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Anal. Chem. 1988, 6 0 , 2322-2328
Capillary Zone Electrophoretic Separation of Peptides and Proteins Using Low pH Buffers in Modified Silica Capillaries Randy M. McCormick E. I . du Pont de Nemours and Co., Inc., Central Research and Development Department, Experimental Station, Building 228, Wilmington, Delaware 19898 High-efflciency capillary zone electrophoretic separations of peptldes and protelns In modifled sillca caplllarles have been achieved In low pH aqueous buffers. Caplilarles were modIfled with phosphate moieties from the separation buffer as well as with conventlonal danes. Separations of proteins wlth molecular weights ranglng from 12K to 77K and lsoelectrlc polnt values of 4.5-1 1 have been achleved In under 25 mln. Ylxtures of octapeptMe homologues that M e r by the addltbn of methylene groups to the amino acld side chalns of the peptldes have been resolved. Capllary zone electrophoresis (CZE) has also been used to separate mixtures of proteins of hlghiy conserved sequence that dMer by a few amlno acld substltutlons In a total sequence of over 100 amlno aclds. Effects of the magnitude of the applled potentlai on separatlon efflciency in CZE are dlscussed. The rate at which the voltage Is Introduced across the capillary was found to have a significant Impact on the asymmetry and peak wldth of proteln bands in CZE Separations.
The development of capillary zone electrophoresis (CZE) in recent years represents a significant departure from conventional gel electrophoretic separations. As a separation technique, gel electrophoresis of proteins is widely practiced, and is simple and inexpensive to implement; it is also tedious, labor-intensive, time-consuming, and semiquantitative. CZE reduces the tedium of electrophoretic separations by eliminating the preparation of gels for separations and the staining/destaining procedures for band detection. The CZE technique also affords the researcher with the possibility of achieving rapid, high-resolution separations of macromolecules such as proteins. The need for high-resolution protein separations has become more important due to the recent revolution in molecular biology. One of the possible areas of utility of CZE separations is the verification of purity of recombinant protein products to be used as therapeutic agents ( I ) . In addition, such a high-resolution method would be useful for studying various aspects of posttranslational processing, such as glycosylation, phosphorylation, protein folding, etc. ( 2 ) ,as well as for the detection and characterization of isoenzymes (3). Numerous examples of CZE separations of small molecules can be cited, including inorganic ions (4),amino acids (5),small organic ions (6),peptides (3, and oligonucleotides (8). Unfortunately, application of the technique to the separation of proteins is complicated by adsorption of the minute quantities of the protein sample onto the walls of the capillary. Such interactions result in band broadening and tailing, with greatly reduced separation efficiencies. Reported attempts to eliminate this sorption inwlve deactivation of the silica capillaries by physically coating the capillary wall with methylcellulose (9, l o ) , as well as via silane derivatization (11, 12). Addition of chemical agents to the separation buffer ( I I ) , as well as manipulation of the electronic charge on the proteins and the silica capillary wall to prevent adsorption by Coulombic repulsion (13),have also been reported. This study describes ways of deactivating the silica surface to yield capillaries on which a broad range of proteins can be
separated with high resolution. Other aspects of improving the CZE separation of proteins are also reported. The power of capillary zone electrophoresis is demonstrated for the separation of complex protein mixtures as well as for the resolution of several peptide and protein samples containing species that differ by a few amino acids in sequence. EXPERIMENTAL SECTION Apparatus. Separations were done in a capillary electropherograph constructed from an in-house design (Figure 1). All of the high-voltagecomponents of the system are contained in a Lucite cabinet fitted with a safety interlock that interrupts line voltage to the transformer in the power supply when the cabinet door is opened. In addition, a second interlock on the cabinet door controls a normally closed high-voltage relay (Model H-23, Kilovac Corp., Santa Barbara, CA) which clamps the anode to house-ground potential;with this design, the residual capacitive charge stored in the high-voltage circuitry of the power supply is immediately discharged to ground whenever the cabinet door is opened. A high-voltage power supply (ModelLG80P1.5, Glassman High Voltage, Whitehouse Station, NJ) was used to establish the electrical field across the capillary. The output voltage of the power supply was controlled by a Model 273 programmable sweep/function generator (Wavetek, San Diego, CA). The power supply output was connected to 22-gauge platinum wire electrodes immersed along with the ends of the capillary in 25-mL reservoirs of buffer. Separations were done in polyimide-clad fused silica capillaries (Polymicro Technologies, Phoenix, AZ), which were nominally 50 pm i.d. and 375 pm 0.d.; the total length of the capillariesvaried from 75 to 130 cm, with the separation monitored 50-75 cm from the anode. Separated bands were detected on-line with a Model V4 UV absorbancedetector (Isco,Inc., Lincoln, NE)modified by inserting 100-wmpinholes in both the reference and sample optical paths. The conventional flow cell of the detector was replaced with an adjustablepinhole mount attached to a precision translation stage (Oriel Corp., Stratford, CT). This arrangement facilitated alignment of the pinhole with the capillary and allowed the pinhole/capillary assembly to be reproducibly positioned at the focal point of the optical beam in the detector. Detection was accomplished at 190 nm through an unclad section of the capillary used as an absorbancecell (observationvolume of -120 pL). The signal from the detector was fed to a strip chart recorder and to a Model 3357 LAS data acquisition system (Hewlett-PackardCo., Avondale, PA). Electrophoresis. Samples were dissolved in the separation buffer at concentrations of 1-5 mg/mL. Injection of the sample (100-500 pg of each protein) into the capillary was made by electromigration at constant voltage (2.5-5 kV) for a fixed period of time (5-30 9). Starting at the initial lower injection voltage, the applied potential was then increased in a programmed manner to the final running voltage (25-30 kV), where the potential was held constant for the duration of the separation. During electrophoresis, the current through the capillary was not allowed to exceed 125 FA. Between analyses, the capillary was flushed for 5 min with the separation buffer prior to injection of the next sample. Capillary Modification. Capillarieswere cleaned by flushing sequentiallyfor 30 min with 1M KOH and deionized water prior to filling with separation buffer or bonding with silane. For preparation of the poly(vinylpyrro1idinone)-modified (PVP) capillaries, the capillary (1.3 m) was first flushed with several volumes of aqueous acetic acid (pH 3.5). The silane reagent
0003-2700/88/0360-2322$01.50/00 1988 American Chemical Society
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to scintillation fluid and counted. Materials/Supplies. Peptide and protein samples were purchased from Sigma Chemical Co. (St. Louis, MO). Electrophoresis buffers were prepared from in-house reverse-osmosis/ deionized water passed through a Milli-Q Water Purification System (Millipore, Bedford, MA) to yield 15-18 MQ cm water. Phosphoric acid and potassium hydroxide were from Fisher Scientific (King of Prussia, PA). Radioactive [?P]orthophosphoric acid and scintillation fluid (BIOFLUOR)were from New England Nuclear Corp. (Boston, MA).
