Anal. Chem. 1994,66, 4308-4315
TwomDimensional HighmPerformance Liquid Chromatography Using Monodisperse Polymer Beads Containing Segregated Chemistries Prepared by Pore Size Specific Functionalization. SinglemColumn Combinations of Size Exclusion or lon Exchange with ReversedmPhase Chromatography Vladimir Smigol, Frantisek Svec, and Jean M. J. Frechet* Baker Laboratory, Department of Chemistry, Cornel1 University, Ithaca, New York 14853-1301
Separation media for the complete separation of complex samples that require a combination of size exclusion or ion-exchangewith reversed-phase chromatographic modes in a single column have been prepared from size monodisperse 10 pm poly(glycidy1 methacrylate-co-ethylene dimethacrylate) beads using a pore size specific functionalization process. To achieve the first combination of chromatographicmodes, the large pores of the beads were selectively hydrolyzed to diols using aqueous poly(styrenesulfonic acid), while highly hydrophobic octadecyl groups were introduced into the small pores by reaction of the remaining epoxide groups with octadecylamine. These beads provide excellent protein recoveries and may be used for the direct injection separation of samples containing both hydrophilic proteins and hydrophobic drugs. Beads containing diethylaminogroups in the large pores and octadecylfunctionalities in the small pores were also prepared by sue-selective modification. A plot of log k' against ionic strength of the mobile phase for these beads shows the absence of hydrophobic interactions and documents the clean ion-exchange mechanism of protein separation. Examination of the small pores in both types of separation media confirmed that their hydrophobicity was s a c i e n t to allow the separations of small molecules in reversed-phase mode. Column packed with these dualchemistry beads exhibited high efficiencies and were used successfullyfor the separations of proteins and alkylbenzenes or drugs. Multidimensional chromatography provides access to dramatic improvements in peak capacity since capacity in a two-dimensional separation is equal to the product of peak capacities in both dimensions.' In addition, much better resolution is expected in the separation of complex mixture^.^,^ Major fields of application for two-dimensional techniques are thin-layer chromatography and (1) Giddings, J. C. J. High. Resolut. Chromatogr., Chromatogr. Commun. 1987, 10, 319. ( 2 ) Giddings, J. C. Anal. Chem. 1984, 56, 1258A. (3) Little. E. L.; Jeansonne, M. S.; Foley, J. P. Anal. Chem. 1992, 63. 33.
4308 Analytical Chemisfry, Vol. 66, No. 23, December 7, 7994
electrophoretic separation^.^ While two-dimensional separation is readily achieved with the planar medium of thin-layer chromatography by simply rotating the medium 90" after the first separation and proceeding with further elution, this approach cannot be used with a HPLC column. Therefore, chromatographic methods that involve column switching have been developed. A peak containing a class of compounds separated from the rest of the sample in the first column is directed into a second column where it is separated into its individual components! For example, coupled size exclusion and reversed-phase columns have been used for the separation and detection of components in plant extracts5s6and in rubber stock.6 Several columns may also be used to separate different families of compounds, but in this case the instrumentation becomes rather complex. Stationary phases based on modified porous silica with both hydrophilic outer surface and hydrophobic pores (internal surface reversed-phase media, ISRP) have been developed by P i n k e r t ~ n . ~ These columns allow the direct injection of complex matrices such as plasma, serum, saliva, or urine into a column for the determination of drugs and metabolites without any pretreatment, while also preventing the accumulation of proteins and the resultant clogging of the column. ISRP phases are prepared in a multistage process that begins with a complete hydrophobization of the entire surface of small-pore silica beads. In the next step, an enzyme that is totally excluded from the inner pores cleaves the hydrophobic moieties only at the outer surface of beads rendering it hydrophilic. The hydrophobic groups within the pores remain unchanged. In separation processes involving the ISRP packings, all proteins are eluted as one peak at V, followed by the other separated analytes. Following this initial work, several varieties of these restricted access media have been The main advantage of these packings is that fouling of the beads by accumulation of (4) Multidimensional Chromatography: Techniques and Applications. Cortes, H.
