,torespectively,
theirouter phases inchannels
N1minorand N3
thatphases
were close
channel N2 combinedat points 1atand2,
respectively.
Afterthethecombined
two minorphases
outer formed
phases were,respectively,
their
combining points,
oval ballsat points 1combinedand 2, as atshownin Fig. 7b. points,
After athetime,
the ballsphases
were formed
releasedoval
fromthe atpoints
werecombiningcombinedballs
pointsand
1 and2, delivered
as shown
intochannelW underflow
conditions.
They thediffused
W and
in Fig.
7b. Aftera time,laminar
the balls
werereleased frompointsinto
and channel
were delivered
intochannelW underThelaminarflow conditions.
Theyofdiffused
intodissolved
channel W
finally
disappeared.
fluorescencephotographs
the dyesin andthesolventsdisappeared.
strongly supported
the fact thatphotographs
the tube radial
distributionof the ternary
finally
The fluorescenceof thedyes dissolvedin thesolvents
strongly
supported
the factout
thatinthetube radial such
distributionof the ternary
mixed carrier
solvents
was carriedmicro-spaces
as the capillary
tubes
and microchannels,
underlaminarmixed
carrier solventswascarriedflow
out conditions.
in micro-spaces such as the capillary tubes
and microchannels, under laminar flow conditions.
In conclusion, the inner and outer phase formation, based on the tube radial
distributionof the thecarriersolvents,
was phase
consideredthroughthe elutionof
In conclusion,
innerand outerformation,
basedon the behavior
tube radial
distribution
solvents, wasconsideredthroughthedyes
elutionbehavioroftheanalytes ofon thethe carrier
chromatograms,
and thedistributionof thedissolved
in theaqueous-organic
solvent
mixture carrier
solutionin the capillarytube.
We observed
theanalytes on thechromatograms,
and thedistributionof the dyes
dissolved
in thethetube radial distributionof the solvents,
TRDP, inthrough
the fluorescentin theaqueous-organic
solvent mixture
carrier solution
the capillary
tube. Wedyes
observed
the tuberadial distribution
the solvents,
TRDP,
through theFurthermore,
fluorescent dyes
realcapillarytubes underofvarious
analyticalconditions.
whenin the
carriersolventstubes
containing
dyes were
fed intoconditions.
the microchannels
in the microchip,realcapillaryunderthevariousanalytical
Furthermore,when thecarrier
containing
the dyesphenomena
were fed into
in themicrochip,
we alsosolvents
observed
the distributionof thethe microchannels
carrier solvents,
TRDP,
in themicrochannels,
includingboth thephenomena
narrow andofwide
scales. solvents,
The fluorescence
we also observed
the distribution
the carrier
TRDP, indata
theobtained experimentally
separation
performancebaseddataon
microchannels,
includingwere
both consistent
the narrowwith
andthewide
scales. The
fluorescenceobtained
experimentallyconsistent
with theSuch
separation
performancebased onthe tube radial
distributionwereof thecarrier solvents.
visual dataof fluorescenceincapillary
and the microchannels
should bea useful,and powerful
the tuberadialtube
distributionof the carrier solvents.
Suchvisualeffective,
data of fluorescenceinthe capillary
the study.
microchannels should be a useful, effective, and powerful
tool
for TRDPtube
andandTRDC
tool for TRDP and TRDC study.
−

−

−

−

−

18

Chapter 218
18

Figure 1. Illustration of a newly designed microchip incorporating microchannels.

Figure 2. Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidobtained by the TRDC system via absorption detection. Conditions: Capillary tube, 120
cm (effective length: 100 cm) of 75 µm i.d. fused-silica; carrier, (a)water–acetonitrile–ethyl acetate (3:8:4 v/v/v) mixture solution and (b)water–acetonitrile–ethyl acetate (15:3:2 v/v/v) mixture solution; sample injection, 20 cm
height (gravity) × 30 s; flow rate, 0.8 µL min 1; tube temperature, 15 °C; and analyte
concentration, 1 mM.
−

19

Observation and characterization of TRDP

19

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3. Fluorescence photographs and profiles of the fluorescent dyes dissolved in the
aqueous-organic solvent mixture carrier solution in a fused-silica capillary tube.
Conditions: Capillary tube, 120 cm (effective length: 100 cm) of 75 µm i.d. fused-silica;
carrier, (a) water–acetonitrile–ethyl acetate (3:8:4, v/v/v) mixture, including 0.1 mM
perylene and 1 mM Eosin Y and (b) water–acetonitrile–ethyl acetate (15:3:2, v/v/v)
mixture, including 1 mM Eosin Y; and flow rate, 0.8 µL min 1.
−

Figure 4. Fluorescence photographs and profiles of the fluorescent dyes at effective
lengths of 30 and 60 cm in fused-silica capillary tubes. Conditions: Capillary tube, 120
cm (effective length: 30 and 60 cm) of 75 µm i.d. fused-silica; carrier,water–acetonitrile–ethyl acetate (3:8:4, v/v/v) mixture, including 0.1 mM perylene and
1 mM Eosin Y; and flow rate, 0.8 µL min 1.
−

20

Chapter 220

Figure 5. Fluorescence photographs and profiles of the fluorescent dyes dissolved in the
aqueous-organic solvent mixture carrier solution at the various flow rates. Conditions:
Capillary tube, 120 cm (effective length: 100 cm) of 75 µm i.d. fused-silica; carrier,water–acetonitrile–ethyl acetate (3:8:4, v/v/v) mixture, including 0.1 mM perylene and
1 mM Eosin Y; and flow rate, 0.5–3.0 µL min 1.
−

Figure 6. Fluorescence photographs and profiles of the fluorescent dyes dissolved in the
aqueous-organic solvent mixture carrier solution without ethyl acetate. Conditions:
Capillary tube, 120 cm (effective length: 100 cm) of 75 µm i.d. fused-silica; carrier, thewater–acetonitrile (1:4, v/v) mixture, including 0.2 mM perylene and 1 mM Eosin Y;
and flow rate 0.8 µL min 1.
−

21

Observation and characterization of TRDP

21

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 7. Fluorescence photographs of the fluorescent dyes dissolved in the
aqueous-organic solvent mixture carrier solution in a microchannel. Conditions:
Microchannels, channels N1, N2, and N3 (100 µm-wide × 40 µm-deep, each) andchannel W (300 µm-wide × 40 µm-deep) made of glass; carrier,water–acetonitrile–ethyl acetate (3:8:4, v/v/v) mixture, including 0.1 mM perylene and
1 mM Eosin Y; and flow rates (a) 5.0 and (b) 2.0 µL min 1 for channels N1–N3.
−

22

Chapter 222

2.2 Observation and characterization of TRDP with various flow rates
and capillary tube lengths
When ternary mixed solutions of water-hydrophilic/hydrophobic organic solvents are
fed into a microspace under laminar flow conditions, the solvent molecules are radiallydistributed in the microspace. The specific fluidic behavior of the solvents is termed
tube radial distribution phenomenon (TRDP). Here, water–acetonitrile–ethyl acetate
mixed solutions (3:8:4 volume ratio) containing fluorescent dyes, perylene (0.1 mM)
and Eosin Y (1 mM), were fed into fused-silica capillary tubes (75 µm i.d.) forinvestigating the TRDP with a fluorescence microscope-CCD camera under various
analytical conditions. The pressure at the observation point for the fluorescence on the
tube was changed by altering the flow rates, capillary total lengths, and capillaryeffective lengths. The obtained fluorescence images showed that the TRDP in thecapillary tube created the inner and outer phases, i.e., the organic solvent-rich major
inner and the water-rich minor outer phases in this case, providing the kinetic
liquid-liquid interface. The formation of the inner and outer phases in the capillary tube
was observed at flow rates of 0.5–2.0 µL min−1 under the present analytical conditions.
We also discuss the specific formation locations of the major and minor solvents.
Introduction
Various types of aqueous–organic solvent mixed solutions are used in dissolution [9,10],cleaning [11], preservation [12], and as reaction solvents [13,14]. Such mixed solutions
are also useful in separation science [15-17] as extraction solvents and in liquid
chromatography. In liquid chromatography, hydrophilic and hydrophobic organic
solvent mixtures are used as carrier solutions in normal phase chromatography, whilewater and hydrophilic organic solvent mixtures are used in reverse-phase
chromatography.
However,todate,ternary
mixed
solutionsofwater–hydrophilic/hydrophobic organic solvents have not been examined in detail for
use as carrier solutions in liquid chromatography, or even for the other way around.
When ternary mixed solutions of water–hydrophilic/hydrophobic organic solventsare fed into a microspace, such as microchannels in a microchip or capillary tubes,
under laminar flow conditions, the solvent molecules are radially distributed, regardlessof the section shape and the inner wall material in the microspace. This is called thetube radial distribution phenomenon (TRDP). In the TRDP, with the organic
solvent-rich solution, the organic solvent-rich major phase is generated around the
middle of the microspace as an inner phase, while the water-rich minor phase is formednear the inner wall as an outer phase. On the other hand, with the water-rich solution thewater-rich major phase is generated as an inner phase, while the organic solvent-richminor phase is formed as an outer phase. The phenomenon has been supported by
23

Observation and characterization of TRDP

23

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

fluorescence studies of dyes dissolved in ternary mixed solvents and the elution
behavior of hydrophilic and hydrophobic solutes in capillary tubes under laminar flow
conditions.
The phase diagrams for ternary mixed solvents of water, acetonitrile, and ethyl
acetate were examined for TRDP and TRDC in our paper, and the results indicated thatthe pressure change in the capillary tube under laminar flow conditions might alter the
carrier solution from homogeneous to heterogeneous, thus affecting the tube radial
distribution of the solvents in the capillary tube. Therefore, here, we investigated theconditions needed for the formation of the inner and outer phases created through TRDP
in the capillary tubes.
Experimental
Materials
Fused-silica capillary tubes (75 µm i.d.), PTFE capillary tubes (100 µmi.d.), and a microchip made of glass with an incorporated microchannel line (100 µm
wide × 40 µm deep) were used.
Pressure applied in batch vessels Water–acetonitrile–ethyl acetate homogeneous
solutions were prepared in different volume ratios (3:8:4, 3:8:3, and 3:8:2). Eachhomogeneous ternary mixed solution (3 mL) was put in a glass open batch vessel (5mL volume), which was then placed in a pressure-resistant glass vessel (500 mL
volume). Pressures of 1.1 – 1.4 × 105 Pa (cf. atmospheric pressure, 1.01 × 105 Pa)
were applied to the solution with nitrogen gas.
Fluorescence images The fluorescence microscopy setup with either a fused-silica
capillary tube, PTFE capillary tube, or a microchip incorporating a microchannel isshown in Fig. 1. The fluorescence in the capillary tubes or microchannels wasmonitored at various points using a fluorescence microscope equipped with an Hg lamp,
a filter, and CCD camera. The effective capillary length, indicated in Fig. 1, is the
distance from the fluorescence observation point to the capillary outlet. The ternary
mixed solvent solutions were delivered into the capillary tube or the microchannel using
a microsyringe pump.
Results and discussion
Dye fluorescence Our studies6,14) showed that fluorescence from both dyes could bemonitored in organic solvent-rich solutions of water–acetonitrile–ethyl acetate with a
3:8:4 volume ratio. However, in water-rich solutions of water–acetonitrile–ethyl acetatewith a 15:3:2 volume ratio, only the fluorescence from Eosin Y (1 mM) was observed as
the perylene was only slightly dissolved into the solution. Therefore, here, for both dyes
to be dissolved in water–acetonitrile–ethyl acetate mixed solutions, the water-richsolution had an 80:20:9 volume ratio. The fluorescence photographs in a microchannel

24

Chapter 224for the organic solvent-rich solution (3:8:4 volume ratio) containing perylene (0.1 mM)
and Eosin Y (1 mM), and the water-rich solution (80:20:9 volume ratio) containing
perylene (0.1 mM) and Eosin Y (0.1 mM) are shown in Figs. 2a and 2b, respectively.
The water-rich solution (80:20:9 volume ratio) with perylene (0.1 mM) and Eosin Y(0.5 mM) in a PTFE capillary tube is shown in Fig. 2c. In the water-rich solutions, thecomparatively hydrophilic Eosin Y was distributed around the middle of the microspaceof the microchannel and the capillary tube, and the hydrophobic perylene wasdistributed near the inner wall. The fluorescence photographs, in Figs. 2b and 2c,
illustrate TRDP with two-colors in water-rich solutions in a microspace.
Effect of pressure in a batch vessel A phase diagram of the water–acetonitrile–ethyl
acetate mixed solution is shown in Fig. 3. Solutions with volume ratios of 3:8:4, 3:8:3
and 3:8:2 are marked as solutions A, B, and C, respectively. It can be clearly seen thatupon decreasing the ethyl acetate volume fraction in the solution, the distance between
the homogeneous–heterogeneous solution boundaries increased. Under pressures of 1.1– 1.4 × 105 Pa, the phase behaviors of the solutions in open batch vessels wereobserved; the results are summarized in Table 1. In solution A the homogeneous
solution changed to a suspension solution in the pressure range of 1.1 – 1.15 × 105 Pa,
and then changed to a heterogeneous solution between pressures of 1.2 – 1.4 × 105 Pa.
In solution B, the homogenous solution changed to a suspension solution at pressures of
1.1 – 1.4 × 105 Pa, and solution C remained homogeneous. The suspension andhomogeneous solutions in solutions B and C, respectively, under these pressures werestable for ca. 10 min. From these results, it is clear that pressures above atmosphericpressure greatly influence any ternary mixed solvent solutions that lie near the
homogeneous–heterogeneous boundary curve in the phase diagram. As solution Achanges from homogeneous to heterogeneous under the applied pressures in the bulk
vessel, there is a possibility that pressure changes imposed on the solution in thecapillary tube under laminar flow conditions might alter the carrier solution fromhomogeneous in a microsyringe to heterogeneous in a capillary tube; this is discussed in
the following section.
Effects
capillary tube
tube Fluorescence
Effects ofof flow
flow rates in the capillary
Fluorescencephotographs
photographsand
and profiles,
profiles, shown
shown
in Fig. 4, were observed for a capillary tube in which the organic solvent-rich carrier
solution (volume ratio 3:8:4) containing perylene (0.1 mM) and Eosin Y (1.0 mM) was
delivered at flow rates of 0.5 – 3.0 μL min−1. The hydrophobic perylene molecules(blue) were distributed around the middle of the tube, while the comparativelyhydrophilic Eosin Y molecules (green) were distributed near the tube’s inner wall atflow rates of 0.5 – 2.0 μL min−1 (the inflections were observed on the fluorescenceprofiles of Eosin Y). However, at flow rates of 2.5 and 3.0 μL min−1, the tube radial
distributions of the dyes were not observed and the fluorescence profiles showed that25

Observation and characterization of TRDP

25

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

the dyes were homogeneously distributed in the ternary mixed solvent. Also, in the
carrier solution at flow rates of 0.1 and 0.2 μL min−1, we did not observe the tube radial
distributions; also, the fluorescence photographs in Fig. 5 show that the dissolved dyes
were distributed in the axial direction.
Relationships between the flow rates, pressures, and phase formation
The data
obtained in the previous section using an organic solvent-rich carrier solution (3:8:4,
volume ratio) containing perylene (0.1 mM) and Eosin Y (1 mM) in a capillary tube
are used to investigate the relationships between the flow rates, pressures, and phaseformation. The pressure in the capillary tube under laminar flow conditions wascalculated at each flow rate (0.1, 0.2, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, and 3.0 μL min−1)
using the following Hagen–Poiseuille equation:Q=

πa 4 Δp,
8µ L

where Q is the flow rate, a the inner radius, Δp the pressure (or pressure loss), µ the
viscosity, and L the effective capillary length (the length from the observation point
to the capillary outlet). The pressures in the capillary tube were calculated using Q, a,µ, and L values of 0.1 – 3.0 μL min−1, 37.5 µm, 6.89 × 10−4 kgm−1s−1, and 2.0 × 10−1
m, respectively. The formation/non-formation of the inner and outer phases
indicating TRDP creation and non-creation, respectively, are plotted in the pressurevs flow rate graph in Fig. 6. The capillary tube of length (110 cm) and the effective
length (20 cm) is represented as (110-20) in Fig. 6. From the data, it can be seen thatthe flow rate and pressure in a capillary tube, as well as the component ratios of themixed solvents play important roles in determining TRDP in the solvents.
Effects of flow rates and pressures in capillary tubes
The relationships between theflow rates, pressures, and phase formation created through TRDP were examined undervarious conditions in capillary tubes. The pressures were changed by altering the flow
rates as well as the effective capillary lengths. A 110 cm-long capillary tube was used,
and the effective length was varied over 5 – 80 cm (Fig. 6). A capillary tube of length
110 cm with different effective lengths of 5 – 80 cm are represented as (110-effective
length) in the figure. For flow rates of 0.5 – 2.0 µL min−1, at all effective variablecapillary lengths, the inner and outer phases formed in the capillary tube, even at very
low pressures (1.02 – 1.03 × 105 Pa; effective length 5 cm). However, at low flow rates
of 0.1 and 0.2 μL min−1 and at high flow rates of 2.5 and 3.0 μL min−1 there was no
inner and outer phase formation. Different capillary tubes of total length, 290 cm(effective length, 200, 230, and 260 cm), 350 cm (effective length, 260, 290, and 320cm), and 500 cm (effective length, 410, 440, and 470 cm) were also used. A longer
effective capillary length than 470 cm could not be used because of the26

Chapter 226

limitation of the microsyringe pump capacity.due to the limitations incapacity of the the microsyringe pump. From these results in Fig. 7, TRDP was
observed for flow rates of 0.5 – 1.5 μL min−1, and not at low flow rates of 0.1 and 0.2
μL min−1, or at high flow rates of 2.5 and 3.0 μL min−1. At a flow rate of 2.0 µL min−1,some capillary combinations of length-effective lengths of (290-230), (350-320), and(500-440) did not form the inner and outer phases. In addition, the capillary tube oflength-effective length of (290-260) and that of (350-260) showed data quite similar to
Fig. 7. That is, the same effective length that imposed the same pressure at theobservation point in the capillary tube provided the same distribution behavior of thedyes in the tube, in spite of the different total capillary lengths. We summarise that the
majority (with the exception of 3 results) of results with flow rates of 0.5 – 2.0 µL min−1
created the formation of the inner and outer phases in the capillary tube, i.e., the kinetic
liquid–liquid interface through TRDP. Flow rates above and below this range did notprovide phase formation through TRDP.
TRDP in a microspace under laminar flow conditions
Fig. 8 shows the linear
velocity and the shearing stress of the fluid in a capillary tube under laminar flow
conditions at different phases (axial distribution, TRDP, and homogeneous distribution).
At low flow rates (0.1 and 0.2 μL min−1), the axial distribution of the fluorescent dyes
was observed. When the ternary mixed solvent solution is fed into the capillary tube, atflow rates greater than 0.5 µL min−1, the TRDP creates a kinetic liquid–liquid interface
in the capillary tube. If the flow rates become lower than 0.5 µL min−1 or zero, thesurface stress around the liquid–liquid interface, which naturally causes shrinking in therelated interface area, changes the distribution pattern of the dyes and solvents fromradial to axial. Although the lengths of colors, blue and green, in the axial direction in
the axial distribution pattern, as shown in Fig. 5, were irregular, the length of theorganic solvent-rich phase (blue) was much longer than that of the water-rich phase(green). At flow rates greater than 2.0 μL min−1, the kinetic liquid–liquid interface
fluctuates and the shearing stress opposes the radial force. Consequently, the fluidic
behavior at high flow rates leads to homogeneous solutions, where the molecularaggregates are dispersed homogeneously into the solvent. The pressure through thelaminar flow in the capillary tube, induced by changes in the flow rate, trigger the phasechange from homogeneous to heterogeneous in these ternary mixed solvents. However,with the experimental data we can show that the phase formation in the TRDP wascontrolled with the flow rates and not the pressures, at least under the present conditions.
Although the reason has not been made clear yet, new information concerning the
TRDP is interesting, and will lead the way to the next step in TRDP research. Briefly,
we would like to discuss why the major solvents distribute around the middle of the
tube and the minor solvents distribute near the inner wall, regardless of whether the27

Observation and characterization of TRDP

27

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

carrier solutions are organic solvent-rich or water-rich. Fig. 9 shows the linear velocity
in the radial face section of the capillary tube (circle curves) under laminar flow
conditions, based on the following equation:
1 dP 2u=−a − r2 ,4 µ dx()

where u is the linear velocity, μ the viscosity, a the inner radius, r the radial length, anddP/dx the differential of pressure with the axial length. Each circle in the radial face
expresses the same velocity, and the spaces between the circles have the same velocity
interval. The spaces between the curves become smaller from the center to the innerwall, that is, the inside area has a lower gradient in velocity change than does theoutside area. Strictly speaking, Fig. 9 represents an ideal velocity curve; the realvelocity curve in an aqueous–organic solvent mixed solution may deviate from these,
however. The possible distribution patterns of the phase formation in the radial profile
in a capillary tube are schematically depicted in Fig. 9 as patterns I – III. The kinetic
liquid–liquid interface created in TRDP form along one curve of a certain velocity;
therefore, it must be generated along one of the circular curves. For TRDP, distribution
patterns II or III are preferred over pattern I. Furthermore, the major solvents must
occupy the inside area in the capillary tube, with consideration to fluidic stability basedon the linear velocity gradients of the radial profile under laminar flow conditions.
Therefore, the TRDP has distribution pattern III.
In conclusion, the TRDP creates a kinetic liquid–liquid interface in a microspacefrom homogeneous aqueous–organic mixed solutions under laminar flow conditions.
The formation of the inner and outer phases in the capillary tube, examined using
fluorescence microscopy, was observed at flow rates of 0.5 – 2.0 μL min−1. The TRDP
was controlled with the flow rates, not with pressures under the present conditions. Thisfinding is both interesting and useful for proceeding to the next step in the TRDP
research.

28

Chapter 228

Table 1. Effects of pressures on the ternary mixed solvent solutions in a batch vessel.

Figure 1. Schematic diagram of the TRDP system for fluorescence observations. The
pressure in a capillary tube is estimated using the Hagen–Poiseuille equation with theeffective capillary length (the length from the observation point to the capillary outlet).

29

Observation and characterization of TRDP

29

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2. Fluorescence photographs of (a) perylene (0.1 mM) and Eosin Y (1.0 mM)
dissolved in the water–acetonitrile–ethyl acetate mixture (3:8:4 volume ratio) in a
microchannel, at a 1.0 μL min−1 flow rate. (b) Perylene (0.1 mM) and Eosin Y (0.1 mM)
dissolved in the water–acetonitrile–ethyl acetate mixture (80:20:9 volume ratio) in a
microchannel, at a 1.0 μL min−1 flow rate. (c) Perylene (0.1 mM) and Eosin Y (0.5 mM)
dissolved in the water–acetonitrile–ethyl acetate mixture (80:20:9, volume ratio) in a
PTFE capillary tube at a 3.0 μL min−1 flow rate.

Figure 3. Phase diagram for the water–acetonitrile–ethyl acetate mixture. Solution
volume ratios of (A) 3:8:4, (B) 3:8:3, and (C) 3:8:2 are marked and thehomogeneous–heterogeneous solution boundary curve is represented by the dottedcurve.

30

Chapter 230

Figure 4. Fluorescence photographs and profiles of fluorescent dyes, perylene (0.1 mM)
and Eosin Y (1.0 mM), dissolved in the water–acetonitrile–ethyl acetate mixture (3:8:4,
volume ratio) at various flow rates in a capillary tube. The arrows point out the
inflections on the fluorescence profiles of Eosin Y.

Figure 5. Fluorescence photographs of the fluorescent dyes perylene (0.1 mM) and
Eosin Y (1.0 mM) dissolved in the water–acetonitrile–ethyl acetate mixture (3:8:4,
volume ratio) at low flow rates in a capillary tube.

31

Observation and characterization of TRDP

31

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 6. Phase formation of the inner and outer phases, formation ( ○ ) or
non-formation (×), at different flow rates and pressures in a capillary tube of length
110 cm with different effective lengths of 5 – 80 cm, represented as (110-effective
length) in the figure.

Figure 7. Phase formation of the inner and outer phases, formation ( ○ ) or
non-formation (×), at different flow rates and pressures in various capillary tubes. a)capillary length 290 cm (effective length: 200, 230, and 260 cm), b) capillary length 350
cm (effective length: 260, 290, and 320 cm), and c) capillary length 500 cm (effective
lengths: 410, 440, and 470 cm), represented as (capillary length-effective length) in the
figures.

32

Chapter 232

Figure 8. Illustration of the linear velocity and shearing stress of the fluid in thecapillary tube under laminar flow conditions together with the fluorescencephotographs.

Figure 9. Schematic depictions of the possible distribution patterns in a capillary tube.

33

Observation and characterization of TRDP

33

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

2.3 Observation and characterization of TRDP with various innerdiameters of capillary tubes
We have developed a capillary chromatography system using an open-tubular capillary
tube and a ternary water-hydrophilic/hydrophobic organic mixture carrier solution. We
call the system tube radial distribution chromatography (TRDC). Here, fluorescencephotographs of the dyes dissolved in the ternary mixed carrier solvents in the TRDC
system were examined under various conditions concerning the inner diameters, tube
temperatures, tube effective lengths, and flow rates (average linear velocities). The
TRDC system used an untreated fused-silica capillary tube and a ternarywater-acetonitrile-ethyl acetate mixture carrier solution (3:8:4 volume ratio; the organic
solvent-rich solution). The tube radial distribution of the carrier solvents, which
generated the organic solvent-rich major inner phase and the water-rich minor outerphase, could be observed in 50 – 700 µm inner diameter of the tubes at the average
linear velocity of 11 cm min-1 through the fluorescence microscope-CCD camera. The
model
analytesolutionof
1-naphthol,
1-naphthalenesulfonicacid,
2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid was subjected to
the TRDC system. They were separated and detected in this order with 50, 75, and 100µm inner diameters of the open-tubular capillaries.
Introduction
Recently, our group reported on an interesting and useful microfluidic behavior of thesolvents; tube radial distribution of ternary mixed solvents in a microspace,or the tube
radial distribution phenomenon (TRDP) for short. When the ternary mixed
homogeneous solution of water–hydrophilic/hydrophobic organic solvents is delivered
into a microspace, such as a microchannel or a capillary tube, the carrier solvent
molecules are radially distributed in the microspace, generating inner and outer phases.
For example, when a homogeneous carrier solution of water–acetonitrile–ethyl acetate(3:8:4 volume ratio; the organic solvent-rich carrier solution) was fed into a fused-silica
capillary tube, an organic solvent-rich major phase was generated around the middle ofthe tube as an inner phase far from the inner wall, while a water-rich minor phaseformed near the inner wall as an outer phase, or capillary wall phase. A capillarychromatography system, where the outer phase functions as a pseudo-stationary phase
under laminar flow conditions, has been developed based on the TRDP. We call thistube radial distribution chromatography (TRDC). Otherwise, we have investigatedchemical reactions, extraction, and mixing procedures on the basis of the TRDP.
The TRDP, i.e., the creation of kinetic liquid-liquid interface in a microfluidic flow,
while interesting, is an involved procedure by it`s very nature.. The TRDP has leftseveral subjects to be investigated in order to better clarify the microfluidic behavior.

34

Chapter 234

Here the tube radial distribution of the carrier solvents was examined under various
conditions through the fluorescence microscope-CCD camera system. Also, separation
performance was examined for the model mixture analytes in the capillarychromatography; TRDC.
Experimental
Fluorescence photographs
A capillary tube was set up for the fluorescencemicroscope-CCD camera system to confirm the tube radial distribution of the carrier
solvents, i.e., TRDP. The water–acetonitrile-ethyl acetate mixture (3:8:4 volume ratio)
including 0.1 mM perylene and 1 mM Eosin Y solutions was delivered into the capillary
tube. The fluorescence in the capillary tube was monitored using a fluorescencemicroscope equipped with an Hg lamp, a filter, and a CCD camera.
Open-tubular capillary chromatography
The present capillary chromatographysystem comprised of an open fused-silica capillary tube, microsyringe pump, andfluorescence detector. The tube temperature was controlled by dipping the capillary tube
in water maintained at a fixed temperature in a beaker by stirring.
Water-acetonitrile-ethyl acetate mixtures with volume ratios of 3:8:4 were used as
carrier solutions. The homogeneous carrier solutions could be used independent fromthe left-time after the preparation. Analyte solutions were prepared along with thecarrier solutions. The analyte solution was introduced directly into the capillary inlet
side by the gravity method. After analyte injection, the capillary inlet was connected
through a joint to a microsyringe. The syringe was set on the microsyringe pump. The
carrier solution was fed into the capillary tube at a definite flow rate under laminar flow
conditions. On-capillary fluorescence detection (ex. 495 nm and em. 520 nm) was
performed with the detector.
Results and discussion
The phase
phase diagrams that
Phase diagram forfor water-acetonitrile-ethyl
water-acetonitrile-ethyl acetate
acetate mixture
mixture The
indicated the component ratios of the solvents in homogeneous and heterogeneous
solutions are useful tools to explain the TRDP and examine the conditions for the TRDP
and TRDC performance. A phase diagram for the ternary mixture solvents ofwater-acetonitrile (hydrophilic organic solvent)-ethyl acetate (hydrophobic organic
solvent) was examined in a vessel at 0 and 20 °C under a constant pressure oratmospheric pressure (Fig. 1 a)) as well as at atmospheric pressure (1.0 x 105 Pa) and1.3 x 105 Pa at a constant temperature of 20 °C (Fig. 1 b)). The obtained phase diagrams
are shown in Fig. 1. The dotted curves in the diagrams indicate the boundary between
homogeneous and heterogeneous phases. The phase diagram showed that eachcomponent ratio of the solvents made a homogeneous (one homogeneous phase) or aheterogeneous (two homogeneous phases) solution. As shown in Fig. 1, lower
35

Observation and characterization of TRDP

35

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

temperatures and higher pressures expanded the boundary curves outwards in the phase
diagrams.
TRDP created with an organic
proposeda viewof theorganic solvent-richsolvent-rich carrier
carrier solution
solution We We
proposeda viewof
inner
andandouter
phaseformationin the
TRDP,
based
on on
thetheabove
experimental
data.
A
the inner
outer
phase
formationin the
TRDP,
based
above
experimental
data.
specific
homogeneous
solution,
whose
component
ratio wasthenearboundary
betweenA specifichomogeneous
solution,
whose
component
rationearwasthe boundary
homogeneousand heterogeneousin the phasecooledwas
to lowerthan°C
between homogeneousand heterogeneousin diagram,the phasewas
diagram,cooled
to 20
lower
(room
or pressurized
than greater
atmosphericpressure in apressurebatch vessel
than 20temperature)°C (room temperature)or greaterpressurized
than atmospheric
in a
where
it was where
under itthewascontrol
gravity.
In ofan gravity.
organic In
solvent-rich(e.g.,
batch vessel
underof thecontrolan organicsolution
solvent-richwater-acetonitrile-ethyl-acetate
mixture; 3:8:4 volume
ratio),3:8:4
the homogeneous
solutionsolution (e.g., water-acetonitrile-ethyl-acetate
mixture;volume ratio),
the
first
became ansolution
emulsionor became
nontransparent
solutionorthat
included minor
solventthatorhomogeneous
first
an emulsion
nontransparent
solutionwater-richtiny droplets
andthen changed
to a heterogeneous
solutionincluded an
included minor
solvent or
water-richtiny droplets
and then changed
to thata heterogeneous
upper
major
solutionand asolvent-richlower minorsolution
water-richtosolutionthat organic
includedsolvent-rich
an upper majororganic
and solution
a lower due
minor
the
influenceof gravity.
water-richsolution
due to the influence of gravity.
The phase formation of the ternary mixed solvents in a capillary tube under thechange of temperature and pressure is estimated as follows, where it is under little
control of gravity. A specific homogeneous solution with a component ratio near theboundary between homogeneous and heterogeneous in the phase diagram is delivered
with a constant pressure to the radial face and at a lower temperature. In an organic
solvent-rich solution, the homogeneous solution must first become an emulsion solutionthat includes minor solvent or water-rich tiny droplets. Although generation of emulsion
was observed in a batch vessel as described above, it could not be observed in acapillary tube with the present fluorescence microscope-CCD camera system; because
the present system was unable to catch either the emulsion formation or tiny droplets inthe microfluidic flow in a capillary tube. The water-rich tiny droplets start to aggregate
near the inner wall where the linear velocity is lowest under laminar flow conditions.
Consequently, the ternary mixed solvent solution in a tube changes to a heterogeneous
solution that includes a major inner organic solvent-rich solution and a minor outerwater-rich solution, where it is under minimal gravitational control..
In order to create the TRDP, we have to take advantage of the phase separation fromthe homogeneous to the heterogeneous solution with changeing pressure andtemperature, referring to the phase diagrams (Fig. 1), although the volume ratios of theupper and lower phases in a vessel would not necessarily be equal to those of the innerand outer phases in a capillary tube. That is, a homogeneous solution is delivered into a
capillary tube where it then changes into a heterogeneous solution, via emulsion
solution, under higher pressure or lower temperature in the tube. The consideration of
the TRDP process is illustrated in Fig. 2.
TRDP created with a water-rich carrier solution
Similarly, in a water-rich solution,
the homogeneous solution first became an emulsion solution that included minor

36

Chapter 236

solvent or organic solvent-rich tiny droplets, and then changed to a heterogeneous
solution that included an upper minor organic solvent-rich solution and a lower majorwater-rich solution in a batch vessel. The phase formation of the ternary mixed solventsin a capillary tube kept at a low temperature and under pressure due to laminar flow
with minimal gravitational control is estimated as follows. In a water-rich solution, thehomogeneous solution becomes an emulsion solution that includes minor solvent ororganic solvent-rich tiny droplets. Under laminar flow conditions, the organic
solvent-rich tiny droplets start to aggregate near the inner wall where the velocity islowest under laminar flow conditions. The homogeneous solution had not changed
before the delivery to the capillary tube. After introduction to the tube, thehomogeneous solution shifted to a heterogeneous solution including inner and outerphases under higher pressure or lower temperature in the capillary tube, referring to thephase separation in the diagrams (Fig. 2). That is, the ternary-mixed solvent solution ina tube changes to a heterogeneous solution that includes a major inner water-richsolution and a minor outer organic solvent-rich solution where it is under minimal
gravitational control.
Effects of flow rates in the capillary tube at tube temperature 20 °C
Fluorescencephotographs were observed for a capillary tube in which the organic solvent-rich carrier
solution (3:8:4 volume ratio) containing perylene (0.1 mM) and Eosin Y (1.0 mM) was
delivered at flow rates of 0.1 – 4.0 µL min−1 with the inner diameter of 50, 75, and 100µm at a tube temperature of 20 °C (the obtained data is shown in Fig. 4). The
hydrophobic perylene molecules (blue) were distributed around the middle of the tube,
while the relatively hydrophilic Eosin Y molecules (green) were distributed near the
tube’s inner wall at relatively intermediate flow rates of 0.5 – 2.5 µL min−1 with 50 µm,
0.5 – 2.0 µL min−1 with 75 µm, and 0.5 – 1.0 µL min−1 with 100 µm, respectively. The
inflections on the fluorescence profiles of Eosin Y in the outer phase were generated
through the liquid-liquid interface created with TRDP. Inflections were observed in theabove flow rate ranges for every inner diameter. Comparatively, at lower flow rates
where surface tension might be predominant, we did not observe the tube radial
distributions; the dissolved dyes were distributed in the axial direction, while, at higher
flow rates where fluctuation at the liquid-liquid interface might become larger, the tube
radial distributions of the dyes were not observed and the fluorescence profiles showedthat the dyes were homogeneously distributed in the ternary mixed solvents. That is,
fluidic behavior at high flow rates leads to homogeneous solutions, where the molecularaggregates are dispersed homogeneously in the solvent. The data obtained using theorganic solvent-rich carrier solution in the capillary tubes were used to investigate therelationships between flow rates, pressures, and phase formation. The pressure in thecapillary tube under laminar flow conditions was roughly calculated at each flow rate
(0.1 – 4.0 µL min−1) using the following Hagen–Poiseuille equation:
37

Observation and characterization of TRDP

37

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

πa 4 ΔpQ=,
8µ L
where Q is the flow rate, a the inner radius, Δp the pressure (or pressure loss), µ the
viscosity, and L the effective capillary length (the length from the observation point tothe capillary outlet). The pressures in the capillary tubes were calculated using Q, a, µ,and L values of 0.1 – 4.0 µL min−1, 25.0, 37.5, or 50 µm, 6.89 × 10−4 kgm−1s−1, and 2.0
× 10−1 m, respectively.
The formation/non-formation of the inner and outer phases indicating TRDP
creation/non-creation, respectively, is plotted in the pressure vs flow rate graph in Fig. 3.
The TRDP creation/non-creation of the inner and outer phases is presented with symbolof ○ and ×, and partially TRDP observation is presented with symbol of △. From thedata, it can be seen that the flow rate and pressure in a capillary tube play importantroles in determining TRDP in the solvents. In addition, although we were unable toobserve emulsion in the microfluidic flow in the capillary tubes with the present
fluorescence microscope-CCD camera system, we may assume suitable flow rates foremulsion formation that leads to TRDP as another point of interest.
Effects of temperature and pressure on TRDP Fluorescence photographs wereobserved similarly to the above section, but at tube temperature of 0 °C instead of 20 °C.
In contrast to the data shown in Fig. 3, the TRDP could be observed at up to 50 µL
min−1, with all capillary inner diameters, 50, 75, and 100 µm and 20, 50 and 80 cm
effective lengths, where the limitation flow rates, 50 µL min−1, were determined in thepresent microsyringe pump’s capacity. Thinking of the phase diagrams shown in Fig. 1,
the effects of both temperature and pressure must lead to stable TRDP in a large rangeof flow rates up to 50 µL min−1. Relationships between the average linear velocities andpressure are shown in Fig. 5. The TRDP creation and non-creation are presented withthe symbols of ○ and ×, respectively. It was discovered from the data that border
average linear velocity in the TRDP between TRDP and non-TRDP was the same for all
conditions of effective lengths and inner diameters. The energy generated by theaverage linear velocity, 1.13 cm min-1, might be required to break the axial distributionof the solvents that was observed at lower velocities.
Effects of tube inner diameter on fluorescence photographs
Although the innerdiameter of the tube is one of the most important factors in the TRDP, there has notbeen enough data about it yet. Here we examined the effects of the tube inner diameteron fluorescence photographs in the TRDC system. We attempted to use commerciallyavailable fused-silica capillary tubes with various inner diameters; the capillary tubes
with 50 – 700 µm inner diameters were used here. The obtained fluorescencephotographs are shown in Fig. 5 together with the analytical conditions. The flow rates

38

Chapter 238

for all the capillary tubes were adjusted to provide the same average linear velocity ofca. 11 cm min-1. We observed the inner and outer phase formation, i.e., TRDP, in the
fluorescence photographs in all the tubes.
Separation of mixture solution including four analytes
We examined a mixture
analyte solution of 1-naphthol, 1-naphthalenesulfonic acid, 2,6-naphthalenedisulfonicacid, and 1,3,6-naphthalenetrisulfonic acid using the present TRDC system with organic
solvent-rich carrier solution. The obtained chromatograms are shown in Fig. 6 alongwith the analytical conditions. 1-Naphthol, 1-naphthalenesulfonic acid,
2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid were eluted in thisorder, leading to good separation with the organic solvent-rich carrier solution in the
inner diameters of 50, 75 and 100 µm (Fig. 6). The elution order seemed to beconsistent with the hydrophilic character. In the 200, 250, and 320 µm inner diametertubes, 2,6-naphthalenedisulfonic acid and 1,3,6-naphthalenetrisulfonic acid were notseparated. With a 530 µm inner diameter, 1-Naphthol and 1-naphthalenesulfonic acid aswell as 2,6-naphthalenedisulfonic acid and 1,3,6-naphthalenetrisulfonic acid were notseparated on the chromatogram.
In conclusion, we investigated capillary chromatography on the basis of the TRDP.
The tube radial distribution of the carrier solvents was examined by changing tube innerdiameters, flow rates, effective lengths, and temperature through the fluorescencemicroscope-CCD camera system. Consequently, useful information was obtained tounderstand the TRDP and TRDC. Also, separation performance was examined for themodel mixture analytes in the TRDC. The TRDC has potential as a separation tool that
does not require using high voltage supply and/or any specific capillary columns, suchas packed and monolithic.

39

Observation and characterization of TRDP

39

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Phase diagram for the ternary water-acetonitrile-ethyl acetate mixture solution.
a) Pressure constant (atmospheric pressure) and b) temperature constant (20 °C).

Figure 2. Illustration of tube radial distribution phenomenon (TRDP) appearance in a
microfluidic flow.

40

Chapter 240

Figure 3. Relationships between flow rates and pressures in tube radial distribution
phenomenon of the ternary mixed solvents at tube temperature of 20 °C, with capillary
inner diameters of 50, 75, and 100 µm. The formation/non-formation of the inner andouter phases are presented with the symbols ○ and ×, and partially TRDP observation ispresented with the symbol △. Conditions: Capillary, 110 cm total length and 20 cm
effective length; carrier, water-acetonitrile-ethyl acetate (3:8:4 volume ratio) containing
perylene (0.1 mM) and Eosin Y (1.0 mM).

41

Observation and characterization of TRDP

41

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Relationships between average linear velocities and pressures in tube radial
distribution phenomenon of the ternary mixed solvents at tube temperature of 0 °C. The
formation/non-formation of the inner and outer phases are presented with symbol of ○and ×. Conditions: Capillary, 110 cm total length and 20, 50, and 80 cm effective
length; carrier, water-acetonitrile-ethyl acetate (3:8:4 volume ratio) containing perylene(0.1 mM) and Eosin Y (1.0 mM).

42

Chapter 242Figure 5. Fluorescence photographs in the capillary tubes having various innerdiameters. Capillary inner diameter, 50, 75, 100, 200, 250, 320, 530, and 700 µm.
Conditions: Capillary, 110 cm total length and 30 cm effective length fused-silica;
carrier, water-acetonitrile-ethyl acetate (3:8:4 volume ratio) containing perylene (0.1mM) and Eosin Y (1.0 mM); and average linear velocity of 11 cm min-1 for all tubes.

43

Observation and characterization of TRDP

43

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 6. Chromatograms of a mixture analyte solution of 1-naphthol,
1-naphthalenesulfonicacid,
2,6-naphthalenedisulfonic
acid,
and
1,3,6-naphthalenetrisulfonoc acid obtained by the present TRDC system. Capillary innerdiameter, 50, 75, 100, 200, 250, 320, and 530 µm. Conditions: Capillary tube, 120 cm
total length and 20 cm effective length fused-silica; carrier, water-acetonitrile-ethyl
acetate (3:8:4 volume ratio) mixture solution; sample injection, gravity method from 20
cm height (5.4 mm sample length); average linear velocity, 11 cm min-1; temperature,
20 °C; detection, fluorescence, ex. 290 nm and em. 355 nm; and 1-naphthol,
1-naphthalenesulfonic acid (1-NS), 2,6-naphthalenedisulfonic acid (2,6-NDS), 1 mM,
1,3,6-naphthalenetrisulfonic acid (1,3,6-NTS), 2.0 mM.

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Chapter 244

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[24] R. B. Bird, W. E. Stewart, and E. N. Lightfoot, Transport Phenomena, 2nd edn.,
Chap. 2, Wiley, Toronto, Canada, 2002.
[25] H. Small, J. Colloid Interface Sci., 1974, 48, 147.
[26] Ch. -H. Fischer, M. Giersig, Analysis of Colloids, J. Chromatogr., A, 1994, 688,97.
[27] J. C. Giddings, M. N. Myers, G. -C. Lin and M. Martin, J. Chromatogr., A, 1977,
142, 23.

45

Observation and characterization of TRDP

45

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Chapter 3 Consideration
phenomenon (TRDP)

of

tube

radialdistribution

Chapter 3

TRDP is specific fluidic behavior observed in a microspace under laminar flow
conditions. The TRDP creation was considered through experimental data from various
viewpoints, including chromatograms obtained with tube radial distribution
chromatography (TRDC), phase diagram, tie lines, dimensionless numbers, andsolution properties. The part of this chapter is reconstructed and rewritten based on therelated manuscripts that have been published.27,35,39,51,52)

3.1 Experimental consideration through the phase diagram in TRDC
and TRDP
Capillary chromatography using an untreated open tubular capillary tube and a ternarysolvent mixture consisting of water-hydrophilic/hydrophobic organic solvent as a carrier
solution has been developed. The system is called tube radial distribution
chromatography (TRDC). Separation performance of the TRDC system using a
fused-silica capillary tube was examined through the phase diagram for the ternarywater-acetonitrile-ethyl acetate solvent mixture. The TRDC system required
homogeneous carrier solutions with solvent component ratios around the boundary
curve between homogeneous and heterogeneous solution in the phase diagram. The dataobtained using the fused-silica capillary tube were compared with those obtained usinga polytetrafluoroethylene capillary tube in our study.
Introduction
Capillary tubes with inner diameters less than several hundred micrometers that provide
micro-flow are known to exhibit interesting and useful physical or hydrodynamic
phenomena, such as electro-osmotic flow and laminar flow. The electro-osmotic flow ina capillary tube promotes capillary electrophoresis [1,2] and capillaryelectrochromatography [3], while laminar flow conditions enable hydrodynamic
chromatography [4,5]. Recently, our group reported the tube radial distribution
phenomenon of carrier solvents in a micro-flow, which we call the tube radial
distribution phenomenon (TRDP).
The TRDP can be phenomenologically described as follows. When the ternary mixed
solvents of water-hydrophilic/hydrophobic organic solvent mixtures are delivered into a
micro-space, such as a micro-channel or a capillary tube under laminar flow conditions,the solvent molecules are radially distributed in the micro-space, generating inner and46

Chapter 3

46

outer phases. The TRDP creates a phase interface or kinetic aqueous–organic interface
in the micro-space. That is, in a water-rich solution we would observe a water-richmajor inner phase and organic solvent-rich minor outer phase, while in an organic
solvent-rich solution we would observe an organic solvent-rich major inner phase andwater-rich minor outer phase in micro-flow under certain conditions.
An untreated open tubular capillary chromatography system where the outer phasefunctions as a pseudo-stationary phase under laminar flow conditions has beendeveloped based on the TRDP, which we call tube radial distribution chromatography(TRDC). According to the inner and outer phase formation based on the TRDP, with awater-rich carrier solution, the hydrophilic analyte, which is dispersed in the water-richmajor phase (around the middle of the capillary tube), is eluted with a nearly average
linear velocity. The hydrophobic analyte, which is dispersed in the organic solvent-richminor phase near the inner wall of the tube (pseudo-stationary phase), is eluted at alower than average linear velocity. In contrast, with an organic solvent-rich carrier
solution, the hydrophobic analyte dispersed in the organic solvent-rich major phase iseluted with a nearly average linear velocity. The hydrophilic analyte dispersed in thewater-rich minor phase near the inner wall of the tube is eluted with a lower velocity.
Therefore, the elution times of the analytes can be reversed by altering the component
ratio of the solvents in the carrier solution.
Separation performance in the TRDC system was examined from the viewpoint ofpressure in a capillary tube under laminar flow conditions and the phase diagram of theternary mixed solvents of water-acetonitrile-ethyl acetate mixture. The component ratiosof the solvents that were positioned near the boundary between homogeneous andheterogeneous solution in the diagram were required for TRDC separation. The datasuggested that the phase transformation from homogeneous to heterogeneous solution in
the capillary tube under pressure in micro-flow was important for TRDP and TRDC.
That is, experiments and considerations based on the phase diagram are useful for the
study of TRDP or TRDC.
In our study,15) when using polytetrafluoroethylene (PTFE) capillary tubes, withwater-rich carrier solution, hydrophilic 2,6-naphthalenedisulfonic acid and hydrophobic
1-naphthal as model analytes were separated in this order, while with the organic
solvent-rich carrier solution, the reverse elution order was observed—i.e., 1-naphthol
was first detected, followed by 2,6-naphthalenesulfonic acid—although they showed
split-separation. Here, the separation performance of the analytes was examined by the
TRDC in a similar way to the study through phase diagrams,15) using a fused-silica
capillary tube instead of a PTFE capillary tube.
Experimental
Fused-silica capillary tubes with an inner diameter of 50 µm were used. The present
TRDC system comprised of a fused-silica capillary tube (120 cm total length and 100
47

Consideration of TRDP

47

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

cm effective length), micro-syringe pump, and absorption detector. The tube
temperature was controlled by dipping the capillary tube (ca. 60 cm) in water
maintained at a fixed temperature (0 °C or 20 °C) in a beaker by stirring. The ternary
mixed solvents of water-hydrophilic/hydrophobic organic solvent mixture(water-acetonitrile-ethyl acetate) solutions with various volume ratios were used as
carrier solutions. Analyte solutions containing 1-naphthol and 2,6-naphthalenedisulfonicacid (1 mM each) as models were prepared with the carrier solutions. The analyte
solution was introduced directly into the capillary inlet side by the gravity method (20cm height for 30 s). After analyte injection, the capillary inlet was connected through a
joint to a micro-syringe. The syringe was placed on the micro-syringe pump. The carrier
solution was fed into the capillary tube at 0.2 µL min-1 flow rate under laminar flow
conditions. On-capillary absorption detection (254 nm) was performed with thedetector.
Results and discussion
Phase diagram for water-acetonitrile-ethyl acetate mixture
The phase diagram forthe ternary mixed solvents of water-acetonitrile (hydrophilic organic solvent)-ethyl
acetate (hydrophobic organic solvent) in a vessel at a temperature of 20 °C wasconstructed. The obtained phase diagram is shown in Fig. 1. The dotted curve in the
diagram indicates the boundary between homogeneous and heterogeneous phases. The
phase diagram showed that each component ratio of the solvents formed a homogeneous(one homogeneous phase) or heterogeneous (two homogeneous phases) solution. The
solutions with component ratios of a) – t) were used as carrier solutions in the TRDC
system in the following sections. The component ratios of the solvents or the carrier
solutions a) – t) are plotted in the diagram based on separation performance of analytes
described below, where circles (○), triangles (Δ), and crosses (×) indicatedbaseline-separation, split-separation (not baseline-separation), and non-separation for
the analytes on the chromatograms, respectively.
Chromatograms obtained at various component ratios of water-acetonitrile-ethyl
acetate mixture carrier solution Acetonitrile was added to a solution with a constantwater-ethyl acetate volume ratio of 5:7 in a vessel at a temperature of 20 °C to prepare
carrier solutions (organic solvent-rich carrier solutions) with the following
compositions: a) water-acetonitrile-ethyl acetate 5:13:7 volume ratio; b) 5:15:7 volume
ratio; c) 5:17:7 volume ratio; d) 5:19:7 volume ratio; and e) 5:20:7 volume ratio. The
five carrier solutions a) – e) were all homogeneous solutions. The analyte mixtures of
1-naphthol and 2,6-naphthalenedisulfonic acid were examined with the present TRDC
system using carrier solutions a) – e). The obtained chromatograms are shown in Fig. 2.
Carrier solutions a) – c) could separate the analytes in the mixture. 1-Naphthol(hydrophobic) was eluted first with near the average linear velocity in the capillary tube

48

Chapter 348

under laminar flow conditions and 2,6-naphthalenedisulfonic acid (hydrophilic) was
eluted second at a lower than average linear velocity. The elution order was reasonable
in the TRDC system with the organic solvent-rich carrier solution as mentioned in the
Introduction. The other homogeneous carrier solutions, d) and e), ofwater-acetonitrile-ethyl acetate showed split-separation and non-separation of theanalytes, respectively, under the present analytical conditions. The separation
performances obtained with carrier solutions a) – e) are plotted with the following
symbols: circles (○), triangles (Δ), and crosses (×) in the phase diagram in Fig. 1. The
separation performance decreased from carrier solution a) to e) as shown in Fig. 2.
However, even carrier solutions b) and c) in which the compositions were a little far
from the boundary in the diagram also showed baseline-separation for the analytes.
TRDP must occur with such allowance for the solvents compositions around theboundary in this area of the diagram. The chromatograms of the analytes, the mixtureof 1-naphthol and 2,6-naphthalenedisulfonic acid, were examined with thehomogeneous carrier solutions f) – t). The compositions for the solutions f) – t) werepositioned near the boundary in the phase diagram; f) water-acetonitrile-ethyl acetatewith a 0.25:0:10 volume ratio, g) 0.4:1:9 volume ratio, h) 0.55:2:8 volume ratio, i)
0.8:3:7 volume ratio, j) 1.1:4:6 volume ratio, k) 1.5:5:5 volume ratio, l) 2.05:6:4 volume
ratio, m) 14:30:15 volume ratio, n) 4:6:2.4 volume ratio, o) 5:5:1.75 volume ratio, p)
6:4:1.3 volume ratio, q) 7:3:1.1 volume ratio, r) 8:2:0.95 volume ratio, s) 9:1:0.95
volume ratio, and t) 10:0:0.9 volume ratio. The carrier solutions f) – i) and p) – t)showed no separation behavior and presented just single peaks on the chromatogramswith near the average linear velocity (data not shown). The carrier solutions j) – o)showed separation behavior as indicated in Fig. 3. The component ratios of the solvents
for solutions f) – t) were plotted in the phase diagram in Fig. 1 with the symbols basedon the chromatographic data. As shown in Fig. 1, the TRDC system was used withseveral homogeneous carrier solutions with component ratios of the solvents around theboundary curve in the phase diagram. Such specific carrier solutions caused a tube
radial distribution of the carrier solvents in the capillary tube under laminar flow
conditions, i.e., the TRDP. The pressure imposed on the carrier solution in the capillary
tube under laminar flow conditions might alter the carrier solution from homogeneousin the batch vessel to heterogeneous in the tube, affecting the tube radial distribution of
the solvents in the capillary tube.
Optimization of the component ratio of solvents for separation As shown in Fig. 3,
high resolutions of the analytes in the TRDC system were observed around the carrier
solutions of l) and m) in the phase diagram. The chromatograms were examined in moredetail with carrier solutions u) – w) that had compositions between solutions l) and m)along the boundary curve in the phase diagram (Fig. 1). They were u)
water-acetonitrile-ethyl acetate with a 5:14.5:8.5 volume ratio, v) 5:13:7 volume ratio,
49

Consideration of TRDP

49

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

and w) 6:13.5:6.5 volume ratio. The obtained chromatograms are shown in Fig. 4
together with their resolutions (Rs). Carrier solutions v) and w) showed the best
resolutions, ca. 14, under the present analytical conditions.
Reversing of the elution order of the analytes
As characteristic separation behaviorof the TRDC system using the PTFE capillary tube, we reported that a water-rich carrier
solution elutes the more hydrophilic analyte first and an organic solvent-rich carrier
solution elutes the more hydrophobic analyte first.15) However, the reverse elution
behavior of the analytes was not observed in Fig. 3 under these analytical conditions.
The analytical conditions, such as tube temperature, tube length, and flow rate, weresignificant and critical for separation performance in the TRDC system. Tentatively, thechromatograms were examined by the TRDC system using carrier solutions f) – t)where the capillary tube was dipped in the beaker controlled at 0 °C instead of 20 °C.
The separation performance with solutions f) – t) were plotted with the symbols in the
diagram in Fig. 5. The chromatograms obtained with carrier solutions l) – q) that show
separation are presented in Fig. 6. Water-rich carrier solutions p) and q) gave the reverse
elution order, 2,6-naphthalenedisulfonic acid first and then 1-naphthol second, although
they showed split-separation. Furthermore, the analyte solutions were subjected to the
TRDC system with the water-rich carrier solution of water-acetonitrile-ethyl acetate in a
15:3:2 volume ratio (water-rich carrier solution) under the analytical conditions of tube
temperature 0 °C instead of 20 °C, a tube effective length of 200 cm instead of 100 cm,
and a flow rate of 0.8 µL min-1 instead of 0.2 µL min-1. The obtained chromatogram isshown in Fig. 7. 2,6-Naphthalenedisulfonic acid and 1-naphthol were eluted in thisorder with baseline-separation. The individual peaks were identified with thechromatogram of each analyte.
Consideration of the influence of the tube materials on separation
Almost the same
elution behavior or elution order in the TRDC system with the fused-silica capillary
tube was observed as that with the PTFE capillary tube reported in our work.15)
However, there were also differences in separation behavior between them. In thefused-silica capillary tube, the resolutions obtained with the organic solvent-rich carrier
solution were better than those of the water-rich carrier solution. On the other hand, in
the PTFE capillary tube, the resolutions obtained with the water-rich carrier solution
were better than those of the organic solvent-rich carrier solutions as reported.15) The
interaction between the outer phase of water-rich and fused-silica inner wall as well as
the outer phase of organic solvent-rich and PTFE (polymer material) inner wall may
give prior conditions for making the outer phase (pseudo-stationary phase) in thecapillary tube, while the interaction between the outer phase of water-rich and polymer
material inner wall as well as the outer phase of organic solvent-rich and fused-silica
inner wall may result in conditions inconvenient for forming the outer phase in the50

Chapter 350capillary tube.
In conclusion, chromatograms of model analytes were examined by the TRDC
system using a fused-silica capillary tube from the viewpoint of the phase diagram ofternary mixed solvents. The separation performances of the analytes were similar to
those obtained with a PTFE capillary tube described in our work.15) The organic
solvent-rich carrier solution, model analytes, hydrophobic 1-naphthol and hydrophilic
2,6-naphthalenedisuflonic acid, were separated in this order, while they were eluted in
the inverse order with the water-rich carrier solution, 2,6-naphthalenedisuflonic acidwas detected first, followed by 1-naphthol detection. However, the fused-silica capillary
tube resolutions obtained with the organic solvent-rich carrier solution were better thanthose of the water-rich carrier solution. The interaction between the outer phase of thewater-rich and fused-silica surface may provide better conditions than that between theouter phase of organic solvent-rich and fused-silica surface for forming the outer phasein fused-silica capillary tubes.

51

Consideration of TRDP

51

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Phase diagram for water-acetonitrile-ethyl acetate mixture (20 °C) and thecomponent ratios of the solvents for the chromatograms obtained by the TRDC systemwith tube temperature 20 °C. The component ratios of the carrier solutions a) – e)related to the experiments shown in Fig. 2 as well as the carrier solutions f) – t) related
to the experiments in Fig. 3 are plotted in the diagram with the symbols ○, Δ, and ×,indicting baseline-separation, split-separation, and non-separation, respectively, for the
analytes.

52

Chapter 352

Figure 2. Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidby the TRDC system using various component ratios of water-acetonitrile-ethyl acetate
mixture as carrier solutions. Tube temperature 20 °C. (1) 1-Naphthol and (2)2,6-naphthalenedisulfonic acid were eluted in this order with carrier solutions a) – d).

Figure 3. Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidby the TRDC system using various component ratios of water-acetonitrile-ethyl acetate
mixture as carrier solutions that were positioned near the boundary curve in the phase
diagram. Tube temperature 20 °C. (1) 1-Naphthol and (2) 2,6-naphthalenedisulfonicacid were eluted in this order with the carrier solutions j) – o).

53

Consideration of TRDP

53

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

igure
Figure4.4.Chromatograms
Chromatogramsofofaamixture
mixtureofof 1-naphthol
1-naphthol andand 2,6-naphthalenedisulfonic
2,6-naphthalenedisulfonic acidacidby the TRDC system using various component ratios of water-acetonitrile-ethyl acetate
mixture as carrier solutions that were positioned between solutions l) and m) in Fig. 3and the resolutions (Rs) for the analyte peaks. Tube temperature 20 °C. (1) 1-Naphtholand (2) 2,6-naphthalenedisulfonic acid were eluted in this order with carrier solutions l)
– m).

54

Chapter 3

54

Figure 5. Phase diagram for water-acetonitrile-ethyl acetate mixture (20 °C) and thecomponent ratios of the solvents for the chromatograms obtained by the TRDC systemwith tube temperature 0 °C. The component ratios of the carrier solvents f) – t) areplotted in the diagram with the symbols (○, Δ, and ×). 1-Naphthol and
2,6-naphthalenedisulfonic acid were eluted in this order with carrier solutions l) – n),while they were eluted in the reverse order with the carrier solutions p) and q).

55

Consideration of TRDP

55

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 6. Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidby the TRDC system using various component ratios of water-acetonitrile-ethyl acetate
mixture as carrier solutions that were positioned near the boundary curve in the phase
diagram. Tube temperature 0 °C. (1) 1-Naphthol and (2) 2,6-naphthalenedisulfonic acid
were eluted in this order with carrier solutions l) – n), while they were eluted in thereverse order with carrier solutions p) and q). The component ratios of carrier solutionsl) –p) used for the chromatograms are plotted in the phase diagram of Fig. 5.

Figure 7. Chromatograms of a mixture of (1) 1-naphthol and (2)2,6-naphthalenedisulfonic acid by the TRDC system. Capillary effective length, 200 cm(total length 220 cm); carrier, water-acetonitrile-ethyl acetate of 15:3:2 volume ratio;
tube temperature, 0 °C; and flow rate, 0.8 µL min-1.

56

Chapter 356

3.2 Experimental consideration through the phase diagram including
solubility curves at different temperatures in TRDC and TRDP
Open-tubular capillary chromatography using a ternary solvent mixture consisting ofwater–hydrophilic–hydrophobic organic solvent as a carrier solution has beendeveloped. When the ternary carrier solution is fed into the capillary tube, the carrier
solvents are radially distributed and generate inner and outer phases in the tube. The
outer phase functions as a pseudo-stationary phase in chromatography. Here,investigations proceeded with reference to the solubility curves on the phase diagram of
the ternary mixed solvents at 5 and 20 °C. Model analytes, 1-naphthol and
2,6-naphthalenedisulfonic acid (2,6-NDS), were examined with ternarywater–acetonitrile–ethyl acetate solvent mixtures as carrier solutions and a fused-silica
capillary tube (50 µm inner diameter). They were separated in this order with organic
solvent-rich carrier solutions, while, in the reverse order with the water-rich carrier
solutions at a tube temperature of 5 °C.
Introduction
Since the nineteenth century, microfluidic flow has been known to exhibit interesting
and useful physical and hydrodynamic phenomena such as electroosmotic flow andlaminar flow. Our group reported the tube radial distribution phenomenon of carrier
solvents in 2009, which we call the tube radial distribution phenomenon (TRDP). Whenthe ternary mixed solvents of water–hydrophilic/hydrophobic organic solvent mixtures
are delivered into a microspace such as a microchannel or a capillary tube under laminarflow conditions, the solvent molecules are radially distributed in the microspace andgenerate inner and outer phases.
The TRDP creates a phase interface or a kinetic liquid-liquid interface in a
microspace. We are currently investigating the TRDP from the viewpoint ofchromatography,19,24) extraction,16) mixing,26) and chemical reaction space.28) A
capillary chromatography system in which the outer phase acts as a pseudo-stationary
phase under laminar flow conditions has been developed based on the TRDP. We call it
tube radial distribution chromatography (TRDC).
The TRDP appears through phase separation from homogeneous solution toheterogeneous solution including two phases with pressure and temperature changes.
The phase separation with associated changes forms an upper and lower phase in abatch vessel under the control of gravity. At the same time, the phase separation
introduces TRDP, including inner and outer phases, in a micro-flow under laminar flow
conditions with minimal control from gravity. During the TRDP and TRDC
investigation, we considered that the solubility curves on the phase diagram must be
useful tools for investigation. Here, various component ratios of the ternary mixed
solvents on the phase diagram including solubility curves were used as carrier solutions
57

Consideration of TRDP

57

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesin the TRDC, and the obtained chromatograms are considered together along with thedata provided through the solubility curves.
Experimental
Fused-silica capillary tubes (50 µm inner diameter) were used. A schematic diagram ofthe present capillary chromatography system (TRDC system) comprised an
open-tubular fused-silica capillary tube having (120 cm total length and 100 cm
effective length), microsyringe pump, and absorption detector. The tube temperature
was controlled by dipping the capillary tube in water maintained at a definite
temperature (5 or 20 °C) in a beaker with stirring. Water–acetonitrile–ethyl acetate
solvent mixtures were used as carrier solutions. Analyte solutions including 1-naphtholand 2,6-NDS (1 mM each) were prepared using the carrier solutions. The analyte
solution was introduced directly into the capillary inlet using the gravity method (20 s ×
30 cm height). After analyte injection, the capillary inlet was connected through a joint
to a microsyringe. The syringe was set on the microsyringe pump. The carrier solution
was fed into the capillary tube at a definite flow rate (0.2 µL min−1) under laminar flow
conditions. On capillary absorption detection (254 nm) was performed using thedetector.
Results and discussion
Phase diagram for ternary solvents A phase diagram for the ternary mixture ofwater–acetonitrile (hydrophilic organic solvent)–ethyl acetate (hydrophobic organic
solvent) was examined in a batch vessel at temperatures of 5 and 20 °C. The obtainedphase diagram is shown in Fig. 1. The curves expressed in the diagram (◆ : 5 °C and□: 20 °C) indicate the solubility curves. The inside and outside of the curves represent
heterogeneous and homogeneous solutions, respectively. The phase diagram shows thateach component ratio of the solvents forms a homogeneous (one homogeneous phase)or heterogeneous (two homogeneous phases) solution. The heterogeneous solutions
include the upper (organic solvent-rich solution) and lower (water-rich solution) phasesin the batch vessel. The component ratios of the solvents in the solutions 1–10 are
plotted in the diagram.
Chromatograms obtained with tube temperatures of 20 °C
The model analytes,
hydrophobic 1-naphthol and hydrophilic 2,6-NDS, were examined with the present
TRDC system at a tube temperature of 20 °C. The ternary mixture ofwater–acetonitrile–ethyl acetate homogeneous solution 1 (the volume ratio of 3:9:4) and7 (60:13:7) were used as the organic solvent-rich and water-rich carrier solutions,
respectively. The compositions of solvents 1 and 7 were positioned close to thesolubility curve at 20 °C (Fig. 1). 1-Naphthol and 2,6-NDS were separated in this orderwith the organic solvent-rich carrier solution, but were not separated with the water-rich

58

Chapter 358

carrier solution (Fig. 2).
Chromatograms obtained with tube temperatures of 5 °C
The analytes were
examined with the TRDC system at a tube temperature of 5 °C. The homogeneous
carrier solutions 1–6 and 7–10 were used as the organic solvent-rich and water-richcarrier solutions, respectively. The obtained chromatograms are shown in Fig. 3. The
solvent compositions are described in the figure captions. 1-Naphthol and 2,6-NDS
were separated in this order with the organic solvent-rich carrier solution (1–5), but they
were not separated with carrier solution 6, for which the solvent composition was
positioned outside the solubility curve at 5 °C (homogeneous region). With thewater-rich carrier solution, 1-naphthol and 2,6-NDS were separated in the reverse
elution order with carrier solutions 7 and 8, but they were not separated with carrier
solutions 9 and 10, for which solvent compositions were placed outside the solubility
curve at 5 °C (homogeneous region). The elution order of the analytes could be changedby altering the component ratios of the solvents, organic solvent-rich or water-rich, at a
tube temperature of 5 °C. The experimental data were consistent with the TRDC
separation performance and the principles, which were reported.2,14,17) With the organic
solvent-rich solution, the organic solvent-rich major phase was generated around the
middle of the capillary tube as an inner phase, whereas a water-rich minor phase wasformed near the inner wall as an outer phase (pseudo-stationary phase). In contrast, withthe water-rich solution, the water-rich major phase was generated as an inner phase,whereas the organic solvent-rich minor phase was formed as an outer phase. Whileundergoing chromatographic separation, the hydrophobic and hydrophilic model
analytes were distributed in the inner and outer phases because of their nature.
The organicorganic solvent-rich
Consideration throughthrough phase
phase diagram
diagram andandchromatogram
chromatogram The
carrier solutions (1–5), for which solvent compositions existed in a large domain
between the solubility curves of 5 and 20 °C as shown in Fig. 1, showed better
resolution than the water-rich carrier solutions (7 and 8), for which solvent
compositions existed in a more narrow domain. It was suggested that separation
performance in the TRDC might be related to the data provided through the tie lines on
the phase diagram.

In conclusion, a phase diagram including solubility curves at 5 and 20 °C wasconstructed and the component ratios of the solvents near the solubility curves wereused as carrier solutions in the TRDC to expand our knowledge of the TRDP and TRDC.
Model analytes, 1-naphthol and 2,6-NDS, were separated in this order with organic
solvent-rich carrier solutions, and in the reverse order with the water-rich carrier
solutions at a tube temperature of 5 °C. The organic solvent-rich carrier solutions, forwhich solvent compositions existed in a large domain between the solubility curves of 5
59

Consideration of TRDP

59

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesand 20 °C, showed better resolution than the water-rich carrier solutions, for whichsolvent compositions existed in a narrow domain. Introducing the phase diagram
concept into the TRDC and TRDP will improve to ease and clarity of related
investigations in the future.

Figure 1. Phase diagram for water–acetonitrile–ethyl acetate mixture at 5 and 20 °C.
The curves labeled with symbols ◆ and □ in the diagram indicate the solubility
curves at 5 and 20 °C, respectively. The symbol ●	  shows the component ratios ofsolutions 1-10 that gave the chromatograms as shown in Fig. 3.

60

Chapter 360

Figure 2. Chromatograms of 1-naphthol (peak 1) and 2,6-NDS (peak 2) by the TRDC
system with tube temperature of 20 °C. a) Organic solvent-rich and b) water-richcarrier solution have water–acetonitrile–ethyl acetate mixture volume ratio of a) 3:8:4
and b) 60:13:7, respectively.

Figure 3. Chromatograms of 1-naphthol (peak 1) and 2,6-NDS (peak 2) by the TRDC
system with tube temperature of 5℃. The water–acetonitrile–ethyl acetate mixture
volume ratio of ① 3:9:4, ② 3:10:4, ③3:11:4, ④ 3:12:4, ⑤ 3:13:4, ⑥ 3:14:4 in a)organic solvent-rich carrier solutions and ⑦ 60:13:7, ⑧60:14:6, ⑨ 60:15:5, ⑩ 60:16:4
in b) water-rich carrier solutions.
61

Consideration of TRDP

61

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

3.3 Experimental consideration through the phase diagram including
tie lines and solubility curves in TRDC and TRDP
Open-tubular capillary chromatography using a ternary solvent mixture consisting of awater–hydrophilic–hydrophobic organic solvent as a carrier solution has beendeveloped. When the ternary carrier solution is fed into the capillary tube, the carrier
solvents are radially distributed and generate inner and outer phases in the tube. The
outer phase functions as a pseudo-stationary phase in chromatography. Here,investigations proceeded with reference to the tie lines and solubility curves on thephase diagram of the ternary mixed solvents. Model analytes, 1-naphthol and
2,6-naphthalenedisulfonic acid, were separated in this order with ternarywater–acetonitrile–ethyl acetate solvent mixtures (organic solvent-rich solutions) thatpossessed various solvent compositions on the tie lines. In addition, fluorescencephotographs of the dyes dissolved in the ternary solvents in the capillary tubes wereobserved with a fluorescence microscope-CCD camera system. It was found that the
separation performance on the chromatograms and the phase formation observed in the
fluorescence photographs were related to data provided through the tie lines andsolubility curves on the phase diagram. The solvent compositions on the same tie linethat gave different volume ratios of upper and lower phases in a vessel influenced thechromatographic separation, or the resolutions of the analytes, and also the inner andouter phase formation in the chromatography.
Introduction
The TRDP creates a phase interface or a kinetic liquid-liquid interface in a microspace.
We are currently investigating the TRDP from the viewpoint of chromatography,
extraction, mixing, and chemical reaction space. A capillary chromatography system inwhich the outer phase acts as a pseudo-stationary phase under laminar flow conditions
has been developed based on the TRDP. We call it TRDC.
The TRDP appears through phase separation from a homogeneous solution to aheterogeneous solution including two phases with pressure and temperature changes.
The phase separation with associated changes forms an upper and lower phase in abatch vessel under the influence of gravity. At the same time, the phase separation
introduces TRDP, including inner and outer phases in a micro-flow under laminar flow
conditions where gravity has no influence. During the TRDP and TRDC investigation,
we considered that tie lines and the solubility curves on the phase diagram would be
useful tools for investigation. Here, various component ratios of the ternary mixed
solvents on the tie lines were used as carrier solutions in the TRDC, and the obtained
chromatograms and fluorescence photographs were considered together along with data
provided through the tie lines and solubility curves.

62

Chapter 362

Experimental
TRDC system Fused-silica capillary tubes (50 µm inner diameter) were used. A
schematic diagram of the present capillary chromatography system (TRDC system)comprised an open-tubular fused-silica capillary tube (120 cm total length and 100 cm
effective length), a microsyringe pump, and an absorption detector (Fig. 1 A)). The tube
temperature was controlled by dipping the capillary tube in water maintained at a steady
temperature (5 °C) in a beaker with stirring. Water–acetonitrile–ethyl acetate solvent
mixtures were used as carrier solutions. Analyte solutions including 1-naphthol and
2,6-naphthalenedisulfonic acid (1 mM each) were prepared using the carrier solutions.
The analyte solution was introduced directly into the capillary inlet using the gravity
method (20 s × 30 cm height). After analyte injection, the capillary inlet was connected
through a joint to a microsyringe. The syringe was set on the microsyringe pump. The
carrier solution was fed into the capillary tube at a definite flow rate (0.2 µL min−1)
under laminar flow conditions. Upon capillary absorption, detection (254 nm) was
performed using the detector.
Fluorescence photographs We set up a capillary tube of the same size as thatused in the TRDC system for the fluorescence microscope-CCD camera
system (Fig. 1 B)). The fluorescence in the capillary tube was monitored at
approximately 100 cm from the capillary inlet using a fluorescencemicroscope equipped with an Hg lamp, a filter, and a CCD camera. The
carrier solution contained 0.1 mM perylene (blue) and 1 mM Eosin Y (green).
The carrier solution was delivered into the capillary tube at a definite flowrate (0.2 µL min−1) using a microsyringe pump.
Results and Discussion
Tie lines in the phase diagram
Fig. 2 shows the tie lines at 5 °C on the phase diagram.
The tie lines were constructed using the solvent composition of the homogeneous
solution at 20 °C, the water content of the lower phase (the water-rich phase) in a batch
vessel at 5 °C, and the solubility curve at 5 °C. The plots of i–vi are on the same tie line,
and the volume ratios of the upper and lower phases at 5 °C in the batch vessel changein accordance with the ratios of the line lengths, which are separated by the plots on thetie line (although the solvent compositions of the upper and lower phases are all thesame for the plots of i–vi, water-acetonitrile-ethyl acetate volume ratios of upper andlower phases are 17:64:19 and 58:36:6, respectively). For example, the solvent
composition of i changes from a homogeneous solution at 20 °C to a heterogeneous
solution including upper (organic solvent-rich) and lower (water-rich) phases with thevolume ratio of the upper and lower phases at 6:4 at 5 °C in a vessel. Similarly, thevolume ratios of the upper and lower phase for plots ii–vi are 7:3, 8:2, 9:1, 9.5:0.5, and63

Consideration of TRDP

63

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

9.8:0.2, respectively. In addition, the plots of v and vii–ix are on different tie lines on
the phase diagram. However, the solutions of the plots of v and vii–ix at 20 °C are
separated into a 9.5 (upper phase):0.5 (lower phase) volume ratio at 5 °C in the batch
vessel (although the solvent compositions of the upper and lower phases are quite
different for the plots of v and vii–ix).
Effects of volume ratios of the upper and lower phases in a batch vessel on separation
performance in TRDC As described in the introduction, the phase separation withpressure and temperature changes forms between upper and lower phases in a batch
vessel under the control of gravity. At the same time, the phase separation introduces
TRDP, including inner and outer phases in a micro-flow where it is under laminar flow
conditions and not controlled by gravity. The volume ratios of the upper and lower
phases in a vessel that were estimated using the tie lines would not necessarily be equal
to those of the inner and outer phases in TRDP. However, we believe that the volume
ratios of the upper and lower phases in a vessel may be related to the formation of innerand outer phases in the TRDP. First, we attempted to examine the effects of the solvent
compositions on the same tie line, or phase volume ratios in a vessel, on the separation
performance in the TRDC. The solvent compositions provide different volume ratios of
the upper and lower phases in a batch vessel (from 6:4 to 9.8:0.2), as described above.
The analytes, 1-naphthol and 2,6-naphthalenedisulfonic acid, were examined with the
TRDC system using carrier solutions i–vi. The obtained chromatograms are shown in
Fig. 3 together with the resolutions (Rs), whereas the solvent compositions are describedin the figure captions. 1-Naphthol and 2,6-naphthalenedisulfonic acid were separated inthis order with carrier solutions ii–vi, but they were not separated with carrier solution i.
Improved resolutions were observed around carrier solutions iv and v; Rs, 6.3–7.0. In
addition, fluorescence photographs were observed with a fluorescence microscope-CCD
camera system. The obtained results are shown in Fig. 4. The inner and outer phases in
the capillary tube were formed with carrier solutions ii–vi, although the distribution
patterns of the dyes differed from each other to some extent. However, the formation
was not observed with carrier solution i. The observed TRDP was almost consistent
with the chromatographic separation, as shown in Fig. 3. The solvent compositions on
the same tie line that gave different volume ratios of upper and lower phases in thevessel, influenced the inner and outer phase formation in the TRDC, and also changed
Rs on the chromatograms.
Effects of solvent compositions on the different tie lines on the separation performancein TRDC Next, the analytes, 1-naphthol and 2,6-naphthalenedisulfonic acid, were
examined with the TRDC system using carrier solutions v and vii–ix on different tielines. The solvent compositions of these systems provided the same volume ratios of theupper and lower phases (9.5:0.5) in the batch vessel, as described above. The obtained64

Chapter 364chromatograms are shown in Fig. 5 together with Rs, whereas the solvent compositions
are described in the figure captions. 1-Naphthol and 2,6-naphthalenedisulfonic acid
were separated in this order with carrier solutions v and vii–ix at similar resolutions; Rs,
5.6–7.0. Furthermore, fluorescence photographs were observed with a fluorescencemicroscope-CCD camera system. The inner and outer phases in the capillary tube wereformed with carrier solutions v and vii–ix with similar distribution patterns of the dyes
(Fig. 6). The observed TRDP was consistent with the chromatographic separation
shown in Fig. 5. The experimental data indicated that the TRDC proceeds through phaseseparation from a homogeneous solution to a heterogeneous solution, little influenced
by the solvent compositions of the inner and outer phases in the capillary tube.
In conclusion, the component ratios of the solvents on the tie lines were used as
carrier solutions in the TRDC to expand our knowledge of the TRDP and TRDC. The
different solvent compositions on the same tie line that gave different volume ratios of
the upper and lower phases in a vessel influenced the Rs of analytes on thechromatograms and the formation of inner and outer phases in the TRDC. Furthermore,
if the different solvent compositions on the different tie lines provided the same volume
ratio of the upper and lower phases in a vessel, then the obtained chromatograms andfluorescence photographs would be similar to each other in the TRDC. The
experimental data supported that TRDC was performed through the phase separationfrom a homogeneous solution to a heterogeneous solution. Introducing the tie lines
concept into the TRDC and TRDP will increase both the ease and clarity of related
investigations in the future.

65

Consideration of TRDP

65

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Schematic diagrams of A) open-tubular capillary chromatographic system(TRDC system) and B) fluorescence microscope-CCD camera.

Figure 2. Tie lines in the phase diagram at 5 °C. The ○s express the prepared component
ratios of the solvents in the heterogeneous solutions at 20 °C.

66

Chapter 366

Figure 3. Chromatograms of 1-naphthol and 2,6-naphthalenedisulfonic acid by the
TRDC system with tube temperature of 5 °C. 1; 1-Naphthol and 2;
2,6-naphthalenedisulfonic acid. Carrier, water–acetonitrile–ethyl acetate mixture
volume ratio of (i) 34:53:13, (ii) 30:56:14, (iii) 26:58:16, (iv) 21:62:17, (v) 19:63:18,
and (vi) 18:64:18.

