Anal. Chem. 2007, 79, 1243-1250
Hydrophilic Interaction Chromatography Using Methacrylate-Based Monolithic Capillary Column for the Separation of Polar Analytes Zhengjin Jiang,†,‡,§ Norman W. Smith,*,† Paul D. Ferguson,‡ and Mark R. Taylor§
Pharmaceutical Sciences Research Division, King’s College London, London, SE1 9NH, UK, and Analytical Research & Development and Structural and Separation Sciences Group, Pfizer Global R & D, Sandwich, Kent, CT13 9NJ, UK
A porous zwitterionic monolith was prepared by thermal copolymerization of N,N-dimethyl-N-methacryloxyethylN-(3-sulfopropyl)ammoniumbetaineandethylenedimethacrylate inside a 100-µm-i.d. capillary. The resulting monolith was evaluated as a hydrophilic liquid chromatography (HILIC) stationary phase. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical HILIC mechanism was observed at higher organic solvent content (ACN% > 60%). The poly(SPE-co-EDMA) monolith showed very good selectivity for neutral, basic, and acidic polar analytes. For charged analytes, both hydrophilic interactions and electrostatic interactions contributed to their retention, which provide chromatographers more choice to optimize the separations. Reversed-phase liquid chromatography (RPLC) is the most often used and powerful separation mode because of its versatility and ability to retain and resolve a number of different types of compounds. However, for polar analytes, highly aqueous mobile phases are often required in order to achieve adequate retention, which can cause a number of issues such as stationary-phase collapse1 and decreased sensitivity in electrospray ionization mass spectrometry.2,3 Normal-phase liquid chromatography is a useful separation technique for providing effective retention for polar molecules,4,5 but the poor solubility of polar analytes in nonaqueous mobile phases, together with lower peak efficiency, reduced selection of stationary phases, and decreased reproducibility, has extremely limited its application. Hydrophilic interaction chromatography (HILIC), which was first named and investigated systematically by Alpert,6 is a useful * Corresponding author. Tel: +44-20-78483944. Fax: +44-20-78484462. E-mail:
[email protected]. † King’s College London. ‡ Analytical Research & Development, Pfizer Global R & D. § Structural and Separation Sciences Group, Pfizer Global R & D. (1) Reid, T. S.; Henry, R. A. Am. Lab. 1999, (July), 24-28. (2) Voyksner, R.D. In Electrospray Ionization Mass Spectrometry; Cole, R. B., Ed.; John Wiley & Sons: Hoboken, NJ, 1997; pp 323-324. (3) Weng, N.; Shou, W.; Chen, Y. L.; Jiang, X. J. Chromatogr., B 2001, 754, 387-399. (4) Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. Practical HPLC Method Development; John Wiley & Sons: New York, 1988; pp 114-120. (5) Synder, L. R.; Kirkland, J. J. Introduction to modern liquid chromatography, 2nd ed.; Wiley: Boca Raton, FL, 1979; Chapter 8. (6) Alpert, A. J. J. Chromatogr. 1990, 499, 177-196. 10.1021/ac061871f CCC: $37.00 Published on Web 12/20/2006
© 2007 American Chemical Society
alternative and rival technique to RPLC for separating polar compounds. It has been successfully used for carbohydrates,7-9 peptides,6,10-12 proteins,13 natural product extracts,14 polar pharmaceuticals,15,16 and some small polar analytes.17 Normally, HILIC is run using polar stationary phases and a high-organic, lowaqueous mobile phase in order to achieve retention of very polar compounds that could not be retained using reversed-phase methods.6,9,11,18,19. Silica,3,14,20,21 amino,12,14,22-24 diol,15,25 polyhydroxyethyl aspartamide, and cyclodextrin-based packings6,14 are most often used as HILIC stationary phases. More recently, commercial HILIC columns, such as ZIC-HILIC and ZIC-pHILIC, have also been developed by SeQuant.17,26-28 Both of these stationary phases, first developed by Irgum et al.,29-31 have highly (7) Alpert, A. J.; Shukla, M.; Shukla, A. K.; Zieske, L. R.; Yuen, S. W.; Ferguson, M. A. J.; Mehlert, A.; Pauly, M.; Orlando, R. J. Chromatogr., A 1994, 676, 191-202. (8) Churms, S. C. J. Chromatogr., A 1996, 720, 75-91. (9) Churms, S. C. In Carbohydrate analysis: high performance liquid chromatography and capillary electrophoresis; El Rassi, Z., Ed.; Journal of Chromatography Library 58; Elsevier: New York, 1995; Chapter 3. (10) Yoshida, T. J. Biochem. Biophys. Methods 2004, 60, 265-280. (11) Yoshida, T. Anal. Chem. 1997, 69, 3038-3043. (12) Oyler, A. R.; Armstrong, B. L.; Cha, J. Y.; Zhou, M. X.; Yang, Q.; Robinson, R. I.; Dunphy, R.; Burnisky, D. J. J. Chromatogr., A 1996, 724, 378-383. (13) Linder, H.; Sarg, B.; Meraner, C.; Wilfried, H. J. Chromatogr., A 1996, 743, 137-144. (14) Strege, M. A. Anal. Chem. 1998, 70, 2439-2445. (15) Olsen, B. A. J. Chromatogr., A 2001, 913, 113-122. (16) Strege, M. A.; Stevenson, S.; Lawrence, S. M. Anal. Chem. 2000, 72, 46294633. (17) Guo, Y.; Gaiki, S. J. Chromatogr., A 2005, 1074, 71-80. (18) Zhu, B. Y.; Mant, C. T.; Hodges, R. S. J. Chromatogr. 