Original

CHROMATOGRAPHY, Vol.32 No.3 (2011)

Original

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
Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University,
Kyotanabe, Kyoto 610-0321, Japan

Abstract
A capillary chromatography called the tube radial distribution chromatography (TRDC) system has been developed using an open capillary
tube and a water-hydrophilic/hydrophobic organic solvent mixture carrier solution. In this study, we examined the effects of the analyte injection volume (injection time) and concentration on the chromatograms in the TRDC system using a fused-silica capillary tube (50 µm i.d. and100 cm effective length) and a water-acetonitrile-ethyl acetate mixture carrier solution (volume ratio, 3: 8: 4). Analyte solutions of 1-naphtholand 2,6-naphthalenedisulfonic acid (1 mM each) were injected into the system with various injection times of 10-1500 s from a height of 20
cm by 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) were analyzed with the definite injection time of 30 s from a height of 20 cm by gravity. They were separated and detectedwith a baseline separation and their calibration curves showed linearity up to 1.5 mM. It was confirmed that the TRDC system worked well asa quantitative analysis.
Keywords : chromatography, tube radial distribution, laminar flow conditions, analyte injection volumes, analyte concentrationphy (TRDC) system.

Introduction
Analytical methods using capillary tubes have attracted a great

In the TRDC system [6,7], 1) various types of capillary tube

deal of interest since the last century [1-3]. Well-known methods

are available, such as fused-silica, polyethylene, and poly

include capillary electrophoresis, capillary electrochromatography,(tetrafluoroethylene), 2) a ternary mixed solution, i.e., water-

micellar electrokinetic capillary chromatography, and high-hydrophilic/hydrophobic organic mixture carrier solution (homoge-

performance liquid capillary chromatography using packed andneous) must be delivered into the capillary tube under laminar flowmonolithic columns. However, only a few new concepts concern-

conditions, 3) the elution times of analytes, hydrophobic and hy-

ing capillary chromatography have been reported in the last decade

drophilic, are easily reversed by changing the component ratios of[4,5]. Related research has been discussed in detail in our previous

the carrier solvents, i.e., by using an organic solvent-rich and apapers [6,7]. We have developed a capillary chromatography sys-

water-rich carrier solution, and 4) the first peak appears with al-

tem using an open capillary tube and a water-hydrophilic/hydro-

most the average linear velocity and the other peaks are eluted withphobic organic mixture carrier solution [6-11]. This system works

lower velocities than the average linear velocity under laminar flow

under laminar flow conditions and does not require any packed re-conditions in both organic solvent-rich and water-rich carrier solu-

agents in the capillary tube or application of high voltage to thetions.
Separation performance in the TRDC system is explainedends of the tube. It is called a tube radial distribution chromatograCorrespondence: Kazuhiko Tsukagoshi

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CHROMATOGRAPHY, Vol.32 No.3 (2011)

based on the obtained experimental data as follows. First, aqueous

raphy separation and flow-injection analysis. In this study, we ex-

and organic solvents in the carrier solution are dispersed non-amined separation performance with various analyte injection vol-uniformly at a specific flow in the capillary tube under laminarumes and concentrations in the TRDC system to expand our

flow conditions, generating organic solvent-rich and water-richknowledge regarding this new separation system.

phases in the tube. An organic solvent-rich carrier solution generates an organic solvent-rich major inner phase and a water-rich mi-

Experimental

nor outer phase in the capillary tube, while a water-rich carrier so-

Chemicals and capillary tubes

lution results in a water-rich major inner phase and an organic

Water was purified with an Elix 3 UV (Millipore Co.,solvent-rich minor outer phase in the tube. That is, a major innerBillerica, MA). All reagents used here were commercially available

phase is formed around the center of the tube away from the innerand of analytical grade. 1-Naphthol, 2,6-naphthalenedisulfonicwall and a minor outer or capillary wall phase is generated near theacid, acetonitrile, and ethyl acetate were purchased from Wako

inner wall. The tube radial distribution of the solvent molecules inPure Chemical Industries, Ltd. (Osaka, Japan). A fused-silica capil-

the carrier solution is thus caused by the flow in the capillary tube

lary tube (50 µm i.d. and 150 µm o.d.) was purchased from GL

under laminar flow conditions. Consequently, the analytes that are

Science (Tokyo, Japan).

delivered through the capillary tube are distributed between the inApparatus and procedures

ner and outer phases, and undergo chromatographic separation in

The TRDC system equipped with absorption on-line detection

the capillary tube.
The tube radial distribution of the carrier solvents in thewas comprised of an open fused-silica capillary tube (50 µm i.d.,