67

Consideration of TRDP

67

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Fluorescence photographs of the fluorescent dyes dissolved in the ternary
mixed carrier solvents. Carrier, water–acetonitrile–ethyl acetate mixture volume ratio of(i) 34:53:13, (ii) 30:56:14, (iii) 26:58:16, (iv) 21:62:17, (v) 19:63:18, and (vi) 18:64:18
including 0.1 mM perylene and 1.0 mM Eosin Y.

Figure 5. Chromatograms of 1-naphthol and 2,6-naphthalenedisulfonic acid by the
TRDC system with tube temperature 5 °C. Carrier, water–acetonitrile–ethyl acetate
mixture volume ratio of (v) 19:63:18, (vii) 16:59:25, (viii) 13:54:33, and (ix) 11:43:46.

68

Chapter 3

68Figure 6. Fluorescence photographs of the fluorescent dyes dissolved in the ternary
mixed carrier solvents. Carrier, water–acetonitrile–ethyl acetate mixture volume ratio of(v) 19:63:18, (vii) 16:59:25, (viii) 13:54:33, and (ix) 11:43:46; including 0.1 mM
perylene and 1.0 mM Eosin Y.

69

Consideration of TRDP

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

3.4 Experimental consideration through the estimation of thedimensionless Weber number in TRDP
A ternary mixed solvents solution of water-hydrophilic/hydrophobic organic solvent is
an example of a two-phase separation, mixed solvent solution system. Upon temperaturechange, the homogeneous solution (single-phase) changes to a heterogeneous solution(two-phases), generating upper (organic solvent-rich) and lower (water-rich) phases in abatch vessel. When the ternary mixed solution is fed into a microspace, the solvent
molecules are radially distributed in the microspace under laminar flow conditions,generating inner and outer phases. This process is coined as tube radial distribution
phenomenon (TRDP). Here, the dimensionless number, Weber number, of the inner andouter phases in the TRDP process was estimated for the water–acetonitrile–ethyl acetate
and water–acetonitrile–chloroform systems. The relationship between the Weber
number and TRDP formation was examined. TRDP was observed in both systems when
the Weber numbers of the organic solvent-rich and water-rich phases were different.
However, an either excessively large or small difference in the Weber numbers did not
facilitate TRDP. TRDP formation was compared with an annular flow formation using
the immiscible mixed solvent solutions.
Introduction
Phase separation of aqueous systems containing polymers, micelles, and ionic liquid
solutions is well known and has been used in the many fields of analytical science since
the last century [15,18,19]. However, a novel two-phase separation system involving
ternary mixed solvents has been reported by our group.17,24) Upon temperature and/orpressure changes, a ternary mixed solvents solution of water-hydrophilic/hydrophobic
solvents changes from a homogeneous (single-phase) to a heterogeneous (two-phase)
solution. Accordingly, we demonstrated that the two-phase separation systemcomprising a ternary mixed solvents solution in a batch vessel could be used for the
extraction of metal ions.41)
Furthermore, interesting and unique fluidic behaviors of two-phase separation
systems, such as water–hydrophilic/hydrophobic organic solvents, micelle aqueous
solvent, and ionic liquid aqueous solvent solutions, in a microspace under laminar flow
conditions were observed. When the homogeneous solutions of two-phase separation
systems are delivered to a microspace, such as glass microchannels and capillary tubes(e.g., fused-silica, polyethylene, and polytetrafluoroethylene), the homogeneous
solutions change to the heterogeneous solutions, generating a hydrodynamic
liquid-liquid interface with inner and outer phases. We termed such a process as tube
radial distribution phenomenon (TRDP).
Liquid–liquid flows have a wide range of applications in lab-on-a-chip devices andmicroreactors. Liquid–liquid flows using immiscible mixed solvents, oil and water, inconventional tubes and mixing procedures have been studied [14,16,17,20]. When themixed solvents flows are successfully controlled, various types of flow patterns, such as
droplet, slug, and annular flows, are observed in the tube. Flow patterns generated from
immiscible mixed solvents were discussed in terms of dimensionless numbers,
especially, the Weber number [14,16]. Here the dimensionless number, the Weber70

Chapter 3

70number, was estimated for microfluidic flows in TRDP using a ternary mixed solvents
solution of water-hydrophilic/hydrophobic organic solvent, and the relationship between
the Weber number and TRDP formation was investigated.
Experimental
Phase diagrams Phase diagrams were constructed for the ternary mixed solvents ofwater–hydrophilic/hydrophobic organic solvents (water–acetonitrile–ethyl acetate andwater–acetonitrile–chloroform systems) at 20 °C in a batch vessel. The phase diagram
included the solubility curve that separates the component ratios of the solvents into
homogeneous (single-phase) and heterogeneous (two-phase) areas.
Viscosity measurement
The homogeneous solutions of all the mixed solventschanged to heterogeneous solutions that comprised two phases, the upper and lower
phases, in a batch vessel by changing the temperature. The viscosities of the upper(organic solvent-rich) and lower (water-rich) solutions were measured with a viscometer(HAAKE RheoScope 1; Thermo Scientific, Sydney, NSW, Australia).
Interfacial tension calculation
The surface tensions of the upper (organic
solvent-rich) and lower (water-rich) solutions (upon conversion of the homogeneous
solution into the heterogeneous solution) were measured with a surface tension balance.
The interfacial tension (Σ) was calculated using the following equation [13] :Σ=!! !!! !! !! !!
!!!.!"# !! !!

,where σo is the surface tension of the organic solvent-rich phase (O) and σw is thesurface tension of the water-rich phase (W).
Fluorescence microscope-CCD camera system
Fused-silica capillary tubes (innerdiameter: 50 µm) were used. To monitor the TRDP, the capillary tube was set up
accordingly and observed under a fluorescence microscope-CCD camera system.
Ternary water–hydrophilic/hydrophobic organic solvent systems with various
component ratios and comprising perylene (0.05–0.1 mM) and Eosin Y (0.1–1.0 mM)
were used. The fluorescence in the capillary tube (total length: 120 cm) was monitored
at 20 cm from the tube outlet using a fluorescence microscope equipped with an Hglamp, a filter, and a CCD camera. Perylene and Eosin Y emit light at 470 and 550 nm,
respectively. Accordingly, the resulting fluorescence observed was mainly blue andgreen.
Results and discussion
Estimation of weber number Various types of oil–water (immiscible solvents) flows
in a tube were examined and discussed in terms of dimensionless numbers such as the
Capillary, Reynolds, and Weber numbers [14,16,17,20]. More specifically, the Weber
number has been used to examine the flow patterns of droplet, slug, and annular flows
in a tube [14,16]. The Weber number, We, is expressed according to the following
equation:
71

Consideration of TRDP

71

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

We =!! !! ! !
!

,where ρi is the density, Vi is the superficial velocity, D is the inner diameter, andΣ is theinterfacial tension (subscript i refers to the organic solvent-rich solution (O) or
water-rich solution (W)). The value of ρiVi2D corresponds to the inertial force. Hence,
the Weber number is defined as the ratio of inertial force to the interfacial tension(interfacial force). The inertial force tends to extend the interface in the direction of theflow and maintain a continuous fluid. In contrast, the interfacial force tends to minimize
the interfacial energy by reducing the oil–water interfacial area, leading to the formationof droplets and plugs. Fig. 1 shows illustrations of the flow systems, immiscible mixed
solvent and TRDP, and their typical flow patterns in a tube. For typical immiscible
mixed solvent solutions, the superficial velocities are obtained by dividing the flow
rates in the individual delivery tubes, oil and water lines, by the cross–section of thetube, mixing single line (Fig. 1a) [14]. In contrast, in TRDP, the apparent flow rates ofthe organic solvent-rich and water-rich phases in the capillary single tube are obtainedby dividing the flow rate in the capillary tube by the volume ratio of the upper(organic-solvent-rich) and lower (water-rich) phases in a batch vessel. The superficial
velocities are then calculated by dividing the apparent flow rates by the cross-section of
the capillary tube (Fig. 1b).
Flow patterns of ternary mixed solvents of water-hydrophilic/hydrophobic organic
solvents
Foroughi et al. reported the relationship between the flow patterns andassociated Weber numbers for immiscible mixed solvents solutions [14]. Generally,
when immiscible solutions (i.e., O and W) were fed into the mixing single tube with thetwo individual flow lines, as shown in Fig. 1a, and the flow rate of solution O wasconstant and that of solution W gradually increased, specific flow patterns in the single
tube were generated. A droplet flow was observed when the flow rate and the Weber
number of solution W were relatively small and the interfacial tension in the droplet of
W was dominant relative to the inertial force. A slug flow was observed when the flowrate and the Weber number of solution W were at intermediate values and the inertial
force and interfacial tension in the slug of W were comparable. An annular flow wasobserved when the flow rate and the Weber number of solution W were relatively large
and the inertial force in the inner phase of W was dominant relative to the interfacial
tension. When the ternary mixed solution of water–hydrophilic/hydrophobic organic
solvent was introduced into a capillary tube, the solvents molecules were radiallydistributed in the tube under certain flow conditions, that is, TRDP. As shown in Fig. 1b,
we examined flow patterns using varying component ratios of the ternary solvents in the
TRDP flow system in a manner similar to that of the immiscible mixed solvents system.
Specific flow patterns in the capillary tube were observed, as depicted in the phase
diagrams in Fig. 2: TRDP (organic solvent-rich inner phase and water-rich outer phase(○), and water-rich inner phase and organic solvent-rich outer phase (●)) andnon-TRDP (axial distribution ( △ ) and heterogeneous distribution ( × )) for thewater–acetonitrile–ethyl acetate and water–acetonitrile–chloroform systems. The
relationship between the flow patterns and Weber numbers is discussed in the following
section.

72

Chapter 372

Relationship between the volume ratios, weber numbers, and flow patterns in the TRDP
flow system The volume ratios of the generated upper (organic solvent-rich) andlower (water-rich) phases of the ternary mixed solvents at varying component ratios in abatch vessel at low temperatures were examined. Additionally, the Weber numbers of
the inner (organic solvent-rich or water-rich) and outer (water-rich or organic
solvent-rich) phases in the TRDP flow system were calculated. The flow patterns,
TRDP and non-TRDP, in the capillary tube were determined on a fluorescencemicroscope-CDD camera system. The following two systems were studied:water-acetonitrile-ethyl acetate and water-acetonitrile-chloroform. The results are
summarized in Fig. 3. The notation of the flow patterns, expressed with symbols, ○,●, △, and ×, is similar to that employed in Fig. 2. As observed in Fig. 3, similar
relationships between the volume ratios, Weber numbers, and flow patterns for bothternary mixed solvents systems studied (water–acetonitrile–ethyl acetate andwater–acetonitrile–chloroform) were determined. At the water-rich (organic
solvent-rich) phase volume ratio of ~50% vol where the Weber numbers of the twophases were comparable, TRDP was not observed; an axial distribution flow (△) was
observed. In this case, the inertial force and interfacial tension in the phases werecomparable. When the water-rich (organic solvent-rich) phase volume ratios wereconsiderably small (<5% vol) or large (>95% vol), TRDP was not observed; ahomogeneous flow (×) was observed. The significantly small Weber number or inertial
force were unable maintain a continuous flow. In contrast, when the water-rich (organic
solvent-rich) phase volume ratios were 5–30% vol and 60–95% vol, TRDP was clearlyobserved; an organic solvent-rich inner phase and water-rich outer phase (○) or awater-rich inner phase and organic solvent-rich outer phase (●) were formed. In thiscase, the inner phase that had a larger volume ratio possessed a relatively larger Weber
number than that of the outer phase, leading to a dominant inertial force relative to theinterfacial tension. Similar relationships between the Weber numbers (the ratio of theinertial force to the interfacial tension) in the larger volume phase and flow patterns(annular flow or TRDP flow) in immiscible mixed solvents solutions [14] and ternary
mixed solvents solution of water–hydrophilic/hydrophobic organic solvent wereobserved.
In conclusion, the dimensionless number, the Weber number, was used to examine
TRDP formation, including the inner and outer phases, in the water–acetonitrile–ethyl
acetate and water–acetonitrile–chloroform systems. An interesting relationship between
the Weber number and inner and outer phase formation in TRDP was determined.
TRDP was clearly observed in the capillary tube when the Weber numbers of theorganic solvent-rich and water-rich phases differed. The current findings are an
extension of the application of the TRDP concept to investigate microfluidic behaviors,
and thus present potential in developing new systems based on microfluidic technology
relying on liquid–liquid interfacial processes such as chromatography and extraction.

73

Consideration of TRDP

73

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Nomenclature; D = inner diameter [m], V = superficial velocity [m s −1], We = Weber
Number [-],Σ = interfacial tension (interfacial force) [mN m−1], σ = surface
tension [mN m−1], and ρ= density [kg m −3].

Figure 1. Illustration of the flow systems and typical flow patterns. (a) Immiscible
mixed solvent flow and (b) TRDP flow systems.

74

Chapter 374

Figure 2. Phase diagram of (a) water–acetonitrile–ethyl acetate and (b)water–acetonitrile–chloroform systems determined at 20 °C. TRDP conditions: capillarytube, 50 µm inner diameter and 100 cm effective length; flow rate, 0.20 µL min−1. The
flow patterns are denoted as follows: TRDP, organic solvent-rich inner phase andwater-rich outer phase (○), and water-rich inner phase and organic solvent-rich outerphase (●); non-TRDP, axial distribution (△) and homogeneous distribution (×).

75

Consideration of TRDP

75

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3. Relationship between the water phase volume ratio and Weber number for thefollowing
systems:(a)water–acetonitrile–ethyl
acetateand(b)water–acetonitrile–chloroform. TRDP conditions: capillary tube, 50 µm inner diameterand 100 cm effective length; flow rate, 0.20 µL min−1. The flow patterns are denoted asfollows: TRDP, organic solvent-rich inner phase and water-rich outer phase (○) as wellas water-rich inner phase and organic solvent-rich outer phase (●) and non-TRDP, axial
distribution (△) and homogeneous distribution (×).

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Chapter 376

3.5 Experimental consideration through the inner and outer phaseformation in TRDP using various types of mixed solvent solutions
When mixed solvent solutions, such as ternary water–hydrophilic/hydrophobic organic
solvents, water–surfactant, water–ionic liquid, and fluorous–organic solvents, aredelivered into a microspace under laminar flow conditions, the solvent molecules areradially distributed in the microspace, generating inner and outer phases. This specificfluidic behavior is termed tube radial distribution phenomenon (TRDP). Here, thefactors influencing the formation of inner and outer phases in the TRDP using theabove-mentioned mixed solvent solutions were investigated. We examined phase
diagrams, viscosities of the two phases (upper and lower phases in a batch vessel),volume ratios of the phases, and bright-light or fluorescence photographs of the TRDP.
When the difference in viscosities between the two phases was large (> ca. 0.73 mPa·s),
the phase with the larger viscosity formed an inner phase regardless of the volume ratios.
Conversely, when the difference was small (< ca. 0.42 mPa·s), the phase with the larger
volume formed an inner phase. The TRDP using a water–surfactant mixed solution was
also investigated in capillary chromatography based on TRDP.
Introduction
The phase separation of aqueous systems containing polymers, micelles, and ionicliquids is well known, and has been used in the fields of analytical chemistry andseparation science since the last century [15,18,21-23]. For example, aqueous micellar
solutions of specific non-ionic surfactants separate into two distinct phases when heated
above a certain temperature (cloud point), i.e., a temperature-induced phase separation
occurs [24-28]. One phase behaves as an almost surfactant-free aqueous solution(aqueous phase), whereas the other phase is a concentrated surfactant solution(surfactant-rich phase). Hydrophobic compounds dissolved in the aqueous micellar
solution flow into the surfactant-rich phase, whereas hydrophilic compounds remain in
the aqueous phase.
Fluorous or fluorocarbon chemistry, which involves phase separation processes, hasalso been investigated since the study reported by Horváth and Rábai in 1994 [29]. The
phase separation of fluorous (fluorocarbon)–organic (hydrocarbon) solvent mixed
solutions has been applied in separation science [30,31]. Mixed solutions offluorous–organic solvents separate into two distinct phases when cooled below a certain
temperature in a batch vessel [32]. In which the lower phase is comprised of a nearlypure fluorous solvent and the upper phase consisting of an organic solvent. Based on
this phase separation system, liquid–liquid and liquid–solid extractions have beenreported using a variety of fluorous solvents [33-35].
A novel phase separation system involving a ternary mixed solvent system has beenreported by our group.14,17,38) A ternary mixed solvent solution of77

Consideration of TRDP

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

water–hydrophilic/hydrophobic solvents changes from a homogeneous (single-phase) toa heterogeneous (two-phase) system in a batch vessel according to temperature and/orpressure changes. Furthermore, when the ternary mixed solvent solution is introduced
into a microspace, such as a microchannel on a microchip or a capillary tube, underlaminar flow conditions, the solvent molecules are radially distributed in the microspace,
generating inner and outer phases. This process is termed as tube radial distribution
phenomenon (TRDP). The TRDP creates a kinetic liquid-liquid interface in a
microspace. We developed new types of open-tubular capillary chromatography, i.e.,
tube radial distribution chromatography (TRDC) and microreactors based on the TRDP.
More interestingly, such specific fluidic behavior, TRDP, could also be observed in a
microspace, involving the other two-phase separation solutions such as water–surfactant,water–ionic liquid, and fluorous-organic solvents.
However, comprehensive information about the factors influencing the formation of
the inner and outer phases in the TRDP is still lacking for various types of mixed
solutions. To this effect, the phase diagrams, viscosities of the two phases (upper andlower phases) in a batch vessel, volume ratios of the phases, and bright-light or
fluorescence photograph of the TRDP were examined by using six kinds of phaseseparation solutions. General discussion for the TRDP creation has been done on thebasis of the experimental data, and the TRDP with water–surfactant mixed solution wasapplied as an attempt to a capillary TRDC.
Experimental
Phase diagrams Phase diagrams were constructed for the six types of mixed
solvent solutions: ternary water–hydrophilic/hydrophobic organic solvents(water–acetonitrile–ethyl acetate), water–surfactant (water–Triton X-100–KCl),water–ionic liquid where 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and1-ethyl-3-methylimidazolium methylphosphonate ([C2mim]MP) were used(water–[C4mim]Cl–KOH,
water–[C4mim]Cl–K2HPO4,
andwater–[C2mim]MP–K2HPO4),
andfluorous-organic
solvents(tetradecafluorohexane–hexane). All phase diagrams, regardless of the composition
system, included solubility curves that separated the component ratio areas into
homogeneous (single-phase) and heterogeneous (two-phase) areas in a batch vessel.
Viscosity measurement The homogeneous solutions of all the mixed solvent systems
were converted to heterogeneous solution systems comprised of two phases, upper andlower, by changing the temperature. The viscosities of the upper and lower solutions
were measured with a viscometer (HAAKE RheoScope 1; Thermo Scientific, Sydney,
NSW, Australia).
Bright-field or fluorescence microscope–CCD camera system

78

Chapter 3

78

Fused-silica capillarytubes (50 or 75 µm i.d.) were used. The bright-field or fluorescence microscope–CCD
camera system was equipped with the fused-silica capillary tube. The fluorescentdye-containing mixed solvent solution that was introduced in the capillary tube wasobserved using a microscope and a CCD camera for bright-field and fluorescenceimaging. For the latter imaging, the microscope was equipped with an Hg lamp and afilter (U-MWU2; excitation wavelength: 330–385 nm, emission wavelength: > 420 nm).
The temperature of the capillary tube was maintained using a thermo-heater (Thermo
Plate MATS-555RO; Tokai Hit Co., Shizuoka, Japan).
Capillary TRDC The open-tubular capillary chromatography, TRDC, was mainlycomprised of a microsyringe pump, a fused-silica capillary tube (total length: 150 cm
and effective length: 100 cm), a thermo-heater, and a fluorescence detector (excitation
wavelength: 290 nm and emission wavelength: 355 nm). An aqueous solution
containing 2% wt. Triton X-100 and 2.4 M KCl or 2% wt. Triton X-114 was used as acarrier solution. A model analyte solution comprising 1-naphthol (1 mM) and
2,6-naphthalenedisulfonic acid (1 mM) dissolved in the carrier solution was prepared.
The analyte solution was injected into the capillary inlet using the gravity method (from30 cm height and for 30 s), and then fed into the capillary tube at a flow rate of 1.0 µL
min−1.
Results and discussion
Phase diagrams including solubility curves
Phase diagrams were constructed for thefollowing six types of mixed solvent systems studied; water–acetonitrile–ethyl acetate,water–Triton X-100–KCl, tetradecafluorohexane–hexane, water–[C4mim]Cl–KOH,
water–[C4mim]Cl–K2HPO4, and water–[C2mim]MP–K2HPO4 (Fig. 1). The
compositions shown on the axes of the phase diagrams are different depending on thesystem studied. The phase diagram of water–acetonitrile–ethyl acetate was based on thesolvent volume percentage of each component. The phase diagrams of water–Triton
X-100–KCl and tetradecafluorohexane–hexane were constructed according to therelationship between the temperature and concentration of Triton X-100 (% wt.) or mole
fraction of tetradecafluorohexane. The phase diagram of water–ionic liquid–salt wasconstructed based on the relationship between the concentrations of the salt and ionicliquid. All phase diagrams included solubility curves that separated the component ratio
areas of the solvents into the two phases, i.e., homogeneous (single-phase) andheterogeneous (two-phases). In other words, the homogeneous solution changed to aheterogeneous solution or the heterogeneous solution changed to a homogeneous
solution upon alteration of the temperature in the batch vessel. The solubility curves
were constructed based on the experimental data. The homogeneous mixtures, at theassociated component ratios, designated by the numbers in square brackets in theindividual phase diagrams, separated into two phases (upper and lower) in the batch
79

Consideration of TRDP

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

vessel upon change in the temperature. The viscosities and volume ratios of the upperand lower phases were examined; furthermore, the homogeneous solutions weredelivered into the capillary tube to observe the TRDP using either bright-field or
fluorescence microscopy, as discussed in the following sections.
Viscosities of the upper and lower phases
We examined the viscosities of the upperand lower solutions of the different mixed solvent systems following phase separation.
The relationship between the designated solution mixture of a specific component’s
composition and the associated viscosity for each system is shown in Fig. 2. The
differences in viscosities between the upper and lower solutions of thewater–acetonitrile–ethyl
acetate,water–Triton
X-100–KCl,tetradecafluorohexane–hexane, water–[C4mim]Cl–KOH, water–[C4mim]Cl–K2HPO4,
and water–[C2mim]MP–K2HPO4 systems were roughly estimated to be respectively
0.42, 440, 0.36, 1.7, 0.22, and 0.73 mPa·s. The system may be tentatively classified
according to the degree of difference in viscosities of the upper and lower phases for thefollowing discussion: large (> ca. 0.73 mPa·s) and small (< ca. 0.42 mPa·s). A large
viscosity difference between the upper and lower phases was observed for thewater–Triton X-100–KCl, water–[C4mim]Cl–KOH, and water–[C2mim]MP–K2HPO4systems, whereas a small viscosity difference was observed for thewater–acetonitrile–ethyl
acetate,tetradecafluorohexane–hexane,
andwater–[C4min]Cl–K2HPO4 systems.
Volume ratios of the upper and lower phases
The volume ratios of the upper andlower phases of the solvent mixtures designated by the numbers in square brackets inthe phase diagrams were evaluated. The upper/lower phase ratios for the mixed solvent
systems: water–acetonitrile–ethyl acetate (20 → 0 °C), water–Triton X-100–KCl (20 →34 °C), tetradecafluorohexane–hexane (20 → 10 °C), water–[C4mim]Cl–KOH (20 →15 °C), water–[C4mim]Cl–K2HPO4 (20 → 15 °C), and water–[C2mim]MP–K2HPO4 (20→ 15 °C) varied respectively as a function of the composition ratios from 90:10 to 4:96
(organic solvent-rich/water-rich), from 60:40 to 12:88 (surfactant-rich/water-rich), from85:15 to 20:80 (organic solvent /fluorous solvent), from 80:20 to 20:80 (ionicliquid-rich/water-rich), from 95:5 to 20:80 (ionic liquid-rich/water-rich), and from 90:10
to 10:90 (ionic liquid-rich/water-rich). The volume ratios of the upper and lower phases
are shown in Fig. 3.
Evaluation of the TRDP The homogeneous solutions were delivered to the capillary
tube under laminar flow conditions. The solvents’ behaviors, TRDP and non-TRDP,
were assessed using bright-field or fluorescence microscopy. Typical photographs
showing the development of TRDP are shown in Fig. 4. Interestingly, the solution
mixturesofwater–Triton
X-100–KCl,water–[C4mim]Cl–KOH,
and80

Chapter 380

water–[C2mim]MP–K2HPO4 generated the TRDP in the surfactant-rich inner phase andthe ionic liquid-rich inner phase, regardless of the volume ratios of the two phases orcomponent ratios of the solvents. In contrast, the solution mixtures ofwater–acetonitrile–ethyl
acetate,tetradecafluorohexane–hexane,
andwater–[C4min]Cl–K2HPO4 generated the TRDP in the major volume inner phase andminor volume outer phase. An inverse (reversible) distribution pattern was observedwith a change in the volume ratios of the solvents. For example, when considering thewater–acetonitrile–ethyl acetate system, introduction of the organic solvent-rich mixed
solution to the capillary tube generated an organic solvent-rich major inner phase and awater-rich minor outer phase; in contrast, introduction of the water-rich mixed solution
to the capillary tube produced a water-rich major inner phase and an organic
solvent-rich minor outer phase. The TRDP processes in the different mixed solvent
systems are denoted by the symbols ○, ●, and ×, and vary according to the lower andupper phases volume content (Fig. 3). The symbols ○ and ● denote the development of
TRDP, whereas symbol × implies that TRDP was not observed in a given system at aparticular upper/lower volume composition. The mixed solvent systems that include
both symbols ○ and ● in the figures demonstrate the occurrence of a reversible
distribution pattern of the inner and outer phases in accordance with the phasecomposition.
Investigation of the inner and outer phase formation
The factors influencing theformation of the inner and outer phases, i.e., non-reversible TRDP or reversible TRDP,
were evaluated based on the viscosities and volume ratios of the upper and lower phases,
and TRDP observations. When the difference in the viscosities between the two phases
was large (> ca. 0.73 mPa·s), the phase with the larger viscosity developed as the inner
phase regardless of the volume ratios. Conversely, when the difference in the viscosities
between the two phases was small (< ca. 0.42 mPa·s), the phase with the larger volume
content formed as the inner phase. The flow characteristics of two immiscible liquids
with different viscosities in a tube were investigated in the last century [36-39]. The
experimental studies showed that low-viscosity fluids have a tendency to encapsulate
high-viscosity fluids. This behavior is in accordance with the viscous–dissipation
principle, which postulates that the degree of viscous dissipation is smaller for a given
flow rate. Thus, the high-viscosity fluid is located at the core of the tube, away from theinner wall [36-27]. However, in 1984, Joseph et al. reported that if the low-viscosity
fluid occupies an extremely large volume of space in a tube, the low-viscosity fluid islocated at the core of the tube rather than near the wall of the tube, regardless of theviscosity values [40]. The discussions relating to the inner and outer flows in a tube in
the previous papers [36-40] were consistent with the present experimental data. The
flow characteristics of two immiscible liquids with different viscosities in a tube [36-40]
strongly supported the existence of a correlation between the development of TRDP and
81

Consideration of TRDP

81

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

the viscosities of the two phases for the six types of mixed solvents systems examinedherein.
Capillary TRDC
A capillary TRDC system was tentatively developed using a
fused-silica capillary tube as separation column and an aqueous micellar solution (2%wt. Triton X-100 and 2.4 M KCl or 2% wt. Triton X-114 solution) as a carrier solution.
Triton X-114 is a well-known typical micelle aqueous two-phase separation system.
Hydrophobic 1-naphthol and hydrophilic 2,6-naphthalenedisulfonic acid (2,6-NDS) as amodel mixture was examined in the current TRDC system. The obtained
chromatograms are shown in Fig. 5 as an initial result. 1-Naphthol and 2,6-NDS wereseparated and detected in this order; Triton X-114 system without salt gave better
resolutions. The elution order was expected based on the distribution of the aqueous
micellar solution that generated a surfactant-rich inner phase (hydrophobic) and asurfactant-poor outer phase (hydrophilic). The hydrophilic outer phase acted as a
pseudo-stationary phase under laminar flow conditions. The two peaks were identified
by analyzing individual analytes.
In conclusion, the TRDP generates a kinetic liquid–liquid interface in a microspace
upon introduction of a homogeneous mixed solvent solution in a microspace underlaminar flow conditions. The factors determining the formation of the inner and outerphases in the microspace were assessed by examining the phase diagrams incorporating
solubility curves, viscosities of the upper and lower phases in a batch vessel, volume
ratios of the phases, and development of the TRDP using microscopy imaging. Whenthe difference in the viscosities between the two phases was large (> ca. 0.73 mPa·s),
the phase with the higher viscosity formed the inner phase regardless of the volume
ratios, whereas when the difference in viscosities was small (< ca. 0.42 mPa·s), thephase with the larger volume ratio formed the inner phase. This finding is a steppingstone to future work in the TRDP research area.

82

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Figure 1. Phase diagrams incorporating solubility curves of a) water–acetonitrile–ethyl
acetate, b) water–Triton X-100–KCl (2.4 M), c) tetradecafluorohexane–hexane, d)e)water–[C4mim]Cl–K2HPO4,
andf)water–[C4mim]Cl–KOH,
water–[C2mim]MP–K2HPO4. The homogeneous solution mixtures, at the associatedcomponents composition, as denoted by the numbers in square brackets in the phase
diagrams were used for subsequent experiments.

83

Consideration of TRDP

83

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2. Viscosities of the (○) upper and (□) lower phases of the solvent mixtures, withdifferent solvent compositions, denoted by the numbers in square brackets in Fig. 1: a)water–acetonitrile–ethyl acetate, b) water–Triton X-100–KCl (2.4 M), c)e)tetradecafluorohexane–hexane,d)water–[C4mim]Cl–KOH,
water–[C4mim]Cl–K2HPO4, and f) water–[C2mim]MP–K2HPO4.

84

Chapter 384

Figure 3. Relationship between the development of the TRDP and the volume contentsof the lower and upper phases. Symbols ○ and ● denote the observation of the TRDP,
whereas symbol × implies that the TRDP was not observed. Plots containing symbols ○and ● show the reversible distribution patterns of the inner and outer phases. The
different systems studied were: a) water–acetonitrile–ethyl acetate (flow rate: 0.2 µL
min−1; temperature: 0 °C); b) water–Triton X-100–KCl (2.4 M) (flow rate: 5.0 µL
min−1; temperature: 34 °C); c) tetradecafluorohexane–hexane (flow rate: 5.0 µL min−1;
temperature: 10 °C); d) water–[C4mim]Cl–KOH (flow rate: 1.0 µL min−1; temperature:
15 °C); e) water–[C4mim]Cl–K2HPO4 (flow rate: 1.0 µL min−1; temperature: 15 °C);and f) water–[C2mim]MP–K2HPO4 (flow rate: 1.0 µL min−1; temperature: 15 °C).
Capillary tube inner diameter: a) 50 µm and b)–f) 75 µm.

85

Consideration of TRDP

85

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Bright-field and fluorescence photographs showing the development of the
TRDP in the different systems: a) water–acetonitrile–ethyl acetate (flow rate: 0.2 µL
min−1; temperature: 0 °C; and 1 mM Eosin Y and 0.1 mM perylene); b) water–Triton
X-100–KCl (2.4 M) (flow rate: 5.0 µL min−1; temperature: 34 °C; and 5 mM
Rhodamine B); c) tetradecafluorohexane–hexane (flow rate: 5.0 µL min−1; temperature:
10 °C; and 0.1 mM perylene); d) water–[C4mim]Cl–KOH (flow rate: 1.0 µL min−1;
temperature: 15 °C; and 5 mM Rhodamine B); e) water–[C4mim]Cl–K2HPO4 (flow rate:
1.0 µL min−1; temperature: 15 °C; and 2 mM Rhodamine B); and f)
water–[C2mim]MP–K2HPO4 (flow rate: 1.0 µL min−1; temperature: 15 °C; and 5 mM
Rhodamine B). Capillary tube inner diameter: a) 50 µm and b)–f) 75 µm.

86

Chapter 386

Figure 5. Chromatograms of the present capillary TRDC system: a) 2 wt.% Triton
X-100 and 2.4 M KCl at 34 °C and b) 2 wt.% Triton X-114 at 23 °C. Capillary tube:
inner diameter: 75 µm, total length: 150 cm, and effective length: 100 cm; flow rate: 1.0µL min−1; and analyte concentration: 1 mM.

References[1] S. Terabe, Anal. Chem., 2004, 76, 240.
[2] C. A. Lucy, A. M. MacDonald, and M. D. Gulcev, J. Chromatogr. A, 2008, 1184,
81.
[3] K. Otsuka, Chromatography, 2007, 28, 1.
[4] H. Small, F. L. Saunders, and J. Solc, Advances in Colloid and Interface Science,
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[5] R. Umehara, M. Harada, and T. Okada, J. Sep. Sci., 2009, 32, 472.
[6] K. Hata, T. Kaneta, and T. Imasaka, Anal. Chim. Acta., 2006, 556, 178.
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[8] K. Sueyoshi, F. Kitagawa, and K. Otsuka, Anal. Chem., 2008, 80, 1255.
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[10] J. L. Chen, Talanta, 2011, 85, 2330.
[11] C. H. Fischer and M. Giersig, J. Chromatogr. A, 1994, 688, 97.
[12] M. T. Blom, E. Chmela, R. E. Oosterbroek, R. Tijssen, and A. Berg, Anal. Chem.,
2003, 75, 6761.
[13] T. Ami, K. Awata, H. Umekawa, and M. Ozawa, Int. J. Multiphase Flow, 2012, 26,
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[14] H. Foroughi and M. Kawaji, Int. J. Multiphase Flow, 2011, 37, 1147.
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules[15] K. E. Gutowski, G. A. Broker, H. D. Willauer, J. G. Huddleston, R. P. Swatloski, J.
D. Holbrey, and R. D. Rogers, J. Am. Chem. Soc., 2003, 125, 6632.
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[19] C. Wu, J. Wang, H. Wang, Y. Pei, and Z. Li, J. Chromatogr. A, 2011, 1218, 8587.
[20] A. L. Dessimoz, L. Cavin, A. Renken, and L. K. Minker, Chem. Eng. Sci., 2008, 63,4035.
[21] Y. Lu, W. Lu, W. Wang, Q. Guo, and Y. Yang, Talanta, 2011, 85, 1621.
[22] C. Wua, J. Wanga, H. Wanga, Y. Pei, and Z. Li, J. Chromatogr. A, 2011, 1218,
8587.
[23] J. Han, Y. Wang, Y. Li, C. Yu, and Y. Yan, J. Chem. Eng. Data, 2011, 56, 3679.
[24] H. Watanabe and H. Tanaka, Talanta, 1978, 25, 585.
[25] K. Fujinaga, Anal. Sci., 1993, 9, 479.
[26] T. Saitoh, H. Tani, T. Kamidate, and H. Watanabe, Trends Anal. Chem., 1995, 14,213.
[27] T. M. Z. Moattar and R. Sadeghi, Fluid Phase Equilib., 2002, 203, 177.
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A, 2005, 1082, 176.
[29] T. I. Horváth and J. Rábai, J., Science, 1994, 266, 72.
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88

Chapter 3

88Chapter 4 Development
chromatography (TRDC)

of

tube

radialdistribution

Chapter 4

A TRDP-based capillary chromatography, referred to as tube radial distribution
chromatography (TRDC), where the outer phase serves as a pseudo-stationary phase
under laminar flow conditions, was developed as one of the applications of TRDP. The
TRDC was examined in detail from various viewpoints of analytical conditions. The
part of this chapter is reconstructrd and rewritten based on the related manuscripts thathave been published thus far.10,15,20,21,23,49)

4.1 Analytical conditions and separation performance in TRDC
We have developed a capillary chromatography system using an open capillary tube
made of fused-silica, polyethylene, or poly(tetrafluoroethylene), and awater-hydrophilic-hydrophobic organic mixture carrier solution. This tube radial
distribution chromatography (TRDC) system works under laminar flow conditions.
Here, the following analytical conditions in the TRDC system using a fused-silica
capillary tube and a water-acetonitrile-ethyl acetate mixture carrier solution were
examined: tube temperature, 5 – 25 °C; tube inner diameter, 50 - 250 µm; tube effective
length, 100 – 200 cm; and flow rate, 0.2 – 1.5 µL min-1. For example, the effects oftemperature on separation performance in the TRDC system were observed with theorganic solvent-rich carrier solution; 1-naphthol and 2,6-naphthalenedisulfonic acid in amodel mixture were eluted with baseline separation over the temperature range of 5 –23 °C. The resolution, theoretical plate number, and height equivalent to the theoreticalplate were calculated from the experimental data obtained by examining the effects oftube length. The mixture of 1-naphthol, Eosin Y, 1-naphthalenesulfonic acid,
2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid was subjected tothe present TRDC system and the analytes in the mixture solution were eluted in theabove order with the organic solvent-rich carrier solution, providing a good separation
performance on the chromatogram.
Introduction
Miniaturization is one of the most active research directions in analytical chemistry.
Valuable investigations with respect to miniaturization have also been done in the fieldof liquid chromatography: e.g., capillary liquid chromatography or capillarychromatography. Capillary chromatography is generally performed with fused-silica
capillary tubes whose inner diameter (i.d.) is ca. 50 – 100 µm, because of their superiormechanical and optical properties compared to other materials. Various capillarychromatography techniques, including capillary electrochromatography [1,2], micellar
electrokinetic capillary chromatography [3,4], and high-performance liquid capillarychromatography using packed and monolithic columns [5-7], have been investigated as
powerful separation tools. However, they require specific treatments or procedures,such as applying a high voltage, additives (gels, surfactants, etc.), and packing agents.

89

Development of TRDC

89

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Capillary chromatography using open fused-silica capillary tubes has also been
investigated, and the results indicated interesting and unique characteristics. Most ofthese studies, however, used coated or modified capillary tubes. For example, one study
coated the inner wall of the tube with silicone grease SE-30 [8] or modified it with amonolayer particle phase [9]. Porous silica structures were also introduced on the wallby dynamic or static coating techniques [10-12] and various polymeric siloxanes were
immobilized on the walls [13,14]. We modified the inner walls with specific functionalgroups or molecules, such as phenylboronic acid, iminodiacetic acid, and antibodies forcapillary chromatography to take advantage of specific interactions between the solutes
and the modified walls [15-18]. Methods for the preparation of open capillary tubes
coated with molecularly imprinted polymer [19] and cation exchange polymer [20] havealso been reported for use in chromatography. Furthermore, such coating techniques
have been applied to fused-silica capillary tubes less than 10 µm in i.d. to improveseparation performance [21-23].
However, coating the inner walls in the manner required for separation inchromatography is both a time-consuming and laborious procedure, and it is made moredifficult when trying to coat the inside of narrow-bored capillary tubes. Recently,
capillary chromatography using open fused-silica capillary tubes that were not speciallycoated or modified were reported [24-28]. In one example, Tabata et al. developed an
open tubular capillary chromatography method based on microphase separation of
mixed solvents, even though narrow-bored tubes of 10 µm i.d were used [27]. When an
acetonitrile-water mixture with suitable salt concentration was pumped into afused-silica capillary tube in which the inner wall was negatively charged due to thedissociation of its silanol groups, microphase separation occurred near the capillary wall,
resulting in a water-enriched aqueous phase attached to the capillary inner wall.
Furthermore, capillary chromatography using wide-bored tubes of 50 µm i.d. wascarried out together with an ionic liquid and a gel [28].
We developed a capillary chromatography method using an open capillary tube and awater-hydrophilic-hydrophobic organic solvent mixture (homogeneous solution) as acarrier solution; the system worked under laminar flow conditions. We called this thetube radial distribution chromatography (TRDC) system. We have demonstrated theseparation of a mixture of hydrophilic and hydrophobic molecules by capillarychromatography using open capillary tubes made of fused-silica, polyethylene, orpoly(tetrafluoroethylene) (PTFE) (50, 200, or 100 µm i.d., respectively), and awater-acetonitrile-ethyl acetate mixture carrier solution (homogeneous solution). The
elution times of the analytes in the system can be easily reversed by changing thecomponent ratio of the carrier solvents (or by using an organic solvent-rich and awater-rich carrier solution) in all types of the capillary tubes. We proposed that
separation in the capillary chromatography system was performed based on the tube
radial distribution of the carrier solvents under laminar flow conditions, known here as atube radial distribution chromatography (TRDC) system.
The tube radial distribution of the solvents in the TRDC system	
 is illustrated in Fig. 1
and phenomenologically described as follows. Under laminar flow conditions, aqueousand organic solvents in the carrier solution of a water-hydrophilic-hydrophobic organic
mixture are dispersed non-uniformly in a specific flow in the capillary tube generating

90

Chapter 490

an organic solvent-rich phase and a water-rich phase. A major inner phase is formed
around the center of the tube and away from the inner wall, while a minor outer orcapillary wall phase is generated near the inner wall. An organic solvent-rich carrier
solution generates an organic solvent-rich inner phase, while a water-rich carrier
solution results in a water-rich inner phase as shown in Fig. 1. The analytes that aredelivered through the capillary tube are distributed between inner and outer phases,
undergoing chromatographic separation under laminar flow conditions. So far, the tube
radial distribution in the TRDC system has been supported by experimental data using
polymer particles as analytes and phenylboronic acid or iminodiacetic acid-modified
fused-silica capillary tubes. The illustration of the tube radial distribution of the carrier
solvents (Fig. 1) is also drawn based on the data of the fluorescence photographs andprofiles obtained by the fluorescence microscope-CCD camera.
As investigation into the TRDC system has just begun, it is important to examine theelution behavior under various analytical conditions in the system to expand our	 knowledge regarding its separation performance. Here, we examined the analyticalconditions of the TRDC system in detail, including tube temperature, tube innerdiameter, tube length, and flow rate, using fused-silica capillary tubes and awater-acetonitrile-ethyl acetate mixture as a carrier solution. In contrast to polyethylene
and PTFE capillary tubes, fused-silica capillary tubes with various inner diameters arecommercially available for an economical rate. Using the present TRDC system wewere able to demonstrate the separation and detection of a mixture sample solution thatwas comprised of five analytes.
Experimental
The present capillary chromatography system comprised of an open fused-silica
capillary tube, microsyringe pump, and absorption detector. The tube temperature wascontrolled by dipping the capillary tube in water maintained at a fixed temperature in a
beaker by stirring. Water-acetonitrile-ethyl acetate mixtures with volume ratios of 3:8:4
and 15:3:2 were used as carrier solutions. Analyte solutions were prepared with thecarrier solutions. The analyte solution was introduced directly into the capillary inlet
side using the gravity method. After an analyte injection, the capillary inlet wasconnected through a joint to a microsyringe. The syringe was set on the microsyringe
pump. The carrier solution was fed into the capillary tube at a fixed flow rate underlaminar flow conditions. On-capillary absorption detection (254 nm) was performed
with the detector.
Results andand discussion
discussion
Results
Reversal of the elution times of the analytes in the TRDC system
Model mixture
solutions of 1-naphthol and 2,6-naphthalenedisulfonic acid as well as 1-naphthoic acidand 1,3,6-naphthalenetrisulfonic acid were analyzed using the present TRDC system.
The obtained chromatograms are shown in Fig. 2; the analytical conditions aredescribed in the figure caption. Using the organic solvent-rich carrier solution ofwater-acetonitrile-ethyl acetate with a volume ratio of 3:8:4, the mixtures of 1-naphtholand 2,6-naphthalenedisulfonic acid as well as 1-naphthoic acid and1,3,6-naphthalenetrisulfonic acid were separated through the open capillary tube and91

Development of TRDC

91

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

were detected in this order (Fig. 2 a)). On the other hand, using the water-rich carrier
solution of water-acetonitrile-ethyl acetate with a volume ratio of 15:3:2, the two
mixture solutions were separated and detected with the inverse elution times, i.e.,
2,6-naphthalenedisulonic and 1-naphthol as well as 1,3,6-naphthalenetrisulfonic acidand 1-naphthoic acid were detected in this order (Fig. 2 b)). The components of theanalytes on the chromatograms were confirmed by their individual absorption signals.
In both chromatograms, the first peaks were detected with elution times roughly
corresponding to the average linear velocities under laminar flow conditions, and thesecond peaks were eluted with a velocity below the average linear velocity. When theorganic solvent-rich carrier solutions were used, the comparatively hydrophobic
analytes were first eluted, while when the water-rich carrier solutions were used as
carrier solutions, the comparatively hydrophilic analytes were eluted first. That is, thechromatograms clearly indicated the reversibility of the elution times by changing thecomponent ratio of the solvents in the carrier solution. The results shown in Fig. 2 areconsistent with the tube radial distribution behavior of the carrier solvents in the TRDC
system that was proposed in our studies.
Effects of tube temperature on separation
We examined the effects of tube
temperature on separation in the TRDC system with a mixture analyte solution of
1-naphthol and 2,6-naphthalenedisulfuric acid as a model. The following experiments
were performed with the organic solvent-rich carrier solutions because the carrier
solutions provided better resolution on the chromatograms than the water-rich carrier
solutions as shown in Fig. 2. The obtained chromatograms are shown in Fig. 3 together
with analytical conditions.	
  As can be seen in Fig. 3, 1-naphthol and
2,6-naphthalenedisulfuric acid in the mixture solution were detected with baseline
separation in the temperature range of 5 –	
 23 °C, while they were not separated at all at
a temperature of 25 °C. In more detail, the resolutions were improved by increasing thetemperature between 5 °C and 20 °C, but the resolution decreased suddenly at 23 °C.
The data clearly indicated that the tube temperature had a significant and criticalinfluence on separation performance in the TRDC system. From this we can claim thatthe formation of the inner and outer phases of the carrier solution in the tube due to thetube radial distribution of the solvents must change with temperature.
Effects of tube inner diameter on separation We examined the effects of the tube
inner diameter on separation in the TRDC system with a mixture analyte solution of
1-naphthol and 2,6-naphthalenedisulfuric acid as a model. We attempted to use
commercially available fused-silica capillary tubes with various inner diameters.
However, as the tubes with an i.d. of more than 250 µm lacked flexibility, it wasdifficult to fix them in the present absorption detector. In addition, tubes of 25 µm i.d.
generated severe backpressure, making a continous delivery of the carrier solutiondifficult with the current microsyringe pump. Consequently, the capillary tubes with ani.d. of 50	
 –	
 250 µm were used here. The obtained chromatograms are shown in Fig. 4
together with the analytical conditions. The flow rates for all the capillary tubes were
adjusted to provide almost the same average linear velocity of ca. 22 cm min-1. We
observed well-separated peaks on the chromatograms; the first peaks were eluted with

92

Chapter 492

almost average linear velocity and the second peaks were eluted with a smaller than the
average linear velocity. Also, the elution times of the second peaks appeared earlier
with increasing inner diameter, although the elution times of the first peaks were almost
constant. The formation of the inner and outer phases of the carrier solution in the tube
due to the radial distribution of the solvents must change markedly in the wider tubes.
Next, we will examine the TRDC system using the capillary tubes with wider innerdiameters.
Effects of tube effective length on separation We also examined the effects of the
tube effective length on separation in the TRDC system using the model mixture analyte
solution. The obtained chromatograms are shown together with the analytical conditionsin Fig. 5. The first peaks were eluted with almost average linear velocity and the second
peaks were eluted with velocities smaller than the average linear velocity. The
resolution, theoretical plate number, and height equivalent to the theoretical plate were
calculated in the usual manner, and the obtained values are summarized in Table 1. As
shown in Table 1, longer tubes showed a better resolution and theoretical plate number.
On a different note, nearly the same height equivalent to a theoretical plate of 0.13	
 –	
 0.16 mm for 1-naphthol or 1.10	
 –	
 1.29 mm for 2,9-naphthalenedisulfonic acids wasobserved for all tube lengths. From the data, separation in the TRDC system seemed tobe performed based on the usual chromatographic separation procedure, at least under
the present analytical conditions.
Effects of flow rate on separation
The effects of flow rate (0.2	 –	
 1.5 µL min-1) on
separation were examined in the TRDC system. The obtained chromatograms are shownin Fig. 6 together with the analytical conditions. The first peaks were eluted with almost
the average linear velocity and the second peaks were eluted with velocities smaller
than the average linear velocity. Although the second peak showed broadening at lower
flow rates, they showed almost Gaussian peaks with good separation. That is, Fig. 6
meant that the tube radial distribution of the solvents in the capillary tube was
performed even at the minimum flow rate of 0.2 µL min-1 in the present system.
Separation of mixture solution including five analytes
We examined a mixture
analyte solution of 1-naphthol, Eosin Y, 1-naphthalenesulfonic acid,
2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid using the present
TRDC system with organic solvent-rich carrier solution and water-rich carrier solution.
The obtained chromatograms are shown together with the analytical conditions in Fig. 7.
The elution times of the analytes were reversed when using the organic solvent-rich andthe water-rich carrier solutions in a similar way to other reported chromatograms in the
TRDC
system.
1-Naphthol,
Eosin
Y,
1-naphthalenesulfonicacid,
2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid were eluted in thisorder, leading to a good separation from the organic solvent-rich carrier solution (Fig. 7a)). The elution order seemed to be consistent with the hydrophilic character. With thewater-richcarriersolution,thecomparativelyhydrophiliccompounds
1-naphthalenesulfonicacid,
2,6-naphthalenedisulfonic
acid,
and
1,3,6-naphthalenetrisulfonic acid were not separated, but were eluted together with
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Development of TRDC

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

almost average linear velocity, while the hydrophobic compounds Eosin Y and1-naphthol were eluted in this order with velocities smaller than the average linear
velocity (Fig. 7 b)), indicating the reverse elution order compared to that of the organic
solvent-rich carrier solution.
In conclusion, the analytical conditions, such as temperature, tube inner diameter,
tube length, and flow rate, were examined for the first time in the TRDC system. The
resolution, theoretical plate number, and height equivalent to the theoretical plate were
calculated based on the experimental data. The analytes that were delivered through thecapillary tube were distributed between inner and outer phases, undergoing
chromatographic separation under laminar flow conditions. Successful separation of a
mixture solution including five analytes indicates that the TRDC system has potentialthat should be examined further in future studies.

Figure 1. Illustration of a tube radial distribution of the carrier solvents in the TRDC
system that is drawn based on the fluorescence photographs obtained by thefluorescence microscope-CCD camera. 1) Fused-silica (50 µm i.d.), polyethylene (200µm i.d.), and PTFE (100 µm i.d.) capillary tubes were available. 2) For example, awater-acetonitrile-ethyl acetate (2:9:4 v/v/v) mixture solution was used as an organic
solvent-rich carrier solution.	
 3) For example, a water-acetonitrile-ethyl acetate (15:3:2
v/v/v) mixture solution was used as a water-rich carrier solution. 4) The analytes thatare delivered through the capillary tube are distributed between inner and outer phases,
undergoing chromatographic separation under laminar flow conditions.

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Chapter 4

94

a)

b)

Figure 2. Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidas well as 1-naphthoic acid and 1,3,6-naphthalenetrisulfonic acid obtained by thepresent system. Conditions: Capillary tube, 120 cm (effective length: 100 cm) of 75 µm
i.d. fused-silica; carrier, a) water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution
and b) water-acetonitrile-ethyl acetate (15:3:2 v/v/v) mixture solution; sample injection,
20 cm height (gravity)	
 ×	
 30 s; flow rate, 0.8 µL min-1; tube temperature, 20 °C; and1-naphthoic acid, 2,6-naphthalenedisulfonic acid, and 1-naphthol, 1 mM each and1,3,6-naphthalenetrisulfonic acid, 2 mM.
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Development of TRDC

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3. Effects of temperature on separation in the present system. Conditions:
Capillary tube, 120 cm (effective length of 100 cm, where part of it (ca. 80 cm) was
dipped in the temperature-controlled water) of 75 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection, 20 cm
height (gravity)	
 ×	
 30 s; flow rate, 0.8 µL min-1; tube temperature, 5 – 25 °C; and2,6-naphthalenedisulfonic acid and 1-naphthol, 1 mM each.

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Chapter 496

Figure 4. Effects of the inner diameter of the tube on separation in the present system.
Conditions: Capillary tube, 120 cm (effective length: 100 cm) fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection, 20 cm
height (gravity) × 2 – 45 s; flow rate, 0.5 – 8.0 µL min-1; tube temperature, 20 °C; and2,6-naphthalenedisulfonic acid and 1-naphthol, 1 mM.

Figure 5. Effects of the effective length of the tube on separation in the present system.
Conditions: Capillary tube, effective length (100 – 200 cm) of 75 µm i.d. fused-silica;
carrier, water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection,
20 cm height (gravity)×30 s; flow rate, 0.8 µL min-1; tube temperature, 20 °C; and2,6-naphthalenedisulfonic acid and 1-naphthol, 1 mM.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 6. Effects of the flow rate of carrier solution on separation in the present system.
Conditions: Capillary tube, 120 cm (effective length: 100 cm) of 75 µm i.d. fused-silica;
carrier, water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection,
20 cm height (gravity)×30 s; flow rate, 0.2 – 1.5 µL min-1; tube temperature, 20 °C; and2,6-naphthalenedisulfonic acid and 1-naphthol, 1 mM each.

a)

98
98

Chapter 4b)Figure 7. Chromatograms of the mixture analyte solution of 1-naphthol, Eosin Y,
1-naphthalenesulfonicacid,
2,6-naphthalenedisulfonic
acid,
and
1,3,6-naphthalenetrisulfonic acid obtained by the present method. Conditions: Capillarytube, 120 cm (effective length: 100 cm) of 75 µm i.d. fused-silica; carrier, a)water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution and b)
water-acetonitrile-ethyl acetate (15:3:2 v/v/v) mixture solution; sample injection, 20 cm
height (gravity) × 30 s; flow rate, 0.8 µL min-1; temperature, 20 °C; and 1-naphthol,
1-naphthalenesulfonic acid, and 2,6-naphthalenedisulfonic acid, 1 mM,
1,3,6-naphthalenetrisulfonic acid, 2.0 mM, and Eosin Y, 0.1 mM.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

100

Chapter 4100

4.2 Influences of analyte injection volumes and concentrations on thechromatograms in TRDC
We examined the effects of the analyte injection volume (injection time) andconcentration on the chromatograms in the tube radial distribution chromatography(TRDC) system using a fused-silica capillary tube (50	
 µm	 i.d. and 100 cm effective
length) and a water-acetonitrile-ethyl acetate mixture carrier solution (volume ratio,
3:8:4). Analyte solutions of 1-naphthol and 2,6-naphthalenedisulfonic acid (1 mM each)were injected into the system with various injection times of 10 – 1500 s from a heightof 20 cm using gravity. They were separated and detected in this order with good
reproducibility up to an injection time of 150 s. The analyte solutions (0.075 – 3.0 mM)
definite injection
injection time
time ofof 30
30 ss fromfrom aa heightheight of
of 20
20 cm
cm using	 using
were analyzed with the definite
gravity. They were separated and detected with a baseline separation and theircalibration curves showed linearity up to 1.5 mM. It was confirmed that the TRDC
system was effective as a quantitative analysis.
Introduction
Analytical methods using capillary tubes have attracted a great deal of interest since thelast century [29-31]. Well-known methods include capillary electrophoresis, capillaryelectrochromatography, micellar electrokinetic capillary chromatography, andhigh-performance liquid capillary chromatography using packed and monolithic
columns. However, only a few new concepts concerning capillary chromatography havebeen reported in the last decade [25,28]. Related research has been discussed in detail in
our papers.2,10) We have developed a capillary chromatography system using an open
capillary tube and a water-hydrophilic/hydrophobic organic mixture carrier solution.
This system works under laminar flow conditions and does not require any packed
reagents in the capillary tube or application of high voltage to the ends of the tube. It iscalled a tube radial distribution chromatography (TRDC) system.
To date, various mixtures of hydrophilic and hydrophobic analytes have been
separated using the TRDC system. However, we have yet to examine the effects of theanalyte injection volume or injection time and concentration on the chromatograms in
the TRDC system in detail. The analyte injection volume and concentration are
fundamental analytical factors in general chromatography separation and flow-injection
analysis. Here, we examined separation performance with various analyte injection
volumes and concentrations in the TRDC system to expand our knowledge regarding
this new separation system.
Experimental
The TRDC system equipped with absorption on-line detection was comprised of anopen fused-silica capillary tube (50 µm i.d., 120 cm length; 100 cm effective length),
microsyringe pump, and absorption detector.	
 The tube temperature was controlled byimmersing the capillary tube in water maintained at a fixed temperature (0 °C) in a
beaker maintained by stirring. A water-acetonitrile-ethyl acetate mixture with a volume
ratio of 3:8:4 was used as an organic solvent-rich carrier solution. An analyte solution
including 1-naphthol and 2,6-naphthalenedisulfonic acid as a model was prepared with
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Development of TRDC

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

the carrier solution. The analyte solution was introduced directly into the capillary inlet
side by the gravity method (from a height of 20 cm). After analyte injection, thecapillary inlet was connected through a joint to a microsyringe. The syringe was set on
the microsyringe pump and the carrier solution was fed into the capillary tube at a fixed
flow rate (0.2 µL min-1) under laminar flow conditions (Reynolds number was
calculated roughly to be 0.14 in the present system). On-capillary absorption detection(254 nm) was performed with the detector.
Results and discussion
Analyte injection times, volumes, and zone lengths An analyte solution consisting of
1-naphthol and 2,6-naphthalenedisulfonic acid was injected into the capillary tube using
gravity from a height of 20 cm at various injection times of 10 – 1500 s (25 min) in thecurrent TRDC system. In order to examine the analyte injection volumes and zone
lengths in the capillary tube corresponding to the analyte injection times of 10 – 1500 s
effectively, the rate of flow using gravity was estimated	 with the Hagen-Poiseuilleequation. The flow rate in the capillary tube with gravity from a height of 20 cm wascalculated using the Hagen-Poiseuille equation (Eq. 1), which is shown below: Q is theflow rate, a is the radius, Δp is loss of pressure, µ is viscosity, and L is total length.
πa 4 P
Q
8 L 	
 	
 	
 	
 	
 	
 	
 	
 	
 	
 	
 	
 Eq.
	
 	
 	
 	
 	
 	
 	
 	
 
1)

The flow rate for the capillary 50 µm i.d. (a, 25×10-6 m) was calculated to beapproximately 2.67×10-13 m3s-1, where Δp, µ, and L were estimated to be 1.3×103
kgm-1s-2 (calculated from the difference in height or position between the capillary inlet
and outlet, 20 cm), 6.2×10-4 kgm-1s-1 (measured by a viscosity measurement device forthe carrier solution), and 1.2 m, respectively. In addition, the value of the flow rate, Q,
was estimated experimentally by the experimental data from the gravity method.
1-Naphthol analyte (1 mM) was injected into the capillary tube in the usual way (from aheight of 20 cm for 10 s) and then the analyte was delivered with the carrier solution
into the tube by gravity (from a height of 20 cm) instead of the microsyringe pump. The
flow rates for the capillary 50 µm i.d. were calculated with Eq. 2, where t is the elution
time and L’ is the effective length. The value of Q was calculated to be approximately
2.72×10-13 m3s-1 (using t, L’, and a of 120 min, 1.0 m, and 25×10-6 m, respectively).
a 2π L’
Q
t
	
 	
 	
 	
 	
 	
 	
 	
 	
 	
 	
 
	
 
Eq. 2)
There was good correspondence in the flow rate between the value calculated with theHagen-Poiseuille equation and with that of the experimental data using gravity. The
value of the flow rate, 2.7×10-13 m3s-1, was used in the following estimations. The
analyte injection volumes were calculated by multiplying the flow rates and theinjection times, as well as the analyte zone lengths in the tube, were calculated by
dividing the analyte injection volumes by the tube cross-section. That is, the injection
times of 10 – 1500 s (25 min) corresponded to the analyte injection volumes of 2.7 –400 nL and the zone lengths of 1.5 – 200 mm.

102

Chapter 4102

Effects of injection time on the chromatograms
We examined the effects of analyte
injection time on separation in the TRDC system with an analyte solution of 1-naphtholand 2,6-naphthalenedisulfuric acid as a model. The experiments were performed withthe organic solvent-rich carrier solution because it provided better resolution than thewater-rich carrier solution on the chromatograms when the fused-silica capillary tube
was used in our studies [7,10].The analyte solutions of 1-naphthol and
2,6-naphthalenedisufonic acid (1 mM each) were analyzed with various injection timesof 10 – 1500 s (25 min) by the TRDC system. The typical chromatograms obtained at
injection times of 10, 40, 150, 600, 1320, and 1500 s are shown in Fig. 1. As shown in
Fig. 1, 1-naphthol and 2,6-naphthalenedisufonic acid were separated and detected in thisorder with the TRDC system using the organic solvent-rich carrier solution. These
results offered good reproducibility for their peak shapes and elution times with theinjection times of 10 – 150 s (2.7 – 40 nL injection volume or 1.5 – 20 mm zone length).
The first peaks of 1-naphthol were eluted with nearly the average linear velocity in thepresent system, and the second peaks of 2,6-naphthalenedisufonic acid were eluted
lower at a lower than average linear velocity. The capacity factors of the second peaks,
which were calculated using the first peaks as the dead (hold-up) times, at injection
times of 10, 40, and 150 s were 0.57, 0.61, and 0.58, respectively. With injection timesof 150 – 1350 s (22 min) (40 – 360 nL injection volume or 20 – 180 mm zone length),
although they were separated on the chromatograms, the elution times of the second
peaks started to come earlier. With injection times of more than 1400 s, even separation
was not performed as shown in the typical chromatogram (injection time 1500 s (25min) in Fig. 1. It is noted here that such experiments treated with such a wide range of
injection volumes (2.7 – 400 nL) and zone lengths (1.5 – 200 mm) would not be carriedout in the usual capillary chromatography using monolithic or packed columns
combined with a sample injector. Large injection times or analyte injection volumes led
to either non-Gaussian or trapezoidal peaks, as shown in Fig. 1. The relationships
between the injection times and peak heights or peak areas for both analytes were
examined for injection times of 10 – 150 s. The obtained relationships are shown in Fig.
2. The relationships between the injection times and peak areas for both analytes
indicated good linearity, while those between injection times and peak heights indicatedlinearity up to ca. 40 s for 1-naphthol and up to ca. 60 s for 2,6-naphthalendisulfonicacid. The inflection point (ca. 40 s) of 1-naphthol appeared earlier than that of
2,6-naphthalendisulfonic acid (ca. 60 s). The difference in inflection points may havebeen caused by the differences in elution time or diffusion time between 1-naphthol and
2,6-naphthalenedisulfonic acid.
Effects of analyte concentrations on the chromatogram
We examined the effects of
the analyte concentrations (0.075 – 3.0 mM) on separation in the TRDC system with ananalyte solution of 1-naphthol and 2,6-naphthalenedisulfuric acid as a model. The
analyte solution was injected into the capillary tube using gravity from a height of 20
cm for 30 s. The typical chromatograms obtained with concentrations of 0.1, 0.5, 1.5,
2.0, and 3.0 mM are shown in Fig. 3. 1-naphthol and 2,6-naphthalenedisulfuric acid
were separated with a base-line separation and detected up to 1.5 mM. The capacity
factors at concentrations of 0.1, 0.5, and 1.5 mM were 0.54, 0.59, and 0.57, respectively.
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Development of TRDC

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

At concentrations	
 higher than 1.5 mM, the resolutions gradually decreased until they
could no longer be separated on the chromatogram at all at about 3.0 mM, as shown in
Fig. 3. The calibration curves were examined up to 1.5 mM for both analytes with peakheights and peak areas. The obtained calibration curves are shown in Fig. 4. Goodlinearity was observed in the curves shown in Fig. 4. Correlation coefficients were0.996 – 0.998 for all calibration curves. Relative standard deviations of 1-naphthol were
2.5 % (n=8) for both peak heights and areas, while those of 2,6-naphthalenedisulfuric
acid were 3.2 % (n=8) for peak heights and 5.4 % (n=8) for peak areas. It was
confirmed from the obtained data in Fig. 4 that the TRDC system was effective as aquantitative analysis.
In conclusion, we examined the effects of analyte injection volume andconcentration on the chromatograms in the TRDC system. The injection volumes andconcentrations are fundamental analytical factors in chromatographic research. The
analyte solution of 1-naphthol and 2,6-naphthalenedisulfuric acid was used as a model
to first be separated and then detected to varying injection times up to 150 s (injection
volume 40 nL or zone length 20 mm), they were also analyzed up to 1.5 mM with good
reproducibility. Separation in the present TRDC system became difficult at analyte
injection times of more than 150 s or analyte concentrations of more than 1.5 mM. The
difficulty may be due to instability or the collapse of the inner and outer phaseformations based on the tube radial distribution of the carrier solvents in the capillary
tube with extremely large analyte injection volumes or concentrations. Additionally, theranges of analyte injection volume and concentration that provide quantitative analysis
might be influenced by analytical conditions, such as capillary inner diameter, flow
rates, and component ratios of the carrier solvents.

104

Chapter 4104

Figure 1.	
 Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidobtained by the TRDC system with various injection times. Conditions: Capillary tube,
120 cm (effective length: 100 cm) of 50 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection, 20 cm
height (gravity) × 10, 40, 150, 600, 1320, and 1500 s; flow rate, 0.2 µL min-1; tube
temperature, 0 °C; and analyte concentration, 1 mM.

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Development of TRDC

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2. Relationships between injection times and peak heights or peak areas for
1-naphthol and 2,6-naphthalenedisulfonic acid in the TRDC system. Conditions:
Capillary tube, 120 cm (effective length: 100 cm) of 50 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection, 20 cm
height (gravity) × 10 – 150 s; flow rate, 0.2 µL min-1; tube temperature, 0 °C; andanalyte concentration, 1 mM.

106

Chapter 4106Figure 3.	
 Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidobtained by the TRDC system with various analyte concentrations. Conditions:
Capillary tube, 120 cm (effective length: 100 cm) of 50 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection, 20 cm
height (gravity) × 30 s; flow rate, 0.2 µL min-1; tube temperature, 0 °C; and analyte
concentration, 0.1, 0.5, 1.5, 2.0, and 3.0 mM.

Figure 4.	
 Calibration curves of 1-naphthol and 2,6-naphthalenedisulfonic acid obtainedby the TRDC system. Conditions: Capillary tube, 120 cm (effective length: 100 cm) of
50 µm i.d. fused-silica; carrier, water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture
solution; sample injection, 20 cm height (gravity) × 30 s; flow rate, 0.2 µL min-1; tube
temperature, 0 °C; and analyte concentration, 0.1 – 1.5 mM.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

4.3 Investigation of the composition for a ternary mixed solvent system
in TRDC using PTFE capillary tube
The tube radial distribution chromatography (TRDC) system works under laminar flow
conditions. Here, a phase diagram for the ternary mixture carrier solvents of water,acetonitrile, and ethyl acetate was constructed. The phase diagram that included aboundary curve between homogeneous and heterogeneous solutions was considered to
be together with the component ratios of the solvents in the homogeneous carrier
solutions required for the TRDC system where a poly(tetrafluoroethylene) (PTFE)
capillary tube was used. It was found that the TRDC system performed well withhomogeneous solutions having component ratios of the solvents that were positioned
near the homogeneous-heterogeneous solution boundary of the phase diagram. For
preparing the carrier solutions of water-hydrophilic/hydrophobic organic solvents for
the TRDC system, we used methanol, ethanol, 1,4-dioxane, and 1-propanol instead ofacetonitrile (hydrophilic organic solvent), as well as chloroform and 1-butanol, insteadof ethyl acetate (hydrophobic organic solvent). The homogeneous ternary mixture
carrier solutions were prepared near the homogeneous-heterogeneous solution boundary.
Analyte mixtures of 2,6-naphthalenedisulfonic acid and 1-naphthol were separated with
the TRDC system using these homogeneous ternary mixture carrier solutions. The
pressure change in the capillary tube under laminar flow conditions might alter the
carrier solution from homogeneous to heterogeneous in the batch vessel, thus affecting
the tube radial distribution of the solvents in the capillary tube.
Introduction
Capillary tubes, e.g., fused-silica and PTFE, that feature inner diameters less thanseveral hundred micrometers are found to have uniform bores, inert inner surfaces, andgood flexibility. Capillary tubes with these characteristics play an important role inseveral unique micro-flow analyses [27, 32-36]. The capillary tubes are also known to
exhibit interesting and useful physical or hydrodynamic phenomena, such as
electro-osmotic flow and laminar flow. The electro-osmotic flow in a capillary tube has
capillary electrophoresis [37-39] and capillary electrochromatography [40-42] properties
while laminar flow conditions enable hydrodynamic chromatography [43-45].
The tube radial distribution of the solvents, or the TRDP, was also observed in a
microchannel in a microchip under laminar flow conditions.6) A carrier solution
containing fluorescent dyes was introduced into a microchannel (100 µm width by 40µm depth) and the tube radial distribution of the dyes was monitored using afluorescence microscope. For example, when an organic solvent-rich carrier solution(water-acetonitrile-ethyl acetate; 3:8:4 volume ratio) containing perylene and Eosin Y
was fed into the channel, perylene (hydrophobic) and Eosin Y (comparativelyhydrophilic) were distributed around the center and near the inner wall of the
microchannel, respectively. A capillary chromatography system has been developed
based on the tube radial distribution of the carrier solution in the capillary tube underlaminar flow conditions. The chromatography system is called a tube radial distribution
chromatography (TRDC) system.

108

Chapter 4108However, further information on the TRDC system or the TRDP is required forvarious applications, such as in analytical chemistry, physical chemistry, and chemicalengineering laboratories. Here, a phase diagram for the ternary mixture carrier solventsof water, acetonitrile, and ethyl acetate was constructed and is considered to becongruent with the component ratios of the solvents required for the TRDC system
where the PTFE capillary tube was used. Subsequently, various kinds ofwater-hydrophilic/hydrophobic organic solvent mixture solutions, other than the usualwater-acetonitrile-ethyl acetate mixture solution, were prepared and examined as carrier
solutions in the TRDC system.
Experimental
Figure 1 shows a schematic diagram of the present capillary chromatography system,comprised of an open PTFE capillary tube [100 ± 25 µm i.d., 120 cm length (effective
length, 100 cm)], a micro-syringe pump, and an absorption detector. The tube
temperature was controlled by dipping the capillary tube (ca. 80 cm) in water
maintained at a fixed temperature (0 or 15 °C) in a beaker by stirring.
Water-hydrophilic/hydrophobic organic solvent mixture solutions with the various
volume ratios were used as carrier solutions. Analyte solutions were prepared with thecarrier solutions. The analyte solution was introduced directly into the capillary inlet
side by the gravity method (20 cm height for 30 s). After analyte injection, the capillary
inlet was connected through a joint to a micro-syringe. The syringe was placed on the
microsyringe pump. The carrier solution was fed into the capillary tube at 2.0 µL min-1
flow rate under laminar flow conditions. On-capillary absorption detection (320 nm)
was performed using the detector.
Results and Discussion
Preliminary experiments at various component ratios of water-acetonitrile-ethyl acetate
mixture carrier solution
The solution having a constant component ratio ofwater-acetonitrile (hydrophilic) of 100:20 volume ratio was added ethyl acetate(hydrophobic) in a vessel at a temperature of 22 °C to prepare carrier solutions with thefollowing compositions: a) water-acetonitrile-ethyl acetate of 100:20:0 volume ratio; b)
100:20:4 volume ratio; c) 100:20:9 volume ratio; d) 100:20:10 volume ratio; and e)
100:20:11 volume ratio. The first four carrier solutions, a) – d), were homogeneous, but
the last carrier solution, e), was heterogeneous (including two homogeneous phases).
The carrier solution d), the water-acetonitrile-ethyl acetate of the 100:20:10 volume
ratio, was the specific homogeneous solution that possessed the component ratio of thesolvents near the homogeneous-heterogeneous solution boundary region of the phase
diagram. The analyte mixtures of 2,6-naphthalenedisulfonic acid and 1-naphthol were
examined with the capillary chromatography system with the first four homogeneous
carrier solutions a) – d). The obtained chromatograms are shown in Fig. 2. Only the
specific homogeneous carrier solution d), the water-acetonitrile-ethyl acetate of
100:20:10 volume ratio, could separate the analytes in the mixture.
2,6-Naphthalenedisulfonic acid (comparatively hydrophilic) was eluted first with near
the average linear velocity in the capillary tube under laminar flow conditions and1-naphthol (comparatively hydrophilic) was eluted second with lower at a lower than
average linear velocity. The elution order was reasonable in the TRDC system. As
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Development of TRDC

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

shown in Fig. 1, a major inner phase was formed with the water-rich solvent and aminor outer phase, or capillary wall phase, was formed with the organic-rich solvent ina capillary with a water-rich carrier solution. The analytes that were delivered throughthe capillary tube were distributed between the inner and outer phases, undergoing
chromatographic separation under laminar flow conditions. The other homogeneous
carrier solutions a) – c) of water-acetonitrile-ethyl acetate did not show any separation
behavior for the analytes under the current analytical conditions (Fig. 2).
Phase diagram for water-acetonitrile-ethyl acetate mixture and the component ratios
required for TRDC
A phase diagram for the ternary mixture solvents ofwater-acetonitrile (hydrophilic organic solvent)-ethyl acetate (hydrophobic organic
solvent) was examined in a vessel at a temperature of 22 °C. The obtained phase
diagram is shown in Fig. 3. The dotted curve in the diagram indicates the boundary
between homogeneous and heterogeneous phases. The phase diagram showed that eachcomponent ratio of the solvents made a homogeneous (one homogeneous phase) or aheterogeneous (two homogeneous phases) solution. The component ratios of thesolvents in the carrier solutions a) – d) that gave the chromatograms shown in Fig. 2
were plotted in the diagram. Circles (○) and crosses (×)	 indicate baseline-separation andnon-separation for the analytes, respectively. The chromatograms obtained with thehomogeneous carrier solutions that had solvent component ratios positioned near the
homogeneous-heterogeneous solution boundary curve in the phase diagram are shownin Fig. 4. The carrier solution component ratios were as follows: f)water-acetonitrile-ethyl acetate of 100:0:9 volume ratio; g) 80:20:9 volume ratio; h)60:40:12.5 volume ratio; i) 50:50:17 volume ratio; j) 30:70:33 volume ratio; and k)
14.5:50:50 volume ratio. The component ratios of the solvents for the chromatograms in
Fig. 4 were also plotted in the phase diagram with circles and crosses together withtriangles (Δ) which indicate split-separation (not baseline-separation). As is clearlyshown in Figs. 2–4, the TRDC system performed with the specific homogeneous carrier
solutions having the component ratios of the solvents that were positioned almost
directly on the homogeneous-heterogeneous solution boundary curve in the phase
diagram. Specific carrier solutions such as these caused tube radial distribution of thecarrier solvents in the capillary tube under laminar flow conditions, i.e., the TRDP. The
pressure imposed on the carrier solution in the capillary tube under laminar flow
conditions might alter the carrier solution from homogeneous in the batch vessel toheterogeneous (although not necessarily with two clear homogeneous phases), affecting
the tube radial distribution of the solvents in the capillary tube. We reported that,characteristicly, a water-rich carrier solution elutes the more hydrophilic analyte firstand an organic solvent-rich carrier solution elutes the more hydrophobic analyte first ina TRDP system [16-18]. That is, in the case of a water-rich carrier solution, thehydrophilic analyte, which is dispersed in the water-rich major phase (around the
middle of the capillary tube), is eluted with nearly average linear velocity. The
hydrophobic analyte, which is dispersed in the organic solvent-rich minor phase near
the inner wall of the tube (pseudo-stationary phase), is eluted at a lower than average
linear velocity. In the case of an organic solvent-rich carrier solution, the hydrophobic
analyte dispersed in the organic solvent-rich major phase is eluted with nearly average
linear velocity. The hydrophilic analyte dispersed in the water-rich minor phase near the

110

Chapter 4110inner wall of the tube (pseudo-stationary phase) is eluted at a lower than average linear
velocity. Therefore, the elution times of the analytes can be easily reversed by altering
the component ratio of the solvents in the carrier solution. In fact, in Fig. 4, thewater-rich carrier solutions, such as f) water-acetonitrile-ethyl acetate of 100:0:9
volume ratio, g) 80:20:9 volume ratio, h) 60:40:12.5 volume ratio, and i) 50:50:17
volume ratio, separated 2,6-naphthalenedisulfonic acid and 1-naphthol in this order,
while the organic solvent-rich carrier solution, j) 30:70:33 volume ratio, separated themin the reverse order, i.e., 1-naphthol was first eluted and secondly
2,6-naphthalenedisulfonic acid was detected, although baseline separation could not beprovided under the present analytical conditions.
Chromatograms obtained with the carrier solutions using various hydrophilic organic
solvents instead of acetonitrile The ternary mixture carrier solutions consisting ofwater-hydrophilic/hydrophobic organic solvent were prepared by using thewater-acetonitrile-ethyl acetate mixture, as indicated in our reports. Here, instead ofacetonitrile (hydrophilic organic solvent), methanol, ethanol, 1,4-dioxane, and1-propanol were used to prepare the homogeneous ternary mixed carrier solutions that
had the specific component ratios of the solvents near the homogeneous-heterogeneous
solution boundary region. The component ratios of the solvents in the prepared carrier
solutions were as follows: water-methanol-ethyl acetate (100:20:15 volume ratio),
water-ethanol-ethyl acetate (100:20:15 volume ratio), water-1,4-dioxane-ethyl acetate(100:5:10 volume ratio), and water-1-propanol-ethyl acetate (100:10:11 volume ratio).
The chromatograms obtained with the homogeneous carrier solutions are shown in Fig.
5, together with that obtained with the water-acetonitrile-ethyl acetate mixture(100:20:10 volume ratio). It was confirmed from the chromatograms that
2,6-naphthalenedisulfonic acid and 1-naphthol were separated and detected in this orderwith the TRDC system using the water-rich carrier solutions prepared with varioushydrophilic organic solvents.
Chromatograms obtained with the carrier solutions using various hydrophobic organic
solvents instead of ethyl acetate
Here, instead of including ethyl acetate(hydrophobic organic solvent) in the ternary mixture carrier solution ofwater-acetonitrile-ethyl acetate, chloroform, hexane, and 1-butanol were used to prepare
the homogeneous ternary mixed carrier solutions that had the specific component ratiosof the solvents near the homogeneous-heterogeneous solution boundary region. The
component ratios of the carrier solutions were as follows: water-acetonitrile-hexane(100:30:1 volume ratio), water-acetonitrile-chloroform (100:30:1 volume ratio), andwater-acetonitrile-1-butanol (100:15:20 volume ratio). The chromatograms obtainedwith the carrier solutions are shown in Fig. 6, along with those obtained with thewater-acetonitrile-ethyl acetate mixture as well. It was also confirmed that under thepresent analytical conditions, 2,6-naphthalenedisulfonic acid and 1-naphthol wereseparated in this order with the TRDC system using the water-rich carrier solutions thatwere prepared with chloroform, 1-butanol, and ethyl acetate as hydrophobic organic
solvents, but not hexane.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Separation of mixture solution including three analytes We next examined a mixture
analyte solution of 1-naphthalenesulfonic acid, 1-naphthoic acid, and 1-naphthol using
the present TRDC system with PTFE capillary tube and several types of homogeneous
water-rich carrier solutions. The carrier solutions were water-acetonitrile-ethyl acetate(100:20:10 volume ratio), water-ethanol-ethyl acetate (100:20:15 volume ratio), andwater-acetonitrile-chloroform (100:30:1 volume ratio). These carrier solutions wereprepared based on the concept that the homogeneous carrier solutions were nearly on
the homogeneous-heterogeneous solution boundary region. The obtained
chromatograms are shown in Fig. 7, together with the analytical conditions.
1-Naphthalenesulfonic acid (pKa, 0.57), 1-naphthoic acid (pKa, 3.70), and 1-naphthol(pKa, 9.34) were eluted in this order, leading to separation with the water-rich carrier
solution. 1-Naphthalenesulfonic acid was eluted at nearly the average linear velocity
under laminar flow conditions, while the other compounds, 1-naphthoic acid and1-naphthol, were eluted in this order at lower than average linear velocities. The elution
order of 1-naphthalenesulfonic acid, 1-naphthoic acid, and 1-naphthol was reasonable if
one considers the hydrophilicity, or hydrophobicity, based on their molecular structures
or pKa values.
In conclusion, the phase diagram for the water, acetonitrile, and ethyl acetate
mixture was examined and considered to be congruent with the component ratios of thesolvents required for the TRDC system. The TRDC system was performed withhomogeneous solutions having the component ratios of the solvents that werepositioned almost on the homogeneous-heterogeneous solution boundary curve of thephase diagram. Various types of water-hydrophilic/hydrophobic organic solvents, otherthan the usual water-acetonitrile-ethyl acetate mixture, were prepared for the TRDC
system. Thus-prepared carrier solutions were found to be usable for chromatographic
separation in the TRDC system.