J., Ed.; M. Dekker: New York, 1990. (5) Emi, F.; Frei. R W. J. Chromatogr. 1978, 149, 561. (6) Johnson, E. L.; Gloor, R; Majors, R E. J. Chromatogr. 1978, 149, 571. (7) Hagestam, I. H.; Pinkerton, T. C. Anal. Chem. 1985, 57, 1757. (8) Pinkerton, T. C. J. Chromatogr. 1991, 544, 13. (9) Haginaka, J. Trends Anal. Chem. 1991, 10 (l),17. 0003-270019410366-4308$04.50100 1994 American Chemical Society
proteins on their surface is prevented while small molecules penetrate the beads and are separated in normal fashion. We have recently introduced the concept of pore size specific functionalization to prepare porous media in which pores within different size ranges may be endowed with different ~hemistries.l'-~~ This concept relies on the use of catalysts with defined molecular volumes that are able to perform a chemical moddcation 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 for which kinetics factors are the sole consideration. The method is well suited for the preparation of separation media with pores modified according to their size and allows the preparation of beads with different chemistries segre gated in different size pores. This concept also adds a new perspective to chromatographic separation of complex samples. Its feasibility was first documented with the preparation of a separation medium for the directinjection HPLC determination of drugs in biological fluids.14 The concept of pore size specific modification was then extended with the preparation and use of a single packing in which hydrophobic interaction chromatography of proteins and reversed-phase chromatography of small molecules could proceed conse~utively.~~ While our earlier communication was focused on a medium with only phenyl surface chemistry that had different coverage levels segregated in different size pores (gradient of coverage density),I5 this report concerns separation media, in which totally different chemistries are localized separately in small and large pores, respectively (gradient of chemistries), and their application in HPLC separations of complex samples. EXPERIMENTAL SECTION
Materials. A narrow molecular weight distribution sodium poly(styrenesulfonattt) (M, = 5000, M,/M,, = 1.09) was obtained from Scientific Polymer Products, Inc. (Ontario, NY). Prior to its use, the sodium salt was converted to the free acid by an ionexchange process using a AG 5OW-X strong acid cation exchanger @io-Rad, Richmond, CA). Vitamin BIZ (cyanocobalamin, MW 1356),cytochrome c (from bovine heart, MW 13 400), ribonuclease A (from bovine pancreas, MW 13 600), lysozyme (MW 14 loo), myoglobin (from horse heart, MW 17 000), soya bean trypsin inhibitor (MW 22 500), carbonic anhydrase (from bovine erythrocytes, MW 29 OOO), chicken egg albumin (ovalbumin, Grade V, MW 45 000), bovine serum albumin (98-99%, MW 66 700), human serum albumin (essentially fat free, MW 69 000), conalbumin (from chicken egg white, Type I, MW 75 500), alcohol dehydrogenase (from yeast, MW 150 000), P-amylase (from sweet potatoes, MW 200 000), apoferritin (from horse spleen, MW 443 000), bovine thyroglobulin (MW 669 000), hemocyanin (from limulus polyphemus hemolymph, MW 3 750 000), bovine dried plasma, and the anticonvulsant drugs brocainamide, lidocain, carbamazepine, phenytoin, desipramine, phenobarbital) were purchased from Sigma. The hydrocarbon standards were obtained from Aldrich. All solvents were HPLC grade. (10)Anderson, D.J. Anal. Chem. 1993,65, 434R (11) Frechet, J. M. J. Macromol. Chem., Macromol. Symp. 1993,70/71,289. (12)Smigol, V.;Svec, F.; Frechet, J. M. J. Macromolecules 1993,26, 5615. (13)Svec, F.; Frechet, J. M. J. Ado. Mater. 1994,6,342. (14)Smigol, V.;Svec, F.; Frechet, J. M. J.J. Liquid Chromatogr. 1994,17,891. (15)Smigol, V.;Svec, F.; Frechet, J. M. J. Anal. Chem. 1994,66,2129.
