Original

Chromatography, Vol.27 No.3 (2006)

Original

Study of a Capillary LC System using Temperature−Responsive
Polymer−Modified Packing Materials
Masahito Kanezawa1, Eri Ayano1, Hideko Kanazawa1†, Yoshikatsu Akiyama2 and Teruo Okano2
1

2

Kyoritsu University of Pharmacy, 1−5−30 Shibakoen, Minato−ku, Tokyo 105−8512, Japan.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8−1 Kawadacho,
Shinjyuku−ku, Tokyo 162−8666, Japan

Abstract
We have developed a new method of HPLC packing materials modified with a temperature−responsive polymer, poly (N −isopropylacrylamide) (PNIPAAm). The surface properties and functions of the stationary phases are controlled by the external temperature. We could
separate various pharmaceuticals, peptides and proteins by using only the aqueous mobile phase. In this study, we developed a capillary chromatographic system using packed PNIPAAm modified silica beads. We demonstrated the separation of steroids and phenylthiohydantoins(PTH)−amino acids on a capillary column by temperature control. The retention times of the analytes increased with increasing the temperature, and temperature−responsive elution behavior was observed. The result suggested that we can separate a trace amount of samples used in
the pharmaceutical and biomedical fields by only changing the temperature in this system.
Key words: poly (N −isopropylacrylamide) (PNIPAAm); capillary column; temperature−responsive chromatography; steroid; PTH−aminoacid.

necessary to prevent excessively long retention times with conven-

1. INTRODUCTION

tional reverse−phase columns.

The needs of biotechnology for high−resolution purification

Stimuli−responsive polymers, which change their structure

and analytical technology have spurred new methods for chroma-and physical properties in response to external signals, comprise

tographic systems, concerning both columns and equipment.
In the case of the separation of peptides, proteins, and othernew materials with interesting applications in biomaterial science

biological molecules, and for maintaining viable cells, it is fre-

and technology. Such polymers that are responsive to changes inquently necessary to avoid the use of organic solvents in the mobile

the pH [1], temperature, and light have been widely utilized forphase, because these can cause sample denaturation. Completedrug−delivery systems [2, 3], cell culture substrates [4, 5], and bio-

avoidance of organic solvents is also advantageous for environ-conjugates [6]. However, there have been few reports on the use ofmental reasons. Reversed−phase chromatography (RPC) using

these stimulus−responsive polymers in chromatographic separa-chemically modified stationary phases is generally faster and easiertions. Temperature seems to be one of the most attractive tools forthan other modes of high−performance liquid chromatographysuch stimuli. There are technical difficulties associated with sol-

(HPLC), and consequently, has achieved very wide popularity. The

vent−gradient elution in capillary columns. Temperature program-retention and selectivity in RPC are controlled primarily by chang-ming is used in microcolumns and capillary columns [7−10]. Ac-

ing the polarity of the mobile phase. The use of organic solvents iscording to these reports, temperature change has been used as a†

To whom correspondence should be addressed:
Hideko KANAZAWA, Ph.D.
Department of Physical Chemistry, Kyoritsu University of Pharmacy 1−5−30 Shibakoen, Minato−ku, Tokyo, JAPAN
Phone: +81−3−5400−2657
Fax: +81−3−5400−1378
E−mail: kanazawa−hd@kyoritsu−ph.ac.jp―１１１―

Chromatography, Vol.27 No.3 (2006)

technique for changing the mobile−phase properties, for reducing

PAAm by radical telomerization using MPA as a chain−transfer

the solvent viscosity, and for increasing the diffusion coefficient.

agent. PNIPAAm−grafted silica was used as a packing material.

However, in our system, changing the temperature strongly affects

The immobilization reaction involved the formation of an amide

the properties and surface functions of the stationary phase, rather

bond between the carboxylic acid end group of PNIPAAm and sur-than the mobile phase. Also packed capillary columns are expected

face amino groups on the aminopropyl silica support, using acti-

to be, due to their relatively low heat capacity and small diameter,vated ester−amine coupling. This immobilization strategy is themore suitable for temperature programming than conventionalsame as that reported previously [12].