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Chapter 4112

Figure 1. Schematic diagram of the present capillary chromatography system. With awater-rich carrier solution, 1) a major inner phase is formed with a water-rich solvent,and 2) a minor outer phase is formed with an organic-rich solvent.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2. Chromatograms of a mixture of 2,6-naphthalenedisulfonic acid and 1-naphtholwith the capillary chromatography system using various component ratios ofwater-acetonitrile-ethyl acetate mixture as carrier solutions. Conditions: Capillary tube,
120 cm (effective length: 100 cm) of 100 µm i.d. PTFE; carrier, a)water-acetonitrile-ethyl acetate of 100:20:0 volume ratio, b) 100:20:4 volume ratio, c)
100:20:9 volume ratio, and d) 100:20:10 volume ratio; sample injection, 20 cm height(gravity)×30 s; flow rate, 2.0 µL min-1; tube temperature, 15 °C; and2,6-naphthalenedisulfonic acid and 1-naphthol, 2 mM each. The component ratios of thecarrier solutions a) - d) used for the chromatograms are plotted in the phase diagram ofFig. 3.

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Figure 3. Phase diagram for water-acetonitrile-ethyl acetate mixture and the component
ratios of the solvents for the chromatograms obtained with the TRDC system. The
dotted curve in the diagram indicates the homogeneous-heterogeneous solution
boundary curve. The component ratios of the carrier solvents, a) – d) and f) – k), for thechromatograms in Figs. 2 and 4 are plotted in the diagram with the symbols ○,	
 Δ,	
 and
×, to express baseline-separation, split-separation, and non-separation, respectively, forthe analytes. The chromatograms obtained with a) water-acetonitrile-ethyl acetate of
100:20:0 (or 83:17:0 as a percentage) volume ratio, b) 100:20:4 (or 81:16:3) volume
ratio, c) 100:20:9 (or 78:16:7) volume ratio, and d) 100:20:10 (or 77:15:8) volume ratio
are shown in Fig. 2 and the chromatograms obtained with f) water-acetonitrile-ethyl
acetate of 100:0:9 (or 92:0:8) volume ratio, g) 80:20:9 (or 73:18:8) volume ratio, h)
60:40:12.5 (or 53:36:11) volume ratio, i) 50:50:17 (or 43:43:15) volume ratio, j)
30:70:33 (or 23:53:25) volume ratio, and k) 14.5:50:50 (or 13:44:44) volume ratio areshown in Fig. 4.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Chromatograms of a mixture of 2,6-naphthalenedisulfonic acid and 1-naphtholwith the capillary chromatography system using various component ratios ofwater-acetonitrile-ethyl acetate mixture as carrier solutions that were positioned almoston the homogeneous-heterogeneous solution boundary curve of the phase diagram.
2,6-Naphthalenedisulfonic acid and 1-naphthol were eluted in this order with the carrier
solutions of f) – i), while they were eluted in the reverse order with the carrier solutionof j). Conditions: Capillary tube, 120 cm (effective length: 100 cm) of 100 µm i.d.
PTFE; carrier, f) water-acetonitrile-ethyl acetate of 100:0:9 volume ratio, g) 80:20:9
volume ratio, h) 60:40:12.5 volume ratio, i) 50:50:17 volume ratio, j) 30:70:33 volume
ratio, and k) 14.5:50:50 volume ratio; sample injection, 20 cm height (gravity)×30 s;
flow rate, 2.0 µL min-1; tube temperature, 0 °C; and 2,6-naphthalenedisulfonic acid and1-naphthol, 2 mM each. The component ratios of the carrier solutions f) – k) used forthe chromatograms are plotted in the phase diagram of Fig. 3.

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Figure 5. Chromatograms of a mixture of 2,6-naphthalenedisulfonic acid and 1-naphthol
obtained by the TRDC system using various hydrophilic organic solvents instead ofacetonitrile. Conditions: Capillary tube, 120 cm (effective length: 100 cm) of 100 µm
i.d. PTFE; carrier, water-methanol-ethyl acetate (100:20:15 volume ratio),
water-ethanol-ethyl acetate (100:20:15 volume ratio), water-1,4-dioxane-ethyl acetate(100:5:10 volume ratio), water-1-propanol-ethyl acetate (100:10:11 volume ratio), andwater-acetonitrile-ethyl acetate (100:20:10 volume ratio); sample injection, 20 cm
height (gravity)×30 s; flow rate, 2.0 µL min-1; tube temperature, 15 °C; and2,6-naphthalenedisulfonic acid and 1-naphthol, 2 mM each.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 6. Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidobtained by the TRDC system using various hydrophobic organic solvents instead of
ethyl acetate. Conditions: Capillary tube, 120 cm (effective length: 100 cm) of 100 µm
i.d. PTFE; carrier, water-acetonitrile-chloroform (100:30:1 volume ratio),
water-acetonitrile-hexane (100:30:1 volume ratio), water-acetonitrile-1-butanol	 (100:15:20 volume ratio), and water-acetonitrile-ethyl acetate (100:20:10 volume ratio);
sample injection, 20 cm height (gravity)×30 s; flow rate, 2.0 µL min-1; tube temperature,
15 °C; and 2,6-naphthalenedisulfonic acid and 1-naphthol, 2 mM each.

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Chapter 4118

Figure 7. Chromatograms of the mixture analyte solution of 1-naphthalenesulfonic acid,
1-naphthoic acid, and 1-naphthol obtained by the TRDC system. Conditions: Capillarytube, 120 cm (effective length: 100 cm) of 100 µm i.d. PTFE; carrier,water-acetonitrile-ethyl acetate (100:20:10 volume ratio), water-ethanol-ethyl acetate(100:20:15 volume ratio), and water-acetonitrile-chloroform (100:30:1 volume ratio);
sample injection, 20 cm height (gravity)×30 s; flow rate, 2.0 µL min-1; tube temperature,
15 °C; and 1-naphthalenesulfonic acid, 1-naphthoic acid, and 1-naphthol, 2 mM each.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

4.4 Investigation of the composition for a ternary mixed solvent system
in TRDC using fused-silica capillary tube
The composition for the water–hydrophilic organic solvent-hydrophobic organic solventin tube radial distribution chromatography (TRDC) using fused-silica capillary tubes
was investigated using phase diagrams, chromatograms, and fluorescence photographs.
Mixtures of a hydrophilic organic solvent (acetonitrile, ethanol, methanol, 1-propanol,
or 1,4-dioxane) and a hydrophobic solvent (ethyl acetate, hexane, 1-butanol, or
chloroform) were examined as the ternary solvent system, which was the carrier
solution in TRDC. Water–acetonitrile–chloroform and water–acetonitrile–ethyl acetate
systems performed well in TRDC. A model analyte solution of 1-naphthol and
2,6-naphthalenedisulfonic acid was separated by TRDC using the chloroform solvent
system with a range of volume ratios for the solvent components. The elution order ofthese two compounds could be reversed by changing the system from an organic
solvent-rich solvent to water-rich one. A mixture of five analytes, 1-naphthol, Eosin Y,
1-naphthalenesulfonicacid,
2,6-naphthalenedisulfonic
acid,
and
1,3,6-naphthalenetrisulfonic
acid,
was
also
separatedusing
thewater–acetonitrile–chloroform solvent system (32:65:3 volume ratio). These compounds
eluted in the order given above.
Introduction
Micro-flow analyses using microfluidics have been carried out in microspaces such as
capillary tubes (e.g., fused-silica and poly(tetrafluoroethylene)) and microchannels in a
microchip [27,32-36]. In microfluidics, various types of fluidic behavior are observed
for solvents in a microspace based on electro-osmotic and laminar flow [46-48]. The
fluidic behavior of solvents in the microspace is related to the mixing, separation,diffusion, and reaction of solutes. These properties are important and useful in thedesign of micro-reactors or micro-total analysis systems [49-51].
Several groups have reported that water and oil flow separately in a tube with aliquid-liquid radial interface under specific fluidic conditions [52-54]. Our group has
published the reports on the tube radial distribution phenomenon (TRDP) of different
solvent systems since 2009. TRDP has been observed in many mixed solutions,
including ternary water–hydrophilic solvent–hydrophobic solvent, water–surfactant,water–ionic liquid, and fluorous/organic solvents. For example, when a ternary solventof water–acetonitrile–ethyl acetate is introduced into a microspace under laminar flow
conditions, inner and outer phases are generated as the solvent molecules are radiallydistributed in the microspace. Based on TRDP, we have investigated capillarychromatography (tube radial distribution chromatography, TRDC), extraction, reaction
spaces, and mixing processes.
Here, the types of solvents and their volume ratios in a ternary solvent mixture ofwater–hydrophilic organic solvent–hydrophobic organic solvent were investigated for
TRDC. The optimum solvent mixture was determined using phase diagrams,
chromatograms, and fluorescence photographs. A hydrophilic organic solvent(acetonitrile, ethanol, methanol, 1-propanol, or 1,4-dioxane) and hydrophobic organic
solvent (ethyl acetate, hexane, 1-butanol, or chloroform) were mixed with water to

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produce a ternary water–hydrophilic organic solvent–hydrophobic organic solvent
system. The water–acetonitrile–chloroform solvent system was examined in detail andcompared with a water–acetonitrile–ethyl acetate solvent system, which is commonlyused in the TRDC.
Experimental
Open-tubular capillary chromatography
Fused-silica capillary tubes (internal
diameter 50 µm) were used. The TRDC system included an open fused-silica capillary
tube (total length 120 cm and effective length 100 cm), microsyringe pump, andabsorption detector. The tube temperature was controlled by dipping the capillary tube
in water maintained at a fixed temperature in a beaker by stirring. Various
water–hydrophilic organic solvent–hydrophobic organic solvent systems were used as
the carrier solution. Analyte solutions were prepared with the carrier solutions. The
analyte solution was introduced directly into the capillary inlet by the gravity method(from a height of 20 cm and for 30 s). After analyte injection, the capillary inlet wasconnected to a microsyringe attached to a pump. The carrier solution was fed into thecapillary tube at a set flow rate under laminar flow conditions. On-capillary absorption
detection (254 nm) was performed.
Fluorescence microscope-charge coupled device (CCD) camera system
To monitor
TRDP with the solvent system, the capillary tube was set up with a fluorescencemicroscope-CCD
camerasystem.
Ternary
water–hydrophilicorganic
solvent–hydrophobic organic solvent systems, with various volume ratios andcontaining perylene and Eosin Y, were used. The fluorescence in the capillary tube wasmonitored using a fluorescence microscope equipped with an Hg lamp, a filter, and a
CCD camera. Perylene and Eosin Y emit light at 470 and 550 nm, respectively, andbecause of this the resulting fluorescence was mostly blue and green.
Results and discussion
Preliminary experiments with water–hydrophilic organic solvent–ethyl acetate solvent
mixtures The hydrophilic organic solvent was either acetonitrile, methanol, ethanol,
1-propanol, or 1,4-dioxane. Phase diagrams of water–hydrophilic organic solvent–ethyl
acetate ternary solutions were examined at 0 and 20 °C (Fig. 1). The curves in the phase
diagram show the boundary or solubility curves between homogeneous (single phase)and heterogeneous (two phases) solutions. The separation performance for a model
analyte mixture of NA and NDS in TRDC was examined.	
 The volume ratios of thecomponents in the homogeneous ternary solvent solutions were positioned near theboundary curve at 20 °C, with an organic solvent-rich solution, NA eluted before NDS,
and a water-rich solution, NDS eluted before NA. This reversal of the elution order forthe analytes was only observed in the water–acetonitrile–ethyl acetate system. With theother ternary mixtures investigated, NA eluted before NDS with organic solvent-richsolutions and no separation occurred with water-rich solutions.
Preliminary experiments with water–acetonitrile–hydrophobic solvent mixtures
The
hydrophobic organic solvent was ethyl acetate, chloroform, 1-butanol, or hexane. Phase
diagrams of the water–acetonitrile–hydrophobic organic solvent mixtures were121

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

examined at 0 and 20 °C (Fig. 2). The separation performance for a model analyte
mixture of NA and NDS was investigated. The volume ratios of the components in thehomogeneous ternary solvent systems were positioned near the boundary curve at 20 °C.
The separation order of the analytes in TRDC was reversible using different volume
ratios with water–acetonitrile–chloroform and water–acetonitrile–ethyl acetate. With
water–acetonitrile–1-butanol, NA was eluted before NDS when the mixture was anorganic solvent-rich mixture. With the water–acetonitrile–hexane mixture, NA and NDS
were not separated.
Phase
diagrams,
chromatograms,
andfluorescencephotographswithwater–acetonitrile–chloroform
Seeing how water–acetonitrile–chloroform
performed in a similar manner to water–acetonitrile–ethyl acetate, we examined thewater–acetonitrile–chloroform system in more detail using phase diagrams,
chromatograms, and fluorescence photographs. Figure 3 shows the phase diagrams of
the water–acetonitrile–chloroform system. At a constant pressure (1 atm), as thetemperature decreased from 20 to 0 °C, the boundary curve expanded (Fig. 3 a)). The
curve also expanded as the pressure was increased from 1.0 to 1.3 atm at a constanttemperature (20 °C) (Fig. 3 b)). These changes in the boundary curves are similar to
those observed in our study for water–acetonitrile–ethyl acetate. Separation informationof the NA and NDS mixture with the water–acetonitrile–chloroform system is shown in
the phase diagram with the symbols (Fig. 4). With an organic-solvent rich carrier
solution (solutions (f)–(k) in Fig. 4), NA was eluted before NDS, contrary to thewater-rich carrier solutions (solutions (c)–(e)) where NDS eluted before NA. With
solutions (a), (b), and (l)–(r), NDS and NA were not separated under the present
conditions. Typical chromatograms are shown in Fig. 4. Typical fluorescencephotographs are also shown in Fig. 4. TRDP was observed in the mixtures (f)–(k),which had an organic solvent-rich major inner phase and water-rich minor outer phase,and (c)–(e), which had a water-rich major inner phase and organic solvent-rich minor
outer phase. TRDP was not observed in mixtures (a), (b), or (l)–(r). In other words,
when organic solvent-rich solutions ((f)–(k)) were delivered into the capillary tube, themajor phase that formed in the tube was an organic solvent-rich mixture. In contrast,
when water-rich solutions ((c)–(e)) were delivered into the capillary tube, the major
phase that formed in the tube was water-rich. Separation of compounds by TRDC isbased on the distribution of the hydrophilic compounds (e.g. NDS) in the water-richphase and hydrophobic compounds (e.g. NA) in the organic solvent-rich phase. The
outer phase acts as a pseudo-stationary phase under laminar flow conditions. In thepresent study, the elution order in the chromatograms was consistent with thisseparation mechanism. With an organic solvent-rich inner phase, NA eluted before NDS,
but in a water-rich inner phase, NDS eluted before NA. The mixtures that did not show
TRDP did not provide any chromatographic separation in the TRDC. The TRDP and
TRDC results with the water–acetonitrile–chloroform system were the same as thoseobserved for the water–acetonitrile–ethyl acetate system.
Separation of an analyte mixture We examined separation of an analyte mixture (NA,
Eosin Y, NS, NDS, and NTS) using the TRDC system with organic solvent-rich andwater-rich carrier solutions prepared using the water–acetonitrile–chloroform system.

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122

The obtained chromatograms are shown in Fig. 5. The elution orders of the analytes
were different with the organic solvent-rich than they were with the water-rich carrier
solutions, as was reported for other TRDC systems. With the organic-solvent richcarrier solution, the elution order was NA, Eosin Y, NS, NDS, and finally NTS (Fig. 5a)). This elution order is consistent with the increasingly hydrophilic character of thecompounds. In contrast, with the water-rich carrier solution, the comparativelyhydrophilic compound, NTS, was eluted first, followed by co-elution of NS and NDS,
and then the hydrophobic compounds Eosin Y and 1-naphthol (Fig. 6 b)). This elution
order was the reverse of that observed with the organic solvent-rich carrier solution.
Comparison of water–acetonitrile–ethyl acetate and water-acetonitrile–chloroform
solvent systems
Figure 6 shows the phase diagrams (20 °C) and separation
performance for the water–acetonitrile–ethyl acetate and water–acetonitrile–chloroform
solvent
systems.
The
water–acetonitrile–ethyl
acetateandthewater–acetonitrile–chloroform solvent systems have quite different boundaries in thephase diagrams. Therefore, we were unable to draw any relevant comparisons of theirseparation performance with similar ratios for the ternary components of the solvent
system. We, tentatively, investigated the TRDC separation performance through carrier
solutions with same volume ratios for the two phases, organic solvent-rich andwater-rich, at a lower temperature in a batch vessel. The composition positions in thephase diagram, (1)–(5) in Fig. 6, showed the same volume ratios of the two phases bothfor the water-acetonitrile-ethyl acetate and the water-acetonitrile-chloroform solution
systems, and these ratios are also given in the figure captions. The separation
performance for NA and NDS was dependent on the ratio of the organic solvent-richand water-rich phases (Fig. 6). With the ethyl acetate system, the upper and lower
phases were organic solvent-rich and water-rich solutions, respectively, while with thechloroform system, the upper and lower phases were water-rich and organic solvent-richsolutions, respectively. Compared to the ethyl acetate system, TRDC separation wasachieved with the chloroform system with a wider range of organic solvent-rich andwater-rich volume ratios. The reason for this difference in performance has not been
made clear yet. Chloroform is more hydrophobic (logP, 1.97) than ethyl acetate (logP,0.73), and this may generate a more stable liquid-liquid interface between water-richand organic solvent-rich phases through TRDP. Table 1 shows the resolutions for themodel analytes, NA and NDS, obtained with the ethyl acetate and the chloroform
solvent systems. The ethyl acetate system provided better resolution than the chloroform
system, although TRDP was observed over a wider range of volume ratios for thechloroform system than for the ethyl acetate system. We examined the distribution
ratios, D, in the two solvent systems with various compositions ((1)–(5) in Figs. 6 and
7) at a low temperature, generating upper and lower phases in a batch vessel. D is givenby the ratio of analyte concentration in the organic-solvent rich phase to analyte
concentration in the water-rich phase. If D>1, most of the analyte dissolves in theorganic solvent-rich phase, and if D<1, most of the analyte is dissolved in the water-richphase. The concentrations of the analytes in the two phases were determined using
capillary electrophoresis with a calibration curve. For NA, all the D values (solutions(1)–(5)) were greater than one, which indicates it was easier for NA to dissolve in an
organic solvent-rich solution than a water-rich solution. The D values of the ethyl
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

acetate system (4.2–14.5) were larger than those of the chloroform system (1.5–3.0).
For NDS, all the D values (solutions (1)–(5)) were less than one, indicating that it iseasier for NDS to dissolve in a water-rich solution than an organic solvent-rich solution.
The D values of the ethyl acetate system (0.5–0.25) were smaller than those of thechloroform system (0.30–0.65). Consequently, NA dissolves in the organic solvent-richphase and NDS in the water-rich phase with the ethyl acetate system more effectively
than the chloroform system. That is, the trends observed with the D values are
consistent with the better resolution for NA and NDS observed with the ethyl acetate
system compared with the chloroform system.
In
conclusion,
weexaminedvariouswater–hydrophilicorganic
solvent–hydrophobic organic solvent systems for TDRP and TRDC. Phase diagrams,
fluorescence photographs, and chromatograms for a water–acetonitrile–chloroform
solvent system showed particular TRDP and TRDC. The performance of this solvent
system in TRDC was comparable to that of a water–acetonitrile–ethyl acetate system.
The results of this study provide basic data on the microfluidic behavior, TRDP, and theseparation technology, TRDC, with the studied solvent systems.

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Chapter 4124

Figure 1. Phase diagrams for water–hydrophilic organic solvent–ethyl acetate solvent
systems, and separation performance of a NA and NDS mixture on chromatograms. a)
Acetonitrile, b) methanol, c) ethanol, d) 1-propanol, and e) 1,4-dioxane. The
dotted-dashed	 and dotted curves show the boundaries between homogeneous andheterogeneous solutions at 0 and 20 °C. The symbols ○, ●, and × show separation with
NA eluted before NDS, separation with NDS eluted before NA, and no separation,
respectively. Chromatography conditions: capillary tube, 120 cm (effective length, 100cm) of 50 µm i.d. fused-silica; carrier, water–hydrophilic organic solvent–ethyl acetate;
sample injection, 20 cm height (gravity)×30 s; flow rate, 0.2 µL min-1; tube temperature,
3 °C; and NA and NDS concentrations, 2 mmol L-1 each.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2. Phase diagrams for water–acetonitrile–hydrophobic organic solvent systems,
and separation performance of an NA and NDS mixture on chromatograms. a) Ethyl
acetate, b) hexane, c) 1-butanol, and d) chloroform. The dotted-dashed	 and dottedcurves show the boundaries between homogeneous and heterogeneous solutions at 0
and 20 °C. The symbols ○, ●, and × show separation with NA eluted before NDS,
separation with NDS eluted before NA, and no separation. The chromatography
conditions are the same as those in Fig. 1.

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Figure 3. Phase diagrams for the water–acetonitrile–chloroform solvent system. a)
Constant pressure (1 atm) and b) constant temperature (20 °C).

Figure 4. Phase diagrams, chromatograms, and fluorescence photographs for thewater–acetonitrile–chloroform solvent system. Water–acetonitrile–chloroform volume
ratios were as follows: (a) 99.5:0:0.5, (b) 76.2:23:0.8, (c) 66:33:1, (d) 58:41:1, (e)49.5:49.5:1, (f) 39:59:2, (g) 32:65:3, (h) 27:69:4, (i) 22:73:5, (j) 15:77:8, (k) 8:76:16, (l)4:64:32, m) 4:58:38, (n) 2:49:49, (o) 1:41:58, (p) 0.7:33:66.3, (q) 0.5:23:76.5, and (r)0.1:0:99.9, containing perylene (~0.1 mmol L-1) and Eosin Y (~1.0 mmol L). The
symbols ○, ●, and × show separation with NA eluted before NDS, separation with NDS
eluted before NA, and no separation. Chromatography conditions are the same as thosein Fig. 1.
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 5. Chromatograms of the analyte mixture of NA, Eosin Y, NS, NDS, and NTS
obtained by TRDC with the water–acetonitrile–chloroform solvent system.
Chromatography conditions: capillary tube, 120 cm (effective length, 100 cm) of 50 µm
i.d. fused-silica; carrier, water–acetonitrile–chloroform a) organic solvent-rich (20:53:27
volume ratio) and b) water-rich (75:15:10 volume ratio); sample injection, 20 cm height(gravity)×30 s; flow rate, 0.2 µL min-1; tube temperature, 3 °C; and analyte
concentrations, NA, NS, NDS and NTS 1 mmol L-1 each and Eosin Y 0.5 mmol L-1.

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Chapter 4128

Figure 6. Comparison of phase diagrams and TRDC separation performance withwater–acetonitrile–ethyl acetate and water–acetonitrile–chloroform solvent systems.
The volume ratios of the upper and lower phases in a batch vessel are as follows:
Water-rich:organic solvent-rich; (1) 2:98, (2) 10:90, (3) 28:72, (4) 60:40, and (5) 90:10
in the two systems. The symbols ○, ●, and × show separation with NA eluted before
NDS, separation with NDS eluted before NA, and no separation.

Figure 7. The separation performance with various volume ratios of upper and lower
phases in a) water–acetonitrile–ethyl acetate and b) water–acetonitrile–chloroform
solvent systems. The volume ratios of the upper and lower phases in a batch vessel are
as follows: Water-rich:organic solvent-rich; (1) 2:98, (2) 10:90, (3) 28:72, (4) 60:40,
and (5) 90:10 in the two systems. The symbols of ○, ●, and × mean show separation
with NA eluted before NDS, separation with NDS eluted before NA, and no separation.

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4.5 Fundamental experiments for tentative comparison of TRDC andcapillary zone electrophoresis
A capillary chromatography using an untreated open tubular capillary tube and ternary
mixed solvents of a water-hydrophilic/hydrophobic organic solvent as a carrier solution
has been developed; the system is called tube radical distribution chromatography(TRDC). A model mixture analyte solution, including 1-naphthol, 1-naphthoic acid,
1-naphthalenesulfonic acid, 2,6-naphthalenedisulfonic acid, 1,3,6-naphthalenetrisulfonicacid, was examined by the TRDC and capillary zone electrophoretic (CZE) systems thatcomprised mainly a capillary tube and a detector. In the TRDC system the elution orderof analytes could be changed by altering the component ratios of the solvents, as in the
CZE system the elution order was changed by altering the electro-osmotic flow
direction. The experimental data obtained here gave a clue for thinking of the featuresand utility of the TRDC as a new separation method.
Introduction
Capillary tubes with inner diameters less than several hundred micrometers that provide
a micro-flow are known to exhibit interesting and useful physical or hydrodynamic
phenomena, such as electro-osmotic flow and laminar flow. The electro-osmotic flow ina capillary tube promotes capillary electrophoresis [38,39,55], and capillaryelectrochromatography [42,56,57], while laminar flow conditions enable hydrodynamic
chromatography [44,45]. Recently, our group reported the tube radial distribution
phenomenon of carrier solvents, which we call the tube radial distribution phenomenon(TRDP).
When the ternary mixed solvents of water-hydrophilic/hydrophobic organic solvent
mixtures are delivered into a microspace, such as a microchannel or a capillary tube
under laminar flow conditions, the solvent molecules are distributed radially within the
microspace, generating inner and outer phases. The TRDP creates a phase interface or
kinetic aqueous–organic interface in a microspace. A capillary chromatography system
where the outer phase functions as a pseudo-stationary phase under laminar flow
conditions has been developed based on the TRDP. We call it tube radial distribution
chromatography (TRDC). In the TRDC system the analytes are distributed between the
inner and outer phases due to their hydrophobic or hydrophilic nature, undergoing
chromatographic separation. The TRDC system does not require any specific columns,
such as packed and monolithic, or application of high voltage to the ends of thecapillary tube.
As the investigation of TRDP and TRDC has just begun, we have to examine
anything we can imagine in order to expand our knowledge about TRDP and TRDC.
The TRDC and capillary zone electrophoresis (CZE) systems have quite simple
instruments comprising mainly a capillary tube and a detector, although they havedifferent separation mechanisms. After the analyte solution was injected in the inlet
capillary side, the carrier solution was delivered with a microsyringe pump in the TRDC
system, while the running buffer was fed by applying high voltage in the CZE system.
We are interested in examining separation performance of these open-tubular capillary
analyses. The model analyte solution was subjected to the TRDC and CZE systems. The

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obtained chromatograms and electropherograms were compared and discussed eachother through the fundamental experiments.
Experimental
TRDC system
Fused-silica (75 µm inner diameter) and poly(tetrafluoroethylene)(PTFE) (100 µm inner diameter) capillary tubes were used. A schematic diagram of thepresent TRDC system comprised the fused-silica or PTFE capillary tube (120 cm length,
effective length 100 cm), a microsyringe pump, and an absorption detector. The tube
temperature was controlled by dipping the capillary tube (ca. 60 cm) in water
maintained at a specific temperature (0 or 20 °C) in a beaker maintained by stirring.
Water-acetonitrile-ethyl acetate mixture	
 solutions	 (3:8:4 or 15:3:2 volume ratio) wereused as carrier solutions. The component ratios of the solvents were recommended as anorganic solvent-rich and water-rich carrier solution in our works. Analyte solutions wereprepared with the carrier solutions. The analyte solution was introduced directly into thecapillary inlet side by the gravity method (30 cm height for 20 s). After analyte injection,
the capillary inlet was connected through a joint to a micro-syringe. The syringe wasplaced on the microsyringe pump. The carrier solution was fed into the capillary tube at
0.8 µL min-1 for fused-silica tube and 2.0 µL min-1 for PTFE tube under laminar flow
conditions. On-capillary absorption detection (254 nm for fused-silica tube or 320 nm
for PTFE tube 2) was performed with the detector.
CZE system
A schematic diagram of the present CZE system comprised thefused-silica or PTFE capillary tube (120 cm length, effective length	
 100 cm), a DC
power supplier (Model HCZE-30PNO.25, Matsusada Precision Devices Co., LTD.),and an absorption detector. 10 mM Phosphate buffer (pH 7.0) was used as a runningbuffer solution. Analyte solutions were prepared with the running buffer solution. The
analyte solution was introduced directly into the capillary inlet side by the gravity
method (30 cm height for 20 s). After analyte injection, a high voltage of 12 kV wasapplied to the electrodes. On-capillary absorption detection (254 nm for fused-silica
tube or 320 nm for PTFE tube2)) was performed with the detector.
Results and discussion
Separation performance in the TRDC system The molecular structures of five model
analytes are shown in Fig. 1; 1-naphthol, 1-naphthoic acid, 1-NS, 2,6-NDS, and1,3,6-NTS. The hydrophobicity of the analytes decreases from 1-naphthol to 1,3,6- NTS.
The solution including the five model analytes was subjected to the TRDC system withthe organic solvent-rich carrier solution (water-acetonitrile-ethyl acetate, 3:8:4 volume
ratio) and the water-rich carrier solution (15:3:2 volume ratio). The TRDC system withthe organic solvent-rich carrier solution showed the best resolutions at around a 20 °C
tube temperature using the fused-silica tube, while the system with the water-rich carrier
solution showed the best resolutions at around a 0 °C tube temperature using the PTFE
tube.11,10,14) The chromatograms obtained under the recommended analytical conditions
are shown in Fig. 2. With the organic solvent-rich carrier solution 1-naphthol and1-naphthoic acid, 1-NS, 2,6-NDS, and 1,3,6-NTS were eluted in this order due to theirhydrophobic or hydrophilic nature, although 1-naphthol and 1-naphthoic acid were not131

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

separated (Fig. 2 a)). On the other hand, the water-rich carrier solution 1-NS, 2,6-NDS,
and 1,3,6-NTS were not separated; however, after that, 1-naphthoic acid and 1-naphthol
were eluted in this order due to their nature (Fig. 2b)). Roughly speaking, the reverse
elution orders were observed in the TRDC system between the organic solvent-rich andwater-rich carrier solutions. The separations were performed on the chromatograms
within ca. 15 min.
Separation performance in the CZE system using the fused-silica capillary tube
Thesolution including the five model analytes was subjected to the CZE system using the
fused-silica capillary tube with the normal electro-osmotic flow direction where thecathode was set to the capillary outlet and the reverse electro-osmotic flow direction
where the cathode was set to the capillary inlet. The obtained electropherograms areshown in Fig. 3. With the normal electro-osmotic flow direction (Fig. 3 a)), 1-naphthol,
1-naphthoic acid, and 1-NS were eluted in this order on the elecropherogram within ca.
60 min, but 2,6-NDS and 1,3,6-NTS were not detected within 120 min due to their
negative charge using the present analytical conditions. 2,6-NDS and 1,3,6-NTS must
require a large electrophoretic flow to the capillary inlet. However, whenelectro-osmotic flow direction was reversed, none of the analytes were detected within
120 min (Fig. 3 b)). Electro-osmotic flow was larger than electrophoretic flow for the
analytes under the conditions.
Separation performance in the CZE system with the PTFE capillary tube It is wellknown that a PTFE capillary tube shows a much smaller electro-osmotic flow than afused-silica capillary tube. Similarly, the solution including the five model analytes was
subjected to the CZE system using the PTEF capillary tube with the normal and reverse
electro-osmotic flow directions. The obtained electropherogrames are shown in Fig. 4.
With the normal electro-osmotic flow direction (Fig. 4 a)), 1-naphthol, 1-naphthoic acid,and 1-NS were eluted in this order on the elecropherogram within ca. 110 min due tothe small electro-osmotic flow in the PTFE tube. 2,6-NDS and 1,3,6-NTS were notdetected within 120 min due to their negative charge under the analytical conditions. On
the other hand, with the reverse electro-osmotic flow direction (Fig. 4 b)), 1,3,6-NTS
and 2,6-NDS were eluted in this order within ca. 65 min due to their negative charge.
However, 1-naphthol, 1-naphthoic acid, and 1-NS were not detected within at least 120
min. Even in the PTFE capillary tube electro-osmotic flow was larger thanelectrophoretic flow for the three analytes under the conditions.
In conclusion, the elution behavior of the model five analytes was examined throughthe chromatogram obtained by the TRDC system and the electropherogram obtained by
the CZE system. In the TRDC system, the elution order of analytes could be changed byaltering the component ratios of the solvents, the organic solvent-rich and the water-richcarrier solutions, within ca. 15 min, while in the CZE system, as is well known, theelution order was changing by altering the electro-osmotic flow direction to within ca.
60 or 120 min. The TRDC system can easily reverse the elution order of analytes
without applying high voltage within short analytical time. The experimental data may
show the feature of the TRDC system through the fundamental experiments with the
CZE data.

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132

	
 

Figure 1. Molecular structures of analytes and their hydrophobic and hydrophilic nature.

Figure 2. Chromatograms of the analyte mixture with the TRDC system using a) theorganic solvent-rich and b) water-rich carrier solutions. Conditions: Capillary tube, 120
cm (effective length: 100 cm), a) fused-silica and b) PTFE; carrier, a) organic
solvent-rich (water-acetonitrile-ethyl acetate of 3:8:4 volume ratio) and b) water-rich(15:3:2 volume ratio); sample injection, 30 cm height (gravity) × 20 s; flow rate, a) 0.8µL min-1 and b) 2.0 µL min-1; tube temperature, a) 20 °C and b) 0 °C; detection
wavelength, a) 254 nm and b) 320 nm; and analyte concentration, 2 mM each.
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Development of TRDC133

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3. Electropherograms of analyte mixture with the CZE system using the
fused-silica capillary tube. Electro-osmotic flow direction a) from the capillary inlet tothe outlet and b) from capillary outlet to inlet. Conditions: Capillary tube, 120 cm(effective length: 100 cm) of fused-silica; running buffer, 10 mM phosphate buffer (pH7.0); sample injection, 30 cm height (gravity) × 20 s; applied voltage, 12 kV; detection
wavelength, 254 nm; and analyte concentration, 2 mM each.

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Figure 4. Electropherograms of analyte mixture with the CZE system using the PTFE
capillary tube. Electro-osmotic flow direction a) from capillary inlet to outlet and b)
from capillary outlet to inlet. Conditions: Capillary tube, 120 cm (effective length: 100cm) of PTFE; running buffer, 10 mM phosphate buffer (pH 7.0); sample injection, 30
cm height (gravity) × 20 s; applied voltage, 12 kV; detection wavelength, 320 nm; andanalyte concentration, 2 mM each.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Chapter 5 Effects of inner wall characteristics of capillarytubes on tube radial distribution chromatography (TRDC)
TRDP is basically caused through phase transformation of the two-phase separation
solution systems, not through interaction between an inner wall of the capillary tubes
and solvent molecules. However, the stability of TRDP formation、 especially outerphase formation、 seemed to be influenced by the materials of the inner wall. The
effects of the inner wall characteristics of capillary tubes on TRDC performance were
examined. The part of this chapter is reconstructed and rewritten based on the related
manuscripts that have been published.4,5,22)
Chapter 5

5.1 Effects of the inner-wall characteristics of the fused-silica capillary
tube on TRDC
A capillary chromatography system was developed using an open capillary tube and awater–acetonitrile (hydrophilic)–ethyl acetate (hydrophobic) mixture carrier solution.
Here, we examine the effects of the inner-wall characteristics of a fused-silica capillary
tube on the	 separation performance in this system. Untreated (silanol group-intact)	
 andinactivated (silanol group-end-blocked) fused-silica capillary tubes were used. The
mixture analyte of 1-naphthol and 2,6-naphthalenedisulfonic acid was injected into two
types of capillary tubes. They were eluted from both tubes in this order with	 water–acetonitrile–ethyl acetate (volume ratio 2:7:4) mixture carrier solution, and eluted
in reverse order with water–acetonitrile–ethyl acetate (volume ratio 15:3:2). The peak
shapes	 observed here were analyzed while considering the inner-wall characteristics ofthe capillary tubes from the viewpoint of the tube radial distribution of the carrier
solvents in the tubes.
Introduction
We developed a capillary chromatography system using an open capillary tube and an
aqueous–organic mixture carrier solution that worked under laminar flow conditions.
Unlike conventional capillary electrochromatography [1,2], capillary electrophoresis[3,4] and capillary liquid chromatography [5,6], separation in this system was
performed without the use of any specific materials, such as packed capillary tubes,
additives, such as gels, surfactants, host molecules or salts, or high-voltage supply
devices.
Based on our results, we proposed that separation in the capillary chromatographysystem was performed based on the tube radial distribution of the aqueous–organic
mixture solvents in the capillary tubes under laminar flow conditions. We call this atube radial distribution chromatography (TRDC) system. Here, to expand ourknowledge regarding the TRDC system, we examined the effects of the inner-wallcharacteristics of a fused-silica capillary tube on the separation performance in thesystem. Untreated (silanol group-intact) and inactivated (silanol group-end-blocked)

138

Chapter 5138fused-silica capillary tubes were used. Aside from the altered inner-wall characteristicsof the fused-silica capillary tubes, the fowllowing experiments were carried out under
the same analytical conditions used previously. The data obtained here were consideredfrom the viewpoint of the tube radial distribution of the carrier solvents in the tubes
under laminar flow conditions.
Experimental
Fused-silica capillary tubes (50 µm i.d., 150 µm o.d.), untreated (silanol group-intact)and inactivated (silanol group-end-blocked), were used; they were commerciallyavailable. The capillary chromatography system consisted of a fused-silica capillary
tube (80 cm in length; effective length, 60 cm), a microsyringe pump, and an absorption
detector.
Aqueous–organic
solvent
mixturecarriersolutionsofthewater–acetonitrile–ethyl acetate mixture with volume ratios of 2:7:4 and 15:3:2 wereused. A mixture analyte solution of 1-naphthol and 2,6-naphthalenedisulfonic acid was
prepared with the carrier solutions. The analyte solution was introduced directly into thecapillary inlet for 20 s from a height of 20 cm by a gravity method. After analyte
injection, the capillary inlet was connected through a joint to a microsyringe. The
syringe was set on a microsyringe pump. The carrier solution was fed into the capillary
tube at a specified flow rate under laminar flow conditions (setting flow rate of thepump; 0.3 µL min-1). On-capillary absorption detection (254 nm) was performed with adetector.
Results and discussion
The TRDC systems were performed using fused-silica, polyethylene, andpoly(tetrafluoroethylene) capillary tubes in our previous work. However, because they
had quite different inner diameters of 50, 200, and 100 µm, respectively, it was hard tostrictly evaluate the influence of the inner-wall of the capillary tube. Here, we tried to
examine the effects of the inner-wall characteristic on the separation performance in the
TRDC system by using fused-silica capillaries under quite the same analyticalconditions, except for the inner-wall characteristics.
First, we examined an untreated (silanol group-intact) fused-silica capillary tube
with aqueous–organic solvent mixture carrier solutions, an organic solvent-rich carrier
solution (water–acetonitrile–ethyl acetate (volume ratio 2:7:4)) and a water-rich carrier
solution (the water–acetonitrile–ethyl acetate (volume ratio 15:3:2)). The obtained
chromatograms are shown in Fig. 1. 1-Naphthol and 2,6-naphthalenedisuflonic acid
were eluted from the capillary in this order with the organic solvent-rich carrier solution(Fig. 1a)). 1-Naphthol was eluted at ca. 4.2 min with a near average linear velocity(average linear velocity under the laminar flow conditions was confirmed through anexperiment using a normal carrier solution not including organic solvents), and2,6-naphthalenedisulfonic acid was eluted at a lower than average linear velocity. A
water-rich phase (minor solvent phase) was formed near the inner wall as a
pseudo-stationary phase, due to the tube radial distribution of the carrier solvents inwhich phase 2,6-naphthalenedisuflonic acid was dispersed and eluted with a lower than
average linear velocity. 1-Naphthol and 2,6-naphthalenedisuflonic acid were eluted inreverse order when the carrier solution was water-rich (Fig. 1b)). An organic
solvent-rich phase (the minor solvent phase) generated near the inner wall due to the139

Effects of inner wall characteristics on (TRDC)

139

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

tube radial distribution of the carrier solvents in which phase 1-naphthol was dispersed
and eluted at a lower than average linear velocity. The elution times and the peak shapesof the chromatograms shown in Fig. 1 were partly different from those reported in ourwork using the same untreated fused-silica capillary tube. The difference between themmust have been caused by the different analytical conditions, such as the capillary
length and the flow rate.
Next, we examined an inactivated (silanol group-end-blocked) fused-silica capillary
tube with an aqueous–organic solvent carrier solution, an organic solvent-rich carrier
solution and a water-rich carrier solution. The obtained chromatograms are shown in
Fig. 2; an elution behavior similar to that of the untreated capillary tube was observed.
1-Naphthol and 2,6-naphthalenedisuflonic acid were eluted from the capillary in thisorder with an organic solvent-rich carrier solution (Fig. 2a)), and were eluted in reverse
order in the water-rich carrier solution (Fig. 2b)). The inner-wall characteristics of the
fused-silica capillary tubes did not change the elution orders of the model analytes in thepresent TRCD system. However, it should be noted that the second peak of 1-naphthol,obtained through an inactivated tube with the water-rich carrier in Fig. 2b), indicatedbroadening on the peak shape, compared with the other peaks observed in Figs. 1 and 2.
The component of the second peak was dispersed in the minor solvent phase generated
near the inner wall of the capillary tube as a pseudo-stationary phase. When using theinactivated (comparatively hydrophobic) fused-silica capillary	  with the water-richcarrier solution, 1-naphthol (hydrophobic) was eluted as a second peak along the innerwall surface, as shown in Fig. 2b). In this case, the hydrophobic interaction between theanalyte (1-naphthol) and the inner wall of the inactivated fused-silica must have led to apeak broadening of 1-naphthol. That is, the specific broadening of the second peak in
Fig. 2b) consists of the separation performance based on the tube radial distribution of
the carrier solvents in the TRDC system.	
 In addition, in the case of no interaction between the analyte and the inner wall, it
was observed for both analytes, 1-naphthol and 2,6-naphthalenedisuflonic acid, in Figs.
1 and 2 that the peak area of the anylyte as a second peak was smaller than it was as thefirst peak. Although such a phenomenon may have been caused by the light-path lengthsof the major and minor solvent phases in the capillary tube as well as reflection andrefraction of the entrance and transmittance light at the outer and inner wall surfaces of
the capillary tube; the reason has yet to be clarified.

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Chapter 5140

Figure 1. Obtained chromatograms of a mixture analyte of 1-naphthol and
2,6-naphthalenedisulfonic acid by the present TRDC system using an untreatedfused-silica capillary tube. a) Water–acetonitrile–ethyl acetate mixture (2:7:4) carrierand	 b) water–acetonitrile–ethyl acetate mixture (15:3:2) carrier. Conditions: capillarytube, 80 cm (effective length: 60 cm) of 50 µm i.d. fused-silica capillary; sample
injection, 20 cm height (gravity)×20 s; and analyte concentration, 2 mM each.

141

Effects of inner wall characteristics on (TRDC)141

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2. Obtained chromatograms of a mixture of 1-naphthol and
2,6-naphthalenedisulfonic acid by the present TRDC system using an	
 inactivatedfused-silica capillary tube. a) Water–acetonitrile–ethyl acetate mixture (2:7:4) carrierand	 b) water–acetonitrile–ethyl acetate mixture (15:3:2) carrier. Conditions: capillarytube, 80 cm (effective length: 60 cm) of 50 µm i.d. fused-silica capillary; sample
injection, 20 cm height (gravity)×20 s; and analyte concentration, 2 mM each.

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5.2 Effects of inner-wall-modified capillary tubes on TRDC
We tried to introduce inner-wall-modified (e.g., phenylboronic-acid- andiminodiacetic-acid-modified) fused-silica capillary tubes to the tube radial distribution
chromatography (TRDC) system to separate model mixture analytes. The
phenylboronic-acid-modified capillary tube was combined with absorption detection to
analyze a mixture of adenosine and deoxyadenosine. The iminodiacetic-acid-modified
capillary tube was combined with chemiluminescence detection using a luminol
reaction to analyze a mixture of copper(II) and hematin. A water (carbonate
buffer)-acetonitrile-ethyl acetate (2:7:4 v/v/v) and a water (carbonate
buffer)-acetonitrile-ethyl acetate (15:3:2 v/v/v) mixture solution were used as carrier
solutions in the TRDC system, and typical carbonate buffer solutions not containing any
organic solvents were also used as carrier solutions as reference solutions. In bothmodified capillary tubes, the organic-solvent-rich carrier solution successfully improved
the separation of the mixture analytes in the system, and the water-rich carrier solution
greatly depressed their separation, when compared with chromatography using
carbonate buffer carrier solutions containing no organic solvents. Such observed
phenomena were discussed with consideration given to the separation mechanism of the
TRDC system.
Introduction
Miniaturization generally has a number of merits, so it has been one of best-recognizedresearch directions in analytical chemistry. Interesting investigations with respect tominiaturization have also been done in the field of liquid chromatography; e.g., they arecalled open tubular liquid chromatography and capillary liquid chromatography. Opentubular liquid chromatography is generally identified with tube inner diameters less thanseveral decades µm [7-9]. Knox et al. reported in their theoretical study that the
optimum inner diameter of the open tubular column was approximately one-fifth of thediameter of the particles in the optimum packed column [9]. Recently,hydrophilic-interaction chromatography using 10 µm i.d. porous layer open tubular
column was developed for ultra-trace glycan analysis [10]. However, open tubularliquid chromatography naturally has some difficulties in the construction and operationof such very narrowly bored tubes.
Capillary liquid chromatography is performed with wide-bored capillary tubes thatare usually applied up to several hundreds µm i. d. Various capillary chromatography
techniques, including capillary electrochromatography [1,4], micellar electrokinetic
capillary chromatography [11,12], and high-performance liquid capillarychromatography using packed and monolithic columns [6,13], have been investigated as
powerful separation tools. We proposed a novel capillary chromatography using an
open capillary tube (50 – 200 µm i.d.)	
 and a water-hydrophilic/hydrophobic organic
solvent mixture (homogeneous solution) as a carrier solution; the system worked basedon the radial distribution of carrier solvents in the capillary tube under laminar flow
conditions. We called this system of capillary chromatography; a tube radial distribution
chromatography (TRDC) system.

143

Effects of inner wall characteristics on (TRDC)

143

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Data obtained in the TRDC system so far indicates that the properties of thematerials used in the capillary tube, such as fused-silica, polyethylene, andpoly(tetrafluoroethylene), have little influence on separation performance. However,what we are interested in is if the inner wall characteristics of a capillary tube affects or
improves separation performance in the TRDC system. Here, we prepared two types of
inner-wall-modified open fused-silica capillary tubes, phenylboronic-acid- andiminodiacetic-acid-modified, according to the preparation procedures described in ourprevious works [14-15], and tried to introduce the modified tubes into the TRDC systemusing an aqueous-organic solvent mixture carrier solution. The fused-silica,
polyethylene, and poly(tetrafluoroethylene) capillary tubes used so far in the TRDC
system were relatively inert, although they have quite different chemical characteristics
on their inner walls. In contrast, the modified capillary tubes have specific interaction
affinity to certain specific analytes. The phenylboronic acid moieties on the inner wallof the capillary tube have specific interactions with compounds having cis-diol groups
in the molecules, such as nucleosides and saccharides [14,15]. The iminodiacetic acidmoieties on the inner wall have another specific affinity to metal ions through complex
formation [16].
We examined the interaction between the function moieties, phenylboronic acid andiminodiacetic acid, on the inner walls and the model analytes using the modified
capillary tubes, as reflected in the separation performance in the TRDC system. The
TRDC system with the organic-solvent-rich carrier solution improved separation
performance and the system with the water-rich carrier solution did not show any
separation performance under the present analytical conditions. Also, the findings in the
TRDC system using the modified capillary tubes provided us important information thatstrongly supported our proposed separation mechanism in the TRDC system.
Experimental
A fused-silica capillary tube (75	  µm	
  i.d. and 150 µm o.d.) was used.
Phenylboronic-acid- and iminodiacetic-acid-modified fused-silica capillary tubes wereprepared as described in our previous papers [13–15]. TRDC systems equipped withabsorption detection (System 1) and chemiluminescence (CL) detection (System 2) are
shown in Fig. 1 a and b, respectively. System 1 in Fig. 1 a was used with thephenylboronic-acid-modified capillary tube (60 cm length, 40 cm effective length) for
nucleoside analysis. System 2 in Fig. 1 b, taking advantage of the luminol reaction, wasused with the iminodiacetic-acid-modified capillary tube (70 cm length) for metal
catalyst analysis. A batch-type CL detection cell equipped with an optical fiber [17] wasused in System 2. The CL reagent solution [10 mM carbonate buffer solution (pH 10.8),
including 5	
 µM	
 luminol and 50 mM hydrogen peroxide] was delivered to the capillaryoutlet, where the analytes having catalytic activity for the luminol reaction and the CL
reagent were mixed to generate CL light. The aqueous-organic solvent mixture carrier
solutions were prepared with a water (10 mM carbonate buffer, pH 9.5 or
10.8)-acetonitrile-ethyl acetate mixture (2:7:4 v/v/v) and a water (10 mM carbonate
buffer, pH 9.5 or 10.8)-acetonitrile-ethyl acetate mixture (15:3:2 v/v/v) mixture. Ten
mM carbonate buffer solutions of pH 9.5 and 10.8, not including any organic solvents,
were used as reference carrier solutions to the aqueous-organic solvent carrier solution
for Systems 1 and 2, respectively. A mixture of adenosine and deoxyadenosine, as well

144

Chapter 5144

as a mixture of Cu(II) and hematin, were prepared as model analytes by dissolving theminto the carrier solution. The metal catalyst solution included potassium sodium tartratewith a concentration 20 times higher than that of the catalyst. The analyte solutions
were injected into the capillary tube inlet using the gravity method (from a 30 cm heightfor 15 s for System 1 and from a 35 cm height for 5 s for System 2). The analytes were
further delivered in the capillary tubes with the carrier solution by micro-syringe pumps
at a flow rate of 0.13	
 µL min−1 for System 1 and 0.33	
 µL min−1 for System 2. Analytesof nucleosides in System 1 were detected by adsorption (267 nm), and analytes of metalcompounds in System 2 were detected by CL detection.
Results and discussion
Phenylboronic-acid-modified capillary tube
Boronic acids and boronic acidcompounds are known to interact with cis-diol groups included in saccharide andnucleoside molecules through ester formation, leading to separation and molecular
recognition of such biomolecules [18]. We reported capillary liquid chromatography
and capillary electrophoresis using phenylboronic-acid-modified capillary tubes [14,15].
Mixtures of a nucleoside and deoxynucleoside, for example,	  adenosine anddeoxyadenosine, were successfully separated in the modified capillary tube; theirresolution in capillary electrophoresis was better than in capillary liquid
chromatography, where the analytes were delivered in the capillary tube by the gravity
method. The mixture of adenosine and deoxyadenosine as a model was examined by thepresent TRDC system using the phenylboronic-acid-modified capillary tube with awater (10 mM carbonate buffer, pH 9.5)-acetonitrile-ethyl acetate mixture (2:7:4 v/v/v)
and a water (10 mM carbonate buffer, pH 9.5)-acetonitrile-ethyl acetate mixture (15:3:2
v/v/v). The obtained chromatograms are shown in Fig. 2, together with thechromatogram obtained using a carbonate buffer carrier solution which didn’t include
any organic solvents as a reference. The mixture of adenosine and deoxyadenosine wasseparated through the modified capillary tube with the carbonate buffer carrier solution(Fig. 2 a). Deoxyadenosine has no cis-diol group in its molecules but adenosine has one.
Consequently, the former moved first in the capillary tube without any interaction with
the inner wall of the phenylboronic-acid-modified capillary, but the latter moved slowly
in the tube because of the interaction of the cis-diol groups with the phenylboronic acidmoiety on the inner wall, causing a prolonged elution time. Interesting separation
performance was observed in the TRDC system with the organic-solvent-rich (Fig. 2 b)
and water-rich (Fig. 2 c) carrier solutions. The resolution of the nucleosides was
improved with the organic-solvent-rich carrier solution. The obtained chromatogram in
Fig. 2 b provided a complete baseline separation. In contrast, the resolution was
extremely depressed with the water-rich carrier solution; no separation was observed on
the chromatogram in Fig. 2 c.
Iminodiacetic-acid-modified capillary tube
Capillary liquid chromatography using
the iminodiacetic-acid-modified capillary tube was reported; a mixture of Cu(II) or
Co(II) and hematin was	
 separated through the tube and detected with CL detection
where the mixture was delivered in the tube by the gravity method [16]. The experimentusing the iminodiacetic-acid-modified capillary tube in the TRDC system was carriedout similar to the capillary liquid chromatography experiment. The mixture of Cu(II)145

Effects of inner wall characteristics on (TRDC)

145

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

and hematin was examined by the TRDC system using the iminodiacetic-acid-modified
capillary tube with water (10 mM carbonate buffer, pH 10.8)-acetonitrile-ethyl acetate
mixture (2:7:4 v/v/v) and water (10 mM carbonate buffer, pH 10.8)-acetonitrile-ethyl
acetate mixture (15:3:2 v/v/v) carrier solutions. The obtained chromatograms are shownin Fig. 3, along with the chromatogram obtained with a carbonate buffer solution (pH10.8) as a reference. The mixture was separated with the carbonate buffer solution;
hematin and Cu(II) were hence eluted in this order (Fig. 3 a). The metal complexationinteraction between Cu(II) and the iminodiacetic acid moiety on the inner wall mustcause the separation of Cu(II) and hematin. Hematin [Fe(III)-porphyrin complex]
undergoes little complex formation with the iminodiacetic acid moiety. An interesting
separation performance was again observed in the TRDC system with theorganic-solvent-rich and water-rich carrier solutions, as shown in Fig. 3 b and c,
respectively. The TRDC system with the organic-solvent-rich carrier solution showed abetter resolution (Fig. 3 b) than capillary chromatography using the modified capillary
tube and the normal carbonate buffer without organic solvents (Fig. 3a). In contrast, the
TRDC system with the water-rich carrier solution showed no separation on thechromatogram (Fig. 3 c).
Consideration of separation performance
Until now, the TRDC system was operated
with open capillary tubes, such as a fused-silica capillary, polyethylene, andpoly(tetrafluoroethylene) capillary tube, whose inner wall of them were unmodified anduntreated. The two types of modified capillary tubes, phenylboronic-acid- andiminodiacetic-acid-modified, were introduced into the TRDC system with awater-acetonitrile-ethyl acetate mixture carrier solution. In both capillary tubes, the
TRDC system with the organic-solvent-rich carrier solution showed improved
separation performance compared to capillary chromatography using the modified tubes
and carbonate buffer carrier solutions with no organic solvents. In contrast, both
capillary tubes in the TRDC system with the water-rich carrier solution showed no
separation performance despite being modified. As nucleosides are comparativelyhydrophilic, they are dispersed in the water-rich phase. When using theorganic-solvent-rich carrier solution, the minor solvent phase or the water-rich phaseforms near the inner wall of the capillary. Therefore, nucleosides that were dispersed in
the water-rich phase could easily interact with phenylboronic acid moieties, showing
better resolution (Fig. 2 b) than in the chromatogram obtained with the normalcarbonate buffer carrier solution (Fig. 2 a). When using the water-rich carrier solution,
the major solvent phase or the water-rich phase forms around the middle of the tube.
Therefore, nucleosides dispersed in the water-rich phase might exist far from the innerwall, leading to a lower probability of interaction with phenylboronic acid moieties; noseparation was observed on the chromatogram (Fig. 2 c). Similarly, we could explain
the separation performance in the TRDC system using the iminodiacetic-acid-modified
capillary tube. Cu(II) is completely dispersed in the water-rich phase. When using theorganic-solvent-rich carrier solution, the Cu(II) dispersed in the water-rich phase or
minor solvent phase must exist near the inner wall of the capillary, providing better
complex formation with the iminodiacetic acid moieties on the wall, as compared to thechromatography separation mode using a carbonate buffer carrier solution with noorganic solvents. When using the water-rich carrier solution, the Cu(II) in the major

146

Chapter 5146solvent phase or the water-rich phase that formed around the center of the tube
interacted little with the iminodiacetic acid moieties on the inner wall, leading to no
separation from hematin, which had little ability to form complexes with theiminodiacetic acid moieties.
In conclusion, for the first time, we tried to introduce inner-wall-modified(phenylboronic-acid- and iminodiacetic-acid-modified) fused-silica capillary tubes into
the TRDC system with an aqueous-organic solvent carrier solution. We examined theinteraction between the functional moieties on the inner walls and the analytes using themodified capillary tubes, as reflected in the separation performance of the TRDC
system. TRDC with an organic-solvent-rich carrier solution was found to successfully
improve the separation performance, although the system with a water-rich carrier
solution did not show any separation performance under the present conditions. Also,
the findings in the TRDC system using the modified capillary tubes gave us important
information that supported our proposed separation mechanism in the TRDC system.

147

Effects of inner wall characteristics on (TRDC)

147

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesa)b)Figure 1. Schematic diagrams of the TRDC system. a) System 1, equipped withabsorption detection and b) System 2, equipped with CL detection.

148

Chapter 5148

Figure 2. The chromatograms obtained using the phenylboronic-acid-modified capillary
tube. a) Capillary chromatography with the 10 mM carbonate buffer solution (pH 9.5),b) TRDC system with the organic-solvent-rich carrier solution [water (10 mM carbonate
buffer, pH 9.5)-acetonitrile-ethyl acetate; 2:7:4 v/v/v], and c) TRDC system with thewater-rich carrier solution [water (10 mM carbonate buffer, pH 9.5)-acetonitrile-ethyl
acetate; 15:3:2 v/v/v]. Conditions: Capillary tube, 60 cm length (effective length 40 cm)
of 75 µm i.d.; sample injection, 30 cm height (gravity)×15 s; flow rate, 0.13 µL min−1;
detection, 267 nm absorption; and analyte concentration, 19 mM each.

Figure 3. The chromatograms obtained using the iminodiacetic-acid-modified capillary
tube. a) Capillary chromatography with the 10 mM carbonate buffer solution (pH 10.8),b) TRDC system with the organic-solvent-rich carrier solution [water (10 mM carbonate
buffer, pH 10.8)-acetonitrile-ethyl acetate; 2:7:4 v/v/v], and c) TRDC system with thewater-rich carrier solution [water (10 mM carbonate buffer pH, 10.8)-acetonitrile-ethyl
acetate; 15:3:2 v/v/v].	
 Conditions:	
 Capillary tube, 70 cm of 75 µm i.d.; sample injection,
35 cm height (gravity)×5 s; flow rate, 0.33 µL min−1; detection,	
 luminol	
 CL detection;	
 and analyte concentration, 1 mM Cu(II) and 10 µM hematin.
149

Effects of inner wall characteristics on (TRDC)

149

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

5.3 Effects of the inner wall materials of capillary tubes on TRDC
The effects of tube materials on separation performance were examined within the tube
radial distribution chromatography (TRDC) system, by using poly(tetrafluoroethylene)(PTFE; 100-400 µm inner diameter), polyethylene (PE; 200 µm inner diameter), and acopolymer of (tetrafluoroethylene-perfluoroalcoxyethylene) (PTFE-PFAE; 100 µm
inner diameter) capillary tubes. An analyte solution of 2,6-naphthalenedisulfonic acidand 1-naphthol as a model was subjected to the system with a water-acetonitrile-ethyl
acetate carrier solution; a volume ratio of 5:3:2 (water-rich carrier) and a volume ratioof 3:8:4 (organic solvent-rich carrier). The flow rates were set to 0.5 µL min-1 for PTFE
and PTFE-PFAE tubes as well as 2.0 µL min-1 for PE tubes under laminar flow
conditions. The analytes in the solution were separated in this order using the water-richcarrier solution with baseline separation occurring in the three capillary tubes, while
they were eluted in the reverse order or not separated at all with the organic solvent-richcarrier solution. The effects of tube temperature on separation performance were also
examined in the water-rich carrier solution; the best resolutions were observed at a tube
temperature of 0 °C. The obtained results were compared with those for the case of the
fused-silica capillary tube and discussed.
Introduction
Capillary chromatography and CE have received a lot of attention as powerful
separation methods, where fused-silica capillaries are mainly used for separation tubes[19-21]. A fused-silica capillary tube was found to have uniform narrow bore and good
flexibility for micro-analysis. A poly(tetrafluoroethylene) (PTFE) capillary tube thatpossesses similar properties to those of the fused-silica capillary tube is also widelyused not only in analytical chemistry but also in fields including pharmaceuticalchemistry and medicine [22-25], though the nature of the inner wall surface of the PTFE
is different from that of fused-silica. We have developed a novel capillarychromatography system using an open capillary tube and a ternary mixed solvent ofwater-hydrophilic/hydrophobic organic mixture solution. This system works underlaminar flow conditions and does not require any packed reagents in the capillary tube
or the application of high voltage to the ends of the tube. It is called a tube radial
distribution chromatography (TRDC) system. To date, fused-silica capillary tubes havebeen mainly examined in the TRDC system. Here, the effects of polymer tube materials,
such
as
PTFE,
polyethylene
(PE),
and
a
copolymerof(tetrafluoroethylene-perfluoroalcoxyethylene) (PTFE-PFAE), on the separation
performance of the TRDC system were examined.
Experimental
PTFE capillary tubes (100 µm i.d., 200 µm o.d.; 150 µm i.d., 300 µm o.d.; 200 µm i.d.,
350 µm o.d.; and 250 µm i.d., 400 µm o.d.), PE (200 µm i.d., 500 µm o.d.) capillarytubes, and PTFE-PFAE (100 µm i.d., 300 µm o.d.) capillary tubes were used. The
present capillary chromatography system was comprised of an open capillary tube,
micro-syringe pump, and absorption detector. A capillary tube, 110 cm in length

150

Chapter 5150

(effective length: 90 cm), was also placed in the system. The tube temperature wascontrolled by immersing the capillary tube in water maintained at a specific temperature
in a beaker by stirring. Water-acetonitrile-ethyl acetate mixtures with volume ratios of
15:3:2 and 3:8:4 were used as water-rich and organic solvent-rich carrier solutions.
Analyte solutions were prepared with the carrier solutions. The analyte solution was
introduced directly into the capillary inlet side for 10 s from a height of 25 cm for thecapillary tube. After analyte injection, the capillary inlet was connected through a joint
to a micro-syringe which was set on the micro-syringe pump. The carrier solution was
fed into the capillary tube at a flow rate of 0.5 µL min-1 for PTFE and PTFE-PFAE, as
well as at 2.0 µL min-1 for PE. On-capillary absorption detection (254 nm) was
performed by the detector.
Results and discussion
Effects of tube inner diameter on separation We examined the effects of the tube
inner diameter on separation in the TRDC system with a mixture analyte solution of
2,6-naphthalenedisulfuric acid and 1-naphthol as a model. We used commerciallyavailable PTFE capillary tubes with various inner diameters of 100–250 µm i.d. The
obtained chromatograms are shown in Fig. 1 together with analytical conditions. The
flow rates for all the capillary tubes were adjusted to provide almost the same average
linear velocity of ca. 6.4 cm min-1. We observed well-separated peaks on thechromatograms; 2,6-naphthalenedisulfuric acid and 1-naphthol were separated in thisorder with the water-rich carrier solution. The first peaks were eluted with an almost
average linear velocity and the second peaks were eluted at a slower than average linear
velocity. Also, the elution times of the second peaks appeared earlier with increasing
inner diameter, although the elution times of the first peaks were nearly constant.
Additionally, the second peak of 1-naphthol showed broadening on the chromatograms.
The outer phase (the organic solvent-rich solution) worked as a pseudo-stationary phasethat had a larger diffusion coefficient in the TRDC system than that in a usual stationary
phase in liquid chromatography. Hydrophobic 1-naphthol distributed in a specificstationary phase, which lead to the peak broadening.
Effects of tube temperature on separation using polymer capillary tubes The effectsof tube temperature on separation in the TRDC system using PTFE, PE, andPTFE-PFAE capillary tubes were examined with the water-rich and the organic
solvent-rich carrier solutions. First, the chromatograms obtained using PTFE are shownin Fig. 2 together with those obtained using other capillary tubes. As can be seen in Fig.
2, with the water-rich carrier solution, 2,6-naphthalenedisulfuric acid and	
 1-naphthol
were detected in the mixture solution with baseline separation in the temperature range
of 0–30 °C, but the resolutions decreased gradually between 30 °C and 45 °C andfinally they were not separated on the chromatogram at 50 °C (Fig. 2 a)). On the other
hand, with the organic solvent-rich carrier solution they were eluted in reverse order,
but not separated with baseline separation at 0 and 10 °C (Fig. 2 d)).	
 The data clearly
indicated that the tube temperature had a crucially significant influence on separation
performance in the TRDC system. The formation of the inner and outer phases in a tube
based on TRDP must change with temperature. Also, an analyte solution of151

Effects of inner wall characteristics on (TRDC)

151

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

2,6-naphthalenedisulfuric acid and 1-naphthol was examined with the TRDC systemusing PE and PTFE-PFAE with the water-rich and organic solvent-rich carrier solutions
at the tube temperature of 0 °C. They were eluted with baseline separation in this orderwith the water-rich carrier solution in both of the tubes, while, with the organic
solvent-rich carrier solution, they were eluted in the reverse order without baseline
separation in PE and they were not separated in PTFE-PFAE. We examined the effectsof tube temperature on separation in the TRDC system using PE and PTFE-PFAE
capillary tubes with water-rich carrier solutions. The obtained chromatograms are also
shown in Fig. 2 along with the analytical conditions. As can be seen in the figure,through PE capillary tubes 2,6-naphthalenedisulfuric acid and 1-naphthol in the mixture
solution were separated and detected within the temperature range of 0–10 °C, but theresolutions decreased gradually between 15 °C and 25 °C and finally they were notseparated on the chromatogram at 30 °C (Fig. 2 b)). In a similar way
2,6-naphthalenedisulfuric acid and 1-naphthol in the mixture solution were eluted withthe water-rich carrier solution through PTFE-PFAE capillary tube (Fig. 2 c))
Separation of mixture solution including three analytes
We examined a mixture
analyte solution of 2,6-naphthalenedisulfonic acid, Eosin Y, and 1-naphthol using thepresent TRDC system with a water-rich carrier solution. The obtained chromatograms
are shown in Fig. 3. 2,6-Naphthalenedisulfonic acid, Eosin Y, and 1-naphthol were
eluted in this order, leading to good separation with the water-rich carrier solution. The
elution order seemed to be consistent with the substances’ hydrophilic characters. With
the water-rich carrier solution, the hydrophilic compounds 2,6-naphthalenedisulfonicacid were eluted with almost average linear velocity, while the relatively hydrophobic
compounds Eosin Y and 1-naphthol were eluted in this order at a lower than average
linear velocity..
Consideration of the influence of the tube materials on separation Since separationin the TRDC system is performed based on the tube radial distribution of the carrier
solvents as described in the introduction, 2,6-naphthalendisulfonic acid and 1-naphthol
were separated in this order with the water-rich carrier solution, while they were eluted
in reverse order with the organic solvent-rich carrier solution under the present
analytical conditions using polymer capillary tubes, such as PTFE, PE, and PTFE-PFAE,
except for when using PTFE-PFAE with the organic solvent-rich	 carrier solution. The
same elution behavior was observed in the TRDC system using the fused-silica capillary
tube as shown in Fig. 4. However, there was a difference in the separation behavior
between the polymer capillary tubes and fused-silica capillary tubes. First, in polymer
capillary tubes resolutions obtained with the water-rich carrier solution were muchbetter than those with the organic solvent-rich carrier solution. However, as waspreviously reported in fused-silica capillary tubes,10) the resolutions obtained with theorganic solvent-rich carrier solution were better than those of the water-rich carrier
solutions. The interaction between the outer phase of the organic solvent-rich solution
and the polymer surface as well as the outer phase of the water-rich solution and silanol
surface might give prior conditions for creating the inner and outer phases in thecapillary tube, while, the interaction between the outer phase of the water-rich solution
and polymer surface as well as the outer phase of the organic solvent-rich solution and152

Chapter 5152silanol surface might create inconvenient conditions for the generation of the inner andouter phases in the capillary tube. Secondly, in polymer tube materials, the resolutions
decreased with the increase of the tube temperature, while, in fused-silica tubes, theresolutions increased with increasing of the tube temperature until up to 20 °C (Fig. 2e)).10) The tendency concerning the effect of tube temperature, tube materials, and thecomponent ratio of the carrier solvents on separation performance is interesting, whereenergy or enthalpy for separation must be complexly influenced by various types of heat,such as dissolution, adsorption, desorption and solvation at the liquid-liquid interface.
In conclusion, the polymer tube materials, such as PTFE, PE, and PTFE-PFAE, were
examined in the TRDC system and the obtained results were discussed together withthose concerning the fused-silica capillary tube that we reported previously. Although
there is a difference in the nature of the wall`s inner-surface between polymer andfused-silica capillary tube, the fundamental elution behavior in the TRDC system is thesame in all capillary tubes. However, the resolutions provided by the water-rich carrier
are better than organic solvent-rich carrier in polymer capillary tubes, conversely, theresolutions provided bye the organic solvent-rich carrier solution are better than thosewater-rich carrier solution in fused-silica capillary tubes.

153

Effects of inner wall characteristics on (TRDC)

153

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Effects of the inner diameter of the tube on separation in the TRDC systemusing PTFE capillary tube. Conditions: Capillary tube, 110 cm (effective length: 90 cm)
PTFE of 100, 150, 200, and 250 µm i.d.; carrier, water-acetonitrile-ethyl acetate (15:3:2
v/v/v) mixture solution; sample injection, 25 cm height (gravity)×10 s; flow rate,
0.5–3.0 µL min-1; tube temperature, 0 °C; and 2,6-naphthalenedisulfonic acid and1-naphthol, 1 mM.

154

Chapter 5154

Figure 2. Effects of tube temperature on separation in the TRDC system using various
types of capillary tube. Conditions for a) and d): Capillary tube, 110 cm (effective
length of 90 cm, the part of it (ca. 60 cm) was dipped in the temperature-controlledwater) of 100 µm i.d. PTFE; carrier, water-acetonitrile-ethyl acetate (15:3:2 v/v/v)
mixture solution; sample injection, 25 cm height (gravity)×10 s; flow rate, 0.5 µL min-1;
and tube temperature, 0–50 °C. Conditions for b) and c): Capillary tube, 110 cm(effective length of 90 cm, the part of it (ca. 60 cm) was dipped in thetemperature-controlled water) of 200 µm i.d. PE and 100 µm i.d. PTFE-PFAE; carrier,water-acetonitrile-ethyl acetate (15:3:2 v/v/v) mixture solution; sample injection, 20 cm
height (gravity)×10 s; flow rate, 2.0 µL min-1 for PE and 0.5 µL min-1 for PTFE-PFAE;
and tube temperature, 0–30 or 50 °C. Conditions for e): Capillary tube, 120 cm(effective length of 100 cm, the part of it (ca. 80 cm) was dipped in thetemperature-controlled water) of 75 µm i.d. fused-silica; carrier, water-acetonitrile-ethyl
acetate (3:8:4 v/v/v) mixture solution; sample injection, 20 cm height (gravity)×30 s;
flow rate, 0.8 µL min-1; and tube temperature, 5–25 °C. Analyte concentrations for a) –e): 2,6-Naphthalenedisulfonic acid and 1-naphthol, 1 mM each.

Figure 3. Chromatograms of the mixture analyte solution of 2,6-naphthalenedisulfonicacid, Eosin Y, and 1-naphthol obtained by the TRDC system. Conditions: Capillary tube,
110 cm (effective length: 90 cm) of 100 µm i.d. PTFE, 200 µm i.d. PE, and 100 µm i.d.
PTFE-PFAE; carrier, water-acetonitrile-ethyl acetate (15:3:2 v/v/v) mixture solution;
sample injection, 20 cm height (gravity)×10 s; flow rate, 0.5 µL min-1 for PTFE and
PTFE-PFAE, and 2.0 µL min-1 for PE; temperature, 0 °C; and 1-naphthol,
2,6-naphthalenedisulfonic acid, 1 mM, and Eosin Y, 3 mM.

155

Effects of inner wall characteristics on (TRDC)

155

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Chromatograms of the mixture analyte solution of 2,6-naphthalenedisulfonicacid and 1-naphthol obtained by the TRDC system using a fused-silica capillary tube.
Conditions: Capillary tube, 120 cm (effective length: 90 cm) of 75 µm i.d. fused-silica;
carrier, water-acetonitrile-ethyl acetate (15:3:2 and 3:8:4 v/v/v) mixture solution;
sample injection, 20 cm height (gravity)×30 s; flow rate, 0.8 µL min-1; temperature,
20 °C; and 1-naphthol and 2,6-naphthalenedisulfonic acid, 1 mM.

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2006, 1125, 144.

157

Effects of inner wall characteristics on (TRDC)

157

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Chapter 6 Detection methods in tube radial distribution
chromatography (TRDC)
In order to expand a range of detectable analytes, fluorescence and chemiluminescence
detection was introduced into the TRDC systems in addition to absorption detection.
The part of this chapter is reconstructed and rewritten based on a related, publishedmanuscript.11)

6.1 Introduction of fluorescence and chemiluminescence detection to
TRDC
Chapter 6

Fluorescence and chemiluminescence detection were introduced into a tube radial
distribution chromatography (TRDC) system using an open fused-silica capillary tube
and a water-acetonitrile-ethyl acetate mixture carrier solution. Model analyte mixture
solutions, such as Eosin Y and perylene as well as dansyl methionine and perylene,
were injected into the capillary tube through the utilization of a gravity method. The
analyte solution was subsequently delivered through the capillary tube with the carrier
solution by a microsyringe pump; the system worked under laminar flow conditions.
The analytes were separated through the tube and detected by on-capillary withfluorescence detection or by end-capillary with chemiluminescence detection by taking
advantage of a peroxyoxalate chemiluminescence reaction. Eosin Y and perylene aswell as dansyl methionine and perylene were detected in this order with a carrier
solution of water-acetonitrile-ethyl acetate (15:3:2 volume ratio), while they weredetected in the reverse order with a carrier solution of water-acetonitrile-ethyl acetate(3:8:4 or 2:7:4 volume ratio) with fluorescence or chemiluminescence detection. The
elution times of the analytes were reversed by changing the component ratio of thesolvents in the carrier solution. A fluorescein isothiocyanate-labeled bovine serum
albumin was also analyzed and separated from the coexisting labeling reagent using thepresent system.
Introduction
Capillary chromatography and related separation technology, including capillaryelectrochromatography [1,2], capillary electrophoresis [3,4], capillary liquid
chromatography [5-7], and hydrodynamic chromatography [8], are among the mostactive areas of research in chemical engineering, analytical technology, and separation
science. A novel capillary chromatography method using an open capillary tube and theuse of a water-hydrophilic-hydrophobic organic mixture as a carrier solution wasdeveloped. This is called a tube radial distribution chromatography (TRDC) system,
which is based on the tube radial distribution of carrier solvents in the capillary tube
under laminar flow conditions.
Fluorescence and chemiluminescence detection, as well as absorption detection, havebeen widely used in many flow analyses, including flow-injection analysis,
high-performance liquid chromatography, and capillary electrophoresis, in order to158

Chapter 6

158

improve the analytical performance with regards to sensitivity and analytical subjects[9-13]. Here, we have attempted to introduce fluorescence and chemiluminescence
detection into the TRDC system.
Experimental
Fused-silica capillary tubes (50 or 75 µm i.d., 150 µm o.d.) were used. Schematic
diagrams of the TRDC systems comprised of an open capillary tube and a microsyringe
pump equipped with a fluorescence detector and a chemiluminescence detector, as
shown in Fig. 1 a) and b), respectively. Fused-silica capillary tubes, 80 cm in length(effective length: 60 cm) and 75 µm i.d. for fluorescence detection, and 90 cm in lengthand 50 µm i.d. for chemiluminescence detection, were installed in the systems. Mixturesof water-acetonitrile-ethyl acetate at different volume ratios (15:3:2, 2:7:4, and 3:8:4)
were used as the carrier solution. Analyte mixture solutions, Eosin Y and perylene
mixture as well as dansyl methionine and perylene mixture, were prepared along withthe carrier solutions. The analyte solution was introduced directly into the capillary inlet
for 10 s from a height of 25 cm for fluorescence detection, or for 10 s from a height of
30 cm for chemiluminescence detection by the gravity method. After analyte injection,
the capillary inlet was connected through a joint to a microsyringe which was set on the
microsyringe pump. The carrier solution was fed into the capillary tube at a flow rate of
0.2 µL min-1 for fluorescence and chemiluminescence detection under laminar flow
conditions. On-capillary fluorescence detection was carried out for Eosin Y andperylene mixture with ex. 430 nm and em. 520 nm as well as with ex. 360 nm and em.
515 nm for dansyl methionine and perylene mixture. End-capillary chemiluminescence
detection was performed through making use of peroxyoxalate chemiluminescence
reactions as follows. A flow-type chemiluminescence detection cell (0.5 mm i.d.
poly(tetrafluoroethylene) tube) was used (Fig. 1 b)). The chemiluminescence reagent
consistingofacetonitrile
solution(2mM
bis[2-(3,6,9-trioxadecayloxycarbonyl)-4-nitrophenyl] oxalate (TDPO) and 200 mMhydrogen peroxide) was delivered at the flow rate of 20 µL min-1 to the capillary outlet
in the detection cell where the chemiluminescence reagent and the analytes were mixed
to induce chemiluminescence.
Results and discussion
Fluorescence detection The analyte mixture solution of Eosin Y and perylene was
examined with the present TRDC system which was outfitted with a fluorescence
detector using an open fused-silica capillary and water-acetonitrile-ethyl acetate mixture
carrier solution. Eosin Y and perylene were eluted through the capillary tube in thisorder with the water-acetonitrile-ethyl acetate carrier solution (15:3:2 volume ratio). In
addition, they were eluted in the reverse order with the water-acetonitrile-ethyl acetatecarrier solution (2:7:4 volume ratio). The chromatograms obtained with the water-richand organic solvent-rich carrier solutions are shown in Fig. 2 a) and b), respectively.
The analyte mixture solution of dansyl methionine and perylene was analyzed in asimilar way using the system equipped with a fluorescence detector. Dansyl methionine
and perylene were separated and detected in this order with the water-rich carrier

159

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

solution and they were detected in the reverse order with the organic solvent-rich carrier
solution. The chromatograms thus obtained are shown in Fig. 3 a) and b), respectively.
Chemiluminescence detection The peroxyoxalate chemiluminescence reaction [14] is
one of the most important reactions used in various flow systems. Oxalate reagents,
such as bis(2,4,6-trichlorophenyl) oxalate (TCPO) and TDPO, react with hydrogen
peroxide to give a dioxetane compound as an immediate, which interacts withfluorescent compounds. Energy transfer occurs from the dioxetane compound to thefluorescent compound through a complex formation, and the excited fluorescentcompound drops to the ground state, emitting chemiluminescence. These fluorescentcompounds can be detected with a high level of sensitivity through chemiluminescenceby taking advantage of the peroxyoxalate chemiluminescence reaction. The analyte
mixture solution of Eosin Y and perylene was subjected to the present TRDC systemequipped with a chemiluminescence detector. They were eluted with thewater-acetonitrile-ethyl acetate carrier solution (15:3:2 volume ratio) in this order and in
the reverse order with the water-acetonitrile-ethyl acetate carrier solution (3:8:4 volume
ratio). The chromatograms obtained with the water-rich carrier solution and the organic
solvent-rich carrier solution are shown in Figs. 4 a) and b), respectively. The analyte
mixture of dansyl methionine and perylene was examined in a similar way using thissystem with the chemiluminescence detector. The chromatograms obtained with thewater-rich and organic solvent-rich carrier solutions are shown in Fig. 5 a) and b),
respectively. Dansyl methionine and perylene were eluted in this order with thewater-rich carrier solution and in the reverse order with the organic solvent-rich carrier
solution. The detection limits of the fluorescent compounds for fluorescence detection
were 10–100 µM order (S/N=3), and those for chemiluminescence detection were 1–10
µM order (S/N=3), respectively. Chemiluminescence detection provided greater
detection sensitivity than fluorescence detection under the present analytical conditions.
The data obtained here through fluorescence and chemiluminescence detection
suggested that the TRDC system could be applied successfully to the separation anddetermination of fluorescence-labeled compounds, such as amino acids, peptides,
proteins, saccharides, and nucleic acids.
Consideration of the elution order Various mixtures consisting of hydrophilic andhydrophobic molecules as analytes have been examined using the TRDC system withfused-silica, polyethylene, or poly(tetrafluoroethylene) capillary tubes and awater-acetonitrile-ethyl acetate mixture carrier solution. The separation performance in
the TRDC system is explained as follows. 1) Water and organic solvents in the carrier
solution are not dispersed uniformly in the capillary tube based on the tube radial
distribution of the solvents under laminar flow conditions. A major solvent phase(water-rich or organic solvent-rich) forms around the middle of the tube far from theinner wall, while a minor solvent phase (organic solvent-rich or water-rich) is generated
near the inner wall of the capillary tube as a pseudo-stationary phase. 2) Hydrophilicmolecules in the analyte mixture are subsequently dispersed in the water-rich phase, andhydrophobic molecules are dissolved in the organic solvent-rich phase. 3) The analyte
dispersed in the major solvent phase around the middle of the capillary tube is eluted

160

Chapter 6160

with near average linear velocity, while the analyte dispersed in the minor solvent phasenear the inner wall of the tube is eluted with smaller than average linear velocity; theseparation is chromatographically performed through distribution between inner andouter phases. The elution times of the analytes can be easily reversed by changing thecomponent ratio of the solvents in the carrier solution. Eosin Y and dansyl methionine
are comparatively hydrophilic compounds and perylene is a hydrophobic compound.
The elution orders observed on the chromatograms in Fig. 2–5 were consistent withseparation performance based on the tube radial distribution of the carrier solvents in
the TRDC system. For example, Eosin Y (hydrophilic) and perylene (hydrophobic)
were eluted in this order with the water-rich carrier solution (Fig. 2 a) and Fig. 4 a)) andin the reverse order with the organic solvent-rich carrier solution (Fig. 2 b) and Fig. 4b)).
Analysis of fluorescence labeled protein
Bovine serum albumin as a model protein
was labeled with fluorescein isothiocyanate (FITC) which is one of the most usefullabeling reagents for biomolecules. Bovine serum albumin (0.5 µmol) and FITC (1.0µmol) were dissolved in a solution of water-triethylamine (95:5 volume ratio) (100 µL).
The mixture was stirred for 1 min and left in the dark for 20 min. After the evaporationof the solvent, the residue was redissolved in the carrier solution (1 mL) to give theanalyte solution including an excess (or free)-FITC and FITC-labeled bovine serum
albumin (BSA). The mixture of FITC and FITC-labeled BSA was subjected to the
TRDC with fluorescence detection. FITC and FITC-labeled BSA were separated anddetected in this order with the organic solvent-rich carrier solution, while they weredetected in the reverse order with the water-rich carrier solution. Clearly, the elution
times of the analytes were changed by altering the component ratios of the carrier
solvents. The elution orders obtained from the mixture of FITC and FITC-labeled BSA
were reasonable considering the free-FITC more hydrophobic than the FITC-labeledprotein. The chromatograms obtained for the mixture of FITC and FITC-labeled BSA as
well as the mixture of FITC, FITC-labeled BSA, and perylene that was added to thesolution after the labeling procedure are shown in Fig. 6, together with the analyticalconditions in the captions. As shown in Fig. 6 b), perylene, FITC, and FITC-labeled
BSA were separated and detected in this order with the organic solvent-rich carrier
solution. The first peak of perylene on the chromatograms roughly appeared with theaverage linear velocity and the others, FITC and FITC-labeled BSA peak, were eluted
with lower velocity under laminar flow conditions. The average linear velocity wasconfirmed by the experiment with a normal aqueous carrier solution not including any
organic solvents. The elution order of perylene, FITC, and FITC-labeled BSA on thechromatogram was consistent with the nature of hydrophilicity or hydrophobicity ofthese analytes including the labeled protein. Separation was performed using an
untreated open fused silica capillary tube and a water-acetonitrile-ethyl acetate mixture
carrier solution without any additives, such as gels, or applying a high voltage. To date,
the TRDC has mostly been applied to the analysis of organic compounds with low
molecular weights. The results obtained for biopolymer analysis here provide insight toexpand the TRDC system to future research.