Polymer Beads. Uniformly sized 10-pm porous 60:40 vol % glycidyl methacrylate-ethylene dimethacrylate copolymer beads (resin I) were prepared by a modified shape template swelling and polymerization method described in detail elsewhere.16J7 Cyclohexanol (60 ~ 0 1 %was ) used as the porogenic solvent. The specific surface 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.14 Preparation of the Sepamtion Media. The beads containing multiple functionalities were prepared by the modification process shown in Scheme 1. Pore Size Specific Hydrolysis of Epoxide Groups. The hydrolysis of resin I containing epoxide groups (Scheme 1, modification step a) was catalyzed with 1 wt.% 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, partly modfied, beads A containing functionalities I1 were then filtered off on a fritted glass filter and washed with water until neutral, washed with acetone, and dried in vacuo at room temperature. Aminolysis of Residual Epoxide Groups with Octadecylamine. Beads A (1.5 g) containing both vicinal hydroxyl groups I1 in large pores and residual epoxide groups in small pores were admixed to 5 g of molten octadecylamine in a 50-mL round-bottom flask at 70 "C, and the mixture was stirred for 16 h (modification step b). The reaction mixture was then diluted with 20 mL of 1,4-dioxane, stirred for 30 min, and filtered. The resulting beads B containing groups I11 were washed with dioxane and water. Any unreacted epoxide groups remaining due to steric constrains were hydrolyzed in 20 mL of 0.1 mol/L sulfuric acid at 60 "C for 4 h. The beads were filtered, washed with water and then with methanol, and dried. The amino group content of the beads was 0.35 mmol/g as determined by elemental analysis of nitrogen as well as by an acid-base titration. Reaction with Epichlorohydrin. Dry beads B from the previous reaction step were dispersed in 20 mL of water and stirred magnetically for 16 h. The excess water was removed on a fritted glass filter, and the beads were redispersed in 20 mL of 50 wt.% aqueous KOH and stirred for 1 h. M e r removal of the liquid, the beads were added to 40 mL of an epichlorohydrinwater (1:l) mixture and stirred at room temperature for 3 h (modfication step c). The product was washed with water and acetone and dried to afford epoxidized beads C containing functionalities lV (1.12 mmol/g epoxide groups determined by volumetric titration). Aminolysis of Epoxide Groups with Diethylamine. The epoxidized beads C (1.5 g) were placed in a 50-mL round bottom flask and 20 mL of diethylamine was added. After heating at reflux (16)Smigol, V.;Svec, F. J. Appl. Polym. Sci. 1992,46, 1439. (17) Smigol, V.;Svec, F. J. Appl. Polym. Sci. 1993,48, 2033. (18)Halasz, I.; Martin, IC Angew. Chem., Int. Ed. Eng. 1978,17,901. Analytical Chemistty, Vol. 66,No. 23, December 1, 1994
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(55 “C) for 6 h, the resin was filtered and washed with water (modification step d). Any remaining unreacted epoxide groups were again hydrolyzed in 20 mL of 0.1 mol/L sulfuric acid at 60 “C for 4 h to afford beads D containing functionalities V. Finally, the beads were filtered, washed with water and then with methanol, and dried. The beads contained 0.62 mmol/g groups V, as determined by elemental analysis of nitrogen after subtraction of the nitrogen introduced with octadecylamino groups. High-Performance Liquid Chromatography. The properties of the beads prepared in this study were determined by chromatography carried out in a 300-mm x 7.8-mm i.d. or 50mm x 8-mm i.d. stainless steel columns 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 W detector. Protein Recovery. The protein recovery from the beads packed in a 100.” x 8-mm i.d. column was measured upon isocratic elution at ambient temperature. Recovery was calculated as the percentage of the peak area of protein leaving the column 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. The mobile phase was 0.15 mol/L sodium chloride in 0.15 mol/L phosphate buffer (PH 7) for the size exclusion mode, while 0.5 mol/L sodium chloride in 0.01 mol/LTRISHCl buffer solution (PH 7.6) was used for the ion-exchange mode. All other chromatographic conditions were identical for both modes: flow rate 1 mL/min, 10 mg/mL protein solution in buffer, injection 20 pL, detection 280 nm. The experimental error is less than 10%. RESULTS AND DISCUSSION
Direct HPLC separations of complex biological samples either require a quite elaborate equipment that involves several columns, pumps, and valves or a less important part of the mixture is eluted as a single peak without any separation of its components. The dual-column two-dimensional HPLC technique falls in the first category4 while the single-column separations that use the restricted access media represent the other gro~p.~-lOObviously, the second approach that involves only a single column is simpler, and therefore, it is preferred in the separation of biological samples. However, this technique only provides limited information as the proteins are simply eluted in a single peak. The recently introduced11-15 approach involving a single column packed with a separation medium that combines two segregated chemistries overcomes this limitation, separates all of the components of the mixture, and provides a more complete analysis. Reversed-phase chromatography is the most frequently used separation mode for small hydrophobic compounds while biological macromolecules such as proteins are typically separated by size exclusion, ion-exchange, or hydrophobic interaction chromat o g r a p h ~ .Therefore, ~~ a combination of reversed-phase chromatography with one of the other modes would be ideally suited for the separation of complex mixtures that contain both small molecules and proteins. In order to avoid contact of the proteins with the highly hydrophobic surface that is required for reversed phase, the pores that are readily accessible to the proteins must not contain any such groups. The highly hydrophobic functionalities must be “hidden” in pores that are smaller than the (19) HPLC of Biological Molecules, Methods and Applications: Gooding, K M.,
Regnier, F. E., Eds.; M. Dekker: New York, 1990.
