Anal. Chem. 1995, 67, 1907-1911
Temperature-Controlled High-Performance Liquid Chromatography Using A Uniformly Sized Temperature-Responsive Polymer-Based Packing Material Ken Hosoya,* Kazuhiro Kimata, Take0 Araki, and Nobuo Tanaka
Department of Polymer Science, Kyoto lnstifute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Jean 1111. J. Fr6chet Department of Chemistry, Baker Laboratory, Come11 University, Ithaca, New Yo& 14853-1301
Uniformly sized polymer-based packing materials modified with a temperature-responsive polymeric selector, poly(N-isopropylacrylamide), on either the internal or the external surface were prepared by an in situ surfaceselective moditication method. These packing materials were utilized as stationaryphases in reversed-phasehighperformance liquid chromatography to evaluate their ability to thermally control Chromatographic separation selectivity. A packing material having a moditied internal surface showed temperature-dependentseparation selectivity in drug separation. Drugs that could not be separated below the phase transition temperature of the poly(N-isoproWlacrytamide)selectorwere separatedfaster and with much better resolution above the phase transition temperature, while the unmodified base packing material afforded worse resolution of the drugs above the phase transition temperature due to the peak overlapping resulting from faster elution of all the solutes employed. In addition, a packing material moditied on the external surface showed a temperature-responsive shift of the elution region for a polypeptide such as BSA, which was thought to be useful in direct injection of a serum sample. Control of chromatographic separation selectivity in HPLC by applying external physical stimuli such as light,'s2 electric or magnetic field?,* or temperature5t6is particularly interesting, because separation selectivity or resolution can be controlled without changing stationary phases and/or mobile phases. Actually, photocontrolled chromatographyhas been examined utilizing photoresponsive cis-trans or nonion-cation transformations of functional polymeric chromatographic selector^.^-^ (1) Shinkai, S.; Kinda, H.; Manabe, 0.1.Am. Chem. SOC.1982,104,2933. (2) Karube, I.; Ishimori, Y.;Suzuki, S.; Sato, T. Biofechnol. Bioeng. 1978,20, 1775. (3) Kwon, I. C. Makromol. Chem., Macromol. Symp. 1990,33,265. (4) Okuzaki, H.; Nagata, Y. Polymerfiefir., Jpn. 1991,40,615. (5) Nozawa, I.; Suzuki, Y.; %to, S.; Sugibayashi, IC; Morimoto, Y. J. Biomed. Mater. Res. 1991,25, 577. (6) Okano, T.;Bae, Y. H.; Jacobs, H.; Kim, S. W.Adu.DrugDeliuey Syt. 1990, 4,255.
(7) Negishi, N.; Ishihara, IC;Shinohara, I.; Okano, T.; Kataoka, IC;Sakurai, Y. Makromol. Chem. Rapid Commun. 1981,2, 95. (8) Negishi, N.; Ishihara, IC; Shinohara, I.; Okano, T.; Kataoka, IC; Sakurai, Y.; Akaike, T. Chem. Lett 1981,681. (9) Ishihara, IC; Shinohara, I.; Okano, T.Kagaku No Ryoiki 1983,37,873. 0003-2700/95/0367-1907$9.0010 0 1995 American Chemical Society
Although the reported photocontrolled chromatography was an interesting example of controlling separation selectivity by applying external physical stimuli, a phototransmittabletube such as a quartz tube should be used as column material, and moreover, the light can hardly pass through the column homogeneously if the column employed has large diameter. Similarly, some special apparatus would be required to apply electric or magnetic field to the column. In contrast, temperature can be applied much more easily with the use of the thermostated column oven commonly utilized in GC or LC. Therefore, temperature will be the easiest and the most applicable physical stimulus for HPLC. The polymer of N-isopropylacrylamide (NIPAM) is a known temperature-responsive polymerlOJ1and has been utilized in temperature-responsive hydrogels for novel drug delivery systemsa6Due to the reported temperaturedependent phase transition of poly (N-isopropylacrylamide) (poly-NIPAM),loJ1the polymer dissolvesin water below its phase transition temperature (32 "C), while it becomes water insoluble above the phase transition temperature. This drastic change in the water solubility of the polymer can be explained on the basis of a conformational transformation of the poly-NIPAM chain between the relatively hydrophilic helix form and the hydrophobic random coil form. Porous glass-based packing material and silica gel modified with poly-NIPAM have been used for temperaturedependent pore size control in size exclusion chromatography12 as well as for separation selectivity in reversed-phase liquid chromatography (RPLC) ,13 but the reported methods of preparation of the stationary phases were rather complicated.12 Recently, we invented a very easy preparation of a uniformly sized polymer-based packing material modified with poly-NIPAM.'