Facilitation of Applicability in Ice Chromatography by Mechanistic

Anal. Chem. 2009, 81, 890–897

Facilitation of Applicability in Ice Chromatography by Mechanistic Considerations and by Preparation of Fine Water-Ice Stationary Phase Yuiko Tasaki and Tetsuo Okada* Department of Chemistry, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan We have, in previous papers, demonstrated that some solute is adsorbed on the ice surface at temperatures below -5 °C by the hydrogen-bond formation between the polar groups in the solute and the dangling bonds on the water-ice surface. However, the separation efficiency of this method, named ice chromatography, was seriously restricted principally due to the large sizes of water-ice particles used as a stationary phase. We have devised a convenient method to prepare finer ice particles with diameters of ∼10 µm. This stationary phase has provided much larger theoretical plate numbers (N ) 1500) than that prepared in the previous way (N ) 250) and has allowed various applications such as separation of amino acid derivatives, poly(oxyethylene) oligomers, and estrogen. The improved separation performance allows us to discuss retention mechanisms in more detail and to see subtle differences in the retentivity between solutes. In analytical chemistry, liquid water has been extensively used as a medium for basic reactions, for example, acid-base equilibria, complex formation, redox reactions, etc. Also, an aqueous solution usually comprises one of two phases for solvent extraction. Chromatographic and electrophoretic experiments may also be carried out with aqueous solutions. Most of the fundamental work has thus been done with usual liquid water because it is easy to deal with, it dissolves many substances, it is nontoxic and nonhazardous, it is ubiquitous and inexpensive, etc. Different facets of water, which emerge under nonambient conditions, are also of fundamental and practical interest. Changing a temperature is a good example. At high temperatures, water shows the properties comparable to some organic solvents, and therefore, superheated water has been employed as a chromatographic mobile phase in place of organic solvents.1,2 The corrosive properties of supercritical water have been utilized to the decomposition of hazardous materials.3 Although water is no longer used as a solvent below its freezing point, water-ice is also an attractive material, which deserves fundamental and applicable * To whom correspondence should be addressed. Phone and Fax: +81-35734-2612. E-mail: [email protected]. (1) Smith, M. R. J. Chromatogr., A 2008, 1184, 441. (2) Vanhoenacker, G.; Sandra, P. J. Sep. Sci. 2006, 29, 1822. (3) Matsumura, Y.; Minowa, T.; Potic, B.; Kersten, S. R. A.; Prins, W.; van Swaaij, W. P. M.; van de Beld, B.; Elliott, D. C.; Neuenschwander, G. G.; Kruse, A.; Antal, M. J., Jr. Biomass Bioenergy 2005, 29, 269.