161

Detection methods in TRDC

161

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

In conclusion, we attempted to introduce fluorescence and chemiluminescence
detection into the TRDC system to extend its analytical performance with regards toboth analyte subjects and sensitivity. The model analyte solutions of Eosin Y andperylene mixture as well as dansyl methionine and perylene mixture were separated anddetected with fluorescence or chemiluminescence detection in the TRDC system.
Chemiluminescence detection was about 10–100 times more sensitive than fluorescence
detection in the present TRDC system. The elution times of the analytes of the mixture
solution could be easily changed by altering the component ratio of the carrier solvents.
The data obtained here suggested that the present TRDC system would be useful for the
separation and determination of fluorescence-labeled compounds.

162

Chapter 6

162

a)

b)

Figure 1. Schematic diagrams of the present TRDC systems with a) fluorescence
detection and b) chemiluminescence detection.

163

Detection methods in TRDC

163

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2. Chromatograms of a mixture of Eosin Y and perylene by the present TRDC
system with fluorescence detection. Conditions: Capillary tube, 80 cm (effective length:
60 cm) of 75 m i.d. fused-silica; carrier, a) water-acetonitrile-ethyl acetate (15:3:2
volume ratio) mixture solution and b) water-acetonitrile-ethyl acetate (2:7:4 volume
ratio) mixture solution; sample injection, 25 cm height (gravity)×10 s; flow rate, 0.2 L
min-1; analyte concentration, 1 mM each.

Figure 3. Chromatograms of a mixture of dansyl methionine and perylene by the present
TRDC system with fluorescence detection. Conditions: Capillary tube, 80 cm (effective
length: 60 cm) of 75 m i.d. fused-silica; carrier, a) water-acetonitrile-ethyl acetate(15:3:2 volume ratio) mixture solution and b) water-acetonitrile-ethyl acetate (2:7:4
volume ratio) mixture solution; sample injection, 25 cm height (gravity)×10 s; flow rate,
0.2 L min-1; analyte concentration, 1 mM each.

164

Chapter 6164

Figure 4. Chromatograms of a mixture of Eosin Y and perylene by the present TRDC
system with chemiluminescence detection. Conditions: Capillary tube, 90 cm of 50 mi.d. fused-silica; carrier, a) water-acetonitrile-ethyl acetate (15:3:2 volume ratio) mixture
solution and b) water-acetonitrile-ethyl acetate (3:8:4 volume ratio) mixture solution;
sample injection, 30 cm height (gravity)×10 s; flow rate, 0.2 L min-1; analyte
concentration, 50 M each.

165

Detection methods in TRDC

165

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 5. Chromatograms of a mixture of dansyl methionine and perylene by the present
TRDC system with chemiluminescence detection. Conditions: Capillary tube, 90 cm of
50 m i.d. fused-silica; carrier, a) water-acetonitrile-ethyl acetate (15:3:2 volume ratio)
mixture solution and b) water-acetonitrile-ethyl acetate (3:8:4 volume ratio) mixture
solution; sample injection, 30 cm height (gravity)×10 s; flow rate, 0.2 L min-1; analyte
concentration, 50 M each.

Figure 6. Chromatograms of a) a mixture of FITC and FITC-labeled BSA as well as b) a
mixture of perylene, FITC, and FITC-labeled BSA by the present TRDC system withfluorescence detection. Conditions: Capillary tube, 120 cm (effective length: 100 cm) of
50 m i.d. fused-silica; carrier, water-acetonitrile-ethyl acetate (3:8:4 volume ratio)
mixture solution; sample injection, 30 cm height (gravity)×30 s; flow rate, 0.2 L min-1;
fluorescence detection, ex. 495 nm and em. 520 nm; analyte concentration, 1 mM
perylene and 0.5 mM FITC-labeled BSA.

References[1] H. J. Issaq, K. C. Chan, J. Blonder, X. Ye, and T. D. Veenstra, J. Chromatogr. A,
2009, 1216, 1825.
[2] A. C. Otieno, and S. M. Mwongela, Anal. Chim. Acta, 2008, 624, 163.
[3] K. Tsukagoshi, N. Jinno, and R. Nakajima, Anal. Chem., 2005, 77, 1684.

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[4] M. C. Breadmore, J. R. E. Thabano, M. Dawod, A. A. Kazarian, J. P. Quirino, and R.
M. Guijt, Electrophoresis, 2009, 30, 230.
[5] M. C. Jung, N. Munro, G. Shi, A. C. Michael, and S. G. Weber, Anal. Chem., 2006,
78, 1761.
[6] J. Urban and P. Jandera, J. Sep. Sci., 2008, 31, 2521.
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[8] T. Okada, M. Harada, and T. Kido, Anal. Chem., 2005, 77, 6041.
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[10] K. Tsukagoshi, M. Tahira, and R. Nakajima, Anal. Sci., 2003, 19, 1019.
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[14] “Chem- and Bioluminescence”, ed. J. G. Burr, 1985, Chap. 5, Marcel Dekker, Inc.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Chapter 7 Development of microchip-tube radial distribution
chromatography (microchip-TRDC)
We tried to apply the TRDC to a microchip device. The TRDC system was well matchedto the concept of micro-total analysis system (µ-TAS) or lab on a microchip. The
microchip-TRDC with chemiluminescence detection was developed. The part of thischapter is reconstructed and rewritten based on related, published manuscripts.36,40)

7.1 Michrochip-TRDC with chemiluminscence detection

Chapter 7

A capillary chromatography system has been developed using a ternary mixed solvents
solution, i.e., water-hydrophilic/hydrophobic organic solvent mixture as a carrier
solution. Here, we tried to carry out the chromatographic system on a microchip
incorporating the open-tubular microchannels. A model analyte solution of isoluminol
isothiocyanate (ILITC) and ILITC-labeled biomolecule was injected to the double
T-junction part on the microchip. The analyte solution was delivered in the separation
microchannel (40 µm deep x 100 µm wide x 22 cm long) with the ternarywater-acetonitrile-ethyl acetate mixture carrier solution (3:8:4 volume ratio; the organic
solvent-rich or 15:3:2 volume ratio； the water-rich). The analyte, free-ILITC andlabeled bovine serum albumin (BSA) mixture, was separated through the microchannel,
where the carrier solvents were radially distributed in the separation channel generating
inner and outer phases. The outer phase acts as a pseudo-stationary phase under laminarflow conditions in the system. The ILITC and the labeled BSA were eluted and detected
through a chemiluminescence reaction.
Introduction
Our group reported the tube radial distribution phenomenon (TRDP) of carrier solvents
under microfluidic flow conditions in 2009. When the ternary mixed solvents ofwater-hydrophilic/hydrophobic organic solvent mixture are delivered into a microspace,such as a microchannel or a capillary tube, the solvent molecules are radially distributedin the microspace generating inner and outer phases. A capillary chromatographysystem based on the TRDP in which the outer phase acts as a pseudo-stationary phase
under laminar flow conditions has been developed. We call the separation method tube
radial distribution chromatography (TRDC). All of TRDC systems have been performedby using various types of capillary tubes. Miniaturization on a microchip incorporating
the microchannels has not been applied to the TRDC system.
Microchip devices are among the most active areas of research in chemicalengineering, analytical	
 technology, and separation science. Such research areas were
well known as micro-total analysis systems (µ-TAS) or Lab on a Chip	
 [1]. As one ofthese, microchip-electrophoresis is widely investigated as a small and rapid separation
method. However, the microchip-electrophoresis necessarily needs a voltage supplierdevice and electrodes. Also, capillary chromatography as a microfluidic analysis
requires specific separation columns, such as monolithic or packed channels or tubes.

168

Chapter 7

168

Although they might improve the separation performance, such as resolutions, they aretime-consuming and boring for the preparation and cost the users a great deal.
Here we tried to carry out the TRDC system on a microchip, i.e., microchip-TRDC
system. Furthermore, detection on the microchip was performed withchemiluminescence (CL) reactions. The ILITC and ILITC-labeled biomolecule
mixtures were used as model analytes, referring to the previous paper.25) Biomolecule
analysis, such as protein and enzyme are interesting and useful in bioscience, however it
has some problems in µ-TAS such as adsorption onto a microspace wall. The
microchip-TRDC system with CL detection will bring out new research area in the
TRDP and TRDC.
Experimental
systemwith
withdetection
A microchip made of glass was
Microchip-TRDC system
CLCL
detection
manufactured
Technology(Kanagawa,Japan). Fig. 1 illustrates
theA
microchip with
madeMicrochemicalof glass was
manufactured
with Microchemical
Technology(Kanagawa,Japan).
Fig. 1 illustrates
the microchip
and microchip-holder
usedherein.
microchip andmicrochip-holder
used herein.
The microchannels
were 100 m
wide
and
The
microchannels
100 µm wide line
and 40µmm
deep,
the microchannel
40 mdeep, exceptwere
the microchannel(400
wideexcept
and 100m deep) for line
thehydrogensolution.
The deep)
microchip-TRDC
system comprised
microchip,(400µm peroxidewide and100 µmfor the hydrogen
peroxide asolution.
Thea
microchip-TRDC
systema microchip,a microsyringe pumps,
andmixturea CL
microsyringe pumps,
and compriseda CL detector.
A water-acetonitrile-ethyl
acetatedetector.
A water-acetonitrile-ethyl
acetate mixture(3:8:4
ratio; theratio;organic(3:8:4 volume
ratio; the organic solvent-richsolutionor volume
15:3:2 volume
thesolvent-richsolution was
or 15:3:2
volumeratio; thewater-richsolution) was usedas thewater-rich solution)used asthe carrier
solutionfor chromatography,wherecarrier
for chromatography,
where the bufferwater component
was including
10 mM carbonate
water solution
component
was 10 mM carbonate
at pH 11.8
4 M
microperoxidase.
buffer
at pH 11.8 including 4 µM microperoxidase.
procedure Bio-macromolecules, such as BSA (Mw; 66000) and alcoholLabeling procedure
Bio-macromolecules,
such (ADH)
as BSA (Mw;
(Mw; 148000),
66000) andalcoholdehydrogenase
from
yeast
dehydrogenase from yeast
werelabeledwith ILITC as
follows:(ADH)(Mw; 148000),
were
as werefollows:
biomolecules(0.5 µmol)
dissolved
in a solutionofbiomolecules(0.5 mol)andlabeled
ILITCwith(1.0ILITC
mol)and
ILITC (1.0 µmol)
werevolume
dissolvedin a(100solutionwater-triethylamine(95:5
water-triethylamine(95:5
ratio)
L). ofThemixture was stirred
forvolume
1 min
and leftin µL).
the Thedarkmixture
for 20 was
min.stirred
Afterforevaporation
solvent,
the20residue
wasratio)(100
1 min and ofleftthein thedark formin. Afterevaporationof thethe residue
wasredissolved
the carrier
solution(1000
redissolved into
thesolvent,carrier solution
(1000
L)
to produceinto
an analyte
solution,includingexcess(or free)-ILITC
andsolution,
ILITC-labeledbiomolecules.
µL)
to produce
an analyte
includingexcess (or free)-ILITC and ILITC-labeledbiomolecules.

ILITC(Isoluminolisothiocyanate)

Biomolecule

ILITC-labeled biomolecule

Hydrophobic-modified microchannel
microchannel
preparation
First, 1.0 vol% trichloro
Hydrophobic-modified
preparation(octadecyl)silane toluene
was fedsilane
into themicrochannel
pointFirst,
1.0 vol%trichlorosolution(octadecyl)
toluenesolutionfrom
wasthefed
intoC atthea-1-1
flow rate of 10fromL minfor 10
Next,rate
toluenea flow
ratetolueneof 100
microchannel
the point
C min.
at a flowof 10solutionµL minwasforfed10atmin.
Next,
-1
-1
L min wasfor 100min.
Then,chloroform
was
fedmin.
at aThen,flow rateof 100 L
min-1100
chloroform
solutionsolution
fed ata flowrateof 100 µLsolution
min forfor 10 min. Finally, the microchannel was heated by thermo-controller at 150 °C for 30
min.
Fluorescence microscope-CCD camera system
The microchip was set up so that the169
fluorescence microscope-CCD camera system could confirm the tube radial Microchip-TRDC
distributionof the carrier solvents, i.e., TRDP. The carrier solution, water-acetonitrile-ethyl acetate
mixture including perylene (em. 470 nm; blue) and Eosin Y (em. 550 nm; green), was169Hydrophobic-modified
preparation
First,Molecules
1.0 vol% trichloro
Tube Radialmicrochannel
Distribution Phenomenon(TRDP) of Solvent(octadecyl) silane toluene solution was fed into the microchannel from the point C at aflow rate of 10 L min-1 for 10 min. Next, toluene solution was fed at a flow rate of 100
L min-1 for 100 min. Then, chloroform solution was fed at a flow rate of 100 L min-1
was
fedmin.
at a Finally,
flow ratetheofmicrochannel
100 µL min-1 wasfor 10min. byFinally,
the microchannel
for 10heatedthermo-controller
at 150was°C heatedfor 30bythermo-controller at 150 °C for 30 min.
min.
Fluorescence microscope-CCD camera
camera systemsystem
The microchip was set up so that theThemicrochip
was set up so camerathat thesystem
fluorescencemicroscope-CCD
camerasystemfluorescencemicroscope-CCD
could confirm
the tube radial
distribution
could
the tube i.e.,radialdistributionof thesolution,
carrier solvents,
i.e., TRDP. The acetatecarrierof the confirm
carrier solvents,
TRDP.
The carrierwater-acetonitrile-ethyl
solution,water-acetonitrile-ethyl
acetateincludingperylene(em.
470
nm; blue)
mixture including
perylene (em. 470
nm;mixtureblue) and
Eosin Y(em. 550
nm;
green),
wasandEosin into
Y (em.
nm; green),
was delivered
into way
the separation
channel inprocedurethe samedeliveredthe550separation
channelin the sameas the preparationway
as the preparation
procedure mentionedThe
at thewas
fourmonitored
points inmentioned
blow. The fluorescenceat the fourblow.
pointsin fluorescencethe microchannel
themicrochannel
was monitored
a fluorescencewitha anHgwith an microscope
Hg lamp, aequipped
filter, and
CCD
using
a fluorescencemicroscopeusing
equippedlamp,
a filter, and a CCD camera.
camera.
Analytical procedure
procedure inin thetheTRDC
TRDC
The analytical procedure was carried out by theTheanalyticalcarried out
the following
steps; 1) The
preparation,
2)
following
threeprocedure
steps; 1) was
preparation,2) bysampleload, andthree3) analysis.
schematic
sampleload,
and3)
analysis.
The
schematic
illustration
is
shown
in
Fig.
2.
The
analyteillustration is shown in Fig. 2. The analyte (sample) and carrier solutions were(sample)solutions werefrom
delivered
the C
microchannels
from pointspumps,
S anddeliveredand
intocarrier
the microchannels
pointsinto
S andwith the microsyringeCwith A
theandmicrosyringe
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A and B, therespectively.
In 1) frompreparation,
microchannel
point Sthetopumps
B, respectively.
1) preparation,
microchannel
from waspointfirstS tofilled
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was solution
first filled
withpump
the analyte
double T-junction
withthe analyte
with
A. Thesolution
carrier
with
pump
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solution
was channel
then fed(frominto double
the separation
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detection(frompoint)and thepoint
wastetochannel
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detection
point W)(fromat thedetection
flow ratepoint
10 L
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B, where
the organic
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min with
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divided into
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divided intoandthe waste
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due channels,
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1:24
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due
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their
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as wellassolution
where thewater-richcarriersolutionpressures
well
where
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dividedinto thetwo channels
dividedintoratio
the twowith back
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due to theirpressuresthat mightbewith 1:9
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be larger
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the bare microchannel.
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the
-1
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10 s,pumpwhile,Bpump
B
load,
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analyte wassolution
waspumpfed bywithanalytefed byA pumpwith 5AL
min5 µL
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did not
did
thesolution.
carrier solution.
After
that,
in 3) analysis,
thesolution
carrier solution
feednot
thefeedcarrier
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analysis,the carrier
was fed wasinto fedtheinto
the separation
waste again
channelsagain as mentioned
in the above-preparation
separation
and wasteandchannels
as mentioned
in the above-preparation
procedure,
procedure,
while,A thepump
didanalyte
not feedthe analytemM hydrogen
while, the pump
did not
feedAthesolution.
50 mMsolution.
hydrogen50
peroxide(10 mM
peroxidemM pH
carbonate
buffer;pH 10.8)
wasdelivered tofrom
the microchannel
fromcarbonate(10buffer;10.8) wasdeliveredto themicrochannel
points H to HW
at-1-1
pointsH rateto HWtheminflow with
rate of
5 µLX.minpump
X. mixed
The analytes
were
mixed
the flowof 5atLpump
The with
analytes
werewith thehydrogenwiththe hydrogen
peroxide
solutionpointin thepoint
to CL
induceThe CLwasa
peroxide
solution in
the detection
to detection
induce CL.
The
wasCL.
detectedwith
detected
with a tube
photomultipliertube (PMT). Microchannels
were icecooledwith theicephotomultiplier(PMT). Microchannels
were cooled withthroughthroughthe experiments
to keepwell.
the TRDP well.
experiments
to keep the TRDP
Results and discussion
The TRDP and the microchip design
The TRDP appears through phase separationfrom homogeneous solution to heterogeneous solution including two phases withpressure and temperature changes. The phase separation with associated changes forms
an upper and lower phase in a batch vessel under the control of gravity. At the sametime, the phase separation introduces TRDP, including inner and outer phases in a
micro-flow where it is under laminar flow conditions with minimal control by gravity.
When the ternary mixed solvent mixture, i.e., water–hydrophilic/hydrophobic organic
solvent mixture, is delivered into a microspace, the solvent molecules are radially1
distributed in the microspace regardless of the material (fused silica, polyethylene, and170

Chapter 7

170

PTFE). In the microspace, with an organic-solvent-rich solution, an organic-solvent-richmajor phase is generated around the middle of the microspace as an inner phase, while awater-rich minor phase is formed near the inner wall as an outer phase. In contrast, witha water-rich solution, the water-rich major phase is generated as an inner phase, while
the organic-solvent-rich minor phase is formed as an outer phase. The microchip
incorporating the microchannels used in this study was designed with reference to ourprevious papers and other groups’ papers regarding microchip-electrophoresis [2-6].
The injection protocol was also introduced on the microchip system with reference toour previous paper where the sample introduction into the capillary was madehydrodynamically by pressure, caused by the flow of the sample solution [7].
Fluorescence photographs and chromatogram with the organic solvent-rich carrier
solution The obtained fluorescence photographs with the organic solvent-rich carrier
solution are	
  shown in Fig. 3 together with the conditions. The carrier solution
containing fluorescence dyes was fed into the separation channel and the waste channel.
The organic solvent-rich major phase including perylene (blue) generated in the middleof the center of the microchannel as an inner phase. While, the water-rich minor phaseincluding relatively hydrophilic Eosin Y (green) was formed near the inner wall of thechannel as an outer phase. A bare or untreated microchannel in glass-microchip wasused because the water-rich outer phase was reported to be more stable on the inner wallof a fused-silica tube than that of a polymer tube in our paper.22) The TRDP with theorganic solvent-rich carrier solution, generating inner organic solvent-rich and outerwater-rich phases, were consistent and reasonable with our previously recorded data andthe TRDP concept.14,15,17) The obtained chromatogram with the organic solvent-richcarrier solution is shown in Fig. 4. The experimental conditions described in theexperimental session were determined, referring to analytical conditions previously
reported by our group.19,24,25) With the organic solvent-rich carrier solution thewater-rich minor outer phase worked as a pseudo-stationary phase under laminar flow
conditions in the TRDC. Relatively hydrophobic free- or excess-ILITC that distributedin the organic solvent-rich major inner phase was eluted first and relatively hydrophilic
ILITC-labeled BSA that distributed in the water-rich minor outer phase was detected on
the chromatogram afterwards. The outer phase constructed with the alkaline water-richsolution (pH of the water component is 11.8) may minimize adsorption of
ILITC-labeled BSA onto the inner wall. The peaks were identified with the individual
analyte solutions. The elution behavior of the analytes was quite similar to that obtainedwith the TRDC system using a fused-silica capillary tube in our previous paper.25)
ILITC-labeled BSA could be determined in the range of 2.5 - 50 mM with the detection
limit of 2.5 mM (S/N=3). The resolutions (Rs) and the theoretical plate numbers (N)were calculated to be 4.4 and 7800, respectively, for ILITC-labeled BSA analysis. CL
detection in a microchip relatively shows poor sensitivity due to a small sample volume[20]. But we will try to improve the sensitivity in the present system through thedetailed examination of the analytical conditions.
Fluorescence photograph and chromatogram with the water-rich carrier solution
The obtained fluorescence photographs with the water-rich carrier solution are shown in
Fig. 5 together with the conditions. A hydrophobic-modified microchannel was used for
171

Microchip-TRDC

171

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

the water-rich carrier solution because the organic solvent-rich outer phase was morestable on the inner-wall of a polymer tube than that of a fused-silica tube.22) The carrier
solution containing fluorescence dyes was fed into the separation and waste channels. In
this case, the water-rich major phase including Eosin Y (green) generated in the middleof the center of the microchannel as an inner phase. While, the organic solvent-richminor phase including perylene (blue) was formed near the inner wall of the channel as
an outer phase. The TRDP with the water-rich carrier solution, in contrast to the organic
solvent-rich carrier solution, generating inner water-rich and outer organic solvent-richphases, were consistent and reasonable with our previous data and the TRDP
concept.14,15,17) The obtained chromatogram with the water-rich carrier solution isshown in Fig. 6. A hydrophobic-modified microchannel was used for the water-richcarrier solution. In contrast to the chromatogram obtained with the organic solvent-richcarrier solution, relatively hydrophilic ILITC-labeled BSA was first detected, followedby relatively hydrophobic free-ILITC detection. But baseline separation was not
obtained between the two peaks under the present analytical conditions. The outer phase
constructed with the organic solvent-rich solution may lead to adsorption of free-ILITC
onto the inner wall and thus peak broadening. The peaks were identified with theindividual analyte solutions.
Chromatogram of ADH
We also analyzed a mixture of free-ILITC andILITC-labeled ADH. We are interested in bio-macromolecules that are generally
difficult to determine with capillary zone electrophoresis comprising an open-tubular
capillary tube and no additive-containing carrier solution. ADH is a much larger
biomolecule than BSA and is easily labeled with ILITC. The obtained chromatogram of
ADH with the organic solvent-rich carrier solution and untreated fused-silica capillary
tube is shown in Fig. 7. Free-ILITC was first eluted and secondly ILITC-labeled ADH
was detected on the chromatogram. As the organic solvent-rich solution was used as acarrier solution, relatively hydrophobic free-ILITC was first detected, followed byrelatively hydrophilic ILITC-labeled ADH detection. The outer phase constructed withthe alkaline water-rich solution (pH of the water component is 11.8) may also minimize
adsorption of ILITC-labeled ADH onto the inner wall. The peaks were identified withindividual analyte solutions. The elution behavior of the analytes was similar to thatobtained with the TRDC system using a fused-silica capillary tube in our paper. ADH
was more insoluble than BSA in water maybe due to the larger molecular weight. ADH
may aggregate partially through the labeling procedure and make larger hydrophobic
macromolecules to give unknown peaks. Then, the unknown peaks appeared in earlier
elution time than ILITC and lacked reproducibility. It was confirmed that the
ILITC-labeling technique was useful for analyzing biomolecules having an amino group,
such as amino acid, peptide, protein, and enzyme, in the TRDC system. We plan to
examine a specific protein, such as a cancer marker, in the simple and economical
separation system, that is, the microchip-TRDC.
In conclusion, the TRDC based on the TRDP was successfully developed on the
microchip, leading to the microchip-TRDC system. The model analyte mixture offree-ILITC and ILITC-labeled BSA as well as free-ILITC and ILITC-labeled ADH
were analyzed with the microchip-TRDC system. The system worked without applying

172

Chapter 7172

a voltage supplier device like a microchip-electrophoresis and specific columns, such as
packed and monolithic, like normal capillary chromatography. In addition, the analytes
were detected with CL reaction. The CL detection did not require any light source or
wavelength discrimination. Consequently, the present microchip-TRDC system with CL
detection was simple and matched to the concept of µ-TAS or Lab on a Chip. We
reported unique extraction and mixing methods based on TRDP on a microchip. If we
combine these techniques of separation, extraction, and mixing based on the TRDP, ona microchip, we will be able to develop a novel type of Lab on a Chip system based on
TRDP.

173

Microchip-TRDC

173

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesa)b)Figure 1. Schematic diagram of a) microchip incorporating the microchannels and b)
microchip-holder. S, sample (analyte) solution delivery point; C, carrier solutiondelivery point; W, analyte or carrier solution waste point; H, hydrogen peroxide solutiondelivery point; and HW, hydrogen peroxide solution waste point.

174

Chapter 7174

Figure 2. Schematic illustration of analytical procedures, 1) preparation, 2) sample load,and 3) analysis. S, sample (analyte) solution delivery point; C, carrier solution delivery
point; W, analyte or carrier solution waste point; H, hydrogen peroxide solution delivery
point; and HW, hydrogen peroxide solution waste point.

175

Microchip-TRDC

175

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3. Fluorescence photographs of the organic solvent-rich carrier solution
including perylene and Eosin Y fed into the separation microchannel. Carrier,water-acetonitrile-ethyl acetate (3:8:4 volume ratio) containing 0.1 mM perylene and 1
mM Eosin Y.

Figure 4. Chromatogram of free-ILITC and ILITC-labeled BSA mixture obtained withthe microchip-TRDC system using the organic solvent-rich carrier solution. Carrier,water-acetonitrile-ethyl acetate (3:8:4 volume ratio) and analyte, 50 µM ILITC-labeled
BSA.

176

Chapter 7176

Figure 5. Fluorescence photographs of the water-rich carrier solution including perylene
and Eosin Y fed into the separation microchannel. Carrier, water-acetonitrile-ethyl
acetate (80:20:9 volume ratio) containing 0.1 mM perylene and 0.5 mM Eosin Y.

Figure 6. Chromatogram of free-ILITC and ILITC-labeled BSA mixture obtained withthe microchip-TRDC system using the water-rich carrier solution. Carrier,water-acetonitrile-ethyl acetate (15:3:2 volume ratio) and analyte, 50 µM ILITC-labeled
BSA.
177

Microchip-TRDC

177

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 7. Chromatogram of free-ILITC and ILITC-labeled ADH mixture obtained withthe	
 microchip-TRDC system using the organic solvent-rich carrier solution. Carrier,water-acetonitrile-ethyl acetate (3:8:4 volume ratio) and analyte, 50 µM ILITC-labeled
ADH.

References[1] Y. Fujii, T. G. Henares, K. Kawamura, T. Endo, H. and Hisamoto, Lab. Chip, 2012,12, 1522.
[2] M. Hashimoto, K. Tsukagoshi, R. Nakajima, K. Kondo, and A. Arai, J.Chromatogr.
A, 2000, 867, 271.
[3] K. Tsukagoshi, N. Jinno, and R. Nakajima, Anal. Chem., 2005, 77, 1684.
[4] K. Tsukagoshi, T. Saito, and R. Nakajima, Talanta, 2008, 77, 514.
[5] B.-F. Liu, M. Ozaki, Y. Utsumi, T. Hattori, and S. Terabe, Anal. Chem., 2003, 75,
36.
[6] R. Su, J.-M. Lin, , K. Uchiyama, and M. Yamada, Talanta, 2004, 64, 1024.
[7] K. Tsukagoshi, K. Nakahama, and R. Nakajima, Anal. Sci., b, 20, 379.

178

Chapter 7

178

Chapter 8 Examination of various types of analytes with tube
radial distribution chromatography (TRDC)
Various types of analytes, such as organic compounds, amino acids, peptides, proteins,
nucleosides, metal ions, metal complexes, fluorescent compounds, lambda-DNA,polymer compounds, and optical isomers were analyzed using the TRDC
system.3,8,9,13,19,24,25,30,33)

8.1 Examination of carboxylated polymer particles as analytes with
TRDC using a polyethylene capillary tube

Chapter 8

A tube radial distribution chromatography (TRDC) system has been developed using an
open capillary tube and an aqueous–organic solvent mixture as a carrier solution underlaminar flow conditions. Here, we examine the elution behavior of carboxylated
polymer particles (1.0 µm diameter) together with fluorescein isothiocyanate in the
TRDC system using a polyethylene capillary tube in order to extend our knowledge
regarding the separation performance of the system. The model analyte solution offluorescein isothiocyanate and carboxylated polymer particles was injected into theopen polyethylene capillary tube utilizing the gravity method, and subsequently
delivered through the capillary tube with the carrier solution of thewater–acetonitrile–ethyl acetate mixture (15:3:2 volume ratio) using a microsyringe
pump. On-capillary detection was performed for the analytes using an absorption
detector, which indicated individual elution times and peak shapes on thechromatograms. We discuss the observed phenomena considering the separation
mechanism of the TRDC system.
Introduction
It is important to examine the elution behavior of various types of analytes in the TRDC
system for expanding our knowledge regarding the separation performance. Here, we
examined a nondiffusive analyte of carboxylated polymer particles with 1.0 µm
diameter together with fluorescein isothiocyanate (FITC) in the TRDC system. The
mixture solution of FITC and carboxylated polymer particles was used as an analyte
solution in our previous study [1] concerning wide-bore hydrodynamic chromatography
in which we used a carrier solution which didn`t include any organic solvents. These
analytes were comparatively hydrophilic, but were quite different with respect to theirsizes and diffusion properties. We compared the data obtained by the present TRDC
system with that obtained by the wide-bore hydrodynamic chromatography system inour previous study [1]. The separation performance of the carboxylated polymer
particles in the TRDC system gave us important information that supported the
explanation of the separation mechanism of the TRDC system that we proposed.
Experimental
Carboxylated polymer particles (1.0 µm diameter) prepared from polystyrene matrix,
FluoSpheres (1.0 µm), were provided as suspension (2% solids, 4 × 1010 particles mL−1)
and diluted with a carrier solution as necessary. A high-density polyethylene capillary
179
Examination of various types of analytes with TRDC

179

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

tube (200 µm i.d., 500 µm o.d.) was used. The present TRDC system was comprised ofan open polyethylene capillary tube (70 cm length, effective length 50 cm),
microsyringe pump, and absorption detector. The solution mixture ofwater–acetonitrile–ethyl acetate (15:3:2 volume ratio) was used as the carrier solution.
We prepared the analyte solutions, FITC, FluoSpheres (1.0 µm), and their mixture withthe carrier solution. The solutions were introduced directly into the capillary inlet sidefor 5 s from a height of 20 cm by the gravity method. After the analyte injection, thecapillary inlet was connected through the joint to the microsyringe, and the syringe wasset on the microsyringe pump. The carrier solution was fed into the capillary tube at aflow rate of 4.0 µL min−1 under laminar flow conditions. On-capillary detection was
performed for the absorption (254 nm) by the detector.
Results and discussion
Discussion
We examined FITC, FluoSpheres (1.0 µm), and the mixture of the two in the present
TRDC system using an open polyethylene capillary tube and a water–acetonitrile–ethyl
acetate carrier solution. The Reynolds number was roughly estimated to be < 1 under
the present analytical conditions, confirming that the present system worked underlaminar flow conditions. As shown in Figs. 1 (a) and (b), FITC and FluoSpheres (1.0µm) were detected at ca. 3.9 min with a Gaussian peak and ca. 1.9 min with a
non-Gaussian peak, respectively. The elution times of 1.9 and 3.9 min almost
corresponded to the maximum and average linear velocities, respectively, in thecapillary tube under laminar flow conditions. The maximum and average linear
velocities were tentatively calculated with the ordinary equations [1] concerning linear
velocity under laminar flow conditions, although the linear velocity of a water-organic
solvent mixture carrier solution in the capillary tube may deviate from an ideal
parabolic curve under laminar flow conditions. When the mixture of FITC andFluoSpheres (1.0 µm) was subjected to the present TRDC system, they separated
through the polyethylene capillary tube, as shown in Fig. 1 (c). Although someoverlapping was observed in Fig. 1 (c), the separation performance was much better
than that reported in our previous study [1], as outlined below.
In our previous study [1], we also examined FITC and FluoSpheres (1.0 µm) bymeans of wide-bore hydrodynamic chromatography, in which we used an aqueous
carrier solution that did not include any organic solvents. Previously, researchers havenoted that under laminar flow conditions, compounds and particles with diffusionindexes greater than 1, such as FITC, fed into a capillary tube can be delivered withaverage linear velocities and show Gaussian peaks. In contrast, those with diffusionindexes less than 0.1, such as FluoSpheres (1.0 µm), can be delivered with maximum
linear velocities and show non-Gaussian peaks, while analytes with diffusion indexes
between 0.1 and 1 are better characterized by intermediate behavior. The
chromatograms of FITC and FluoSpheres (1.0 µm) reported in the previous study wereconsistent with the estimated chromatograms based on their calculated diffusion indexes.
FluoSpheres (1.0 µm) and FITC were eluted in this order with the maximum linear
velocity and showed non-Gaussian peaks and with the average linear velocity with
Gaussian peaks, respectively. We should emphasize that, in principle, these peaks arenever separated by wide-bore hydrodynamic chromatography; a Gaussian peaknecessarily appears on the slope of a non-Gaussian peak.

180
180

Chapter 8

The data of FITC and FluoSpheres (1.0 µm) obtained with the present TRDC systemwere different from those obtained by wide-bore hydrodynamic chromatography.
Although the peak of the FluoSpheres (1.0 µm) appeared with the apex of the maximum
linear velocity in the capillary tube, the slope of the non-Gaussian peak did not last
longer than the elution time at the average linear velocity in the tube (see Fig. 1 (b)),
leading to the separation of FITC and the FluoSpheres (1.0 µm) in the mixture (see Fig.
1 (c)). The data obtained here were considered in the TRDC system as follows: nosolvent (composed of aqueous and organic components) in the carrier solution wasdispersed fully uniformly inside the capillary tube. A major solvent phase or awater-rich phase formed around the middle of the tube far from the inner wall, while aminor solvent phase or an organic solvent-rich phase occurred near the inner wall of thecapillary tube. FITC and FluoSpheres (1.0 µm) were comparatively hydrophilic anddispersed in the water-rich phase around the middle of the tube.
For further consideration of the obtained chromatograms, we calculated the diffusion
coefficients and indexes. The Einstein–Stokes equation (see Eq. (1)) and the diffusionindex equation (see Eq. (2)) are shown below, where D is the diffusion coefficient, τ thediffusion index, k the Boltzmann constant, T the absolute temperature, µ the viscosity, dthe particle diameter, L the length, a the radius, and uav the average linear velocity.

D
τ

kT
3π d 	  (1)DL

a 2 av	  (2)Table 1 summarizes the roughly calculated results concerning the diffusion coefficients,
D, and diffusion indexes, τ, for FITC and FluoSpheres (1.0 µm) under the present
analytical conditions. FITC, diffusive solute, and FluoSpheres (1.0 µm), non-diffusive
solute, were delivered with the average linear velocity in the capillary tube with
Gaussian peak and with the maximum linear velocity with non-Gaussian peak,
respectively. However, the carboxylated FluoSpheres (1.0 µm) were mainly dispersedin the water phase (around the middle of the tube) and not in the organic phase (near the
inner wall of the tube), providing the disappearance of the non-Gaussian peak aroundthe elution time at the average linear velocity in the tube.
In conclusion, the chromatogram of the mixture of FITC and FluoSpheres (1.0 µm)
obtained using the TRDC system had a much better resolution than that obtained bywide-bore hydrodynamic chromatography. They were almost completely separated in
the TRDC. The peak of FluoSpheres (1.0 µm) appeared with the apex at the maximum
linear velocity in the capillary tube, but the slope of the non-Gaussian peak did not last
longer than the elution time at the average linear velocity in the tube. The observed
phenomena was explained by means of the tube radial distribution of the carrier
solvents in the tube within the TRDC system.
Nomenclature; a = inner diameter [m], D = diffusion coefficient [m2s−1], d = particle
diameter [m], k = Boltzmann constant [NK−1], L = length [m], T = absolute temperature[K], uav = average linear velocity [ms−1], µ = viscosity, [kgm−1s−1], and τ = diffusionindex [-]
181
Examination of various types of analytes with TRDC

181

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Chromatograms of FITC, FluoSpheres (1.0 µm), and their mixture as obtainedby the present TRDC system. (a) 0.5 mM FITC, (b) 200 times dilution of FluoSpheres,and (c) the mixture of 0.5 mM FITC and 200 times dilution of FluoSpheres. Conditions:
Capillary, 70 cm (effective length 50 cm) with 200 µm i.d. polyethylene capillary;
carrier, water–acetonitrile–ethyl acetate mixture (15:3:2 volume ratio); sample injection,
20 cm height (gravity) × 5 s; and flow rate, 4.0 µL min−1.

182
182

Chapter 8

Table 1.	
 Diffusion coefficients and diffusion indexes.

183
Examination of various types of analytes with TRDC

183

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

8.2 Examination of polymer compounds as analytes with TRDC using
a fused-silica capillary tube
Fluidic behavior of the polymer compounds, FluoSpheres (1.0 µm diameter), wasexamined by tube radial distribution chromatography (TRDC) system. The ternary
mixture of water-acetonitrile-ethyl acetate, 15:3:2 (water-rich) or 3:8:4 (organic
solvent-rich) volume ratio, as a carrier solution was fed into the fused-silica capillary
tube under laminar flow conditions. The peak of the FluoSpheres appeared with the
apex at the maximum linear velocity and the slope of the non-Gaussian peak did not last
longer than the elution time at the average linear velocity with the water-rich carrier
solution. With the organic solvent-rich carrier solution FluoSpheres was eluted at near
the average linear velocity.
Introduction
Although various types of analyte were analyzed using the TRDC system, we have nothad enough information about fluidic behavior of the polymer compounds in the TRDP.
Here, we examined the fluidic behavior of the polymer compounds, FluoSpheres (1.0µm diameter), through the TRDP in fused-silica capillary tubes by use of the TRDC
system.
Experimental
Lambda-DNA (48502 bp, 32,300,000 molecular weight) was used. Carboxylated polymer
particles prepared from polystyrene matrix, FluoSpheres (1.0 µm diameter), from
Molecular Probes Inc., were provided as suspension (2% solids, 4 × 1010 particles mL−1)
and diluted with a carrier solution as necessary. The present chromatography systemcomprised of an open fused-silica capillary tube (50 µm inner diameter), microsyringe
pump, and absorption detector. The tube temperature was controlled by dipping thecapillary tube in water maintained at a specific temperature of 20 °C in a vessel.
Water-acetonitrile-ethyl acetate mixtures with volume ratios of 15:3:2 and 3:8:4 wereused as water-rich and organic solvent-rich carrier solutions in the TRDC system. The
component ratios of 15:3:2 and 3:8:4 were recommended in our reports.15,17,19) Analyte
solutions were prepared with the carrier solutions. Here, 200 times-diluted FluoSheresand 0.8 nM lambda-DNA (MW 32,300,000; 48502bp) were used as analyte solutions.
The analyte solution was introduced directly into the capillary inlet side by the gravity
method. After analyte injection, the capillary inlet was connected through a joint to a
microsyringe which was then set on the microsyringe pump. The carrier solution was
fed into the capillary tube at a definite flow rate under laminar flow conditions.
On-capillary absorption detection (260 nm) was performed with the detector.
Results and discussion
When the present capillary chromatography is carried out with a water carrier solution,
the chromatographic system inevitably works as a wide-bore hydrodynamic
chromatography for polymer compounds. The wide-bore hydrodynamic
chromatography works by a different mechanism originating from the solute
distribution coupled with the laminar flow conditions [2-4]. A diffusive solute gives an
elution curve with an apex at the time required for the average linear velocity to thedetector (i.e., such a curve is characterized by a Gaussian peak), whereas anon-diffusive solute yields an asymmetric elution curve with the apex at the maximum
184
184

Chapter 8linear velocity in a capillary (i.e., such a curve is characterized by a non-Gaussianpeak).
As preliminary experiments, FluoShperes as artificial polymer particles andlambda-DNA as a natural biopolymer, were examined by the wide-bore hydrodynamic
chromatography mode using a water carrier solution (not containing any chemicals or
additives). The obtained chromatogram is shown in Fig. 1 together with analyticalconditions. FluoSpheres were eluted first at the maximum flow rate under the laminarflow conditions with non-Gaussian peak and then lambda-DNA was eluted with almost
average linear velocity with Gaussian peak. They showed typical elution patterns on thechromatograms by the wide-bore hydrodynamic chromatography as described above.
FluoroShere was non-diffusive and lambda-DNA was diffusive solutes in the water
carrier.
Lamda DNA that is rather large molecular weight features hydrophilic and partiallyhydrophobic character, although it depends on the conditions such as temperature,solved solvent, and added additives. The solubility of lambda-DNA was examined in
the carrier solutions for the TRDC system. Lambda-DNA was dissolved with thewater-rich carrier solution but could not be dissolved with the organic solvent-richcarrier solution due to its hydrophilic nature. In addition the absorption spectra oflambda-DNA gradually changed in the water-rich carrier solution. Lambda-DNA wouldchange from double helix conformation to single coil one with ethyl acetate. Such achange was not observed with a water-acetonitrile mixture solution. Only FluoSpheres
were examined by the present system with a water carrier solution (wide-borehydrodynamic chromatography mode), a water-acetonitrile-ethyl acetate mixture
solution (15:3:2; volume ratio) (TRDC mode using the water-rich carrier solution), anda water-acetonitrile-ethyl acetate mixture solution (3:8:4; volume ratio) (TRDC modeusing the organic solvent-rich carrier solution). The obtained chromatograms are shownin Fig. 2 along with analytical conditions. With the water carrier solution, FluoSpheres
showed a typical chromatogram of non-diffusive solute in a wide-bore hydrodynamic
chromatography (Fig. 2 a)). The data of FluoSpheres obtained with the TRDC system(Fig. 2 b) and c)) were different from those obtained by the wide-bore hydrodynamic
chromatography (Fig. 2 a)). In the TRDC system using the water-rich carrier solution,although the peak of the FluoSpheres appeared with the apex at the maximum linear
velocity in the capillary tube, the tailing slope of the non-Gaussian peak did not last
longer than the elution time at the average linear velocity in the tube (Fig. 1 b)).
FluoSpheres were comparatively hydrophilic and dispersed largely around the middle ofthe tube in the water-rich phase with only a little in the organic solvent-rich outer phase.
On the other hand, with the organic solvent-rich carrier solution FluoSpheres were
eluted at near the average linear velocity with nearly Gaussian peaks. It might be
attributed to few dissociated carboxyl groups of FluoSpheres in the organic solvent-richsolution and higher diffusion coefficient of organic solvents (acetonitrile and ethyl
acetate) to the solutes than that of water. We will try to consider the chromatographic
data together with computer simulation and fluorescence microscopy observation in the
future.
In conclusion, we tried to examine the fluidic behavior of the polymer compounds,
FluoSheres (1.0 µm diameter), with the TRDC system based on the TRDP. FluoSpheres
showed specific fluidic behavior with the TRDC system compared to the chromatogram
obtained the wide-bore hydrodynamic chromatography. The data also meant that the185
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

TRDC system had potential to deal with various types of analyte, from low molecularweight ones to polymer compounds, with the same system and a similar procedure.
Information about the fluidic behavior of the polymer compounds obtained here isinteresting in the TRDP or TRDC study, and will be useful to design a microreactor or
micro total analysis systems related with polymer compounds.

Figure 1. Chromatograms of Fluospheres and lambda-DNA obtained by wide-borehydrodynamic chromatography. Conditions: Capillary tube, 110 cm (effective length:
90 cm) of 50 µm i.d. fused-silica; carrier, water; sample injection, 50 cm height(gravity) × 20 s; flow rate, 0.5 µL min-1; tube temperature, 20 °C; and 200 times-diluted
FluoSpheres and 0.8 nM lambda-DNA. Vmax and Vaverage mean the maximum linear
velocity and the average linear velocity under the present laminar flow conditions,
respectively. The plots on the horizontal line mean the estimated elution times with
Vmax and Vaverage.

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Figure 2. Chromatograms of FluoSpheres obtained by the present chromatographic
system. a) Wide-bore hydrodynamic chromatography mode, b) TRDC mode with thewater-rich carrier solution, and c) TRDC mode with the organic solvent-rich carrier
solution. Conditions: Capillary tube, 110 cm (effective length: 90 cm) of 50 µm i.d.
fused-silica; carrier, a) water, b) water-rich carrier solution (water-acetonitrile-ethyl
acetate, 15:3:2 volume ratio), c) organic solvent-rich carrier solution(water-acetonitrile-ethyl acetate, 3:8:4 volume ratio); sample injection, 50 cm height(gravity) × 20 s; flow rate, 0.5 µL min-1; tube temperature, 20 °C; and 200 times-diluted
FluoSpheres. Vmax and Vaverage mean the maximum linear velocity and the average
linear velocity under the present laminar flow conditions, respectively. The plots on thehorizontal line mean the estimated elution times with Vmax and Vaverage.

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8.3 Examination of metal compounds as analytes with TRDC
We examined metal compounds such as copper(II) sulfate, hematin, and hemoglobin
using tube radial distribution chromatography (TRDC) system equipped with achemiluminescence detector. The model mixture solutions of hematin–copper(II) andhemoglobin–copper(II) were injected into a fused-silica capillary tube using a gravity
method and subsequently delivered with the carrier solutions of water–acetonitrile–ethyl
acetate (2:7:4 volume ratio) and water–acetonitrile–ethyl acetate (15:2:1 volume ratio),
respectively, using a microsyringe pump. The mixture of hematin–copper(II) as well as
hemoglobin–copper(II) was well separated and eluted with the organic solvent-richcarrier solution based on the tube radial distribution and detected by chemiluminescencein the present TRDC system.
	 Introduction
We have just begun a series of investigations of the TRDC system in order to broaden
our knowledge about its capabilities. In our studies, model mixtures of hydrophilic andhydrophobic molecules were mainly analyzed using the TRDC system. Here, we haveattempted to examine the separation characteristics of metal compounds, such as Cu(II),hematin, and hemoglobin, in the TRDC system with an untreated open capillary tube
and also the effects of pH value of the aqueous component in the carrier solution on theseparation performance of the TRDC system.
Experimental
A fused-silica capillary tube (75 µm i.d. and 150 µm o.d.) was used. The present TRDC
system consists of a fused-silica capillary tube (70 cm in length), a microsyringe pump,
and a chemiluminescence (CL) detector that takes advantage of the luminol reaction.
The flow-type CL detection cell (0.5 mm i.d. poly(tetrafluoroethylene) tube) [5] wasused in the system. Three water-hydrophilic-hydrophobic organic solvent mixture
carrier solutions were prepared. In the first solution (the organic solvent-rich carrier
solution), the water or aqueous component was 10 mM carbonate buffer at pH 10.8,
containing 25	
 µM luminol; the water–acetonitrile–ethyl acetate mixture was in a
volume ratio of 2:7:4. In the second solution (the water-rich carrier solution, containing
pH 10.8 carbonate buffer), the aqueous component was the same as in the first, but thewater–acetonitrile–ethyl acetate mixture volume ratio was 15:2:1. In the third solution(the water-rich carrier solution, containing pH 7.0 phosphate buffer), the aqueouscomponent was 10 mM phosphate buffer at pH 7.0, containing 25 µM luminol; thevolume ratio of the water–acetonitrile–ethyl acetate mixture was 15:2:1. The mixture ofhematin and copper(II) sulfate (Cu(II)), as well as the mixture of hemoglobin and Cu(II),as models were dissolved in the carrier solutions. The metal catalyst solution included
potassium sodium tartrate at a concentration 20 times higher than that of the catalyst.
The analyte solutions were injected into the capillary tube inlet by the gravity method(from a 25 cm height for 10 s). The analytes were then delivered in the capillary tube
with the carrier solution using a microsyringe pump at a flow rate of 0.5 µL min−1. The
oxidant reagent solution of 50 mM hydrogen peroxide (10 mM carbonate buffer, pH
10.8) was delivered at a flow rate of 10 µL min−1 to the capillary outlet where theanalyte having catalytic activities for luminal reaction, the luminol, and the oxidant
reagent were mixed to generate CL light.
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Results and discussion
The TRDC system with the organic solvent-rich carrier solution First, we examined
the mixture analytes of hematin–Cu(II) or hemoglobin–Cu(II) using the present TRDC
system with the organic solvent-rich carrier solution, where the aqueous component was
10 mM carbonate buffer at pH 10.8, containing 25 µM luminol, and thewater–acetonitrile–ethyl acetate was in a 2:7:4 ratio. Cu(II) is hydrophilic, and hematin
and hemoglobin were comparatively hydrophobic. The mixture of hematin and Cu(II)was eluted in this order at ca. 6.5 min and 7.8 min, respectively (Fig. 1a). They weredelivered at near the average linear velocity and a slower than the average velocity,respectively, under laminar flow conditions. Similarly, the mixture of hemoglobin andCu(II) was eluted in this order (Fig. 1b). The chromatogram of the mixture of hematinor hemoglobin (hydrophobic) and Cu(II) (hydrophilic) in Fig. 1a or b was consistent
with the separation principle of the TRDC system with the organic solvent-rich carrier
solution.
The TRDC system with the water-rich carrier solution We examined the mixture
analytes of hematin–Cu(II) or hemoglobin–Cu(II) with the present TRDC system using
the water-rich carrier solution. The aqueous component was 10 mM carbonate buffer at
pH 10.8, containing 25 µM luminol; the water-acetonitrile-ethyl acetate volume ratio
was 15:2:1. The obtained chromatograms showed no separation; all peaks appeared atabout 6.5 min with near average linear velocity. Despite the slight hydrophobicity ofhematin and hemoglobin in comparison to Cu(II), they should be also dissolved due tothe presence of carboxylate anion groups or protonated amino groups with thewater-rich phase or the major solvent phase under the present analytical conditions. We
tried to increase the hydrophobicity of hematin and hemoglobin by reducing the pH
value of the aqueous component from pH 10.8 to 7.0 in the carrier solution; the mixture
analytes were dissolved with the carrier solution prepared with 10 mM phosphate buffer(see the water-rich carrier solution, containing pH 7.0 phosphate buffer, above).
Hematin was soluble in this carrier solution at 0.5 µM, but hemoglobin, with an
isoelectric point of 6.8-7.0, did not dissolve in the carrier solution. The obtained
chromatogram of the mixture of hematin and Cu(II) with the carrier solution is shown in
Fig. 2. They were eluted in the reverse order, although the hematin peak featured a little
broadening. Cu(II) was eluted with near average linear velocity and hematin with avelocity slower than the average linear velocity. The carboxyl groups of the hematin
molecular structure might become protonated in such an aqueous–organic mixture
solvent because of the lower pH value, leading to an increase in the hydrophobicity ofhematin. Such hematin was easily dissolved in the organic solvent-rich phase or theminor solvent phase in the water-rich carrier solution, and delivered with the velocity
slower than the average linear velocity. In consideration of the separation behavior ofCu(II) and hematin, the chromatogram shown in Fig. 2 was consistent with theseparation principle of the TRDC system with the water-rich carrier solution described
previously.
In conclusion, metal compounds, such as Cu(II), hematin, and hemoglobin, which
are of great interest as biocompounds, were analyzed through the TRDC system with achemiluminescence detector based on a luminal reaction. Cu(II) is hydrophilic, andhematin and hemoglobin are comparatively hydrophobic. The mixtures were separated
through a capillary tube with the help of the tube radial distribution of carrier solvents in
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

the TRDC system and detected through their catalytic activity for the luminol
chemiluminescence reaction. The data obtained herein suggest the possibility to extend
the application of the TRDC system to other analytes.

Figure 1. Chromatograms of mixtures of a) hematin and Cu(II) and b) hemoglobin andCu(II) obtained using the present TRDC system with the organic solvent-rich carrier
solution (with 10 mM carborate buffer, pH 10.8). Conditions: capillary tube, 70 cm of
75 µm i.d. fused-silica; carrier, water (10 mM carborate buffer, pH 10.8, containing 25
mM luminol)–acetonitrile–ethyl acetate (2:7:4 volume ratio) mixture solution; sample
injection, 25 cm height (gravity) × 10 s; carrier flow rate, 0.5 µL min−1; oxidant reagent
flow rate, 10 µL min−1; analyte concentration, 0.1 µM hematin, 0.05 µM hemoglobin,
and 2 mM Cu(II).

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Figure 2. Chromatogram of a mixture of hematin and Cu(II) obtained using the present
TRDC system with a chemiluminescence detector (with 10 mM phosphate buffer, pH
7.0). Conditions: capillary tube, 70 cm of 75 µm i.d. fused-silica; carrier, water (10 mM
phosphate buffer, pH 7.0, containing 25 mM luminol)-acetonitrile-ethyl acetate (15:2:1
volume ratio) mixture solution; sample injection, 25 cm height (gravity) × 10 s; carrier
flow rate, 0.5	 µL min−1; oxidant reagent flow rate, 10	
 µL min−1; analyte concentration,
0.5 μM hematin and 1 mM Cu(II).

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

8.4 Examination of biomolecules as analytes with TRDC
We examined the elution behavior of isoluminol isothiocyanate (ILITC)-labeledbiomolecules (α-amino acids, peptides, and proteins) in an open-tubular capillarychromatography system using an untreated fused-silica capillary tube and awater-acetonitrile-ethyl acetate mixture carrier solution. Such an open-tubular capillarychromatography is called tube radial distribution chromatography (TRDC). A mixtureof ILITC and ILITC-labeled biomolecules was analyzed using TRDC withchemiluminescence detection that provided simple instrument without a light source
and complex optical devises. The ILITC and the twenty labeled α-amino acids wereseparated, in this order or the reverse order, or not separated with an organic
solvent-rich and water-rich carrier solution. Their elution behavior was considered to beof a hydrophilic or hydrophobic nature of ILITC and the labeled α-amino acids. The
ILITC and the labeled protein, alcohol dehydrogenase and bovine serum albumin, wereseparated in this order with an organic solvent-rich carrier solution, while they were
eluted in reverse order given a water-rich carrier solution, based on the TRDC
separation performance. The TRDC system worked with the untreated open-tubular
capillary tube not using any specific capillary tubes, such as coated, packed, ormonolithic.
Introduction
To date, various mixtures of hydrophilic and hydrophobic analytes have been separatedusing the TRDC system. However, most of these have been organic compounds withlow molecular weights: 1-naphthol and 2,6-naphthalenedisufonic acid are typical model
analytes. Here, we examined the elution behavior of biomolecule analytes, isoluminol
isothiocyanate (ILITC)-labeled α–amino acids, peptides, and proteins, in the TRDC
system with chemiluminescence (CL) detection. We briefly described the preliminary
results of this investigation in a previous communication.9)
Experimental
A fused-silica capillary tube (untreated or silanol group-intact; 75 µm i.d. and 150 µmo.d.) was used. A schematic representation of the present TRDC system with CL
detection is shown in Fig. 1. The system consists of a fused-silica capillary tube (70 cm
in length), a microsyringe pump, and a CL detector that takes advantage of the luminol
reaction. A flow CL detection cell (0.5 µm i.d. polytetrafluoroethylene (PTFE) tube) [6]
was used in this system. A water-acetonitrile-ethyl acetate mixture was prepared in a
volume ratio of 3:8:4 as an organic solvent-rich carrier solution or in a volume ratio of
15:3:2 as a water-rich carrier solution, where the water component was 10 mM
phosphate buffer at pH 7.0 or 10 mM carbonate buffer at pH 10.8 or pH 11.8, including
4 µM microperoxidase. α-Amino acids, peptides, and proteins were labeled withisoluminol isothiocyanate (ILITC) as follows. Biomolecule (0.5 µmol) and ILITC (1.0µmol) were dissolved in a solution of water-triethylamine (95:5 volume ratio) (100 µL).
The mixture was stirred for 1 min and left in the dark for 20 min. After the evaporationof the solvent, the residue was redissolved in the carrier solution (1 mL) to give ananalyte solution, including excess (or free)-ILITC and ILITC-labeled biomolecules.
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ILITC

Biomolecule

ILITC-labeled biomolecule(Isoluminolisothiocyanate)
The analyte solution was injected into the capillary tube inlet by gravity from a heightof 25 cm for 10 s. The analytes were then delivered into the capillary tube with thecarrier solution using a microsyringe pump at a flow rate of 0.5 µL min−1. An oxidant
reagent solution of 50 mM hydrogen peroxide (10 mM carbonate buffer, pH 10.8 or
11.8) was delivered at a flow rate of 10 µL min−1 to the capillary outlet where theanalytes were mixed with the reagents to generate CL. The analytes of the mixture wereconfirmed with the individual peaks on the chromatograms.
Results and discussion
Preliminary experiments
In the TRDP an organic solvent-rich carrier solution
generates an organic solvent-rich inner phase and a water-rich outer phase, while awater-rich carrier solution results in a water-rich inner phase and an organic solvent-richouter phase. That is, a major inner phase is formed around the center of the tube awayfrom the inner wall and a minor outer or capillary wall phase is generated near the innerwall. In the TRDC separation system, the analytes that are delivered through thecapillary tube are distributed between the inner and outer phases created by the TRDP,
undergoing chromatographic separation where the outer phase works as a
pseudo-stationary phase in chromatography. The analytes distributed in the outer phasewould also interact with an inner-wall surface of the capillary tube. As preliminary
experiments, an excess (or free)-ILITC and ILITC-labeled aspartic acid mixture was
examined by the present TRDC system with CL detection. The CL detection features asimple instrument without a light source and complex optical devises, and is useful for
analytes possessing a low absorption properties. The carrier solutions, the organic
solvent-rich and the water-rich solutions, were prepared by using three different
aqueous solutions: 10 mM phosphate buffer (pH 7.0) and 10 mM carbonate buffer (pH
10.8 and 11.8). ILITC and ILITC-labeled aspartic acid were not separated with bothcarrier solutions using the 10 mM phosphate buffer (pH 7.0). Although, ILITC andILITC-labeled aspartic acid were separated with the organic solvent-rich solution in thisorder, they were not separated with the water-rich carrier solution using a 10 mM
carbonate buffer (pH 10.8 and 11.8). From the data, ILITC-labeled aspartic acid wasfound to be more hydrophilic than ILITC in the TRDC system with the organic
solvent-rich solution using a 10 mM carbonate buffer (pH 10.8 and 11.8). A labelingreaction between ILITC and biomolecules possessing an amino group, such as aminoacids, peptides, and proteins, is performed; the isothiocyanate group of ILITC reacts
with the amino group to produce the labeled molecules under the conditions describedin the experimental sections. The pH value influences the hydrophilic and hydrophobic
natures of the analytes, ILITC and ILITC-labeled biomolecules, through dissociationand protonation of the functional groups of the molecules. However, the pH in an
aqueous-organic solvent solution cannot be treated in a similar way as the one in an
aqueous solution. In the following experiments, ternary mixed carrier solutions were
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

prepared using a 10 mM carbonate buffer (pH 11.8), which might effect dissociation of
the carboxyl groups of biomolecules.
Typical chromatograms obtained for α-amino acids Twenty α-amino acids werelabeled with ILITC. The mixtures including free-ILITC and ILITC-labeled α-aminoacid were subjected to the present TRDC system. Typical chromatograms obtained for
cysteine, aspartic acid, phenylalanine, and tyrosine are shown in Fig. 2. ILITC andILITC-labeled cysteine were eluted with the organic solvent-rich carrier solution in thisorder, while they were separated in the reverse order with the water-rich carrier solution.
Also, ILITC and ILITC-labeled aspartic acids were eluted with the organic solvent-richcarrier solution in this order, while they were not separated with the water-rich carrier
solution. From the results and the TRDC separation performance, ILITC-labeled
cysteine and aspartic acid could be more hydrophilic than ILITC under the present
TRDC conditions. On the other hand, ILITC and ILITC-labeled phenylalanine were
eluted with the organic solvent-rich carrier solution in the reverse order, while they
were separated in this order with the water-rich carrier solution. Also, ILITC andILITC-labeled tyrosine were separated with the organic solvent-rich carrier solution in
the reverse order, while they were not separated with the water-rich carrier solution.
From the results and the TRDC separation performance, ILITC-labeled phenylalanine
and tyrosine could be more hydrophobic than ILITC under the present TRDC
conditions.
Separation performance of α-amino acids From the obtained chromatograms, thetwenty ILITC-labeled α-amino acids were classified according to their nature into thethree types: more hydrophilic than ILITC, like LITC-labeled cysteine and aspartic acid;
more hydrophobic than ILITC, like ILITC-labeled phenylalanine and tyrosine; andalmost equal hydrophilic or hydrophobic nature to ILITC, which showed no separation
between ILITC and ILITC-labeled α-amino acid on the chromatograms. The three types
were denoted as “hydrophilic”, “hydrophobic”, and “neutral” were compared to ILITC
nature for convenience. The classification of ILITC-labeled α-amino acids nature issummarized in Table 1. Here, excess ILITC was estimated to almost react with activefunction groups of α-amino acids through a labeling reaction. α-Amino acids other thanalanine, leucine, and asparagine that were expressed by “neutral” were separated by the
TRDC system with the organic solvent-rich carrier solution. Also, α-amino acids otherthan alanine, isoleucine, leucine, asparagine, serine, methionine, tyrosine, aspartic acid,glutamic acid, lysine, and arginine, which were “neutral”, were separated by the systemwith the water-rich carrier solution. As a rough estimation for the labeled α-amino acids
separation performance in the present TRDC system, shown in the table, neutral aminoacids, such as aliphatic amino acids, aromatic amino acids, and heterocyclic compoundamino acids were labeled “hydrophobic”. However, neutral amino acids, such as oxy
amino acid and sulfur-containing amino acid, as well as, acidic amino acid and basic
amino acids were labeled “hydrophilic”. The classification of the data in Table 1 wasreasonable and consistent with the molecular structures of ILITC-labeled α-aminoacids.
Separation of peptides
Glycine, glycylglycine, glycylglycylglycine, andglycylglycylglycylglycine were reacted with ILITC, and the obtained mixture solutions
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Chapter 8were examined by the TRDC system. The chromatograms are shown in Fig. 3. With theorganic solvent-rich carrier solution, ILITC-labeled glycine and peptides were firsteluted; secondly, ILITC was detected. However, in the water-rich carrier solution they
were eluted in the reverse order, but mixtures of ILITC and ILITC-labeledglycylglycylglycylglycine were not separated. From the results, roughly speaking,
ILITC was more hydrophobic than the ILITC-labeled glycine and peptides. Tentatively,
the mixture including ILITC, the labeled glycine, and the three labeled-peptides was
subjected to the TRDC system with the organic solvent-rich carrier solution. The
obtained chromatogram is shown in Fig. 4. The elution order was as follows: first,
ILITC-labeledglycylglycylglycylglycine,secondly
ILITC-labeledglycine,
ILITC-labeled glycylglycine, and ILITC-labeled glycylglycylglycine (these three
analytes were not separated), thirdly ILITC; the order was consistent with the elution
behavior observed in Fig. 3.
Separation of protein
The chromatograms of the mixture of ILITC andILITC-labeled bovine serum albumin (BSA) (Mw 66000) were examined by TRDC withthe organic solvent-rich and water-rich carrier solutions. ILITC and ILITC-labeled BSA
were separated and detected in this order with the organic solvent-rich carrier solution,
while they were detected in the reverse order with the water-rich carrier solution.9)
Clearly, the elution times of the analytes were changed by altering the component ratiosof the carrier solvents. The mixture of ILITC and ILITC-labeled alcohol dehydrogenasefrom yeast (ADH) (Mw 140000) was also analyzed with the TRDC system; thesecomponents showed similar elution behaviors to the labeled BSA on the chromatograms.
The elution orders obtained for the mixture of ILITC and ILITC-labeled BSA or ADH
were reasonable, considering the free-ILITC to be more hydrophobic than the
ILITC-labeled protein.
Separation of biomolecules mixture
We examined the mixture of ILITC,ILITC-labeled ADH, and ILITC-labeled BSA with the present TRDC system. The
obtained chromatograms are shown in Fig. 5a). ILITC, ILITC-labeled ADH, and
ILITC-labeled BSA were detected in this order with the organic solvent-rich carrier
solution, while they were eluted in the reverse order with the water-rich carrier solution.
The orders of analyte elution on the chromatograms indicated that the hydrophilicity of
the ILITC-labeled BSA must be larger than that of the ILITC-labeled ADH. The
isoelectric points of BSA and ADH are 4.8 and 5.8, respectively [7]. It is difficult to
describe the dissociation and protonation of carboxyl groups and amino groups onlabeled proteins in an aqueous-organic solvent mixture. However, for the moment, thedifference in the gaps between the isoelectric points (4.8 for BSA and 5.8 for ADH) andpH 11.8 of the water component in the carrier solution seemed consistent with thenature of the hydrophilicity or hydrophobicity of the labeled proteins observed on thechromatograms. The mixture of ILITC, ILITC-labeled glutamic acid, ILITC-labeled
ADH, and ILITC-labeled BSA was also examined with the TRDC system with luminol
CL detection. The obtained chromatograms are shown in Fig. 5b). ILITC,ILITC-labeled glutamic acid, ILITC-labeled ADH, and ILITC-labeled BSA were detectedin this order with the organic solvent-rich carrier solution, while they were eluted in thereverse order with the water-rich carrier solution. The elution orders obtained for the
mixture of ILITC and ILITC-labeled biomolecules were reasonable, considering the195
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

free-ILITC to be more hydrophobic than the ILITC-labeled biomolecules. The elution
orders for ILITC-labeled glutamic acid, ILITC-labeled ADH, and ILITC-labeled BSA
on the chromatograms could indicate the hydrophilicity or hydrophobicity of these
ILITC-labeled biomolecules.
In conclusion, biomolecules, α-amino acids, peptides, and proteins, were labeledwith ILITC for analysis using the TRDC with the CL detection system. Separation was
carried out using an untreated open-tubular fused-silica capillary tube and awater-acetonitrile-ethyl acetate mixture carrier solution without any additives, such asgels; specific columns, such as packed and monolithic; or applying a high voltage.
Detection was also simply performed using CL detection without a light source andcomplex optical devices. The separation performance of free-ILITC and ILITC-labeledbiomolecules gave interesting information about the hydrophilic or hydrophobic naturein the labeling biomolecules. ILITC, ILITC-labeled α-amino acid, ILITC-labeledprotein in the mixture solution were separated and confirmed by the individual peaks on
the chromatograms. TRDC has mostly been applied to the analysis of organic
compounds with low molecular weights. The results obtained for biomolecules,
especially, biopolymer analysis considered here provide insight to expand the TRDC
system to future research.
	
 
	
 

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Figure 1. Schematic diagram of the present TRDC system with CL detection.

Figure 2. Chromatograms of a mixture of ILITC and ILITC-labeled a) cysteine, b)
aspartic acid, c) phenylalanine, and d) tytosine obtained by the present TRDC system.
Conditions: Capillary tube, 70 cm length of 75 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution (organic solvent-rich) andwater-acetonitrile-ethyl acetate (15:3:2 v/v/v) mixture solution (water-rich); sample
injection, 25 cm height (gravity) × 10 s; flow rate of the carrier solution, 0.5 µL min-1;
and ILITC-labeled α-amino acid concentration, 50 mM.
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3. Chromatograms of a mixture of ILITC and ILITC-labeled glycine or peptides
obtained by the present TRDC system. ILITC-labeled glycine and peptides
concentration, 50 mM. Other conditions are the same as in Fig. 2.

Figure 4. Chromatograms of a mixture of ILITC, ILITC-labeled glycine, and threekinds of peptides obtained by the present TRDC system. Carrier,water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution (organic solvent-rich).
Other conditions are the same as in Fig. 2.
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Chapter 8

Figure 5. Chromatograms of mixtures of a) ILITC, ILITC-labeled ADH, and
ILITC-labeled BSA as well as b) ILITC, ILITC-labeled glutamic acid, ILITC-labeled
ADH, and ILITC-labeled BSA obtained by the present TRDC system. ILITC-labeledanalytes concentration, 50 mM. Other conditions are the same as in Fig. 2.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

8.5 Examination of lambda-DNA molecules as analytes with TRDC
The elution behavior of lambda-DNA (48502 bp) as a biopolymer was examined by the
tube radial distribution chromatography TRDC system. The ternary mixture ofwater-acetonitrile-ethyl acetate, 15:3:2 or 3:8:4 volume ratio, as a carrier solution was
fed into the capillary tube made of PTFE or fused-silica. The mixture of hydrophobic
1-naphthol and hydrophilic lambda-DNA was subjected to the TRDC system using thewater-rich carrier solution. Lambda-DNA and 1-naphthol were distributed between the
inner and outer phases due to their hydrophilic and hydrophobic nature, and then eluted
in this order, undergoing chromatographic separation. The mixture of hydrophilic
2,6-naphthalenedisulfonic acid and hydrophobic lambda-DNA that was treated withsurfactants was also examined with the organic solvent-rich carrier solution. The
modified hydrophobic DNA and 2,6-naphthalenedisulfonic acid were distributed andeluted in this order due to their nature.
Introduction
The TRDP is a new phase-interface concept and can be applied in various technological,
engineering, and other scientific fields. We are currently investigating the TRDP fromthe viewpoints of chromatography, extraction, and chemical reaction space. In the
open-tubular capillary chromatography called tube radial distribution chromatography(TRDC), the outer phase created based on the TRDP in a capillary tube acts as a
pseudo-stationary phase. The TRDC system works without applying high voltages orusing specific columns, e.g., monolithic or packed. Although various types of analytes
have been analyzed using the TRDC system, there has not been enough information
about the elution behavior of polymer compounds in the TRDC system or the TRDP.
We were interested in the elution behavior of such large biomolecules, such as
lambda-DNA, and here we tentatively examined such behavior by the TRDC system.
Experimental
Polytetrafluoroethylene (PTFE) capillary tubes with 100 µm inner diameter andfused-silica capillary tubes with 50 µm inner diameter were used. A schematic diagramof the present capillary chromatography system comprises an open-tubular capillarytube, microsyringe pump (55-1111, Harvard Apparatus; Bioanalytical Systems, Inc.,
West Lafayette, IN), and absorption detector (Fig. 1). The tube temperature wascontrolled by dipping the capillary tube in water that was maintained at a constanttemperature in a vessel. Water-acetonitrile-ethyl acetate mixtures with volume ratios of
15:3:2 and 3:8:4 were used as carrier solutions. The component ratios of 15:3:2 and3:8:4 were recommended as water-rich and an organic solvent-rich carrier solutions in
our previous reports [5,6,12]. Analyte solutions were prepared with the carrier solutions.
The analyte solution was introduced directly into the capillary inlet side through thegravity method. After analyte injection, the capillary inlet was connected through a joint
to a microsyringe. The syringe was set on the microsyringe pump. The carrier solution
was fed into the capillary tube at a definite flow rate under laminar flow conditions.
On-capillary absorption detection (254 or 260 nm) was performed by using the detector.
Results and discussion

200
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Chapter 8

Preliminary experiments concerning lambda-DNA
Lambda-DNA	
 (MW 32300000;
48502 bp) that has a relatiely large molecular weight features hydrophilic and partiallyhydrophobic characters, although the details depend on circumstances and conditions,such as temperature, the dissolved solvent, and which additives were added. We tried to
examine the elution behavior of lambda DNA with the TRDC system with thewater-rich and organic solvent-rich carrier solutions. First, the solubility oflambda-DNA was examined in the carrier solutions for the TRDC system.
Lambda-DNA was dissolved by the water-rich carrier solution but was not dissolved by
the organic solvent-rich carrier solution due to its hydrophilic nature. The absorption
spectra of lambda-DNA were examined in the water-rich carrier solution. The spectrachanged gradually up until 48 h (the absorbance at 260 nm increased) and after that, it
did not change, maintaining its shape. As far as we know, there have been no reports
regarding the absorbance change of lambda-DNA in water-hydrophilic/hydrophobicorganic solvent mixture solutions. The absorbance change of lambda-DNA must be
attributed to the change from a double helix conformation to a single coil one with ethyl
acetate or water-acetonitrile-ethyl acetate mixture. That type of change was notobserved with a water-acetonitrile (15:3 volume ratio) mixture solution. We willcontinue investigating further details in the future.
Chromatograms of lambda-DNA obtained by the TRDC system with the water-richcarrier solution
The carrier temperature is critical to the performance of TRDC
separation. The PTFE capillary tube showed overall better resolutions than thefused-silica ones in the TRDC system with the water-rich carrier solution.15) Typicalmodel analytes for the TRDC separation, hydrophobic 1-naphthol and hydrophilic
2,6-naphthalenedisulfonic acid, were subjected to the TRDC system with the water-richcarrier solution and a PTFE capillary tube at a tube temperature of 0 - 50 °C (Fig. 2).
The TRDC system with the water-rich carrier solution creates the water-rich major
inner phase and organic solvent-rich minor outer phase (pseudo-stationary phase), as
described in the Introduction. Hydrophilic 2,6-naphthalenedisulfonic acid, which wasdispersed in the inner phase, and hydrophobic 1-naphthol, which was dispersed in theouter phase, were eluted in this order, undergoing chromatographic separation. The
resolutions were improved at lower temperatures and showed the best resolution at 0 °C.
In the TRDC system using the water-rich carrier solution, the peaks of lambda-DNA on
the chromatogram gradually changed up until 48 h and after that ceased to change, as
shown in Fig. 3. The times in Fig. 3 mean the standing-times of the analyte solution
before injection into the system. The change-tendency on chromatograms was similar tothat on the absorption spectra mentioned above. Then, the mixture of hydrophobic
1-naphthol and hydrophilic lambda-DNA that was left for 48 h in the carrier solution
was examined with the TRDC system with the water-rich carrier solution at a tube
temperature of 0 °C. Other analytical conditions, such as flow rate, an analyte injection
volume, and a capillary length were recommended through our reports. The obtained
chromatograms are shown in Fig. 4. Lambda-DNA and 1-naphthol were completely
separated on the chromatogram. They were distributed between the inner and outerphases due to their hydrophilic and hydrophobic nature, and then eluted in this order,
Chromatograms of lambda-DNA obtained by the TRDC system with the organic
solvent-rich carrier solution When the organic solvent-rich carrier solution was usedin the TRDC system, the fused-silica capillary tube showed better resolutions than the201
Examination of various types of analytes with TRDC

201

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

PTFE tube.10) Then, the model analytes for the TRDC, hydrophobic 1-naphthol andhydrophilic 2,6-naphthalenedisulfonic acid, were subjected to the TRDC system withthe organic solvent-rich carrier solution and a fused-silica capillary tube at tube
temperatures 0 - 65 °C (Fig. 5). The TRDC system with the organic solvent-rich carrier
solution creates an organic solvent-rich major inner phase and a water-rich minor outerphase. Hydrophobic 1-naphthol dispersed in the inner phase and hydrophilic
2,6-naphthalenedisulfonic acid dispersed in the outer phase were eluted in this order,
undergoing chromatographic separation. The resolutions changed with the tube
temperatures and showed their best resolutions at 20 °C. As lambda-DNA was notdirectly dissolved by the organic solvent-rich carrier solution as mentioned above,
lambda-DNA was dissolved in 20 mM sodium dodecylsulfonate (SDS) solutions to
prepare the lambda-DNA analyte solution. The treatments that surrounded
lambda-DNA with surfactants made lambda-DNA stable hydrophobically. The mixtureof hydrophilic 2,6-naphthalenedisulfonic acid and surfactant-modified hydrophobic
lambda-DNA was examined through the TRDC system with the organic solvent-richcarrier solution at a tube temperature of 20 °C. The other analytical conditions wererecommended through our papers. The obtained chromatograms are shown in Fig. 6.
Surfactant-modified lambda-DNA and 2,6-naphthalenedisulfonic acid were completed
and separated on the chromatogram. They were distributed between the inner and outerphases due to their hydrophobic and hydrophilic natures, and then eluted in this order.
In conclusion, the elution behavior of lambda-DNA was examined with the TRDC
based on the TRDP. 1-Naphthol and bare lambda-DNA mixture as well as
2,6-naphthalenesulfonic acid and surfactant-modified lambda-DNA were separated with
the TRDC system, due to their hydrophilic and hydrophobic natures. The elution
behavior of lambda-DNA was consistent with the separation performance of the TRDC
system. The elution behavior observed for lambda-DNA, a rather large biomolecule, isinteresting and will be important in future applications of the TRDP or TRDC.

