Anal. Chem. 1994,66, 2129-2138
High-Performance Liquid Chromatography of Complex Mixtures Using Monodisperse Dual-Chemistry Polymer Beads Prepared by a Pore-Size-Specific Functionalization Process. A Single Column Combination of Hydrophobic Interaction and Reversed-Phase Chromatography Vladlmlr Smlgol, Frantisek Svec, and Jean M. J. Frhhet’ Baker Laboratory, Department of Chemistry, Cornel1 Universiv, Ithaca, New York 14853-130 1
A novel separation medium for HPLC combining hydrophobic interaction and reversed-phase separation modes in a single column has been prepared from monodisperse 10-pm poly(glycidyl methacrylate-~ethyleaedimethacrylate)beadsusing a pore-sizespecific functionellzation process. In this approach, the large pores of each bead were provided with phenyl groups interspersed among hydrophilic functionalities while a much higher surface concentration of hydrophobic phenyl groups was introduced into the small pores. Due to the size-specific character of the modification process, no protein interaction with any highly hydrophobic surface was observed during chromatography. The beads were used for the separation of samples containing both proteins and small hydrocarbon or drug molecules. A plot of log k’ against salt concentration in the mobile phase clearly documents the clean hydrophobic interaction mechanism of protein separation and the absence of charged groups while the linear plot of log k’ against acetonitrile concentration for numerous compounds demonstrates the reversed-phase separation ability. No decrease of the efficiency of the test column (23 000 plates/m) was observed in long-term experiments during which more than 1000 injectionsand many changes betweenthe modeswere performed,
The number of commercially available HPLC columns for different chromatographic modes is enormous, but much duplication and overlap exists in the chemistries of these columns. Various modes of HPLC, such as normal-phase, reversed-phase, ion-exchange, bioaffinity, size-exclusion,can theoretically be distinguished according to their retention mechanisms. These are related to particular types of solutestationary phase surface group interactions. In contrast, typical chromatographic media contain multiple organic and inorganic surface functionalities and retention by more than one mechanism is observed particularly for silica-based separation media. Two or more functionalities can also be introduced intentionally in the separation system in order to improve the selectivity of separationsthrough the simultaneous use of various types of interactions. Therefore, approaches (a-d) have been described in the literature. (a) The tandem connection of two columns packed with anion- and cation-exchange stationary phases, respectively, with the same mobile phase running through both columns increasesthe selectivity of separationof proteins.’ Alternately, 0003-27O0194IO306-21298O4.5OlO 0 1994 American Chemlcal Society
a single column may be first partially packed with one phase and then completely filled with another one (“twin” phase).2 Finally, the two phases used in the tandem columns can also be blended and packed into a mixed-bed c0lumn.3~~ However, only media that are “mobilephase compatible”can be blended. (b) Stationary phases for mixed-interaction chromatography of biopolymers with multiple retention mechanismsused in parallel have been prepared by the simultaneous bonding of two or more ligands (mixed ~hemistry).~ Unlike the “twin” phases and “mixed-bed”phases, the distance between different functionalities attached to the surface in the medium must be shorter than the size of the solutes to ensure the Occurrence of multisite interactions. (c) A refinement of the mixed-interactionchromatography technique has resulted in the design of separation media that segregate the various retention mechanisms occurring within a mixed-mode c o l ~ m n .A~ multimodal column operates in more than one independent mode determined by the mobile phase. Separations of proteins in which the mode is dictated by the composition of the mobile phase were termed “multimodal chromatography” by Regnier.s-7 The chemistries of mixed mode media are not segregated but distributed in all pores. (d) Finally, stationary phases based on modified porous silica with both a hydrophilic outer surface and hydrophobic pores (internal-surface reversed-phase media, ISRP) were developed by Pinkertod to allow the direct injectionof complex matrices such as plasma, serum, saliva, and urine into a column for the determination of drugs and metabolites without any pretreatment while preventing accumulation of proteins and clogging of the column. The preparation of ISRP phases begins with a complete hydrophobization of the entire surface of small-pore silica beads. In the next step, a protein that is totally excluded from the inner pores cleaves the hydrophobic (1) El Rassi, 2.;Horwath, C. J. Chromatogr. 1986,359, 255. ( 2 ) Clement, A.; Loubinoux, B. J . Liq. Chromatogr. 1983, 6, 1705. (3) Maa, Y. F.; Antia, F. D.; El Rassi, 2.;Horwath, C. J. Chromatogr. 1988,452, 331. (4) Floyd,T. R.; Hartwick, R. A. in High-PerformanceLiquid Chromatography,
Advances and Perspectiws; Horvath, C., Ed.; Academic Press: New York, 1986; Vol. 4, p 45. (5) Kennedy, L. A.; Kopaciewicz, W.; Regnier, F. E. J. Chromarogr. 1986,359, 13. (6) Heinitz,M. L.; Kennedy,L. A.; Kopaciewicz, W.; Regnier, F. E. J. Chromatoat. 1988,443. 173. (7) Miller, N . T.; Feibush, B.; Karger, B. L. J. Chromarogr. 1965, 316, 519. (8) Hagcstam, I. H.; Pinkerton, T. C. AMI. Chem. 1985.57, 1757.