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Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
hydrodynamic size of the protein molecules. The restricted access media solve this problem by using porous silica beads in which essentially all pores are smaller than the proteins. Once the external surface of the beads has been made hydrophilic, the proteins have no tendency to adhere to it and they leave the column without being separated since they cannot penetrate the beads7 In our approach, we use beads with a relatively broad pore size distribution; therefore, the proteins penetrate those pores that are large enough to accommodate them but they encounter a surface chemistry that has been optimized for their separation. This is because only the pores smaller than the proteins are functionalized with the hydrophobic chemistry. Therefore no proteins can stick to the column, yet the reversed-phase separation of small molecules is still possible. In order to document the feasibility of the pore size specific modification process for the preparation of such a dual-chemistry medium, we prepared beads containing C18 groups in small pores and either hydroxyl or diethylamino groups in the large pores. These materials can be used for the separation of mixtures of proteins and small hydrophobic molecules using consecutive reversed-phase and either size exclusion or ion-exchange chromatographic modes. Design of the Separation Medium with Bimodal Chemistry. The concept of the pore size specific modification has been described in detail p r e v i o ~ s l y . ~ l - Briefly, ~ ~ J ~ the concept relies on the restricted access of molecules to porous structure as a function of their hydrodynamic volume, a phenomenon that is also the basis of the technique of size exclusion chromatography. Therefore only large pores are modified when a polymer catalyst is used. For example, the epoxide groups of the poly(glycidy1 methacrylate-methylene dimethacrylate) are modified in a pore size specific fashion by a hydrolysis reaction that is catalyzed by poly(styrenesulfonic acid). The small pores remain completely unchanged during this reaction but they can be modified later by reaction with a low molecular weight reagent that is small enough to penetrate them, such as octadecylamine. Since the large pores are already “deactivated”, the later reaction takes place exclusively in the small pores and both chemistries are segregated. The process used for the preparation of the media with bimodal distribution involves only a few steps shown in Scheme 1. Hydrolysis of poly (glycidylmethacrylate-coethylene dimethacw late) beads catalyzed by poly (styrenesulfonic acid) (MW 5000; modification step a) introduces vicinal hydroxyl groups in pores large enough to accommodate the polymeric acid. The molecular weight of the polymeric catalyst chosen was lower than the typical molecular weight of proteins to be separated (> lo4) in order to be on the safe side and avoid any contact of the proteins with the highly hydrophobic functionalities contained in the smaller pores. In the second reaction step (modification step b, Scheme l), the remaining epoxide groups react with octadecylamine in dioxane to afford beads B. The content of the hydrophobic octadecyl groups is 0.35 mmol/g according to N analysis. These beads can be immediately used for the separation of mixtures using a combination of size exclusion and reversed-phase chromatography. Two additional steps are required for the preparation of a different separation medium that combines the characteristics needed for both ion-exchange and reversed-phase chromatography. First, the hydroxyl groups are “activated” again20by abase(20) Turkova, J.: Blaha, IC;Horacek, J.: Vajcner, J.: Frydrychova, A; Coupek. J. J. Chromatogr. 1981,215, 165.
Scheme 1
1.6
1
Resin I
Modification step (a)
acid) + water
O.O
BEADSA
-0.8
O A OI
1
I
I
I
I
0
10
20
30
40
~
50
Acetonitrile, 9b Mod/ficationstep (b) Octadecyiamine
BEADSB
Modification step (c)
1
C
I
A
+KOH
Figure I. Retention factor K of a 50-mm x 8-mm i.d. column packed with beads D (open points) and a 300-mm x 7.8-mm i.d. column packed with beads B (closed points) as a function of acetonitrile concentration in the mobile phase: mobile phase, acetonitrile-water; flow rate, l mumin; analytes, benzene (squares) and ethylbenzene (triangles).