Figure 1. Schematic diagram of the present TRDC system.

202
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Chapter 8

Figure 2. Chromatograms of 1-naphthol and 2,6-naphthalenedisulfonic acid obtained by
the TRDC system using the water-rich carrier solution. Conditions: Capillary tube, 110
cm (effective length: 90 cm) of 100 µm i.d. PTFE; carrier, the water-rich solution(water-acetonitrile-ethyl acetate, 15:3:2 volume ratio); sample injection, 50 cm height(gravity) × 20 s; flow rate, 1.3 µL min-1; tube temperature, 0 - 50 °C; absorption
detection, 254 nm; and 1 mM 1-naphthol and 2,6-naphthalenedisulfonic acid.

Figure 3. Chromatograms of lambda-DNA obtained by the TRDC system using thewater-rich carrier solution. a) 0 h, b) 24 h and c) 48 h after analytes preparation.
Conditions: Capillary tube, 110 cm (effective length, 90 cm) of 50 µm i.d. fused-silica;
carrier, the water-rich carrier solution (15:3:2, volume ratio); sample injection, 50 cm
height (gravity) × 20 s; flow rate, 0.5 µL min-1; tube temperature, 20 °C; absorption
detection, 260 nm; and lambda-DNA, 0.8 nM.
203
Examination of various types of analytes with TRDC

203

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Chromatograms of 1-naphthol and lambda-DNA obtained by the TRDC
system using the water-rich carrier solution. a) 0.8 nM lambda-DNA and 1 mM
1-naphthol, b) 0.8 nM lambda-DNA, c) 1 mM 1-naphthol. Conditions: Capillary tube,
110 cm (effective length, 90 cm) of 100 µm i.d. PTFE; carrier, the water-rich solution(water-acetonitrile-ethyl acetate, 15:3:2 volume ratio); sample injection, 50 cm height(gravity) × 20 s; flow rate, 1.3 µL min-1; tube temperature, 0 °C; absorption detection,
260 nm.

Figure 5. Chromatograms of 1-naphthol and 2,6-naphthalenedisulfonic acid obtained by
the TRDC system using the organic solvent-rich carrier solution. Conditions: Capillarytube, 110 cm (effective length: 90 cm) of 50 µm i.d. fused-silica; carrier, the organic
solvent-rich solution (water-acetonitrile-ethyl acetate, 3:8:4 volume ratio); sample
injection, 50 cm height (gravity) × 20 s; flow rate, 0.5 µL min-1; temperature, 0 - 65 °C;
absorption detection, 254 nm; and 1 mM 1-naphthol and 2,6-naphthalenedisulfonic
acid.
204
204

Chapter 8

Figure 6. Chromatograms of 2,6-naphthalenedisulfonic acid and SDS-modified
lambda-DNA obtained by the TRDC system using the organic solvent-rich carrier
solution. a) 0.8 nM lambda-DNA and 1 mM 2,6-naphthalenedisulfonic acid, b) 0.8 nM
lambda-DNA, c) 1 mM 2,6-naphthalenedisulfonic acid. Conditions: Capillary tube, 110
cm (effective length, 90 cm) of 50 µm i.d. fused-silica; carrier, the organic solvent-richsolution (water-acetonitrile-ethyl acetate, 3:8:4 volume ratio); sample injection, 50 cm
height (gravity) × 20 s; flow rate, 0.5 µL min-1; tube temperature, 20 °C; absorption
detection, 260 nm; and analyte solution includes 20 mM SDS.

205
Examination of various types of analytes with TRDC

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

8.6 Examination of metal ions as analytes with TRDC using absorption
reagent
Chrome Azurol S as an absorption reagent was introduced into the tube radial
distribution chromatography (TRDC) system for metal ion separation and on-line
detection. The fused-silica capillary tube (75 µm i.d. and 110 cm length) and awater-acetonitrile-ethyl acetate mixture (3:8:4 volume ration) including 20 mM Chrome
Azurol S as a carrier solution were used. Metal ions, i.e., Co(II), Cu(II), Ni(II), Al(III),and Fe(III), as models were injected into the present TRDC system. Characteristic
individual absorption characteristics and elution times were obtained as a result ofcomplex formation between the metal ions and Chrome Azurol S in thewater-acetonitrile-ethyl acetate mixture solution. The elution times of the metal ionswere examined based on their absorption behavior; Co(II), Ni(II), Al(III), Fe(III), andCu(II) were eluted in this order over elution times of 4.7 - 6.8 min. The elution orders
were determined from the molar ratios of the metal ions to Chrome Azurol S and
Irving-Williams series for bivalent metal ions.
Introduction
Various types of analytes, such as organic compounds, amino acids, proteins,
nucleosides, metal ions, metal complexes, fluorescent compounds, and optical isomers,
were analyzed using the TRDC system. However, metal ions and metal complexes wereseparated and detected by our TRDC system which was equipped withchemiluminescence detection. The metal ions and metal complexes show catalytic
activity for luminol-hydrogen peroxide chemiluminescence reactions. Althoughchemiluminescence detection has several advantages, including no requirement for alight source and high sensitivity, it requires a hydrogen peroxide delivery line and pump,mixing device, and flow detection cell to perform chemiluminescence analysis in thesystem.
Here, Chrome Azurol S was introduced as an absorption reagent into the TRDC
system for metal ion separation and on-line detection. Metal ions, i.e., Co(II), Cu(II),
Ni(II), Al(III), and Fe(III), as models were examined by the TRDC system.
Characteristic individual absorption characteristics and elution times were obtained as
the results of complex formations between the metal ions and Chrome Azurol S even in
the water-acetonitrile-ethyl acetate mixture solution. The successful use of theabsorption reagent in the TRDC system with the water-hydrophilic/hydrophobic organic
solvent mixture for on-line metal ion analysis indicated that the TRDC system and
TRDP are attractive and applicable in separation science.
Experimental
Fused-silica capillary tubes (75 µm i.d. and 150 µm o.d.) were utilized. A
fused-silica capillary tube (75 µm i.d., 110 cm length; 90 cm effective length), a
microsyringe pump, and an absorption detector made up the TRDC system withabsorption detection. Tube temperature was controlled by immersing the capillary
tube (40 cm) in water maintained at 20 °C in a beaker by stirring. A
water-acetonitrile-ethyl acetate mixture in volume ratios of 3:8:4 including 20 mM
Chrome Azurol S was used as carrier solution. Analyte solutions, including
1-naphthol, 2,6-naphthalenedisulfonic acid, and the metal ions (Co(II), Cu(II), Ni(II),206
206

Chapter 8

Cu(II), Al(III), and Fe(III) (chloride salts) as well as Ni(II) (nitrate salt), wereprepared along with the carrier solution. The analyte solution was introduced
directly into the capillary inlet side via the gravity method (30 cm height for 20 s),
then, the capillary inlet was connected through a joint to a microsyringe. The syringe
was set on the microsyringe pump. The carrier solution was fed into the capillary
tube at a definite flow rate (ca. 0.8 µL min-1) under laminar flow conditions.
On-capillary absorption detection was performed with the detector; 254 nm for
1-naphthol and 2,6-naphthalenedisuffonic acid or 600 nm for metal ions.
Results and discussion
Absorption spectra for metal ion-Chrome Azurol S complexes
The molecular
structure of Chrome Azurol S is shown in Fig. 1. Chrome Azurol S is used as anabsorption reagent for metal ion detection through a complex formation reaction withmetal ions in aqueous solution [8-10]. First, whether Chrome Azurol S reacts with metal
ions to produce a complex in the ternary mixed solvents of water-acetonitrile-ethyl
acetate (3:8:4 volume ratio) as well as whether the complex shows characteristic
absorption in the ternary mixed solvent solution were examined. Absorption spectra
based on metal ion-Chrome Azurol S complex formation in the ternary mixed solventsof water-acetonitrile-ethyl acetate (3:8:4 volume ratio) are shown in Fig. 2 together withanalytical conditions. It was clearly shown that the metal ions, Co(II), Cu(II), Ni(II),
Al(III), and Fe(III), had specific absorption spectra through complex formation with
Chrome Azurol S in the ternary mixed solvent solution, compared to the absorption
spectrum of Chrome Azurol S itself, in the absence of any metal ions. A wavelength of
600 nm was used for on-line metal detection in the TRDC system equipped withabsorption detection in the following experiments.
Phase diagram of the ternary mixed solvents including Chrome Azurol S A phase
diagram for the ternary mixture solvents of water-acetonitrile (hydrophilic organic
solvent)-ethyl acetate (hydrophobic organic solvent) including 20 mM Chrome Azurol
S was examined in a vessel at a temperature of 22 °C. The obtained phase diagram isshown in Fig. 3. As shown in the diagram, 20 mM Chrome Azurol S was not dissolvedin the ternary mixed solvent solutions with a water component ratio of less than ca. 12
volume %. The phase diagram showed that each component ratio of the solvents made ahomogeneous (one homogeneous phase) or heterogeneous (two homogeneous phases)
solution. The curve in the diagram indicates the boundary between the homogeneousand heterogeneous phases. The TRDC system was used with certain homogeneous
carrier solutions with component ratios of solvents that were positioned near the
homogeneous-heterogeneous solution boundary curve in the phase diagram. Suchspecific carrier solutions caused a tube radial distribution of the carrier solvents in thecapillary tube under laminar flow conditions, i.e., the TRDP. In addition, in the TRDC
system using the fused-silica capillary tube, the organic solvent-rich carrier solution had
better resolution than the water-rich carrier solution.10) Here, the homogeneous solutionof water-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio) including 20 mM
Chrome Azurol S, the component ratio of which was near the boundary curve in thephase diagram (Fig. 3), was used as a carrier solution in the present TRDC system using
the fused-silica capillary tube.

207
Examination of various types of analytes with TRDC

207

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Separation and detection of 1-naphthol and 2,6-naphthalenedisulfonic acid
The
TRDP leads to the formation of inner and outer phases, i.e., a kinetic liquid-liquidinterface, in a capillary tube under laminar flow conditions. In this case using thewater-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio) including 20 mM Chrome
Azurol S carrier solution (organic solvent-rich carrier solution), the organic solvent-richmajor inner phase was generated around the middle of the tube, while the water-richminor outer phase was formed near the inner wall. In the TRDC system, the outer phasethat moved relatively little under laminar flow conditions acted as a pseudo-stationary
phase. Analytes were distributed between the inner and outer phases due to their nature,undergoing chromatographic separation. To confirm that the TRDP occurred in thecapillary tube under the present analytical conditions, a mixture of 1-naphthol and
2,6-naphthalenedisulfonic acid was injected into the present TRDC system which was
designed for the separation and detection of metal ions through complex formations
between a metal ion and Chrome Azurol S. The mixture of them has been used as amodel analyte for the TRDC system. The obtained chromatogram is shown in Fig. 4.
1-Naphthol (hydrophobic) and 2,6-naphthalenedisulfonic acid (hydrophilic) wereseparated and detected (254 nm) in this order. The first peak of 1-naphthol was eluted
near the average linear velocity under laminar flow conditions. The elution behavior
observed on the chromatogram supported the occurrence of TRDP in the present TRDC
system.
Chromatograms of metal ions
The metal ions Co(II), Cu(II), Ni(II), Al(III), andFe(III) were examined in the present TRDC system. The obtained individual
chromatograms are shown in Fig. 6 together with analytical conditions. The elution
times and peak areas of the metal ions are summarized in Table 1 with those of
1-naphthol and 2,6-naphthalenedisulfonic acid shown in Fig. 5. As shown in the table,
the elution times of Co(II), Ni(II), Al(III), Fe(III), and Cu(II) metal ions increased inthis order. Elution order will be discussed later. Tentatively, the Al(III) and Cu(II)mixture as well as the Co(II) and Cu(II) mixture were analyzed in the present TRDC
system. The obtained chromatograms are shown in Fig. 7. As shown in the figures, they
were completely separated and detected on the chromatograms. To obtain quantitative
information, the calibration curves of Al(III) and Cu(II) were examined by the TRDC
system. Al(III) and Cu(II) were linearly determined over the range of 0.2 – 5 mM
(coefficient of correlation 0.997) and 0.2 – 4 mM (coefficient of correlation 0.994),
respectively.
Molar ratios of metal ions to Chrome Azurol S on complex formation The molar
ratios of the metal ions to Chrome Azurol S at the point of complex formation in the
ternary mixed solvents of water-acetonitrile-ethyl acetate mixture were examined using
a standard molar ratio method. To each, 1.0 mM metal ion solution (0.5 mL) was added
to various concentrations of Chrome Azurol S solution (4.5 mL) to obtain a metal
ion-Chrome Azurol S mixture solution including 0.1 mM metal ions and 0.025-0.400
mM Chrome Azurol S, where all solutions were prepared with the ternary mixed
solvents of the water-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio). Fig. 7
shows the relationship between the molar ratio of Chrome Azurol S to Fe(III) and theabsorption due to complex formation. From the figure, it was clearly shown that themolar ratio of Fe(III) to Chrome Azurol S was 1:2. In a similar way, the molar ratios of208
208

Chapter 8the other metal ions, Co(II), Ni(II), Cu(II), and Al(III), to Chrome Azurol S were
examined, and respective values were 1:1, 1:1, 1:2, and 1:1.
Elution order and separation mechanism The TRDP in the present TRDC systemcreated an organic solvent-rich major inner phase and a water-rich minor outer phase in
the capillary tube. Hydrophilic Chrome Azurol S was distributed in the water-richminor outer phase and acted as a pseudo-stationary phase on chromatography. Complex
formation between the metal ion and Chrome Azurol S must make the complex morehydrophobic through charge counteraction concerning the positive charge of the metal
ion, negative charge of Chrome Azurol S, or the counteranion (chloride ion). That is,the complexes would be effectively distributed in the organic solvent-rich major inner
phase that acts as a mobile phase, leading to earlier elution times in the TRDC system.
However, due to the naturally hydrophilic nature of Chrome Azurol S, the complexes
with lower stabilities instead showed earlier elution times. Furthermore, the molar ratioof metal ions to Chrome Azurol S of 1:1 would elute earlier than that of 1:2. With
regard to bivalent metal ions, the elution order of Co(II), Ni(II), and Cu(II) was
consistent with the Irving-Williams series (Co(II) < Ni(II) < Cu(II)) and the molar ratios
calculated above (the ratio of metal ion to Chrome Azurol S; 1:1 for Co(II) and Ni(II) as
well as 1:2 for Cu(II)). For trivalent metal ions, the elution order of Al(III) and Fe(III)
was also consistent with the molar ratios calculated above (1:1 for Al(III) and 1:2 forFe(III)).
Different elution behavior between nickel (II) chloride and nickel (II) nitrate In theexperiments performed in the present study, the nickel (II) nitrate solution as an analyte
showed a later elution time (7.5 min) than the nickel (II) chloride (5.2 min). The
difference in elution times between nickel (II) chloride and nickel (II) nitrate exhibited
excellent reproducibility. Although the reason has not yet been clarified, the electron
density and the ion diameter of the counteranion, chloride or nitrate anion, may
influence hydrophobicity of the complexes. By taking advantage of the elution behaviorof nickel (II) nitrate, we attempted to separate a metal ion mixture solution including
Co(II), Cu(II), and Al(III) (chloride salts) as well as Ni(II) (nitrate salt) using thepresent TRDC system. The obtained chromatogram is shown in Fig. 8. Co(II), Al(III),
Cu(II), and Ni(II) were completely separated and detected with good reproducibility by
the present TRDC system.
In conclusion, metal ions and metal complexes were separated and detected by a
TRDC system equipped with chemiluminescence detection. Althoughchemiluminescence detection has several advantages, such as lack of light source
requirements and high sensitivity, it requires an oxidant delivery line and pump, asolution mixing device, and a flow detection cell for performing chemiluminescence
flow analysis. Here, Chrome Azurol S as an absorption reagent was introduced into the
TRDC system for metal ion separation and on-line absorption detection. Characteristic
individual absorption characteristics and elution times were obtained as a result ofcomplex formation between the metal ions and Chrome Azurol S in thewater-acetonitrile-ethyl acetate mixture solution. Co(II), Al(III), Cu(II), and Ni(II) werecompletely separated and detected in this order on-line by the TRDC system withabsorption detection. The system worked without requiring the application of a highvoltage and without the use of any specific columns. Metal separation was performed
209
Examination of various types of analytes with TRDC209

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

using an untreated open tubular fused-silica capillary. The successful utilization of theabsorption reagent in the TRDC system with the water-hydrophilic/hydrophobic organic
solvent mixture for on-line metal ion analysis has great potential to expand the use of
TRDP in further research.

Figure 1. The molecular structure of Chrome Azurol S as an absorption reagent.

210
210

Chapter 8

Figure 2. Absorption spectra of the metal ion-Chrome Azurol S complex in the ternary
mixed solvents of water-acetonitrile-ethyl acetate (3:8:4 volume ratio). Conditions: 0.1
mM Chrome Azurol S and 0.1 mM metal ion (Co (II), Cu (II), Ni (II), Al (III), and Fe(III); chloride salts).

Figure 3. A phase diagram for the ternary mixture solvents of water-acetonitrile-ethyl
acetate mixture including 20 mM Chrome Azurol S. Chrome Azurol S (20 mM) was notdissolved in ternary mixed solutions with water component ratios of less than ca.12
volume %. The curve in the diagram indicates the boundary between the homogeneousand heterogeneous phases.
211
Examination of various types of analytes with TRDC

211

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Chromatograms of the mixture analyte solution of 1-naphthol and
2,6-naphthalenedisulfonic acid obtained by the TRDC system. Conditions: Capillarytube, 110 cm (effective length: 90 cm) of 75 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 volume ratio) including 20 mM Chrome Azurol
S; sample injection, 30 cm height (gravity) × 20 s; flow rate, 0.8 µL min-1; and1-naphthol and 2,6-naphthalenedisulfonic acid, 1 mM each.

Figure 5.Chromatograms
Chromatograms ofof thethe metal
metal ions
ions obtained
obtained byby thethe TRDC
TRDC system.
system. Conditions:Figure5.
Conditions:
Capillary tube, 110 cm (effective length: 90 cm) of 75 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 volume ratio) including 20 mM Chrome Azurol
S; sample injection, 30 cm height (gravity) × 20 s; flow rate, 0.8 µL min-1; and metal
ions, 1 mM each.

212
212

Chapter 8

Figure 6. Chromatograms of the mixtures of metal ions, Al(III) and Cu(II) mixture aswell as Co(II) and Cu(II) mixture obtained by the TRDC system. Conditions: Capillarytube, 110 cm (effective length: 90 cm) of 75 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 volume ratio) including 20 mM Chrome Azurol
S; sample injection, 30 cm height (gravity) × 20 s; flow rate, 0.8 µL min-1; and Co(II),Cu(II), and Al(III), 1 mM each.

Figure 7. The relationship between the molar ratio of Chrome Azurol S to Fe(III) andabsorption due to complex formation. To each 1.0 mM of metal ion solution (0.5 mL)various concentrations of Chrome Azurol S solution (4.5 mL) were added in order toproduce a metal ion-Chrome Azurol S mixture solution including 0.1 mM metal ionsand 0.025-0.400 mM Chrome Azurol S; all solutions were prepared with the ternary
mixed solvents of water-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio).
213
Examination of various types of analytes with TRDC

213

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 8. Chromatograms of the mixtures of metal ions, Co(II), Cu(II), and Al(III)(chloride salts) as well as Ni(II) (nitrate salt). Conditions: Capillary tube, 110 cm(effective length: 90 cm) of 75 µm i.d. fused-silica; carrier, water-acetonitrile-ethyl
acetate (3:8:4 volume ratio) including 20 mM Chrome Azurol S; sample injection, 30
cm height (gravity) × 20 s; flow rate, 0.8 µL min-1; and metal ions, 1 mM each.

Table 1 Elution times and peak areas of analytes
Analyte1-Naphtol
2,6-NDS
Cu(Ⅱ)
Co(Ⅱ)
Ni(Ⅱ)
Al(Ⅲ)
Fe(Ⅲ)

Elution time [min]
5.0
8.1
6.8
4.7
5.2
5.4
6.3

Analyte concentration, 1.0 mM.
2,6-NDS means 2,6-naphthalenedisulfonic acid.

214
214

Chapter 8

Peak area [mV・s]
11.6
20.9
25.5
8.3
13.1
50.9
46.5

8.7 Examination of dansyl-DL-amino acids as analytes with TRDC
using cyclodextrin
The separation of enantiomers, dansyl-DL-amino acids, was carried out throughopen-tubular capillary chromatography based on the tube radial distribution of thecarrier solvents. An untreated poly(tetrafluoroethylene) capillary tube (100 µm innerdiameter and 90 cm effective length) was used as a separation column and awater–acetonitrile–ethyl acetate mixture containing cyclodextrin used as a carrier
solution were utilized for the chromatography. An analyte solution of dansyl-DL-aminoacids, such as dansyl-DL-methionine, was injected into the capillary tube using a gravity
method, and then the analyte solution was subsequently delivered through the capillary
tube with the carrier solution using a microsyringe pump. The ternary mixed carrier
solution (water-rich carrier solution) was radially distributed in the capillary tube basedon the tube radial distribution phenomenon, causing the formation of inner (water-rich)and outer (organic-solvent-rich) phases. The outer or capillary wall phase acted as a
pseudo-stationary phase in the chromatography. The analytes were separated throughthe capillary tube with on-capillary detection by an absorption or a fluorescence
detector. D-Enantiomer and L-enantiomer were eluted in this order through a baseline
separation. The separation mechanism of the enantiomers in the open-tubular capillaryusing cyclodextrin was discussed.
Introduction
Various types of aqueous-organic solvent mixture solutions are used for the processesof dissolution [11, 12], cleaning [13], and preservation [14] and as reaction solvents[15,16]. Such mixtures are also useful in processes involved in separation science[17-19], such as solvent extraction and liquid chromatography. In liquid
chromatography, a hydrophilic and hydrophobic organic solvent mixture is delivered
into a separation column as a carrier solution in a normal-phase chromatography, whilea water and hydrophilic organic solvent mixture is delivered into the column as a carrier
solution in a reverse-phase chromatography. Wang et al. examined sorption isomers ofternary eluents of water–methanol–acetonitrile in reverse-phase liquid chromatography[20]. Bhushan et al. reported direct enantiomeric TLC resolution with the ternarysolvent system of water–methanol–ethyl acetate mixture [21]. However, to the best of
our knowledge, the use of ternary mixed solvents consisting ofwater–hydrophilic/hydrophobic organic solvent mixture solutions, such as
water–acetonitrile–ethyl acetate mixture, being used as carrier solutions in liquid
chromatography has not yet been examined in great detail.
In addition, with the progress of microfabrication technology, fluid flow in a
microspace, such as in a microchannel in a microchip or capillary tube, has received
much attention. Capillary tubes are known to exhibit interesting and useful physical andhydrodynamic phenomena, such as electro-osmotic and laminar flows. Electro-osmotic
flow in a capillary tube has established capillary electrophoresis [22-24] and capillaryelectrochromatography [24-26] properties, while laminar flow conditions enablehydrodynamic chromatography [27-29]. Recently, we investigated the specific fluid
behavior of a ternary mixed solvent solution in a microspace under laminar flow
conditions.
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Examination of various types of analytes with TRDC

215

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

In contrast to other capillary separation techniques, such as capillary electrophoresis
or capillary electrochromatography, the TRDC system performs without requiring theapplication of high voltages or the use specific columns, e.g., monolithic or packed.
Enantiomer separation is one of the most interesting and attractive aspects of analyticalchemistry and separation science [30-33]. Here, we attempted to apply the system toenantiomer separation for the first time, although we have briefly described thepreliminary results of this investigation in a previous communication.8) A combinationof cyclodextrin as host molecule (chiral selector) and dansyl-DL-amino acids as guest
molecules (enantiomers) was adopted as a model for enantiomer separation withreference to previous reports [32,33].
Experimental
PTFE capillary tubes (100 µm inner diameter, 200 µm outer diameter) were used. The
capillary chromatography system consisted of an open-tubular PTFE capillary tube (110cm length, 90 cm effective length), a microsyringe pump, and an absorption or afluorescence detector. A mixture of water–acetonitrile–ethyl acetate containing
cyclodextrin was used as a carrier solution. The tube temperature was controlled bydipping the tube (ca. 40 cm) in a beaker filled with water maintained at a specifictemperature (0 °C) and stirring. The analyte solution was prepared with the carrier
solution. The analyte solution was introduced directly into the capillary inlet side for 10s from a height of 25 cm by a gravity method. After analyte injection, the capillary inlet
was connected through a joint to the microsyringe, which was set on a microsyringe
pump. The carrier solution was fed into the capillary tube at a flow rate of 0.8 µL min−1
under laminar flow conditions. Using this apparatus, on-capillary absorption detection(254 nm) or fluorescence detection (ex. 335 nm and em. 520 nm) was performed.
Results and discussion
Preliminary experiments
In preliminary experiments, a solution of
2,6-naphthalenedisulfonic acid (1 mM) and dansyl-DL-methionine (500 µM) was
analyzed using a normal TRDC system with a water–acetonitrile–ethyl acetate carrier
solution with a volume ratio of 75:15:10 (15:3:2 volume ratio) and not containing
cyclodextrin. 2,6-Naphthalenedisulfonic acid and dansyl-DL-methionine were eluted
with baseline separation in this order, although enantiomer separation ofdansyl-DL-methionine was not performed. With the water-rich carrier solution,
2,6-naphthalenedisulfonic acid (hydrophilic) was eluted with a velocity near the average
linear velocity and dansyl-DL-methionine (relatively hydrophobic) was eluted at a lower
than average linear velocity under the laminar flow conditions.
Phase diagram for ternary mixed solvents including cyclodextrin Phase diagrams forthe ternary mixed solvents of water–acetonitrile (hydrophilic organic solvent)–ethyl
acetate (hydrophobic organic solvent) containing 1 or 2 mM β-cyclodextrin, 10 mM
α-cyclodextrin, and 10 mM γ-cyclodextrin in a vessel were examined at a temperatureof 22 °C. It was difficult to construct the phase diagrams for the ternary mixed solventswith cyclodextrin at higher concentrations because of their solubility. For example, thephase diagrams for mixed solvents including 1 and 2 mM β-cyclodextrin are shown in
Fig. 1. In the diagrams, 1 and 2 mM β-cyclodextrin were not dissolved in ternary mixed
solvents solutions with a water component ratio of less than ca. 32 and 52 volume %,216
216

Chapter 8

respectively. The phase diagram shows that each component ratio of the solvents
comprised a homogeneous (one homogeneous phase) or a heterogeneous (twohomogeneous phases) solution. The curves in the diagrams indicate the boundary
between homogeneous and heterogeneous phases. In the phase diagram of the mixed
solvents including 1 mM β-cyclodextrin, the component ratios of the solvents that werepositioned extremely close to the boundary curve in the homogenous phase are plotted
as I–VIII in Fig. 1 a. In the phase diagram of the mixed solvents containing 2 mM
β-cyclodextrin, the component ratios of the solvents positioned close to the boundary
curve in the homogeneous phase are also plotted in I–VI in Fig. 1 b. The ternary mixed
solvents of water–acetonitrile–ethyl acetate including 10 mM α-cyclodextrin and 10
mM γ-cyclodextrin showed phase diagrams similar to those of the mixed solvents
including 1 and 2 mM β-cyclodextrin shown in Figs. 1 a) and b), although 10 mM
α-cyclodextrin and 10 mM γ-cyclodextrin were not dissolved in ternary mixed solvent
solutions with water component ratios of less than ca. 42 or 52 volume %, respectively(data not shown). In the phase diagram of the mixed solvents including 10 mM
α-cyclodextrin, the component ratios of the solvents positioned close to the boundary
curve in the homogeneous phase are plotted as I–VII. In addition, in the diagram of themixed solvents including 10 mM γ-cyclodextrin, the component ratios of the solvents
positioned close to the boundary curve in the homogeneous phase are plotted as I–VI.
Similar boundary curves between homogeneous and heterogeneous phases wereobserved along the plots in all phase diagrams, except for the component ratio of waterthat indicated the limitation of the dissolution of cyclodextrin. In our study, the TRDC
system used specific homogeneous carrier solutions with component ratios of solvents
positioned near the homogeneous–heterogeneous solution boundary curve in the phase
diagram. Such specific carrier solutions caused a tube radial distribution of the carrier
solvents in the capillary tube under laminar flow conditions, i.e., the TRDP. In addition,in the TRDC system using the PTFE capillary tube, the water-rich carrier solution had
better resolution than the organic-solvent-rich carrier solution. Here, the homogeneous
solution of water–acetonitrile–ethyl acetate mixture including cyclodextrin, with acomponent ratio near the boundary curve (plots I–VIII for 1 mM β-cyclodextrin, plots
I–VI for 2 mM β-cyclodextrin, plots I–VII for 10 mM α-cyclodextrin, and plots I –VI
for 10 mM γ-cyclodextrin), was used as a carrier solution.
Separation of 1-naphthol and 2,6-naphthalenedisulfonic acid with the ternary mixed
solvents including cyclodextrin The TRDP leads to the formation of inner and outerphases, i.e., a kinetic liquid–liquid interface, in a capillary tube under laminar flow
conditions. In this case, using the water–acetonitrile–ethyl acetate mixture including
cyclodextrin as a carrier solution (water-rich), an organic-solvent-rich major inner phase
was generated around the middle of the tube, while a water-rich minor outer phase wasformed near the inner wall. In the TRDC system, the outer phase, which had negligible
movement under laminar flow conditions, acted as a pseudo-stationary phase. Analytes
were distributed between the inner and outer phases because of their natures, and they
underwent chromatographic separation. First of all, we confirmed that adding
cyclodextrin as an additive molecule in the ternary mixed solvents carrier solution did
not influence the TRDP or TRDC performance as follows. A solution of
2,6-naphthalenedisulfonic acid (1 mM) and 1-naphthol (1 mM), typical analytes for the
TRDC system, was analyzed using the water–acetonitrile–ethyl acetate carrier solution
217
Examination of various types of analytes with TRDC217

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

containing cyclodextrin in order to examine the influence of cyclodextrin in the carrier
solution on TRDP performance. The component ratios of the solvents were positioned
near the boundary curves in every phase diagram. The obtained chromatograms for
carrier solutions including α, β, and γ-cyclodextrin are shown in Fig. 2. For example, inthe ternary mixed solvents including 1 mM β-cyclodextrin (Fig. 2 a)),2,6-naphthalenedisulfonic acid (hydrophilic) and 1-naphthol (hydrophobic) wereseparated and detected in this order based on their TRDC separation performance withcarrier solutions containing 1 mM β-cyclodextrin. The component ratio of the solvents
plotted in I, II, and VII exhibited split separation. The component ratios of the solvents
showed baseline separation at III, IV, V, and VI and non-separation at VIII. In theseparation cases, 2,6-naphthalenedisulfonic acid and 1-naphthol were separated anddetected in this order. The first peak of 2,6-naphthalenedisulfonic acid was eluted with avelocity near the average linear velocity under laminar flow conditions. Similar elution
behaviors in 2,6-naphthalenedisulfonic acid and 1-naphthol were observed using ternary
mixed solvents containing 2 mM β−, 10 mM α−, and 10 mM γ-cyclodextrin as shownin Fig. 2 b) – 2 d). Reasonable elution behavior observed on the chromatogramsupported the occurrence of TRDP in the present TRDC system containing cyclodextrin.
Fig. 3 shows the chromatograms of 2,6-naphthalenedisulfonic acid (1 mM) and
1-naphthol (1 mM) obtained by the TRDC system using carrier solutions with andwithout 1 mM β-cyclodextrin. Under the present analytical conditions, cyclodextrin(additive molecule) appears to exert very little influence on the chromatograms or the
TRDC system.
Separation of dansyl-DL-methionine with ternary mixed solvents including 1 mM
β-cyclodextrin8)
The solutions of 500 µM dansyl-DL-methionine, 250 µM
dansyl-L-methionine, and 500 µM dansyl-DL-methionine plus 250 µM
dansyl-L-methionine were subjected to the present TRDC system utilizing awater–acetonitrile–ethyl acetate (15:3:2 volume ratio) carrier solution containing 1 mM
β-cyclodextrin. The chromatograms obtained for the above three analyte solutions areshown in Fig. 4. The elution times and the peak shapes clearly show that the
D-enantiomer and L-enantiomer in dansyl-DL-methionine were separated and detected inthis order. As shown in Fig. 4, the D-enantiomer was eluted with a near average linear
velocity and the L-enantiomer was eluted with a lower than average linear velocity. As
shown in preliminary experiments, dansyl-DL-methionine was eluted with a lower than
average linear velocity based on the nature of hydrophobicity with a carrier solution that
did not contain β-cyclodextrin; that is, dansyl-DL-methionine was relativelyhydrophobic. In the water-rich carrier solution containing 1 mM β-cyclodextrin,β-cyclodextrin was primarily distributed in the major inner phase or the water-richphase. Dansyl-D-methionine has a stronger interaction with β-cyclodextrin as a chiralselector than with dansyl-L-methionine [33]. The interaction between
dansyl-D-methionine and β-cyclodextrin must alter the distribution of the D-enantiomerfrom the outer phase to the inner phase, leading to an earlier elution time of the
D-enantiomer than the L-enantiomer for separation. Separation performance for theenantiomers in the TRDC system is illustrated in Fig. 5.
Separation of dansyl-DL-methionine with the ternary mixed solvents including various
cyclodextrins Dansyl-DL-methionine was examined using the ternary mixed solvents
218
218

Chapter 8

containing α, β, and γ-cyclodextrin. The carrier solutions were specified by thecomponent ratios of the solvents that showed separation performance for the mixture of
2,6-naphthalenedisulfonic acid and 1-naphthol, as shown in Fig. 2. The obtained
chromatograms are shown in Fig. 6. Enantiomer separation was observed in ternary
mixed solvents including cyclodextrin in a manner similar to that of the chromatograms
shown in Fig. 4 and the illustration in Fig. 5. D-Enantiomer and L-enantiomer wereseparated and detected in this order. In carrier solvents with 1 mM β-cyclodextrin, thecomponent ratio of the solvents, plot III, showed enantiomer separation (Fig. 6 a). In
carrier solvents with 2 mM β-cyclodextrin, plots III–V, showed separation (Fig. 6 b). In
carrier solvents with 10 mM α-cyclodextrin, plots III–VI, showed separation (Fig. 6 c).
Finally, in carrier solvents with 10 mM γ-cyclodextrin, plots III–V, showed separation(Fig. 6 d). Resolutions (Rs) were calculated in the usual manner in chromatograms
exhibiting enantiomer separation. The relationships between the component ratios of thesolvents and the Rs are shown in Fig. 7. The resolutions decreased as the component
ratios of the organic solvents in the carrier solutions increased, which might affect theformation of inner and outer phases in the capillary tube. Furthermore, α-cyclodextrin
showed better resolutions than the other cyclodextrin chromatograms.
Comparison of absorption and fluorescence detection
Enantiomer separation ofdansyl-DL-methionine was examined using the ternary mixed solvents containing
α, β, and γ-cyclodextrin under the same conditions as in Fig. 6, except that fluorescence
detection was used instead of absorption detection. As an example, the chromatograms
obtained with 10 mM α-cyclodextrin are shown in Fig. 8. The chromatograms obtainedwith 2 mM α− and 10 mM γ-cyclodextrin showed similar elution behavior (data notshown). D-enatiomer and L-enantiomer were separated and detected in this order. Peak
areas of D-enantiomer and L-enantiomer in absorption and fluorescence detection aresummarized in Table 1. Clearly, the peak area ratios of D-enantiomer to L-enantiomer
with fluorescence detection were larger than those with absorption detection for the dataobtained with α, β, and γ-cyclodextrin. D-enantiomers that were effectively included in
cyclodextrin might indicate larger fluorescence yields than L-enantiomers influorescence detection.
Separation of the mixture of dansyl amino acids with ternary mixed solvents including
cyclodextrin
Dansyl amino acids other than dansyl-DL-methionine, such as
dansyl-DL-aspartic acid and dansyl-DL-phenylalanine, were examined using 10 mM
α-cyclodextrin, 2 mM β-cyclodextrin, and 10 mM γ-cyclodextrin. Dansyl-DL-aspartic
acid showed optical separation with the elution order of D-enantiomer and L-enantiomer,
but dansyl-DL-phenylalanine did not show any separation (data not shown). The
experimental data were consistent with the interaction abilities between dansyl aminoacids and cyclodextrin [33]. Fig. 9 shows the chromatograms of the mixtures ofdansyl-DL-aspartic acid and dansyl-DL-phenylalanine obtained with absorption
detection. The four isomers were separated on the chromatograms.
In conclusion, enantiomer separation of dansyl-DL-amino acids, such as
dansyl-DL-methionine and dansyl-DL-aspartic acid, was performed by the TRDC systemwith a water-rich carrier solution containing cyclodextrin. Cyclodextrin was not
immobilized on the inner wall surface but simply included in the carrier solution. The
219
Examination of various types of analytes with TRDC

219

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

observed elution order of D-enantiomer and L-enantiomer was explained based on the
tube radial distribution of the carrier solvents, providing the major water-rich and minor
organic solvent-rich phases. The results obtained here regarding enantiomer separation
provide important insights that indicate directions for future research to expand the
TRDC system.

a)

b)

220
220

Chapter 8

Figure 1. Phase diagrams for the ternary mixture solvents of water–acetonitrile–ethyl
acetate including a) 1 mM and b) 2 mM β-cyclodextrin. 1 and 2 mM β-cyclodextrin
were not dissolved in ternary mixture solutions with water component ratios of less thanca. 32 and 52 volume %, respectively. The curves in the diagrams indicate the boundary
between the homogeneous and heterogeneous phases. The component ratios of thesolvents for plots (I–VIII) that provide homogeneous solutions were positioned almoston the homogeneous–heterogeneous solution boundary curves of the phase diagrams.
The component ratios for the plots (I–VIII) in the diagrams are I)
water–acetonitrile–ethyl acetate of 100:0:10.5 volume ratio, II) 90:10:10.5 volume ratio,III) 75:15:10 volume ratio (15:3:2 volume ratio), IV) 80:20:10.5 volume ratio, V)
70:30:11.5 volume ratio, VI) 60:40:13.5 volume ratio, VII) 50:50:17.5 volume ratio,
and VIII) 40:60:23.5 volume ratio.

a)

b)

221
Examination of various types of analytes with TRDC

221

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesc)d)Figure 2. Chromatograms of a mixture of 2,6-naphthalenedisulfonic acid and 1-naphtholwith the capillary chromatography system using various component ratios ofwater–acetonitrile–ethyl acetate mixture including a) 1 mM and b) 2 mM β-cyclodextrin,c) 10 mM α-cyclodextrin, and d) 10 mM γ-cyclodextrin as carrier solutions. The
component ratios of the solvents for the plots (I–VIII) are described in Fig. 1.
2,6-Naphthalenedisulfonic acid and 1-naphthol were eluted in this order with certain
carrier solutions. Conditions: Capillary tube, 110 cm (90 cm effective length) of 100 µm
i.d. PTFE; carrier, water–acetonitrile–ethyl acetate mixture solution containing
cyclodextrin; sample injection, 20 cm height (gravity) × 5 s; flow rate, 0.8 µL min−1andanalyte concentration, 1 mM.
222
222

Chapter 8

Figure 3. Chromatograms of a mixture of 2,6-naphthalenedisulfonic acid and 1-naphtholwith a capillary chromatography system using water–acetonitrile–ethyl acetate mixture
carrier solution with 1 mM β-cyclodextrin and one without β-cyclodextrin. Conditions:
Capillary tube, 110 cm (90 cm effective length) of 100 µm i.d. PTFE; carrier,water–acetonitrile–ethyl acetate (15:3:2 volume ratio) mixture solution containing 1mM β-cyclodextrin and not containing β-cyclodextrin; sample injection, 20 cm height(gravity) × 5 s; flow rate, 0.8 µL min−1; and analyte concentration, 1 mM.

Figure 4. Chromatograms of the mixture of a) 500 µM dansyl-DL-methionine, b) 250µM dansyl-L-methionine, and c) 500 µM dansyl-DL-methionine plus 250 µM
dansyl-L-methionine mixture. Conditions: Capillary tube, 110 cm (90 cm effective
length) of 100 µm i.d. PTFE; carrier, water–acetonitrile–ethyl acetate (15:3:2 volume
ratio) mixture solution containing 1 mM β-cyclodextrin; sample injection, 20 cm height(gravity) × 5 s; and flow rate, 0.8 µL min−1.
223
Examination of various types of analytes with TRDC

223

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 5. Illustration of separation performance in the capillary chromatography systemusing water–acetonitrile–ethyl acetate mixture carrier solution including cyclodextrin.

a)

b)

224
224

Chapter 8c)d)Figure 6. Chromatograms of dansyl-DL-methionine obtained using various component
ratios of water–acetonitrile–ethyl acetate mixture including a) 1 mM and b) 2 mM
β-cyclodextrin, c) 10 mM α-cyclodextrin, and d) 10 mM γ-cyclodextrin as carrier
solutions. The component ratios of the solvents for plots (I–VII) are described in Fig. 1.
D-Enantiomer and L-enantiomer were eluted in this order with the certain carrier
solutions. Conditions: Capillary tube, 110 cm (90 cm effective length) of 100 µm i.d.
PTFE; carrier, water–acetonitrile–ethyl acetate mixture solution containing
cyclodextrin; sample injection, 20 cm height (gravity) × 5 s; flow rate, 0.8 µL min−1;
and analyte concentration, 500 µM.

225
Examination of various types of analytes with TRDC

225

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 7. Relationships between component ratios of the carrier solvents and resolutions(Rs) for the data calculated in Fig. 6.

Figure 8. Chromatograms of dansyl-DL-methionine obtained using various component
ratios of water–acetonitrile–ethyl acetate mixture including 10 mM α-cyclodextrin as
carrier solutions with fluorescence detection. The component ratios of the solvents forthe plots (I–VII) are described in Fig. 1. Conditions: Capillary tube, 110 cm (90 cm
effective length) of 100 µm i.d. PTFE; carrier, water–acetonitrile–ethyl acetate mixture
solution containing cyclodextrin; sample injection, 20 cm height (gravity) × 5 s; flow
rate, 0.8 µL min−1; and analyte concentration, 500 µM.
226
226

Chapter 8

Figure 9. Chromatograms of mixtures of dansyl-DL-amino acids obtained using
water–acetonitrile–ethyl acetate mixture including 10 mM α-cyclodextrin, 2 mM
β-cyclodextrin, and 10 mM γ-cyclodextrin as carrier solutions with absorption detection.
Conditions: Capillary tube, 110 cm (90 cm effective length) of 100 µm i.d. PTFE;
carrier, water–acetonitrile–ethyl acetate (15:3:2 volume ratio) mixture solution
containing cyclodextrin; sample injection, 20 cm height (gravity) × 5 s; flow rate, 0.8µL min−1; and analyte concentration, 500 µM.

227
Examination of various types of analytes with TRDC

227

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

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Chapter 9 Consideration
chromatography (TRDC)

of

tube

radialdistribution

TRDC was developed based on TRDP. The TRDC separation was considered fromvarious viewpoints, including temperature, outer phase formation, adding surfactant,and computer simulation. The part of this chapter is reconstructed and rewritten basedon related, published manuscripts.12,29,31,34)

9.1 Experiments and consideration through temperature effect in
TRDC

Chapter 9

We have developed a capillary chromatography system using an open capillary tube
made of fused-silica, polyethylene, or poly(tetrafluoroethylene), and awater-hydrophilic/hydrophobic organic mixture carrier solution. This so-called tube
radial distribution chromatography (TRDC) system works under laminar flow
conditions. Previously, the effects of capillary temperature on separation performance in
the TRDC system had been examined using a fused-silica capillary tube and awater-acetonitrile-ethyl acetate mixture carrier solution; 1-naphthol and
2,6-naphthalenedisulfonic acid in a model mixture were eluted with baseline separation
over a temperature range of 5–23 °C with the organic solvent-rich carrier solution. Here,based on the special assumption that the phase distribution of inner and outer phases in
the capillary tube induced by the tube radial distribution of the carrier solvents did notchange with temperature over a limited range, we attempted to apply the van’t Hoff
equation to the chromatographic data obtained at various capillary temperatures.
Introduction
Since the investigation of the TRDC system is still at the preliminary stage, it isimportant to examine elution behavior under various analytical conditions in the system
to expand our knowledge regarding its separation performance. In our study,10) we
examined the analytical conditions of the TRDC system in detail, including tube
temperature, tube inner diameter, tube length, and flow rate, using fused-silica capillarytubes and water-acetonitrile-ethyl acetate mixture as the carrier solution. Here, we tried
to examine the effects of capillary temperature on separation performance in the TRDC
system through the van’t Hoff equation on the assumption that the phase distribution of
the inner and outer phases did not change under a certain condition.
Experimental
The present capillary chromatography system comprised an open fused-silica capillarytube, 120 cm (effective length of 100 cm) of 75 µm i.d., microsyringe pump, and229
Consideration of TRDC

229

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

absorption detector. The tube temperature was controlled by dipping the capillary tube
in water preset at a definite temperature in a beaker while stirring. A
water-acetonitrile-ethyl acetate mixture with volume ratio of 3:8:4 was used as thecarrier solution. Analyte solutions were prepared with the carrier solutions. The analyte
solution was introduced directly into the capillary inlet by the gravity method (20 cm
height × 30 s). After the analyte injection, the capillary inlet was connected through a
joint to a microsyringe. The syringe was set on the microsyringe pump. The carrier
solution was fed into the capillary tube at a flow rate (0.8 µL min-1) under laminar flow
conditions. On-capillary absorption detection (254 nm) was performed with thedetector.
Results and discussion
We previously examined the effects of tube temperature on separation in the TRDC
system with a mixture analyte solution of 1-naphthol and 2,6-naphthalenedisulfuric acidas a model. The experiments were performed with the organic solvent-rich carrier
solution (water-acetonitrile-ethyl	
 acetate; 3:8:4 v/v/v) because the carrier solution
provided better resolution on the chromatograms than the water-rich carrier solution(water-acetonitrile-ethyl acetate; 15:3:2 v/v/v). The obtained chromatograms in ourwork are shown in Fig. 1 together with analytical conditions as a reference. As can beseen in Fig. 1, 1-naphthol and 2,6-naphthalenedisulfuric acid in the mixture solution
were detected with baseline separation within the temperature range of 5–23 °C, while
they were not separated at all at a temperature of 25 °C. More specifically, theresolution was improved with an increase in temperature from 5 °C to 20 °C but
suddenly decreased at 23 °C. The data clearly indicated that the tube temperature had asignificant, critical influence on the separation performance of the TRDC system.
In our chromatographic investigation, enthalpy (ΔH) is often calculated by using the
van’t Hoff equation as a standard method, leading to thermodynamical discussion, suchas exothermic (ΔH<0) or endothermic (ΔH>0) in the separation system. We attempted
to apply the van’t Hoff equation to the chromatographic data of TRDC system (Fig. 1),
for the first time. The equation is shown below, where ΔH, ΔS, T, R, and Φ are enthalpy,
entropy, absolute temperature, gas constant, and phase ratio, respectively.

ln k ʹ′ = −ΔH ΔS++ ln Φ
RT
R

Here the capacity factor (k') was calculated using the first peak as dead (hold-up) time.
In addition, we assumed that the phase distribution of the inner and outer phases of thecarrier solution in the tube due to the tube radial distribution of the solvents did notchange with temperature over the range of 5–20 °C.
The values of ln	
 k' were plotted against 1/T for the data obtained with the organic
solvent-rich carrier solutions (Fig. 2). Obviously, Fig. 2 does not show a simple linear
plot based on the separation performance observed on the chromatograms in Fig. 1.
Tentatively, we examined the value of enthalpy (ΔH) from the line over the temperature
230
230

Chapter 9

range of 10–20 °C where separation was observed with increasing resolution. The valueof enthalpy was roughly estimated to be 37.1 kJ mol-1. The enthalpy is generally
influenced by various types of heat, such as dissolution, adsorption, desorption, andsolvation. At the present it is difficult to discuss interactions between the fused-silica
inner-wall surface and the analytes that may contribute to separation performance in the
TRDC system. However, the obtained data for the fused-silica capillary tube would givea clue to further account for the separation performance (including the contribution of
the activity on the inner-wall surface) of the TRDC system from the viewpoint ofthermodynamics, comparing the theory with the data for polyethylene orpoly(tetrafluoroethylene) capillary tube.
	 Figure 1.
Figure.
1. Chromatograms
Chromatograms obtained
obtained at
at various
various capillary
capillary temperatures in the TRDC
10)system. Conditions: Capillary tube, 120 cm (effective length of 100 cm, the part of it(ca. 80 cm) was dipped in the temperature-controlled water) of 75 µm i.d. fused-silica;
carrier, water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection,
20 cm height (gravity)×30 s; flow rate, 0.8 µL min-1; tube temperature, 5–25 °C; and2,6-naphthalenedisulfonic acid and 1-naphthol, 1 mM each.

231
Consideration of TRDC

231

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2.
Figure.
2. TheThe van’t
van’t Hoff
Hoff plots
plots obtained
obtained inin the TRDC system. Conditions: Capillarytube, 120 cm (effective length: 100 cm) of 75 µm i.d. fused-silica; carrier,water-acetonitrile-ethyl acetate (3:8:4 v/v/v) mixture solution; sample injection, 20 cm
height (gravity)×30 s; flow rate, 0.8 µL min-1; tube temperature, 5–23 °C; and2,6-naphthalenedisulfonic acid and 1-naphthol, 1 mM each.

232
232

Chapter 99.2 Experiments and consideration through adding surfactants to an
analyte solution in TRDC
The influences on separation performance of adding surfactants to an analyte solution was
examined in a chromatographic system, tube radial distribution chromatography (TRDC),
using a fused-silica capillary tube (75 µm inner diameter and 100 cm effective length) and aternary mixture of water–acetonitrile–ethyl acetate (3:8:4 volume ratio) carrier solution.
Sodium dodecyl sulfate (anionic), ethylhexadecyldimethylammonium bromide (cationic), andTriton X-100 (nonionic) were used as surfactants. Model analytes, 1-naphthol and
2,6-naphthalenesulfonic acid, were separated in this order by adding the anionic and nonionicsurfactants. These surfactants in the analyte solution extremely improved the separation
performance (theoretical plate numbers for 2,6-naphthalenesulfonic acid; >10,000) comparedwith the same separation performed in the absence of surfactants. On the other hand, theanalytes were not separated at all using a cationic surfactant.
Instruction
Introduction
Various types of analytes, such as miscellaneous organic compounds, amino acids, peptides,
proteins, nucleosides, lambda-DNA, metal ions, metal complexes, fluorescent compounds,
polymer compounds, and optical isomers, were analyzed using the TRDC system. Throughall experimental data, we can notice a tendency for the analytes distributed in the outer phaseor pseudo-stationary phase to possess lower elution velocity and a consequential broadeningof their peak shapes. Here, we tried to improve the separation performance for the later peaks
by adding surfactants to the analyte solution. It was observed that the addition of anionic andnonionic surfactants could improve the resolution and theoretical plate numbers for the model
analytes.
Experimental
Fused-silica capillary tubes (75 µm inner diameter) were used. The TRDC system compriseda fused-silica capillary tube (120 cm total length and 100 cm effective length), a microsyringe
pump, and a fluorescence detector. The tube temperature was controlled by immersing thecapillary tube (40 cm) in water maintained at 15 °C in a beaker while stirring. A
water–acetonitrile–ethyl acetate mixture (3:8:4 volume ratio) was used as the carrier solution.
Analyte solutions of 1-naphthol and 2,6-naphthalenedisulfonic acid (1 mM each) wereprepared using the carrier solution either with or without surfactants. The analyte solution was
introduced directly into the capillary inlet side by the gravity method (30 cm height for 25 s).
After analyte injection, the capillary inlet was connected through a joint to a microsyringe.
The syringe was set on the microsyringe pump. The carrier solution was fed into the capillary
tube at a constant flow rate (0.5 µL min−1) under laminar flow conditions. On-capillary
fluorescence detection of the analytes was performed with the detector; ex. 290 nm and em.
355 nm.
233
Consideration of TRDC233

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Results and discussion
Figure 1 shows the chromatograms obtained through the use of the present TRDC system,
where the analytes were prepared with the carrier solution containing the surfactants, 20 mM
sodium dodecyl sulfate (SDS) (anionic), 10 mM ethylhexadecyldimethylammonium bromide(EHDAB) (cationic), and 2.0 wt% Triton X-100 (nonionic). On the other hand, the analytes
prepared with a surfactant-free carrier solution were used as a reference. Hydrophobic
1-naphthol and hydrophilic 2,6-naphthalenedisulfonic acid were separated in this order in theabsence and presence of surfactants (SDS and Triton X-100). The first peak (1-naphthol) was
eluted with an average linear velocity, while the second peak (2,6-naphthalenedisulfonic acid)was detected with a velocity lower than the average linear velocity under the laminar flow
conditions. The elution behavior of the analytes was reasonable because of their hydrophobic
and hydrophilic natures and also because of the TRDP created by the organic solvent-richcarrier solution generating major organic solvent-rich inner phase and minor water-rich outerphase. However, the analytes were not separated with the carrier solution containing EHDAB.
Furthermore, as is clearly shown in Fig. 1, the addition of SDS and Triton X-100 to theanalyte solutions improved the separation of the analytes significantly. The resolution andtheoretical plate numbers are calculated in the usual manner and summarized in Table 1
together with other experimental data. The theoretical plate numbers for
2,6-naphthalenedisulfonic acid with added SDS and Triton X-100 were 10,100 and 22,800,
respectively. These data are generally competitive with those obtained using capillaryelectrophoresis. However, it is significant that the present TRDC system featured suchsuperior separation performance without the necessity of applying high voltages or using any
specific columns, such as those of the monolithic or packed varieties.
The reason why the addition of surfactants to the analyte solutions provided such highresolution has not yet been determined. However, we tentatively suggest the following
explanation of our findings. The tentative suggestion is illustrated in Fig. 2. First, thebroadening of the second peak in the general system without surfactant might be caused by
the greater thickness of the micrometer order of the outer phase that functions as a
pseudo-stationary phase. The analyte having an anionic nature, 2,6-naphthalenedisulfonicacid, is easily surrounded by the surfactants, SDS (anionic) and Triton X-100 (nonionic), to
generate a micellar assembly. The formation of hydrophilic micelles might reduce adsorption
on the inner wall resulting in very sharp peaks. On the other hand, the anionic analyte wasstrictly surrounded with the cationic surfactant, EHDAB, generating reverse micellar
assemblies. The hydrophobic micelle containing the analyte must behave similarly to thehydrophobic analyte, 1-naphthol, in the capillary tube leading to no separation. As anothersuggestion, there is some turbulence nearby the interface between the two phases moving
downward in the capillary tube, since a shear stress is generated due to differences in physicalproperties of these phases. The addition of surfactants may suppress the influence of the
turbulence by the decrease in the friction coefficient between the two phases, leading todecrease in peak broadening or solute diffusion.
234
234

Chapter 9

Figure.
1. Chromatograms
Chromatograms ofof 1-naphthol
1-naphthol and 2,6-naphthalenedisulfonic acid (2,6-NDS)
Figure 1.
obtained with the present TRDC system using analyte solutions with or without
surfactants.

235
Consideration of TRDC

235

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2.
Figure.
2. Illustration
Illustration ofof thethe analyteanalyte distribution
distribution between
between thethe inner and outer phases withor without surfactants.

Table 1. Experimental data of the elution times (t1 and t2), the peak widths(W1 and W2), the capacity factors (k’), the resolutions (Rs), the theoreticalplate numbers (N), and the height of equivalent theoretical plate (H) in thepresent TRDC system. The subscript 1 and 2 are for 1-naphthol and
2,6-naphthalenedisulfonic acid, respectively.
Rs

N

H
/mm

0.47

3.32

625

1.60

0.28

0.22

5.84

22800

0.04

0.60

—

—

—

—

—

0.41

0.41

0.21

4.37

10100

0.10

Surfactant

t1
/min

t2
/min

W1
/min

W2
/min

k’

None

8.85

13.00

0.42

2.08

SDS

8.65

10.58

0.38

EHDAB

8.80

—

Triton
X-100

8.50

10.29

236
236

Chapter 99.3 Theoretical consideration through outer phase formation in TRDC
A capillary chromatography system that consists of an open capillary tube composed offusedsilica,polyethylene,andpoly(tetrafluoroethylene),andawater–hydrophilic/hydrophobic organic mixture carrier solution has been developed.
We call this system the tube radial distribution chromatography (TRDC) system. Here,variance, theoretical plate number, and height equivalents to a theoretical plate for asolute peak in the chromatogram were expressed using theoretical equations derived
from the differential equation for the secondary moment of a solute. The relationshipbetween the thickness and diffusion coefficient in the outer phase in the capillary tube
in the TRDC system was calculated from theoretical equations using numerical values
for analytical conditions and experimental data. The outer phase that featured the TRDC
system had a larger diffusion coefficient and a larger thickness than those of thestationary phase in standard liquid chromatography. We confirmed the separation
performance obtained on the basis of the tube radial distribution of the carrier solvents
under laminar flow conditions in the TRDC system by considering the inner and outerphase generation in the capillary tube.
Introduction
It is important to experimentally and theoretically examine separation performances in
the TRDC system. Here, for a solute peak in a capillary chromatogram, variance,
theoretical plate number, and height equivalent to a theoretical plate were successfully
expressed using theoretical equations derived from the differential equation for thesecondary moment of a solute reported by Aris [1]. The relationship between the
thickness and the diffusion coefficient in the outer phase in the capillary tube in the
TRDC system was estimated from the theoretical equations using the numerical values
for the analytical conditions and experimental data. We discuss the TRDC feature thatas a stationary phase, the outer phase consists of carrier solvents and has a larger
diffusion coefficient and thickness than those in standard liquid chromatographysystems.
Experimental
TRDC system with absorption detection
The TRDC system with UV absorption
spectrometer consisted of an open fused-silica capillary tube (75 µm i.d., 120 cm length;
100 cm effective length), a microsyringe pump, and an absorption detector. The tube
temperature was controlled by immersing the capillary tube in water maintained at atemperature of 15 °C in a beaker while stirring. Water–acetonitrile–ethyl acetate
mixtures with volume ratios of 3:8:4 and 15:3:2 were used as carrier solutions, andanalyte solutions including 1-naphthol and 2,6-naphthalenedisulfonic acid wereprepared with them. The analyte solution was directly introduced into the capillary inlet
side using the gravity method (20 cm height for 30 s). Then, the capillary inlet wasconnected through a joint to a microsyringe. The joint (PEEK-tough-connector) was
237
Consideration of TRDC

237

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

purchased from GL Science. It was used for the connection between the capillary tube
and from the PTFE tube to the microsyringe. The syringe was set on a microsyringe
pump. The carrier solution was fed into the capillary tube at a flow rate of 0.8 µL min−1
under laminar flow conditions. On-capillary absorption detection (254 nm) was
performed using a detector.
Fluorescence microscope–charge coupled device (CCD) camera systemA
fused-silica capillary tube of the same size as that used in the TRDC system withabsorption detection (75 µm i.d., 120 cm length) was set up for a fluorescencemicroscope–CCD camera system. The fluorescence in the capillary tube was monitored
using a fluorescence microscope equipped with an Hg lamp, a filter, and a CCD camera.
The organic solvent-rich carrier solution (water–acetonitrile–ethyl acetate; 3:8:4 volume
ratio) contained 0.1 mM perylene and 1 mM Eosin Y, and the water-rich carrier solution(water–acetonitrile–ethyl acetate; 15:3:2 volume ratio) contained 1 mM Eosin Y(perylene was only slightly dissolved in the solution). The carrier solution was
introduced into the capillary tube at a specific flow rate using a microsyringe pump.
Results and discussion
Differential equation of the secondary moment of a solute in a capillary tube Aristheoretically studied the dispersion in capillary chromatography and expressed it as adifferential equation that represents the secondary moment of a solute; the secondary
moment of a solute m(2) represents the variance of the solute [1]. The model conditions
for a fluid in a capillary tube are determined by considering the dispersion of the solute
dissolved in the fluid, which is introduced into the capillary tube under laminar flow
conditions. The inner and outer phases (corresponding to phases 1 and 2, respectively)
flow in the annular space between two coaxial cylinders of radii r0 and r2 in the capillarytube, the interface being a cylinder of equal axis and radius r1 (0 ≤ r0 < r1 < r2). A
schematic diagram of the inner and outer phases in a capillary tube is shown in Fig. 1.
We suppose that both phases move with a linear velocity that is a function of theradial distance from the axis, r, and the solute diffuses in both phases with a diffusion
coefficient that may also be a function of r (average the diffusion coefficient are used in
the following equations, though). At equilibrium, the solute concentration in phase 2, c2,
is a constant multiple of that in phase 1, i.e., c1 (c2 = αc1), and at any instant, the transfer
rate across the interface is proportional to the distance from the equilibrium at that point,i.e., c2 − αc1. Dispersion of the solute arises from three processes: (i) the combined
effects of diffusion and convection in phase 1; (ii) the finite transfer rate at the interface;and (iii) the combined effects of diffusion and convection in phase 2.
The standard equations (I) for diffusion and flow under the above mentioned
boundary conditions are derived as follows.

238
238

Chapter 9∂c1$= 0 (r = r0 ) (a)
!
∂r
!
∂c1
∂c1 )
∂ 2 c1
∂c1
1 ∂ ,
= D1
(r0 < r < r1 ) (b) !!
* rψ 1 ( r )' + D1ψ 1 (r ) 2 − U 1φ1 (r )r ∂r +
∂t
∂r (
∂x
∂x
!
∂c1
∂c2
!
D1ψ 1 (r )= D2ψ 2 (r )= k (c2 − αc1 ) (r = r1 ) (c)
# (I)
∂r
∂r
!
!
∂c2
∂c2 )
∂ 2 c2
∂c2
1 ∂ ,
= D2
(r1 < r < r2 ) (d)!
* rψ 2 ( r )' + D2ψ 2 (r ) 2 − U 2φ2 (r )r ∂r +
∂t
∂r (
∂x
∂x
!
!
∂c2
= 0 ( r = r2 ) (e)	  D2ψ 2 (r )!
∂r"The standard equations (I) are a set of five equations labeled (a), (b), (c), (d), and (e).
The differential equations expressing the three processes (i), (ii), and (iii) are (b), (c),and (d), respectively. Equations (a) and (e) represent the condition where there is noflow over the boundaries at r = r0 and r = r2.
The following notations are used, and unless otherwise stated, the suffixes 1 and 2
refer to quantities in the inner and outer phases, respectively: ci, solute concentration; Ui,
average linear velocity; Uiφ (r), linear velocity at radius r; Di, average diffusion
coefficient; Diψi(r), diffusion coefficient at radius r; x, the coordinate along the common
axis; r, radial coordinate; and t, time. Furthermore, for simplicity, the variable of length
is changed along the axis so that its origin moves with constant speed V. The quantity V
is later chosen to move at the speed of the center of gravity of the solute (y = x − Vt).
Finally, the following differential equation for the secondary moment of a solute wasintroduced in the paper by Aris [1].
D1ψ 1 (r )

ι

(

)

(

)

2
*
*
1 dm ( 2 )U 2 r 2 − r02 'U 22 r22 − r12 ' {β (1 − β )s(U1 − U 2 )}()
1= β ) D1 + κ1 1 1
+
−
+
+
β
D
κ
&
) 2
&
2
2 dt
2kαr1
D1
D2
(
%
(
%
Here,

dr '
R r 2 − r02 *κ1 = ∫ r(Φ1 (r) −+0 2rψ (r)γ r12 − r02 ,
)
1
r1

€

2

dr '
R r22 − r 2 $κ2 = ∫
(
r
)
Φ
−
#
& 2
r1 2rψ ( r )γ − 1 r22 − r12 "
2
%
r2

(1)

2

r

∫ 2r "φ (r ")dr "Φ (r ) =
1

1

r0

2
1

r − r02r2∫ 2r "φ (r ")dr "Φ (r ) =
2

r

1

2
2

r − r12r2 − r2
β = 1 2 0 : fraction of the solute in the inner phase at equilibriums

239
Consideration of TRDC

239

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesγ=

U1: ratio of the inner phase velocity to the relative velocity
U1 − U 2

(

) (

)

s 2 = r12 − r02 + α r22 − r12 .
Variance of solute in capillary chromatography
The equation expressing thevariance of a solute in capillary chromatography is derived from the differential
equation (Eq. 1) for the secondary moment of a solute under the following conditions:(i) the the diffusion coefficient are constant in the phases, (ii) the inner phase (mobile
phase) moves under laminar flow conditions, (iii) there is no flow in the outer phase(stationary phase), and (iv) r0 = 0.
As the mobile phase moves under laminar flow conditions, the distribution of theflow rate is expressed as a parabolic curve. Then, the following equation is obtained.

(

	
  	
  	
  U 1φ1 (r ) = C r12 − r 2)Here, U1 is the average linear velocity; therefore,
r1

r1∫ U φ (r ) ⋅ 2πrdr = 2C ∫ (r − r )rdr = 1 Cr .
	
  	
  	
  U =
0

1

1 1

Accordingly,

2
1

0

2
1

πr

2

2
1

r

(

2

2
1

)

1 2
Cr1 φ1 (r ) = C r12 − r 2
2
	
  	
  	
 
⎛ r 2 ⎞
φ1 (r ) = 2⎜1 − 2 ⎟.
⎜ r ⎟1 ⎠
⎝
From the above equation, the following equation is obtained for κ1.
U1φ1 (r ) =

r2 &
dr )
κ1 = ∫(Φ1 (r ) − R 2 %0 2 rψ ( r )
r1 $
1
'
r1

	
  	
  	
 

2

r1
r1
r2
dr
dr
dr r 4 2
Φ1 ( r ) 2 − ∫Φ1 ( r ) 2 R + ∫R
0 2 rψ ( r )0 rψ ( r )0 2rψ ( r ) r 4
r1
1
1
1
1

=∫

r1

The first term in the above equation is calculated as follows.

2' r 2r (2 1 − r 2 r 2 dr ( $
r1
r
1
dr
dr ! ∫0
11
1
!
	
  	
  	
  ∫
Φ1 ( r ) 2 = ∫# =&2
0 2 rψ ( r )0 2 rψ ( r )
r1
48
1
1
!"
!%
The second term is written as follows.

()( r 2r )2 1 − r 2 r 2 dr ) % 2
1rdr
dr " ∫0
1
"r
	
  	
  	
  − ∫
Φ1 ( r ) 2 R = − ∫$ 2R=− R'20 rψ ( r )0
r1
rψ 1 ( r ) "
r1
3
1
"# r1
&
The third term is written as follows.
r1

2

(

r1

240
240

Chapter 9)

r1

r 4 2 &1 r 4 # 2 1 2
dr
	
  	
  	
  ∫
R =$ 4! R = R
0 2 rψ ( r ) r 4
81
1% 8 r1 " 0
r1

Finally, the following equation is obtained.
11 1
1
1
	
  	
  	
  κ1 =− R + R2 =11 − 16 + 6 R 2
48 3
8
48

(

)

The condition of no flow in the stationary phase provides the equation U2 = 0. Then,
U1Uγ== 1 =1U1 − U 2 U1
	
  	
  	
 

U1

γ

=

U2
γ −1

U2
.
γ →1 γ − 1∴U1 = lim

As Φ2(r) expresses the flow from r to r2, it is clear that Φ2(r) = 0 for U2 = 0. Then,⎛ R r22 − r 2 ⎞⎟⎜ −
κ2 = ∫
r1 2rψ 2 (r ) ⎜ γ − 1 r 2 − r 2 ⎟
2
1 ⎠
⎝
r2

	
  	
  	
 

dr

2

2

2
⎛ R ⎞ r2
dr ⎛⎜ r22 − r 2 ⎞⎟⎟⎟ ∫.
= ⎜⎜
⎝ γ − 1 ⎠ r1 2rψ 2 (r ) ⎜⎝ r22 − r12 ⎟⎠In the second term of Eq. 1, the component in the parentheses is rewritten as

(

	
  	
 

2

)

(

2
U 2 r 2 − r 2 ⎛ R ⎞ r2
dr ⎛⎜ r22 − r 2 ⎞⎟ U 22 r22 − r12⎟⎟ ∫⋅
κ 2 2 2 1 = ⎜⎜D2D2⎝ γ − 1 ⎠ r1 2rψ 2 (r ) ⎜⎝ r22 − r12 ⎟⎠)2r22 − r12 r2
dr ⎜⎛ r22 − r 2 ⎞⎟.
=R(γ − 1)2 D2 ∫r1 2rψ 2 (r ) ⎜⎝ r22 − r12 ⎟⎠2

U 22

From γ →1, the following equation is obtained.

(

)

(

)

2

U 2 r2 − r2
U 2 r2 − r2 r
dr ⎛⎜ r22 − r 2 ⎞⎟
	
  	
  	
  κ 2 2 2 1 = R 2 1 2 1 ∫ 2.
r1 2rψ ( r ) ⎜ r 2 − r 2 ⎟D2
D22
⎝ 2 1 ⎠

Then,

241
Consideration of TRDC

241

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules2⎛ r22 − r 2 ⎞ 2⎜⎟ R
κ2 = ∫
r1 2rψ ( r ) ⎜ r 2 − r 2 ⎟2
⎝ 2 1 ⎠
3 ⎞
r2 dr ⎛ r24
1⎜ − r22 r 2 + r ⎟R 2
	
  	
  	
 
=
2 ∫r1 ψ (r ) ⎜ 2r
2 ⎟⎠2⎝r22 − r12
dr

r2

(

	
  	
  	
 

)

2
⎧ r 4 r 4⎛ r2 ⎞ ⎛⎜ r22 ⎞⎟⎫⎪ 2⎪21=ln ⎜⎜ ⎟⎟ − 3 − 1 ⎬ R .
⎨2
8 r22 r12 − 1 ⎪⎩ r22 r12 − 1 ⎝ r1 ⎠ ⎜⎝ r12 ⎟⎠⎪⎭

(

1

)

r
By substituting ρ = 2 ,
r1

κ2 =
	
  	
  	
 

=

P=

ρ 2 −1, the following equation is obtained.
ln ρ 2⎧⎪ ρ 4⎫⎪ 2
2
2
2
ln
ρ
−
3
ρ
−
1
⎨
⎬ R
⎪⎭
8 ρ 2 − 1 ⎪⎩ ρ 2 − 1

(

1

)

(

(

)

)R2

2 ρ 4 P −1 − 3ρ 2 − 1()

8 ρ 2 −1
From the above, Eq. 1 is rewritten as follows.

(

)

(

)

)
&
)
1 dm ( 2)U 2r 2U 2 r 2 − r 2 2 ρ 4 P −1 − 3ρ 2 − 1 2 &
R %= R ( D1 + 1 1 11 − 16 + 6 R 2 % + (1 − R )( D2 + 1 2 1
2
2 dt
48
D
D
8
ρ
1
−
1
2
'
$
'
$

(

+

)

(

)

{R(1 − R )sU1}22kαr1
By integrating the above equation with t, the following equation is obtained.

(

)

(

)

'
U 12 r12
U 12 r22 − r12 2 ρ 4 P −1 − 3ρ 2 − 1
2(1 − R )R 2
m = t &2 RD1 +R 11 − 16 + 6 R + 2(1 − R )D2 + 2
2
24 D1
D2
8 ρ −1%2{
R(1 − R )sU 1} #
+
"
kαr1
!
The secondary moment of a solute expresses the variance (σ2) of the solute on t.
When the capillary length is L, the time (tR) required to reach the outlet is given by
L
L
	
  	
  	
  tR = =.
V U1 R

(

( 2)

)

(

)

The variance σ2(L) of a solute at the outlet is then expressed as follows.	
  	
 

(

)

(

)

L '
U12 r12
U 12 r22 − r12 2 ρ 4 P −1 − 3ρ 2 − 1
2(1 − R )R 2
R 11 − 16 + 6 R + 2(1 − R )D2 + 2
σ ( L) =&2 RD1 +
2
U1R %
24 D1
D2
8 ρ −1

(

2

)

2
{
R(1 − R )sU 1} #
+

(

)

"
!
This equation represents the variance σ2(L) of solute distribution in a real capillarycolumn.
kαr1

242
242

Chapter 9Variance, theoretical plate number, and the height equivalent to a theoretical plate fora solute peak in a chromatogram The variance σ2(tR) for a solute peak eluted in thechromatogram is expressed by changing the variable as follows.
L11
σ 2 (tR ) = σ 2 ( ) = 2 σ 2 ( L) = 2 2 σ 2 ( L)V
V
U1 R
Accordingly, the variance σ2(tR) is expressed by the following equation.

(

)

(

)

L '
U12 r12U12 r22 − r12 2 ρ 4 P −1 − 3ρ 2 − 1
2(1 − R )R 2
R 11 − 16 + 6 R + 2(1 − R )D2 + 2
σ (tR ) = 3 3 &2 RD1 +
2
U1 R %
24 D1
D2
8 ρ −1

(

2

)

(

)

2
{
R(1 − R )sU1} #+"!
The theoretical plate number and the height equivalent to a theoretical plate,respectively, are expressed as follows.
kαr1

2

2

& t # &t #
&t #
	
  	
  	
  N = 16$ R ! = 16$ R ! = $ R !
% 4σ " % σ "
%W "
	
  	
  	
  H =

2

2 2
L
L
L
2 L
2
2 U1 R
=
=
σ
=
σ
=
σ
N (tR σ )2
tR2
L(L U1R )2

When σ2(tR)isintroduced

in

the

above

(

)

equation,

(

we

)

get	
 

D U 2r 2(1 − R ) D2 + 2 U1 r22 − r12 2 ρ 4 P −1 − 3ρ 2 − 1 (1 − R )R
H = 2 1 + 1 1 11 − 16 + 6 R 2 + 2
R U1
D2
U1 24 D1
8 ρ 2 −1()

RU1 (1 − R )2 s 2.
kαr1
Using the capacity factor k', where	
  	
  	
 
1
	
  	
  	
  R =
,
1 + k!
the height equivalent to a theoretical plate H is expressed as follows.