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studies.4 Liquid water dissolves many substances, and similarly, water-ice adsorbs various compounds on the surface. It is known that adsorption on water-ice plays important roles in a number of natural processes. For example, the adsorption of antifreeze proteins on the ice surface prevents the development of ice crystals in biological cells; thereby, a number of organisms are alive, e.g., even in the Antarctic Sea, where temperatures are often lower than the freezing point of water.5-10 The formation of the radical species causing the depletion of the ozone layer in the stratosphere is initiated by the adsorption of HCl on ice particles.11 Water-ice is thus an adsorbent of potential use. We have focused on waterice as a solid adsorbent from these perspectives and have shown that it is suitable for a chromatographic stationary phase.12-14 Handling of the ice stationary phase is less convenient than usual chromatographic stationary phases, because low temperatures should always be maintained and utilizable mobile phases are also limited. However, water-ice has various physicochemical features of fundamental interest that have not been elucidated so far or are even unknown. Ice chromatography has high potential to reveal such aspects; this is an optional advantage of this method. Of interest is, for example, whether the surface of water-ice is a real solid or liquid-like. Although various surface-selective methods have been applied to the water-ice surface to elucidate this issue, there is still a debate on its physical and physicochemical nature.15-19 In previous work, we revealed some surface processes involved in ice chromatography.12-14 A solute is adsorbed on the water-ice surface by the hydrogen bonding with the dangling bonds at the temperatures of < -5 °C as illustrated in Figure 1 (the ice surface is solid-like). In contrast, when the temperature (4) Petrenko, V. F.; Whitworth, R. W. Physics of Ice; Oxford University Press: Oxford, U.K., 1999. (5) Strom, C. S.; Liu, Y. X.; Jia, Z. Biophys. J. 2005, 89, 2618. (6) Knight, A. C.; Wierzbicki, A.; Laursen, A. R.; Zhang, W. Cryst. Growth Des. 2001, 1, 429. (7) Knight, A. C.; Wierzbicki, A. Cryst. Growth Des. 2001, 1, 439. (8) Mao, Y.; Ba, Y. J. Chem. Phys. 2006, 125, 091102. (9) Du, N.; Liu, Y. X.; Hew, L. C. J. Biol. Chem. 2003, 278, 36000. (10) Dalal, P.; Knickelbein, J.; Haymet, D. J. A.; Sonnichsen, D. F.; Madura, D. J. PhysChemComm 2001, 7, 1. (11) Parent, P.; Laffon, C. J. Phys. Chem. B 2005, 109, 1547. (12) Tasaki, Y.; Okada, T. Anal. Chem. 2006, 78, 4155. (13) Tasaki, Y.; Okada, T. J. Chromatogr., A 2008, 1189, 72. (14) Tasaki, Y.; Okada, T. J. Phys. Chem. C 2008, 112, 2618. (15) Dash, G. J.; Rempel, W. A.; Werrlaufer, S. J. Rev. Mod. Phys. 2006, 78, 696. (16) Ikeda-Fukazawa, T.; Kawamura, K. Chem. Phys. Lett. 2006, 417, 561. (17) Ewing, E. G. J. Phys. Chem. B 2004, 108, 15953. (18) Doppensemidt, A.; Butt, J. H. Langmuir 2000, 16, 6709. (19) Pittenger, G.; Fain, C. S. Phys. Rev. B 2001, 63, 134102. 10.1021/ac802229t CCC: $40.75  2009 American Chemical Society Published on Web 01/08/2009

Figure 1. Schematic illustration of the basal plane of water-ice (0001) and of possible interactions of polyether (left) and resorcinol (right) with the dangling bonds, which are drawn by black spheres.

of water-ice is raised higher than a particular threshold point (in most cases at a temperature of > -5 °C), the partition of a solute into the quasi-liquid layer at the interface between water-ice and a mobile phase becomes a dominant retention mechanism (the surface is liquid-like). The threshold temperature depends on the type and property of a polar component added in the mobile phase.23 Ice chromatography is thus an efficient probe of the water-ice surface. In a series of our previous work, the ice stationary phase was prepared by freezing water droplets produced with a nebulizer.12-14 The typical diameters of the water-ice particles were 50 to 75 µm. Although this preparation has allowed reasonably reproducible ice chromatographic measurements, the relatively large sizes of the stationary phase often resulted in low retentivity of solutes and limited separation performance. These problems can be resolved by the preparation of finer water-ice particles. High separation performance should allow more detailed discussions on ice chromatographic retention mechanisms. EXPERIMENTAL SECTION The chromatographic system was composed of a Tosoh highperformance liquid chromatography (HPLC) pump model DP8020, a Rheodyne injection valve equipped with a 100 µL sample loop, a Shimadzu multichannel photodiode array detector model SPD-M10AVP or a JASCO fluorescence detector model FP-2020 Plus, and a Thermo Scientific low-temperature bath model HAAKE Phoenix II P1-C41P. The surface tension of a solution was determined by a pendant drop method with homemade apparatus. Water-ice particles were prepared by introducing water droplets into liquid nitrogen. Water droplets were produced by two methods, i.e., with a glass-made nebulizer and with an ultrasound humidifier. The preparation of the water-ice particles with the nebulizer was the same as that previously reported.12 Ice particles with the sizes smaller than 75 µm were collected by sieving and were put into a PEEK column (7.5 mm i.d. × 150 mm length) immersed in liquid nitrogen and lightly tapped down with a PTFE rod. This stationary phase was used mainly for fundamental studies. An ultrasound humidifier allowed the preparation of finer water-ice particles (the diameters of ∼10 µm, see below). The stationary phase was packed according to the procedure illustrated

Figure 2. Column packing procedure.