TRDC system is also supported by other experimental data using

120 cm length; 100 cm effective length), micro-syringe pump (MF-polymer particles as analytes [8], phenylboronic acid or iminodi-

9090; Bioanalytical Systems, Inc., West Lafayette, IN), and ab-

acetic acid-modified fused-silica capillary tubes [9], and double

sorption detector (modified SPD-10AV spectrophotometric detec-

fused-silica capillary tubes with different inner diameters [10], as

tor; Shimadzu Co., Kyoto, Japan). The schematic diagram is shown

well as by visual observation that the dyes dissolved in the carrierin Fig. 1. The tube temperature was controlled by immersing thesolution were radially distributed based on their hydrophilic or hy-

capillary tube in water maintained at a definite temperature (0°C) indrophobic nature in the micro-channel in a micro-chip or in thea beaker with stirring. A water-acetonitrile-ethyl acetate mixture

capillary tube [11,12]. We also tried to discuss the mechanism ofwith a volume ratio of 3: 8: 4 was used as an organic solvent-richthe formation of the inner and outer phases in the tube radial distri-

carrier solution. An analyte solution including 1-naphthol and 2,6-

bution of the solvents [13].

naphthalenedisulfonic acid as a model was prepared with the carrier solution.

To date, various mixtures of hydrophilic and hydrophobic
analytes have been separated using the TRDC system [6,7]. How-

The analyte solution was introduced directly into the capillary

ever, we have not examined the effects of the analyte injection vol-inlet side by the gravity method (from a height of 20 cm). Afterume or injection time and concentration on the chromatograms inanalyte injection, the capillary inlet was connected through a joint

the TRDC system in detail. The analyte injection volume and con-

to a micro-syringe. The syringe was set on the micro-syringe pumpcentration are fundamental analytical factors in general chromatog-

and the carrier solution was fed into the capillary tube at a definite

Fig. 1 Schematic diagrams of the TRDC system.

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CHROMATOGRAPHY, Vol.32 No.3 (2011)

Yusuke Tanigawa, Naoya Jinno, Masahiko Hashimoto and Kazuhiko Tsukagoshi

flow rate (0.2 µL min-1) under laminar flow conditions (Reynolds

cross-section. That is, the injection times of 10-1500 s (25 min)number was calculated roughly to be 0.14 in the present system).

corresponded to the analyte injection volumes of 2.7-400 nL andOn-capillary absorption detection (254 nm) was performed with thethe zone lengths of 1.5-200 mm.

detector.
Effects of injection time on the chromatograms
We examined the effects of analyte injection time on separa-

Results and discussion

tion in the TRDC system with an analyte solution of 1-naphthol

Analyte injection times, volumes, and zone lengths
2,6-

and 2,6-naphthalenedisulfuric acid as a model. The experiments

naphthalenedisulfonic acid was injected into the capillary tube bywere performed with the organic solvent-rich carrier solution be-

gravity from a height of 20 cm at various injection times of 10-

cause the carrier solution provided better resolution on the chroma-

1500 s (25 min) in the present TRDC system. To examine the ana-

tograms than the water-rich carrier solution when the fused-silica

lyte injection volumes and zone lengths in the capillary tube corre-

capillary tube was used in our previous studies [7,10].

Analytesolution

consistingof

1-naphthol

and

The

sponding to the analyte injection times of 10-1500 s, the flow rate

analytesolutionsof

1-naphthol

and

2,6-

by gravity was first estimated with the Hagen-Poiseuille equation

naphthalenedisufonic acid (1 mM each) were analyzed with various

theoretically.

injection times of 10-1500 s (25 min) by the TRDC system. The

The flow rate in the capillary tube with gravity from a heighttypical chromatograms obtained at injection times of 10, 40, 150,

of 20 cm was calculated using the Hagen-Poiseuille equation (Eq.

600, 1320, and 1500 s are shown in Fig. 2. As shown in Fig. 2, 1-

is the radius, ∆p is

naphthol and 2,6-naphthalenedisufonic acid were separated and de-1), which is shown below: Q is the flow rate,

loss of pressure, μ is viscosity, and L is total length.
Q=

tected in this order with the TRDC system using the organic
solvent-rich carrier solution with good reproducibility for their