(

)

+

H =2

(

)

(

)

D1 U12 r12 1 + 6k ʹ′ + 11k ʹ′2
D
U r 2 − r 2 2 ρ 4 P −1 − 3ρ 2 − 1
k ʹ′
U s 2 k ʹ′2++ 2k ʹ′ 2 + 2 1 2 1
+ 1
2
2
2
U1 24 D1
U1
D2
8 ρ −1(1 + k ʹ′)(1 + k ʹ′) kαr1 (1 + k ʹ′)3

(

)

(2)
The above equation represents the height equivalent to a theoretical plate for capillarychromatography with an inner phase (mobile phase) and an outer phase (stationary
phase); D1 is the inner phase diffusion coefficient; D2 is the outer phase diffusion
coefficient; r1 is the inner radius of the capillary tube (from the axis to the inner–outerphase surface); r2 is the outer radius of the capillary tube (from the axis to the capillarywall surface); U1 is the average linear velocity, and k' is the capacity factor.
Basic experimental data for the TRDC system Basic experimental data for the TRDC
system, i.e., chromatograms and fluorescence images, were examined in our studies.
These data must again be used to examine the relationship between the thickness and243
Consideration of TRDC

243

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

diffusion coefficient in the outer phase obtained from the theoretical equations in thisstudy. The data are summarized in Fig. 2.
A model mixture solution of 1-naphthol and 2,6-naphthalenedisulfonic acid wasanalyzed using the TRDC system with absorption detection. The obtained
chromatograms are shown in Fig. 2A. Using an organic solvent-rich carrier solution ofwater–acetonitrile–ethyl acetate with a volume ratio of 3:8:4, the mixture of 1-naphtholand 2,6-naphthalenedisulfonic acid was separated through an open capillary tube andthe order of detection was 1-naphthol and 2,6-naphthalenedisulfonic acid (Fig. 2A-1). In
contrast, using a water-rich carrier solution of water–acetonitrile–ethyl acetate with avolume ratio of 15:3:2, the mixture solution was separated and detected with inverse
elution times, i.e., the order of detection was 2,6-naphthalenedisulfonic and 1-naphthol(Fig. 2A-2). In both chromatograms, the first peaks were detected with elution times
roughly corresponding to average linear velocities, and the second peaks were detectedwith elution times roughly corresponding to below average linear velocities. The
chromatograms clearly indicated the reversibility of the elution times by changing thesolvent ratio in the carrier solution.
In addition, we examined the fluorescence images and profiles of the fused-silica
capillary tube. The experiment with the fluorescence–CCD camera system and thefused-silica capillary tube was performed under almost the same analytical conditions(capillary tube length, tube inner diameter, and flow rate) as those used for the TRDC
system with absorption detection (i.e., the chromatograms shown in Fig. 2A). Fig. 2Bshows the fluorescence images and profiles obtained for a fused-silica capillary tube
into which dye-containing mixed aqueous–organic solvent carrier solutions wereintroduced.
The fluorescence images and profiles for the organic solvent-rich carrier solution
showed that the hydrophobic perylene molecule (blue) was distributed around the tube
center, whereas the hydrophilic Eosin Y molecule (green) was distributed near the tube
inner wall (Fig. 2B-1). In contrast, the fluorescence images and profiles showed that
Eosin Y was distributed around the capillary tube center for the water-rich carrier
solution (Fig. 2B-2). The tube radial distribution of perylene and Eosin Y present in the
carrier solutions in the capillary tube confirmed a partition reversal between the organic
solvent-rich and water-rich carrier solutions (Fig. 2B).
The distributions of perylene and Eosin Y at the capillary tube center and near its
inner wall, respectively, supported the idea of nonuniform dispersion of the aqueous andorganic components of the carrier solutions in the capillary tube in the TRDC system.
They generated a major inner phase (organic solvent-rich or water-rich) and a minor
outer phase or capillary wall phase (water-rich or organic solvent-rich) in the capillary
tube. As a result, the analytes were also distributed between the inner and outer phases
depending on their nature and were separated by chromatography (i.e., corresponding to
the chromatograms shown in Fig. 2A).
Relationship between the thickness and diffusion coefficient in the outer phase
244
244

Chapter 9As

described in the previous sections, the elution behaviors of the analytes and the
distributions of the dyes dissolved in the carrier solvents in the capillary tube, illustrated
by the chromatograms, supported the separation performance obtained on the basis of
the tube radial distribution of the carrier solvents in the TRDC system, as proposed in
our papers. Here, we examined the relationship between the thickness and the diffusion
coefficient in the outer phase from theoretical equations using experimental dataobtained from the chromatogram (Fig. 2A) and the fluorescence image and profile (Fig.
2B) obtained for the organic solvent-rich carrier solution. These data were used because
for the fused-silica capillary tube in the TRDC system, the chromatographic resolution
with the organic solvent-rich carrier solution was better than that using the water-richcarrier solution. In addition, the organic solvent-rich carrier solution gave clear
fluorescence images and profiles with perylene and Eosin Y.
As shown in Fig. 2A-1, the baseline separation of the analytes 1-naphthol and 2,
6-naphthalenedisulfonic acid was achieved completely with the organic solvent-richcarrier solution. The chromatogram shown in Fig. 2A-1 provided the following
separation performance for the second peak of 2,6-naphthalenedisulfonic acid under the
analytical conditions of a tube inner diameter of 75 µm and an average linear velocity of
3.0 × 10−3 ms−1: resolution, 6.0; theoretical plate number, 900; height equivalent to atheoretical plate, 1.1 mm; and capacity factor, 0.87. By fluorescent dyes observation,formation of inner and outer phases due to the distribution of the carrier solvents was
similarly observed as in a capillary tube with an effective length of 20–100 cm.
Although a capillary tube with an effective length of less than ca. 20 cm could not be seton the fluorescence microscope stage for fluorescence observations, the radial
distribution of fluorescent dyes in the microchannels (effective lengths 0.5–3 cm) of a
microchip were clearly observed. With reference to the experimental data, the heightequivalent to a theoretical plate was estimated using the capillary effective length of 100
cm.
Using Eq. 2 along with the above-mentioned numerical values such as the theoreticalplate number, height equivalent to a theoretical plate, capacity factor, tube innerdiameter, and average linear velocity, we attempted to calculate the relationshipbetween the thickness and diffusion coefficient in the outer phase, or capillary wall
phase. We used a diffusion coefficient of 2.0 × 10−9 m2 s−1 of a solute in ethyl acetate
for the inner phase (although we also used this in acetonitrile, 2.5 × 10−9 m2 s−1, for the
inner phase, there was little difference in the calculated results; the diffusion coefficientof the inner (mobile) phase generally has little influence on the separation performance).
Furthermore, it was assumed that in the TRDC system, the outer phase moved slightly
and its movement could be neglected under laminar flow conditions. The relationshipbetween the thickness and diffusion coefficient in the outer phase is shown in Fig. 3.
The outer phase, in the TRDC system, functions similarly to the stationary phase instandard liquid chromatography. However, the outer phase in TRDC is a very specificstationary phase. It comprises the carrier solvents and the organic solvent-rich orwater-rich solution. Therefore, the outer phase in the system has a larger diffusion
245
Consideration of TRDC

245

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

coefficient and a larger thickness (in the order of microns) than those of the stationary
phase in standard chromatography. In fact, the large diffusion coefficient and thicknessof the outer phase, which acts as the stationary phase in the TRDC system, might resultin broadening of the second peaks shown in Fig. 2A.
At present, it is impossible to directly measure the diffusion coefficient and thickness
in the outer phase in the TRDC system. However, if the process used to derive therelated equations is accurate, the diffusion coefficient and thickness values in the outerphase should theoretically exist on the relationship curve in Fig. 3. We attempted toobtain the values by assuming that the relationship (Fig. 3) is valid and using thefollowing numerical values of the diffusion coefficient and thickness. First, thediffusion coefficient of poly(dimethylsiloxane), a high viscosity liquid which has also
been used as a stationary phase in coated-capillary chromatography [2], is 8.9 × 10−13
m2 s−1. The real diffusion coefficient in the present TRDC system must be larger thanthat of poly(dimethylsiloxane). Second, the fluorescence image and profile shown in
Fig. 2B-1 obtained with the organic rich-solvent carrier solution clearly indicatedformation of inner and outer phases in the capillary tube. We estimated the thickness ofthe outer phase from the inflection of the fluorescence profiles in Fig. 2B-1 and found to
be < ca. 8 µm.
These two numerical values, i.e., the diffusion coefficient of poly(dimethylsiloxane)(8.9 × 10−13 m2 s−1) and the estimated thickness (8 µm), are clearly shown on the curvein Fig. 3. If the process used to plot the relationship curve in Fig. 3 is inaccurate, such a
specific part of the curve between the two values would not appear. The diffusion
coefficient and thickness in the relevant part of the curve were both larger than those fora stationary phase in standard chromatography. In conclusion, the relationship between
the thickness and the diffusion coefficient in the outer phase estimated from Eq. 2 using
the present analytical conditions and experimental data was consistent with the featuresof the outer phase in the TRDC system, i.e., a large diffusion coefficient and a large
thickness of the order of microns. Therefore, in the TRDC system, the relationshipbetween the thickness and the diffusion coefficient in the outer phase theoreticallysupports the chromatographic separation performance obtained on the basis of the tube
radial distribution of the carrier solvents in the capillary tube.
In conclusion, using experimental data, inner and outer phase formation due to thetube radial distribution of the carrier solvents was considered from the elution behaviorsof the analytes in chromatograms, the distributions of dyes dissolved inaqueous–organic solvent mixture carrier solutions in the capillary tube, and theoreticalestimations of the thickness and diffusion coefficient in the outer phase. The dataobtained both experimentally and theoretically were consistent with our proposed
separation performance explained on the basis of tube radial distribution of the carrier
solvents.

246
246

Chapter 9

Figure 1.
Figure.
1. Schematic
Schematic diagram
diagram ofof thethe inner
inner andand outerouter phases
phases inin aa capillary
capillary tube.
tube. The
The inner
inner
and outer phases, phase 1 and phase 2, respectively, flow in the annular space between
two coaxial cylinders of radii r0 and r2 in the capillary tube, the interface being acylinder of equal axis and radius r1 (0 ≤ r0 < r1 < r2). The standard equations (I) are
derived based on this model conditions for a fluid in a capillary tube (the details aredescribed in the text).

247
Consideration of TRDC

247

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2.
2,6-naphthalenedisulfonic
Figure.
2. A)A) Chromatograms
Chromatograms ofof aa mixture
mixture ofof 1-naphthol
1-naphthol andand 2,6-naphthalenedisulfonicacid obtained by the TRDC system with absorption detection; B) fluorescence images
and profiles of fluorescent dyes dissolved in ternary mixed carrier solutions observed by
a fluorescence microscope–CCD camera system. Conditions A: Capillary tube, 120 cm(effective length: 100 cm), i.d. 75 µm, fused silica; carrier 1) water–acetonitrile–ethyl
acetate (3:8:4 v/v/v) solution, and 2) water–acetonitrile–ethyl acetate (15:3:2 v/v/v)
solution; sample injection, 20 cm height (gravity) × 30 s; flow rate, 0.8 µL min−1; tube
temperature, 15 °C; and analyte concentration, 1 mM. Conditions B: Capillary tube, 120
cm (effective length: 100 cm), 75 µm i. d., fused silica; carrier, 1)
water–acetonitrile–ethyl acetate (3:8:4 v/v/v) mixture containing 0.1 mM perylene and 1
mM Eosin Y, and 2) water–acetonitrile–ethyl acetate (15:3:2 v/v/v) mixture containing
1 mM Eosin Y; and flow rate, 0.8 µL min−1.

248
248

Chapter 9

Figure 3.
icient in
Figure.
3. Relationship
Relationship between
between thethe thickness
thickness andand diffusion
diffusion coeff
coefficientin thethe outerouterphase.

249
Consideration of TRDC

249

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

9.4 Consideration through computer simulation in TRDC
When the ternary mixed solvents of a water-hydrophilic/hydrophobic organic solvent
mixture are delivered under microspace under laminar flow conditions, the solvent
molecules are radially distributed in the microspace, and generate a major inner phaseand a minor outer phase. We call this fluidic phenomenon as the tube radial distribution
phenomenon (TRDP). Here, phase formation in the TRDP was collectively consideredbased on experimental data, such as the inner and outer phase formation in a
microchannel under laminar flow conditions, the phase diagram for the ternary mixed
solvents, the solvent-component ratios required for the TRDP, and the phasetransformation in a batch vessel above atmospheric pressure, which were mainly
reported in our papers. Furthermore, the formation of inner and outer phases in acapillary tube was simulated with the two-component solvents mixture model of waterand ethyl acetate. Phase formations in capillary tubes were expressed through computer
simulations.
Introduction
The TRDP creates a liquid–liquid (aqueous–organic) interface in a microflow from ahomogeneous aqueous–organic mixture solution in a batch vessel. The phase interface
is specific; it is kinetic, not static. That is, the TRDP is a new phase interface concept,
and can be applied to various technological, engineering, and scientific fields. We are
currently investigating the TRDP from the viewpoints of chromatography, extraction,and chemical reaction space. Here, the formation of inner and outer phases in the TRDP
is considered based on experimental data that were reported in our papers. Also, theresults of computer simulations agree well with the phase formation observed throughthe TRDP.
Experimental
A microchip made of glass incorporating a microchannel line (100 µm wide × 40 µmdeep) was used. The fluorescence in the microchannel was monitored using afluorescence microscope equipped with an Hg lamp, a filter, and a CCD camera. The
ternary mixed solvent solutions were delivered into the microchannel using a
microsyringe pump. Fluorescence photographs, which contained mainly blue andgreen color because perylene and Eosin Y emit light at 470 and 550 nm, respectively,
were transformed into line drawings to assess the color depth (red, green, and blue
(RGB)). The line drawings were then digitized, and the numbers were standardized
to the line-drawing data to give the fluorescence profiles. The phase formation in acapillary tube was computer-simulated with the volume of the fluid method (VOF)(Fluent program; ANSYS, Inc., Canonsburg, USA). The simulation conditions wereas follows. The carrier solution was a water-ethyl acetate mixture with a 3:7 volume
ratio (organic solvent-rich) and a water-ethyl acetate mixture with a 7:3 volume ratio
250
250

Chapter 9(water-rich). The capillary length was 1,000 µm, the capillary inner diameter was 50µm, the contact angles of 30° and 150° of water (water-rich outer phase) on the innerwall expressed the hydrophilic and hydrophobic nature of the inner wall against theouter phase, respectively; as well as contact angles of 30° and 150° of the organic
solvent (organic solvent-rich outer phase) on the inner wall expressed thehydrophobic and hydrophilic nature of the inner wall against the outer phase,respectively. The linear velocity was 3.7 mm s-1, the outlet pressure was atmosphericpressure (1.0 × 105 Pa), the total mesh number was 807,500 (radial size length, 1.1µm; axial size length, 20 µm), and the capillary was filled with the carrier solution inadvance.
Results and discussion
Under specific microflow conditions of the ternary carrier solvents of awater-hydrophilic/hydrophobic organic solvent, an organic solvent-rich major inner
phase and a water-rich minor outer phase were generated by the TRDP in an organic
solvent-rich carrier solution. In contrast, with a water-rich carrier solution, a water-richmajor inner phase and an organic solvent-rich minor outer phase were generated,regardless of the inner wall material in a microspace. However, specific homogeneous
solutions which of the compositions of the ternary solvents are positioned near to thehomogeneous-heterogeneous boundary in the phase diagram are required for the TRDP.
It is possible that pressure changes imposed on the solution in a microspace underlaminar flow conditions might alter the carrier solution from a homogeneous one in abatch vessel to a heterogeneous one in a microspace.
In our previous paper [9], microchannel fluorescence photographs for the organic
solvent-rich solution (water-acetonitrile-ethyl acetate; 3:8:4 volume ratio) containing
perylene (0.1 mM) and Eosin Y (1 mM) and the water-rich solution (80:20:9 volume
ratio) containing perylene (0.1 mM) and Eosin Y (0.1 mM) were examined with afluorescence microscope-CCD camera (Figs. 1(a) and 1(b), respectively). A phase
diagram of the ternary mixed solvents is also shown in Fig. 2, and the component ratiosof the above-mentioned solutions are plotted in the diagram. For an organic solvent-richsolution, hydrophobic perylene was distributed around the middle of the microspace inthe microchannel, and the relatively hydrophilic Eosin Y was distributed near the innerwall (Fig. 2(a)). In the water-rich solution, Eosin Y was distributed around the middleof the channel and perylene was distributed near the inner wall (Fig. 2(b)).
We considered why major solvents are distributed around the middle of the tube,
whereas minor solvents are distributed close to the inner wall, irrespective of whether
the carrier solutions are organic solvent-rich or water-rich [9]. The linear velocity in theradial face section of the capillary tube (circle curves) under laminar flow conditions
indicates that the inside area of a tube has a lower velocity-change gradient than theoutside area. The real velocity curve in an aqueous-organic solvent mixed solution may
deviate from an ideal velocity curve. Considerations of fluidic stability based on the251
Consideration of TRDC251

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

linear velocity gradients in the radial profile under laminar flow conditions imply thatthe major solvents must occupy the inside area instead of the outside area in thecapillary tube (Fig. 3).
Here, we tentatively tried to propose another view of the inner and outer phaseformation in the TRDP, based on the above and below experimental data. A specifichomogeneous solution, whose component ratio was near the boundary between
homogeneous and heterogeneous in the phase diagram, was pressurized greater thanatmospheric pressure with nitrogen gas in a batch vessel where it was under the controlof gravity. In an organic solvent-rich solution (e.g., water-acetonitrile-ethyl-acetate
mixture; 3:8:4 volume ratio), the homogeneous solution first became an emulsion or
nontransparent solution that included minor solvent or water-rich tiny droplets and thenchanges to a heterogeneous solution that included an upper major organic solvent-richsolution and a lower minor water-rich solution under the control of gravity. In a similarway, in a water-rich solution (e.g., water-acetonitrile-ethyl-acetate mixture; 80:20:9
volume ratio), the homogeneous solution first became an emulsion solution that
included minor solvent or organic solvent-rich tiny droplets, and then changed to aheterogeneous solution that included an upper minor organic solvent-rich solution and alower major water-rich solution (Fig. 4).
The phase formation of the ternary mixed solvents in a capillary tube under pressure
due to laminar flow is estimated as follows, where it is not under the control of gravity.
A specific homogeneous solution with a component ratio near the boundary between
homogeneous and heterogeneous in the phase diagram is delivered under laminar flow
conditions that produce a constant pressure to the radial face. In an organic solvent-richsolution, the homogeneous solution first becomes an emulsion solution that includes
minor solvent or water-rich tiny droplets. The water-rich tiny droplets start to aggregate
near the inner wall where the linear velocity is lowest under laminar flow conditions.
Consequently, the ternary mixed solvent solution in a tube changes to a heterogeneous
solution that includes a major inner organic solvent-rich solution and a minor outerwater-rich solution, where it is not under the control of gravity. Similarly, in awater-rich solution, the homogeneous solution becomes an emulsion solution that
includes minor solvent or organic solvent-rich tiny droplets. The organic solvent-richtiny droplets start to aggregate near the inner wall where the velocity is lowest underlaminar flow conditions. The ternary mixed solvent solution in a tube changes to aheterogeneous solution that includes a major inner water-rich solution and a minor outerorganic solvent-rich solution where it is not under the control of gravity (Fig. 5).
In order to better understand the tube radial distribution of the solvents in a capillarytube, the formation of inner and outer phases in a capillary tube was simulated with amixture of the two-component solvents, a water and ethyl acetate mixture, which were
not homogeneously mixed. As the volume of fluid method (VOF) (Fluent program)functions for multi-phase flow model and actual fluid in the capillary tube generates thetwo phases, the above mixture of the two-component solvents was adopted as a modelin this simulation. Phase-formation images in the organic solvent-rich and water-rich
252
252

Chapter 9

carrier solutions were obtained with the simulation program under the conditions
described in the Experimental section. They are shown in Figs. 6 and 7, respectively.
These images are generated at 0.25, 0.50, 1.00, 2.00, and 3.00 s from the start of theflow. The blue color is composed of 100% volume ratio water per a mesh and the redcolor is 100% volume ratio ethyl acetate per a mesh. The volume ratios between 100%water (blue) and 100% ethyl acetate (red) per a mesh are expressed with 18 color
gradients from blue to red. The color gradient bars are also shown in Figs. 6 and 7.
As shown in Fig. 6, with the organic solvent-rich carrier solution, a minor water-richthin layer (blue) is first generated very close to the inner wall, following which theaqueous–organic mixed solvents become a heterogeneous solution that includes minor
water-rich outer (blue) and major organic solvent-rich inner (from red to yellow green)phases. Such distributions are observed in the simulation images, regardless of whether
the inner-wall characteristics were hydrophilic (contact angle 30°) or hydrophobic(contact angle 150°). With the water-rich carrier solution (Fig. 7), regardless of whether
the inner wall is hydrophilic or hydrophobic, a minor organic solvent-rich thin layer(red) is first generated near the inner wall, following which the aqueous–organic mixed
solvents become a heterogeneous solution that includes minor organic solvent-rich outer(red) and major water-rich inner (from blue to yellow) phases. The simulation images
for phase formation in a capillary tube are consistent with the phase formation observed
experimentally through the TRDP. From experimental data reported in our previous
papers, the TRDP must be constructed under rather complicated conditions, including
the composition ratios of solvents, flow rates, the inner diameter of the tubes, andpressure. The results obtained here imply that the computer simulation is useful for ourinvestigation of the TRDP, such as setting the conditions and designing the microfluidic
device.
In conclusion, the phase formation in the TRDP was explained in one point of view
based on the experimental data as follows. The ternary mixed homogeneous solution ina microflow first becomes an emulsion solution that includes minor tiny solvent
droplets. The tiny droplets then start to aggregate near the inner wall where the linear
velocity is lowest under laminar flow conditions, leading to a heterogeneous solutionthat includes a phase interface in a microflow. Also, the results of our computer
simulation were consistent with the concept of TRDP, i.e., inner and outer phaseformations in microspaces.

253
Consideration of TRDC

253

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1.
Fluorescence photographs
photographs ofof a)a) organicorganic solvent-richsolvent-rich andand b)b) water-richwater-rich
Figure.
1 Fluorescencesolutions in the microchannel. Conditions: Carrier, a) perylene (0.1 mM) and Eosin Y(1.0 mM) dissolved in water-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio) andb) perylene (0.1 mM) and Eosin Y (0.1 mM) dissolved in water-acetonitrile-ethyl
acetate mixture (80:20:9 volume ratio) and flow rate, 1.0 μL min-1.

Figure.
2 Phaseacetate mixture
mixture andand thetheFigure 2.
Phase diagram
diagram forfor aa water-acetonitrile-ethyl
water-acetonitrile-ethyl acetate
component ratios of the solvents. Water-acetonitrile-ethyl acetate volume ratio (a) 3:8:4
and (b) 80:20:9. The homogeneous-heterogeneous solution boundary curve isrepresented by the dotted curve.

254
254

Chapter 9

Figure 3.
Schematic depictions
depictions of
the possible
possible distribution
distribution patterns
minor
Figure.
3 Schematicof thepatterns ofof major
major andand minor
9phases in a capillary tube.

Figure.
of phasephase formation
formation inin ternary
ternary mixed
mixed solvents
solvents inin aa batch
batch vessel
vesselFigure 4.4 Illustration
Illustration ofunder pressure with nitrogen gas where it is under the control of gravity.
255
Consideration of TRDC

255

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure.
5 Illustration
Figure 5.
Illustration ofof phasephase formation
formation ofof ternary
ternary mixed
mixed solvents
solvents inin aa capillary
capillary tube
tube
under pressure due to laminar flow where it is not under the control of gravity.

Figure 6.
Phase formation
formation inin capillary
capillary tubes
tubes obtained
obtained byby aa computer
computer simulation.
simulation. Organic
Organic
Figure.
6 Phasesolvent-rich carrier solution.

256
256

Chapter 9

Figure 7.
Phase formation
formation inin capillary
capillary tubes
tubes obtained
obtained byby aa computer
computer simulation.
simulation.
Figure.
7 PhaseWater-rich carrier solution.

References[1] R. Aris, Proc. R. Soc. London, Ser. A, 1959, 252, 538.
[2] K. Hibi, D. Ishii, I. Fujishima, T. Takeuchi, T. Nakanishi, J. High Resolut.
Chromatogr., Chromatogr. Commun., 1978, 1, 21.

257
Consideration of TRDC

257

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Chapter 10 Development
extraction (TRDE)

of

tube

radialdistribution

A TRDP-based extraction in a microspace, referred to as tube radial distribution
extraction (TRDE), has been performed as one of the applications of TRDP. The TRDE
was developed using a microchip having a triple-branched microchannel and using adevise having double tubes of different inner diameters. The part of this chapter isconstructed rewritten based on related, published manuscripts.16,32,50)

10.1 TRDE using a microchip that has triple-branched microchannels

Chapter 10

Microfluidic behavior of ternary mixed carrier solvents of water–acetonitrile–ethyl
acetate (2:3:1 volume ratio) was examined by use of a microchip incorporating
microchannels in which one wide channel was separated into three narrow channels, i.e.,
triple-branched microchannels. When the ternary carrier solution containing thefluorescent dyes, hydrophobic perylene (blue) and relatively hydrophilic Eosin Y(green), was fed into the wide channel under laminar flow conditions, the carrier solvent
molecules or fluorescence dyes were radially distributed in the channel, forming inner(organic solvent-rich major; blue) and outer (water-rich minor; green) phases in thewide channel. And then, in the narrow channels, perylene molecules mostly appeared toflow through the center narrow channel and Eosin Y, which is distributed in the outerphases in the wide channel, flowed through the both side narrow channels. A metal ion,Cu(II) as a model, dissolved in the ternary mixed carrier solution was also examined.
The Cu(II) showed fluidic behavior, transferring from the homogeneous carrier solution
to the water-rich solution in the side narrow channels through the triple-branchedmicrochannels.
Introduction
The development of micro total analysis systems that include a microchip or
microfluidic device technology is an interesting aspect of analytical chemistry [1,2].
Microfluidics exhibit various types of fluidic behavior in solvents in a microchannel.
The microfluidic behavior has been examined by varying the channel configuration andflow rate of the solvents, by using aqueous–organic solvent mixtures, and by
introducing specific obstacles into the microchannel [3-5]. The fluidic behavior of thesolvents in the microchannel is related to the mixing, separation, diffusion, and reactionof the solutes. Information regarding the flow pattern of the solvents is important anduseful to design microreactors or micro total analysis systems [6,7].
We reported that when ternary mixed solvents of water–hydrophilic/hydrophobicorganic solvents are fed into a microspace, such as microchannels in a microchip or
capillary tubes, the solvent molecules are radially distributed under laminar flow
258
258

Chapter 10

conditions. This is called the tube radial distribution phenomenon (TRDP). In TRDP, inan organic solvent-rich carrier solution, the organic solvent-rich major phase is formed
around the center of the microspace as an inner phase, while the water-rich minor phase
is formed near the inner wall as an outer phase. On the other hand, in the water-richcarrier solution, the water-rich major phase is formed as an inner phase and the organic
solvent-rich minor phase as an outer phase. TRDP forms a specific liquid–liquidinterface, which is not static but kinetic, in a microspace.
Until now, TRDP has been investigated by the use of mainly capillary tubes andstraight single line microchannels. The triple-branched microchannels in which one
wide channel was separated into three narrow channels in a microchip was designed andmanufactured in our study to conform TRDP, where the ternary mixed carrier solution
was fed from the three narrow channels to the one wide channels.14) Here microfluidic
behavior of the ternary mixed carrier solution in the triple-branched microchannels was
examined with the reverse flow direction against the previous paper,14) i. e., the flow
direction form the one wide channel to the three narrow channels. By these experiments,
we could observe unique fluidic behavior of the solvents and solutes in the
microchannels based on TRDP. The preliminary results were reported in acommunication previously.16)
Experimental
Materials
Figure 1 illustrates the setup of the microchip in the microchip holder (Fig.
1 a)) and the enlarged view of the microchip incorporating the triple-branchedmicrochannels (Fig. 1 b)). As shown in Fig. 1 b), one wide channel (300 µm wide × 40
µm deep) denoted as channel W was separated into three narrow channels (each 100 µm
wide × 40 µm deep) designated as channels N1–N3.
Fluorescence microscope equipped with a CCD camera system
A microchip
incorporating the microchannels was set up for the fluorescence microscope–CCD
camera system. Fluorescence in the microchannel was monitored using a fluorescencemicroscope equipped with an Hg lamp, a filter, and a CCD camera. The ternary mixed
solvents of the water–acetonitrile–ethyl acetate mixture (3:8:4 volume ratio) contained
0.1 mM perylene and 1 mM Eosin Y. The carrier solution was fed into themicrochannels using a microsyringe pump.
Cu(II) analysis
The homogenous carrier solution of the water–acetonitrile–ethyl
acetate mixture (2:3:1 volume ratio) including 2.0 mM Cu(II) was fed into channel W ata definite flow rate. 100 µL of the solution in channels N1–N3 was collected throughpolytetrafluoroethylene (PTFE) tubes into the corresponding vessel. The solution wasdried under a vacuum, and 0.5 M ammonia solution (100 µL) was added to the residue
for flow absorption measurement at 600 nm (modified SPD-6AV spectrophotometricdetector, Shimadzu Co., Kyoto, Japan).
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Results and discussion
Fundamental experiments for TRDP in a microchannel
Microchips withsingle-branched microchannels having widths of 100, 200, 300, and 400 µm andidentical channel depths of 40 µm were also manufactured. The influences of channel
width on TRDP in the microchannels were examined with fluorescent dyes, perylene
and Eosin Y, dissolved in an organic solvent-rich carrier solution(water–acetonitrile–ethyl acetate; 3:8:4 volume ratio). The fluorescence images of thedyes in the solution fed into the microchannels were observed with the fluorescencemicroscope–CCD camera system (Fig. 2). The flow rates in all microchannels (100–400µm width) were adjusted to achieve the same average linear velocity of 25 cm min−1. It
is evident from Fig. 2 that TRDP was observed in all microchannels regardless of thechannel width. Hydrophobic perylene molecule (blue) was distributed around the centerof the channel away from the side inner walls of the channel, while relativelyhydrophilic Eosin Y molecule (green) was distributed near the side inner walls of thechannel. That is, the major inner phase (organic solvent-rich phase) and the minor outerphase (water-rich phase) were formed in the microchannels of 100–400 µm width.
Effects of flow rates on TRDP in triple-branched microchannels The effects of flowrate on TRDP in the triple-branched microchannels was examined with an organic
solvent-rich carrier solution (water–acetonitrile–ethyl acetate, 3:8:4 volume ratio)
containing Orange G by using the bright-field microscope–CCD camera system. The
Orange G-containing carrier solution was fed at the flow rates of 2.0–50.0 µL min−1.
The images are shown in Fig. 3. The hydrophilic Orange G molecule was distributednear the channel side inner walls, and the interface between the water-rich outer phaseand the organic solvent-rich inner phase was distinguished because of the color of
Orange G (Fig. 3). The TRDP was observed for the first time with a bright-fieldmicroscope–CCD camera system in our TRDP investigation. The system wasconfirmed to be useful for TRDP observation in a similar way to the fluorescencesystem. At the low flow rates of 2.0 and 5.0 µL min−1, the liquid–liquid interface wasunstable, considerably changed, or appeared to instantaneously disappear, however,stability was observed at the flow rates higher than 5 µL min−1. Thus, flow rates higher
than 5 µL min−1 created a stable liquid–liquid interface in the triple-branchedmicrochannels under the present conditions.
Fluorescence images of the dyes based on TRDP in triple-branched microchannels
The microchip incorporating triple-branched microchannels was shown in Fig. 1 b) with
their respective measurements. The organic solvent-rich carrier solution(water–acetonitrile–ethyl acetate, 3:8:4 volume ratio) containing Eosin Y and perylene
was fed into channel W of the microchip at the flow rate of 6.3 µL min−1. The mixture
carrier solution excluding ethyl acetate (water–acetonitrile, 1:4 volume ratio) but
containing the dyes was also fed into the channel as a reference. Fig. 4 shows the260
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fluorescence images of the dyes in the triple-branched microchannels of the microchip.
Figs. 4 a) and b) were obtained with the water–acetonitrile mixture as a reference andthe water–acetonitrile–ethyl acetate mixture as ternary mixed solvents, respectively. The
distribution behavior of the dyes, i.e., TRDP, was not observed in the image of Fig. 4 a)with the water–acetonitrile mixture. Conversely, the image of Fig. 4 b) with the ternarysolvent mixture shows TRDP, in which the hydrophobic perylene molecule (blue) was
distributed around the center of channel W away from the channel side inner walls,
while relatively hydrophilic Eosin Y molecule (green) was distributed near the channelside inner walls. The distribution phenomena of the ternary mixed carrier solvents fed
into the microchannels was evident in the wide channel. And then, perylene molecules
mostly appeared to flow through the center channel (channel N2) and Eosin Y, which is
distributed in the outer phases in channel W, flowed through channels N1 and N3, asshown in Fig. 4 b).
Distribution of Eosin Y based on TRDP in triple-branched microchannels The
homogenous carrier solution of the water–acetonitrile–ethyl acetate mixture (3:8:4
volume ratio) including 0.5 mM Eosin Y was fed into channel W at a flow rate of 10 µL
min−1. 100 µL of the solution in channels N1–N3 was collected through PTFE tubes
into the corresponding vessel. The solution was subjected to flow absorption
measurement at 517 nm. Eosin Y concentrations in the center channel (channel N2) andthe two side channels (channels N1 and N3) were 0.36, 0.59, and 0.58 mM, respectively(Fig. 4). The concentrations were averages for five measurements. The experimental
data indicated that relatively hydrophilic Eosin Y was distributed from thehomogeneous carrier solution to the water-rich solution in the side narrow channels
rather than the organic solvent-rich solution in the central narrow channel. The
distribution of the fluorescent dyes in the triple-branched microchannels might suggest
the possibility of extraction or separation for some solutes by utilizing the phaseinterface formed by TRDP in these channels.
Distribution of Cu(II) based on TRDP in triple-branched microchannels Distributionof Cu(II) was examined in the triple-branched microchannels by utilizing the kinetic
liquid–liquid or aqueous–organic interface formed by TRDP. First, the calibration curveof Cu(II) was examined in a manner similar to that described in the experimental
section. Cu(II) was determined over the concentration range of 0.5–10 mM with good
linearity (R2 = 0.999). The homogenous carrier solution of the water–acetonitrile–ethyl
acetate mixture (2:3:1 volume ratio) including 2.0 mM Cu(II) was fed into channel W at
the flow rate of 40 µL min−1. 100 µL of the solution in channels N1–N3 was collectedwith PTFE tubes into the corresponding vessel. Cu(II) concentrations in the center widechannel (channel N2) and the two side channels (channels N1 and N3) were 0.8, 2.5,
and 2.7 mM, respectively (Fig. 6 a)). The concentrations were averages for fivemeasurements. As a reference, Cu(II) concentrations were examined with thewater–acetonitrile (2:3 volume ratio) carrier solution in a similar manner. Cu(II)261
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

concentrations in channels N1–N3 were 2.1, 2.1, and 2.0 mM, respectively (Fig. 6 b)).
The experimental data indicated that Cu(II) was extracted to the water-rich solution in
the side narrow channels from the homogeneous solution by the formation of a phaseinterface in the microchannel; we refer to this process as tube radial distribution
extraction (TRDE). Moreover, we examined the Cu(II) distribution by TRDE at otherflow rates of 32 and 63 µL min−1 in channel W. At 32 µL min−1, Cu(II) concentrationsin the center channel (channel N2) and the two side channels (channels N1 and N3)
were 0.8, 3.0, and 2.9 mM, whereas, at 63 µL min−1, those were 1.1, 2.9, and 2.7 mM,
respectively. Comparing with the Cu(II) concentration, 2.0 mM, in the homogeneous
carrier solution, the Cu(II) concentration in the solution collected from the centerchannel (channel N2) decreased, while that from the two side channels (channels N1
and N3) increased at every flow rates. The obtained data showed that TRDE occurred in
the triple-branched microchannels of the microchip.
In conclusion, TRDP has been investigated by the use of mainly capillary tubes andstraight single line microchannels. The TRDP of the ternary mixed carrier solution in
the triple-branched microchannels was observed with a fluorescence microscope-CDD
camera system. Consequently, perylene molecules mostly appeared to flow through thecenter narrow channel and Eosin Y flowed through channels in the side narrow channels.
The TRDP was also observed through bright-light microscope-CCD camera system. We
tried to apply the phase interface formed by TRDP, i.e., inner and outer phases, to the
distribution or extraction procedure of Cu(II) in the triple-branched microchannels. This
process will be helpful for investigating separation and extraction in a microfluidic
device.

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a)

b)

Figure 1.
Figure.
1. Schematic
Schematic representation
representation ofof a)a) setup of the microchip in the microchip holder
and b) the enlarged view of the microchip incorporating triple-branched microchannels.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2.
uorescent dyes
Figure.
2. Fluorescence
Fluorescence images
images ofof ﬂfluorescentdyes dissolved
dissolved inin a ternary mixed carrier
solvent in microchannels with channel widths of 100, 200, 300, and 400 mm andidentical channel depth of 40 µm. Conditions: Carrier, water–acetonitrile–ethyl acetate(3:8:4 v/v/v) mixture including 0.1 mM perylene and 1 mM Eosin Y; and flow rates, 1,
2, 3, and 4 µL min−1 for microchannel widths of 100, 200, 300, and 400 µm,
respectively.

Figure.
3. Effects
Effects ofof ﬂflow
rates of the carrier solution on TRDP. Conditions: Carrier,Figure 3.
ow rateswater–acetonitrile–ethyl acetate (3:8:4 v/v/v) mixture containing 2 mM Orange G andflow rate, 2.0–50 µL min−1 for channel W.

Figure 4.
uorescent dyes
Figure.
4. Fluorescence
Fluorescence images
images ofof ﬂfluorescentdyes dissolved
dissolved inin a mixed carrier solventin triple-branched microchannels. a) Water–acetonitrile (1:4 v/v) and b)
water–acetonitrile–ethyl acetate (3:8:4 v/v/v) mixtures. Conditions: Carrier,aqueous–organic mixtures including 0.1 mM perylene and 1 mM Eosin Y; and flow rate,
6.3 µL min−1 for channel W.

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Figure 5.
Figure.
5. Distribution
Distribution ofof Eosin
Eosin YY fromfrom thethe homogeneous
homogeneous ternary
ternary mixed solution into
the outer phases in the side narrow channels by TRDP. Conditions: Carrier,water–acetonitrile–ethyl acetate (3:8:4 v/v/v) mixture; flow rate, 10 µL min−1 forchannel W; Eosin Y concentration in the homogeneous solution, 0.50 mM.

Figure 6.
Figure.
6. Distribution
Distribution ofof Cu(II)Cu(II) in triple-branched microchannels. a)
Water–acetonitrile–ethyl acetate (2:3:1 v/v/v) mixture and b) water–acetonitrile (2:3v/v) mixture carrier solution. Conditions: Flow rate, 40 µL min−1 for channel W andCu(II) concentration in the homogeneous solution, 2.0 mM.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules10.2 TRDE using a device having double tubes of different innerdiameters
A novel microflow-extraction system was proposed using double capillary tubes having
different inner diameters in tube radial distribution phenomenon (TRDP). The tubes
were fused-silica capillary tubes with 75 and 250 µm inner diameters; the smaller tube
was inserted into the larger one through a T-type joint. A homogeneous aqueous
solution containing 12 wt% Triton X-100 as a non-ionic surfactant and 2.4 M potassium
chloride was fed into the larger tube at a flow rate of 20 µL min-1, where the tube wasmaintained at a temperature of 34 °C. The homogeneous aqueous solution changed to aheterogeneous solution with two phases in the tube; the surfactant-rich phase was
generated in the middle of the tube as an inner phase, while the aqueous phase
containing little of the surfactant was formed near the tube wall as an outer phase. In the
TRDP Rhodamine B dissolved in the homogeneous solution was distributed into theinner surfactant-rich phase. The distributed Rhodamine B (red color) was observed witha bright-field microscope-CCD camera system. In the present microflow-extraction
system, taking advantage of the TRDP, the inner phase containing Rhodamine B flowed
inside the smaller tube, while the outer phase flowed outside the tip of the smaller tube
into the larger tube, which was made using double tubes having inner diameters. Thisobservation showed that Rhodamine B dissolved in the homogeneous solution was
separated or extracted into the inner surfactant-rich phase through the double capillarytubes based on the specific microfluidic behavior of TRDP.
Introduction
The fluidic behavior of the solvents in the microspace is related to the mixing,
separation, diffusion, and reaction of the solutes. Extraction is, generally, one of themost useful and effective separation methods; however, recently, microflow solvent
extraction systems have been investigated by a liquid-liquid interface between an
aqueous and organic solvent solution in a microspace [8-10]. The microflow solvent
extraction has several advantages over conventional macroscale solvent extraction. For
example, the microflow system has a larger surface/interface area per unit volume thanthat of the conventional macroscale system and the increase in the interface area
provides enhancement of extraction efficiency [8].
We reported that when homogeneous solutions that feature a two-phase separation
property, such as a water-acetonitrile-ethyl acetate mixed solvent solution and non-ionicsurfactant aqueous solution, are fed into a microspace, such as microchannels in a
microchip or capillary tubes, the solvent molecules are radially distributed in the
microspace under laminar flow conditions. This is called the tube radial distribution
phenomenon (TRDP). For example, in the TRDP with a non-ionic surfactant aqueous
solution, through the two-phase separation property, the surfactant-rich phase is formed
around the center of the microspace as an inner phase, while the aqueous, or almost
surfactant-free, phase is formed near the inner wall as an outer phase.44) The TRDP
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creates a specific liquid–liquid interface that is not static but kinetic in a microspace,providing an inner and outer phase.
Here, a novel microflow-extraction system was proposed using double capillarytubes having different inner diameters: smaller and larger ones.45) A homogeneous
aqueous solution containing a non-ionic surfactant was fed into the larger tube, leading
to the TRDP that created the inner surfactant-rich phase and the outer almost
surfactant-free aqueous phase. At the tip of the smaller tube in the larger one, the inner
phase flowed inside the smaller tube, while, the outer phase flowed outside the smaller
tube. The specific flow showed the possibility that a certain substance dissolved in thehomogeneous solution was extracted into the inner phase, or surfactant-rich phase,through the double capillary tubes, based on the TRDP.
Experimental
Fused-silica capillary tubes (75 µm inner diameter and 150 µm outer diameter; 250 µm
inner diameter and 350 µm outer diameter) were used. A schematic diagram of thepresent microflow extraction system using double capillary tubes, having different innerdiameters, is shown in Fig. 1. The smaller tube (75 µm i.d.) was inserted into the larger
one (250 µm i.d., 120 cm length) through a T-type joint. A homogeneous aqueous
solution containing 12 wt% Triton X-100 and 2.4 M KCl was fed into the larger tube ata flow rate of 20 µL min-1, where the tube was maintained at a temperature of 34 °C
with a thermo-heater (Thermo Plate MATS-555RO; Tokai Hit Co., Shizuoka, Japan).
The homogeneous aqueous solution changed to a heterogeneous solution with twophases in the capillary tube. The radial distribution of the surfactant was observed
through Rhodamine B dissolved in the homogeneous aqueous solution with abright-field microscope-CCD camera system that was comprised of a microscope and a
CCD camera.
Results and discussion
Two-phase separation in a batch vessel Homogeneous aqueous solutions of specificnon-ionic surfactants separate into two distinct phases in a batch vessel when heated
above a certain temperature (i.e., cloud point); a temperature-induced phase separation
occurs. One phase behaves as a concentrated surfactant solution containing considerable
amounts of water (surfactant-rich phase), whereas the other phase is an almost
surfactant-free aqueous solution. The hydrogen bond between an oxygen atom of an
ether bond in a non-ionic surfactant and water molecule leads to good solubility of thesurfactant in water. However, a high temperature above the cloud point attempts to cut
the hydrogen bond, leading to separation of surfactant-rich and aqueous phases. In
addition, the cutting or dihydration increases in the presence of salts. Hydrophobic
compounds dissolved in the homogeneous aqueous solution are extracted into thesurfactant-rich phase, whereas hydrophilic compounds remain in the aqueous phase.
Clouding points of an aqueous solution containing 2 wt% Triton X-100 as a non-ionicsurfactant were examined through inspection with various concentrations of KCl, 0–3.0
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

M. The obtained data, or phase diagram, is shown in Fig. 2. The homogeneous andtransparent solution at 25 °C changed through a clouding point into the heterogeneous
solution including two phases: the upper surfactant-rich suspension phase and theaqueous lower phase. The clouding points gradually decreased from 65 °C to 30 °C
with an increasing KCl concentration from 0 to 3.0 M as shown in Fig. 2. Also,
clouding points of an aqueous solution containing 2.4 M KCl were examined withvarious concentrations of Triton X-100, 0–30 wt%. The homogeneous solution at 25 °Cchanged through a clouding point to the heterogeneous solution including two phases,
the surfactant-rich phase and the aqueous phase, at 34 °C for all concentrations of
Triton X-100 (data not shown).
TRDP observation
Homogeneous aqueous solutions containing Triton X-100 (2, 12,22, and 30 wt%) and 2.4 M KCl possessed a clouding point at 34 °C, as described above.
The homogeneous solutions that included 5 mM Rhodamine B were fed into thecapillary tube (250 µm i. d.) at a flow rate of 20 µL min-1 under laminar flow conditions,where the tube was maintained at temperatures of 25 °C or 34 °C. The bright-field
photographs of the aqueous solution were examined with the microscope-CCD camera
system. Non-TRDP (homogeneous) was observed at 25 °C and TRDP was created at
34 °C for all aqueous solutions. Typical photographs obtained from the aqueous
solution containing 12 wt% Triton X-100 and 2.4 M KCl are shown in Fig. 3. The
homogeneous red solution as a result of Rhodamine B was observed at a temperature of
25 °C, which was under the clouding point. That is, the distribution of the surfactant in
the tube did not occur at a temperature of 25 °C (non-TRDP). At a temperature of 34 °C,
the solution phase containing the surfactant was generated in the middle of the tube as
an inner phase (the surfactant-rich phase) and the solution phase containing little of thesurfactant was formed near the inner wall as an outer phase (the aqueous phase).
Rhodamine B dissolved into the solution was distributed in the surfactant-rich phase(red color). The homogeneous aqueous solution containing 12 wt% Triton X-100 and
2.4 M KCl with a clouding point of 34 °C clearly brought about phase transformation to
the heterogeneous solution with the surfactant-rich phase and the aqueous phase throughthe tube, the temperature of which was controlled at 34 °C (TRDP).
Microflow extraction with TRDP The homogeneous aqueous solution containing 12
wt% Triton X-100, 2.4 M KCl, and 5 mM Rhodamine B was fed at a flow rate of 20 µL
min-1 into the larger capillary tube. The aqueous solution containing 12 wt% Triton
X-100 was selected mainly because of the volume ratio of the two phases and because
the viscosity of the solution could be easily delivered into the capillary tube. The
photographs obtained at the tip of the smaller tube within the larger one at 25 °C and
34 °C are shown in Fig. 4. At 25 °C, the homogeneous red solution resulting from
Rhodamine B was fed without any specific fluidic behavior. On the other hand, notable
and specific fluidic behavior was observed on the tip of the smaller tube at 34 °C. The
inner phase (red color) containing Rhodamine B flowed inside the smaller tube, while
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Chapter 10the outer phase flowed outside the small tube, as shown in Fig. 4. This observation
implies that Rhodamine B dissolved into the homogeneous solution was separated or
extracted into the inner phase (surfactant-rich phase) through the double capillary tubes
based on specific microfluidic behavior, TRDP.
In conclusion, a novel microflow extraction was demonstrated through double
capillary tubes having different inner diameters, a smaller and larger tube, in thespecific microfluidic behavior of TRDP. A homogeneous aqueous solution containing
Triton X-100 as a non-ionic surfactant and KCl was fed into the larger tube, where thetube was maintained at the temperature of the clouding point. The homogeneous
aqueous solution changed to a heterogeneous solution through two phases in the tube:
the surfactant-rich inner phase and nearly surfactant-free outer phase (TRDP).
Rhodamine B dissolved into the homogeneous solution was distributed into thesurfactant-rich inner phase in the TRDP. Rhodamine B was extracted into thesurfactant-rich phase through the double capillary tubes based on the specificmicrofluidic behavior of TRDP.

Figure 1.
ow extraction
Figure.
1. Schematic
Schematic diagram
diagram ofof microﬂmicroflow
extraction systemsystem using double capillarytubes having different inner diameters.

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Development of TRDE

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 2.
Figure.
2. Phase
Phase diagram
diagram ofof aqueousaqueous solution
solution containing
containing Triton
Triton X-100
X-100 and KCl. 2 wt%
Triton X-100 and various concentrations of KCl. The solubility curve between
homogeneous and heterogeneous solutions is depicted with the clouding points.

Figure 3.3. Bright-f
ield photographs
Figure.
Bright-field
photographs inin thethe larger
larger capillary tube (250 µm i.d.) at 25 °C
and 34 °C. The homogeneous aqueous solution containing 12 wt% Triton X-100, 2.4 M
KCl, and 5 mM Rhodamine B was fed at a flow rate of 20 µL min-1 into the larger
capillary tube.

Figure.
4. Bright-f
Bright-field
photographs at
at thethe tip
tip of the smaller capillary tube (75 µm i.d.)Figure 4.
ield photographs
within the larger tube (250 µm i.d.) at 25 °C and 34 °C. The homogeneous aqueous
solution containing 12 wt% Triton X-100, 2.4 M KCl, and 5 mM Rhodamine B was fed
at a flow rate of 20 µL min-1 into the larger capillary tube.
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References[1] K. Ueno, F. Kitagawa, and N. Kitamura, Bull. Chem. Soc. Jpn., 2011, 77, 1331.
[2] D. R. Reyes, D. Iossifidis, P. A. Auroux, and A. Manz, Anal. Chem., 2002, 74, 2623.
[3] Y. Kikutani, H. Hisamoto, M. Tokeshi, and T. Kitamori, Lab. Chip, 2004, 4, 328.
[4] A. Hibara, M. Tokeshi, K. Uchiyama, H. Hisamoto, and T. Kitamori, Anal. Sci.,
2001, 17, 89.
[5] N. Kaji, Y. Okamoto, M. Tokeshi, and Y. Baba, Chem. Soc. Rev., 2010, 39, 948.
[6] H. Nakamura, Y. Yamaguchi, M. Miyazaki, H. Maeda, and M. Uehara, Chem.
Commun., 2002, 23, 2844.
[7] H. Kawazumi, A. Tashiro, K. Ogino, and H. Maeda, Lab. Chip, 2002, 2, 8.
[8] M. Maeki, Y. Hatanaka, K. Yamashita, M. Miyazaki, and K. Ohto, Solent. Extr. Res.
Dev., Jpn., 2014, 21, 77.
[9] Z. X. Cai, Q. Fang, H. W. Chen, and Z. I. Fang, Anal. Chem. Acta, 2006, 556, 151.
[10. A. Aota, M. Nonaka, A. Hibara, T. Kitamori, Angew. Chem. Int. Ed, 2007, 46, 878.
[11] K. Fujinaga, Anal. Sci., 1993, 9, 479.
[12] T. Saitoh, H. Tani, T. Kamidate, H. Watanabe, Trends Anal. Chem., 1995, 14, 213.
[13] P. I. Trindade, M. M. Diogo, D. M. F. Prazeres, C. J. Marcos, J. Chromatogr. A,
2005, 1082, 176.

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Chapter 11 Development of tube radial distribution mixing(TRDM)A TRDP-based specific mixing of solvents in a microspace, referred to as tube radial
distribution mixing (TRDM), has been developed as an application of TRDP. The
TRDM was developed using a microchip on which three narrow channels werecombined to form one wide channel. The part of this chapter is constructed andrewritten based on the related manuscript that has been published.26)

11.1 TRDM using a microchip having three-to-one line microchannels
The mixing process of ternary solvents (water–hydrophilic/hydrophobic organic
mixture) prepared in microchannels in a microchip was examined by fluorescenceobservation of the dyes dissolved in the solvents under laminar flow conditions. A
microchip incorporating microchannels was used. In it, three narrow channels werecombined to form one wide channel. Water–acetonitrile (hydrophilic) mixture
containing relatively hydrophilic Eosin Y (green) was fed into the narrow centerchannel and an acetonitrile–ethyl acetate (hydrophobic) mixture containing hydrophobic
perylene (blue) was fed into the two narrow side channels in the microchip. The
mixtures in the narrow channels combined in the wide channel to prepare the ternarysolvents of water–acetonitrile–ethyl acetate, causing the tube radial distribution of thesolvents. We observed the mixing process of the ternary solvents in the wide channel
through fluorescence of the green and blue dyes, including an aqueous–organicinterface. For example, the green dye that was fed into the center channel was
distributed near the inner side walls and the blue dye that was fed into the two sidechannels was distributed around the center area in the wide channel. Such specificmixing behavior was not observed for two-component solvents in the wide channel,
such as water–acetonitrile mixture and water–ethyl acetate mixture.
Chapter 11

Introduction
The development of micro total analysis systems that include a microchip or
microfluidic device technology is an interesting aspect of analytical science [1,2].
Microfluidics exhibits various types of fluidic mixing processes of solvents in a
microchannel. Solvent mixing has been examined by varying the channel
configuration and flow rate of the solvents, by using aqueous–organic solvent
mixtures, and by introducing specific obstacles into the microchannel [3-5]. Fluidic
mixture of the solvents in the microchannel is related to the separation, diffusion,and reaction of the solutes. Information regarding the mixing process of the solventsis important and useful to design microreactors or micro total analysis systems [6,7].
Various types of aqueous-organic solvent mixture solutions are used in dissolution
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Chapter 11[8,9], cleaning [10], preservation [11], and as reaction solvents [12,13]. Such mixtures
are also useful in separation science [14-16]. However, to our knowledge, the use of
ternary mixed solvents of water–hydrophilic/hydrophobic organic solvent mixture
solutions has not been examined in detail. When ternary mixed solvents ofwater–hydrophilic/hydrophobic organic solvents were fed into a microspace, such as
microchannels in a microchip or capillary tubes, under laminar flow conditions, thesolvent molecules were radially distributed in a microspace regardless of the section
shape and the inner wall material; we call it the “tube radial distribution phenomenon”(TRDP) . To be concrete, with an organic solvent-rich solution, the organic solvent-richmajor phase was generated around the center of the microspace as an inner phase, while
the water-rich minor phase was formed near the inner wall as an outer phase. On theother hand, with a water-rich solution, water-rich major inner and organic solvent-richminor outer phases were formed, respectively.
Here, fluidic mixing of the ternary solvents (water–acetonitrile–ethyl acetate
mixture) was examined through fluorescence observation of the dyes in a microchannel.
A microchip was designed with three narrow channels that combine to form one widechannel. The water–acetonitrile mixture and acetonitrile–ethyl acetate mixture solutions
were fed into the narrow channels to prepare the ternary solvent solution of thewater–acetonitrile–ethyl acetate mixture in the wide channel. The ternary solvents
solutions caused specific fluidic mixing process of the solvents in the wide channel,
based on the tube radial distribution of the ternary solvents, i.e., the TRDP.
Experimental
Figure 1 illustrates the design and setup of the microchip in the microchip holder usedherein. The three narrow channels (each 100 µm wide × 40 µm deep) denoted aschannels N1 – N3 combine to form one wide channel (300 µm wide × 40 µm deep),which is denoted as channel W in the microchip.
A microchip incorporating the microchannels was set up for the fluorescencemicroscope–CCD camera system. The water–acetonitrile mixture (3:2 volume ratio)
containing 3.0 mM Eosin Y and acetonitrile–ethyl acetate mixture (3:2 volume ratio)
containing 0.15 mM perylene were fed into channel N2 and channels N1 and N3,
respectively, using microsyringe pumps. The fluorescence in the microchannel wasmonitored around the combining point and 3 cm from the combining point in channel
W using a fluorescence microscope equipped with an Hg lamp, a filter, and a CCD
camera.
Results and discussion
Mixing process of ternary solvents prepared in microchannels
Thewater–acetonitrile mixture (3:2 volume ratio) containing 3.0 mM Eosin Y and theacetonitrile–ethyl acetate mixture (3:2 volume ratio) containing 0.15 mM perylene were
fed into channel N2 and channels N1 and N3, respectively, to prepare the ternarysolvents of water–acetonitrile–ethyl acetate in channel W. The mixture solutions were273
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

fed into channels N1 – N3 at a flow rate of 2.0 µL min−1. The volume ratio of thewater–acetonitrile–ethyl acetate mixed solution in channel W was 3:8:4 (the organic
solvent-rich solution). Fig. 2 shows the fluorescence photographs of the solvents at the
combining point and 3 cm from the combining point in channel W. The specific mixing
process was observed clearly in the microchannel in Fig. 2, based on the TRDP with theorganic solvent-rich solution. That is, the photographs indicate that the hydrophobic
perylene (blue) fed from the side channels N1 and N3 was distributed around the centerof channel W away from the side inner walls of the channel. On the other hand,hydrophilic Eosin Y (green) fed from the center channel N2 was distributed near theside inner walls. The TRDP of the water–acetonitrile–ethyl acetate mixture solution (theorganic solvent-rich solution) occurred in the wide channel W by feeding thewater–acetonitrile mixture and acetonitrile–ethyl acetate mixture solutions from thenarrow channels.
Mixing process of ternary solvents prepared in microchannels through various
mixing procedures The fluorescence photographs and profiles of the solvents were
examined at a distance of 3 cm from the combining point in channel W through various
mixing procedures (Fig. 3). The water–acetonitrile mixture (3:2 volume ratio)
containing 3.0 mM Eosin Y and the acetonitrile–ethyl acetate mixture (3:2 volume
ratio) containing 0.15 mM perylene were fed into channel N2 and channels N1 and N3,
respectively. The mixture solutions were fed into channels N1 – N3 by varying mixing
flow rates ranging from 2.0 to 50 µL min−1. The details of the combined flow rates areshown in Fig. 3. Lines a - f indicate flow rates of channel N2 (ranging from 2.0 to 50µL min−1), and rows 1 – 6 indicate flow rates of channels N1 and N3 (ranging from 2.0
to 50 µL min−1). All of the fluorescence photographs in Fig. 3 were named using line
letters and row numbers. For example, the fluorescence photograph positioned at Line aand Row 2, which was denoted “photograph a-2,” was obtained from water-acetonitrile
mixture containing Eosin Y delivered in channel N2 at 2.0 µL min−1 andacetonitrile-ethyl acetate mixture containing perylene delivered in channels N1 and N3
at 5.0 µL min−1. It was confirmed that the fluorescence photographs in Fig. 3 did notchange for at least 20 min. Specific microfluidic mixing process of the solvents was
performed based on the TRDP in the microchannels. For example, photographs a-1, b-1,
c-2, d-1, e-1, and f-1 show photographs of the mixture solutions fed into channels N1
and N3 at a flow rate of 2.0 µL min−1 and in channel N2 at flow rates ranging from 2.0
to 50 µL min−1. The volume ratios of the water–acetonitrile–ethyl acetate mixed
solution in channel W were changed from 3:8:4 (the organic solvent-rich solution) to
56:41:3 (the water-rich solution). With the TRDP, the mixing process of the solventschanged from the patterns of the organic solvent-rich major inner phase (blue) and thewater-rich minor outer phase (green) to the patterns of the water-rich major inner phase(green) and the organic solvent-rich minor outer phase (blue) via the transition mixing
processes. However, from the phase diagram mentioned below, photographs f-1 could
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not show the TRDP. As shown in photographs d-1. d-2, d-3, d-4, d-5, and d-6, themixture solutions were fed into channels N2 at a flow rate of 10 µL min−1 and inchannels N1 and N3 at flow rates ranging from 2.0 to 50 µL min−1. The volume ratiosof the water–acetonitrile–ethyl acetate mixed solution in channel W were changed from
43:46:11 to 6.0:58:36 (the organic solvent-rich solution). With the TRDP, the mixing
process changed from the transition mixing patterns to the patterns of the organic
solvent-rich major inner phase (blue) and the water-rich minor outer phase (green).
Various fluidic mixing processes of the solvents or fluorescent dyes were observed byvarying flow rates and mixing ratios of the water–acetonitrile and the acetonitrile-ethyl
acetate mixture solutions, based on the TRDP of the water–acetonitrile–ethyl acetate
mixture in channel W. However, the ternary solvents in channel W did not always
create the specific mixing process as described in the last section. The microfluidic
mixing process of the solvents based on the TRDP is called “tube radial distribution
mixing” (TRDM). The fluorescence photographs and profiles that had the same
component ratios of the solvents (water–acetonitrile–ethyl acetate volume ratio are
10:57:33, 3:8:4, and 33:49:18) in Fig. 3 are rearranged with the residence times inchannel W from the combining point (Fig. 4). The TRDM observed on the fluorescencephotographs proceeded with increasing residence times or mixing times from the
combining point.
Mixing process of two-component solvents in the microchannels The fluorescencephotographs of the solvents that dissolved the dyes, hydrophobic perylene (blue) andrelatively hydrophilic Eosin Y (green), were examined at a distance of 3 cm from the
combining point in channel W by feeding two-component solvents, such as
water–acetonitrile and water–ethyl acetate mixtures. The fluorescence photographs of
the water–acetonitrile mixture in channel W were examined. Water containing 3.0 mM
Eosin Y in channel N2 and acetonitrile containing 0.15 mM perylene in channels N1
and N3 were delivered at flow rates of 2.0 – 50 µL min−1, similar to those shown in Fig.
3. Eosin Y (green) flowed around the center of the channels and perylene (blue) flowed
near both the side inner walls of the channels, according to the volume ratios of waterand acetonitrile (Fig. 5). When hydrophobic perylene was deposited in channel W, i.e.,
in relatively water-rich solutions, the deposition disturbed a microfluidic flow in thechannel. In these cases, fluorescence photographs are not presented in Fig. 5. The
fluorescence photographs of the water–ethyl acetate mixture in channel W were also
examined. Water containing 1.0 mM Eosin Y in channel N2 and ethyl acetate
containing 0.1 mM perylene in channels N1 and N3 were delivered at flow rates ranging
from 2.0 to 50 µL min−1, similar to those shown in Fig. 3. At relatively low flow rates,
Eosin Y in water and perylene in ethyl acetate separated with vertical interfaces inchannel W, while at high flow rates they separated with a horizontal interface (Fig. 6).
No specific mixing process as observed with the ternary solvents solution in Figs. 2 and3 were created using the two-component solvents, such as water–acetonitrile mixture orwater–ethyl acetate mixture. The fluorescence photographs using the two-component
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

solvents were normal because of the nature of the solvents and dyes used: i.e.,acetonitrile, hydrophilic; ethyl acetate; hydrophobic; Eosin Y, relatively hydrophilic;and perylene, hydrophobic.
Phase diagram for water–acetonitrile–ethyl acetate mixture solution and thecomponent ratios required for TRDM caused by TRDP
A phase diagram for the
ternary mixture solvents of water–acetonitrile-ethyl acetate had been examined in a
vessel at a temperature of 22 °C. The phase diagram is shown in Fig. 7. The dottedcurve in the diagram indicates the boundary between the homogeneous andheterogeneous phases. The phase diagram showed that each component ratio of thesolvents made either a homogeneous (one homogeneous phase) or a heterogeneous (twohomogeneous phases) solution. As indicated in our previous paper, the TRDP was
performed with specific homogeneous carrier solutions having the component ratios ofthe solvents positioned around the homogeneous-heterogeneous solution boundary
curve in the phase diagram. Such carrier solutions caused the TRDP of the ternarysolvents in a microflow through phase transformation from homogeneous toheterogeneous under pressure. The fluorescence photographs shown in Fig. 8 can beroughly classified into several typical mixing processes: three typical TRDM caused by
the TRDP and two typical mixing patterns not based on the TRDP (Fig. 8). All thecomponent ratios of the solvents in channel W in Fig. 3 are plotted in the phase diagramof Fig. 7, where open circles (○) and closed circles (●) indicate TRDM images andnon-TRDM (or doubtful cases) images, respectively. The closed circle signals were faraway from the dotted line of the homogeneous-heterogeneous solution boundary curve.
The solutions of the component ratios of the solvents that were indicated by the closed
circles did not show the TRDM at low flow rates of 0.1 – 1.0 µL min−1 for channels N1– N3, which provided long residence times or mixing times in channel W from the
combining point. The plotted data in Fig. 7 were close to the previous data,15,17) where
the specific homogeneous carrier solutions having the component ratios of the solvents
around the boundary in the phase diagram led to the TRDP.
In conclusion, the ternary solvents of water–acetonitrile–ethyl acetate mixture in thewide channel were prepared by combining the water–acetonitrile mixture andacetonitrile–ethyl acetate mixture through the narrow channels in the microchip. Some
specific and interesting mixing processes of the solvents with the dissolved fluorescentdyes could be observed in the wide channel based on the TRDP of ternary solvents.
This unique mixing process was denoted as “tube radial distribution mixing” (TRDM).
The mixing process based on the TRDP was not observed using the two-component
solvents, i.e., the water–acetonitrile mixture or the water–ethyl acetate mixture. The
TRDM data visualized with the dyes are expected to be useful for developing a mixing
technique to create a phase interface and a chemical reaction space in a microspace.

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Figure 1.
Figure.
1. Schematic
Schematic representations
representations ofof thethe setup
setup ofof thethe microchip in the microchip
holder and the enlarged view of the microchip incorporating the three narrow channels
combined to form one wide channel.

Figure 2.
Figure.
2. Fluorescence
Fluorescence photographs
photographs ofof thethe solvents
solvents with the dissolved
fluorescent dyes at a) the combining point of the channels and b) 3 cm fromthe combining point in channel W. The white dashed line indicates the
combining point between channels N1 – N3 and channel W. Conditions:
Carrier, water–acetonitrile (3:2, volume ratio) containing 3.0 mM Eosin Y inchannel N2 and acetonitrile–ethyl acetate (3:2, volume ratio) containing 0.15
µM perylene in channels N1 and N3; flow rate, 2.0 µL min−1 for channels N1– N3. The volume ratio of water–acetonitrile–ethyl acetate in channel W is
3:8:4.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3.
iles ofFigure.
3. Fluorescence
Fluorescence photographs
photographs andand prof
profilesof the solvents with the dissolved
fluorescent dyes at 3 cm from the combining point in channel W where thewater–acetonitrile–ethyl acetate mixture was fed with various mixing procedures.
Conditions: Carrier, water–acetonitrile (3:2, volume ratio) containing 3.0 mM Eosin Y
in channel N2 and acetonitrile–ethyl acetate (3:2, volume ratio) containing 0.15 mM
perylene in channels N1 and N3; flow rates, 2.0 - 50 µL min−1 for channels N1 - N3.
The volume ratios of water–acetonitrile–ethyl acetate in channel W and the residence
times from the combining point are indicated below each photograph.

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Figure 4.
iles ofFigure.
4. Fluorescence
Fluorescence photographs
photographs andand prof
profilesof thethe solvents with the dissolved
fluorescent dyes at 3 cm from the combining point in channel W where thewater–acetonitrile–ethyl acetate mixture was fed with the same component ratios of thesolvents. Conditions: Carrier, water–acetonitrile (3:2, volume ratio) containing 3.0 mM
Eosin Y in channel N2 and acetonitrile–ethyl acetate (3:2, volume ratio) containing 0.15
mM perylene in channels N1 and N3. The volume ratios of water–acetonitrile–ethyl
acetate in channel W are 10:57:33, 3:8:4, and 33:49:18. The photograph titles that areused in Fig. 3 and the residence times from the combining point are indicated below
each photograph.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure.
5. Fluorescence
Fluorescence photographs
photographs ofof thethe solvents with the dissolved fluorescentFigure 5.
ﬂuorescent dyes
at 3 cm from the combining point in channel W where the water–acetonitrile mixture
was fed. Conditions: Carrier, water containing 3.0 mM Eosin Y in channel N2 andacetonitrile containing 0.15 mM perylene in channels N1 and N3; flow rates, 2.0 – 50
µL min−1 for channels N1 – N3. The volume ratios of water–acetonitrile in channel W
and the residence times from the combining point are indicated below each photograph.
When perylene was deposited in channel W, fluorescence photographs are not shown in
the figure.

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Figure.
6. Fluorescence
Fluorescence photographs
photographs ofof thethe solvents
solvents with the dissolved fluorescentFigure 6.
ﬂuorescent dyes
at 3 cm from the combining point in channel W where the water–ethyl acetate mixture
was fed. Conditions: Carrier, water containing 1.0 mM Eosin Y in channel N2 and ethyl
acetate containing 0.1 mM perylene in channels N1 and N3; flow rates, 2.0 – 50 µL
min−1 for channels N 1– N3. The volume ratios of water–ethyl acetate in channel W andthe residence times from the combining point are indicated below each photograph.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 7.
Figure.
7. Phase
Phase diagram
diagram forfor aa water–acetonitrile–ethyl
water–acetonitrile–ethyl acetate mixture and thecomponent ratios of the solvents that provided the TRDM based on the TRDP inchannel W. The dotted curve in the diagram indicates the homogeneous-heterogeneous
solution boundary curve. The symbols ○ and ● indicate the TRDM photograph and the
non-TRDM (or doubtful case) photograph, respectively.

Figure 8.
Figure.
8. Typical
Typical TRDM
TRDM photographs
photographs based
based onon thethe TRDP
TRDP andand typical mixing patterns
not based on the TRDP.

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References[1] P. J. Asiello and A. J. Baeumner, Lab Chip, 2011, 11, 1420.
[2] D. R. Reyes, D. Iossifidis, P. A. Auroux, and A. Manz, Anal. Chem.,
2002, 74, 2623.
[3] Y. Kikutani, H. Hisamoto, M. Tokeshi, and T. Kitamori, Lab Chip, 2004, 4, 328.
[4] A. Hibara, M. Tokeshi, K. Uchiyama, H. Hisamoto, and T. Kitamori, Anal. Sci.,
2001, 17, 89.
[5] N. Kaji, Y. Okamoto, M. Tokeshi, and Y. Baba, Chem. Soc. Rev., 2010, 39, 948.
[6] H. Nakamura, Y. Yamaguchi, M. Miyazaki, H. Maeda, and M. Uehara, Chem.
Commun., 2002, 23, 2844.
[7] H. Kawazumi, A. Tashiro, K. Ogino, and H. Maeda, Lab Chip, 2002, 2, 8.
[8] G. Hefter, Pure Appl. Chem., 2005, 77, 605.
[9] G. R. Castro and T. Knubovets, Crit. Rev. Biotechnol., 2003, 23, 195.
[10] J. Niu and B. E. Conway, J. Electroanal. Chem., 2003, 546, 59.
[11] D. M. Ruiz and R. E. De Castro, J. Ind. Microbiol. Biotechnol., 2007, 34, 111.
[12] H. Ogino, K. S. Siddiqui, and T. Thomas, “Protein Adaptation in Extremophiles”,
2008, Nova Science Publishers, Inc., New York.
[13] M. Tjahjono, C. Huiheng, E. Widjaja, K. Sa-Ei, and M. Garland, Talanta, 2009, 79,856.
[14] M. K. Yeh, S. L. Lin, M. I. Leong, S. D. Huang, and M. R. Fuh, Anal. Sci., 2011,
27, 49.
[15] T. Maruyama, H. Matsushita, J. Uchida, F. Kubota, N. Kamiya, and M. Goto, Anal.
Chem., 2004, 76, 4495.
[16] F. Torrens, Chromatographia., 2001, 53, 199.

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Chapter 12 Development of tube radial distribution reaction(TRDR)A TRDP-based chemical reaction in a microspace, referred to as a tube radial
distribution reaction (TRDR), has been performed as one of the applications of TRDP.
The derivatization reaction of bovine serum albumin (BSA) with fluorescamine (FR),using the TRDR system, was evaluated and compared with conventional batch-based
methods. The part of this chapter is constructed and rewritten based on related,published manuscripts.18,28)

12.1 TRDR featuring the liquid-liquid interface created with TRDP
A micro-flow reaction system was developed in which liquid-liquid interface wascreated based on the tube radial distribution of ternary mixed carrier solvents. The
system was constructed from double capillary tubes having different inner diameters(100 and 250 µm i.d.). The smaller tube was inserted into the larger one through aT-type joint. The reaction of a protein with a fluorescence derivatizing reagent was
adopted as a model. A water–acetonitrile mixture (3:1 volume ratio) including bovine
serum albumin (hydrophilic) was delivered into the large tube from the inside throughthe small tube and an acetonitrile–ethyl acetate mixture (7:4 volume ratio) containing
fluorescamine (hydrophobic) as a derivatizing reagent was delivered from the outside
through the joint. Solutions were mixed through the double capillary tubes to promoteternary mixed carrier solvents (water–acetonitrile–ethyl acetate; 1:2:1 volume ratio).
The liquid-liquid interface was created based on the tube radial distribution of ternarysolvents in the larger tube. The derivatization reaction was performed in the larger, or
reaction, tube in the micro-flow system. The fluorescence intensity of thefluorescamine-derivatized bovine serum albumin obtained by the system, which
specifically included the kinetic liquid–liquid interface in the tube, was greater than thatobtained through a batch reaction using a homogeneous solution of water–acetonitrile(1:2 volume ratio).
Chapter 12Introduction
Micro-flow systems are an active area of research in chemistry and biochemistry. Novelcomponents of micro-flow systems are frequently sought to improve performance fordifferent types of uses and users [1-3]. Capillary tubes with inner diameters of less thanseveral hundred micrometers, which provide a micro-flow, are also known to exhibit
interesting and useful physical or hydrodynamic phenomena, such as electro-osmotic
flow and laminar flow. The electro-osmotic flow in a capillary tube promotes capillaryelectrophoresis [4-6] and capillary electrochromatography [7], while the laminar flow
conditions enable hydrodynamic chromatography [8,9].
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Recently, our group reported on the tube radial distribution phenomenon of ternary
mixed carrier solvents in a micro-flow, which we abbreviate as the “tube radial
distribution phenomenon (TRDP).” When the ternary mixed carrier solvents ofwater–hydrophilic/hydrophobic organic solvent mixtures are delivered into a
micro-space, such as a micro-channel or a capillary tube under laminar flow conditions,the carrier solvent molecules are radially distributed in the micro-space, generating
inner and outer phases. A capillary chromatography system where the outer phasefunctions as a pseudo-stationary phase under laminar flow conditions has beendeveloped based on the TRDP. We call this “tube radial distribution chromatography(TRDC)”.
There is much research interest in carrying out chemical reactions inaqueous–organic solvent mixtures [10,11], and at the liquid-liquid interfaces [12-14];
interesting and useful outcomes have been reported. TRDP creates a phase interface, i.e.,
it is a kinetic, not static, liquid–liquid interface in a micro-space under laminar flow
conditions. Here, we applied the phase interface to a chemical reaction space in a
micro-flow system. The formation of the liquid-liquid interface based on TRDP using
double capillary tubes having different inner diameters was strictly confirmed throughseveral experiments. Also, the results obtained with the present micro-flow reaction
system were carefully compared with those of a batch reaction system. We briefly
described preliminary results of this investigation in a previous communication.18) A
chemical reaction takes place in the micro-flow featured with the kinetic liquid–liquidinterface created under the TRDP. The derivatization reaction of bovine serum albumin(BSA) with fluorescamine (FR) as a model was carried out in the present micro-flow
reaction system.
Experimental
Apparatus and procedures
A schematic diagram of the present micro-flow reaction
system is shown in Fig. 1. The smaller tube (100 µm i.d.) was inserted into the larger
one (250 µm i.d., 150 cm length) through a T-type joint. A water (10 mM borate buffer,
pH 9.0)–acetonitrile mixture (3:1 volume ratio) including 25 µM bovine serum albumin(BSA) was delivered into the large tube from the inside through the small tube, and an
acetonitrile–ethyl acetate mixture (7:4 volume ratio) including 1 mM Fluorescamine(FR) was delivered from the outside through the joint with micro-syringe pumps at
various specified flow rates. The carrier solutions delivered from inside and outside into
the larger tube were called Carriers A and B, respectively, as shown in Fig. 1. The
carriers were fed at flow rates of 7.5, 12, and 15 µL min-1 (Carrier A and B: 2.5 and 5µL min-1; 4 and 8 µL min-1; and 5 and 10 µL min-1, respectively). Carriers A and B
were then mixed in the large tube to perform the derivatization reaction in thewater–acetonitrile–ethyl acetate (1:2:1 volume ratio) solution. The solution including
FR-derivatized BSA was collected at the outlet of the large tube until 1 mL was
obtained for fluorescence spectroscopy (FP-6500, JASCO Corporation, Tokyo, Japan).
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Fluorescence photographs
A large capillary tube (250 µm i.d.) was set up for the
fluorescence microscope-CCD camera system (Fig. 1) to confirm the tube radial
distribution of the carrier solvents, i.e., TRDP. The water–acetonitrile mixture (3:1
volume ratio) including 1 mM Eosin Y and acetonitrile–ethyl acetate mixture (7:4
volume ratio) including 0.1 mM perylene solutions was delivered into the large
capillary tube. The fluorescence in the capillary tube was mainly monitored from a
position 30 cm from the outlet using a fluorescence microscope equipped with an Hglamp, a filter, and a CCD camera.
Results and discussion
Tube radial distribution phenomenon of fluorescent dyes in a capillary tube Fig. 2
shows fluorescence photographs and profiles observed in the capillary tube (250 µmi.d.) at 20, 40, 80, and 120 cm positions from the smaller capillary outlet in which thefluorescent dye-containing aqueous–organic solvent carrier solution was delivered at aflow rate of 15 µL min-1. The photographs and profiles showed that the hydrophobic
perylene molecule (blue) was distributed around the middle of the tube and away fromthe tube inner wall, whereas the relatively hydrophilic Eosin Y molecule (green) was
distributed near the tube inner wall. Thus, the kinetic liquid–liquid interface createdthrough the TRDP was clearly confirmed in the present micro-flow system using double
capillary tubes.
Effects of the flow rate on the tube radial distribution
The effects of the flow rate on
the tube radial distribution of the carrier solvents were examined using a fluorescencemicroscope-CCD camera system. Fig. 3 shows photographs taken at the smaller
capillary tube outlet, where the water-acetonitrile mixture, including Eosin Y (green)from the inside and the acetonitrile-ethyl acetate including perylene (blue) from theoutside, were mixed. As shown in Fig. 3, at all flow rates examined (7.5, 12, and 15 µL
min-1), the tube radial distribution started to form at the smaller tube outlet with anorganic solvent-rich major inner phase (blue) and water-rich minor outer phase (green)
based on the TRDP. That is, the water-rich solvents (green) and the organic solvents(blue) that were fed to the smaller tube outlet quickly changed from the inside andoutside deliveries to the outer (green) and inner (blue) phases in the larger tube. We
could observe the moment of TRDP creation at the smaller tube outlet for the first time.
Fig. 4 shows fluorescence photographs and profiles of the dyes in the larger capillary
tube 30 cm from the outlet at flow rates of 7.5, 12, and 15 µL min-1. As shown in Fig. 4,
TRDP was observed at all flow rates, and the outer water-rich phase became thicker
with increasing flow rates.
Separation of 1-naphthol and 2,6-naphthalendisulfonic acid
As described in the
Introduction, the TRDC system has been developed based on the TRDP. We examined
the separation performance of the TRDC using the present flow system with double
capillary tubes of different inner diameters. The model analytes, hydrophobic
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1-naphthol and hydrophilic 2,6-naphthalendisulfonic acid, were injected into the smaller
capillary tube inlet by a gravity method (for 20 s from a height of 30 cm).
Fluorescence detection was used. The thus obtained chromatograms are shown in Fig. 5
together with the analytical conditions. Since the water-rich outer phase acted as a
pseudo-stationary phase in this case, hydrophobic 1-naphthol was first eluted near the
average linear velocity, and second hydrophilic 2,6-naphthalendisulfonic acid waseluted with a lower velocity than the average linear velocity at all flow rates of 7.5, 12,
and 15 µL min-1. In more detail, the second peaks became broader and the resolution(Rs; numbers in Fig. 5) decreased with increasing flow rates. The experimental data
were consistent with the observation that the water-rich outer phase as a
pseudo-stationary phase in the flow system became thicker, or included more organic
solvents, with an increasing flow rate. It was suggested that the liquid-liquid interface
based on TRDP in the capillary tube changes with the flow rates as a chemical reaction
space, creating different fluidic property and component ratios of the solvents in the
inner and outer phases.
Derivatization in batch reaction
FR, which has no fluorescence itself before
derivatization, is commonly used as a fluorogenic reagent in analytical chemistry. The
reagent reacts readily under alkaline conditions with primary amines to form fluorescent
substances, providing the basis for a rapid and sensitive assay of amino acids, peptides,
proteins, and other primary amines [15]. FR is also quickly hydrolyzed in aqueous
solution to become fluorescently inactive. The reaction schemes are shown in Fig. 6. Anaqueous solution including a biomolecule, such as a protein, and an organic solution
including FR are mixed and stirred for the derivatization reaction. FR, reacts
competitively with BSA and water (hydrolysis) in an aqueous–organic solvent mixture
solution. First, the derivatization reaction of BSA with FR was confirmed in a batch
vessel using an aqueous–organic solvent solution in the usual manner as a reference. A
10 mM borate buffer solution (0.66 mL, pH 9.0) of 25 mM BSA was mixed with anacetonitrile solution (1.34 mL) of 1 mM FR, and stirred in the batch vessel(water–acetonitrile, 1:2 volume ratio and 8.3 mM BSA, 0.67 mM FR). The fluorescence
due to the derivative in the solution was measured by fluorescence spectroscopy (ex 390
nm). The fluorescence spectrum exhibited a maximum at around 30 min after mixing,
which then gradually decreased. The obtained spectrum at 30 min is shown in Fig. 7.
The maximum intensity was ca. 200 (arbitrary units) (ex 390 nm, em 470 nm). The
derivatization reaction of BSA with FR did not occur in a batch vessel using anaqueous–organic solvent solution (water–acetonitrile–ethyl acetate; 1:2:1 volume ratio)
due to protein deposition.
Derivatization in tube radial distribution reaction The fluorescence spectrum of thesolution collected through the capillary tube in the micro-flow reaction system(water–acetonitrile–ethyl acetate; 1:2:1 volume ratio and 8.3 mM BSA, 0.67 mM FR) is
also shown in Fig. 7. The maximum intensities were ca. 340, 670, and 950 (arbitrary
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

units) (ex 390 nm, em 470 nm) at flow rates of 7.5, 12, and 15 µL min-1, respectively.
The fluorescence intensities, migration times in the large tube (140 cm from the small
capillary tube outlet), and collection times up to 1 mL in a vessel are summarized inTable 1 together with the fluorescence intensities obtained in the batch reaction. The
micro-flow reaction system with a flow rate of 15 µL min-1 provided the highest
fluorescence intensity, 950 (arbitrary units), among the experiments conducted. The
reaction space created by the TRDP with 15 µL min-1 must bring out a high derivatizing
efficiency with the suitable fluidic property and composition of the solvents in thephases. The fluorescence intensity of the FR-derivatized BSA obtained under thepresent flow system in the capillary tube (950 arbitrary units) was about 4 times higher
than that obtained in the batch-vessel using a water–acetonitrile homogeneous solution.
Because the intensity of the fluorescence in the flow system did not increase in thecollecting vessel, which is explained in the next section (Table 1 and Fig. 8), the greater
fluorescence intensity in the system was attributed to the specific reaction area, i.e., theliquid-liquid phase interface created through the TRDP in the capillary tube where thehydrolysis of FR must be inhibited for the derivatization reaction to proceed. A
chemical reaction that takes place in a micro-flow under the TRDP performance iscalled a “tube radial distribution reaction (TRDR)”.
Effects of the reaction time on the fluorescence intensity
Figure 8 shows therelationships between the reaction times and the fluorescence intensities (ex 390 nm, em
470 nm) in a batch reaction. A reaction between BSA and FR was not carried out in thewater–acetonitrile–ethyl acetate mixture solution because of protein deposition, due toethyl acetate, which is shown with the plots (◯) on the horizontal line in Fig. 8. The
reaction between them occurred in the water–acetonitrile mixture solution with the
maximum fluorescence intensity at ca. 30 min, as described above. To determine its
influence on the reaction, ethyl acetate was added at 20 and 30 min after starting thereaction in a water–acetonitrile mixture solution. As shown in Fig. 8, the addition of
ethyl acetate did not cause any marked changes in the fluorescence intensity in the
aqueous–organic solvent mixture solutions. As shown in Table 1, the micro-flow
reaction or TRDR system with a flow rate of 15 µL min-1 required a migration time of
4.6 min in a capillary tube of 140 cm in length, and a collecting time of 67 min at theoutlet of the tube. Although the migration time and the collecting time in the TRDR
system were quite different from the reaction time of 30 min, which provided the
maximum fluorescence intensity in the batch reaction system, the data in Fig. 8 showed
no influence compared with the fluorescence intensity or derivatizing efficiency
between the TRDR and batch reactor systems. This was because the fluorescenceintensity in the flow system did not increase in the collecting vessel. Fig. 9 shows therelationship between the migration times and the fluorescence intensities in the TRDR
system as well as the relationship between the reaction time and the fluorescenceintensity in the batch reaction system. At all flow rates, the present TRDR systemprovided higher fluorescence intensity than that obtained by the batch reaction system
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conducted for 30 min.
In conclusion, the TRDP creates a specific phase interface, i.e., a kinetic, and notstatic, aqueous–organic interface in micro-space under laminar flow conditions. We
applied the phase interface created through the TRDP to a chemical reaction space in a
micro-flow system. A chemical reaction that takes place in a micro-flow under TRDP
performance is called a “tube radial distribution reaction (TRDR)”. The derivatization
reaction of BSA with FR was carried out in the present TRDR system as a model. The
fluorescence intensity of the FR-derivatized BSA obtained under the tube radial
distribution phenomenon, which included a kinetic liquid–liquid interface, was greaterthan that obtained through batch reaction using a homogeneous solution ofwater–acetonitrile (1:2 volume ratio). The data obtained here will facilitate thedevelopment of novel micro-flow reactor devices.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure.
1. Schematic
Schematic diagram
diagram ofof the present micro-ﬂ
micro-flow
Figure 1.
ow system with a fluorescence
ﬂuorescencemicroscope-CCD camera.