in Figure 2. The ice particles dispersed in liquid nitrogen were poured into a stainless chromatographic packer (volume ) 40 cm3) connected to the PEEK column (the same as the one stated above), and then the entrance of the packer was closed with the other end of the column kept open. Keeping the column and packer in the air allowed the vaporization of liquid nitrogen, which moderately raised the pressure in the packer. The expanded nitrogen was released from the open end of the column and pushed the ice particles down the column. A salt solution was also used for ice stationary phase preparation to study an effect of an added salt on the shape and size distribution of ice particles. The stationary phase prepared with the ultrasonic humidifier was used to study separation efficiency of ice chromatography. The sizes of ice particles were measured on micrographs; more than 200 particles on the focal plane of a micrograph were randomly selected, and their sizes were measured. The packed column was immersed in the low-temperature bath maintained at -10.0 °C unless otherwise stated. However, temperature effects were negligible in the range of -5 to -10.0 °C. An operation temperature was measured just outside of the Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

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Chart 1. Structures of Solute Molecules

column with a digital thermometer equipped with Pt-100 thermoresister; the temperature fluctuation was smaller than 0.1 °C. The mobile phase was precooled by passing through a coiled tube connected between the pump and the sample injector. A silica gel column (GL-PACK LiChrosorb SI60 5 µm, GL Science) was used for comparison of solute retention with that in ice chromatography. Hexane containing tetrahydrofuran (THF) or diethyl ether (DEE) as a polar modifier was used as a mobile phase. The water content in the mobile phase was lower than the detection limit of the Karl Fischer titration. The concentrations of solutes were 10-100 µM. Benzo-18crown-6 (B18C6) and dibenzo-24-crown-8 (DB24C8) were synthesized according to the literature.20 Monodispersed poly(oxyethylene(p)nonylphenyl ethers) (p; the number of repeating oxyethylene units), POE(4)NPE, POE(5)NPE, and POE(6)NPE, were isolated from a polydispersed material (the average number of repeating oxyethylene units was 5) by HPLC and were assigned by mass spectrometry. o-Phthalaldehyde (OPA) derivatives of amino acid (20) Pedersen, C. J. J. Am. Chem. Soc. 1967, 89, 7017.

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esters (methyl ester of Ala, Val, Phe, Ser, Cys, and His, and ethyl ester of Gly and Tyr) were prepared with mercaptoethanol or hexanethiol according to the literature.21,22 The products prepared in an aqueous solution were transferred into acetic acid. An aliquot of the acetic acid solution was then added to hexane and was used as a sample. The fluorometric detection of these compounds were carried out at λex ) 330 nm and λem ) 400 nm. The structures of solute compounds are summarized in Chart 1. Well-packed columns were selected in terms of peak shapes; the columns giving skewed or split peaks were not used. Nevertheless, unlike usual HPLC stationary phases, the repeatability of the retention time was not very high mainly because of the difficulty in column preparation. Since retention factors were much more reproducible than retention times (the ratio of the surface to the interstitial volume was constant), discussions were based on the former. As shown below, though solute retention can be measured over 24 h on the same column, retention data measured with freshly prepared columns are reported to avoid (21) Turiak, G.; Volicer, L. J. Chromatogr., A 1994, 668, 323. (22) Donzanti, A. B.; Yamamoto, K. B. Life Sci. 1988, 43, 913.

Table 1. Number of Interacting Points (n) Determined by Eq 1a compounds

n

DEE concn range/% (v/v)

hydroquinone resorcinol POE(4)NPE POE(5)NPE POE(6)NPE B18C6 DB24C8 OPA-Gly-ME OPA-Ser-ME

2.06 2.17 1.56 2.30 2.37 1.77 2.61 1.54 2.87

4-11 4-11 5-12 5-12 6-13 4-11 7-14 3-9 17-22

a

Figure 3. log k-log[DEE] plots for resorcinol and hydroquinone.