moieties only at the outer surface of beads and renders the surface hydrophilic. The hydrophobic groups within the pores remain unchanged. In the ISRP packings, proteins are eluted at Vi followed by other separated analytes. Following this initial work, several varieties of these restricted-access media have been d e v e l ~ p e d . ~ J ~ "Multidimensional" separations may be used to carry out more complex analyses. The multidimensional approach was first introduced in the area of thin-layer chromatography (TLC). l J 2 A multidimensional HPLC system typically involves several columns in series. A peak containing a class of compounds separated from the rest of the mixture in the first column is directed into the a second column, where it is separated into its individual components.13 However, the instrumentation used is complex and rather costly. A slightly different approach involving single column "pseudo-multidimensional" HPLC simplifies the concept of multidimensional HPLC to effect sequential separations in two different elution modes.14 In contrast to mixed-mode stationary phases, which are multimodal with respect to the stationary phase, the sequential multimodal elution is multimodal with respect to the mobile phase. The phase used was conventional reversed-phase C18 silica. While the feasibility of mixed-mode, multimodal, multidimensional, and direct injection chromatographicseparations have been demonstrated, the media used in these studies were only optimized for very specific targets. The surface groups responsible for the separation are introduced randomly in all the pores or, in the case of ISRP, only on the outer surface. We have recently introduced the concept of pore-sizespecificfunctionalization of porous polymers to prepare media in which the surface of pores within different size ranges may be endowed with different chemistries.l5 This concept relies on the use of catalysts with defined molecular volumes that are able to perform a chemical modification process only in those pores large enough to allow their access. Overall, control of the modification process relies on control of the hydrodynamic volume of the catalyst, as opposed to the usual modification process when kinetics factors are the sole consideration. The method allows the preparation of beads with different chemistries segregated in different size pores. This concept adds a new perspective to multidimensional chromatographic methods. This report extends the concept of pore-size-specific modification to the first preparation and useof a singlepacking in which hydrophobic interaction chromatography of proteins and reversed-phase chromatography of small molecules can proceed consecutively. EXPERIMENTAL SECTION Materials. A narrow polydispersity sodium poly(styrenesulfonate) (Mw = 5 000, M w / M n = 1.09) was obtained (9) Pinkerton, T . C. J. Chromarogr. 1991, 544, 13. (10) Haginaka, J. Trends Anal. Chem. 1991, 10 (l), 17. (11) Randerath, K. Thin b y e r Chromatography; Academic Press: New York, 1986; p 51. (1 2 ) Multidimensional Chromatography: Techniques and Applications; Cortes. H. J., Ed.; M.Dekker: New York, 1990. (13) Roth, W.; Beschke. K.;Jauch, R.; Zimmcr, A,; Kcss, F. W. J. Chromatogr. 1981, 222, 13. (14) Little, E. L.; Jeansonne, M.S.;Foley, J. P. Anal. Chem. 1992, 63, 33. (15) Smigol, V.; SVCC,F.; Frkhet. J. M.J. Macromolecules 1993, 26, 5615.
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Table 1. Propertkr of UnHormly S l z d Poiy(glycldyl methacrylate-coathylene dfmethacrylate) hads
particle size, pm 10.0 median pore diameter! nm 10.9 epoxide groups, mmol/g 2.7 polystyrene exclusion limit," 3.4 specific pore volume," mL/g 1.1 M W X 10-5 median pore diameter," nm 13.8 specific surface area! m2/g 114 a Calculated from size-exclusion-chromatographydata. b Calculated from BET measurement.
from Scientific Polymer Products, Inc. (Ontario, NY). Prior to its use, the sodium salt was converted to the free acid by an ion-exchange process using a AG 5OW-X strong acid cation exchanger (Bio-Rad, Richmond, CA). Human serum albumin (HSA, essentiallyfat free, MW 69 000), bovine serum albumin (BSA, 98-9996, MW 66 700), chicken egg albumin (ovalbumin, Grade V, MW 45 000), conalbumin (from chicken egg white, Type I, MW 75 SOO), ribonuclease A (from bovine pancreas, MW 13 600), cytochrome c (from bovine heart, M W 13 400), lysozyme (MW 14 loo), myoglobin (from horse heart, MW 17 000), chymotrypsinogen (MW 24 000), soya bean trypsin inhibitor (MW 22 500), and bovinedried plasma were purchased from Sigma. The hydrocarbon standards were obtained from Aldrich and anticonvulsant drugs from Sigma. All solvents were HPLC grade. Polymer Beads. Uniformly sized 10-pm porous 6040 vol 96 glycidylmet hacrylate-ethylene dimethacrylate copolymers (resin I)were prepared by a modified shape template swelling and polymerization method described in detail elsewhere.16J7 Cyclohexanol(60 vol96) was used as the porogenic solvent. The specificsurface area was calculated from nitrogen sorption using the BET method (combined BET sorptometer and mercury porosimeter, Porous Materials Inc., Ithaca, NY). The pore-size distribution, pore volume and median pore diameter were calculated from the retention volume of polystyrene standards (Polymer Laboratories, Church Stretton, UK) in THF.18 The content of epoxide groups was determined by volumetric titration as follows: the beads were dispersed in 0.1 mol/L tetramethylammonium bromide solution in acetic acid and titrated with 0.1 mol/L perchloric acid solution in acetic acid until the crystal violet indicator indicated the blue-green end point. Table 1 lists the characteristics of the beads. Nonspecific Modification of the Polymer Beads. Total Hydrolysis. Hydrolysis of the epoxide groups of resin I into vicinal diol groups (eq 1) proceeds under catalysis with mineral acid. Resin I (10 g) was suspended in 50 mL of 0.1 mol/L aqueous sulfuric acid, stirred occasionally, and kept at 60 OC for 10 h. The product was washed thoroughly with water to afford diol resin I1 used for further modifications. Additional Hydrophilization of A11 Pores. Dry diol resin I1 (5 mL) was dispersed in 20 mL of water and stirred with a magnetic bar for 16 h. The excess water was removed on a fritted-glass filter and the beads redispersed in 20 mL of 50 wt % aqueous KOH and stirred for 1 h. The liquid was removed, and the beads were transferred to 40 mL of an (16) Smigol, V.;Svec, F. J. Appl. Polym. Sei. 1992, 46, 1439. (17) Smigol, V.; Svec,F. J . Appl. Polym. Sci. 1993, 48, 2033. (18) Halasz, I.; Martin, K.Angew. Chem., Inr. Ed. Eng. 1978, 17, 901.