1991, 548, 13-24. (19) Hemstro ¨m, P.; Irgum, K. J. Sep. Sci. 2006, 29, 1784-1821. (20) Grumbach, E. S.; Wagrowski-Diehl, D. M.; Mazzeo, J. R.; Alden, B.; Iraneta, P. C. LC-GC N. Am. 2004, 10, 1010-1023. (21) Weng, N.; Shou, W. Z.; Addison, T.; Maleki, S.; Jiang, X. Rapid Commun. Mass Spectrom. 2002, 16, 1965-1975. (22) Tolstikov, V. V.; Fiehn, O. Anal. Biochem. 2002, 301, 298-307. (23) Guo, Y.; Huang, A. J. Pharm. Biomed. Anal. 2003, 31, 1191-1201. (24) Neue, U. D. HPLC Columns Theory, Technology, and Practice; John WileyVCH: Hoboken, NJ, 1997; pp 217-223. (25) Tanaka, H.; Zhou, X.; Masayoshi, O. J. Chromatogr., A 2003, 987, 119125. (26) Grumbach, E. S.; Diehl, D. M.; McCabe, D. R.; Mazzeo, J. R.; Neue, U. D. LC-GC N. Am. 2003, (Suppl. 53). (27) Dell’Aversan, C.; Eagleham, G. K.; Quilliam, M. A. J. Chromatogr., A 2004, 1028, 155-164. (28) Tobias, J.; Patrik, A. LC-GC Eur, 2004, 40-41. (29) Jiang, W.; Irgum, K. Anal. Chem. 2001, 73, 1993-2003. (30) Viklund, C.; Sjo ¨gren, A.; Irgum, K.; Nes, I. Anal. Chem. 2001, 73, 444452. (31) Jiang, W.; Irgum, K. Anal. Chem. 2002, 74, 4682-4687.
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polar zwitterionic functional groups of sulfobetaine covalently attached to silica-based or polymer-based beads, and they take advantage of weak electrostatic interactions between charged analytes and zwitterionic functional groups combined with the high efficiency and selectivity of hydrophilic interaction. However, to produce these HILIC columns in a microformat requires a tremendous amount of skill because of the small internal diameters and small particles used. Additionally, there are several other drawbacks associated with packed capillaries, such as the need for frits, problems with back pressure, stability of silica gel at extreme pH conditions, etc. Monolithic columns have been extensively studied for use in micro-HPLC32-34 since they were first introduced for capillary liquid chromatography in 1989 by Hjerte´n and Liao.35 This was due to their high stability even under extreme pH conditions, fast and simple preparation, and the wide selection of monomers available with different functional groups. However, few studies have been reported for the application of monolithic columns in the HILIC mode.36-38 The lack of studies of monolithic columns for HILIC is the result of several factors. First, the limited solubility of very polar monomers in most commonly used porogens requires a completely new optimization of the polymerization solution mixture and, second, there is a lack of commercially available polar monomers. Sulfoalkylbetaine polymers constitute a class of zwitterionic surface with permanent cationic and anionic charges in proximity as pendent moieties attached to the polymer chain. They have been extensively studied39 since they were first synthesized in 1958 by Hart and Timmerman.40 Viklund prepared porous zwitterionic monoliths by photoinitiated copolymerization of N,Ndimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine (SPE) and ethylene dimethacrylate (EDMA), as well as by surface grafting of electrolyte-responsive poly(SPE) onto a rigid carrier poly(trimethylolpropane trimethacrylate) monolith inside a 2.7-mm-i.d. glass column in order to characterize their interaction with proteins.41 Electrostatic interaction was observed on these sulfobetaine-type monoliths, but no hydrophilic interaction has been reported. In theory, these type of polar zwitterionic monoliths, which are similar to commercial ZIC-HILIC and ZIC-pHILIC, could be effective HILIC stationary phases. This report demonstrates the suitability for HILIC of a porous poly(SPE-co-EDMA) monolith column prepared by thermal-initiated copolymerization of SPE and EDMA inside a 100-µm-i.d. fused-silica capillary. The composition of the polymerization mixture was optimized in order to obtain satisfactory column permeability, efficiency, and separation performance. The optimized monolithic column was applied to the separation of neutral, acid, and basic analytes in a HILIC mode. The effects of pH, salt (32) Fields, S. M. Anal. Chem. 1996, 68, 2709-2712. (33) Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1992, 64, 820-822. (34) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem. 1996, 68, 3498-3501. (35) Hjerte´n, S.; Liao, J. L.; Zhang, R. J. Chromatogr. 