π 4 ∆p8μ L

peak shapes and elution times with the injection times of 10-150 s

Eq. 1)(2.7-40 nL injection volume or 1.5-20 mm zone length). The first
The flow rate for the capillary 50 µm in i.d. (a, 25×10-6 m) was cal-peaks of 1-naphthol were eluted with near the average linear veloc-

m s , where ∆p, μ, and L

ity in the present system and the second peaks of 2,6-

were estimated to be 1.3×103 kgm-1s-2 (calculated from the differ-naphthalenedisufonic acid were eluted with lower velocity than theence in height or position between the capillary inlet and outlet, 20

average linear velocity. The capacity factors of the second peaks,-13

culated to be approximately 2.67×10

-4

3 -1

-1 -1

cm), 6.2×10 kgm s (measured by a viscosity measurement de-

which were calculated using the first peaks as the dead (hold-up)

vice for the carrier solution), and 1.2 m, respectively.

times, at injection times of 10, 40, and 150 s were 0.57, 0.61, andAlso, the value of the flow rate, Q , was estimated experimen-

0.58, respectively. With injection times of 150-1350 s (22 min) (40tally by the experimental data of gravity method. 1-Naphthol ana-

-360 nL injection volume or 20-180 mm zone length), althoughlyte (1 mM) was injected into the capillary tube in the usual way

they were separated on the chromatograms, the elution times of the(from a height of 20 cm for 10 s) and then the analyte was deliv-

second peaks became earlier. With injection times of more thanered with the carrier solution into the tube by gravity (from a

1400 s, even separation was not performed as shown in the typicalheight of 20 cm) instead of the micro-syringe pump. The flow rates

chromatogram (injection time 1500 s (25 min) in Fig. 2. It is noted

for the capillary 50 µm in i.d. were calculated with Eq. 2, where there that such experiments treated with the wide ranges of the in-is the elution time and L’ is the effective length. The value of Q

jection volume (2.7-400 nL) and the zone length (1.5-200 mm)-13

was calculated to be approximately 2.72×10
and

3 -1

would not be carried out in the usual capillary chromatography us-

m s (using t, L’ ,of 120 min, 1.0 m, and 25×10-6 m, respectively).

ing monolithic or packed columns together with an sample injector.
Large injection times or analyte injection volumes led to non-

2

Q=

πL’
t

Gaussian peaks or trapezoid-type peaks, as shown in Fig. 2. The re-Eq. 2)

lationships between the injection times and peak heights or peak arThere was good correspondence in the flow rate between theeas for both analytes were examined for injection times of 10-150

value calculated with the Hagen-Poiseuille equation and that withs. The obtained relationships are shown in Fig. 3. The relationships-13

the gravity experimental data. The value of the flow rate, 2.7×10

between the injection times and peak areas for both analytes indi-

m3s-1, was used in the following estimations. The analyte injection

cated good linearity, while those between injection times and peakvolumes were calculated by multiplying the flow rates and the in-heights indicated linearity up to ca. 40 s for 1-naphthol and up tojection times as well as the analyte zone lengths in the tube wereca. 60 s for 2,6-naphthalendisulfonic acid. The inflection point (ca.

calculated by dividing the analyte injection volumes by the tube

40 s)

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of

1-naphthol

appearedearlier

than

that

of

2,6-

CHROMATOGRAPHY, Vol.32 No.3 (2011)

Fig. 2 Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acid obtained 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.

Fig. 3 Relationships between injection times and peak heights or peak areas for 1-naphthol and 2,6naphthalenedisulfonic acid in the TRDC system.
Conditions: Capillary tube, 120 cm (effective length: 100 cm) of 50 µm i.d. fused-silica; carrier, wateracetonitrile-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; and analyte concentration, 1 mM.

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CHROMATOGRAPHY, Vol.32 No.3 (2011)

Yusuke Tanigawa, Naoya Jinno, Masahiko Hashimoto and Kazuhiko Tsukagoshi

naphthalendisulfonic acid (ca. 60 s). The difference in inflection

curves shown in Fig. 5. Correlation coefficients were 0.996-0.998

points may have been due to the differences in elution time or dif-

for all calibration curves. Relative standard deviations of 1-

fusion time between 1-naphthol and 2,6-naphthalenedisulfonicnaphthol were 2.5% (n=8) for both of peak heights and areas asacid.

well as 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 fromthe obtained data in Fig. 5 that the TRDC system worked well as a

Effects of analyte concentrations on the chromatogram

quantitative analysis.