epichlorohydrin-water (1:l) mixture and stirred at rwm temperature for 3 h. The resulting beads (III) (eq 2) containing epoxide groups (1.9 mmol/g) were washed with water and acetone. The epoxidized beads were then hydrolyzed and worked up using the same procedure as described above to afford beads containing groups IV. Complete Hydrophobization of All Pores. Resin I was suspended in a melt of 8 g of phenol and 0.01 g of KOH and stirred at 70 OC for 6 h. Thereactionmixture was thendiluted with 20 mL of dioxane and mixed for 30 min while cooling to ambient temperature. The resulting beads containing functionalities with structure V (eq 3) were washed with dioxane, acetone, water, acetone again, and dried. Preparation of Beads with Two Segregated Chemistries (Scheme 1). Partial Hydrophobization of All Pores (Modification Step a). Poly(glycidy1 methacrylate-co-ethylene dimethacrylate) beads I (1.6 g, 2.7 mmol epoxide groups) were immersed in a solution of 0.01 g of KOH (0.17 mmol) and 1.O g of phenol (10.2 mmol) in 60 mL of dioxane and refluxed for 2 h. The resulting beads (A) were washed with dioxane, acetone, water, and acetone again and dried. No residual epoxide groups could be detected in beads A by IR spectroscopy. Reaction of All Pores with Epichlorohydrin (Modification Step b). Dry beads A were dispersed in 20 mL of water and stirred with a magnetic bar for 16 h. The excess water was removed on a fritted-glassfilter, and the beads were redispersed in 20 mL of 50 wt % aqueous KOH and stirred for 1 h. The liquid was removed, and the beads were transferred to 40 mL of a epichlorohydrin-water (1:1) mixture and stirred at room temperature for 3 h. The product was washed with water and acetone and dried to afford beads (B) containing 1.36 mmol/g epoxide groups. Pore Size-Specific Hydrolysis (ModificationStep c). The hydrolysis of the epoxide groups contained in beads B was catalyzed with a 1 wt 7% aqueous solution of poly(styrenesulfonic acid) (Mw 5000) containing 0.054 mol/L sulfonic groups. The epoxide resin (1.6 g) was placed in a 50-mL beaker, 10 mL of aqueous catalyst solution was added, and the beaker was sealed with Parafilm. The dispersion was stirred magnetically at ambient temperature for 48 h. The resulting modified beads were then filtered off on a frittedglass filter and washed with water until neutral. The resulting beads (C) were washed with acetone again and dried in vacuo at room temperature. Additional Hydrophobization of the Small Pores (Modification Step d). Beads resulting from the previous reaction (1.6 g) were suspended in a melt of 8 g of phenol and 0.01 g of KOH and stirred at 70 OC for 6 h. The reaction mixture was diluted with 20 mL of dioxane and mixed for 30 min while cooling to the ambient temperature. The monodisperse beads with dual chemistry (C)were worked up as in reaction step a above. Methods. IR Spectroscopy. IR spectra were recorded on a Nicolet FT-IR spectrometer from KBr pellets. High- Performance Liquid Chromatography. The properties of polymers prepared in this study were determined by chromatography carried out in a 100-mm X 8-mm i.d. or 150-mm X 4.6-mm i.d. stainless steel column using a commercial HPLC chromatograph (IBM-Nicolet ternary
gradient liquid chromatograph LC 9560) equipped with a Rheodyne 7125 loop injector and a Hewlett-Packard 1050 UV detector. Protein Recovery and Relative Protein Hydrophobicity. The protein recovery from the beads packed in a 100-mm X 8-mm i.d. column was obtained upon isocratic elution at a flow rateof 1 mL/min and ambient temperature. The volume injected was 20 pL. Recovery was calculated as the percentage of protein peak area leaving the column under standard chromatographic conditions (mobile phase, 0.15 mol/L ammonium sulfate in 0.02 mol/L phosphate buffer, pH 7; flow rate, 1 mL/min; 10 mg/mL protein solution in the phosphate buffer; detection, 254 nm) with respect to the peak area of the same amount of the protein injected into a system from which the column was removed, and the inlet and outlet capillaries were connected with an empty column tube having a volume similar to the void volume of the packed column. The experimental error does not exceed 10%. For the determination of relative hydrophobicities, all proteins were injected into the column and eluted under standard conditions using a linear gradient from 2 mol/L ammonium sulfate solution in 0.02 mol/L phosphate buffer (pH 7) to the buffer only within 60 min. The relative hydrophobicity of cytochrome c was arbitrarily set to be 0 while a protein eluted at the end of the gradient (retention time, 60 min) would have a relative hydrophobicity of 1.O. The relative hydrophobicities, RH, of the various proteins were calculated accordingto the equation RH = t,/60, where t , is the retention time of a specific protein. HSA Test (Relative Hydrophilicity of Packings). A solution of human serum albumin (10 wt 5%) in 0.1 mol/L phosphate buffer (pH 3.8) containing 0.15 mol/L NaCl was injected into the column and the peak of the nonadsorbed protein was monitored. The mobile phase was changed to a different 0.1 mol/L phosphate buffer (pH 7) containing 0.15 mol/L NaCl and the peak released under these conditions was monitored. Finally, the mobile phase was changed to a 10% ethylene glycol solution in 0.1 mol/L phosphate buffer (pH 7) and the peak was monitored. The percentage of the HSA released in each step was calculated and used for relative comparison of various beads. The total quantity of released protein was also compared to that obtained in a system from which the column was removed.