1989, 473, 273-275. (36) B Pack, W. D.; Risley, S. J. Chromatogr., A 2005, 1073, 269-275. (37) Ruth, F. J. Chromatogr., A 2004, 1033, 267-273. (38) Xie, S.; Svec, F.; Fre´chet, J. M. J. J. Polym. Sci. Polym. Chem. 1997, 35, 1013-1021. (39) Schulz, D. N.; Peiffer, D. G.; Agarwal, P. K.; Larabee, J.; Kaladas, J. J.; Soni, L.; Handwerker, B.; Garner, R. T. Polymer 1986, 27, 1734-1742. (40) Hart, R.; Timmerman, D. J. Polym. Sci. 1958, 28, 638-640. (41) Viklund, C.; Irgum, K. Macromolecules 2000, 33, 2539-2544.
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concentration, and organic solvent content on separation have also been investigated. MATERIALS AND METHODS Materials. The monomer SPE was a kind gift of Raschig GmbH (Ludwigshafen, Germany). EDMA, 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS), azobisisobutyronitrile (AIBN), ammonium hydroxide, benzoic acid (B), 2-hydroxybenzoic acid (2-HB), 3-hydroxybenzoic acid (3-HB), 3,4-dihydroxybenzoic acid (3,4-DHB), 3,5-dihydroxybenzoic acid (3,5-DHB), 2,6-dihydroxybenzoic acid (2,6-DHB), 3,4,5-trihydroxybenzoic acid (3,4,5-THB), methacrylamide, acrylamide, N,N-dimethacrylamide, N,N′-dimethylenebisacrylamide, toluene, thymine, uracil, adenine, cytosine, and thiourea were all purchased from Aldrich Chemical (Poole, UK). Ammonium formate and formic acid were obtained from BDH Laboratory Supplies (Poole, UK). HPLC-grade methanol and acetonitrile (ACN) were obtained from Fisher Scientific (Leicestershire, UK). The water used throughout all experiments was purified using an Elga water purifier (Bucks, UK). The fusedsilica capillaries with a dimension of 50- or 100-µm i.d. (375-µm o.d.) were purchased from Composite Metal Services Ltd. (Hallow, Worcestershire, UK). Instrumentation. A Shimadzu CTO-6A column oven (Kyoto, Japan) was used for thermal polymerization. Experiments were carried out with a laboratory-built HPLC system that comprised an Applied Biosystems 757 absorbance detector (Ramsey), a fourport injection valve with a 100-nL internal loop from Valco (Houston, TA), a Thermo Separations Constametric 4100 solvent delivery system (Riviera Beach, FL), and a Kontron Deg 104 degasser (Tokyo, Japan). In order to split the mobile phase, a stainless steel tee with flow-split capillary (50 µm i.d. × 30 cm length) was installed before the injector. The actual volume flow rate was determined from the volume of eluent collected during a certain period of time. Detection wavelength was 214 nm. A Data Apex chromatographic Clarity data station (Aston Scientific Ltd., Bucks, UK) was used for data acquisition and data handling. Chromatograms were converted to an ASCII file and redrawn using Microcal Origin 6.0. Preparation of the Monolithic Column. In order to provide anchoring sites for the polymer, the capillaries were treated with γ-MAPS, a bifunctional reagent, prior to polymerization using a method described elsewhere.42 The monomers (SPE and EDMA), the polymerization initiator (AIBN, ∼1 wt % with respect to the monomer), and porogen (methanol) were mixed ultrasonically to yield a homogeneous solution. The monomer mixture and the porogenic solvent were mixed in various ratios in order to prepare the monolith with various porosities, as detailed in Table 1. After sonication and bubbling with nitrogen for 10 min in order to remove dissolved gases, the polymerization mixture was introduced into the pretreated capillary. Both ends of the capillary were sealed with GC septa, and the capillary was kept at 60 °C in the HPLC oven for 12 h (reaction as in Figure 1). The capillary column was then rinsed with methanol to remove the porogenic solvents and any other unreacted soluble compounds. A 2-3-mm detection window was created at a distance of 5 cm from the end of the column using a thermal wire stripper. Monolithic material at this (42) Jiang, Z.; Smith, N. W.; Ferguson, P.; Taylor, M. J. Biochem. Biophys. Methods, accepted for publication.