Figure.
2. Fluorescence
Fluorescence photographphotograph andand prof
profilesof the ﬂfluorescentFigure 2.
iles ofuorescent dyes dissolved in the
ternary mixed carrier solvents at 20, 40, 80, and 120 cm from the smaller capillaryoutlet. Conditions: carrier, water–acetonitrile–ethyl acetate mixture (1:2:1, v/v/v),
including 0.067 mM perylene and 0.33 mM Eosin Y; flow rate, 15 µL min-1.
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Figure 3.
uorescent dyes
Figure.
3. Fluorescence
Fluorescence photographphotograph ofof thethe ﬂfluorescentdyes dissolved
dissolved in the ternary
mixed carrier solvents around the smaller capillary outlet. Conditions: carrier,water–acetonitrile–ethyl acetate mixture (1:2:1, v/v/v), including 0.067 mM perylene
and 0.33 mM Eosin Y; flow rate, 15 µL min-1.

Figure.
4. Fluorescence
Fluorescence photographphotograph andand prof
profilesof the ﬂfluorescentdyes dissolved in the
Figure 4.
iles ofuorescent dyes
ternary mixed carrier solvents at flow rates of 7.5, 12, and 15 µL min-1. Conditions:carrier, water–acetonitrile–ethyl acetate mixture (1:2:1, v/v/v), including 0.067 mM
perylene and 0.33 mM Eosin Y and observed point, 30 cm from the larger capillaryoutlet.

291
Development of TRDR

291

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure.
5. Chromatograms
Chromatograms ofof 1-naphthol
1-naphthol andand 2,6-naphthalenedisulfonic
2,6-naphthalenedisulfonic acid by the
Figure 5.
micro-flow system using double capillary tubes of different inner diameters.
Conditions: carrier, water–acetonitrile–ethyl acetate mixture (1:2:1, v/v/v); observed
point, 30 cm from the larger capillary outlet; analyte injection, 20 s × 30 cm heightgravity method from the smaller tube inlet; detection (ex. 290 nm and em. 335 nm);analyte concentrations, 1 mM each.

Figure.
6.Reaction
Reactionschemes
schemesof
ofthe
thederivatization
derivatizationof
ofBSA
BSAwith
withFR.
FR.
Figure 6.

292
292

Chapter 12

Figure.
7. Fluorescence
Fluorescence spectra
spectra ofof thethe FR-derivatized
FR-derivatized BSA.
BSA. The solutions included 8.3
Figure 7.
mM BSA and 0.67 mM FR for derivatization in the systems. Curves a), b), and c):
micro-flow reaction (tube radial distribution reaction; TRDR) system(water–acetonitrile–ethyl acetate; 1:2:2 v/v/v) at 7.5, 12, and 15 µL min-1, respectively.
Curve d): batch reaction (BR) system (water–acetonitrile; 1:2 v/v).

Figure 8.
uorescence intensities (exFigure.
8. Relationships
Relationships between
between thethe reaction
reaction times
times and the ﬂfluorescence390 nm, em 470 nm) in the batch reaction system. ◯, Water–acetonitrile–ethyl acetate(1:2:1 volume ratio; 8.3 µM BSA and 0.67 mM FR); , water–acetonitrile (1:2 volume

ratio; 8.3 µM BSA and 0.67 µM FR), and ▲, water–acetonitrile (1:2 volume ratio; 11
mM BSA and 0.88 mM FR).
293
Development of TRDR

293

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 9.
ﬂuorescence intensities in
Figure.
9. Relationship
Relationship between
between thethe migration
migration times and the fluorescencethe micro-flow reaction (tube radial distribution reaction; TRDR) system as well as
between the reaction time and the fluorescence intensity in the batch reaction system.
The solutions included 8.3 µM BSA and 0.67 mM FR for derivatization in the systems.
TRDR system (water–acetonitrile–ethyl acetate; 1:2:1 v/v/v) at 7.5, 12, and 15 µL min-1
and batch reaction system (water–acetonitrile; 1:2 v/v).

References[1] M. H. Ghanim and M. Z. Abdullah, Talanta, 2011, 85, 28.
[2] P. Hrncirik and J. Nahlik, Chem. Biochem. Eng., 2010, 24, 489.
[3] C.-G. Yang, Z.-R. Xu, and J.-H. Wang, Trends Anal. Chem., 2010, 29, 141.
[4] E. Guihen and W. T. O’Connor, Electrophoresis, 2010, 31, 55.
[5] S. Terabe, Anal. Chem., 2004, 76, 240.
[6] C. A. Lucy, A. M. MacDonald, and M. D. Gulcev, J. Chromatogr., A, 2008, 1184,
81.
[7] K. Otsuka, Chromatography, 2007, 28, 1.
[8] H. Small, F. L. Saunders, and J. Solc, Adv. Colloid Interface Sci., 1976, 6, 237.
[9] R. Umehara, M. Harada, and T. Okada, J. Sep. Sci., 2009, 32, 472.
[10] M. Tjahjono, C. Huiheng, E. Widjaja, K. Sa-Ei, and M. Garland, Talanta, 2009, 79,856.
[11] G. El-Subruiti, G. Younes, and M. Jaber, Prog. React. Kinet. Mech., 2011, 36, 73.
[12] Y. M. A. Yamada, T. Watanabe, T. Beppu, N. Fukuyama, K. Torii, and Y. Uozaki,
Chem.-A Eur. J., 2010, 16, 11311.

294
294

Chapter 12[13] J. Jovanovic, E. V. Rebrov, T. A. Nijhuis, V. Hessel, and J. C. Schouten, Ind. Eng.
Chem. Res., 2010, 49, 2681.
[14] K. Tsukagoshi, Y. Hattori, T. Hayashi, R. Nakajima, K. Yamashita, and H. Maeda,
Anal. Sci., 2008, 24, 1393.
[15] K. Tsukagoshi, A. Tanaka, R. Nakajima, and T. Hara, Anal. Sci., 1996, 12, 525.

295
Development of TRDR

295

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Chapter 13 Two-phase separation systems of mixed solutions
and TRDP
TRDP is caused by phase transformation of the two-phase separation systems of mixed
solvent solutions, such as ternary water–hydrophilic/hydrophobic organic solvents,
water–surfactant, water–ionic liquid, and fluorous–organic solvents, in a microspace
under laminar flow conditions. The part of this chapter is constructed and rewritten
based on related, published manuscripts.37,41,44,46,48)

13.1 Two-phase extraction of metal ionswater-acetonitrile-ethyl acetate mixed solution

using

a

ternary

A ternary mixed-solvent solution of water-acetonitrile-ethyl acetate changes from ahomogeneous (single-phase) to a heterogeneous (two-phase) system with changes intemperature and/or pressure. Here, we used this system in a batch vessel to extract metal
ions. Water-acetonitrile-ethyl acetate at a volume ratio of 3:8:4 containing
8-hydroxyquinoline was used as a ternary mixed-solvent solution, changing fromhomogeneous at 25 °C to heterogeneous after 30 min at 0 °C. The two-phase systemcomprised an upper (organic solvent-rich) phase and a lower (water-rich) phase at avolume ratio of 6:1. Fe(III), Co(II), and Ni(II) were used as model metal ions dissolvedin the homogeneous solution at 25 °C. The distribution constants and the extraction
percentages were determined by measuring the metal ion concentrations in the upperand lower phases with inductively coupled plasma atomic emission spectroscopy. The
metal ions were extracted through complexation with 8-hydroxyquinoline in theorganic-rich phase with distribution constants and extraction percentage values,
respectively, of 0.47 and 74 for Fe(III), 0.15 and 47 for Co(II), and 0.08 and 32 forNi(II).

Chapter 13

Introduction
The process of phase separation of aqueous systems containing polymers, micelles, andionic liquid solutions has been well known and used in the fields of analytical chemistry
and separation science since the last century [1-5]. For example, aqueous micellar
solutions of some non-ionic surfactants separate into two distinct phases when heated
above a certain temperature (the cloud point); that is, a temperature-induced phaseseparation occurs [6-10]. One phase is an almost micelle-free aqueous solution (theaqueous phase), while the other is a concentrated surfactant solution containing
considerable amounts of water (the surfactant-rich phase). Hydrophobic compounds
dissolved in the aqueous micellar solution are extracted into the surfactant-rich phase,
while hydrophilic compounds remain in the aqueous phase.
296
296

Chapter 13

Fluorous chemistry, which can undergo phase separation, has been heavily
investigated since the seminal work of Horváth and Rábai in 1994 [11]. Two-phaseseparation of fluorous-organic solvent mixed solutions has been applied in separation
science [12,13]. When cooled below a certain temperature [14], mixed solutions offluorous-organic solvents separate into two distinct phases in a batch vessel; the lower
phase comprising an almost pure fluorous solvent, and the upper phase comprising anorganic solvent. Based on this two-phase system, liquid-liquid and liquid-solid
extractions have been reported using a variety of fluorous solvents [15-17].
A novel two-phase separation system involving ternary mixed solvents has beenreportedbyourgroup.
A
ternary
mixed
solvent
solutionofwater-hydrophilic/hydrophobic solvents changes from a homogeneous (single-phase) toa heterogeneous (two-phase) system with temperature and/or pressure changes. Thisphenomenon causes a specific fluidic behavior in a microspace, such as a microchannel
on a microchip or a capillary tube, leading to new types of open-tubular capillarychromatography and microreactors. Here, for the first time we have used a two-phaseseparation system comprising a ternary mixed-solvent solution in a batch vessel toextract metal ions.
Experimental
8-hydroxyquinoline (8-HQ), iron (1000 ppm, 0.275 M HNO3), nickel (1000 ppm, 0.1 M
HNO3), and cobalt (1000 ppm, 0.1 M HNO3) standard solutions were purchased from
Nacalai Tesque, Inc. (Kyoto, Japan). A homogeneous ternary mixed-solvent solution ofwater-acetonitrile-ethyl acetate (at a volume ratio of 3:8:4) containing 8-HQ was
prepared at 25 °C. Metal ions were dissolved in the homogeneous solution, and thesolution was then cooled to 0 °C and held to obtain a heterogeneous solution including
both upper (organic solvent-rich) and lower (water-rich) phases. The metal
concentrations were determined in both phases. Each phase was subjected toevaporation, and the resultant residue was dissolved in 0.1 M nitric acid (10 mL) for
inductively coupled plasma atomic emission spectroscopy (ICP, 8100-Type instrument,
Shimadzu Co.). Distribution constants (D) and extraction percentages (E%) were
calculated using the following equations:
D = Co / Cw,
E(%) = 100 D / (D + Vw/Vo),in which Co and Cw are the metal concentrations in the upper and lower phases,
respectively; Vo and Vw are the volumes of the upper and lower phases, respectively.
Results and discussion
Phase diagram and tie line
The phase diagram of the ternary mixed solvent solutionof water-acetonitrile-ethyl acetate is shown in Fig. 1. The solid curve indicates theboundary between the heterogeneous and homogeneous systems at 25 °C. The dottedcurve is the boundary between these systems at 0 °C. The plot (○) near the boundary
curve at 25 °C locates the component volume ratio of 3:8:4 for water-acetonitrile-ethyl
297
Two-phase separation systems of mixed solutions and TRDP

297

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

acetate. The tie-line of the plot at 0 °C on the phase diagram was constructed using thesolvent composition of the homogeneous solution at 25 °C, the water content of thelower phase in the batch vessel at 0 °C (where Karl Fischer moisture titrator (MKC-610;
API Co.) was used), and the boundary curve at 0 °C. The volume ratio of the upper andlower phases at 0 °C in the batch vessel corresponds to the ratio of the line lengths,
which are separated by the plotted point on the tie line. In this case, the volume ratio of
the upper and lower phases was 6:1, as shown in Fig. 1. The phase diagram of theternary mixed solvent solution containing 10 mM 8-HQ was similar to that shown in
Fig. 1, although 10 mM 8-HQ was only sparingly dissolved in a mixed solvent solution
containing water at less than ca. 50 vol%.
Preliminary extraction experiments Two preliminary experiments (Experiments 1
and 2) are illustrated in Fig. 2. In Experiment 1, a solution of ethyl acetate containing 10
mM 8-HQ and an acetic acid solution (pH 2.8) containing 0.34 mM metal ions (at avolume ratio of 6:1; total volume 15 mL) in a batch vessel was held for 30 min without
stirring. This system, as might be expected, consisted of two phases. The metal
concentrations of the two phases were then measured with ICP. D and E(%) were bothzero for all metal ions (Fe(III), Co(II), and Cu(II)). That is, Experiment 1 showed no
extraction behavior for these metal ions under the conditions used. In Experiment 2, thesame conditions were used as in Experiment 1, but the system was well stirred in thebatch vessel for 30 min, and then held without stirring for 30 min. The metal
concentrations in the two phases were then measured by ICP. The D and E(%) values
were, respectively, 0.46 and 73 for Fe(III), 0.15 and 47 for Co(II), and 0.08 and 32 forNi(II) (the numbers were averages for 3 – 5 times measurements). Thus, as a general
result, Experiment 2 showed extraction behavior for the metal ions. After 3 hours ofstirring, D and E(%) remained constant at the above values for all metal ions.
Extraction experiment using the ternary mixed solvent solution Experiment 3 using
the ternary mixed solvent solution was carried out as illustrated in Fig. 2. A
homogeneous ternary mixed solvent solution of water-acetonitrile-ethyl acetate (volume
ratio 3:8:4; total volume 15 mL) containing 10 mM 8-HQ and 0.34 mM metal ion
solution in a batch vessel was cooled from 25 to 0 °C, and held for 30 min. The metal
concentrations of the two phases were then measured by ICP. D and E(%) were,respectively, 0.47 and 74 for Fe(III), 0.15 and 47 for Co (II), and 0.08 and 32 for Ni(II)(the numbers were averages for 3 – 5 times measurements). That is, the two-phasesystem of the ternary mixed solvent solution performed similarly to the extraction
system of Experiment 2. After 3 hours, D and E(%) remained constant at the above
values for all metal ions. It is worth emphasizing that Experiment 3 using the two-phasesystem performed metal ion extraction with 8-HQ without stirring at almost the same D
and E(%) values as those obtained in Experiment 2, although the extraction conditions,such as the pH values of the water-rich phase, have to be examined in detail in order toimprove the extraction efficiency. The order of extraction performance obtained with298
298

Chapter 13

8-HQ in Experiments 2 and 3, Fe(II)>Co(II)>Ni(II), was consistent with that reported
previously [18,19].
Extraction of mixed metal ions Mixtures of metal ions containing Fe(III) and Co(II),
Fe(III) and Ni(II), and Co(II) and Ni(II) were extracted in the same way as inExperiment 3 (each metal concentration was 0.18 mM). The values of D and E(%) for
the Fe(III) and Co(II) mixture were, respectively, 0.40 and 70 for Fe(III); 0.14 and 45
for Co(II). For the Fe(III) and Ni(II) mixture, the respective D and E(%) values were0.33 and 66 for Fe(III) and 0.08 and 32 for Ni(II). Finally, the respective D and E(%)
values for the Co(II) and Ni(II) mixture were 0.16 and 50 for Co(II) and 0.11 and 39 forNi(II) (the numbers were averages for 3 – 5 times measurements). These values for the
metal ions were very similar to those obtained in Experiments 2 and 3.
In conclusion, a two-phase separation system comprising a ternary mixed-solvent
system was reported. The water-acetonitrile-ethyl acetate system changed from ahomogeneous (single-phase) to a heterogeneous (two-phase) system with temperature
and/or pressure changes. Here, for the first time, we used a two-phase separation systemcomprising a ternary mixed solvent solution in a batch vessel to extract metal ions.
Model metal ions of Fe(III), Co(II), and Ni(II) were successfully extracted with thetwo-phase system. The experimental data obtained here suggest that the new phaseseparation system offers new capabilities in separation science, similar to othertwo-phase systems consisting of polymers, micelles, ionic liquids, and fluorous
solutions.

299
Two-phase separation systems of mixed solutions and TRDP

299

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Phase diagram of the ternary mixed-solvent solution comprisingwater-acetonitrile-ethyl acetate. The solid curve represents 25 °C and the dotted curve
represents 0 °C. The component ratio of water-acetonitrile-ethyl acetate for the plotted
point (○) is a 3:8:4 volume ratio. The straight line on the plot represents the tie line at
0 °C.

Figure 2. Illustration of extraction experiments in the batch vessel. The extraction
conditions are described in detail in Experiments 1–3 in the text.

13.2 TRDP created with an aqueous ionic liquid mixed solution
300
300

Chapter 13

The tube
radialdistribution
solvents
was ionic
observedin anmixed
aqueoussolution
ionic liquid mixed
13.2
TRDP
createdwithofanaqueousliquidsolution of 1-butyl-3-methylimidazolium chloride fed into a capillary tube. A phase
diagram
1-butyl-3-methylimidazolium
chloride
potassiumThe
tube wasradialconstructed
distributionwithof solvents
was observed in an aqueous
ionicandliquidmixedhydroxide,which
includedboundary
curvesbetweenhomogeneousandheterogeneous
solution of 1-butyl-3-methylimidazolium chloride fed into a capillary tube. A phasesolutions was
at 15constructedand 20 °C. As
example, an aqueous ionic liquid
mixedandhomogeneous
diagram
withan1-butyl-3-methylimidazolium
chloride
potassium
solution,comprising
the
ratio
of
1-butyl-3-methylimidazolium
chloride:
potassiumhydroxide, which included boundary curves between homogeneous and heterogeneoushydroxideat(24.0: 20.0wt/wt%),
which werepositioned
the mixedboundaryat 20 °C,
solutions
15 and20 °C.
As an example,
an aqueous
ionicnearliquidhomogeneous
were delivered
into a fused-silica
tube (75 µm inner diameter,
110: cm
length,solution,comprising
the ratio ofcapillary
1-butyl-3-methylimidazolium
chloride
potassium-1hydroxide: 20.0 wt/wt%),whichboundary atsolution
20 °C,
15 °C tube(24.0
temperature)
at a flowrate wereof 1.0positionedµL min .near
Thethehomogeneouschangedto a heterogeneous
solutionwith two
inner(the ionic
were
deliveredinto a fused-silica
capillary
tubephases:(75 mthe
innerdiameter,
110 liquid-rich)
cm length,-1
and°C
thetubeouter(the aqueousthe. ionicliquid) phases,solution
in theThe homogeneous
15
temperature)
at aphaseflow merely
rate of containing
1.0 L min
capillary totube.
The radial distributionof thetwo
solvents
the inneraqueousmixedchangeda heterogeneous
solution withphases:in the(theionic
ionicliquid
liquid-rich)solutionthrougha bright-lightmicroscope-CCD
camerasystem.
andthe wasouterobserved(the aqueous
phase
merely containing
the ionic
liquid)phases, in thecapillary tube. The radial distribution of the solvents in the aqueous ionic liquid mixed
Introduction
solution
was observed through a bright-light microscope-CCD camera system.
Since the last century, phase separation in aqueous two-phase systems of ionic liquids(ILs) has been well known in the field of analytical chemistry and separation science
Introduction[1-5]. theFor last
example,solutions ofseparate
into two
distinctwhen
Since
century,aqueous
phase separationin ILsaqueous
two-phasesystemsof phases
ionic liquids
cooledhas
below
certaintemperature;a temperature-induced
phase separation(ILs)
been awellknown
in the fieldthatof is,
analyticalchemistry and separation
scienceoccursFor[12].example,
One phaseis an almost
IL-poor
whiledistinct
the otherphasewhenis a[1-5].
aqueoussolutionsof ILsaqueous
separatephase,
into twophasesconsiderable(IL-rich phase).
an application, it wasreportedthatcooledbelowILa solution
certain temperature;that Also,is, a astemperature-induced
phase
separation
cytochromesolubilized
IL-pooraqueoussolution
arewhile
extracted
into theIL-richoccurs[12]. cOne
phase is inanthealmostIL-pooraqueousphase,
the other
phase
is a
phase by takingof the phaseseparation
two-phaseof thatILs
considerable
IL advantage
solution (IL-richphase).
Also, as inanaqueous
application,
it wassystems
reported[9].
cytochromec solubilized in the IL-poor aqueous solution are extracted into the IL-richHere,an aqueous
IL mixedthat showed
phase two-phaseseparation systems
as mentionedphaseby takingadvantageof the solution
phase separation
in aqueousof ILs
above
was
fed
into
a
capillarytube
toexplore
a
new
type
of
TRDP
that
is
not
used
with
[9].
theHere,ternary
solvents
of water-hydrophilic/hydrophobic
solvents.
The tube
anmixedaqueous
IL mixed
solution that showed phaseorganic
separation
as mentioned
radial was
distribution
the solvents
the aqueous
mixed
solution
successfully
above
fed into ofa capillary
tube into explore
a newILtypeof TRDP
thatwasis notused withobservedinthecapillarytube,
indicatingthekinetic
liquid-liquidinterface
betweenthethe ternary mixed solvents of water-hydrophilic/hydrophobic organic solvents. The tube
IL-poordistribution
aqueous andfinding
expandthe TRDP
concept toradialof thethe IL-richsolventsphases.
in theThis
aqueous
IL willmixed
solution
was successfully
a new researchof tube,microfluidic
behavior,
and liquid-liquidallow the possibility
developing
observedin the subject
capillaryindicating
the kinetic
interfaceofbetween
thenew typesof chromatography,
micro-reaction,and mixing
processwith theIL-pooraqueousand the IL-richextraction,phases. Thisfinding will expand
the TRDP
concept
to
IL mixed
solutions.
aaqueous
new researchsubjectof microfluidic behavior, and allow the possibility of developing
new types of chromatography, extraction, micro-reaction, and mixing process with theExperimental
aqueous
IL mixed solutions.
The purity of potassium hydroxide (KOH) reagent was confirmed to be 87 wt% withtitration. Acid Orange 10 was used as a dye for a bright-field microscope observation. A
Experimental
fused-silica
capillarytubehydroxide(75 µm i.d.
and 150µm o.d.,cm totaltolength)
was used.
The
purity ofpotassium(KOH)
reagent
was 110
confirmed
be 87 wt%
with
The microscopy
was setup withtube.
Bright-lightmicroscope
titration.
Acid Orange
10 wasusedaasfused-silica
a dye for acapillarybright-fieldmicroscopeobservation.
A
observation capillaryin the capillary
wasandmonitored
a length
90 cm
fromwasthe used.
inletfused-silica
tube (75tubes
m i.d.
150 m ato.d.,
110 cmoftotal
length)usingmicroscopy
a microscope
equippedcamera. capillary
The parttube.
of Bright-lightthe capillarymicroscopetube forThe
was setup with CCD
a fused-silica
observation inthroughthe bright-fielda temperatureofthe capillarytubes wasmicroscope
monitored was
at a maintained
length of 90atcm
from the inlet
301
Two-phase separation systems of mixed solutions and TRDP

301

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesusing
a microscope
equipped(MATS-555RO;
CCD camera. Tokai
The Hit
partCo.,of Shizuoka,
the capillary
tube for15 °C witha thermo-controller
Japan).
observation through the bright-field microscope was maintained at a temperature of15°C withthermo-controller (MATS-555RO; Tokai Hit Co., Shizuoka, Japan).
Resultsanda discussion
As described in the introduction, an aqueous solution of ionic liquid,
Results
and discussion
1-butyl-3-methylimidazolium
chloride ([C4mim]Cl), including a salt, such as potassiumAs
described
in carbonate,
the introduction,
aqueous potassium
solution hydrogenphosphateof ionic liquid,
citrate,potassium
potassium anphosphate,includingsalt, suchas potassium
1-butyl-3-methylimidazolium([Cdistinct(K2HPO4) or KOH, separateschloride
into twophaseswhen acooledbelow
a certain
4mim]Cl),
citrate,potassium
carbonate, experiments,
potassium phosphate,potassiumhydrogenphosphate
temperature.
As preliminary
we examined
the aqueous
solutions of
HPO4) orKOH,
separates
twowt%)
distinctphasescooled(24.0
below
a certain(K[C42mim]Cl(21.3wt%)–K
mixtureand when[C4mim]Clwt%)–
KOH
2HPOinto
4 (13.7
temperature.
As preliminary
we phase
examined
the aqueous
solutionsof(20.0 wt%) mixture
through theexperiments,
upper and lower
separation
in a batch
vessel and(21.3wt%)–K
HPO4 (13.7inwt%)
mixtureandConsequently,[C4mim]Cl (24.0wt%)–[C
the4mim]Cl
inner andouterphase 2separationa capillarytube.
nearlythe KOHsame(20.0wt%)ormixture
throughthe upper
lower phasein aqueous
a batch vessel
andseparation
distribution
behavior
wasandobserved
for theseparation
two mixed
solutions.
the
inner
outer solution
phase separation
in a capillary tube.
Consequently,
nearlysame
Here,the andaqueousof a [C4mim]Cl–KOH
mixture,where KOH
had athesimpler
separation
or distribution
for thetwo mixed
aqueous solutions.
, wasobserved
investigatedin detailas a model.
chemical construction
thanbehavior
K2HPO4was
where
KOH had a andsimplerHere,aqueoussolutiona [C4mim]Cl–KOH
A the
phase
diagramwas ofconstructed
between mixture,
the KOH
concentration
thewasin detail aswas
a model.
chemicalconstruction
thanatK15[C4mim]Cl
concentration
and4,20°Cinvestigated(KOH concentration
estimated based on
2HPOphase diagram
betweentheobtained
KOH phase
concentration
andthetheAassumptionthat thewas
purityconstructedlevel was 100%). The
diagram isshownmim]Clconcentration
at are15 and20 °C (KOHconcentration
was estimatedbased on[C
in 4Fig.
1. Thedotted curves
the boundary
betweenhomogeneousand heterogeneoustheassumptionthat the purity solution
level was100%) two
. Thephases:
obtaineddiagramis andshown
solutions.
The heterogeneous
includedan phase
IL-pooraqueousaninFig. 1.
The dottedcurves are
the°C
boundary
betweenhomogeneous
IL-richphase.
The boundaryat 15
is positioned
slightly
lower thanand
thatheterogeneousat 20 °C. The
solutions.
heterogeneous
solution
an IL-pooraqueous forand theancomponentThe
ratiosof [C4mim]Cl
andincluded
KOH intwo
thephases:
homogeneous
solutionsIL-richphase.
The
boundary
at 15
°C is
positioned
slightly
than thatcurveat 20 at°C.20Thesymbols,○, ofNos.
1 – 7 thatwere
positioned
almoston lower
the boundary°Cmim]Cl
and
KOH
in
the
homogeneous
solutionsforthecomponent
ratiosof[Cwere as follows: (No. 1) 4[C4mim]Cl:KOH, 52.3:6.5 wt/wt%; (No. 2) 44.8:9.3; (No. 3)
symbols,of 4)
Nos.
1 – 7 that(No.
were5)positioned
on18.4:24.0;
the boundary
at 20 °C
37.2:12.8;○,(No.
30.5:16.5;
24.0:20.0;almost(No. 6)(No.curve7) 14.0:27.0.
52.3:6.5
wt/wt%;
2) 44.8:9.3;
(No.
3)
were
follows: (No.
[C4mim]Cl:KOH,
Phaseasseparationin the1)homogeneous
solutionsof Nos.
1 – 7(No.
by changing
them
from
37.2:12.8;(No.
(No. 5)24.0:20.0; below(No. 6)and
18.4:24.0;
20 °C to 15°C4)in30.5:16.5;
a batch-vessel
is describedshown in(No.
Fig.7)1.14.0:27.0.
The salt
Phase
separationin the homogeneousNos.
1 – 7ratios
by changing
fromdepositions
were observedfor Nos. 1 –solutions
3 and alsoofthevolumeof upperthemand lower
20°C to
15 not°C exactly
in a batch-vessel
is Nos.
described
and shownin Fig.
Thelowersaltphaseswereestimated for
1 – 4. below
The viscositiesof theupper1.and
depositions
observed
for Nos. 1The– 3 phaseand also
the volume
upperphases werewere
examinedqualitatively.
separation
was ratios
carriedofout
withandan lower
upper
phasesnot andexactlyestimated
foraqueousNos. 1 –solution
4. The viscosities
upper
and lower
IL-rich were
solutiona lower
IL-poorfor Nos. 4 of
– 7.theThevolumeratiosphaseswere examined
The phasewas 80:20,
carried 59:41,
out with
an upperof the upperand lowerqualitatively.
phases for Nos.
4, 5, separation
6, and 7 were
30:70,
and
2IL-richsolution and aGenerally
lower IL-pooraqueoussolutionfor Nos. ions
4 – 7.(OH
The, volume
21:79, respectively.
speaking,
addingkosmotropic
HPO4 ,ratios
etc.)
of
the
upper
and
lower
phases
for
Nos.
4,
5,
6,
and
7
were
80:20,
59:41,
30:70,
into an aqueous IL solution leads to an increase in the interaction between ions and221:79,respectively.generating
Generally the
speaking,
adding kosmotropic(OH-, HPO
water molecules,
two separated
phases [4].ions
However,throughthe
4 , etc.)
into
an aqueous
IL solution
leadshow
to many
an increasein the interactionionsandinteraction,
it is difficult
to know
water molecules
leave in between
the IL-richphase,
water
molecules,
generating
twoto separated[4].
However,theand howmany watermoleculesthe
move
the IL-poorphasesaqueousphase.
That is,throughthe volumeinteraction,is difficult
to knowhowmaymany
molecules
leavein the IL-richphase,
ratio of the itupperand lower
phasebewater
decidedonly withobtained
experimental
anddata.how many water molecules move to the IL-poor aqueous phase. That is, the volume
ratioof the upperaqueousand lower
phase atmay
be containing
decided only
obtained
experimental
Homogeneous
solutions
20 °C[C4withmim]Cland KOH,
Nos. 4 –data.
7, were delivered into the capillary tube which of the observation point was controlled
and KOH,
Nos.
4–
Homogeneous
aqueoussolutions
at 20
at 15
°C. The solutionof No.
3 could
not°C
be containing
fed into the[Ccapillary
becauseof salt
4mim]Cl tube
7,
were delivered
into thecapillarytubeexaminedwhich ofwith
the observation
was controlled
deposition.
The TRDP
in thetube wasa bright-fieldpoint
microscope
for the302
302

Chapter 13

at
15 °C. The
solutionof TheNo. homogeneous
3 could not besolutions
fed into thecapillary
becauseof salta
solutionsof Nos.
4 - 6.
of No.
4 andtube
7 did
not create
deposition.
TRDP
in the tube was
examinedwith aconditions.
bright-fieldTypicalmicroscope
for the
TRDP withThe
goodreproducibility
under
the present
bright-field
solutionsof Nos.
4 - 6.
homogeneous
solutionsof No. 4solution
and 7 did
not5create
photographsof TRDP
areTheshown
in Fig. 2. The
homogeneousof No.
createda
TRDP
reproducibility
the present
conditions.
Typicalbright-fielda TRDPwith
withgooda majorinner phase underof the IL-richsolution,where Acid
Orange10 wasphotographsof TRDPare minor
shownouterin Fig.
2. Thehomogeneous
solutionof No.existed
5 created
mainly dissolved,
and thephaseof theIL-poor aqueous
solution
at a
-1
aflow
TRDP
a majorinner
phase of the IL-richsolution,where Acid
Orange10 wasratewithof 1.0µL min. However,homogeneous
solutionsof No.
6 createdthemainlydissolved,
and
the
minor
outer
phase
of
the
IL-pooraqueoussolutionexistedat
TRDP with a minor inner phase of the IL-rich solution, where Acid Orange 10 wasa
-1
. However,homogeneous
solutionsNo. 6 created
thea
flow
rate
of 1.0 Lmainlydissolved,
andminthe major
outer thephaseof the IL-pooraqueousofsolution
existed at
-1
-1
TRDP
with
a minorinner
phase
of wasthe partiallyIL-rich solution,
Orangewasflow rateof 3.0µL min(theTRDPcreated atwhere
a flowAcid
rate of
1.0 µL10min).
mainlydissolved,
the major
outerbutphaseof the in
IL-pooraqueous[Csolution
existedAlso itat isa
Acid Orange
10 isandsolublein water,insoluble
ionic liquid,
4mim]Cl.
-1
-1
(the
TRDP
was
partially
created Thus,
at a flowof 1.0 Lofmin
flow
rate solubleof 3.0 Lmina 50
scarcely
with
wt%
KOH
aqueoussolution.
the rate
distribution
Acid).
Also ittheisAcid
Orangeis soluble
water, aqueous
but insoluble
in mustionic beliquid,[C4mim]Cl.
Orange10 to10IL-richand inIL-poorphasesjudgedby comparing
scarcelysolublewithina 50
KOHInaqueous
solution.
distribution
Acidyellow color
depthsbothwt%phases.
the batchvessel,Thus,
Acid theOrange10 wasofmostly
Orange10theto IL-poor
IL-rich andIL-pooraqueousmust However,be judged inbyNos.
comparingsoluble inaqueousphasein No. phases
4 solutions.
5, 6, andthe7
yellow
color
in 10
both
phases.
In the batch
vessel,Orange10 wasmostlysolutions
Aciddepths
Orangewasvery slightly
solublein theAcid
IL-pooraqueousphase,
but
solublein thesoluble
IL-poorinaqueous
phasephase,in No. by
4 solutions.
However,Nos. depth.
5, 6, and7
was mostly
the IL-rich
judging theyellowincolorThesolutions
Aciddata
Orangewas vessels
very slightly
solublein theIL-poor photographs
aqueous phase,butexperimental
in the10batch
consistedof thebright-light
shown
was
mostlysoluble in the IL-rich phase, by judging the yellow color depth. The
in Fig.
2.
experimental
datawithin thebatchwater-hydrophilic/hydrophobic
vessels consisted of the bright-lightshown
In the TRDP
ternaryorganicphotographs
solvents solutions
inFig. 2. by us previously, major inner and minor outer phases were observed in various
reported
In theTRDP withternary
water-hydrophilic/hydrophobic
solvents
solutionstypes
of capillarytubes.
We didnot observe minor inner andorganic
major outerphasesin thereportedby us
major innerand However,minor outer
wereTRDP
observed
in various
TRDP withthepreviously,
ternary solvents
solution.
thephases
observed
patternof thetypes
of capillarytubes.inWe
not observeminor
innerwasandnotmajor
outerconsistent
phases inwith
theinner andouter phases
thedidaqueous
IL mixed
solutionalwaysTRDP
with the pattern
ternary insolvents
solution.
However,observed
TRDP
patternreports.
of thethe distribution
the ternary
mixedsolventsthe
solutionsin ourprevious
innerand outerin the aqueous
mixed
was not always
consistent
The reason
for phases
the differencein the IL
inner
andsolution
outer formation
patterns
betweenwith
thethedistribution
patternin the andternary
our previous
reports.
aqueous
IL mixed
solution
the mixed
ternarysolvents
mixed solutions
solvents insolution
has notbeen
Thereason for the difference in the inner and outer formation patterns between theclarified.
aqueous IL mixed solution and the ternary mixed solvents solution has not beenclarified.
In conclusion, we have developed a new type of TRDP, i.e., a kinetic liquid-liquidinterface, in a capillary tube, by the delivery of an aqueous homogeneous solution of IL,
conclusion,
we havedeveloped
a new typeof TRDP,i.e., a kineticmim]Cl,including
KOH.
The previous
TRDP
using ternary
mixed liquid-liquidsolvents of[C4In
interface,in a capillary tube, by thedeliveryof an aqueous
solutionwater-hydrophilic/hydrophobicorganic
mixtureshave homogeneous
been examined
fromof IL,
the4mim]Cl,including
KOH.
The
previousTRDP
using
ternary
mixed
solventsof[C
viewpointsof chromatography, extraction, micro-reaction, and the mixing process. The
4water-hydrophilic/hydrophobic
havesolution
been will
examinedfromthenew type of TRDP with theorganic
aqueousmixtures
IL mixed
lead tofurther
4extraction,
micro-reaction,
andandtherelated
mixingtechnologies.
process. The
viewpointsof chromatography,
developmentsof both fundamentals
and applicationsof TRDPnew type of TRDP with the aqueous IL mixed solution will lead to further developmentsof both fundamentals and applications of TRDP and related technologies.

303
Two-phase separation systems of mixed solutions and TRDP

303

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Phase diagram of an aqueous IL mixed solution consisting of [C4mim]Cl and
KOH at 15 (■) and 20 °C (●). The phase separation in a batch vessel at 15°C is also
illustrated along with the volume ratio of upper and lower phases in the figure; blue,
IL-poor aqueous and yellow, IL-rich. The compositions of the homogeneous solutions(20°C), Nos. 1- 7, represented by the symbols (○) are (No. 1) [C4mim]Cl : KOH,
52.3:6.5 wt/wt%; (No. 2) 44.8:9.3; (No. 3) 37.2:12.8; (No. 4) 30.5:16.5; (No. 5)
24.0:20.0; (No. 6) 18.4:24.0; (No. 7) 14.0:27.0.

Figure 2. Typical photographs obtained with the bright-field microscope-CCD camera
system for solutions of Nos. 5 and 6 at a tube temperature of 15 °C. The arrows indicate
the interface between the IL-rich and IL-poor aqueous phases. Conditions: Capillarytube, 110 cm total length of 75 µm i.d. fused-silica; solution composition, (No. 5)[C4mim]Cl:KOH, 24.0:20.0 wt/wt% and (No. 6) 18.4:24.0; flow rate, 1.0 µL min-1 forNo. 5 and 3.0 µL min-1 for No. 6.

304
304

Chapter 13

13.3 TRDP created with an aqueous non-ionic surfactant mixed
solution
A new type of tube radial distribution phenomenon was observed in an aqueous micellar
solution of non-ionic surfactant that was fed into a microspace. A homogeneous
aqueous solution containing 2 wt% Triton X-100 and 2.0 M sodium chloride was fed
into a microchannel (40 µm in depth and 200 µm in width) in a microchip at a flow rateof 4.0 µL min-1, where the microchip was maintained at a temperature of 34 °C. The
homogeneous aqueous solution changed to a heterogeneous solution with two phases in
the microchannel; the surfactant-rich phase was generated around the middle of thechannel, while the aqueous phase merely containing the surfactant was formed near the
inner wall. The radial distribution of the surfactant was observed through Rhodamine B
dissolved in the aqueous micellar solution with a bright-field microscope-CCD camera
system. An open-tubular capillary chromatographic system was also tried to develop
using the fused-silica capillary tube (75 µm inner diameter and 120 cm length) as aseparation column and the aqueous micellar solution as a carrier.
Introduction
Since the last century, phase separation in aqueous polymer or micellar solutions has
been well known in the field of analytical chemistry and separation science [6-10,20].
Aqueous micellar solutions of some non-ionic surfactants separate into two distinct
phases when heated above a certain temperature (clouding point), that is, atemperature-induced phase separation occurs [6-10]. One phase is an almost
micelle-free aqueous solution (aqueous phase), while the other phase is a concentrated
surfactant solution containing considerable amounts of water (surfactant-rich phase).
Hydrophobic compounds that are solubilized in the aqueous micellar solution are
extracted into the surfactant-rich phase, while hydrophilic compounds remain in the
aqueous phase.
Here, an aqueous micellar solution of non-ionic surfactant that showed phaseseparation as mentioned above was fed into a microspace to explore a new type of
TRDP that was not used with the ternary mixed solvents ofwater-hydrophilic/hydrophobic organic solvents. The tube radial distribution of thesurfactant was successfully observed in the microchannel in a microchip and thecapillary tube, indicating the kinetic liquid-liquid interface. We also tried to develop an
open-tubular capillary chromatographic system, i.e., TRDC, using the fused-silica
capillary tube as a separation column and the aqueous micellar solution as a carrier.
Experimental
Materials
A microchip incorporating a straight-line microchannel (40 µm deep and200 µm wide) was manufactured with Microchemical Technology (Kanagawa, Japan).
The microchip was set up in a microchip holder. A fused-silica capillary tube (75 µm
305
Two-phase separation systems of mixed solutions and TRDP

305

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

inner diameter and 150 µm outer diameter) was used.
Bright-field microscope-CCD camera system
The bright-field microscope-CCD
camera system was setup with the microchip or the fused-silica capillary tube. The reddye, Rhodamine B, dissolved in the aqueous micellar solution of 2 wt% Triton X-100
and 2.0 M sodium chloride was observed using the microscope and CCD camera. The
microchip or capillary tube was maintained at temperatures of 20 °C and 34 °C on athermo-heater.
Open-tubular capillary chromatography The open-tubular capillary chromatography
comprised mainly the microsyringe pump (Model 11 Single syringe 55-1199; Harvard
Apparatus, Inc., Massachusetts, U.S.A), the analyte injector (17.7 nL injection volume)(Chemco Co., Osaka, Japan), the fused-silica capillary tube (150 cm total length and100 cm effective length), the thermo-heater (20 °C and 34 °C) (Thermo Plate
MATS-555RO; Tokai hit Co., Shizuoka, Japan), and the fluorescence detector (ex. 290
nm and em. 355 nm) (Fig. 1). An aqueous solution containing 2 wt% Triton X-100 and
2.0 M sodium chloride was used as a carrier solution. Model analyte solution of
1-naphthol and 2,6-naphthalenedisulfonic acid (1 mM each) that was prepared with thecarrier solution was fed into the capillary tube at a flow rate of 2 µL min-1.
Results and discussion
Discussion
Phase diagram of aqueous micellar solution Clouding points of an aqueous solution
containing 2 wt% Triton X-100 as a non-ionic surfactant were examined throughinspection with various concentrations of sodium chloride, 0–3.0 M. The obtained datais shown in Fig. 2. The homogeneous and transparent solution at 20 °C changed througha clouding point to the heterogeneous solution including the two phases, the aqueous
phase and the surfactant-rich suspension phase. The clouding points gradually decreased
from 65 °C to 25 °C with increasing of sodium chloride concentration from 0 to 3.0 M
as shown in Fig. 2.
Tube radial distribution of aqueous micellar solution in a microchannelAhomogeneous aqueous solution containing 2 wt% Triton X-100 and 2.0 M sodium
chloride possessed a clouding point at 34 °C as indicated in Fig. 2. The homogeneous
solution that also included 5 mM Rhodamine B was fed into the microchannel in a
microchip at a flow rate of 4 µL min-1 under laminar flow conditions, where themicrochip was maintained at temperatures of 20 °C or 34 °C. The bright-field
photographs of the aqueous micellar solution were examined with the microscope-CCD
camera system. The obtained photographs are shown in Fig. 3. At a temperature of
20 °C, which was under the clouding point, the homogeneous red solution due to
Rhodamine B was observed at a flow rate of 4 µL min-1. That is, the distribution of thesurfactant in the microchip was not observed at a temperature of 20 °C (Fig. 3 a)). At atemperature of 34 °C and a flow rate of 4 µL min-1, the solution phase containing the306
306

Chapter 13surfactant was generated around the middle of the microchannel as an inner phase (thesurfactant-rich phase) and the solution phase not containing the surfactant was formednear the inner wall as an outer phase (the aqueous phase) (Fig. 3 b)). Rhodamine B
dissolved in the solution was distributed in the surfactant-rich phase (red) [21]. The
homogeneous aqueous solution containing 2 wt% Triton X-100 and 2.0 M sodium
chloride with a clouding point of 34 °C must bring about phase transformation to theheterogeneous solution with the aqueous phase and the surfactant-rich phase through themicrochannel, the temperature of which was controlled at 34 °C.
Tube radial distribution of aqueous micellar solution under various conditions
Figure 4 shows the photographs of aqueous solution containing 2 wt% Triton X-100
and 2.0 M sodium chloride at 33 - 36 °C at various flow rates of 0.8 – 8.0 µL min-1. The
microchannel was suitable to the microscope observation because of the rectangle
cross-section. At 33 °C, which was under the clouding point, the aqueous solution
containing Rhodamine B indicated homogeneous red solution under all conditions. On
the other hand, at 34, 35 and 36 °C, which were just over the clouding point, the TRDP
was observed at 0.8 – 4.0 , 0.8 – 8.0, and 3.2 – 8.0 µL min-1, respectively, although it
was difficult to clearly distinguish between TRDP and non-TRDP in photographobservation. In the TRDP, the solution phase containing most of the surfactant was
generated around the middle of the microchannel as an inner phase (the surfactant-richphase) and the solution phase containing a small amount of the surfactant was formednear the inner wall as an outer phase.
Tube radial distribution of aqueous micellar solution in a capillary tube
The
distribution of the surfactant and Rhodamine B in the aqueous solution containing 2
wt% Triton X-100 and 2.0 M sodium chloride was also observed in the capillary tube
under and over the clouding point. The photographs are shown in Fig. 5; the TRDP is
not observed at 20 °C and observed at 34 °C. The microfluidic flow in the capillary
tube at 34 °C where gravity has little effect must produce a specific radial distributionof the surfactant featuring the surfactant-rich inner phase and the aqueous outer phasesimilar to that in the microchannels of a microchip. We developed a new type of TRDP,
i.e., the kinetic liquid-liquid interface, in the capillary tube, by delivery of an aqueous
micellar solution of non-ionic surfactant, Triton X-100, together with sodium chloride.
Temperature control regarding the clouding point played an important role in thepresent TRDP using an aqueous micellar solution. The previous TRDP using ternary
mixed solvents of water-hydrophilic/hydrophobic organic mixtures have been examinedfrom the viewpoints of chromatography (TRDC), extraction (TRDE), mixing (TRDM),
and as chemical reaction space (TRDR). The findings with the new TRDP of theaqueous micellar solution will lead to the further development of both fundamentals andapplications of TRDP and related technologies, such as TRDC, TRDE, TRDM, and
TRDR.
307
Two-phase separation systems of mixed solutions and TRDP

307

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Open-tubular capillary chromatography Tentatively, we tried to develop the TRDC
system using the fused-silica capillary tube as a separation column and the aqueous
micellar solution containing 2 wt% Triton X-100 and 2.0 M sodium chloride as a carrier.
Hydrophobic 1-naphthol and hydrophilic 2,6-naphthalenedisulfonic acid as a model
mixture was subjected to the present TRDC system (Fig. 1). The obtained
chromatograms are shown in Fig. 6. They were not separated at a tube temperature of
20 °C, while, they were separated and detected in this order at a tube temperature of
34 °C. The elution order was reasonable because of the distribution of the aqueous
micellar solution generating the surfactant-rich inner phase (hydrophobic) and thesurfactant-light outer phase (hydrophilic). The two peaks were identified with individual
analyte analysis, although they did not show baseline separation under the present
conditions.
In conclusion, a new type of TRDP was successfully created by feeding the aqueous
micellar solution (containing 2 wt% Triton X-100 and 2.0 M sodium chloride) into themicrospace over the clouding point. The TRDP generated the surfactant-rich inner
phase and the surfactant-little outer phase. The TRDC system was also developed basedon the TRDP. The new type of TRDP supported our consideration that the TRDP isgenerally caused through phase transformation from homogeneous to heterogeneous(including two phases) with some changes in pressure and/or temperature in a
microspace.

308
308

Chapter 13

Figure 1. Schematic diagram of an open-tubular capillary chromatography.

Figure 2. Phase diagram of an aqueous micellar solution containing sodium chloride.
The solubility curve is constructed along the clouding points. The concentration of
Triton X-100 is 2.0 wt%.

Figure 3. Bright-field photographs in a microchannel. a) 20 °C and b) 34 °C. The
homogeneous aqueous solution containing 2 wt% Triton X-100, 2.0 M sodium chloride,and 5 mM Rhodamine B was fed at a flow rate of 4 µL min-1 into the microchannel.

309
Two-phase separation systems of mixed solutions and TRDP

309

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Bright-field photographs under various conditions. The temperature of 33 36 °C and the flow rates of 0.8 – 8.0 µL min-1. The homogeneous aqueous solution
containing 2 wt% Triton X-100, 2.0 M sodium chloride, and 5 mM Rhodamine B was
fed into the microchannel.

Figure 5. Bright-field photographs in a capillary tube. a) 20 °C and b) 34 °C. The
homogeneous aqueous solution containing 2 wt% Triton X-100, 2.0 M sodium chloride,and 5 mM Rhodamine B was fed into the capillary tube (75 µm inner diameter and 120
cm long; the observed point was 100 cm from the inlet) at a flow rate of 2 µL min-1.

310
310

Chapter 13

Figure 6. Chromatograms obtained by the present TRDC system. a) 20 °C and b) 34 °C.
Capillary, 75 µm inner diameter, 150 cm total length and 100 cm effective length;
carrier, aqueous solution containing 2 wt% Triton X-100 and 2.0 M sodium chloride;flow rate, 2 µL min-1; and the analyte concentration, 1 mM each.

311
Two-phase separation systems of mixed solutions and TRDP

311

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

13.4 TRDP created with a fluorocarbon and hydrocarbon organic
solvent mixed solution
A fluorocarbon and hydrocarbon organic solvent mixture is known as atemperature-induced phase-separation solution. When a mixed solution oftetradecafluorohexane as a fluorocarbon organic solvent and hexane as a hydrocarbon
organic solvent (e.g., 71:29 volume ratio) was delivered into a capillary tube that wascontrolled at 10 °C, the tube radial distribution phenomenon (TRDP) of the solventswas clearly observed through fluorescence images of the dye, perylene, dissolved in themixed solution. The homogeneous mixed solution (single phase) changed to aheterogeneous solution (two phases) with inner tetradecafluorohexane and outer hexane
phases in the tube under laminar flow conditions, generating the dynamic liquid-liquidinterface. We also tried to apply TRDP to a separation technique for metal compounds.
A model analyte mixture, copper(II) and hematin, was separated through the capillarytube, and detected with a chemiluminescence detector in this order within 4 min.
Introduction
An investigation of fluorocarbon organic solvent solutions, or fluorous chemistry, hasbeen reported since seminal work done by Horváth and Rábai in 1994 [11]. Two-phaseseparation in fluorocarbon and hydrocarbon organic solvent mixed solutions is known
in the field of analytical chemistry and separation science [12,13]. Mixed solutions offluorocarbon-hydrocarbon organic solvents separate into two distinct phases in a batch
vessel when cooled below a certain temperature; that is, a temperature-induced phaseseparation occurs [14]. The lower phase is a fluorocarbon organic solvent phase, while
the upper phase is a hydrocarbon organic solvent phase. Based on this two-phase system,
liquid-liquid and liquid-solid extractions have been reported using different
fluorocarbon organic solvents [15,16,17].
When ternary mixed solvents of water–hydrophilic/hydrophobic organic solvents(e.g., water-acetonitrile-ethyl acetate mixtures) are fed into a microspace, such as glass
microchannels or capillary tubes (fused-silica, polyethylene, and PTFE for example)
under laminar-flow conditions, the solvent molecules are radially distributed in the
microspace. This “tube radial distribution phenomenon” (TRDP) creates inner and outerphases in the microspace, generating a specific dynamic liquid-liquid interface. Theinterface forms radially with a specific linear velocity in a capillary tube. We haveinvestigated TRDP in the chromatography, extraction, chemical reaction, and mixing
processes. The TRDP with the other two-phase separation aqueous solution systems,
such as water-surfactant and water-ionic liquid mixed solutions, were also reported in
our previous paper. However, these experimental data seemed to be just initial data in
the field of the TRDP and related research, because TRDP involves a quite novel
microfluidic behavior that has never been investigated. We must continue examining
fundamental facts about the TRDP as much as we can. Here, a312
312

Chapter 13

fluorocarbon-hydrocarbon organic solvent mixed solution, not an aqueous solution,
which showed two-phase separation in a batch vessel, was fed into a capillary tube to
explore the TRDP, for the first time. The finding here is that it is important to know the
TRDP capacity, and to expand the related technology.
Experimental
Reagents
Tetradecafluorohexane (C6F14; Mw 338.04 and density 1.67 g cm-3) as amodel fluorocarbon organic solvent and hexane (C6H14; Mw 86.18 and density 0.66 gcm-3) as a model hydrocarbon organic solvent were used. Also, cobalt(II) sulfate(Co(II)), copper(II) sulfate (Cu(II)), and hematin were used.
Fluorescence microscope-CCD camera system A fused-silica capillary tube (75 µm
inner diameter and 150 µm outer diameter) was used. A fluorescence microscope-CCD
camera with a fused-silica capillary tube (100 cm total length) was set up. Capillary
tube observations were monitored at a length of 80 cm from the inlet using afluorescence microscope equipped with an Hg lamp and a filter. Blue fluorescencephotographs were obtained because perylene emits light at 470 nm. The capillary tube
used for observation was maintained at a temperature of 10 °C with a thermo-controller(MATS-555RO; Tokai Hit Co., Shizuoka, Japan).
Separation system of metal compounds using an open-tubular capillary tube The
microspace separation system of metal compounds was developed using an
open-tubular capillary tube (100 cm length), a microsyringe pump, and a batch-type
chemiluminescence (CL) detection cell. The mixed solution of tetradecafluorohexaneand hexane (71:29 volume ratio) containing 50 µM luminol as a carrier solution wasdelivered into the tube at 1.8 µL min-1. The oxidant solution (40 mM hydrogen peroxide
dissolved in 10 mM carbonate buffer solution, pH 10.8) was put in a CL detection cell
equipped with an optical fiber [22]. The analyte solutions were prepared with thecarbonate buffer solution-acetonitrile (1:2 volume ratio) and introduced directly into thecapillary inlet side by the gravity method (from 20 cm height and for 20 s). The analytes,
metal compounds, having catalytic activity for the luminol-hydrogen peroxide CL
reaction, were mixed with the CL reagents at the tip of the capillary outlet in the cell togenerate CL light. The CL was detected with the photomultiplier tube (PMT)(H5783-20, Hamamatsu Photonics K. K., Shizuoka, Japan).
Results and discussion
Phase
diagram
Tetradecafluorohexane-hexane
mixtureisafluorocarbon-hydrocarbon organic solvent mixed solution; its phase-separation
temperature is under ca. 30 °C [14]. The phase diagram shown in Fig. 1, wasconstructed for varying the tetradecafluorohexane mole fractions. The curve in the
diagram marks the boundary between homogeneous (single phase) and heterogeneous(two phases) solutions. The tetradecafluorohexane-hexane mixed homogeneous313
Two-phase separation systems of mixed solutions and TRDP

313

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

solutions Nos. 1 – 9 in Fig. 1 were investigated. The heterogeneous solution includes
two phases: the lower tetradecafluorohexane and upper hexane phases in a batch vessel.
For example, the 0.36 mole fraction tetradecafluorohexane-hexane solvent mixed
solution (No. 4) was cooled from 30 to 10 °C. Phase separation occurred at 10 °C,
generating an upper hexane phase (blue solution with perylene) and a lower
tetradecafluorohexane phase. The lower-to-upper phase volume ratio was estimated tobe 8:9. Similarly, the volume ratios of the tetradecafluorohexane to hexane phases in theheterogeneous solutions were (No. 1) 15:65; (No. 2) 29:71, (No. 3) 42:58, (No. 4) 47:53,(No. 5) 50:50, (No. 6) 63:37, (No. 7) 71:29, (No. 8) 78:22, and (No. 9) 80:20. We also
examined
homogeneous
mixturesofoctafluorotoluene-toluene
andtrifluorotoluene-toluene with fluorocarbon organic solvent mole fractions of 0.3, 0.5,
and 0.7 in the temperature range of 0 – 20 °C. Under these conditions, no two-phasesystem was observed. Consequently, in the present work we examined the TRDP in thetetradecafluorohexane-hexane mixture in detail.
TRDP creation
The homogeneous tetradecafluorohexane-hexane solvent mixed
solutions Nos. 1 - 9 were delivered into a capillary tube at a temperature of 10 °C and atflow rates of 0.5 – 20 µL min-1. No tube radial distribution of the solvents was observed
for solutions Nos. 1 – 6; the solvents were distributed in the axial direction, as shown in
Fig. 2 a). On the other hand, for solutions Nos. 7 – 9 the tube radial distribution of thesolvents was clearly observed in the capillary tube at all flow rates. The fluorescence
images shown in Fig. 2 b) indicate that the TRDP creates inner major
tetradecafluorohexane and outer minor hexane phases; perylene (blue) was dissolved inhexane, but little dissolved in tetradecafluorohexane. Fluorocarbon organic solvent does
not mix with hydrocarbon organic solvent or water at a lower temperature. Then, thevolume ratios of the two phases in a batch-vessel and a capillary tube at a lower
temperature agreed with the mixing ratios of the solvents. Also, the TRDP creation was
confirmed in the microchannel (100 µm width and 45 µm depth) on a microchip madeof glass. Our TRDP for solutions Nos. 7 – 9 followed the same trend as the TRDP froma ternary mixed solvent solution of the water-acetonitrile-ethyl acetate previously
reported; major solvents were distributed around the middle of the tube, whereas minor
solvents were distributed near the inner wall, irrespective of whether the carrier
solutions were organic solvent-rich or water-rich phases. The linear velocity in theradial face section of the capillary tube under laminar flow conditions is circular,
indicating that the inside area of the tube has a lower velocity-change gradient than theoutside area; however, the real velocity curve in an aqueous-organic solvent mixed
solution may deviate from an ideal velocity curve. Fluidic stabilities based on linear
velocity gradients in the radial profiles under laminar flow conditions infer that the
major solvents must occupy the inside area instead of the outside area in the capillary
tube. Because fluorocarbon organic solvents have high viscosities, stiff structures, andlow surface tensions compared with hydrocarbon organic solvents, these specificproperties might support TRDP, thus allowing for major inner fluorocarbon organic
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solvent phase formation. The fluidic behavior of the TRDP was also in accordance withthe viscous–dissipation principle, which postulates that the degree of viscous dissipationis smaller for a given flow rate. Thus, the high-viscosity fluid is located at the core of
the tube, away from the inner wall [23-26]. However, why an axial distribution of thesolvents occurs for solutions Nos. 1 - 6 has not yet been clarified.
Microspace separation system for metal compounds
The retention times of Cu(II),Co(II), and hematin as model metal compounds were examined with the microspace
separation system using the fused-silica capillary tube and the mixed solvent solution oftetradecafluorohexane-hexane as a carrier. The analyte solution of each metalcompound was fed through the capillary tube and mixed with the reagents at the tip ofthe capillary outlet to generate CL. The retention times of Cu(II), Co(II), and hematinwere 2.2, 2.2, and 2.7 min, respectively. The retention times of Cu(II) and Co(II) were
the same; they were delivered with almost the average linear velocity, while, that ofhematin was delayed; it was fed with a lower velocity than the average linear velocity.
The analytes (Cu(II), Co(II), and hematin) were dissolved in the aqueous andacetonitrile mixture to prepare the analyte solutions. The Cu(II) and Co(II) were notdissolved by either tetradecafluorohexane and hexane, and then they were notdistributed to the carrier solution in the capillary tube. The hematin was dissolved inhexane, but not dissolved by the tetradecafluorohexane. It was then distributed to thehexane outer phase in the capillary tube. In the TRDP with the tetradecafluorohexaneand hexane mixed solvent, the hexane phase as an outer phase moved with much lower
velocity than the average linear velocity which was easily estimated as being a parabolic
curve in the tube under laminar flow conditions. Consequently, Cu(II) and Co(II) were
eluted with the average linear velocity, followed by the elution of hematin with a lower
velocity than the average linear velocity. Tentatively, the mixed analyte solution ofCu(II) and hematin was subjected to the present separation system. The obtained CL
separation profile is shown in Fig. 3. They were completely separated; Cu(II) was firstdetected, and next hematin was detected. The separation in the capillary tube was
performed without applying any high-voltage like capillary electrophoresis and using
specific columns, such as monolithic and packed ones, like conventional capillarychromatography.
In conclusion, the tube radial distribution phenomenon (TRDP) with aphase-separated solution of a fluorocarbon (tetradecafluorohexane)-hydrocarbon
organic solvent (hexane) mixture, for the first time, was successfully observed in acapillary tube under laminar flow conditions. A dynamic liquid-liquid interface between
the inner tetradecafluorohexane and the outer hexane phases was generated. The
separation system of metal compounds was also developed based on the TRDP with thefluorocarbon-hydrocarbon organic solvent mixed solution. The Cu(II) and hematin asmodel analytes were separated through the capillary in this order and detected with CL
detection. Our findings expand the TRDP concept to investigate microfluidic behaviors,315
Two-phase separation systems of mixed solutions and TRDP

315

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

and indicate the possibility of developing new types of separation technology, such as
chromatography, extraction and micro-reaction, using a fluorocarbon-hydrocarbon
organic solvent mixed solution in a microspace.

Figure 1. Phase diagram of a tetradecafluorohexane-hexane mixed solution that was
examined in a batch vessel.

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Figure 2. Fluorescence images of the microfluidic behavior of atetradecafluorohexane-hexane mixed solution. a) Non-TRDP (No. 2 in Fig. 2,
tetradecafluorohexane mole fraction 0.2) and b) TRDP (No. 7 in Fig. 2,
tetradecafluorohexane mole fraction 0.6). Flow rate 5.0 µL min-1.

Figure 3. CL separation profile of Cu(II) and hematin.
Conditions: Capillary, fused-silica capillary tube of 75 µm inner diameter and 100 cm
length; carrier, tetradecafluorohexane and hexane mixture (71:29 volume ratio)
containing 50 µM luminol; the oxidant solution in the CL detection cell, 40 mMhydrogen peroxide dissolved in 10 mM carbonate buffer solution (pH 10.8); analyte
injection, the gravity method (from 20 cm height and for 20 s); flow rate, 1.8 µL min-1;
and metal compound concentration, 4.5 mM Cu(II) and 0.5 µM hematin.
317
Two-phase separation systems of mixed solutions and TRDP

317

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules13.5 TRDP created with a water-acetonitrile containing sodium
chloride mixed solution
Open-tubular capillary chromatography was developed based on tube radial distributionof the water-acetonitrile mixture solvents containing sodium chloride under laminarflow conditions. The specific homogeneous carrier solvents were radially distributed in
the capillary tubes, generating inner and outer phases. This distribution behavior wasobserved with a fluorescence microscope-CCD camera system and considered with thephase diagram constructed with the solvents composition and sodium chloride
concentration. Hydrophilic 1-naphthol and hydrophobic 2,6-naphthalenedisulfonic acidas model analytes were separated in this order with the fused-silica capillary tube (100µm inner diameter), while, in the reverse order with the PTFE capillary tube (100 µm
inner diameter). The outer phase created in the capillary tube functioned as a
pseudo-stationary phase in chromatography. The mixture solution including five
analytes was examined with the present capillary chromatography. The opposite elution
order was observed between the fused-silica and PTFE tubes.
Introduction
Until now, the TRDP has been investigated by using the ternary mixed solvents ofwater-hydrophobic/hydrophilic organic mixtures. The TRDP appears through phaseseparation from a homogeneous solution to a heterogeneous solution including the twophases with pressure and temperature change. The phase separation with their change
makes an upper and lower phase in a vessel under the control of gravity. The phaseseparation introduces TRDP including inner and outer phases in a micro-flow where it
is not under the control of gravity and laminar flow conditions. We were interested inwater-acetonitrile mixture solvents. The homogeneous mixture solvents changed toheterogeneous including the two phases by adding sodium chloride (NaCl) in a vessel[27]. An open-tubular capillary chromatography was also reported by using the mixed
solvents in the narrow-bored fused-silica tubes of 10 µm inner diameter [28]. Here thewater-acetonitrile
mixturecontaining
NaCl
insteadofthewater-hydrophobic/hydrophilic organic mixture was, for the first time, examined fromthe viewpoint of TRDP or TRDC by using wide-bore capillary tubes with fused-silica
and PTFE (100 µm inner diameter each).
Experimental
Open-tubular capillary
Open-Tubular
Capillary chromatography
Chromatography(TRDC(TRDC system)
system) Fused-silica
Fused-silica andand PTFE
capillary tubes (100 µm inner diameter) were used. The present capillarychromatography system comprised of an open-tubular capillary tube, microsyringe
pump, and absorption detector. The tube temperature was controlled by dipping thecapillary tube in water maintained at a definite temperature in a beaker with stirring.
Water-acetonitrile mixtures containing NaCl were used as carrier solutions. Analyte
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Chapter 13

solutions were prepared with the carrier solutions. 1-Naphthol, 1-naphthalenesulfonicacid(1-NS),
2,6-naphthalenedisulfonicacid(2,6-NDS),
and1,3,6-naphthalenetrisulfonic acid (1,3,6-NTS) were used as analytes. The analyte
solution was introduced directly into the capillary inlet side by the gravity method (30cm height × 30 s; ca. 30 nL). After analyte injection, the capillary inlet was connected
through a joint to a microsyringe. The syringe was set on the microsyringe pump. The
carrier solution was fed into the capillary tube at a definite flow rate under laminar flow
conditions. On-capillary absorption detection (254 nm) was performed with thedetector.
We setset upup a
Fluorescence microscope
Microscope equipped
Equipped with
with aa CCD
CCD camera
Camera system
System We
capillary tube the same size as that of the TRDC system with absorption
detection for the fluorescence microscope-CCD camera system. The tube
temperature was controled with a thermo-plate (MATS-555S, Tokai Hit Co.
Ltd.; Shizuoka, Japan). Fluorescence in the capillary tube was monitored at
approximately 100 cm from the capillary inlet using a fluorescencemicroscope equipped with an Hg lamp, a filter and a CCD camera. The
carrier solution contained 0.1 mM perylene and 1 mM Eosin Y. We delivered
the carrier solution into the capillary tube at a definite flow rate using the
microsyringe pump.

Results and discussion
diagram for Water-Acetonitrile
water-acetonitrile containing
Phase Diagram
Containing NaCl
NaCl AA phase
phase diagram
diagram forfor thewater-acetonitrile mixture containing NaCl was examined in a vessel at a temperature of
20 °C. The obtained phase diagram is shown in Fig. 1. The curve in the diagram
indicates the boundary between homogeneous and heterogeneous solutions including
the two phases. The phase diagram showed that each composition of the solvents and
NaCl made a homogeneous (one homogeneous phase) or a heterogeneous (twohomogeneous phases) solution. Fluorescence photographs and chromatograms were
examined with the solutions possessing compositions of (a) – (g) in the diagram as
carrier solutions in the following sections. The compositions of (a) – (g) are noted in thecaption of Fig. 1.
photographs andand Profiles
profiles inin Fused-Silica
fused-silica and PTFEPTFE Capillary
capillary Tubes
tubes
Fluorescence Photographs
The fluorescence photographs and profiles of the fluorescent dyes, hydrophobic
perylene (blue) and relatively hydrophilic Eosin Y (green), dissolved in thehomogeneous carrier solutions, (a) – (e), were examined with the fluorescencemicroscope-CCD camera system. As the carrier solutions of (f) and (g) (acetonitrile-richsolution) did not dissolve Eosin Y, they were not used as carrier solutions for the
fluorescence observation. With the fused-silica capillary tube the TRDP was observed
for the carriers (d) and (e) (acetonitrile-rich solution), partially observed for (c), and notobserved for (a) and (b) (water-rich solution) (Fig. 2 A)). The acetonitrile-rich inner319
Two-phase separation systems of mixed solutions and TRDP

319

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

phase (blue) and the water-rich outer phase (green) were generated with the carrier
solutions (d) and (e). The water-rich outer phase was suitable to the fused-silica innerwall. On the other hand, with the PTFE capillary tube, the TRDP was observed for the
carrier solutions, (a) – (c) (acetonitrile-rich solution) and partially observed for the
carriers (d) and (e) (Fig. 2 B)). The water-rich inner phase (green) and theacetonitrile-rich outer phase (blue) were generated with carrier solutions (a) – (e). The
acetonitrile-rich outer phase was suitable to the PTFE inner wall.
obtained by Using
using Fused-Silica
fused-silica andcapillary tubes
Chromatograms Obtained
and PTFE Capillary
Tubes The
chromatograms of hydrophobic 1-naphthol and hydrophilic 2,6-NDS, as model analytes,
were examined with the present TRDC system with the homogeneous carrier solutions,(a) – (f). As the carrier solution of (g) (acetonitrile-rich solution) did not dissolve
2,6-NDS, it was not used as a carrier solution. With the fused-silica capillary tube thebase-line separation of the analytes was observed for carriers (d), (e), and (f)(acetonitrile-rich solution), splitted separation for the carrier (c), and not separated forthe carriers (a) and (b) (water-rich solution) (Fig. 3 A)). The hydrophobic 1-naphthol
was first detected and then hydrophilic 2,6-NDS was detected with the carrier solutionsof (c) – (f). Because 2,6-NDS was distributed in the outer phase (water-rich) as apeudo-stationary phase. The separation information was expressed on the phase
diagram; the symbles, ○, △, and x indicated baseline separation, splitte separation, andnon-separation, respectively (Fig. 4 A)). Retention factor, theoretical number (EMG,
exponential modified Gaussian, method was mainly used), and height of theoreticalplate were calcurated in the usual manner (Fig. 5 A)). On the other hand, with the PTFE
capillary tube the baseline separation was observed for carrier (a) - (d), not separated for(e) and (f) (Fig. 3 B)). In this case the hydrophilic 2,6-NDS was first detected, followedby hydrophobic 1-naphthol detection with the carrier solutions of (a) – (d). Because
1-naphthol was distributed in the outer phase (acetonitrile-rich). The separation
information was expressed on the phase diagram with the symbles, ○ and x, respectively(Fig. 4 B)). Retention factor, theoretical number, and height of theoretical plate were
calcurated in the usual manner (Fig. 5 B)). The chromatographic data shown in Fig. 3
were well consistent with the fluorescent photographic ones shown in Fig. 2. The
coincidence was also reasonable for the separation concept of the TRDC.
Effects of
Photographs AAphase
phasediagram
diagram forfor theof Tubetube Temperaturetemperature on Fluorescence
fluorescence photographs
water-acetonitrile mixture containing NaCl was examined in a vessel at a tube
temperature of 0 °C. The obtained phase diagram is shown in Fig. 6 together with thatobtained at 20 °C. The curves at a tube temperature of 0 °C in the diagram also indicate
the boundary between homogeneous and heterogeneous solutions. The compositions of(a’) –(g’) are noted in the caption of Fig. 6. Fluorescence photographs were further
examined with the solutions possessing compositions of (h) – (n) in the diagram as
carrier solutions at a tube temperature of 20 °C. The plotes of (h) – (n) are shown in Fig.
7 and the compositions of the homogeneous solutions are noted in the caption of Fig. 7;
320
320

Chapter 13they are water-acetonitrule mixutre (3 : 7 volume ratio) containing 0.129 – 0.0096 M
NaCl. As the solution of water-acetonitrule mixutre (3 : 7 volume ratio) containing
0.129 M NaCl created the TRDP and showed separation performance with the
fused-silica capillary tube but did not with the PTFE capillary tube at a tube temperatureof 20 °C as shown in Figs. 2-4, the fused-silica capillary tube was used in the following
experiments. First, with the fused-silica capillary tube the TRDP was observed for the
carrier solution (h) and not observed for the carrier solutions (i) – (n) (Fig. 8 A)) at a
tube temperature of 20 °C. The acetonitrile-rich inner phase (blue) and the water-richouter phase (green) were generated with the carrier solution (h). The water-rich outerphase was suitable to the fused-silica inner wall. Subsequently, at a tube temperature of
0 °C instead of 20 °C with the same fused-silica capillary tube the TRDP was observed
for the carrier solutions (h) - (k), partially observed for the carrier (l), and not observed
for the carriers (m) and (n) (Fig. 8 B)). The acetonitrile-rich inner phase (blue) and thewater-rich outer phase (green) were generated with the carrier solutions (h) - (k). The
water-rich outer phase was suitable to the fused-silica inner wall. The phase separation
easily occurred from homogeneous solution to heterogeneous solution at 0 °C to lead
the TRDP observation compared to 20 °C. The results were well consistent with thephase diagram of 0 and 20 °C shown in Fig. 7.
Effects ofThechromatograms
chromatograms ofof hydrophobicof Tubetube Temperaturetemperature on Chromatograms
chromatograms The
1-naphthol and hydrophilic 2,6-NDS, as model analytes, were examined with thepresent TRDC system with the homogeneous carrier solutions, (h) – (n), at a tube
temperature of 20 °C. With the fused-silica capillary tube the base-line separation wasobserved for the carrier (h) and not separated for the carriers (i) - (n) (Fig. 9 A)). The
hydrophobic 1-naphthol was first detected and then hydrophilic 2,6-NDS was detectedwith the carrier. Because 2,6-NDS was distributed in the outer phase in the tube. The
separation information was expressed on the phase diagram with the symbols of ○ and x(Fig. 10 A)). On the other hand, at a tube temperature of 0 °C instead of 20 °C thebaseline separation was observed for the homogeneous carrier solutions (h) - (m), notseparated for the carrier (n) (Fig. 9 B)). The hydrophobic 1-naphthol was first detected,
followed by hydrophilic 2,6-NDS detection with the carrier solutions of (h) – (m). The
separation information was expressed on the phase diagram with the symbols, ○ and x,
respectively (Fig. 10 B)). The carrier solution (m) did not show the TRDP at theobservation point (at 20 cm from the capillary outlet) in Fig. 8 B)), but the TRDP might
be created, at least partially, in other parts of the capillary tube which were far awayfrom the capillary outlet and were exposed with higher pressure. The phase separation
easily occurred from homogeneous solution to heterogeneous solution at 0 °C to lead
the TRDC separation compared to 20 °C. The results were well consistent with thephase diagram of 0 and 20 °C as shown in Fig. 7.
Separation of Mixture
Analytes We
We examinedexamined a mixture
mixture Solution
solution Including
including Five
five analytes
analyte solution of 1-naphthol, Eosin Y, 1-NS, 2,6-NDS, and 1,3,6-NTS using the321
Two-phase separation systems of mixed solutions and TRDP

321

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

present TRDC system with the fused-silica and PTFE capillary tubes. The obtained
chromatograms are shown in Fig. 11 together with the analytical conditions. The elution
times of the analytes were reversed when using the fused-silica and the PTFE tubes in
the TRDC system. 1-Naphthol, Eosin Y, 1-NS, 2,6-NDS, and 1,3,6-NTS were eluted inthis order with the fused-silica capillary tube and water-acetonitrile (3 : 7 volume ratio)
containing 0.129 M NaCl carrier solution (Fig. 11 A)), although Eosin Y and 1-NS
were not separated with baseline. The elution order seemed to be consistent with thehydrophilic or hydrophobic character of the analytes. With the PTFE capillary tube andwater-acetonitrile (7 : 3 volume ratio) containing 1.23 M NaCl carrier solution, thecomparatively hydrophilic compounds 1-NS, 2,6-NDS, and 1,3,6-NTS were notseparated but were eluted together with almost average linear velocity, while thehydrophobic compounds Eosin Y and 1-naphthol were eluted in this order with velocity
smaller than the average linear velocity (Fig. 11 B)), indicating a reverse elution ordercompared to that of the fused-silica capillary tube.
in thethe TRDP
TRDP between
betweenTernary
ternary Water-Hydrophilic/Hydrophobic
water-hydrophilic/hydrophobic Carrier
Difference in
Solvents and Water-Acetonitrile Containing NaCl Carrier Solvents
The ternarywater-hydrophilic/hydrophobic carrier solutions are classified into two types, organic
solvent-rich and the water-rich carrier solutions. The organic solvent-rich solutions
generate the organic solvent-rich major inner phase and the water-rich minor outerphase, while, the water-rich solutions form the water-rich major inner phase and theorganic solvent-rich minor outer phase. The tube radial distribution of the solvents
brings out regardless with the tube materials, such as fused-silica, polyethylene, andPTFE, although the stability of the outer phase is influenced by the tube materials undersome cases.22) On the other hand, in the TRDP with water-acetonitrile containing NaClthe organic solvent-rich carrier solution create the organic solvent-rich major inner
phase and the water-rich minor outer phase with the fused-silica capillary tube. The
water-rich carrier solution does not create the TRDP with the fused-silica capillary tubeat least under the present analytical conditions. The water-rich carrier solution creates
the water-rich major inner phase and the organic solvent-rich minor outer phase with thePTFE capillary tube. The organic solvent-rich carrier solution does not create the TRDP
with the PTFE capillary tube. Judging from the information of fluorescence images,
chromatograms, and phase diagrams, the TRDP with both carrier solutions of theternary water-hydrophilic/hydrophobic mixture and water-acetonitrile mixture(hydrophilic) containing NaCl must be caused by the phase separation fromhomogeneous to heterogeneous solution in a microspace or not be influenced by gravity.
However, the TRDP with the ternary mixed carrier solvents is little influenced by the
inner wall materials in a microspace, while, the TRDP with the water-acetonitrile mixed
solvents is influenced by the inner wall materials. The reason has not been clear yet. But,
the carrier solvents with or without hydrophobic solvent (ethyl acetate) may play animportant role in inner and outer phase generation. In other words, the carriercomposition constructed with only water and hydrophilic solvents without hydrophobic

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solvents may introduce sensitivity against the inner wall nature into the stable outerphase formation in the TRDP. The above description is consistent with ourconsideration of the TRDP proposed in our paper. The phase formation in a capillary
tube under pressure due to laminar flow is estimated as follows, where it is not under
the control of gravity. A specific homogeneous solution with a component ratio near theboundary between homogeneous and heterogeneous in the phase diagram is delivered
under laminar flow conditions that produce a constant pressure to the radial face. The
homogeneous solution first becomes an emulsion solution that includes minor solvent
or tiny droplets. The tiny droplets start to aggregate near the inner wall where the linear
velocity is lowest under laminar flow conditions. And then, the mixed solvent solution
in a tube changes to a heterogeneous solution that includes a major inner solution and aminor outer solution, where it is not under the control of gravity. From the above
viewpoint, the carrier composition constructed with only water and hydrophilic solvents
without hydrophobic solvents may be sensitive against the inner wall nature for making
an outer phase in the TRDP. That is, in the emulsion mentioned above, tiny
water-droplets in a acetonitrile-rich carrier solution are suitable on a fused-silica innerwall to form the outer phase, while, tiny acetonitrile droplets in a water-rich carrier
solution is suitable on a PTFE inner wall.
In conclusion, we tried to create the TRDP with water-acetonitrile containing NaCl
carrier solution. The fluorescence photographs, chromatograms, and phase diagram
information supported TRDP creation in the water-acetonitrile containing NaCl carrier
solution through the fused-silica and PTFE capillary tubes. With the fused-silica
capillary tube the TRDP created organic solvent-rich major inner and water-rich minor
outer phases. With the PTFE capillary tube, water-rich major inner and acetonitrile-richminor outer phases developed. The outer phase acted as pseudo-stationary phase in thecapillary chromatography (TRDC system). 1-Naphthol and 2,6-NDS as model wereseparated in this order with fused-silica tube and in the reverse order with the PTFE
tube. The obtained experimental data expanded concept and application, as well as
provided new information in the TRDP and TRDC.