RESULTS AND DISCUSSION Generally,multidimensional separation techniques provide a tool for an increase in the resolving power of chromatographic methods. The concept of pore-size-specificfunctionalization we recently developed15 can be used advantageously for the preparation of novel separation media that exhibit some features of single-column multidimensional liquid chromatography. According to the definition given by Giddings,19 a multidimensionalseparation requires that all compounds to be separated be subjected to two or more independent separative steps where none of these steps nullifies the separation achieved in the previous steps. (19) Giddings, J. C. J. High. Resolut. Chromatogr., Chromatogr. Commun. 1987, 10, 319.
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Foley14 introduced the term “pseudo-multidimensional” for sequential separations carried out in a single column, where part of the sample is “stored” at the top of the column in the first step (“pseudo-first” dimension) while the other part is separated into its individual components. In the following step (second dimension), the chromatographic conditions are changed and the stored part of the sample is separated. The term “pseudo-multidimensional” was used to account for the fact that components separated in the first step were not subjected to more than one separative mode. Our objective was to prepare a single separation medium combining the two different types of chemistries that are required for (a) the hydrophobic chromatography of proteins and (b) the reversed-phase chromatography of small molecules. Such a combination of separation modes is not feasible unless the two chemistries are segregated from each other. In particular, the proteins must not be able to come into contact with the highly hydrophobic surface that is required for reversed-phase chromatography. Using pore-size-specific chemistry, the readily accessible surface of the large pores of a standard polymeric medium can be made hydrophilic, while the small pores can be provided with the hydrophobicity that is required for their use in the reversed-phase mode. In order to demonstrate the success of our approach to single-column separation of complex mixtures, it is necessary to also prepare comparative batches of beads that each contain only one of the two types of chemistries. Evaluation of Completely Hydrolyzed Beads. First, it was assumed that a complete hydrolysis of the epoxide groups of copolymer beads I to diol beads 11(eq 1) would be sufficient
-a
0.0 -0.5 0
(20) Cornell, C. N.; Caplan, L. J. Biochemisrry 1978, 17, 1755. (21) Steiner, R. F.;Edelhoch, H.Nature 1961, 192, 873.
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1 2 Ammonium sulfate, moi/L
3
Flgure 1. Effect of ammonium sulfate concentratlon on the retentbn factor for various proteins in columns packed wlth beads containing segregated chemistries (1, 3) and wlth completely hydrolyzed beads (2, 4): column size 100-mm X 8-mm 1.d.: mobile phase 0.02 mol/L phosphate buffer soiutlon (pH 7); flow rate, 1 mL/min: UV detectlon; analytes, lysozyme (1, 2) and cytochrome c (3, 4). 1.0
0.5
L
-
0 0
0.0
-0.5
I IO
30 40 50 Acetonitrile, % Figuro 2. Retentbn factor K of a column packed with completely hydrolyzed beads as a function of acetonttrile concentration in the mobile phase: column size, 1 0 0 ” X &” 1.d.; mobile phase, acetonitrile-water: flow rate, 1 mL/min; analytes, benzene(l), toluene (2), and ethylbenzene (3).
20
In an ideal hydrophilic packing, HSA must not be retained Separation media exhibiting a lower hydrophilicity than this ideal packing will partly retain HSA. Application of the HSA test on the hydrolyzed diol beads I1 revealed that 42% of total HSA was retained on the column at pH 3.8 while 94% was eluted at pH 7, the remaining 6% of HSA being eluted only with an eluent containing ethylene glycol. This confirms the fairly good, but not ideal, hydrophilicity of the hydrolyzed surface. However, a problem arises at higher concentrations of ammonium sulfate in the aqueous mobile phase. Various proteins are retained on the relatively hydrophilic surface even at pH 7, as documented by the plots of log k’ us salt concentration in the mobile phase (Figure 1). This can be attributed to the presence of some small hydrophobic areas that are only able to interact with the proteins when the mobile phase has a high ionic strength. These hydrophobic areas likely originate with the hydrocarbon portion of the chains within copolymer I1 because these are insufficiently shielded by the hydroxyl groups. Though the hydrolyzed beads contain some hydrophobic domains, the hydrophobicity of the pore surface is still insufficient for separations in the reversed-phase mode. Figure 2 showsalmost no retention of substituted benzeneswith mobile phases containing more than 60% of acetonitrile in water and no difference in retention times for benzene, toluene, and at anypH.
to prevent the adsorption of protein molecules on the surfaces of the pores. While these beads would not be suitable for the required separation, they would allow a useful comparison with other media prepared within this study. Human serum albumin (HSA) is an excellent probe for the determination of the relative hydrophilicities of chromatographic packings. HSA has a hydrophobic character when it is dissolved in a buffer at pH 5, the protein conformation changes and the newly exposed functionalities on the surface of the protein coil are more hydrophilic.20+21 At pH 3.5, HSA is weakly bound on small hydrophobic patches of the surface that are surrounded with hydrophilic groups. An increase in pH from 3.5 to 7 causes its release from the beads. In contrast, if strong multipoint interactions occur between protein groups and larger hydrophobic areas of the packing surface, then a glycol must be added to the mobile phase to allow complete elution.
0.5
0
2.0
,
I
Y -
Phenol, melt
(3)
KOH, 70 0
O
?