effects of column degradation when the retention mechanism is discussed. The retention data were measured at least five times with different columns under the same conditions. In addition, the peak width much more strongly depended on the column conditions. Therefore, the best values for the theoretical plate numbers are reported. RESULTS AND DISCUSSION Separation Selectivity of the Water-Ice Stationary Phase. We indicated that a polar component (e.g., THF, DEE, and dodecanol) added to the hexane-based mobile phase competes with solutes for the adsorption sites on the water-ice surface and also that hydrogen bonds are dominantly formed between a solute and the -OH dangling bonds. Thus, the hydrogen-bond acceptors can be retained on a water-ice stationary phase. In actuality, retention has been confirmed for a compound with an -OH, -NH2, -O-, or -CO- group in a part of the structure; above all, a hydroxyl compound is strongly retained. The adsorption model on the -OH sites on the ice surface has allowed the derivation of eq 1, which represents the retention factor (k) as a function of several parameters:12,13

k)

t n Ks AΓOH

V(1 + KPC[PC])n

(1)

where KPC and KS are the adsorption constants for a polar component (PC) added to the mobile phase and a solute on the ice surface, respectively, n is the number of the -OH sites required for the solute retention (alternatively, the number of hydrogen bonds simultaneously formed between the solute and the water-ice surface), A is the surface area of the stationary t phase, V is the void volume of the column, and ΓOH is the total surface concentration of the OH sites. When KPC[PC] > 1, the logarithmic form of eq 1 implies that the log k-log[PC] plot is linear and its slope is equal to the negative value of n. Figure 3 shows the log k-log[PC] plots for resorcinol and hydroquinone. Table 1 lists n for several solute compounds with DEE as a polar additive. For hydroquinone and resorcinol, n is almost equal to 2; this possibly validates the present model, because two polar (23) Balcan, B.; Anghel, D.-F. Colloids Surf., A 2003, 221, 1.

The number of measurement points was 6-8.

groups are contained in each solute molecule. For B18C6 and DB24C8, n is equal to 1.77 and 2.61, respectively, indicating that all of the ethereal oxygen atoms do not participate in the formation of hydrogen bonds with the water-ice surface and that 2-3 hydrogen bonds are formed at a time. A solute with one polar group in a molecule showed very weak or no retention (results are not shown). Simultaneous hydrogen-bond formation is thus required for measurable retention. The retention ability is, in general, enhanced with increasing number of polar groups contained in a solute molecule due to a slight increase in n and possibly to entropic effects. Hence, two or more hydrogen-bond acceptor groups should be contained in a molecule for a solute to give ice chromatographic retention. Poly(oxyethylene) (POE) is suitable for systematically studying an effect of the number of ethereal oxygens on ice chromatographic retention because compounds with various POE chain lengths are available. Also, since POE is usually used in a polydispersed form in nonionic or anionic detergents, separation in terms of the chain length of POE has been an important task in surfactant industries and for assessing their environmental influences.23 Ice chromatography is capable of separating POE oligomers as shown in Figure 4. Their retention increases with increasing number of ethereal oxygen atoms in molecules. The n values for POE(p)NPE also increase with increasing p as listed in Table 1; the corresponding value for POE(3)NPE was not determined because of its too weak retentivity. Similar to ice chromatography of crown ethers, all of the ethereal groups in a solute molecule do not interact simultaneously with the dangling

Figure 4. Separation of POE(p)NPE, p ) 3, 4, and 5. Mobile phase, 3% DEE in hexane. Detection, UV (275 nm). Temperature, -5.0 °C. Column, 7.5 mm i.d. × 150 mm packed with ∼75 µm ice particles as a stationary phase. Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

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Table 2. Comparison of Ice Chromatographic Retention Factors of Methoxyphenols and ω-(4-Hydroxyphenyl)-1-alkanols with Those with Silica Gel Chromatography concn THF in hexane

o-MP

m-MP

p-MP

ice silicagel

0 10%(v/v)

0 (0) 1.23 (0.65)

1.80 (1.00) 1.91 (1.00)

1.64 (0.91) 2.12 (1.11)

ice silicagel

Figure 5. Separation of estrogen. Mobile phase, 5% DEE in hexane. Detection, UV (275 nm). Temperature, -10 °C. Column, 7.5 mm i.d. × 150 mm packed with ∼75 µm ice particles as a stationary phase.