BEADS C
IV
H
1
I
1.0
OH
L a
ModiNcarion step (d)
I
1.5
-
0.5
C BEADS 0
0.0
j
1
I
I
I
I
0
1
2
3
4
5
I
Number of alkyl C atoms
catalyzed reaction with epichlorohydrin (modification step c, Scheme 1). The epoxide content determined by volumetric titration was 1.12 mmol/g. Though there are some hydroxyl groups present even in the small pores, their reaction is unlikely both because of the shielding effect of the bulky substituent and because of their hydrophobic environment that impairs reaction in the aqueous medium. The final reaction step is the aminolysis of the epoxy-activated beads with diethylamine (mod~cationstep d, Scheme 1) to afford beads D containing N,N-diethylamino groups (0.62 mmol/g). As a final precaution, the beads are treated again with dilute sulfuric acid to ensure that no epoxide groups remain because these might react with the proteins and interfere with their separation. At this stage, the beads contain segregated hydrophobic and ionizable groups useful for a combination of ionexchange and reversed-phase chromatography. Because all the above modification reactions proceed under relatively mild conditions, the appearance of the products monitored by optical microscopy does not change. Chromatographic Evaluation of Beads with Bimodal Chemistry. The dualchemistry beads can be used not only to separate complex mixtures but also for the simple separation of either family of compounds. Therefore, their chromatographic properties were evaluated first in a single mode. Reversed-Phase Chromatographyof Small Molecules. A characteristic of reversed-phase chromatography is the linearity
Figure 2. Variation of retention factor K with the number of carbon atoms in a homologous series of substituted aromatic hydrocarbons (benzene to amylbenzene): columns, 50-mm x 8-mm i.d., beads D (A) and 300-mm x 7.8-mm i.d., beads B (m); mobile phase, acetonitrile-water (2575); flow rate, 1 mumin.
of the plot of retention factor k' vs volume fraction of acetonitrile in the mobile phase. This provides for a simple test of the retention mechanism. Indeed, the dependency was found to be linear for various compounds including alkylbenzenes and anticonvulsant drugs. Figure 1 shows the effect of mobile-phase composition on the retention factors of benzene and ethylbenzene for the columns packed with beads B and D and documents the linearity of the dependency. Retention factors in a 25:75 vol % acetonitrile-water mixture were plotted against the number of carbon atoms in the alkyl chain of the alkylbenzenes. Figure 2 shows that these plots are also linear with a slope of 0.155 and 0.185 for the beads having 12-diol and diethylamino chemistry in their large pores, respectively. The slopes represents the retention increase caused by one methylene group in the alkyl chain of alkylbenzenes (selectivity a) and compare favorably to those of C18alkylated silica and alkylated polymer gels.21Surprisingly, since both beads have the same chemistry in their small pores, the selectivity of beads D with the diethylamino chemistry in the large pores is higher than that of the hydrolyzed 1,Zdiol (21) Tanaka, N.;Araki, M. Adu. Chromotogr. 1989, 30,81.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4311
5
300
E
2oo
Table I. Properties of Uniformly Sized Poly(Glycidy1 Methacrylate.coEthylene Dimethacrylate) Beads
1j
particle size, pm epoxide groups, mmol/g specific pore volume,a mL/g median pore diameterf nm median pore diameter,bnm polystyrene exclusion limit? MW specific surface area,b m2/g
i
10.0 2.7 1.1 13.8 10.9 3.4 105 114
Calculated from size exclusion chromatography data. Calculated kom BET measurement. 0
1 0
,
I
1
1
2
'
1
'
1
'
4
3
Table 2. Protein Recoveries from Columns Packed with Beads Containing Bimodal Chemistries
Llnear flow veloclty, cm/mln
300
11
0
Drotein recovenr. %
A
~~~
A
A A
1
2
3
4
5
Linear flow veloclty, cm/min Figure 3. Effect of flow rate on efficiency and back pressure of a column packed with beads containing segregated chemistries: (a, top) column, 300-mm x 7.8-mm i.d., beads 8; mobile phase, acetonitrile; analyte, toluene (0);mobile phase, 0.15 moVL NaCl in 0.1 mol/L phosphate buffer (pH 7); analytes, uracil (A),cytochrome c (e), and conalbumin (W). (b, bottom) Column, 50" x 8-mm i.d., beads D; mobile phase, acetonitrile; analyte, toluene (0);mobile phase, 0.15 mol/L NaCl in 0.01 mol/L TRIS-HCI buffer (pH 7.6); analyte, myoglobin (A).