I V
0
1
1
5
4
5
6
Number of C atom8
Varletion of retention feetor K with the number of carbon atoms In a homdogous series 07 "JWed ammatic hydrocarbons (benzeneto amyhnzene): columti sk6,lCiWbn X-8-" i.d.; mobile phase,acetonitrile-water(3070);fiow rate, fflrVtWC aokmnpmkhg, completely hydrolyzed beads (11, two Wregeted chembtrles (e), and completely hydrophobized beads (3). Figure 3.
ethylbenzene even in 35% acetonitrile. The slopes of the straight lines shown are 2 ordets of magnitude lower than those for poly(styrene-co-divinylbenzene).22 A plot of the logarithm of the retention factor in acetonitrile-water mixture (30:70) us the number of carbon atoms of the substituents of aromatic hydrocarbon solutes (Figwre 3) confirms this very low hydrophobicity since its slope is only 0.076. Evaluation of Beads with Enhanced Hydrophilicity. Though the hydrolyzed beads are not suitable for reversed-phase separations, their pore surface still,,contains some small hydrophobic areas that induce the undesired protein-surface interactions. The reaction path designed for the preparation of our novel separation medium includes an additional hydrophilization step consisting aetion of the hydroxyl groups of diol beads XI with ohydrin followed by another hydrolysis of the newly imoduced epoxide groups, as shown in eq 2. This additional hydrophilizationstep results
0
II
in
N
in a better shielding of the hyd c main chains of the polymer, preventing their contac he protein molecules. The HSA test clearly documents thb high increase in the hydrophilicity: only 13% of HSA is $till retained at pH 3 8 with only 2% at pH 7 (well within the range of experimental error of the analysis method). Evaluation of Beads HydrophobiZliPd ib Phenol Melt. In contrast to the totally hydrolyzed beads, modification of the original glycidyl methacrylate beads'with phenol in the melt (eq 3) should afford a separation medium with a high surface monitoring of the concentration of phenyl groups. I modified beads by IR shows astrong t 690 cm-*assigned to a vibration of monosubstituted benkne rings. Reaction of epoxide beads I wit henol in the melt is expected to provide beads with a tophobicity that is (22) Lloyd, L. L. J . Chtomatogr. 1991, 544, 201.
'90
sufficient for the reversed-phase chromatography of small molecules. A plot of the logarithm of the retention factor in an acetonitrile-water mixture (30:70) us the number of carbon atoms in substituents of the aromatic hydrocarbon solutes (Figure 3) has a slope of 0.199, which is almost 3 times higher than that for the hydrolyzed beads. Due to the high hydrophobicity, a strong adsorption of protein moleculesshould also occur. The HSA test confirmed that all of the protein is absorbed at pH 3.5 and 81% at pH 7. Very low recoveries of other proteins at pH 7 (Table 2) also confirm the vastly enhanced hydrophobicity of this packing. Design of the Multimodal Medium. The preceding evaluation of model packings functionalized with a single type of chemistry leads to the following conclusions. (a) Beads with hydrophilized pores have sufficient hydrophilicity to prevent nonspecific interactions with proteins. While they are suitable for use with proteins, they do not interact with small molecules and are unsuitable for reversedphase chromatography. (b) Beads with pores functiofialized with phenol in the melt are sufficiently hydrophobic for the separation of small moleculesby reversed-phaseHPLC. These beads also interact strongly with proteins and could only be used for their separation if strong eluents such as acetonitrile were used. However, tbe use of acetonitrile causes denaturation of pioteins, hence a gentle method of separation such as hydrophobic interaction chromatography is preferred. Hydrophobic-interactionHPLC is an excellent method for the separation and purification of proteins because it provides for high recovery of proteins while preserving their biological activity, in contrast to reversed-phase chromatography. S u p ports for hydrophobic-interaction chromatography typically contain a low density of C1, Cq, or phenyl substituents located on a hydrophilic surface.23 The hydrolyzed beads described above provide the hydrophilic surface, but they do not carry the necessary low density of hydrophobic groups. However, the original epoxide groups of resin I can be reacted first with phenol under very mild conditionsto afford a relativelylow coverageof the hydrophilic surface with the hydrophobic phenyl groups that are required for protein separation. Though the degree of hydrophobicity achieved in this reaction is expected to be sufficient for hydrophobic interaction chromatography, it does not meet the requirements of reversed-phase separations that are only achieved for epoxide beads modified under forcing conditions in molten phenol. Therefore, the chemistry selected for both modes of the separation process is a s follows: a few hydrophobic sites (23) Shansky,R.E.Wu,S. L.;Figueroa, A.; Karger, B.L.In HfLCof Biological Molecules, Methods and Applications; Gooding, K.M., Regnicr, F. E.,Eds.; Dckker: New York, 1990; p 95.
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Table 2. Protein Recoveries from Columns Packed with Beads Containing Dlfferent Chemistries relative protein recovery, 76 protein MW hydrophobicity 1" 2b cytochrome c myoglobin ribonuclease A ovalbumin