bonds on the water-ice surface. Unfortunately, isocratic elution cannot resolve oligomers in a wider range of the POE chain length because of a large difference in retentivity between POE(p)NPE and POE(p+1)NPE. Estrogen is the name of a group of human hormones having the common multiring structure. Estron, estradiol, and estriol are the members of estrogen. Estron has one hydroxyl group and carbonyl group in a molecule. Estradiol and estriol have two and three hydroxyl groups in individual molecules, respectively. From their structures, the ice chromatographic retention was expected to increase in the order of estron, estradiol, and estriol. Figure 5 shows ice chromatographic separation of estron and estradiol. Unfortunately, estriol was so insoluble in a hexane-based solvent that its ice chromatographic behavior was not studied. However, its retention should be stronger than the others. Although a carbonyl group contributes to solute retention to some extent, its affinity to the water-ice surface is much weaker than that of a hydroxyl group. The separation factor between estron and estradiol is ca. 5 under the experimental condition given in Figure 4; this reflects a difference in the affinity to the ice surface between a hydroxyl group and a carbonyl group. The corresponding value obtained with a silica gel column was 2.3 though the separation condition was different from that for ice chromatography. A waterice stationary phase is likely to recognize the number of polar groups more strongly than a usual stationary phase. As noted above, the ice stationary phase differs in separation selectivity from silica gel, albeit hydrogen bonding is a dominant molecular interaction for both stationary phases. Since the molecular processes involved in either system have not been completely elucidated and experimental conditions are also different, it is insignificant to say too much about the differences. Nevertheless, phenomenological comparisons for some solutes are of use to discuss the characteristics of the ice stationary phase. Table 2 compares the ice chromatographic retention factors of methoxyphenols and ω-(p-hydroxyphenyl)-alkanols with those obtained with silica gel chromatography. Interestingly, the ice chromatographic elution orders are quite different from those with silica gel. For silica gel, solute retention becomes stronger with increasing distance between two polar groups in solutes of both 894


k (relative k)

stationary phase

3%(v/v) 25%(v/v)

HPM

HPE

HPP

2.43 (1.00) 2.89 (1.00)

2.07 (0.85) 3.10 (1.07)

2.26 (0.93) 3.43 (1.19)

homologous series. In contrast, such relation is not necessarily applicable to ice chromatographic retention orders. Silica gel is an inorganic polymeric material, and therefore the surface atoms, in turn the active sites as well, are tightly fixed on particular positions. In contrast, water molecules on the ice surface can rotate even if the temperature is lower than the melting point.16 The movements of the surface molecules may lead to the flexibility of the surface adsorption sites and eventually result in the selectivity different from that of silica gel chromatography. The flexibility of surface water molecules is thought to be related to the formation of a quasi-liquid layer on the water-ice surface.15,16 Enhanced Applicability with Fine Ice Stationary Phase. Ice chromatography is capable of recognizing the number of polar groups in solute molecules and separating some solutes as shown above. However, the separation performance of this method is not very high; the theoretical plate number was not more than 250. Low separation performance of ice chromatography mainly came from a use of large water-ice particles (50 to 75 µm in diameter). This methodological limitation can obviously be overcome by reducing particle sizes. An ultrasonic humidifier has proven an effective alternative to a nebulizer and allows the preparation of fine water-ice particles. Figure 6 shows micrographs of ice particles prepared with an ultrasonic humidifier with different magnifications. A histogram in Figure 6 shows the distribution of the diameters of ice particles prepared in this way. The average diameter of ice particles is 6.24 µm (σ ) 3.34 µm), which is equivalent to that of a usual chromatographic stationary phase. This stationary phase actually allowed an improvement of the theoretical plate number, e.g., N ) 710 for DB24C8. Water-ice doped with an electrolyte possibly brings about further improvements in ice chromatographic separation performance because the surface tension, which is expected to affect the size of water mist, is modified and the surface nature of waterice should be affected by incorporating a salt. On the basis of this idea, several salts including NaCl, NaBr, KCl, and tetrabutylammonium bromide (TBAB) were tested as a dopant. The concentration of the salt was kept lower than 1 mM to avoid effects of a liquid phase coexistent with salt-doped ice on solute retention. Most of the salts tested did not improve separation performance probably due to low concentrations, which do not cause drastic change in the physical properties of water and ice. However, a positive effect was confirmed for TBAB doping; the largest N value for DB24C8 was 1500. Microscope observations and the measurements of particle size distribution have revealed that the improved performance with TBAB-doped ice does not originate from its