protein
SEW
IEbb
cytochrome c myoglobin ribonuclease A ovalbumin bovine serum albumin human serum albumin conalbumine lysozyme chymotrypsinogen trypsin inhibitor bovine plasma
108 93 105 107 91 93 101 107 105 89 92
103 98 102 104 96 98 101 100 99 94
Column SEW was packed with beads containing vicinal diol groups in the large pores and octadecylamino groups in the small pores. Column IERP was packed with beads containing diethylamino groups in the large pores and octadecylamino groups in the small pores. lo7 A
A A
4 A'
beads B. This suggests that the ethyl substituents in the large pores also make a small contribution to the overall hydrophobicity of the separation medium. A Van Demteer plot obtained for the column packed with the l@pm beads B (Figure 3a) shows the effect of flow rate on column efficiency. The maximum plate number of 28 000 corresponding to a reduced plate height of 3.6 particle diameters is achieved with toluene in acetonitrile at a flow rate of 1 mL/min. The efficiency obtained for the column packed with beads D is lower than that for beads B. A maximum of about 20 000 plates that corresponds to a reduced plate height of 5.2dpis obtained for toluene at a flow rate of 0.5 mL/min (Figure 3b). Size Exclusion Chromatography of Proteins. Because the molecular weights of proteins range from thousands to millions, it is possible to separate some of them according to their hydrodynamic volumes by size exclusion chromatography. Typically, the separation is achieved in an aqueous medium. The surface of pores in which the separation takes place must be hydrophilic, otherwise recovery of the proteins is poor and the stationary phase quickly loses its separation ability. The vicinal hydroxyl groups in the large pores of beads A obtained by hydrolysis of the epoxide groups should endow the surface with sufficient hydrophilicity. Indeed, Table 2 documents the almost 4312
Analytical Chemistry, Vol. 66,No. 23,December 1, 1994
A AAA
A
1021
4
,
,
,
,
6
0 Volume, ml
,
10
12
Figure 4. Size exclusion calibration curve for column 300-mm x 7.8-mm i.d. packed with beads B: mobile phase, 0.15 mol/L NaCl in 0.1 mol/L phosphate buffer (pH 7); flow rate, 1 mumin; analytes, (down from the upper left corner) hemocyanine, thyroglobulin, apoferritin, ,!?-amylase, alcohol dehydrogenase, conalbumine, bovine serum albumin, carbonic anhydrase, myoglobin, cytochrome c, ribonuclease A, and vitamin Bl2.
complete recovery of all the proteins tested. Figure 4 shows a calibration curve obtained for the series of proteins. Though the beads were not speczcally designed for separation by size exclusion, the calibration curve is rather linear in the range of molecular weights from about 13 000 to almost 2 x lo6. In contrast, the polystyrene exclusion limit observed for the original unmodified epoxide beads was only 3.4 x lo5. This apparent discrepancy is actually not a concern since the comparison should
1
0 -
A
-7
-
4
L
Y
3
~
0.0
0.5
1.0
1.5
2.0
NaCI, mol/l
Figure 5. Effect of sodium chloride concentration on the retention factor K for various proteins in columns packed with beads D containing segregated chemistries: column, 50-mm x 8-mm id.; mobile phase, 0.01 mol/L TRIS-HCI buffer solution (pH 7.6); flow rate, 1 mumin; UV detection; analytes, bovine serum albumin (l), human serum albumin (2), soya bean trypsin inhibitor (3), myoglobin (4), cytochrome c (5), and lysozyme (6).