BSA HSA conalbumine lysozyme chymotripsinogen trypsin inhibitor bovine plasma
13 400
17 000 13 700 45 000 66 700 69 000 75 500 14 100 24 000 21 500
0.0 0.03 0.1 1 0.23 0.24 0.27 0.29 0.38 0.52 0.57
88 89 34 49 23 19 51 48 19 2 49
99 103 97 96 93 94 94 104 93 88 101
1
Polymeric reagent
0 Column 1contains beads hydrophobized in the phenol melt. Column 2 contains beads D with a bimodal chemistry.
dispersed in a hydrophilic medium to allow for the hydrophobic interaction chromatography of proteins and strongly hydrophobic sites for reversed-phaseseparation of small molecules. The next requirement is the incorporation of both types of chemistries in different pores of the medium in such a way that proteins will never come into contact with the highly hydrophobic surface of reversed-phase sites. Concept of the Pore-Size-SpecificModification. Almost all porous polymers are characterized by a relatively broad pore-size distribution. Access to the different pores is controlled by the hydrodynamic volume of the dissolved molecules. Molecules will only penetrate those pores that are able to accommodate their size while smaller pores remain inaccessible for steric reasons. The size-restricted access of molecules to a porous structure, which is also the basis of the technique of size-exclusion chromatography, can be used to prepare a new generation of polymeric separation media. The functionalization of the pore surface is carried out in a sizespecific fashion that allows the introductionof different surface chemistries within well-defined families of pores in a single porous bead. The concept is shown schematically in Figure 4, where a hypothetical pore is depicted as a planar projection of a cone. The surface of this pore is covered with reactive groups (e.g. epoxides) that may be modified by a reaction (hydrolysis) that is catalyzed by a polymeric catalyst. The ability of the functional groups contained in different pores to react is controlled by the relative size of the catalyst molecule and the pores. The reaction occurs only in those parts of the hypothetical pores that are accessible to the large catalyst molecules while reactive groups located in the inaccessible parts remain unchanged. These can be used in a subsequent reaction step for a different modification reaction that leads to creation of different surface chemistry in the small pores. Although the pores of macroporous beads are not cones but irregular voids,24this broad concept applies to all types of porous materials regardless of the shape of the pores. Preparation and Characterizationof the SeparationMedium with Bimodal Chemistry. The process used for preparation of the separation medium with bimodal distribution of chemistries consists of four steps (Scheme 1): reaction of groups in all
pores with phenol under mild conditions, activation of the newly created hydroxyl groups with epichlorohydrin, poresize-specific hydrolysis of the epoxide groups in large pores, and additional hydrophobization of the small pores with phenol under forcing conditions. Reaction of poly(glycidy1 methacrylate-co-ethylene dimethacrylate) beads with aqueous phenol (Scheme 1, modification step a) introduces a low density of phenyl groups throughout the beads. The reaction is base induced and the degree of substitution depends on the molar ratio of phenol to epoxide groups.25 Since the reaction proceeds in alkaline water, a significant proportion of the original epoxide groups are hydrolyzed and the product contains both phenyl and hydroxyl groups. IR analysis of the beads shows that ca. 13% of all available epoxide groups are in fact substituted with phenol as calculated from the ratio of absorbances at 690 cm-l (vibration of monosubstituted benzene ring) and at 2940 cm-l (valence vibration of aliphatic CH bonds).25 The IR spectrum also shows a large hydroxyl band at 3100-3800 cm-1.
(24) Sherrington,D. C.; Hodge, P. Syntheses and Separations Using Functional Polymers; J. Wiley: New York, 1989.
(25) Horak, D.; Straka, J.; Stokr, J.; Schneider, B.; Tennikova, T. B.; Svec, F. Polymer 1991, 32, 1135.
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Low molecular weight reagent
Figure 4. Schematic view of a pore-size-specific modification.
&h.rm 1
Y
O
?
RESIN I
BEADS A
BEADS C
SLIGHTLY HYDROPHOBIC
BEADS D
HKiHLY HYDROPHOBIC
In the second modification step (b) the entire surface of the beads is activated again by a base-catalyzed reaction of its hydroxyl groups with epichlorohydrin.26 As a result, the beads again contain epoxide groups as confirmed by IR spectrometry (peaks at 906 and 852 cm-l). The epoxide content, determined by volumetric titration, was 1.% mmol/ g* The third step (modification step c) is a pore-size specific hydrolysis. As demonstrated earlier,*5a polymeric acid such as poly(styrenesu1fonic acid) will only hydrolyze the epoxide (26) Turkova, J.; Blaha, K.;Horacek, J.; Vajcncr, J.; Frydrychova, A.; Coupek, J. J. Chromatogr. 1981, 215, 165.
groups in pores large enough to accommodate it. Since our aim is to prepare a separation medium for mixtures containing proteins, the epoxide groups located in pores large enough to allow permeation of proteins should be hydrolyzed. The range of pores permeable for a particular protein will of coursedepend on its size. For example, the radius of gyration of human serum albumin (MW 69 000) is 3.1 nm, which corresponds to a "solid sphere" radius of ca. 4 nmS8Therefore, HSA would be able to penetrate pores with an apparent diameter of >8 nm. The smaller the proteins, the smaller the pores that are penetrated. The smallest protein used in this study is cytochrome c, with a molecular weight of 13 400. Therefore, to remain on the safe side, a poly(styrenesu1fonic acid) with molecular weight of 5 000 was chosen as the catalyst for the pore-size-specific hydrolysis process. After the pore-size-specific hydrolysis, the remaining epoxide groups are located only in the small pores. Since these pores have to be used for the separation of small molecules in the reversed-phase mode, their hydrophobicity should be as high as possible. Therefore, the last reaction step transforms the remaining epoxide groups into phenyl ethers. To obtain maximum conversion without concurrent hydrolysis, the reaction is done under forcing reaction conditions (KOHin molten phenol). The additional hydrophobization of small pores is reflected by an increase of the IR absorption maximum at 690 cm-l. Despite the several consecutive reaction steps, the beads did not change in their appearance and no crushed or colored beads were detected by optical microscopy. Evaluation of Beads with Bimodal Distribution of Chemistries. Because the bead surface chemistry has been incorporated in two different surface density levels that are segregated in pores of different sizes, the packing material may be used for two different modes of HPLC. The hydrophilic surface of the large pores is modified with only enough hydrophobic phenyl groups to enable a gentle hydrophobic interaction chromatography of proteins while the smaller pores are functionalized with a high density of phenyl groups providing them with the level of hydrophobicity that is required for reversed-phase chromatography. Since two types of segregated surface chemistries are present in each bead of the packing, it may be used for the separation of samples in either of the two single chromatographic modes, or in the separation of complex samples using both modes in a consecutive fashion. Test of the Small Pores under Reversed-Phase Conditions. The reversed-phase properties of the beads were evaluated first in a single chromatographic mode. Figure 5 shows the effect of the volume fraction of acetonitrile in the mobile phase on the retention factor for various compounds including anticonvulsant drugs and aromatic hydrocarbons. This data suggests that the resolution will be sufficient for the separation of hydrophobic compounds even in the isocratic elution mode. The linearity of the plot confirms that despite the bimodal chemistry the separation is controlled by the type of interactions that are characteristic of reversed-phasechromatography.The straight line of the dependency of the logarithm of retention factor k' on the number of carbon atoms in the benzene substituents has a slope of 0.158 (Figure 3), which is close to that of theunimodal beads that had been totally hydrophobized Ana!ytical Chemistry, Vd. 88, No. 13, July 1, 1994
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Acetonitrile, W Figure 5. Retention factor k‘ of a column for the separation of small molecules as a function of acetonitrile Concentration in the moblle phase: column size, 100-mm X &”1.d.; mobile phase, acetonitrllewater; flow rate, 1 mL/min; analytes, barbital (l), phenobarbltal (2), benzene (3), toluene (4), and ethylbenzene (5).