Table 1. Compositions of the Polymerization Mixtures Used for the Preparation of Poly (SPE-co-EDMA) Monolithic Columns and Their Porosities monomers
porogens
column SPE EDMA methanol monomers porogens porosity C1 C2 C3 C4 C5 C6 C7 C8
43 43 43 43 43 43 53 33
57 57 57 57 57 57 47 67
100 100 100 100 100 100 100 100
30 32.5 33.25 34 35 50 33.25 33.25
70 67.5 66.75 66 65 50 66.75 66.75
0.840 0.762 0.740 0.713 No No 0.775 0.724
point was pyrolyzed and then flushed out with methanol. Finally, the column was cut to a total length of 35 cm with an effective length of 30 cm. A 2-cm length of the capillary containing the monolith inside was cut for scanning electron microscopy (SEM) analysis (FEI Quanta 200 ESEM FEG, Hillsboro, OR). The bulk polymerization of the mixtures was performed in vials in parallel to the polymerizations in the capillaries. The resulting bulk polymer was then removed from the container, cut into small pieces, Soxhlet extracted with methanol for 16 h, dried in vacuum at 60 °C, and the pore size distribution measured using a Quantachrome Poremaster 60 instrument (Hook, UK). Chromatographic Conditions. For much of the reported work on HILIC materials, phosphate buffer was selected because of its UV transparency. However, it was not recommended in this work due to its poor solubility in high organic mobile phases. Stock solutions of ammonium formate (1 M) were prepared and adjusted to the desired pH value with 5 M formic acid or ammonium hydroxide. The mobile phase was prepared by mixing the desired amount of ammonium formate solution, ACN, and water (note that unless otherwise stated, the mobile-phase pH values refer to the aqueous portion only). RESULTS AND DISCUSSION Column Characterization. (1) Optimization of Polymerization Mixture Composition. It is known that the porosity of monolithic phases can be varied by minor changes to the composition of the polymerization mixture.43 In order to investigate the porous properties of the monoliths prepared in our laboratory, mercury intrusion porosimetry, SEM, and micro-HPLC were used to measure the monolithic structures. The total porosity was calculated using the volume flow rate and void volume. The pore size distribution profile of optimized poly(SPE-co-EDMA) was measured in the dry state and may not fully correspond to those of the monolith under conditions used in the HPLC experiments. Unfortunately, no better method for the determination of porous properties of monolithic polymers within the capillary columns is currently available in our laboratory. Normally, porogen composition is a very important variable affecting the porosity. Unfortunately, solvents such as 1-propanol, 1,4-butandiol, toluene, and cyclohexanol, which are most often used for preparing methacrylate-based monoliths, could not be used here because of the immiscibility of the monomer SPE. (43) Ueki, Y.; Umemura, T.; Iwashita, Y.; Odake, T.; Haraguchi, H.; Tsunoda, K. J. Chromatogr., A 2006, 1106, 106-111.