be based on molecular volumes rather than on molecular weights. The efficiency of the column packed with beads B in the size exclusion mode is rather high. The plate number of 24 000 (reduced plate height 3.8dJ measured for uracil in an aqueous mobile phase is very high at a flow rate of 0.2 mL/min. The plate number for proteins is 21 000 and 13 500 platedm for cytochrome c and conalbumin, respectively, at a flow rate of 0.05 mL/min (Figure 3a). However, this flow rate is unrealistic because the separation would take too long to be achieved. Though the efficiency decreases with increasing flow rate, the column still provides remarkable values of 14 000 and 10 000 plates/m for cytochrome and conalbumin, respectively, at a more acceptable flow rate of 0.5 mL/min. Myoglobin is not retained in beads D (Figure 5, curve 4) and can be used for the determination of the efficiency of a column packed with these beads operating in the size exclusion mode. The maximum plate number is about 7200 plates/m at a flow rate of 0.1 mL/min. Ion-Exchange Chromatography of Proteins. The separation of proteins by ion-exchange chromatography results from the interaction of the charged surface of the separation medium with charged groups of the protein. Here again, hydrophobicity of the surface would have a very detrimental effect on both recovery and column stability. Table 2 shows that beads D with the diethylamino chemistry in the large pores also afford very good recoveries for all proteins tested. Figure 5 shows the plot of retention factor against concentration of salt in the mobile phase. The curves have a shape typical for the ion-exchange chromatography of protein^'^ and clearly document the absence of hydrophobic interactions that would be characterized by retention curves with a concave ~ h a p e . ~ ~ , ~ ~ Separations of Complex Mixhrres. The hydrophobic groups in the media described above are completely hidden in the small pores and are therefore inaccessible to large protein molecules. Therefore, a column packed with beads B can be used for the (22) Kennedy, L. A; Kopaciewicz, W.; Regnier, F.E.]. Chromutogr. 1986,359, 73. (23) Heinitz, M.L;Kennedy, L. A; Kopaciewicz, W.; Regnier, F.E.]. Chromutog. 1988,443,173.
0
20 40 Tlme, mln
Figure 6. Direct-injection separation of bovine serum plasma and anticonvulsantdrugs in a column packed with beads B: column, 150mm x 4.6-mm id.; mobile phase, 0.15 mol/L NaCl in 0.1 mol/L phosphate buffer (pH 7)-acetonitrile 80:20; injected volume, 20 mL; UV detection, 254 nm; peaks (1) bovine serum plasma (70 mg/mL), (2) procainamide (10 pg/mL), (3) lidocain (20 pg/mL), (4) carbamazepine (8pg/mL), (5) phenytoin (40 pg/mL), (6) desipramine (10 CcglmL).
direct-injection separation of hydrophobic drugs in blood plasma. Figure 6 shows an excellent separation of five drugs from a sample containing a large excess of plasma proteins. This separation is actually quite comparable to one achieved with a typical restricted access medium. However, the great advantage of the beads modified in pore s u e specific fashion is seen in processes where each of two entirely different families of compounds present in a sample have to be completely separated. The chromatograms of Figure 7 illustrate separations of mixtures consisting of proteins and alkylbenzenes or drugs in a column packed with beads B. After injection, the hydrophobic part of the sample is adsorbed at the top of the column in the small hydrophobic pores while the other part, the proteins, remains in solution. Elution of the proteins proceeds in size exclusion mode at a flow rate suitable for a good separation. This flow rate is a tradeoff between column efficiency and retention time of the smallest nonadsorbed molecule. We have chosen a flow rate of 0.2 mL/min for which the separation of four proteins (thyroglobulin MW 669000, human serum albumin MW 69 000, bovine carbonic anhydrase MW 29 000, ribonuclease A MW 13 600) and vitamin B12 (MW 1356) occurs within 60 min while column efficiency remains sufficiently high. After 65 min in size exclusion mode, the elution mode is changed (this point is shown as a vertical line in the chromatograms). Acetonitrile is admixed to the original mobile phase, and the hydrophobic compounds initially adsorbed at the top of the column are separated in the isocratic reversed-phase mode. The separations of both families of compounds shown in Figure 7 are sufficient even for a quantitative analysis and document the feasibility of the concept. The other set of chromatograms shown in Figure 8 illustrates separations obtained for a column packed with beads D operating first in the ion-exchange mode for the separation of proteins and then followed by reversed-phase mode for the separation of small hydrophobic molecules. The sample is injected into a flow of a Analytical Chemistw, Vol. 66, No. 23, December 1, 1994
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0 20 40 6 0 80 100
0
20 40 60 80 100
Time, min Time, mln Figure 7. Separation of proteins and alkylbenzenes or anticonvulsant drugs in consecutive size exclusion (a) and reversed-phase (b) chromatographic modes in a column packed with beads B containing segregated chemistries: column, 300-mm x 7.8-mmi.d.; mobile phase, 0.15 moVL NaCl in 0.1 mol/L phosphate buffer solution (pH 7) for 65 min at a flow rate of 0.2 mUmin followed by bufferacetonitrile mixture (65:35v/v); flow rate, 1 mUmin; UV detection, 254 nm; peaks (left-hand panel) thyroglobulin ( l ) , human serum albumin (2), carbonic anhydrase (3),ribonuclease A (4), vitamin B12 (5), benzene (6),toluene (7),ethylbenzene (a), propylbenzene (9), butylbenzene (lo),and amylbenzene (11); peaks (right-hand panel) thyroglobulin ( l ) , human serum albumin (2), carbonic anhydrase (3), ribonuclease A (4), vitamin BIZ (5), phenobarbital (6),carbazepamine ( 7 ) ,and phenytoin (8).