HPLC columns[11].
2.3. Instrumentation

Poly (N −isopropylacrylamide) (PNIPAAm) has the sharpest
phase transition in the class of thermosensitive N−alkyl acrylamide

The polymer−modified silica support was packed into a stain-

polymers. This feature makes it the most suitable for studies andless−steel capillary column (250 mm × 0.5 mm i.d.) and a PEEK-

practical applications. PNIPAAm exhibits a thermally reversible

silTM tube (100 mm × 0.15 mm i.d.; Upchurch Scientific, WA, USA

soluble−insoluble change in response to temperature changes) by a Capillary column packer (Nano baume; Western Fluids, CA,across a lower critical solution temperature (LCST) at 32℃ inUSA) at 5 MPa (8 hr at room temperature).

aqueous solutions. Polymer chains of NIPAAm hydrate to form expanded structures below the LCST. At higher temperature thanA Model MP 710 plunger pump controller for the constant−

LCST, however, the chains form compact structures by dehydra-

flow mode (GL Sciences Inc. Tokyo, Japan) was used as the eluent

tion. The remarkable phase transition of PNIPAAm in aqueous me-delivery system. The liquid chromatograph embodied a Model C 4

dia is due to rapid hydration and dehydration changes of the poly-−1344−.1 internal sample injector with an injection volume of 100

mer chain.

nl (Valco Instruments, Houston, TX, USA), a Model MU 701 UV

In this study, we prepared PNIPAAm−modified silica as col-detector (GL Sciences Inc.) and an EZChrom Elite data processor

umn packing materials. Using capillary columns packed with PNI-(GL Sciences Inc.). The packed capillary column was housed in a

PAAm−modified silica, we demonstrated the analysis of steroids

model MO 706 column oven (GL Sciences Inc.). Milli−Q gradeand phenylthiohydantoins (PTH)−amino acids using only an aque-water was used as the mobile phase. The elution behaviors of theous solution as a mobile phase without an organic solvent.

samples were recorded at a flow−rate of 1 ~ 5µL/min at various
temperatures.

2. EXPERIMENTAL
2.1. Chemicals

3. RESULTS AND DISCUSSION

N −isopropylacrylamide (NIPAAm) was kindly provided by

Temperature−responsive chromatographic analysis of steroids and

KOHJIN (Tokyo, Japan) and was purified by recrystallization fromPTH−amino acids on a stainless−steel capillary column.

hexane and dried at room temperature in vacuo. 3−Mercaptopropi-

Using a capillary column packed with PNIPAAm modified

onic acid (MPA), 2, 2’−azobisisobutyronitrile (AIBN), N, N −di-silica connected to an HPLC system, the separation of steroids was

methylformamide (DMF), ethyl acetate and dioxane were pur-carried out by changing the temperature. Milli−Q water was usedchased from Wako Pure Chemicals (Tokyo, Japan). N, N’ −dicyclo-

as the sole mobile phase. Figure 1 shows typical chromatograms ofhexylcarbodiimide (DCC) and N −hydroxysuccinimide were ob-

steroids on a PNIPAAm terminally−grafted silica column packed

tained from Wako Pure Chemicals. (Aminopropyl) silica (averaged

into a stainless−steel capillary (250 mm × 0.5 mm i.d.). The reten-

diameter of 3µm, pore size 120Å) was purchased from Nishiotion times of hydrocortisone and dexamethasone were 14.87 andKogyo (Tokyo, Japan). HPLC−grade uracil and hydrocortisone

18.07 min at 10℃, respectively, and 20.79 and 28.71 min at 50℃,

were purchased from Wako Pure Chemical. Dexamethasone, tes-

respectively. With increasing the temperature, increased interac-tosterone and prednisolone were of HPLC grade and purchased

tions between the solutes and the PNIPAAm terminally−graftedfrom Sigma (St. Louis, MO, USA). PTH−amino acids were ob-

surfaces of the stationary phases were observed. The results indi-tained from Wako Pure Chemicals. 6β−Hydroxytestosterone wascated that a surface modified with PNIPAAm of the stationary

purchased from Sumitomo Chemical (Tokyo, Japan).

phase exhibited temperature−controlled hydrophilic−hydrophobic

Water was distilled and passed through a Milli−Q purification

changes. The surface was hydrophilic at a lower temperature andsystem (Millipore, Bedford, MA, USA).

hydrophobic at an elevated temperature. The surface properties andfunctions of the stationary phases enabled to control by an external

2.2. Temperature−responsive polymer

temperature; thus, the separation selectivity and retention of theWe synthesized carboxyl semitelechelic polymers of NI-

solute were controlled by changes in the column temperatures

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Chromatography, Vol.27 No.3 (2006)

Masahito Kanezawa, Eri Ayano, Hideko Kanazawa,
Yoshikatsu Akiyama and Teruo Okano

Figure 1. Chromatograms of steroids at two temperatures. Column, 250 mm × 0.5 mmi.d., packed with PNIPAAm−
modified silica; flow rate, 5.0 µL/min; eluent, Milli−Q
water; Samples; (A) hydrocortisone (100 µg/mL); (B)dexamethasone (60 µg/mL), column temperature; (1)10℃ and (2) 50℃; detection, UV, 254 nm, injection
volume, 0.1 µL.