Figure 6. Micrographs of ice stationary particles prepared with a humidifier: (a) ×900 and (b) ×400 magnification. The histogram shows the distribution of the diameters of 243 randomly selected ice particles.

narrower size distribution (7.00 µm with σ ) 3.58 µm). There are two possible reasons for this improvement though not understood completely. One is the surface adsorption of TBAB solutions. In actuality, the surface tensions of TBAB solutions were γ ) 71.67 (σrel ) 1.1%), 70.40 (σrel ) 2.2%), and 66.76 (σrel ) 4.7%) for 0.5, 1, and 2 mM TBAB, respectively, relative to γ ) 71.67 for water. Although such a marginal decrease in surface tension does not result in smaller sizes of water mist, TBAB-doped ice must differ in the surface properties from plain ice. The second possible reason is the formation of TBAB hydrate clathrate.24,25 This hydrate, which is stable even above the melting point of water-ice, possibly modifies the surface properties of ice stationary phase. Although the main cause has not been elucidated, a phenomenological difference between plain ice and TBAB-doped ice was found in micrographs. White parts seen in Figure 6 are massive aggregates of ice particles, which interfere with the homogeneous packing of the ice particles into a column. Such aggregation, which more frequently occurs for the plain ice rather than for the TBAB-doped ice, interferes with tight column packing and results in lower theoretical plate numbers. However, the column pressure was very low, typically 0.1 to ∼0.2 MPa, even with the TBAB-doped ice stationary phase. If the column was very well packed with 5-10 µm particles, the pressure drop should be higher than these values by at least 1 order of magnitude. This implies that ice particles are not well packed in a column in comparison with usual HPLC packing materials. The less adherent nature of the TBAB-doped ice thus leads to a slightly better packing condition and results in better separation efficiency. Separation of amino acids gives a good implication of the improved separation efficiency of ice chromatography with TBABdoped ice. Ice chromatographic separation was attempted with TBAB-doped ice for eight amino acid esters, i.e., Gly, Ala, Val, (24) Oyama, H.; Shimada, W.; Ebinuma, T.; Kamata, Y.; Takeya, S.; Uchida, T.; Nagao, J.; Narita, H. Fluid Phase Equilib. 2005, 234, 131. (25) Lipkowski, J.; Komarov, V. Y.; Rodionova, T. V.; Dyadin, Y. A.; Aladko, L. S. J. Supramol. Chem. 2002, 2, 435.

Phe, Cys, Tyr, His, and Ser. Gly, Ala, Val, and Phe have no polar groups in their side chains, whereas Cys, Tyr, His, and Ser have polar groups. Figure 7 shows ice chromatographic separation of the OPA-mercaptoethanol (ME) derivatives of the amino acid esters with step-gradient elution. The first groups, Val, Phe, Ala, and Gly were eluted in this order with 5.4% (v/v) DEE in hexane, and then Cys, Tyr, and Ser were eluted with 26.5% (v/v) DEE in hexane. His was not eluted with these mobile phases but appeared when the DEE concentration was increased up to 50.0% (v/v). Although the same separation was attempted with the plain ice stationary phase, separation between Gly and Ala was not possible. In contrast, separation between these amino acid derivatives was always possible with the TBAB-doped ice stationary phase. Separation between groups is basically explained by the number of polar groups involved in the solutes as discussed above. From the separation shown in Figure 7, some rules can be found in the solute retention on the water-ice stationary phase: (i) the affinity of a phenolic hydroxyl group to the water-ice surface is similar to that of an alcoholic hydroxyl group despite their different acidities, (ii) a thiol group has similar ice affinity to a hydroxyl group, and (iii) an imidazole ring has much higher ice-affinity than a hydroxyl group. It should be noted that water-ice recognizes the structural difference in inert side chains, such as -H, -CH3, -CH(CH3)2, and -CH2-C6H5, which are not involved in the direct interaction with the ice surface. Although the details have not been understood, the elution orders, Val < Phe < Ala < Gly and Cys < Tyr < Ser, are consistent with increasing the partition coefficients of amino acids between water and cyclohexane.26 The enhanced separation performance of ice chromatography thus visualizes subtle differences in the physicochemical characters of solutes at the interface between water-ice and hexane-based solvents. (26) Radzicka, A.; Wolfenden, R. Biochemistry 1988, 27, 1644.