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in the phenol melt (0.199). It also documents the generally small contribution of large pores to the total hydrophobicity of separation media. A Van Demteer plot (Figure 6) obtained for the column packed with the 10-pm beads shows that the plate height achieved with the beads at a flow rate of 1.5 mL/min is 35 pm for a plate number of 29 000 and a reduced plate height of 3.5 d,. Due to the monodispersity of the beads, the back pressure in the column is low over the entire range of flow rates (Figure 6). For example, a back pressure of only 9.5 MPa was observed at a flow rate of 6 mL/min. The linearity of the back pressure us linear mobile-phase velocity documents the very good mechanical stability of the beads. The high efficiency of the column and its low back pressure compare favorably to those of commercial poly(styrene-codivinylbenzene) phases.22 The performance of the column remains unchanged even after more than 1000 repetitive injections. The testing protocol involved 200 injections of toluene in acetonitrilewater (80:20) interspersed with 50injections of bovine plasma solution in 0.01 mol/L phosphate buffer (no retention). The results shown in Figure 7 clearly confirm the excellent stability of both column efficiency and retentivity even after numerous changes of the chromatographic mode. Test of the Large Pores in Hydrophobic-Interaction Chromatography. The retention of various proteins under conditions typical for hydrophobic-interaction chromatography (Figure 8) confirms the separation ability of the large 2136
400
Number of injections Flgure 7. Stability of column for two-dimensional separation as measuredfrom changesof plateheight (O)andtoluene retention factor k‘ C)upon multiple Injections; column size 100-mm X 8-mm 1.d.; mobile phase, water-acetonitrile (80:20); flow rate, 1mL/mln; analyte, toluene.
O.O
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Am&tkdchemistty, Vol. 66, NO. 13, July 1, 1994
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Ammonlum sulfate, mol/L Flgure8. Effectof ammonium sulfate concentratbnon retentkn factor of varlousproteinsina columnpacked with beadscontainingw e g a t e d chemlstrles; column size 1 0 0 ” X &mm 1.d.; mobile phase, 0.02 mol/L phosphate buffer solution (pH 7); flow rate, 1 mllmin; UV detection; analytes, cytochrome c (I), myogloblne(2), ribonucleaseA (9,bovlne serum albumln (4), conalbumln(5), chicken egg albumin (6), human serum albumln (7), lysozyme (8), chymotrypslnogen (9), and soya bean trypsin Inhibitor (10).
pores. All of the proteins tested can be easily separated in a salt gradient except for the group of albumins which have very similar k’at the same concentration of the antichaotropic salt in the mobile phase. The retention curves of the proteins do not exhibit the concave shape that is often characteristic of silica-based media,2’ confirming the absence of charged groups at the surface of the large pores. The protein recoveries obtained with the bimodal beads are not very different from those of unimodal beads in which uniform hydrolysis of all epoxide groups had been carried out (Table 2). Figure 1 clearly documents that the retention factors of proteins in the beads D containing a low density of phenyl chemistry in the large pores are higher at any measured salt concentration as compared to completely hydrolyzed beads A. This measurement confirms the assumption that the hydrophobicity of diol beads A should be lower than that of the phenyl beads D,and therefore, it also confirms the positive effect ofthe phenyl groups present in the large pores. Test in the Separation of Complex Mixtures. The pore sizeselectivity of the modification process and the segregation of both chemistries in different pores is confirmed by the experiments shown in Figure 9 in which the mobile phase was (27) Kennedy, L. A.; Kopaciewicz, W.; Rcgnier, F. E.J. Chromarogr. 1986,359, 73.
’
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Flgure Q. Hydrophoblc Interaction and reversebphase consecutive elution of bovine serum albumin from a column padced wlth beads contalnlng segregated chemistries (A) and from a column completely modffled wlth hlgh coverage of phenyl groups (9): column size, 100mm X 6” Ld.; hydrophobic Interaction chromatography, mobile phase gradient decreasing from 1.7 to 0 moi/L ammonium sulfate In 0.02 mol/L phosphate buffer soluth (pH 7) In 15 mln; reverse phase chromatography, mobilephase of 0.02 mol/L phosphate buttersokrtkn (pH 7)-acetonltrlle mixture (65:35 v/v); flow rate, 1 mL/mln; UV detection.