I
1
The adsorption is size selective,and the components are separated into two groups according to their hydrodynamic size and their affinity toward the functionalitiesof the pore surface. The proteins are adsorbed by ion exchange in large pores while the small molecules are retained within small pores by hydrophobic interactions. The pore size selective, chemistry-driven adsorption represents the first step of the separation. Both parts of the original sample are then separated independently in two different chromatographicmodes. The proteins are eluted in ion-exchange mode using an increasing sodium chloride gradient while the small molecules remain adsorbed at the top of the column during this separation and are not eluted until the mobile phase containing the organic modifier (acetonitrile) is applied and isocratic reversed-phase mode is used. The separation of proteins shown in Figure 8 also confirms that the ionexchange mechanism must be operative. The proteins (myoglobin MW 17 000, cytochrome A MW 13 400,bovine serum albumin MW 66 700, soya bean trypsin inhibitor MW 22 500) are eluted regardless of their molecular weight according to their net charges. This documents the absence of size exclusion effects in the protein separation. Clearly, the flow rate of 1 mWmin is too high and the length of the column (150 mm) is too short for an efficient separation by size exclusion. The higher flow rate together with the mobile-phase gradient results in an acceleration of the protein separation compared to the size exclusion chromatography with beads B and decrease in the total time required for analysis. However, the separation of hydrophobic small molecules that follows the ionexchange chromatography is poorer than that observed with beads B. The peaks are broader because the efficiency of the shorter column is lower. One might wonder whether or not single-column separations of complex mixtures such as those shown in Figures 7 and 8 would be feasible with other separation media. It is likely that some of the restricted access media with optimized pore size distribution would indeed be able to separate a sample using consecutive size exclusion and reversed-phase modes. However, the combination of ionexchange with reversed-phase chromatography is not easily matched. The mixed-mode phases with nonsegregated chemistries as well as the blended phases do not provide for the first step separation into component families, one of which is held into the column while the other is separated, and the elution is not specific for each of these families. We believe our media are unique in their ability to separate a complex sample in consecutive ion-exchange and reversed modes. The experimental results also demonstrate that the separations shown in Figures 7 and 8 have all the attributes of a twcdimensional The first step separates the components of the sample into two classes that are stored separately in different pores. Both classes of compounds are then subjected to a separation into individual components. This is particularly true for the combination of ion-exchange and reversed-phase chromatography as both families of compounds separated in the first step remain at the top of column until they are separated in the second step. CONCLUSION
low ionic strength buffer solution (0.01 mol/L TRISHC1). In contrast to the combined size exclusion and reversed-phase chromatography described above, all of the components of the sample are initially adsorbed at the top of the column in this case. 4314 Analytical Chemistry, Vol. 66,No. 23, December 7, 1994
Separations of different families of compounds in two different chromatographic modes confirms that the pore size specitic functionalization provides separation media with two segregated chemistries. The chromatographic results clearly document that
the separation of complex samples in two consecutive modes is feasible. Though this report describes media with only two different surface chemistries located in the large pores and used for the separation of proteins contained in the sample, the concept of the pore size specific modification of porous materials allows the design of many other chemistries exclusively within the large pores without compromising the properties of the small pores and vice versa. The use of a reaction path involving modfications controlled by polymeric reagents or catalysts with different molecular sizes can result in separation media accommodating more than two segregated chemistries. These media are currently under investigation.
ACKNOWLEDGMENT
Financial support of this research by the National Institutes of Health (GM 44885-05) is gratefully acknowledged. This work also made use of Come11 Material Science Center MLR Central Facilities (Polymer Research Facility) supported by the National Science Foundation under Award DMR-9121654. Received for review June 30, 1994. Accepted September 17, 1994.@ @Abstractpublished in Advance ACS Abstracts, October 15, 1994.
Analytical Chemistty, Vol. 66, No. 23, December 1, 1994
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