* The modification was achieved surface ~electively,'~ where polyNIPAM was combined selectively onto either the external or the (10) Fujishige, S. Polym. J 1987,19,297. (11) Fujishige, S.; Kubota, IC;Ando, I.J. Phys. Chem. 1989,93,3311. (12) Gewehr, M.; Nakamura, IC; Ise, N.; Kitano, H. Makromol. Chem. 1992, 193,246. (13) Yamamoto, IC;Kanazawa, H.; Matsushima, Y.; Takai, N.; Okano, T.; Sakurai, Y. Proceedings of the 114th National Meeting of The Pharmaceutical Society of Japan, Tokyo, 1994, p 160. (14) Hosoya, IC;Sawada, E.; Kimata, IC;Araki, T.; Tanaka, N.; Frkchet, J. M. J. Macromolecules 1994,27,3973. (15) Frkchet, J. M. J.; Hosoya, IC US.Patent 5,306,561, 1994.
Analytical Chemistry, Vol. 67, No. 11, June 1, 1995 1907
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Figure 1. Preparation of uniformly sized macroporous polymer packing materials modified with poly-NIPAM.
internal surface of the macroporous uniformly sized base packing material. In this paper, we report temperature-dependent chromatographic separation selectivity in reversed-phase liquid chromatography using the temperature-responsive polymer-based packing material. EXPERIMENTAL SECTION
Packing Material. Uniformly sized temperature-responsive polymer-based packing materials were prepared according to the previously reported method, namely, in situ surface-selective m~dification,'~ as described below. Uniformly sized polystyrene seed particles were prepared by an emulsifier-freeemulsion polymerization and purified according to the previously reported method.16 The seed particle was -1 pm in diameter. Preparation of uniformly sized macroporous polymer particles by a two-step swelling and polymerization method was carried out as follows. As shown in Figure 1,the 1.4 mL water dispersion of the uniformly sized polystyrene seed particles (9.5 x lop2 g/mL) was admixed with a microemulsion prepared from 0.95 mL of dibutyl phthalate, activating 0.085 g of benzoyl peroxide, 0.04 g of sodium dodecyl sulfate, and 10 mL of distilled water by sonication. This first swelling step was carried out at room temperature with stirring at 125 rpm. Completion of the swelling step was determined by observing the vanishing point of the oil droplets in the added microemulsion through an optical microscope. A dispersion of 9 mL of ethylene dimethacrylate and 10 mL of porogenic solvent such as toluene or cyclohexanol in 90 mL of water containing 1.92 g of poly(viny1 alcohol) (dp = 500, saponification value = 86.5-89 mol %) as dispersion stabilizer was added to the dispersion of swollen particles. The second swelling step was carried out at room temperature for 2 h with stirring at 125 rpm. After the second swelling step was completed, the polymerization procedure was started at 80 "C under argon atmosphere (16) Smigol, V.; Svec, F.; Hosoya, K.; Wang, Q.; Frechet, J. M. J. Angew. Mukromol. Chem. 1992,195, 151. (17) Ugelstad, J.; Kaggerud, K. H.; Hansen, F. K.; Berge, A.Mukromol. Chem. 1979,180, 737. 1908 Analytical Chemistry, Vol. 67, No. 11, June
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with slow stirring. After 4 h, 1 g of N-isopropylacrylamide (NIPAM) and 0.02 g of potassium peroxodisulfate were added directly to the aqueous polymerization medium. After an additional 20 h, the dispersion of polymerized particles was poured into 250 mL of cold water to remove the unbound poly-NIPAM, and the supernatant was discarded after sedimentation of the particles. The polymer particles were redispersed into methanol, and the supernatant was discarded after sedimentation. This procedure was repeated three times in methanol and twice in tetrahydrofuran, after which the polymer particles were filtered on a membrane filter, washed with THF and acetone, and then dried at room temperature to determine yields. When cyclohexanol is utilized as the porogenic solvent, the added NIPAM and poly-NIPAM dissolve in the cyclohexanol phase, which fills pores of the partly polymerized base particles to afford a packing material modified on the internal surface. On the contrary, the external surface of a packing material is modfied with poly-NIPAM when toluene is used as the porogenic solvent, because poly-NIPAM does not dissolve into toluene phase which fills pores of the base particle^.'^ Unmodified macroporous particles (base particles) were also prepared according to the same procedure but without the addition of NIPAM. The calculated yield based on the amount of the monomers used was 94-100%. The prepared particles were packed into a stainless steel column (4.6 mm i.d. x 150 mm) by a slurry technique, using a mixture of glycerol and water (8:2 v/v) as a slurry medium, to evaluate their chromatographic characteristics. Chromatography. All the chromatographic solvents were purchased from Nacalai Tesque and used as received. HPLC was performed with a Jasco 880-PU intelligent HPLC pump equipped with a Rheodyne 7125 valve loop injector and a Waters Model 440 W detector set at 254 nm. Chromatographywas carried out at 30 f 0.5 or 50 f 0.5 "C, and a Shimadzu GR4A recorder was utilized. RESULTS AND DISCUSSIONS