We examined the effects of analyte concentrations (0.075-3.0mM) on separation in the TRDC system with an analyte solution ofConclusions

1-naphthol and 2,6-naphthalenedisulfuric acid as a model. The analyte solution was injected into the capillary tube by gravity from a

We developed a novel capillary chromatography method

height of 20 cm for 30 s. The typical chromatograms obtained withcalled the tube radial distribution chromatography (TRDC) system,

concentrations of 0.1, 0.5, 1.5, 2.0, and 3.0 mM are shown in Fig.

which uses an open capillary tube and an aqueous-organic solvent

4. 1-Naphthol and 2,6-naphthalenedisulfuric acid were separated

carrier solution under laminar flow conditions. The separation per-with a base-line separation and detected up to 1.5 mM. The capac-

formance in the system was explained based on the tube radial dis-

ity factors at concentrations of 0.1, 0.5, and 1.5 mM were 0.54,

tribution of the carrier solvents in the capillary tube. In this study,

0.59, and 0.57, respectively. At concentrations higher than 1.5 mM,

we examined the effects of analyte injection volume and concentra-

the resolutions gradually decreased and finally they could not betion on the chromatograms in the TRDC system. The injection vol-separated on the chromatogram at all at about 3.0 mM, as shown inumes and concentrations are fundamental analytical factors in chro-Fig. 4.

matographic research. The analyte solution of 1-naphthol and 2,6-

The calibration curves were examined up to 1.5 mM for both

naphthalene as a model was separated and detected in this order up

analytes with peak heights and peak areas. The obtained calibration

to the injection time of 150 s (injection volume 40 nL or zone

curves are shown in Fig. 5. Good linearity was observed in thelength 20 mm) and they were also analyzed up to 1.5 mM with

Fig. 4 Chromatograms of a mixture of 1-naphthol and 2,6-naphthalenedisulfonic acid obtained 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.

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CHROMATOGRAPHY, Vol.32 No.3 (2011)

Fig. 5 Calibration curves of 1-naphthol and 2,6-naphthalenedisulfonic acid obtained by the TRDC system.
Conditions: Capillary tube, 120 cm (effective length: 100 cm) of 50 µm i.d. fused-silica; carrier, wateracetonitrile-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.
good reproducibility. Separation in the present TRDC system be-[2]

came difficult at analyte injection times of more than 150 s or ana-

[3]

Pyell, U. Electrophoresis 2010, 31, 814-831.
Cucinotta, V.; Contino, A.; Giuffrida, A.; Maccarrone, G.;
Messina, M. J. Chromatogr. A 2010, 1217, 953-967.

lyte concentrations of more than 1.5 mM. The difficulty may be[4]

due to instability or collapse of the inner and outer phase formation

Okada, T.; Harada, M.; Kido, T. Anal. Chem. 2005, 77,
6041-6046.

based on the tube radial distribution of the carrier solvents in the[5]capillary tube with extremely large analyte injection volumes orCharoenraks, T.; Tabata, M.; Fujii, K. Anal. Sci. 2008, 24,
1239-1244.

concentrations. Also, the ranges of analyte injection volume and[6]concentration that provide quantitative analysis might be influ-

Jinno, N.; Itano, M.; Hashimoto, M.; Tsukagoshi, K. Talanta
2009, 79, 1348-1353.

enced by analytical conditions, such as capillary inner diameter,
flow rates, and component ratios of the carrier solvents.

[7]

Acknowledgments[8]

Jinno, N.; Murakami, M.; Hashimoto, M.; Tsukagoshi, K.
Anal. Sci. 2010, 26, 737-742.
Jinno, N.; Hashimoto, M.; Tsukagoshi, K. J. Chem. Eng. Japan 2009, 42, 767-770.

This work was supported by a Grant-in-Aid for Scientific Re[9]search (C) from the Ministry of Education, Culture, Sports, Sci-

Jinno, N.; Tsuji, K.; Shikatani K.; Hashimoto, M.;
Tsukagoshi, K. J. Sep. Sci. 2009, 32, 4099-4100.

ence, and Technology, Japan. This work was also supported by[10] Yamada, K.; Jinno, N.; Hashimoto, M.; Tsukagoshi, K.

“Advanced Study for Integrated Particle Science and Technology,”

Anal. Sci. 2010, 26, 507-510.

Strategic Development of Research Infrastructure for Private Uni-

[11] Murakami, M.; Jinno, N.; Hashimoto, M.; Tsukagoshi, K.

versities, the Ministry of Education, Culture, Sports, Science andChem. Lett. 2010, 3, 272-273.

Technology, Japan.

[12] Jinno, N.; Murakami, M.; Mizohata, K.; Hashimoto, M.;
Tsukagoshi, K. Analyst 2011, 136, 927-932.

References[1][13] Murakami, M; Jinno, N.; Hashimoto, M.; Tsukagoshi, K.

Koike, R.; Kitagawa, F.; Otsuka. K. J. Sep. Sci. 2009, 32,

Anal. Sci. 2011, 27, 793-798.

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