Figure 2. Retention factors of (A) three steroids and (B) three
PTH−amino acids on a capillary column packed with
PNIPAAm−modified silica with Milli−Q water used asa mobile phase. Flow rate, 2.0 µL/min; injection volume, 0.1 µL; detection, UV, 254 nm. Samples: (A) ○,hydrocortisone (20 µg/mL); □, prednisolone (32 µg/mL);△, dexamethasone (36 µg/mL); (B) ●, PTH−glycine (50 µg/mL); ■, PTH−valine (24 µg/mL); ▲, PTH−leucine (14 µg/mL).

Table 1. Comparison with conventional column and capillary column.
Injection volume(µL)

Flow rate(mL/min)Cloumn volume(mm3)

Conventional column(4.6 φ × 150 mm)

10

1

2492

Capillary column(05 φ × 250 mm)

0.1

0.002

49

k ＝ tR − t０
t０

0.1

0.001

1.77

Where tR is the retention time of steroids and t0 is the retention time

peratures from 10 to 50℃ in 10℃ intervals; k can be expressed as(1)

TM

PEEKsil tubing
capillary column(0.15 φ × 100 mm)

of Uracil. Uracil was used as an inert tracer for measuring the mobile−phase hold−up times. Figure 2 (A) shows the elution behaviorof steroids in Milli−Q water over a variety of column temperatures.

without any change in the eluent with the capillary column.
Table 1 gives a comparison with the conventional column andWith increasing temperature, increased retention factors were ob-capillary column. The sample injection volume was reduced to one

served, despite a reduced back−pressure in the column. It is note-−hundredth, the flow rate was reduced to one−to−five−hundredthworthy that the retention of steroids showed large changes above

and the retention time increased by five times in the case of analyz-

the LCST of PNIPAAm. This implies that the transition of hydro-

ing dexamethasone. By using a capillary column, the consumptions

philic−hydrophobic surface properties at the LCST causes thisof the eluent and the sample injection volume could be achieved toanomalous retention behavior of the steroids. Figure 2 (B) shows

be one−hundredth that using the conventional column.

the elution behavior of PTH−amino acids. The chromatographic
conditions are the same as those in Figure 2 (A).

We previously demonstrated that a hydrophobic interaction
between steroids and PNIPAAm grafted surfaces was readily

Figure 3 shows van’t Hoff plots for steroids (A) and PTH−

modulated by the temperature using conventional−size columns.

amino acids on the capillary column packed with PNIPAAm−

As previously reported, the retention of steroids exhibits a linear

modified silica. A nonlinear relationship between the reciprocal

relationship with the log P values (the partition coefficients in 1−

temperature (1/T) and ln k values is apparent for each steroid andoctanol/water system). The log P values for each steroid are 1.61

PTH−amino acid. This provides evidence for the polymer phasefor hydrocortisone, 1.62 for prednisolone, and 1.83 for dexametha-transition on the stationary−phase surface. Generally, these plotssone. In this study, similar results were obtained; the more hydro-

should be linear for conventional chromatographic processes onphobic steroids showed longer retention times.

commercially available reversed−phase columns under conditions

In order to compare the effects of the temperature in the re-where the retention mechanisms do not change. On the temperature

verse−phase chromatography of steroids with a packed capillary−responsive polymer−modified column, however, deviations fromcolumn, the retention factors (k) were measured at column tem-linearity between the ln k values and the reciprocal temperature

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Chromatography, Vol.27 No.3 (2006)

Figure 3. Van’t Hoff plots of (A) three steroids and (B) three PTH−amino acids on the capillary column. The chromatographic conditions are the same as those in Figure 2.
Samples: (A) ○, hydrocortisone; □, prednisolone;△,dexamethasone; (B) ●, PTH−glycine; ■, PTH−valine;▲, PTH−leucine.