Separation Selectivityin Reversed-PhaseMode. According to the reported temperature-responsiveconformational transi-
tion of the poly-NIPAM chain,I2 hydrated amide groups of the polymer chain are exposed as apparent hydrophilic external functional groups of the helix chain below the phase transition temperature of poly-NIPAM (32 "C), which allows it to dissolve in water. In contrast, above the phase transition temperature, relatively hydrophobic isopropyl substituents and/or the polymer backbone begin to dominate the hydrophobic characteristics of the random coil polymer chain, which changes the hydrophilic water-soluble polymer into water-insoluble polymer. This phase transition of the poly-NIPAM chain between hydrophilic helix and hydrophobic random coil affords a change in the hydrodynamicvolume of the polymer chain. Therefore, in a previous paper,14in order to prove surfaceselective modification with poly-NIPAM, we demonstrated temperature-responsive control of pore size of the prepared uniformly sized polymer-based packing materials modified with poly-NIPAM on the internal or the external surface. In fact, the packing material having the modified internal surface tended to show expansion of the pore size above the phase transition temperature due to shrinkage of the poly-NIPAM chain, while reduction of the pore size was found with the packing material having the modified external surface. Besides the pore size control, this phase transition of the combined polymer chain will presumably lead to a change in the separation selectivity of the stationary phase toward low molecular weight solutes as well as large molecules, because the temperature-responsive transformation of the polymer is caused by the change of the apparent exposed functional groups of the polymer between the hydrophilic amide group and the hydrophobic alkyl substituents, as described above. If it works, on raising the temperature over the phase transition temperature,the hydrophilic amide type stationary phase should be turned into the relatively hydrophobic alkyl type stationaryphase that must have a different separation selectivity. Figure 2 shows a comparison of the separation selectivity in an aqueous acetonitrile mobile phase on changing the column temperature. Since all the plots for the unmodified packing material were placed on the dotted straight line, where retention factors (k') were shorter at higher temperatures, no change in the separation selectivity was obtained with the unmodified packing material. On the other hand, the packing material having the modified internal surface showed a greater affinity for hydrophilic solutes at the lower temperature (30 "C)compared with those at 50 "C as well as a different separation selectivity from that for the unmodified base packing material (see solute 8). This separation selectivity change can be explained on the basis of the temperature-responsive transformation of the characteristics of apparent exposed functional groups of the polyNIPAM selector, because below the phase transition temperature, amide groups are exposed as the apparent functional group of the polymer chain, which preferentially retain relatively hydrophilic solutes, such as acetanilide, acetophenone, methyl benzoate, or cyanobenzene, through interactions between hydrophilic functional groups (e.g., amide-amide interaction). These observations suggest that the separation selectivity of the modified packing material changes above-and below the phase transition temperature. Separation Selectivity in Drug Separation. Another example of a change in separation selectivity with the packing
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material modified on the internal surface can be seen in drug separation, as shown in Figure 3. Three drugs were well separated with the unmodified base packing material at 30 "C, while at 50 "C, barbital and tolbutamide could not be separated due to their peak overlapping resulting from the shorter retention time at 50 "C. On the other hand, with the packing material having the modified internal surface, separation selectivity of the three drugs was found to be different from that with the unmodified base packing material, and barbital and furosemide could not be separated at 30 "C, as shown in Figure 4. However, on raising the column temperature to 50 "C, the three drugs were well separated within 7 min, a shorter analysis time than that with the unmodified base packing material. The faster analysis observed is very important as it saves time and mobile phase. These Analytical Chemistty, Vol. 67,No. 11, June 1, 1995