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Figure 7. Separation of OPA derivatives of amino acid esters with step-gradient elution. Mobile phase, 5.4% DEE in hexane (0-23.8 min) f 26.5% DEE in hexane (23.8-40.0 min) f 50.0% DEE in hexane (40.0-50.0 min). Detection, fluorescence (λex ) 330 nm and λem ) 400 nm). Temperature, -10 °C. Column, 7.5 mm i.d. × 150 mm packed with fine ice particles prepared from an aqueous 1 mM TBAB solution. The terminal hydroxyl group coming from mercaptoethanol enhanced the retentivity of amino acid derivatives.

Figure 8. Changes in k for amino acid derivatives with repeated injection on an ice column.

The OPA derivatization also plays an important role in the determination of the ice chromatographic retention of amino acids, i.e., an additional polar group was introduced during the OPA derivatization with the aid of ME. To reveal this effect, the OPA derivatives of Gly and Ser were synthesized with hexanethiol (HT), and their retentivity on the ice stationary phase was studied. In contrast to the OPA derivatives with ME, those with HT showed very weak retention. OPA-Ser-ME gave k ) 9.8 with 17% DEE in hexane, whereas k for OPA-Ser-HT was 0.21 with 6% (v/v) DEE in hexane. In addition, the retention of OPA-Gly-HT was not confirmed. Thus, the introduction of an additional hydroxyl group effectively enhances the affinity of a solute to the ice stationary phase. As listed in Table 1, the n values for OPA-Ser-ME and OPA-Gly-ME were 2.87 and 1.54, respectively, suggesting that different numbers of polar groups act for the retention of the OPA derivatives. These results suggest that carboxate and 896


isoindole groups involved in the derivatives play auxiliary roles in the adsorption on the water-ice surface. Repeatability and Column Durability. Finally, we would like to mention the repeatability of ice chromatographic separation. Figure 8 shows changes in k with time. The retention factors for the OPA derivatives of Ala, Gly, Phe, and Val were measured by injecting the sample four times every 20 min. Although small deviations can be seen, the retention is relatively stable. The relative standard deviations of retention times were 3.47%, 3.29%, 3.25%, and 6.48% (n ) 4), respectively. The retention of the other amino acids was so strong that the mobile phase should contain high concentrations of a polar component. This caused damage on the ice stationary phase because of the interaction of the polar component in the mobile phase with water-ice. For long-term experiments, the concentration of a polar component in the mobile phase should be kept as low as possible. For this reason, the

repeatability of the other amino acids was not studied. In addition, since the OPA derivatives of amino acids are not stable, the repeatability over the longer time period was not tested. The retention of hydroquinone was studied instead of the amino acid derivatives over 1.5 days; k increased by ca. 4% (21 h later) and by ca. 10% (36 h later). However, the column durability is not very high mainly due to sintering. In general, a column can be used in a day, though depending on a mobile phase composition. CONCLUSION A simple preparation of fine ice particles has allow us to work with higher separation efficiency and to reveal various aspects involved in ice chromatographic retention. Although the present study has opened up the possibilities of ice chromatography in various applications, there remain a number of problems in this method. First, experimental conditions are severely restricted, i.e., temperature should be kept lower than the freezing point of water, a mobile phase that dissolves water-ice cannot be employed, and

sintering of ice particles prevents a long-term use of a separation column. However, a cost for the preparation of an ice column is extremely low, and, in addition, that required for waste treatments is negligibly low. If we can add further functionalities on the ice stationary phase, methodological advantages of ice chromatography can further be emphasized. Such attempts are in progress in our laboratory and will be reported in the near future. ACKNOWLEDGMENT This work was in part supported by Grants-in-Aid for Scientific Research (18205011), for Exploratory Research (19655024), and for JSPS Fellows (19010374) from the Ministry of Education, Culture, Sports, Science and Technology, and from the Japan Society for the Promotion of Science. Received for review July 1, 2008. Accepted December 13, 2008. AC802229T


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Facilitation of Applicability in Ice Chromatography by Mechanistic

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