Based upon our experiments, methanol was selected since it exhibited very good solubility for the water-insoluble cross-linker and water-soluble SPE, as well as yielding monoliths exhibiting uniform structure and good separation selectivity. We also tried binary porogenic solvent systems containing methanol, but no promising improvement was observed. To investigate the influence of the porogen concentration on the preparation of poly(SPE-co-EDMA) monolith, the ratio of SPE/ EDMA (43/57, w/w) was kept constant, while the porogen weight fraction was varied from 70 (column C1) to 50% (column C6). Scanning electron micrographs, Figure 2a and b, show the copolymerized monolith composed of spherical units agglomerated into larger clusters interdispersed by large-pore channels, which are characteristic of monolithic structures. With a decrease of porogen weight fraction in the polymerization mixture, it was found that the microglobules became smaller (figure not shown). This trend is consistent with the results of porosity (T) measured by micro-HPLC. Acetonitrile was pumped through the monolithic column, and toluene was used as the marker of the dead volume. When the porogen weight fraction decreased from 70 to 66%, the T dramatically decreased from 0.840 to 0.713 (Table 1). When the content of porogen solvent was reduced still further (65 to 50%), the permeability became so poor that it was impossible to flush the column even at a pressure of 40 MPa, due to the highly dense polymeric bed formed. At the same time, when the porogen weight fraction decreased from 70 to 66%, the column efficiency increased from ∼4000 to ∼15 000 theoretical plates/m at a linear flow rate of 2 mm/s. However, the back pressure of column C4 is higher than column C3. Therefore, in order to compromise between peak efficiency and pressure drop, a porogen weight fraction of 66.75% was used in further experiments. It was also found that the porosity was sensitive to the EDMA content in the monomer mixture. When the weight content of the porogen solvent methanol was kept constant at 66.75% and the weight content of EDMA in the monomer mixture was varied from 47 (C7) to 67% (C8), the T decreased from 0.775 to 0.724 (Table 1, C3 and C7-C8). As can been in Figure 2, SEM experiments also show that the microglobules became smaller with the increase of EDMA content in the monomer mixture. Conversely, with the increase of EDMA weight content in the polymerization mixture, the column efficiency increased slightly from ∼10 000 to ∼15 000 theoretical plates/m at a linear flow rate of 2 mm/s. However, as expected, because the zwitterionic functional group incorporated in the monolith decreased linearly with the amount of SPE included in the polymerization mixture,41 the hydrophilicity of the monolith also decreased, and therefore, the retention of the polar compound thiourea on this phase decreased (data not shown). Thereafter, the EDMA weight fraction of 57% was selected for all further experiments. For practical uses, the monolith for HPLC should provide macropores with diameters over 50 nm, which are responsible for good permeability, and mesopores with diameters ranging from 2 to 50 nm, which are important to obtain a large surface area. The pore size distribution curve of optimized monolith C3 is shown in Figure 3. It was observed that the majority of the pores have a diameter of 70-450 nm, in addition to a small number of mesopores. Analytical Chemistry, Vol. 79, No. 3, February 1, 2007
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Figure 1. Preparation of poly(SPE-co-EDMA).
Figure 2. Scanning electron microphotographs of monolithic columns prepared with different EDMA content: (a) and (b) column C3; (c) column C7; (d) column C8.
(2) HILIC Retention Mechanism. HILIC separations commonly employ water and acetonitrile as the mobile phase, but require a much higher organic content (>60%) in order to ensure significant hydrophilic interaction. The hydrophilic retention increases with increasing ACN content.6 In order to investigate the HILIC properties of poly(SPE-co-EDMA) monolith, toluene, thiourea, and acrylamide were used as test compounds. The 1246
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content of ACN was varied from 95 to 20% while the ammonium formate concentration was kept constant at 5 mM. Only a low salt concentration was possible due to its limited solubility in the mobile phase with high acetonitrile content. The influence of ACN content in the mobile phase on the retention time of three test compounds is shown in Figure 4. Thiourea, which is normally used as a dead volume marker in RPLC, elutes after toluene and
Figure 3. Pore size distribution curve of monolith C3 measured by mercury intrusion porosimetry.
acrylamide when the ACN concentration was increased from 50 to 95%. The retention time of thiourea decreased initially as the ACN content in the mobile phase decreased from 95 to 60% and then leveled off when the ACN content further decreased to 20%. It is noticeable that thiourea is retained at all ACN concentrations between 20 and 60%. For the nonpolar compound toluene, which was eluted first at high ACN concentration, an opposite curve was observed. The retention time of toluene was approximately constant as the ACN content in the mobile phase decreased from 95 to 65% then rose dramatically at ∼40% ACN. Acrylamide behaved similar to thiourea but with much less retention. These results demonstrated a typical HILIC retention mechanism at higher ACN content (>60%). (3) Permeability. An important characteristic of a column in LC is its permeability K, which represents the resistance to mobilephase flow through the monolithic column. K can be determined by pumping five different solvents through the column at different linear flow rates according to44
K ) (uL/∆P)η where u is the linear velocity of the eluent, η is the dynamic viscosity of the mobile phase, L is the column length, and ∆P is the pressure drop across the column. Toluene was selected as the dead-time marker when ACN, methanol, hexane, or ACN/ water (80/20, v/v) was used as mobile phase. When using water as the mobile phase, acrylamide was selected as the to marker because of the high retention of toluene at the lower ACN concentrations. Good linearity between back pressure and the flow rate for all five cases clearly demonstrates that the monolith was sufficiently mechanically stable to withstand the pressure of the liquid passing through the column up to 30 MPa. The calculated K for column C3 is shown in Table 2. The stability of polar (44) Bristow, P. A.; Knox, J. H. Chromatographia 1977, 10, 279-289. (45) Lide, D. R., Ed. CRC Handbook of Chemistry and Physics, 84th ed.; CRC: Boca Raton, 2003-2004.