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findings strongly suggest that the packing material modified with poly-NIPAM on the internal surface can be used as a stationary phase for temperature-controlled chromatography. In contrast, the packing material modified with poly-NIPAM on the external surface did not show any daerence from the unmodified packing material in separation selectivity toward low molecular weight solutes. A similar result was also obtained in the temperature-responsive pore size ~0ntrol.l~ However, in the case of separating drugs coexisting with a polypeptide such as BSA, the packing material having the modhed external surface shows its possible application. With the unmodified packing material and the packing material modified on the internal surface, in high molecular weight solutes, polypeptide such as BSA was adsorbed due to hydrophobicity of the external surface of the packing materials and could not be eluted before the void volume of the column. On the other hand, BSA was completely excluded with the packing material modified on the external surface with poly-NIPAM and eluted before the void volume of the column used at 30 "C, as shown in Figure 5. Since the unmoditied base packing material could not exclude the polypeptide completely, these findings support the conclusion that the poly-NIPAM selector below the phase transition temper1910 Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
ature is hydrophilic enough to cause elution of polypeptide without hydrophobic adsorption. From those observations, this packing material modified with poly-NIPAM selector on the external surface is applicable as an internal surface reversed-phase type packing material.18 Interestingly, the amount of the excluded polypeptide was drastically decreased on raising the column temperature to 50 "C due to the temperature-responsive transition of hydrophobicity of the external poly-NIPAM layer. Thermal rearrangement of BSA was negligible under the chromatographic conditions employed, and the observed absorption change of polypeptide was completely reversible,Ig which allowed repeated use of the column. The observed phenomenon means that, with the packing material modified on the external surface, control of column temperature can shift the elution region of the polypeptide, which is usually very large and sometime overlaps with the peaks of drug solutes having short retention times. In this case, two drugs used were well separated on the basis of hydrophobic interaction with the unmodified internal surface. (18) Hagestem, I. H.; Pinkerton, T. C. Anal. Chem. 1985, 57, 1757. (19) Hofstee. B. H. J Prep. Biochem. 1975,5, 7 .
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Figure 5. Separation of drugs coexisting with polypeptide with the packing material modified with poly-NIPAM on the external surface. Mobile phase, 10% aqueous acetonitrile-0.02 M phosphate buffer containing 0.1 M sodium sulfate (pH = 7); flow rate, 0.8mumin; detection, UV, 254 nm. Solutes: 1, theophylline (1 mg/mL, 0.3 pL); 2, barbital (1 mg/mL, 6 pL); 3, bovine serum albumin (BSA) (20 mg/mL, 5 pL).
CONCLUSION The uniformly sized packing material surfaceselectively modified with poly-NIPAM was easily prepared and afforded temperature-responsivecontrol of chromatographic separation selectivity among low molecular weight molecules involving drugs as well as larger molecules such as polypeptides. The observed thermal control of separation selectivity was found to be very useful because we may get a different separation selectivity by simply changing column temperature instead of changing columns and mobile phase, which is a time-consuming process. By choosing a packing material having a modified internal surface or a modified external surface, we can select different types of temperature controlled chromatography. In this way, the reported method will allow control of separation selectivity by applying external physical stimuli.
ACKNOWLEDGMENT Partial support from the Japanese Ministry of Education (Grant No. 05740447) is acknowledged with thanks. SUPPLEMENTARY MATERIAL AVAILABLE Additional figure dealing with elution of BSA at 30 and 50 "C (1 page). See any current masthead page for ordering infromation. Received for review November 8, February 18, 1995.@
1994.
Accepted
AC9410880 @
Abstract published in Aduance ACS Abstracts, April 1, 1995.
Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
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