changed during elution. All injected bovine serum albumin is eluted within the salt gradient and no peak is observed after switching the mobile phase to 65% acetonitrile in aqueous buffer (Figure 9A). In contrast, no elution of the protein was achieved in the salt gradient while a sharp peak was obtained only under the reversed-phase conditions when BSA was injected into a column packed with unimodal beads completely modified in the phenol melt and the same elution path was used (Figure 9B). If the separation of a mixture consisting of proteins and alkyl-substituted benzenes is made in a single isocratic reversed-phase mode with 35% acetonitrile in buffer while omitting the hydrophobic interaction mode (Figure lo), all four proteins are eluted in a single peak at retention time close to that of an unretained compound, followed by peaks of wellseparated aromatic hydrocarbons. Figure 10 confirms that the hydrophobic interaction mechanism must be operative for protein separation to occur in the two-dimensional mode. Since there are significant differences between conalbumin (MW 75 000) and the other proteins (MW ca. 14 000) and no separation occurs, this result also confirms the absence of size-exclusion effects in the protein separation. It does not mean that the packing would have no intrinsic ability to separate proteins according to their hydrodynamic volumes. However, the efficiency of the separation at a flow rate of 1 mL/min is low and not sufficient for any size-exclusion separation and much lower flow rates and a longer column would have to be used for this process. The final target, an experimental demonstration of the separationof complex samplesconsistingof proteins and either
40
Flgure 10. Separation of protelns and atkylbenzenes In the reversedphasechromatogmphlcmode In a columnpackedwith beads contalnlng segregated chemlstries: column size, 1 0 0 ” X &mm 1.d.; mobile pheee, 0.02 mol/L phosphatebuffer sdutkn (pH 7)-acetonltrllemlxture (8595 vlv); flow rate, 1 mL/mln; UV detection; peaks, unseparated mlxtwe of cytochrome c, ribonuclease A, conalbumin, and lysozyme (1X benzene (2), tokreMt (31, ethylbenzene (4), propylbenzene (5), butylbenzene (a), and amylbenzene (7).
3
7
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60
Time, min Flgure 11. Separation of proteins and alkylbenzenes In consecutive hydrophobic Interaction and reversebphase chromatographic modes: column size, 1 0 0 ” X 8”1.d.; hydrophobic interaction chromatography, mobile phase gradient decreasing from 1.7 to 0 m o i / ~ ammonlum sutfate In 0.02 mol/L phosphate buffer solution (pH 7) In 15 mln; reversephase chromatography, mobile phase of 0.02 mol/L phosphate buffer solotion (pH 7)-acetonitrlle mixture (8535 v/v); flow rate, 1 mL/min; UV detectlon; peaks, cytochrome c (l), ribonuclease A (2), conalbumin (31, lysozyme (4), soya bean trypsin inhlbitor (5), benzene (B), toluene (7), ethylbenzene (8),propylbenzene (9), butylbenzene (lo), and amylbenzene (11).
aromatic hydrocarbons or drugs is illustrated in Figures 11 and 12. Figure 11 shows the separation of a complex mixture consisting of proteins and aromatic hydrocarbons. After the AMW-1
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does not specify the number of fractions required in each dimension and even a very limited separation into two classes of compounds can be sufficient. The first step includes two different separations: the separation of the two families of components from each other, followed immediately by the separation of one of these families. The remaining part of the sample is then separated in the second step. Obviously, each sample component is subjected to at least two independent separative steps, and we can safely refer to the media reported in this study as "two-dimensional-like". Figure 12 shows a similar separation of a mixture of proteins and hydrophobic drugs. Here again, the same two-dimensional separation mechanism applies. The drugs are first captured within the small pores separately from the proteins and then released and separated by changing the mobile phase after the proteins have been already separated. The excellent separation of both families of compounds in two different chromatographic modes confirms that the poresize-specificfunctionalization provides separation media with two segregated chemistries and clearly documents the feasibility of this new approach to separation of complex mixtures.
Flgurr 12. Separation of proteins and anticonvulsant drugs in consecutive hydrophobic interaction and reversedghase chromatographic modes: column size, 100" X Bmm 1.d.: hydrophabk interaction chromatography, mobile phase gradlent from 0.02 mol/L phosphate buffer solution containing 1.7 moi/L ammonlum sulfate (pH 7) to the buffer in 15 min, reverse-phase chromatography, mobile phase of 0.02 moi/L phosphate buffer solution (pH 7)-acetonltrile (65: 35 vlv) mixture: flow rate, 1 mL/min: UV detection; peaks, cytochrome c (l), ribonuclease A (2), conalbumin (3), lysozyme (4), soya b a n trypsin lnhibkor (51, phenobarbital(61, carbazepamlne(7), and phenytoin (8).
injection,all components of the sample are completelyadsorbed at the top of the column. The adsorption is size selective and the sample is separated into two portions according to the hydrodynamic sizeof the components and their affinity toward the pore surface. The proteins remain in the large pores while the small molecules penetrate the small pores where they interact with the highly hydrophobic surface. This process-the separation of two families of compounds in different pores where they are held by different interactions-is actually the first step of the separation. The proteins are then eluted by a decreasing ammonium sulfate gradient in the hydrophobic interaction mode, whereas the segregated small molecules are retained within the hydrophobic small pores. These are not eluted until a mobile phase containing acetonitrile is applied and isocratic reversed-phase-mode elution is used. These separations of both parts of the original sample represent the second separation step. Therefore, our bimodal columns seem to meet Giddings' definition for multidimensional separation. This definition
2138 Ana~lcaIChemlstry,Vol. 66, No. 13, Ju@ 1, 1994
CONCLUSION The pore-size-specific modification process appears to be a powerful tool for the preparation of separation media with different chemistries segregated in pores of different sizes. The main advantage of the novel HPLC medium appears to be its ease of preparation and its excellent performance. In contrast to existingmaterials, the separation medium developed in this study is bimodal with respect to both the mobile phase and the stationary phase. Though the concept is demonstrated with the preparation of a specific bimodal medium and its use in the separation of proteins in hydrophobic interaction mode and small molecules in reversed-phase mode, many other combinations of chemistries are also feasible. These, together with the preparation of beads containing three different segregated chemistries, are currently under investigation. ACKNOWLEDGMENT Support of this research by a grant of the National Institutes of Health (GM 44885-05) is gratefully acknowledged. This work also made use of MRL Central Facilities supported by the National Science Foundation under Award No. DMR9 12 1654. A useful discussion with Professor Regnier is also acknowledged with thanks. Received for review December 1, 1993. Accepted March 23, 1994.' Abstract published in Advance ACS Abstracts, May 15, 1994.