Figure 4. Relationship between retention time and acetonitrile concentration on poly(SPE-co-EDMA) monolithic column. Conditions: column dimensions, 285 mm × 100 µm i.d.; mobile phase, 5 mM ammonium formate pH 3.0 in ACN/H2O; detection wavelength, 214 nm; flow rate, 800 nL/min; injection, 100 nL.
monoliths is often questionable because of the possible swelling in aqueous buffer. In theory, if the monolith swells, its throughpores will decrease in size, resulting in lower permeability, and vice versa if shrinkage were to occur. As seen in Table 2, though, comparable permeability was observed when different solvents were passed through the poly(SPE-co-EDMA) monolithic column. This indicates that the monolith does not swell or shrink in solvents of different polarity. (4) Reproducibility. The reproducibility of the poly(SPE-coEDMA) monolith was assessed through the percent relative standard deviation (RSD) for the retention factor of three test compounds, namely, toluene, acrylamide, and thiourea (Figure 4a). The run-to-run reproducibility (n ) 10) of acrylamide and thiourea are 0.38 and 0.25%, respectively. The day-to-day reproduc(46) Wode, H.; Seidel, W. Ber. Bunsen. Phys. Chem. 2004, 98, 927-934.
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Table 2. Permeability of the Poly(SPE-co-EDMA) Monolitha mobile phase
relative polarity
viscosity η (×10-3 Pa‚s)
permeability K (×10-14 m2)
acetonitrile hexane methanol ACN/water (80:20; v/v) water
0.460 0.009 0.762 / 1
0.369 0.300 0.544 0.450 0.890
3.41 3.28 2.76 2.76 2.74
a Relative polarity data were from http://virtual.yosemite.cc.ca.us/ smurov/orgsoltab.htm; Viscosity data were from ref 45. The viscosity of ACN/water (80/20, v/v) was obtained from ref 46.
Figure 6. Separation of seven benzoic acid derivatives. Conditions: mobile phase, 50 mM ammonium formate pH 3.0 in ACN/H2O (75/25, v/v). Others as in Figure 4.
Figure 5. Separation of neutral acrylamides. Conditions as in Figure 4. Samples: (1) N,N-dimethylacrylamide; (2) methacrylamide; (3) N,N′-dimethylenebisacrylamide; (4) acrylamide.
ibilities (n ) 3) for the last two peaks are 0.62 and 0.74%. This clearly confirms the robustness of the monolithic column since their separation ability does not appear to deteriorate with either time or number of injections. Ten monolithic columns with the same polymerization composition as that of C3 were prepared from different batches of polymerization mixtures. Column-to-column (n ) 10) reproducibility measurements gave RSD values for retention factors (k) of acrylamide and thiourea of 1.44 and 0.82%, respectively. Applications. (1) Neutral Compounds. The polar zwitterionic poly(SPE-co-EDMA) surface can provide a hydrophilic environment. A mixture of low-molecular-mass amides including acrylamide, methacrylamide, N,N-dimethylacrylamide, and N,N′dimethylenebisacrylamide was used to investigate closely related neutral compounds that are difficult to retain and separate by RPHPLC. As can be seen in Figure 5, good separation was obtained with 98% ACN content. As expected, the retention times of three of the polar compounds acrylamide, methacrylamide, and N,N′dimethylenebisacrylamide decreased with a slight drop in the ACN content from 98 to 92%, while the retention of the relatively less polar compound N,N-dimethylacrylamide increased slightly, which is anomalous since the levels of ACN used here are far higher than are needed to eliminated all hydrophobic interaction. Interestingly, the elution order of methacrylamide and N,N′dimethylenebisacrylamide swapped at 95% ACN. (2) Charged Compounds. As with commercial ZIC-HILC/ pHILIC phases,17 the zwitterionic poly(SPE-co-EDMA) monolith also offers the possibility of weak electrostatic interaction with analytes carrying either positive or negative charges. This provides 1248 Analytical Chemistry, Vol. 79, No. 3, February 1, 2007
Figure 7. Separation of bases and neutral samples. Conditions: mobile phase, 5 mM ammonium formate pH 3.0 in ACN/H2O (95/5, v/v). Others as in Figure 4. Samples: (1) toluene; (2) methacrylamide; (3) acrylamide; (4) thymine; (5) uracil; (6) adenine; (7) thiourea; (8) cytosine.