323
Two-phase separation systems of mixed solutions and TRDP

323

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Phase diagrams for the water–acetonitrile containing NaCl mixture solution.
The curve in the diagrams indicates the boundary between homogeneous andheterogeneous solutions (including two homogeneous phases). The solvents
composition and NaCl concentration for the plots (a) – (g) that provide homogeneous
solutions were positioned almost on the homogeneous–heterogeneous solution
boundary curve of the phase diagrams. The solvents composition and NaCl
concentration for plots (a) – (g) in the diagram are (a) water–acetonitrile of 7 : 3 volume
ratio containing 1.23 M NaCl, (b) water–acetonitrile of 6 : 4 volume ratio containing
0.615 M NaCl, (c) water–acetonitrile of 5 : 5 volume ratio containing 0.364 M NaCl,(d) water–acetonitrile of 4 : 6 volume ratio containing 0.222 M NaCl, (e)water–acetonitrile of 3 : 7 volume ratio containing 0.129 M NaCl, (f) water–acetonitrileof 2 : 8 volume ratio containing 0.0667 M NaCl and (g) water–acetonitrile of 1 : 9
volume ratio containing 0. 0227 M NaCl.

324
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Chapter 13

Figure 2. Fluorescence photographs and profiles of the mixture solution dissolved
fluorescent dyes. A) Fused-silica and B) PTFE capillary tubes. The arrows point out the
inflections on the fluorescence profiles of Eosin Y in fused-silica tube and perylene in
PTFE tube. Conditions: Capillary tube, 110 cm (90 cm effective length) of 100 µm i.d.;carrier, water–acetonitrile mixture solution containing NaCl, (a) water–acetonitrile of
7 : 3 volume ratio containing 1.23 M NaCl, (b) water–acetonitrile of 6 : 4 volume ratio
containing 0.615 M NaCl, (c) water–acetonitrile of 5 : 5 volume ratio containing 0.364
M NaCl, (d) water–acetonitrile of 4 : 6 volume ratio containing 0.222 M NaCl and (e)water–acetonitrile of 3 : 7 volume ratio containing 0.129 M NaCl; tube temperature,
20 °C; flow rate, 0.8 µL min−1; and dye concentration, Eosin Y 0.5 mM for (a) and (b),1 mM for (c) – (e) and perylene 0.1 mM.

325
Two-phase separation systems of mixed solutions and TRDP

325

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3. Chromatograms of 1-naphthol and 2,6-NDS. A) Fused-silica and B) PTFE
capillary tubes. Conditions: Capillary tube, 110 cm (90 cm effective length) of 100 µm
i.d.; carrier, water–acetonitrile mixture solution containing NaCl, (a) water–acetonitrileof 7 : 3 volume ratio containing 1.23 M NaCl, (b) water–acetonitrile of 6 : 4 volume
ratio containing 0.615 M NaCl, (c) water–acetonitrile of 5 : 5 volume ratio containing
0.364 M NaCl, (d) water–acetonitrile of 4 : 6 volume ratio containing 0.222 M NaCl,(e) water–acetonitrile of 3 : 7 volume ratio containing 0.129 M NaCl and (f)water–acetonitrile of 2 : 8 volume ratio containing 0.0667 M NaCl; tube temperature,
20 °C; sample injection, 30 cm height (gravity) × 30 s; flow rate, 0.8 µL min−1; andanalyte concentration, 1-naphtol 1 mM and 2,6-NDS 0.5 mM.
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Chapter 13

Figure 4. Phase diagram for water-acetonitrile mixture containing NaCl and the solvent
composition and NaCl concentration for the chromatograms obtained with the TRDC
system. A) Fused-silica and B) PTFE capillary tubes. The curve in the diagram indicatesthe homogeneous-heterogeneous solution boundary curve. The solvent composition and
NaCl concentration of the carrier solvents, (a) – (f), for the chromatograms in Fig. 4 areplotted in the diagram with the symbols ○, Δ, and ×, to express baseline-separation,
split-separation, and non-separation, respectively, for the analytes.

327
Two-phase separation systems of mixed solutions and TRDP

327

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

328
328

Chapter 13

Figure 5. Retention factor, theoretical plate number (N), and height equivalent to thetheoretical plate (HETP). A) Fused-silica and B) PTFE capillary tubes. They arecalculated with the chromatographic data in Fig. 4 in the usual manner.

329
Two-phase separation systems of mixed solutions and TRDP

329

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 6. Phase diagrams for the water–acetonitrile containing NaCl mixture solution at
0 and 20 °C. The curves in the diagrams indicate the boundary between homogeneousand heterogeneous solutions (including two homogeneous phases) at 0 and 20 °C. The
solvents composition and NaCl concentration for the plots (a’) – (g’) that provide
homogeneous solutions were positioned almost on the homogeneous–heterogeneous
solution boundary curve of the phase diagrams at 0 °C. The solvents composition and
NaCl concentration for the plots (a’) – (g’) in the diagram are (a’) water–acetonitrile of
7 : 3 volume ratio containing 0.800 M NaCl, (b’) water–acetonitrile of 6 : 4 volume
ratio containing 0.200 M NaCl, (c’) water–acetonitrile of 5 : 5 volume ratio containing
0.0556 M NaCl, (d’) water–acetonitrile of 4 : 6 volume ratio containing 0.0357 M NaCl,(e’) water–acetonitrile of 3 : 7 volume ratio containing 0.0192 M NaCl, (f’)
water–acetonitrile of 2 : 8 volume ratio containing 0.0179 M NaCl and (g’)
water–acetonitrile of 1 : 9 volume ratio containing 0. 0156 M NaCl.

330
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Chapter 13

Figure 7. Enlargement of phase diagrams for the water–acetonitrile containing NaCl
mixture solution at 0 and 20 °C around the mixtures of (e) and (e’). The curves in the
diagrams indicate the boundary between homogeneous and heterogeneous solutions(including two homogeneous phases) at 0 and 20 °C. The solvents composition and
NaCl concentration for the plots (h) – (n) that provide homogeneous solutions werepositioned around the mixtures of (e) and (e’) of the phase diagrams of Fig. 7. The
solvents composition and NaCl concentration for the plots (h) – (n) in the diagram arewater–acetonitrile of 7 : 3 volume ratio (h) containing 0.129 M NaCl, (i) containing
0.120 M NaCl, (j) containing 0.111 M NaCl, (k) containing 0.0924M NaCl, (l)containing 0.0284 M NaCl, (m) containing 0.0192 M NaCl and (n) containing 0.0096 M
NaCl.

331
Two-phase separation systems of mixed solutions and TRDP

331

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 8. Fluorescence photographs and profiles of the mixture solution dissolved
fluorescent dyes in the fused-silica capillary tube. Tube temperature of A) 20 °C and B)
0 °C. The arrows point out the inflections on the fluorescence profiles of Eosin Y in
fused-silica tube. Conditions: Capillary tube, 110 cm (90 cm effective length) of 100µm i.d. fused-silica; carrier, water–acetonitrile mixture solution containing NaCl,water–acetonitrile of 7 : 3 volume ratio (h) containing 0.1290 M NaCl, (i) containing
0.1199 M NaCl, (j) containing 0.1107M NaCl, (k) containing 0.0924M NaCl, (l)containing 0.0284 M NaCl, (m) containing 0.0192 M NaCl and (n) containing 0.0096 M
NaCl; flow rate, 0.8 µL min−1; and dye concentration, Eosin Y 1 mM and perylene 0.1
mM.

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Chapter 13

Figure 9. Chromatograms of 1-naphthol and 2,6-NDS. Tube temperature of A) 20 °C
and B) 0 °C. Conditions: Capillary tube, 110 cm (90 cm effective length) of 100 µm i.d.
fused-silica; carrier, water–acetonitrile mixture solution containing NaCl,water–acetonitrile of 7 : 3 volume ratio (h) containing 0.129 M NaCl, (i) containing
0.120 M NaCl, (j) containing 0.111M NaCl, (k) containing 0.0924M NaCl, (l)containing 0.0284 M NaCl, (m) containing 0.0192 M NaCl and (n) containing 0.0096 M
NaCl; sample injection, 30 cm height (gravity) × 30 s; flow rate, 0.8 µL min−1; andanalyte concentration, 1-naphtol 1 mM and 2,6-NDS 0.5 mM.
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Two-phase separation systems of mixed solutions and TRDP333

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 10. Phase diagram for water-acetonitrile mixture containing NaCl and thesolvent composition and NaCl concentration for the chromatograms obtained with the
TRDC system. Tube temperatures of A) 20 °C and B) 0 °C. The curves in the diagram
indicate the homogeneous-heterogeneous solution boundary curves. The solvent
composition and NaCl concentration of the carrier solvents, (h) – (n), for thechromatograms in Fig. 10 are plotted in the diagram with the symbols ○ and × to
express baseline-separation and non-separation, respectively, for the analytes.

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Figure 11. Chromatograms of 1-naphthol,1-NS, 2,6-NDS, 1,3,6-NTS and Eosin Y. A)
Fused-silica and B) PTFE capillary tubes. Conditions: Capillary tube, 110 cm (90 cm
effective length) of 100 µm i.d.; carrier, water–acetonitrile of 3 : 7 volume ratio
containing 0.129 M NaCl for A) and 7 : 3 volume ratio containing 1.23 M NaCl for B);
tube temperature, 20 °C; sample injection, 30 cm height (gravity) × 30 s; flow rate, 0.8µL min−1; and analyte concentration, 1 mM 1-naphthol, 1-NS, 2,6-NDS, 1,3,6-NTS and
0.1 mM Eosin Y for A) and 1 mM 1-naphthol, 1-NS, 2,6-NDS, 1,3,6-NTS and 0.2 mM
Eosin Y for B).

References[1] K. E. Gutowski, G. A. Broker, H. D. Willauer, J. G. Huddleston, R. P. Swatloski, J.
D. Holbrey, and R. D. Rogers, J. Am. Chem. Soc., 2003, 125, 6632.
[2] Y. Lu, W. Lu, W. Wang, Q. Guo, and Y. Yang, Talanta, 2011, 85, 1621.
[3] C. Wua, J. Wanga, H. Wanga, Y. Peia, and Z. Li, J. Chromatogr., A, 2011, 1218,
8587.
[4] Z. Li, Y. Pei, H. Wang, J. Fan, and J. Wang, Trends Anal. Chem., 2010, 29, 1336.
[5] J. Han, Y. Wang, Y. Li, C. Yu, and Y. Yan, J. Chem. Eng. Data, 2011, 56, 3679.
[6] K. Fujinaga, Anal. Sci., 1993, 9, 479.
[7] H. Watanabe and H. Tanaka, Talanta, 1978, 52, 585.
[8] T. Saitoh, H. Tani, T. Kamidate, and H. Watanabe, Trends Anal. Chem., 1995, 14,213.
[9] T. M. Z.-Moattar and R. Sadeghi, Fluid Phase Equilib., 2002, 203, 177.
[10] P. I. Trindade, M. M. Diogo, D. M. F. Prazeres, and C. J. Marcos, J. Chromatogr.,
A, 2005, 1082, 176.
[11] T. I. Horváth and J. Rábai, Science, 1994, 266, 72.
[12] K. Nakashima, F. Kubota, M. Goto, and T. Maruyama, Anal. Sci., 2009, 25, 77.
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules[13] J. Lim and T. M. Swager, Angew. Chem. Int. Ed., 2010, 49, 7486.
[14] H. Matsuda, A. Kitabatake, M. Kosuge, K. Tochigi, and K. Ochi, Fluid Phase
Equilib., 2010, 297,187.
[15] T. Maruyama, K. Nakashima, F. Kubota, and M. Goto, Anal. Sci., 2007, 23, 763.
[16] M. Masato, M. Hasegawa, D. Sadachika, S. Okamoto, M. Tomioka, Y. Ikeya, A.
Masuhara, and Y. Mori, Tetrahedron Lett., 2007, 48, 4147.
[17] C. Dennis and Z. R. Lee, Green Chemistry, 2001, G3.
[18] A. Albert and A. Hampton, J. Chem. Soc., 1954, 505.
[19] W. D. Johnston and H. Freiser, J. Am. Chem. Soc., 1952, 74, 5239.
[20] Taniguchi, Y., Suzuki, K., and Enomoto, T., J. Colloid & Interface Sci., 1974, vol.
46, p. 511.	
 [21] Pourreza, N., Rastegarzadeh, S., Larki, A., Talanta, 2008, vol. 77, p. 733.
[22] K. Tsukagoshi, K. Nakahama, and R. Nakajima, Anal. Chem., 2004, 76, 4410.
[23] A. E. Everagae, Trans. Soc. Rheol., 1973, 17, 629.
[24] H. Sotjthernj and N. L. Ballmar, Appl. Polymer Symp., 1973, 20, 175.
[25] J. L. White and B. L. Lee, Trans. Soc. Rheol., 1975, 19, 457.
[26] D. L. Maclean, Trans. Soc. Rheol., 1973, 17, 385.
[27] M. Tabata, M. Kumamoto, J. Nishimoto, Anal. Sci., 1994, 10, 383.
[28] M. Tabata, Y. G. Wu, T. Charoenraks, S. S. Samaratunga, Bull. Chem. Soc. Jpn.,
2006, 79, 1742.

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Chapter 14 Applications and related techniques withmicrofluidic behavior of mixed solvent solutions
The specific microfluidic behavior of the mixed solvent solutions, TRDP, has led to aunique and novel design of the respective analytical devices, such as for thechromatography, extraction, mixing, and chemical reaction spaces, in a microspace.
The results obtained from them gave clues as to how to apply the TRDP to otherapplications and related techniques. The part of this chapter is constructed andrewritten based on related manuscripts that have been published previously.7,43,47,53)

14.1 TRDP in bent and wound microchannels on microchips
The tube radial distribution of ternary solvents (water–hydrophilic/hydrophobicorganic mixture) fed into bent and wound microchannels in a microchip was examinedby fluorescence observation of dyes dissolved in the solvents under laminar flow
conditions. Four kinds of microchips incorporating bent microchannels were used,
together with a microchip with a straight channel. The microchannels had different
bending times (2, 4, or 12 times), bending radii (0.8, 2.3, or 3 µm), and total channel
lengths (80, 120, 200, or 500 mm). A water–acetonitrile (hydrophilic)–ethyl acetate(hydrophobic) mixture containing relatively hydrophilic Eosin Y (green) andhydrophobic perylene (blue) was delivered into the bent microchannels in the
microchips. The fluorescence of the green and blue dyes enabled us to observe thespecific radial distribution behavior of the ternary solvents in the bent micro channels
at 0 °C, including liquid–liquid interfaces. Further, the radial distribution pattern of thesolvents was clearly observed in the wound microchannel (bending radius, ca. 0.1 µm,
real total channel length, 500 mm, and apparent straight channel length, 40 mm) at
20 °C (a room temperature) as well as 0 °C. It was found that radial distribution
behaviors of the solvents were successfully generated in even the specificmicrochannels including various types of curves under the present conditions.

Chapter 14

Introduction
Since the last century, the development of micro total analysis systems that include a
microchip or microfluidic device has become an interesting and attractive area ofanalytical science [1,2]. It has been reported that microfluidics exhibit various typesof fluidic liquid–liquid interfaces of solvents in a microchannel [3-6]; theseliquid–liquid interfaces have been examined by varying the channel configurations
and flow rates of the solvents, as well as by using various aqueous and organic
solvent combinations. The fluidic liquid–liquid interface of solvents in a
microchannel is related to the separation, diffusion, and reaction of the solutes.
Information on the liquid–liquid interface of solvents is important and useful in the
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

design of microreactors or micro total analysis systems [3,5].
Various types of aqueous–organic solvent mixture solutions are used indissolution [7], cleaning [8], preservation [9], and as reaction solvents [10]. Suchmixtures are also useful in separation science [11,12]. When ternary mixtures ofwater–hydrophilic/hydrophobic organic solvents are fed into microspaces such as
microchannels in microchips or capillary tubes under laminar flow conditions, thesolvent molecules are radially distributed in the microspace; we call this the “tuberadial distribution phenomenon” (TRDP).
Here, the microfluidic liquid–liquid interface of ternary solvents(water–acetonitrile–ethyl acetate mixture) was examined through observation offluorescent dyes in bent and wound microchannels in microchips, in order to expand
our knowledge of the TRDP in microchannels. We used commercial microchips that
had single and Y-type channel ends. The ternary solvent solution was delivered into themicrochannels from the single and Y-type channel ends.
Experimental
The microchips, incorporating microchannels, were made of glass (types ICC-SY05,
ICC-SY10, ICC-SY15, ICC-SY200, ICC-SY500, and ICC-SY500MI, Institute ofMicrochemical Technology Co., Ltd., Kanagawa, Japan). The designs of the
microchannels (100 µm width × 40 µm depth) are shown in Fig. 1; the ICC-SY05,
ICC-SY10, ICC-SY15, ICC-SY200, ICC-SY500, and ICC-SY500MI microchips,
which are commercially available, are called Type A, Type B, Type C, Type D, Type E,
and Type F, respectively, here. Also, the single, bent, or wound microchannels arecalled Channel M (the numbers in parentheses in Fig. 1 mean the total channel lengths).
The radii of the bent parts in the microchannels are 3 mm for Type B and C, 2.3 mm forType D, 0.8 mm for Type E, and ca. 0.1 mm for Type F, respectively. The microchip
was set in a microchip holder for use here (Fig. 2). The microchip set in the holder was
put on a stage for fluorescence microscope–charge-coupled device (CCD) camera
observations. The water–acetonitrile–ethyl acetate mixture (3:8:4 volume ratio)
containing 1.0 mM Eosin Y and 0.1 mM perylene was fed into the microchannel at aflow rate of mainly 2.0 µL min−1 in Channel M, using microsyringe pumps. The
fluorescence in the microchannel was monitored using a fluorescence microscope
equipped with an Hg lamp, a filter, and a CCD camera. The fluorescence photographs
mainly consisted of blue and green because perylene and Eosin Y emit light at 470
and 550 nm, respectively.
Results and discussion
Phase diagram of water–acetonitrile–ethyl acetate mixture solution and TRDP
generation
Our group reported that a ternary mixed solution ofwater–acetonitrile–ethyl acetate is a two-phase separated aqueous solution. Fig. 3 shows
the phase diagram of the ternary mixed solution of water–acetonitrile–ethyl acetate at 0
and 20 °C. The dotted curves show the boundaries between heterogeneous (inside) and338
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homogeneous (outside) regions. The ternary mixed solution of water–acetonitrile–ethyl
acetate (3:8:4, volume ratio; organic-solvent-rich solution) is plotted in the diagram.
When cooled to near 0 °C from 20 °C, the homogeneous ternary mixed solutionchanges to a heterogeneous solution. We viewed inner and outer phase formation in theTRDP, taking advantage of the above-mentioned phase diagram. A specifichomogeneous solution, whose component ratio was near the boundary between thehomogeneous and heterogeneous regions in the phase diagram, was cooled to below
20 °C in a batch vessel, in which it was under the influence of gravity. In an
organic-solvent-rich solution (e.g., water–acetonitrile–ethyl acetate mixture; 3:8:4
volume ratio), the homogeneous solution changes to a heterogeneous solution that
includes an upper major organic solvent-rich solution and a lower minor water-richsolution under the control of gravity. However, the phase formation of ternary mixed
solvents in a microchannel under temperature changes is considered to not be greatly
under the influence of gravity, as follows. In an organic solvent-rich solution, theternary mixed solvent solution in a tube changes to a heterogeneous solution that
includes major inner organic solvent-rich and minor outer water-rich phases (TRDP),
where there is little control by gravity.
Delivery of ternary mixed solvents from single channel inlet The ternary mixture ofwater–acetonitrile–ethyl acetate was delivered from the single channel inlet in a
microchip of Type A. Fluorescence photographs of the fluorescence dyes were
examined in Channel M at 0 and 20 °C (a room temperature). At 20 °C, the TRDP was
not observed, whereas at 0 °C, it was clearly observed; hydrophobic perylene (blue) was
distributed around the middle of the channel, far from the inner wall, and the relativelyhydrophilic Eosin Y (green) was distributed near the inner wall (Fig. 4). Fluorescencephotographs in the bent microchips of Types B–E, at 0 and 20 °C, were also observed.
The observation area and observation points (point 1–4) in the microchips are indicatedin Fig. 5. As an example, the photographs of points 1–4 in the Type E microchip at 0 °C
are also shown in Fig. 5. The TRDP was observed at every point of the microchip ofType E at 0 °C, but was not observed at 20 °C. The TRDP was similarly observed in thebent microchannels of microchips of Types B–D at 0 °C (Fig. 6), i.e., the TRDP wasclearly generated at 0 °C, without any influence of bends in the channels, under thepresent conditions.
Delivery of ternary mixed solvents from Y-type channel inlet The ternary mixture ofwater–acetonitrile–ethyl acetate was delivered from the Y-type channel inlet in the
microchip of Type A. Fluorescence photographs of the fluorescence dyes were observedin Channel M at 0 and 20 °C. At 20 °C, the TRDP was not observed, but it was clearlyobserved at 0 °C (Fig. 7). Fluorescence photographs were observed in the bent
microchip of Type E, into which the ternary mixed solution was similarly deliveredfrom the Y-type channel inlet. Type E was selected as a model for the experiment as thebending of the microchannels in all chips, Types B–E, showed no influence on the
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

TRDP, as described in the above section. The observation area in the Type E chip isshown in Fig. 8. The TRDP was not observed at 20 °C, but it was observed at 0 °C. The
observed TRDP is also shown in Fig. 8. The TRDP was generated at 0 °C in the bent
microchannel, without any influence of the channel inlets, single and Y-type, under thepresent conditions. Tentatively, using the Type E microchip, the TRDP was examined atother flow rates, i.e., 0.1 and 10 µL min−1, at 0 °C, although the TRDP was observed ata flow rate of 2.0 µL min−1 here. The TRDP was observed at flow rates of 0.1 and 10µL min−1 at 0 °C in the Type E microchip (Fig. 9, shows the data at a flow rate of 0.1µL min−1).
Delivery of ternary mixed solvents into wound microchannel in Type F microchip
The ternary mixture of water–acetonitrile–ethyl acetate was delivered into the wound
microchannel from the single channel inlet and Y-type channel inlet in the Type F
microchip (as shown in Fig. 10). Fig. 5 shows fluorescence photographs of thefluorescence dyes, observed in Channel M at 0 and 20 °C, from the single channel inlet(Fig. 11a) and from the Y-type channel inlet (Fig. 11b). As shown in the figures, theTRDP was observed for both flow directions at 0 and 20 °C, although the thickness ofthe outer phase in the TRDP seemed to change slightly at 20 °C. It was also confirmedthat the TRDP appeared almost throughout the entire channels. We do not have any
clear reason for TRDP generation at 20 °C in the wound microchannel. The specificmixing and pressure in the wound microchannel may generate the two separated phases,
i.e., TRDP, even at 20 °C. We will continue to examine the TRDP in wound
microchannels from various viewpoints.
In conclusion, a ternary mixture of water–acetonitrile–ethyl acetate (3:8:4, volume
ratio) was delivered into bent and wound microchannels in microchips. Fluorescencephotographs were observed using a fluorescence microscope–CCD camera system. The
TRDP was clearly observed at 0 °C in the bent and wound microchannels, without any
influence of the bends in the channels. The results obtained with the microchannels
gave clues as to how to apply the TRDP to the design of analytical devices, such as forchromatography, extraction, and chemical reaction spaces, in microchips.

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Figure 1. Designs of microchannels in microchips (Type A–F).

Figure 2. Schematic representation of a microchip in a microchip holder.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 3. Phase diagram of a ternary mixed solution of water–acetonitrile–ethyl acetate
at 0 and 20 °C. The dotted curves show the boundaries between heterogeneous andhomogeneous solutions.

Figure 4. Fluorescence photographs observed in microchannel in Type A microchip at 0
and 20 °C. The ternary mixed solvent solution was delivered from the single channel
inlet.

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Figure 5. Observation areas and observation points in microchannels in microchips(Types B–E). Fluorescence photographs observed in microchannel in Type E at 0 °C.
The ternary mixed solvent solution was delivered from the single channel inlet.

Figure 6. Fluorescence photographs observed in microchannels in microchips of Types
B–D at 0 °C. The ternary mixed solvent solution was delivered from the single channel
inlet.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 7. Fluorescence photographs observed in a microchannel in a Type A microchip
at 0 and 20 °C. The ternary mixed solvent solution was delivered from the Y-typechannel inlet.

Figure 8. Observation areas and observation points in a microchannel in a Type E
microchip. Fluorescence photographs observed in microchip Type E at 0 °C. The
ternary mixed solvent solution was delivered from the Y-type channel inlet.

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Figure 9. Fluorescence photographs observed in microchannel in Type E microchip at
0 °C at flow rate of 0.1 µL min−1. The ternary mixed solvent solution was deliveredfrom the Y-type channel inlet.

Figure 10. Observation areas and solution delivery directions in the microchannel of theType F microchip.

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Applications and related techniques345

Tube Radial Distribution Phenomenon (TRDP) of Solvent Moleculesa)b)Figure 11. Fluorescence photographs observed in microchip Type F at 0 and 20 °C. The
ternary mixed solvent solution was delivered from (a) the single channel inlet and (b)the Y-type channel inlet.
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346

Chapter 1414.2 Introduction of double tubes having different inner diameters to
TRDC system
We introduced double capillary tubes having different inner diameters to the tube radial
distribution chromatography (TRDC) system. The tubes were fused-silica capillarytubes with 100 and 250 µm i.d.; the smaller tube was inserted into the larger one
through a T-type joint. Water–acetonitrile mixture (volume ratio 3:1) and
acetonitrile-ethyl acetate mixture (volume ratio 4:1) solutions were delivered into thelarge tube from the inside through the small tube and from the outside through the joint,respectively, and then mixed through the large tube to provide awater–acetonitrile–ethyl acetate carrier solution. The carrier solution was further fed
into the large tube and then to an absorption detector. By changing the flow rates of the
mixture solutions in both tubes, we could control the component ratio of carrier solventsin the carrier solution, yielding either organic solvent-rich, water–acetonitrile–ethyl
acetate (volume ratio 3:33:8) or water-rich, water–acetonitrile–ethyl acetate (volume
ratio 24:12:1). A model analyte-mixture solution of 1-naphthol and
2,6-naphthalenedisulfonic acid was eluted in this order with the organic solvent-richcarrier solution and eluted in the reverse order with the water-rich carrier solution. We
discussed the chromatographic data together with the analytical conditions from theviewpoint of the tube radial distribution of the carrier solvents.
Introduction
Commercially available capillary tubes, e.g., fused-silica tubes, have uniform narrowbores, inert inner surfaces, and good flexibility, and are convenient for use with various
analytical instruments. Among analytical techniques that make use of these tubes,
various types of capillary chromatography, including capillary electrochromatography[13,14] and capillary high-performance liquid chromatography using packed andmonolithic capillary columns [15,16], have attracted considerable attention for use in
the fields of analytical chemistry and separation science. Capillary chromatographyusing open fused-silica capillary tubes has also been investigated and the results
indicated interesting and unique characteristics [17-20]. Most capillary chromatographysystems feature rapid measurements, easy procedures, inexpensive and small apparatus,
small sample volumes, and low costs.
To date, in the TRDC system, aqueous–organic solvent carrier solutions wereprepared with a water–acetonitrile–ethyl acetate mixture at various volume ratios to
become homogeneous solutions, and were then delivered into a single capillary tube bya microsyringe pump. To change the component ratio of solvents in the carrier solution,
we had to prepare the solution from the beginning and introduce it again into the TRDC
system.
Here, we introduced double capillary tubes having different inner diameters to the
TRDC system, instead of the single capillary tube just mentioned above. The double
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

tubes consisted of a small- and a large-inner diameter fused-silica capillary tube; thesmall tube was inserted into the large tube through a T-type joint. Water–acetonitrile
mixture and acetonitrile–ethyl acetate mixture solutions were delivered into the large
tube from the inside and outside, respectively, and then mixed through the large tube toprovide a water-acetonitrile-ethyl acetate carrier solution. By controlling the flow ratesof the mixture solutions in the two tubes, we could change the component ratio of awater-acetonitrile-ethyl acetate carrier solution, resulting in an organic solvent-richsolution and a water-rich carrier solution. We were thus able to separate and detect amodel analyte-mixture solution of 1-naphthol and 2,6-naphthalenedisulfonic acid.
Separation performance in the TRDC system was discussed based on the obtained
chromatograms and the data concerning analytical conditions.
Experimental
Fused-silica capillary tubes (100 µm i.d. and 200 µm o.d. as well as 250 µm i.d. and350 µm o.d.) were used. Fig. 1 shows a schematic diagram of the TRDC systemcomprising double capillary tubes, two microsyringe pumps, and an absorption detector;
an enlarged image of the tubes and T-type joint is also shown (though the thickness ofthe tubes is not accurately expressed in the enlarged image). The tubes were fused-silica
capillary tubes of 100 and 250 µm i.d. (called Capillaries A and B, respectively);
Capillary A was inserted into Capillary B through a T-type joint. Water–acetonitrile
mixture (volume ratio 3:1) and acetonitrile–ethyl acetate mixture (volume ratio 4:1)
solutions (called Mixtures A and B, respectively) were delivered into Capillary B fromthe inside through Capillary A and from the outside through the joint, respectively, withthe corresponding microsyringe pumps, then mixed through Capillary B. The resulting
water–acetonitrile–ethyl acetate carrier solution in Capillary B was delivered to anadsorption detector; the distance from the outlet of Capillary A to the detector (theeffective capillary length for separation) was 100 or 200 cm. The temperature of
Capillary B having a 200 cm effective length was adjusted by dipping a part of thecapillary tube (ca. 130 cm) in water maintained at a definite temperature in a vessel withstirring. By changing the flow rates of the mixture solutions (Mixtures A and B), we
could control the component ratio of carrier solvents in the carrier solution that was fed
into Capillary B. For example, an organic solvent-rich carrier solution(water–acetonitrile–ethyl acetate, volume ratio 3:33:8) was delivered at flow rates of 1.0
and 8.0 µL min−1 for Mixtures A and B, respectively. A water-rich carrier solution(water–acetonitrile–ethyl acetate, volume ratio 24:12:1) was delivered at flow rates of
8.0 and 1.0 µL min−1 for Mixtures A and B, respectively. An analyte-mixture solution of
1-naphthol and 2,6-naphthalenedisulfonic acid was prepared with Mixture A andintroduced directly into the capillary inlet of Capillary A for 15 s from a height of 20
cm by the gravity method. After analyte injection, the capillary inlet was connected to a
microsyringe set on the microsyringe pump. Mixtures A and B were fed at definite flow
rates, respectively, to yield the desired organic solvent-rich or water-rich carrier solution
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in Capillary B. On-capillary absorption detection (254 nm) was performed with thedetector.
Results and discussion
Separation in the TRDC system Separation in the TRDC system was proposed in ourpaper, briefly repeated below. Water and organic solvent in the carrier solution do notdisperse uniformly in the capillary, leading to the generation of a water-rich phase andan organic solvent-rich phase in the tube. A major solvent phase forms around the
middle of the tube far from the inner wall as an inner phase, while a minor solvent
phase is generated near the inner wall as an outer or capillary wall phase; when using awater-rich carrier solution, a major solvent phase or a water-rich phase forms around the
middle of the tube as an inner phase, while when using an organic solvent-rich carrier
solution, a major solvent phase or an organic solvent-rich phase forms as an inner phase.
The tube radial distribution of the solvent molecules in the carrier solution is thus
caused by the flow in the capillary tube under laminar flow conditions. Subsequently,
the analytes are delivered through the tube, being distributed between the inner andouter (capillary wall) phases. The analyte lightly partitioned in the outer phase near the
inner wall of the tube is eluted with near average linear velocity, while the analytelargely partitioned in the outer phase is eluted with a smaller velocity than the average
linear velocity. The elution times of the analytes can be easily reversed by changing thecomponent ratio of solvents in the carrier solution.
Analytical conditions
The analyte-mixture solution must be injected into thecapillary inlet of Capillary A, because the analyte zone in Capillary B is never formed
exactly through the T-type joint if the analyte solution is injected into the inlet of
Capillary B. Also, 2,6-naphthalenedisulfonic acid that was one of the analytecomponents was not enough dissolved with Mixture B (acetonitrile-ethyl acetate
mixture), even not with acetonitrile alone, to show an absorption signal. Consequently,
the analyte-mixture solution of 1-naphthol and 2,6-naphthalenedisulfonic acid dissolved
with Mixture A (water-acetonitrile mixture) was injected into Capillary A and delivered
toward Capillary B. Mixtures A and B were delivered at the flow rates of 1.0 and 8.0,
2.0 and 6.0, 4.0 and 4.0, 6.0 and 2.0, as well as 8.0 and 1.0 µL min−1, respectively. The
baselines monitored by the absorption detector were stable for the flow rates of 1.0 and
8.0, 4.0 and 4.0, as well as 8.0 and 1.0 µL min−1 for Mixtures A and B. But they were
not stable for the flow rates of 2.0 and 6.0 or for 6.0 and 4.0 µL min−1. The carrier
solution (water-acetonitrile-ethyl acetate mixture) provided by mixing Mixtures A and
B at the end of Capillary A in Capillary B must be homogeneous for the stable baselines
observed through absorption detection, while it must be heterogeneous for the unstablebaselines. Furthermore, as a preliminary experiment, when Mixtures A and B were fed
at the flow rates of 4.0 and 4.0 µL min−1 respectively into the capillaries, the analytes
in the mixture solution were not separated; the carrier solution obtained by the flow
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

rates comprised the water-acetonitrile-ethyl acetate mixture with the volume ratio of
3:5:1. The volume ratio that did not possess a large unbalance in water-organic solvent
ratio must not lead to separation in the present system, although it could provide a stablebaseline. As a result, we adopted the flow rates of 1.0 and 8.0 as well as 8.0 and 1.0 µL
min−1 for Mixtures A and B in the following experiments.
Effect of the temperature of Capillary B First, Capillary B having a 100 cm effective
length for separation was used in the present TRDC system. This capillary length wasvery commonly used in our works, but we had not used a capillary tube of 250 µm i.d,(the largest inner diameter of the tube that we had used in the TRDC system was 200µm of polyethylene tube). When Capillary B, with an effective length of 100 cm, wasused, the obtained chromatographic data lacked reproducibility; therefore the details are
not described here. Then, we tried to use Capillary B with an effective length of 200 cm,
and dipped one part of it (ca. 130 cm) in water to control the capillary temperature. The
data were obtained with good reproducibility at a capillary temperature of 15 - 20 °C. In
a large inner diameter tube of 250 µm i.d. that was necessitated for developing the
double capillary tubes, the capillary temperature control must be useful for providing
stable tube radial distribution of carrier solvents in the capillary tube. In addition theanalytes were never separated at a capillary temperature of more than 25 °C. The
following experiments were carried out using Capillary B of 200 cm effective length at
the capillary temperature of 15 - 20 °C.
Separation performance
Mixture A (water–acetonitrile mixture) and Mixture B(acetonitrile–ethyl acetate mixture) were delivered into the large Capillary B from theinside and outside, respectively, at the flow rates of 1.0 and 8.0 as well as 8.0 and 1.0µL min−1, and then mixed through the tube to provide a water–acetonitrile–ethyl acetatecarrier solution. We could control the component ratio of carrier solvents in the carrier
solution, that is, we could determine if the solution was organic solvent-rich orwater-rich, by controlling the flow rates of the mixture solutions. The following
component ratios of carrier solvents were demonstrated according to the flow rates
mentioned above: organic solvent-rich (water–acetonitrile–ethyl acetate, volume ratio
3:33:8) and water-rich (water–acetonitrile–ethyl acetate, volume ratio 24:12:1). A
model analyte mixture (1-naphthol and 2,6-naphthalenedisulfonic acid) was subjected to
the TRDC system that was adjusted to feed the organic solvent-rich carrier solution.
The two components were eluted in the order listed under laminar flow conditions anddetected by the absorption detector. Similarly, the model analyte mixture was subjectedto the TRDC system that was adjusted to feed the water-rich carrier solution. The twocomponents were eluted in the reverse order. Obtained chromatograms for these two
experiments are shown in Fig. 2. The components of the analytes, 1-naphthol and
2,6-naphthalenedisulfonic acid, in the chromatograms were confirmed with individualabsorption signals. The elution times of the model analyte mixtures were easily changed
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by altering the component ratio of the carrier solvents in the carrier solution. We altered
the component ratio by adjusting the flow rates of the mixture solutions in the double
capillary tubes. Additionally, instead of Mixture B of acetonitrile-ethyl acetate mixture,
we attempted to use a solution containing acetonitrile alone that was fed into Capillary
B. Such an analyte-mixture solution was never separated at any flow rates. Ethyl acetate(hydrophobic organic solvent) made an important role for separation performance in the
TRDC system.
Tube radial distribution of carrier solvents It should be emphasized that analyteseparation was achieved with even the organic solvent-rich carrier solution. Even when
the water–acetonitrile (water-rich) solution was fed from the inner tube and theacetonitrile–ethyl acetate (organic solvent-rich) solution from the outside through theT-type joint, the organic solvent-rich carrier solution that resulted upon their mixing
through Capillary B was delivered to the detector, providing that the organic
solvent-rich phase formed in the middle of the tube far from the inner wall (the inner
phase) and the water-rich phase formed near the inner wall of the tube (the outer orcapillary wall phase). That is, the data obtained here supported that the tube radial
distribution of carrier solvents in a carrier solution was appropriately performed underlaminar flow conditions in the present TRDC system.
In conclusion, we have developed a TRDC system equipped with double capillarytubes having different inner diameters. We could change the component ratio of carrier
solvents in the carrier solution by adjusting the flow rates of the two mixture solutionsdelivered into the tubes in the system, not preparing carrier solutions beforehand andexchanging them. A model analyte mixture solution of 1-naphthol and
2,6-naphthalenedisulfonic acid was eluted, in the order listed, with the organic
solvent-rich carrier solution and eluted in the opposite order with the water-rich carrier
solution. The demonstration shown here supported the specific solvent distribution in
the capillary tube, i.e., the tube radial distribution of the carrier solvents, in the TRDC
system together with the experimental data reported previously.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Schematic of the present TRDC system equipped with double capillary tubes.

Figure 2. Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acidobtained using the present TRDC system equipped with double capillary tubes: a)Organic-solvent-rich carrier solution (water–acetonitrile–ethyl acetate, volume ratio
3:33:8), and b) water-rich carrier solution (water–acetonitrile–ethyl acetate, volume
ratio 24:12:1). Analyte concentrations are each 2 mM.
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352

Chapter 1414.3 Capillary chromatography using an annular and sluggish flow inthe ternary water–acetonitrile–ethyl acetate mixed solution
An open-tubular capillary chromatography system was developed using an annular andsluggish flow system in a ternary water–acetonitrile–ethyl acetate mixed solvent as
carrier solution. Fused-silica capillary tubes with different inner diameters of 75 and200 µm were used. The smaller tube was inserted into the larger tube using a T-type
joint. Water–acetonitrile and acetonitrile–ethyl acetate mixed solutions were delivered
to the smaller and larger tubes, respectively, and then combined at the other end of thesmaller tube located in the larger tube, generating annular and sluggish flows in thewater–acetonitrile–ethyl acetate ternary mixed solution (2:5:3 and 2:4:3 (v/v/v),
respectively). The annular flow comprised the organic solvent-rich inner andwater-rich outer phases and the sluggish flow comprised the organic solvent-richsluggish domain and water-rich continuous phases. A model analyte mixed solution of
1-naphthol (hydrophobic) and 2,6-naphthalenedisulfonic acid (hydrophilic) was
injected into the smaller tube and proceeded to the annular or sluggish flow in thelarger tube, where they were separated and detected in that order. As observed, theannular and sluggish flows in the microspace could be used for separation without any
specific treatments such as tube packing or coating.
Introduction
The TRDP results in a dynamic liquid–liquid interface or inner and outer phases
through phase transformation from a homogeneous (single phase) to a heterogeneous(two phases) mixed solution in a microspace. A capillary chromatography system
based on the TRDP in which the outer phase acts as a pseudo-stationary phase underlaminar flow conditions has been developed. Recently, an annular and sluggish flow
system was investigated in a tube using immiscible organic and aqueous mixed
solutions [21-24]. However, to our knowledge, capillary chromatography systems
based on an annular and sluggish flow system in a microspace have not been reported
yet. Also, a sluggish flow must be thought not to apply it to capillary chromatography,
though it can be easily constructed in a microspace.
Here, an open-tubular capillary chromatography system was developed based on anannular and sluggish flow system in a microspace. The flows were generated using adual capillary tube system with different tube inner diameters. The smaller tube wasinserted into the larger tube. Water–acetonitrile and acetonitrile–ethyl acetate mixed
solutions were separately fed to the small and large tubes, respectively. Both mixtures
combined in the larger tube, generating an annular and sluggish flow in thewater–acetonitrile–ethyl acetate ternary mixed solution serving as carrier solution.
Model analytes were separated in the annular and sluggish flow in a microspace.
Experimental
Fused-silica capillary tubes (the smaller one: inner diameter, 75 µm and outer diameter,
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

150 µm; the larger one: inner diameter, 200 µm and outer diameter, 350 µm) wereused. The capillary chromatography system mainly comprised microsyringe pumps,
capillary tubes, and a fluorescence detector. A schematic of the chromatographysystem is shown in Fig. 1. Water–acetonitrile and acetonitrile–ethyl acetate mixtures
were delivered to the smaller (75 µm i.d.) and larger tube (200 µm i.d.), respectively.
The mixtures were combined at the other end of the smaller tube located in the larger
tube, generating a water–acetonitrile–ethyl acetate ternary mixed solution as carrier
solution: a) annular flow, 2:5:3 (v/v/v) (water–acetonitrile, 3:2 and acetonitrile–ethyl
acetate, 11:9); and b) sluggish flow, 2:4:3 (v/v/v) (water–acetonitrile, 2:1 andacetonitrile–ethyl
acetate, 1:1). An analyte solution of 1-naphthol and
2,6-naphthalenedisulfonic acid (1 mM each) was prepared using the water–acetonitrile
mixed solution. The analyte solution was directly introduced into the smaller capillary
inlet using the gravity method (20 cm height for 20 s; ca. 0.9 mL). Following analyte
injection, the capillary inlet was connected through a joint to a microsyringe on the
microsyringe pump. The mixed solutions were fed to the capillary tubes at constantflow rates of 2.5 µL min−1 (smaller tube) and 5.0 µL min−1 (larger tube) under laminarflow conditions. Fluorescence detection of the analytes was performed with on-line
capillary (effective length, 140 cm) using excitation (ex.) and emission (em.)
wavelengths of 290 nm and 355 nm, respectively. The fluorescence in the larger
capillary tube was monitored at approximately 140 cm from the smaller capillaryoutlet using a fluorescence microscope equipped with an Hg lamp, a filter, and a CCD
camera.
Results and discussion
Figure 2 shows the phase diagram of the ternary water–acetonitrile–ethyl acetate
mixed solvent. The diagram included the boundary curve between the homogeneous(single phase) and heterogeneous (two phases) regions. The component volume ratiosof the ternary water–acetonitrile–ethyl acetate solution 2:5:3 and 2:4:3, as shown in
Fig. 2, are denoted as a) and b), respectively. The mixed heterogeneous solutions a)
and b) comprised an upper (organic solvent-rich) and lower (water-rich) phase solution
in a batch vessel.
The water–acetonitrile and acetonitrile–ethyl acetate mixed solutions wererespectively fed to the capillary tubes and combined to give the ternarywater–acetonitrile–ethyl acetate solution in the larger tube. Fluorescence photographsof the ternary mixed solutions a) and b) in the larger capillary tube are shown in Fig. 3.
The blue region corresponding to perylene (hydrophobic) revealed the organic
solvent-rich solution and the green region corresponding to Eosin Y (hydrophilic)revealed the water-rich solution. As observed in Fig. 3, the heterogeneous solution a),whose component ratio is located near the boundary curve in the phase diagram,displayed two phases i.e., inner (blue, organic solvent-rich) and outer (yellow–green,water-rich) phases, generating an annular flow. In contrast, the heterogeneous solutionb) resulted in two phases positioned along the axial direction, generating an organic
354
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solvent-rich sluggish domain (ca. 510 µm length) and a water-rich continuous phase(ca. 150 µm length), resulting in a sluggish flow.
Separation of model analytes 1-naphtol and 2,6-naphthalenedisulfonic acid using
the present system featuring an annular and sluggish flow system was investigated.
The obtained chromatograms are shown in Fig. 4. 1-Naphtol and
2,6-naphthalenedisulfonic acid were separated and detected in that order, with bothtypes of flows featuring microfluidic behaviors. The linear velocities in the tube areexpressed as a parabolic function under laminar flow conditions. The outer phase of
the annular flow of solution a) and the partial outer phase of the sluggish flow ofsolution b) moved at a very slow rate in the tube, and the outer phase acted as a
pseudo-stationary phase in the current chromatography separation system.
Hydrophobic 1-naphthol and hydrophilic 2,6-naphthalenedisulfonic acid were
distributed in the organic solvent-rich inner phase region or sluggish region and in thewater-rich outer phase region (pseudo-stationary phase), respectively. Consequently,
1-naphthol was eluted first at an average linear velocity and 2,6-naphthalenedisulfonicacid was subsequently eluted at a lower velocity than the average linear velocity,
providing base-line separation on the chromatograms. The difference in separation
performance between a) and b) in Fig. 4 may be caused by the difference of thecomponent ratios of the solvents in the inner and outer phases.
The annular flow was successfully generated in a microspace using the ternary
mixed heterogeneous solution water–acetonitrile–ethyl acetate. The inner and outerphase formation mechanism in a heterogeneous solution using an annular flow isrelatively different from that in a homogenous solution under the TRDP. However,both systems are expected to feature the same chromatography separation mechanism.
On the other hand, separation of the analytes in the sluggish flow was unexpected.
Tentatively, we examined resolutions, theoretical plate numbers, and the height ofequivalent theoretical plate for 2,6-naphthalenedisulfonic acid in the present system
under sluggish flow conditions and varying capillary tube lengths (70, 105, 140, and
175 cm). 1-Naphthol and 2,6-naphthalenedisulfonic acid were separated and detectedin that order, regardless of the capillary tubes studied under sluggish flow conditions.
Separation systems employing capillary tube lengths of 70, 105, 140, and 175 cm
achieved resolutions of 5.8, 8.0, 8.2, and 12, respectively. The corresponding
theoretical plate numbers were 2400, 4000, 5600, and 6800. The associated height ofthe equivalent theoretical plate values were 0.23, 0.21, 0.19, and 0.20 mm. The
resolutions and theoretical plate numbers increased with increasing capillary tube
lengths, whereas the height of equivalent theoretical plate values remained mostly
constant. These results implied that separation under sluggish flow conditions occurredin a similar way with conventional chromatography which of separation ability was
independent of column length.
In conclusion, an annular and sluggish flow system was obtained under microfluidic
flow conditions when heterogeneous ternary solution water–acetonitrile–ethyl acetate
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

was introduced into a capillary tube using a dual capillary tube system featuring
different tube inner diameters. First, we would like to emphasize that the annular flow
was easily generated in the capillary tube using the present system. Secondly, model
analytes 1-naphthol and 2,6-naphthalenedisulfonic acid were separated into the outerphase of the annular flow and partial outer phase of the sluggish flow that acted as
pseudo-stationary phase in the chromatography separation system. It is interesting tonote that separation occurred in both the annular and sluggish flows, generated in a
microspace. The current findings present a new concept towards the development of an
open-tubular capillary chromatography system without any tube packing or coating
requirements.

Figure 1. Schematic diagram of the present capillary chromatographysystem integrating a dual capillary tube system featuring different tube
inner diameters.

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Figure 2. Phase diagram of the ternary water–acetonitrile–ethyl acetate mixed solvent at
20 °C.

Figure 3. Fluorescence photographs of ternary water–acetonitrile–ethyl acetate mixed
solution at volume ratios of a) 2:5:3 and b) 2:4:3.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 4. Chromatograms of 1-naphthol and 2,6-naphthalenedisulfonic acid mixtures
obtained with the present system. The volume ratios of the ternarywater–acetonitrile–ethyl acetate carrier solution are (a) 2:5:3 and (b) 2:4:3. The
separation effective distance between the smaller capillary outlet and the detection pointis 140 cm.

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14.4 Microfluidic inverted flow of aqueous and organic solvent mixed
solution in a microchannel
When two solutions are individually fed into two separated microchannels of a
microchip that combine to form a single channel of a Y-type microchannel, the flows in
the single channel are either parallel for immiscible solutions or initially parallel but
become homogeneous through diffusion for miscible solutions. Nevertheless, a new
type of microfluidic behavior was seen in the Y-type microchannel that was neither
parallel nor homogeneous flow. Water-acetonitrile and acetonitrile-ethyl acetate
mixtures, each marked by a distinctive dye, were delivered at the same flow rate into theY-type microchannel under laminar flow conditions. Under different volume ratio
mixtures, the two phases in the single channel were initially observed to flow in parallel
but then swapped over to the opposite wall while still retaining parallel flow. We callthis type of laminar flow “microfluidic inverted flow”.
Introduction
The development of the micro-total analysis system (µ-TAS) that includes microchip or
microfluidic device technology is a very interesting aspect of analytical science [1,2].
Microfluidic solvents exhibit various fluidic behaviors in microchannels. Their flow
patterns have been examined by varying the channel configuration and flow rate of thesolvents, using aqueous–organic solvent mixtures, and introducing specific obstacles
into microchannels [3,25,26]. Fluidic flow of solvents in microchannels is related toseparation, diffusion, and reaction of solutes. Information regarding their microfluidic
flow and interface formation is important and useful in designing microreactors orµ-TAS [1-3,25,26].
Various types of mixed solutions of aqueous–organic solvents are used in dissolution,cleaning, preservation, and as reaction solvents. Such mixtures are also useful inseparation science [11,12,27]. However, to our knowledge, the use of ternary mixed
solvents of water–hydrophilic/hydrophobic-organic solvents has not been examined indetail. When such ternary mixed solvents were fed into the microspace under laminarflow conditions, the solvent molecules radially disperse in the microspace through aphase transformation; this microfluidic behavior we call the tube radial distribution
phenomenon (TRDP).
Here, the microfluidic behavior of two combining mixtures, water–acetonitrile andacetonitrile–ethyl acetate, was examined under the fluorescence of dyes Eosin Y (green)and perylene (blue) in the respective solutions. A microchip fabricated with a Y-type
microchannel was used, in which two separated channels, labeled channels T1 and T2,
combined to form a single channel, labeled channel S (see Fig. 1). Each channel was
100 µm wide × 40 µm deep. The two mixtures to be combined were fed into the two
separated channels. In the single channel, the combined mixture developed a specificfluidic behavior, i.e., microfluidic inverted flow, which depended on the solvents.
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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Experimental
The water–acetonitrile mixture (20:30 volume ratio) containing 2.0 mM Eosin Y andacetonitrile–ethyl acetate mixture (20:30 volume ratio) containing 0.2 mM perylene
were fed at the same flow rate into channels T1 and T2, respectively, using a
microsyringe pump. A microscope–CCD camera system was set-up to observe
fluorescence from the dyes, Eosin Y and perylene, which emit light at 470 nm (green)
and 550 nm (blue), respectively. The fluorescence was monitored near the combining
point and in channel S using a fluorescence microscope equipped with an Hg lamp, afilter, and a CCD camera. The blue and green fluorescence images give a clear
separation of the two flows. A distinctive feature to be noted is the microfluidic inverted
flow in the Y-type microchannel with the water–acetonitrile and acetonitrile–ethyl
acetate mixed solutions (Fig. 1); the flow conditions are indicated in figure captions.
This inverted flow was unobserved with water and water, acetonitrile and acetonitrile,
ethyl acetate and ethyl acetate, water and acetonitrile mixtures, as well as acetonitrileand ethlyl acetate mixed solutions (miscible solutions); each of these combinations
initially exhibited parallel flow and then homogeneous flow. The immiscible solutionsof water and ethyl acetate mixtures produced parallel or sluggish flow, never inverted
flow, in the Y-type microchannel. Only the ternary mixed solvent of water, acetonitrile,and ethyl acetate could bring about “microfluidic inverted flow”. This information will
be useful for clarifying the creation of “microfluidic inverted flow” in the future.
Results and discussion
Here, with respect to our previous work on ternary mixed solvents ofwater-acetonitrile-ethyl acetate, we tentatively examined the composition ratios near theboundary curve in the phase diagram of the water-acetonitrile-ethyl acetate mixed
solution (Fig. 2). The compositions of the water-acetonitrile-ethyl acetate mixed
solutions were A; 14:43:43, B; 20:50:30, C; 43:43:11, and D; 52:37:11 The
water-acetonitrile and acetonitrile and ethyl acetate mixed solutions were mixed so thatcompositions of A–F were produced in channel S. Mixed solutions with compositions A,
B, and C (organic solvent-rich solutions) showed microfluidic inverted flow, whereas
those of D, E, and F (water-rich solutions) exhibited a parallel-to-homogeneous flow
evolution. Reasons for the microfluidic inverted flow for these mixed solutions remain
unclarified.
Furthermore, we examined the effects of flow rates and channel lengths on the
microfluidic inverted flow; the conditions are given in the captions. The fluorescence
images were observed around the mixing point and in channel S at distances 1, 2, and 3
cm from the mixing point with flow rates 10, 20, and 100 µL min−1 (Fig. 3). As flowrate increased, we observed various stages of inverted flow in the channel. Fig. 4 shows
the inverted flow in a long single microchannel in a Y-type microchip. We also
observed inverted flow in a long wooden microchannel.
Finally, we had a microchip fabricated that included three connecting Y-type mixing
points (Fig. 5). The water-acetonitrile and acetonitrile-ethyl acetate mixed solutions
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were fed into the microchannels via two of the Y-type microchannels (Fig. 5); see
caption for conditions. Downstream from the first two Y-type mixing points,
microfluidic inverted flows formed in the individual channels; the composition ratio of
the water-acetonitrile-ethyl acetate was 20:50:30 in these sections of the channels.
Subsequently, the two inverted flows were mixed at the third Y-type mixing point, fromwhich TRDP formed in the microchannel; the composition ratio after mixing was
20:50:30 in this section channel. This unique microfluidic flow, evident in Fig. 5, has
never been created in a microchannel by any other technique. Such a novel microfluidic
flow pattern or liquid-liquid interface formation might lead to innovation of separation,
extraction, mixing, and chemical reaction in a microspace.
In conclusion, ternary solvents of water–acetonitrile–ethyl acetate mixtures in asingle channel were prepared by combining a water–acetonitrile mixture and an
acetonitrile–ethyl acetate mixture fed from two separated channels in a Y-type
microchannel of a microchip. A microfluidic inverted flow was a specific andinteresting flow seen from the mixing of particular compositions of solvents in a single
channel. This unique inverted flow was produced with the ternary mixed solvent of
combining the two mixtures. The specific microfluidic flow was not observed using
two-component solvents, i.e., a water–acetonitrile mixture or a water–ethyl acetate
mixture. The data of the inverted flow, visualized under dye florescence are expected to
be useful in developing a mixing technique to create a phase interface and a chemical
reaction space in the microspace of a microchip.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 1. Typical microfluidic inverted flow observed in channel S of a Y-type
microchannel. Channel T1, 2.0 mM Eosin Y dissolved in water-acetonitrile (20:30, v/v)
and channel T2, 0.2 mM perylene dissolved in acetonitrile-ethyl acetate (20:30, v/v).
Flow rate, 10 µL min−1 each at 20 °C.

Figure 2. Phase diagram of ternary mixed solvents of water-acetonitrile-ethyl acetate
mixture. The curve represents the boundary between homogeneous (single phase) andheterogeneous (two phases). The component ratios of water-acetonitrile-ethyl acetate;A; 14:43:43 B; 20:50:30, C; 43:43:14, and D; 52:37:11. 20 °C.

362
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Figure 3. Effects of flow rate on the microfluidic inverted flow in channel S. Conditions
in channels T1 and T2 are the same as in Fig. 1. 20 °C.

Figure 4. Effect of single-microchannel length on the microfluidic inverted flow inchannel S. Conditions in channels T1 and T2 are the same as in Fig. 1. 20 °C. The
single microchannel length is 12 cm and included the two bends.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Figure 5. Schematic diagram of a microchip with three Y-type mixing points in the
microchannel. Conditions in channels 1 and 4 are the same as in channel T1 of Fig. 1;channels 2 and 3 have the same conditions as for channel T2 in Fig. 1. Flow rate is 2.0µL min−1 each at 20 °C.

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Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Conclusions
Investigating the microfluidic behaviors of solvents is essential towards the design ofhighly efficient microreactors or micro-total analysis systems, since the microfluidic
behavior of solvents in a microchannel is related to the separation, diffusion, andreaction of the solutes. In this book, we discussed an interesting and unique microfluidic
behavior featured by mixed solvents confined in a microspace that we recently reported,and coined as TRDP. TRDP was observed in diverse mixed solvents systems, such as
ternary water–hydrophilic/hydrophobic organic solvents, water–surfactant, water–ionicliquid, water-nydrophilic solvents containing salts, and fluorous/organic solvents. The
development of TRDP in a ternary mixed solvent solution instigated a two-phaseseparation system that was successfully exploited for chromatography separation,
extraction, and mixing processes as well as for initiating chemical reactions. The
respective developed TRDP-based systems showed great promise as efficient
microreactors and micro-total analysis systems.

A list of the TRDP-related manuscripts reported by theauthor’s group in chronological order
The part of this book is constructed and rewritten based on the following manuscripts
together with new data and consideration.
1)

Micro-Flow Separation System Using an Open Capillary Tube That Works underLaminar Flow Conditions; Naoya Jinno, Masahiko Hashimoto, and Kazuhiko
Tsukagoshi, Analytical Sciences, 25, 145-147 (2009).

2)

Capillary Chromatography Based on Tube Radial Distribution of Aqueous-Organic
Mixture Carrier Solvents; Naoya Jinno, Minoru Itano, Masahiko Hashimoto, andKazuhiko Tsukagoshi, Talanta, 79, 1348-1353 (2009).
3)

Capillary Chromatography Based on Tube Radial Distribution of Aqueous-Organic
Mixture Carrier Solvents: Elution Behavior of Carboxylated Polymer Particles in the
System; Naoya Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Journal ofChemical Engineering of Japan, 42, 767-770 (2009).

4)

Capillary Chromatography Based on Tube Radial Distribution of Aqueous-Organic
Mixture Carrier Solvents: Effect of the Inner Wall Characteristics of the Fused-Silica
Tube on Separation Performance; Naoya Jinno, Ko Hashimoto, Masahiko Hashimoto,and Kazuhiko Tsukagoshi, Analytical Sciences, 25, 1369-1371 (2009).
366

366

5)

Capillary Chromatography Based on Tube Radical Distribution of Aqueous-Organic
Mixture Carrier Solvents: Introduction of Inner-Wall-Modified Capillary tube; Naoya
Jinno, Katsuya Tsuji, Kaoru Shikatani, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
Journal of Separation Science, 32, 4099-4100 (2009).
6)

Distribution of Fluorescent Dyes Dissolved in Ternary Mixed Solvent in a
Micro-Channel under Laminar Flow Conditions; Mari Murakami, Naoya Jinno,
Masahiko Hashimoto, Kazuhiko Tsukagoshi, Chemistry Letters, 39, 272-273 (2010).

7)

Capillary Chromatography Based on Tube Radial Distribution of Aqueous–Organic
Mixture Carrier Solvents: Introduction of Double Tubes Having Different Inner
Diameters to the System; Koju Yamada, Naoya Jinno, Masahiko Hashimoto, andKazuhiko Tsukagoshi, Analytical Sciences, 26, 507-510 (2010).
8)

Separation of Optical Isomers in Capillary Chromatography Using aPoly(tetrafluoroethylene) Capillary Tube and an Aqueous-Organic Mixture Carrier
Solution; Seiji Ishimoto, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
Analytical Sciences, 26, 641-643 (2010).
9)

Elution Behavior of Proteins in Capillary Chromatography Using an Untreated
Fused-silica Capillary Tube and a Water-Hydrophilic-Hydrophobic Organic Mixture
Carrier Solvent; Yuji Masuhara, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko
Tsukagoshi, Chemistry Letters, 39, 688-689 (2010).

10)

Analytical Conditions and Separation performance of Capillary Chromatography
Based on the Tube Radical Distribution of Aqueous -Organic Mixture Carrier Solvents
under Laminar–Flow Conditions; Naoya Jinno, Mari Murakami, Masahiko Hashimoto,and Kazuhiko Tsukagoshi, Analytical Sciences, 26, 737-742 (2010).

11)

Introduction of fluorescence and chemiluminescence detection to capillarychromatography based on tube radial distribution of water–hydrophilic–hydrophobicorganic mixture carrier solvents; Seiji Ishimoto, Yudai Kudo, Naoya Jinno, MasahikoHashimoto and Kazuhiko Tsukagoshi, Analytical Methods, 2, 1377-1381 (2010).

12)

Temperature Effect on Separation Performance in Capillary Chromatography basedon Tube Radial Distribution of Aqueous-Organic Mixture Carrier Solvents; Naoya
Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, The Science and Engineering
Review of Doshisha University, 51, 168-172 (2010).

13)

Metal Compound Analysis by Capillary Chromatography Using an UntreatedCapillary Tube and Water-Hydrophilic-Hydrophobic Solvent Mixture as a Carrier
Solution; Kazuma Yoshida, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko
Tsukagoshi, The Science and Engineering Review of Doshisha University, 51, 198-201(2011).

14)

Fluorescence Observation Supporting Capillary Chromatography Based on Tube
Radial Distribution of Carrier Solvents under Laminar Flow Conditions; Naoya Jinno,
Mari Murakami, Kiyoshi Mizohata, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
367
A list of the TRDP-related manuscripts

367

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Analyst, 136, 927-932 (2011).
15)

Experimental Consideration of Capillary Chromatography Based on Tube Radial
Distribution of Ternary Mixture Carrier Solvents under Laminar Flow Conditions;
Naoya Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Analytical Sciences, 27,259-264 (2011).

16)

Extraction of Cu(II) Based on Tube Radial Distribution of Ternary Mixed Carrier
Solution in Microchannels; Naoya Jinno, Masahiko Hashimoto, and Kazuhiko
Tsukagoshi, Chemistry Letters, 40, 654-655 (2011).

17)

Tube Radial Distribution Phenomenon of Ternary Mixed Solvents in a Microspace
under Laminar Flow Conditions; Mari Murakami, Naoya Jinno, Masahiko Hashimoto,
Kazuhiko Tsukagoshi, Analytical Sciences, 27, 793-798 (2011).
18)

Derivatization of a Protein with Fluorescamine Utilizing the Tube Radial
Distribution Phenomenon of Ternary Mixed Carrier Solvents in a Capillary Tube; Yuji
Masuhara, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Chemistry
Letters, 40, 804-805 (2011).

19)

Use of Tube Radial Distribution of Ternary Mixed Carrier Solvents for Introductionof Absorption Reagent for Metal Ion Separation and On-Line Detection into Capillary;
Satoshi Fujinaga, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
Journal of Separation Science, 34, 2833-2839 (2011).
20)

Components of the Carrier Solvents and Separation Performance in the Tube Radial
Distribution Chromatography Using a Fused-Silica Capillary tube; Naoya Jinno,
Yoichiro Hashimoto, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, The Science andEngineering Review of Doshisha University, 52, 177-180 (2011).
21)

Influences of Analyte Injection Volumes and Concentrations on Capillary
Chromatography Based on Tube Radial Distribution of Carrier Solvents under Laminar
Flow Conditions; Yusuke Tanigawa, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko
Tsukagoshi, Chromatography, 32, 135- 140 (2011).

22)

Effects of Tube Materials on Capillary Chromatography Based on Tube Radial
Distribution of Ternary Mixture Carrier Solvents under Laminar Flow Conditions;
Yudai Kudo, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
Chromatorgaphia, 75, 417-421 (2012).
23)

Tentative Comparison of Tube Radial Distribution Chromatography and CZE;
Kisuke Tabata, Naoya Jinno, Keiichi Noda, Masahiko Hashimoto, Kazuhiko
Tsukagoshi, Chromatorgaphia, 75, 423-428 (2012).

24)

Separation of Dansyl-DL-Amino Acids by Open Tubular Capillary Chromatography
Based on Tube Radial Distribution Phenomenon of the Ternary Mixed Carrier Solvents;
Yudai Kudo, Hyo Kan, Naoya Jinno, Masahiko Hashimoto and Kazuhiko Tsukagoshi,
Analytical Methods, 4, 906-912 (2012).
368
368

25)

Biomolecule Analyses in an Open-Tubular Capillary Chromatography Using Ternary
Biomolecule
Analyses within an Chemiluminescence
Open-Tubular Capillary
Chromatography
Using Ternary
Mixed
Carrier Solvents
Detection;
Naoya Takahashi,
Yuji
Mixed
CarrierSolvents
with
Chemiluminescence
Detection;
Naoya
Takahashi,
Yuji
Masuhara, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Analytical
Masuhara,28,
Naoya
Jinno,
Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Analytical
Sciences,351-357(2012).
Sciences, 28, 351-357 (2012).
26)
Mixing Process of Ternary Solvents Prepared through Microchannels in a Microchip
26)
MixingProcessof Ternary
Solvents
Prepared throughin aHashimoto,
Microchip
under
Laminar
Flow
Conditions;
Kei Nishiyama,
NaoyaMicrochannels
Jinno, Masahiko
underLaminarTsukagoshi,
Flow Conditions;
KeiSciences,
Nishiyama,
Masahiko Hashimoto,and KazuhikoAnalytical
28, Naoya
423-427Jinno,(2012).
and Kazuhiko Tsukagoshi, Analytical Sciences, 28, 423-427 (2012).
27)
Experiments and Considerations through the Phase Diagram in Open Tubular
27)
Experiments
and Considerations
the Phase
Diagram ofin Ternary
Open Tubular
Capillary
Chromatography
Based onthroughTube Radial
Distribution
Mixed
Capillary Using
Chromatography
BasedCapillaryon TubeTube;
RadialYusuke
Distributionof Ternary
Solvents
a Fused-Silica
Tanigawa,
Naoya Mixed
Jinno,
Solvents
Using
a
Fused-Silica
CapillaryTube;
YusukeTanigawa,
Naoya
Jinno,
Masahiko Hashimoto, Kazuhiko Tsukagoshi, American Journal of Analytical Chemistry,
Masahiko
Hashimoto,3,
300-3005(2012). Kazuhiko Tsukagoshi, American Journal of Analytical Chemistry,
3, 300-3005 (2012).
28)
The Micro-Flow Reaction System Featured the Liquid-Liquid Interface Created with
28)
The Micro-Flow
Reaction
System
the Tube;
Liquid-LiquidInterfaceNaoya
CreatedJinno,
withTernary
Mixed Carrier
Solvents
in aFeatured
Capillary
Yuji Masuhara,
Ternary Mixed
Carrier andSolvents
in a Tsukagoshi,
Capillary Tube;
Yuji Masuhara,
Jinno,
Masahiko
Hashimoto,
KazuhikoAnalytical
Sciences, Naoya
28, 439-444
Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Analytical Sciences, 28, 439-444(2012).
(2012).
29)
Consideration of the Tube Radial Distribution of the Carrier Solvents in a Capillary
29)
Considerationof theFlow
TubeConditions
Radial Distributionof theSimulation;
Carrier Solventsin Jinno,a CapillaryTubeunder Laminar
and Computer
Naoya
Yuji
Tube under Tomoya
Laminar Kobayashi,
Flow Conditions
andTakahashi,
Computer Simulation;
Naoya Jinno,
Yuji
Masuhara,
Naoya
Yusuke Tanigawa,
Masahiko
Masuhara,
TomoyaKobayashi,
Naoya
Takahashi,
YusukeTanigawa,
Masahiko
Hashimoto, and Kazuhiko Tsukagoshi, Analytical Sciences, 28, 527-530 (2012).
Hashimoto, and Kazuhiko Tsukagoshi, Analytical Sciences, 28, 527-530 (2012).
30)
Elution Behavior of Lambda-DNA with Ternary Mixed Carrier Solvents in an
30)
Elution Behaviorof underLambda-DNA
Mixed
CarrierNogami,
SolventsSatoshi
in anOpen-Tubular
Capillary
Laminar with
Flow Ternary
Conditions;Takahiro
Open-Tubular
Capillaryunder
Laminar
Flow
Conditions;Takahiro
Nogami,
SatoshiFujinaga, Naoya Jinno, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, AnalyticalFujinaga, 28,
Naoya
Jinno,
Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Analytical
Sciences,
617-620(2012).
Sciences, 28, 617-620 (2012).
31)
Study of Outer Phases in Capillary Chromatography Based on Tube Radial
31)
Study ofofOuterin underCapillary
Chromatography
BasedNaoya
on TubeDistribution
CarrierPhases
Solvents
LaminarFlow Conditions;Jinno,Radial
Mari
DistributionofCarrierSolvents
underLaminar
Flow
Conditions;
Naoya
Jinno,Mari
Murakami, Kiyoshi Mizohata, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
Murakami,
Mizohata, MasahikoHashimoto,and Kazuhiko
Journalof Kiyoshi
Liquid Chromatography& Related
Technologies,
JournalTsukagoshi,
of Liquidof
LiquidChromatography&
RelatedTechnologies,35,
1750–1766(2012).
Journalof
LiquidChromatography&
RelatedTechnologies,
Journalof
LiquidChromatography & Related Technologies, 35, 1750–1766 (2012).
Chromatography & Related Technologies, 35, 1750–1766 (2012).
32)
Microfluidic Behavior of Ternary Mixed Carrier Solvents Based on the Tube Radial32)
Microfluidic
Behavior of Ternary
Mixed Carrier
Solvents Basedon Jinno,
the Tube
RadialDistribution
in Triple-Branched
Microchannels
in a Microchip;
Naoya
Masahiko
Distributionin
Triple-Branched
Microchannels
inaMicrochip;
Naoya
Jinno,
Masahiko
Hashimoto, Kazuhiko Tsukagoshi, Journal of Analytical Sciences, Methods andHashimoto, Kazuhiko
Journal of Analytical Sciences, Methods andInstrumentation,
2, 49-53Tsukagoshi,(2012).
Instrumentation, 2, 49-53 (2012).
33)
Fluidic Behavior of Polymer Compounds in an Open-Tubular Capillary with Ternary
33)
Fluidic
Behaviorof Polymer
in anOpen-Tubular
Capillarywith Ternary
Mixed
CarrierSolvents
under Compounds
Laminar Flow
Conditions;Takahiro
Nogami,
Satoshi
Mixed
CarrierSolvents
under Laminar
Flow Conditions;Nogami,
SatoshiFujinaga,
Masahiko
Hashimoto,and Kazuhiko
Tsukagoshi, Takahiro
Journal of
Flow Injection
Fujinaga,
Masahiko
Hashimoto, and Kazuhiko Tsukagoshi, Journal of Flow Injection
Analysis, 29,
21-24 (2012).
Analysis, 29, 21-24 (2012).
25)

369
369
A list of the TRDP-related manuscripts

369

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

34)

Influence of Adding Surfactants to an Analyte Solution on Separation Performancein Open-tubular Capillary Chromatography Based on the Tube Radial Distribution of
Ternary Mixed Carrier Solvents; Katsuya Unesaki, Masahiko Hashimoto, and Kazuhiko
Tsukagoshi, Chemistry Letters, 41, 855-856 (2012).

35)

Investigation into Tie Lines and Solubility Curves on Phase Diagrams in
Open-Tubular Capillary Chromatography Using Ternary Mixed-Carrier Solvents;
Yusuke Tanigawa, Satoshi Fujinaga, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
Analytical Sciences, 28, 921-924 (2012)36)

Chromatography Using Ternary Water-Acetonitrile-Ethyl Acetate Mixture as a
Carrier Solution on a Microchip Incorporating Microchannels; Takafumi Matsuda,
Naoya Jinno, Kenichi Yamashita, Hideaki Maeda, Akihiro Arai, Masahiko Hashimoto,and Kazuhiko Tsukagoshi, Chemistry Letters, 41, 1448-1450 (2012).
37)

Tube Radial Distribution of Solvents Observed in an Aqueous Ionic Liquid Mixed
Solution Delivered into a Capillary Tube; Yuki Kawai, Masaharu Murata, MasahikoHashimoto, and Kazuhiko Tsukagoshi, Analytical Sciences, 28, 1029-1031 (2012).
38)

Specific microfluidic behavior of ternary mixed carrier solvents ofwater–acetonitrile–ethyl acetate in open-tubular capillary chromatography and thechromatograms; Satoshi Fujinaga, Katsuya Unesaki, Shigeru Negi, MasahikoHashimoto, and Kazuhiko Tsukagoshi, Analytical Methods, 4, 3884-3890 (2012).
39)

Capillary Chromatography Based on Tube Radial Distribution of Ternary Mixed
Solvents: Construction of the Phase Diagram and the Separation Performance; Yusuke
Tanigawa, Satoshi Fujinaga, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, The
Science and Engineering Review of Doshisha University, 53, 167-172 (2013).
40)

Michrochip Chromatography Using an Open-Tubular Microchannel and a Ternary
Water-Acetonitrile-Ethyl Acetate Mixture Carrier Solution; Takafumi Matsuda, Kenichi
Yamashita, Hideaki Maeda, Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Journalof Separation Science, 36, 965-970 (2013).
41)

Two-Phase Extraction of Metal Ions Using a Water-Acetonitrile-Ethyl Acetate
Ternary Mixed-Solvent Separation System; Naoya Takahashi, Masahiko Hashimoto,and Kazuhiko Tsukagoshi, Analytical Sciences, 29, 665-667 (2013).

42)

Examination of Tube Radial Distribution Phenomenon and Its Function Appearance;
Kazuhiko Tsukagoshi, Bunseki Kagaku, 62, 393-407 (2013).
43)

Specific Distribution Behavior of a Ternary Mixture of Solvents Fed into Bent
and Wound Microchannels in Microchips; Kei Nishiyama, Masaharu Murata, MasahikoHashimoto, and Kazuhiko Tsukagoshi, Analytical Sciences, 29, 1003-1008 (2013).
44)

Tube Radial Distribution Phenomenon Observed in an Aqueous Micellar Solution of
Non-Ionic Surfactant Fed into a Microspace and Attempt to Capillary Chromatographic
370
370

Application; Naoya Jinno,
Application；Naoya.
Jinno, Katsuya
Katsuya Unesaki,
Unesaki, Masahiko
Masahiko Hashimoto,
Hashimoto and Kazuhiko
Tsukagoshi, Journal of Analytical Chemistry, 68, 1197-1202 (2013).
45)

Fundamental Research and Application of the Specific Fluidic Behavior of Mixed
Solvents in a Microspace (Invited Review), Kazuhiko Tsukagoshi, Analytical Sciences,30, 65-73 (2014). 	
 
	
 
46)
Tube Radial Distribution Phenomenon with a Two-phase Separation Solution of a
Fluorocarbon and Hydrocarbon Organic Solvent Mixture in a Capillary Tube and Metal
Compounds Separation; Koichi Kitaguchi, Naoya Hanamura, Masaharu Murata,
Masahiko Hashimoto, and Kazuhiko Tsukagoshi, Analytical Sciences, 30, 687-690(2014). 	
 	
 47)
Capillary Chromatography Using an Annular and Sluggish Flow in the Ternary
Water–Acetonitrile–Ethyl Acetate System as Carrier Solution; Yuya Hamaguchi,
Satoshi Fujinaga, Shunpei Murakami, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
Chemistry Letters, 43, 1318-1320 (2014). 	
 
	
 
48)
Open-Tubular Capillary Chromatography Based on Tube Radial Distribution of theWater-Acetonitrile Containing Sodium Chloride Mixture Carrier Solvents; Tomoya
Kobayashi, Hyo Kan, Kisuke Tabata, Masahiko Hashimoto, and Kazuhiko Tsukagoshi,
Journal of Liquid Chromatography & Related Technologies, 38, 44-53 (2015).	
 
	
 
49)
Investigation of the Composition for a Ternary Solvent System in Tube Radial
Distribution Chromatography; Satoshi Fujinaga, Masahiko Hashimoto, and Kazuhiko
Tsukagoshi, Journal of Liquid Chromatography & Related Technologies, 38, 600-606(2015).
	 50)
Microflow-Extraction System Using Double Tubes Having Different Inner
Diameters in Tube Radial Distribution Phenomenon; Katsuya Unesaki, MasahikoHashimoto, and Kazuhiko Tsukagoshi, the Solvent Extraction Research andDevelopment, Japan, 22, 87-93 (2015).
51)

Estimation of the Dimensionless Weber Number in Tube Radial Distribution
Phenomenon under Laminar Flow Conditions; Satoshi Fujinaga, Masahiko Hashimoto,and Kazuhiko Tsukagoshi, Journal of Chemical Engineering of Japan, in pressed

52)

Investigation of Inner and Outer Phase Formation in Tube Radial Distribution
Phenomenon Using Various Types of Mixed Solvent Solutions; Satoshi Fujinaga,
Katsuya Unesaki, Yuki Kawai, Koichi Kitaguchi, Kosuke Nagatanu, MasahikoHashimoto, Kazuhiko Tsukagoshi, and Jiro Mizushima, Analytical Sciences,inpressed53)

Microfluidic Inverted Flow of Aqueous and Organic Solvent Mixed Solution in a
Microchannel under Laminar Flow Conditions; Shunpei Murakami, Satoshi Fujinaga,
Masahiko Hashimoto, and Kazuhiko Tsukagoshi, The Science and Engineering Review
of Doshisha University,
submitted
371
A list of the TRDP-related manuscripts

371

Tube Radial Distribution Phenomenon (TRDP) of Solvent Molecules

Acknowledgements:
This publication was supported by a grant of Doshisha University for research results.

Education and Employment of Professor Kazuhiko Tsukagoshi(Born : November 5, 1959)
B. S. : Doshisha University (1978-1982)
M. S. : Doshisha University (1982-1984)
Fuji Xerox Corporation: Regular member of research institute (1984-1986)
D. S. : Doshisha University (1987-1990); Kogaku Hakushi (Doctor of Engineering),
Doshisha University, 1990
Kyushu University: Research associate (1990-1993)
Kyushu University: Associate Professor (1993-1995)[Indiana University in USA: Post Doctoral Researcher (1994, one year)]
Doshisha University: Associate Professor (1995-2001)
Doshisha University: Professor (2001- )372

The book title; “Investigation of Tube Radial Distribution Phenomenon (TRDP) and Its
Function Appearance”
The author name; Kazuhiko Tsukagoshi
The day of publication; September 25, 2015
Printed and published by Kimura Keibunsha,
110 Umezono-cho, Kawashima, Nishikyo-ku, Kyoto 615-8113, Japan
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