Figure 4. Chromatograms of steroids on a PEEKsilTM capillarycolumn (100 mm × 0.15 mm i.d.) packed with PNIPAAm−modified silica.
Flow rate, 1.0 µL/min; eluent, Milli−Q water; temperature, (A) 10℃ and (B) 50℃; solutes: 1, hydrocortisone(10 µg/mL); 2, testosterone (15 µg/mL); detection, UV,
254 nm; injection volume, 0.1 µL.

were observed. These deviations are consistent with the known
phase transition of the grafted polymer stationary phase. Additionally, the slope of the van’t Hoff plots on the packed with PNIPAAm−modified silica capillary column was negative, which is
opposite to that seen for conventional chromatography. This provides additional evidence that the interaction between steroids or
PTH−amino acids and temperature−responsive surfaces becomesstronger at elevated temperatures.
The results of our studies indicate that the driving force for retention in this system is hydrophobic interactions between the solute molecules and polymer chains on the surface. At temperatureshigher than LCST, PNIPAAm−grafted surfaces exhibited hydrophobic properties and the sensitivity to hydrophobicity of the sol-Figure 5. Chromatograms of testosterone and 6β−hydroxytestosterone at two temperatures. Column, 100 mm × 0.15mm, packed with PNIPAAm−modified silica; flow rate,
1.0 µL/min; eluent, Milli−Q water; temperature, (A) 10℃ and (B) 50℃; solutes: 1, 6β−hydroxytestosterone(50 µg/mL); 2, testosterone (22.5 µg/mL); detection,
UV, 254 nm; injection volume, 0.1 µL.

utes was remarkably increased. In the case of being the column
packed into capillary with a PNIPAAm−modified silica, the analysis of steroids and PTH−amino acids could be achieved with Milli−
Q water as the mobile phase by controlling the external temperature.
Temperature−responsive chromatographic analysis of steroids andPTH−amino acids on a PEEKsil TM capillary column.

The oxidative metabolism of drugs in the liver is catalyzed by

Figure 4 shows typical chromatograms of steroids. The sepa-

substrate−specific or selective cytochrome P 450 (CYP), a super-ration of hydrocortisone and testosterone were achieved by chang-family of haemoproteins that catalyze the metabolism of a large

ing the temperature with Milli−Q water as a mobile phase using a

number of clinically important drugs [13−15]. Hepatic drug oxida-

PEEKsilTM capillary column (100 mm × 0.15 mm i.d.) packed withtion is a major source of interindividual variations in drug pharma-

PNIPAAm modified silica. In reversed−phase HPLC using an ODS

cokinetics and therapeutic response. Testosterone is widely used ascolumn, the retention times should decrease with increasing tem-a probe to measure the activity of CYP 3 A 4, an important mem-

perature. However, the opposite behavior of the retarded retention

ber of the cytochrome P 450 superfamily of drug−metabolizing en-

times was observed with increasing temperature using a PNIPAAm

zymes. Testosterone undergoes oxidative metabolism to one major

modified capillary column. It was considered that a hydrophobic

metabolite, β−hydroxytestosterone, in humans, a pathway whichinteraction exists between steroids and the PNIPAAm−modified

seems to be mediated almost exclusively by CYP 3 A isoforms.

silica.

Figure 5 shows chromatograms of 6 β−hydroxytestosterone and

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Chromatography, Vol.27 No.3 (2006)

Masahito Kanezawa, Eri Ayano, Hideko Kanazawa,
Yoshikatsu Akiyama and Teruo Okano

creased the retention times of the analytes. This is the first reportconcerning a temperature−responsive chromatographic system using a PNIPAAm−modified silica capillary column. A system forcontrolling the function and properties of the HPLC stationary
phases simply by changing the temperature, and furthermore decreasing consumption of the eluent, solutes and packing materials
would be very useful.
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Figure 6. Retention factors of (A) hydrocortisone and testosterone; (B) 6β−hydroxytestosterone and testosterone on a
PEEKsilTM tubing capillary column packed with PNIPAAm−modified silica with Milli−Q water used as amobile phase. Flow rate, 1.0 µL/min; injection volume,
0.1 µL; detection, UV, 254 nm. Samples: (A) ●, hydrocortisone (10 µg/mL); ■, testosterone (15 µg/mL);(B), ○, 6 β−hydroxytestosterone (50 µg/mL); □, testosterone (22.5 µg/mL).

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4. Conclusion
PNIPAAm−modified silica exhibits temperature−controlled[14] F. P. Guengerich, J. Biol. Chem., 1991, 266, 10019−10022.

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ous systems. By using a capillary column packed with PNIPAAm−
modified silica, it was observed that increasing the temperature in-―１１５―