a larger degree of freedom in HILIC method development by varying the pH, ionic strength, or organic solvent concentration. To demonstrate the special selectivity of the poly(SPE-co-EDMA) monolith, seven benzoic acid derivatives and a mixture of four pyrimidines or purines and three neutral compounds were used for further evaluation. Good separations were obtained with optimized chromatographic conditions (Figure 6 and Figure 7). Figure 8a shows the influence of ACN concentration on the retention times of seven acids. As expected, the retention time of all seven acids decreased dramatically initially and then level off on decreasing the ACN concentration from 92 to 70%. Baseline separation was obtained when the ACN concentration is higher than 88% or lower than 75%. Worth noting is the change of elution order of 2-HB and 2,6-DHB with ACN concentration. This unusual behavior was probably related to the ion-exchange interaction between the acids and zwitterionic stationary phase. The four bases, thymine, adenine, uracil, and cytosine, showed a similar behavior but no elution order change was observed (data not shown). Once again, baseline separation was obtained at high ACN concentration (>90%). The effect of salt concentration on the retention of seven acids was also investigated by varying the concentration of ammonium
Figure 8. Influence of mobile phase on separation of seven benzoic acid derivatives. Plot a: effect of ACN content, 5 mM ammonium formate pH 3.0 in ACN/H2O. Plot b: effect of salt concentration, ammonium formate pH 3.0 in ACN/H2O (75/25, v/v). Plot c: effect of pH, 5 mM ammonium formate in ACN/H2O (92/8, v/v). Plot d: effect of pH, 50 mM ammonium formate in ACN/H2O (75/25, v/v); others as in Figure 4.
formate pH 3.0, from 1 to 50 mM in the mobile phase of ACN/ water (75/25, v/v). Due to the solubility limitation of ammonium formate at high ACN concentration (>90%), no investigations have been performed on the four bases. As can be seen in Figure 8b, the seven acids can be divided into two categories depending upon their behavior. The retention of 2-HB and 2,6-DHB increased initially as the ammonium formate concentration increased from 1 to 5 mM, but decreased when the ammonium formate concentration increased further to 50 mM. However, the retention of the other five analytes (B, 3-HB, 3,4-DHB, 3,5-DHB, 3,4,5-THB) almost kept constant when the ammonium formate concentration was lower than 5 mM and then increased slightly when the concentration increased further to 50 mM. B, 3-HB, 3,4-DHB, 3,5-DHB, and 3,4,5-THB have pKa1 values of 4.204, 4.08, 4.48, 4.04, and 4.41, respectively.47 Any electrostatic interactions are unlikely since the analytes would be uncharged in the pH 3.0 buffers. This indicated that the retention increase at higher salt concentrations might be related to a hydrophilic partitioning process. The partitioning model for HILIC assumes the presence of a water-rich liquid layer (47) Dean, J. A. Lange’s Handbook of Chemistry, 15th ed.; McGraw-Hill: New York, 1999; pp 8.24-8.72.
on the packing surface.6 Higher levels of organic content in the mobile phase result in the salt preferring to be in the water-rich liquid layer. A higher salt concentration would drive more solvated salt ions into the water-rich liquid layer. This would result in an increase in volume or hydrophilicity of the liquid layer, leading to stronger retention of the five acids. 2-HB and 2,6-DHB have pKa1 values of 2.98 and 1.30, respectively.47 Therefore, at pH 3.0, they will be negatively charged, so both ion-exchange interaction and hydrophilic interaction might contribute to their retention. With increasing ammonium formate concentration in the mobile phase, the potential electrostatic repulsion from negatively charged sulfonate groups on the sulfobetaine phase decreases, whereas hydrophilic interactions increase, resulting in increased retention of the two acids. On the other hand, a higher salt concentration could decrease the possible weak ion-exchange interaction between positively charged quaternary amine groups and negatively charged acidic analytes via ion suppression, thus leading to decreasing retention. The unusual behavior of 2-HB and 2,6-DHB with increasing ammonium formate concentration was probably related to the balance of opposing effects. Analytical Chemistry, Vol. 79, No. 3, February 1, 2007
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Mobile-phase pH, which can influence solute ionization and polarity of the stationary phases, also plays an important role in affecting selectivity in HILIC. The effect of mobile-phase pH on HILIC separation on the poly(SPE-co-EDMA) monolithic was investigated by changing the pH of stock salt solutions before mixing with ACN. The pH of the stock 1 M ammonium formate solution was adjusted with formic acid or ammonium hydroxide to pH 3.10, 4.5, 6.45, and 8.45. Panels c and d in Figure 8 show the effect of pH on the retention time of seven acids on the poly(SPE-co-EDMA) monolith. Using a mobile phase consisting of 92% ACN and 5 mM ammonium formate, the retention times of 2-B and 2,6-DHB with pKa1 < 3.0 remained almost unchanged because there were no significant changes in ionization, and so the hydrophilicity and ion-exchange interaction in the pH range studied were fairly constant. However, for the other five acids with pKa1 > 4, when the mobile-phase pH increased, they became deprotonated and therefore negatively charged and more hydrophilic, thus leading to stronger hydrophilic interaction and possible ion-exchange interaction and thus longer retention times. To further investigate the effect of pH, ACN/water (75/25, v/v) containing 50 mM ammonium formate with different pH was used. Under these conditions, less ion-exchange interaction will exist due to the higher salt concentration. Figure 8d shows that the retention of five acids (B, 3-HB, 3,4-DHB, 3,5-DHB, 3,4,5-THB) increased slightly initially and then leveled off with the increase in pH from 3.1 to 8.45. However, compared with the behavior of these five acids at 92% ACN, much less increase of retention was observed. This indicated that ion-exchange interaction could contribute significantly to the overall retention of these five acids at a pH above their pKa. Interestingly, the retention of 2-HB and 2,6-DHB, especially the latter, decreased initially and then leveled off with the increase of pH at 75% ACN. Since both acids are completely deprotonated in the pH range studied, this unusual behavior is difficult to explain. Mant and Hodges48 described a situation whereby a series of peptides, whose acid functions had been blocked, showed a lowering of retention time on S300 strong cation-exchange column as the pH was decreased. Their explanation for these results was a neutralization of the sulfonic acid group by the protonated amino function of the polyamine underlayer, which was less pronounced as the pH increased. Thus, as the underlayer became less protonated, the cation-exchange capacity increased. However, our observation cannot be explained by this (48) Mant, C. T.; Hodges, R. S. J. Chromatogr. 1985, 327, 147-155. (49) Kitano, H.; Mori, T.; Takeuchi, Y.; Tada, S.; Gemmei-Ide, M.; Yokoyama, Y.; Tanaka, M. Macromol. Biosci. 2005, 5, 314-321. (50) Kitano, H.; Imai, M.; Sudo, K.; Ide, M. J. Phs. Chem. B 2002, 106, 1139111396.
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process since the N in our monolith is quaternary and therefore remains unaffected by pH. Kitano et al. reported on a sulfoalkylbetaine stationary phase, which they described as a potent osmolyte with a strong ability to bind water to its surface, and the way in which these phases interact with water is unique. The partially negative oxygen atom in the water molecule is orientated toward the positively charged quaternary ammonium, whereas the negatively charged sulfonic acid groups attract the partially positive H-atoms of water, creating their hydration shells.49,50 With the change of pH, the water splitting on two charged position might change and then might result in the change of hydrophilicity of stationary phase. However, more studies need to be performed in the future. For thymine, uracil, adenine, and cytosine, the retention time only fluctuated slightly on poly(SPE-co-EDMA) monolith in the pH range studied (data not shown). CONCLUSION A porous poly(SPE-co-EDMA) monolithic column, which was prepared by thermoinitated copolymerization of SPE and EDMA, has been successfully used as a stationary phase in micro-HILIC. Typical HILIC retention was observed at high organic solvent content (ACN > 60%). The polar zwitterionic monolith provides an environment, not only capable of hydrophilic interaction with polar and charged analytes but also offering the possibility of weak electrostatic interaction with analytes carrying either positive or negative charges. The effects of pH, salt concentration, and ACN content on the separation of acids showed that a HILIC/ionexchange chromatography mechanism on poly(SPE-co-EDMA) could be used to manipulate selectivity when optimizing methods of separation. It is possible that a better monolith for HILIC could be obtained by using a more hydrophilic monomer or cross-linker. Poly(ethylene glycol) diacrylate, which was specially designed and used to decrease unwanted polymer backbone hydrophobicity, could be a good replacement for EDMA as cross-linker. Another possible alternative monomer is the commercially available N,Ndimethyl-N-acryloxyethyl-N-(3-sulfopropyl)ammonium betaine. However, its poor solubility in currently used porogens is a challenge. This work is currently under investigation ACKNOWLEDGMENT We thank Mr. Glen Guermeur of Pfizer UK for providing the porosity measurements and Mr. Phil Roberts of RASCHIG UK Ltd. for kindly providing SPE monomer. Received for review October 5, 2006. Accepted November 15, 2006. AC061871F