LUCITPCABINET INTERLOCK HIGH VOLTAGE RELAY
RESULTS AND DISCUSSION Separations in Low pH Phosphate Buffers. All separations in this study were performed in acidic buffers, as is commonly carried out for the reversed-phase liquid chromatographic separation of proteins using mobile phases containing trifluoroacetic acid (15). Although CZE protein sepI arations typically are performed in neutral (11)or alkaline HIGH VOLTAGE (13) buffers as in conventional electrophoresis, the choice of POWER SUPPLY (0-35KV) acidic buffers has a number of distinct advantages over separations at alkaline pH values. Operation of silica capillaries with acidic pH buffers should yield more reproducible sepaFigure 1. Schematic diagram of capillary electropherograph. rations, as silica has a pronounced solubility in water a t almixture (12),80 pL of [ (3-methacryloyloxy)propyl]trimethoxykaline pH values (16). This aspect is particularly important silane (Serva Fine Biochemicals,Westbury, NY) in 20 mL of pH for capillaries deactivated with silane reagents to yield bonded 3.5 aqueous acetic acid, was pulled through the capillary by house phases on the capillary wall. Operation at low pH values (pH vacuum for 3 h at room temperature. The capillary was next less than the isoelectric point (PI) of all proteins) also ensures purged with distilled water for 3 h. Then, the reagent mixture that the proteins in a sample will have a net positive charge [3% aqueous 1-vinyl-2-pyrrolidinone (Aldrich Chemical Co., and thus will all migrate in the same direction (i.e,, toward Milwaukee, WI) adjusted to pH 6.2 and containing 1 mg/mL the cathode) when the electric field is established across the ammonium persulfate (Polysciences,Inc., Warrington, PA) and capillary. This is in contrast to the situation where neutral 1 pL/mL N,N,”,N’-tetramethylethylenediamine (BioRad, Richmond, CA)] was pulled through the [(3-methacryloy1oxy)or mildly alkaline buffers are used and one depends on the propylltrimethoxysilane-derivatized capillary for 90 min by using velocity of the electroosmotic flow to be greater than the house vacuum; this yielded a polymeric layer on the capillary wall electrophoretic velocity of the proteins (13) and thus carry of hypothetical structure -[Si(0)(CH2),0COCH(CH3)CH2- negatively charged proteins (i.e., those with PI > pH) through (CHzCH(C4H,NO)),],. Unbound reagent was flushed from the the capillary to the cathode (detector). capillary with water. Other capillaries were modified in a similar Use of low pH buffers also substantially reduces the magmanner, substituting acrylamide and acrylic acid for the 1nitude of the electroosmotic flow through the silica capillaries vinyl-2-pprolidinone reagent. Though long-term studies of the (In,as shown in Figure 2. Silanols on the capillary wall stability of these deactivated capillaries have not been conducted, become progressively more protonated as the buffer pH is capillaries prepared in this manner have been used continually made more acidic, thereby reducing the charge on the capillary for several weeks at low pH with no observed deterioration in performance. walls and concomitantly the magnitude of the electroosmotic Glycero-glycidoxypropyl-derivatizedcapillaries were prepared flow. In Figure 2, the flow rapidly decreases as the buffer pH with (3-glycidoxypropyl)diisopropylethoxysilane(Petrarch Sysis reduced to pH -4.5 and then continues to decrease more tems, Inc., Bristol, PA). The reagent mixture [lo0 pL of silane, slowly down to pH 1.5. Protonation of the silica is apparently 50 KL of N,N-diisopropylethylamine(Aldrich), 5 mL of dry not complete a t pH 1.5, as there is still a small residual toluene] was pumped through the heated 1.3 m capillary (100 “C) electroosmotic flow at this pH. This observation is substanat 50 pL/h for 4 days with a syringe pump (Sage Instruments, tiated by reports that the PI of silica is as low as 1.0 (18). Cambridge, MA). After the capillary was sequentially flushed Reduction or elimination of the electroosmotic flow by with toluene and dioxane to remove residual reagent, the epoxide operating a t low pH should yield more reproducible separafunctional group on the silane was opened by reaction at 90 OC tions, since changes in ionic strength, pH, capillary wall with 5 mL of 1.6 mM glycerol (Aldrich) in dry dioxane (Aldrich) containing 80 pL of boron trifluoride etherate (Aldrich), which contamination, etc., can cause substantial changes in the was pumped through the capillary at 140 pL/h. This yielded a magnitude of the electroosmotic flow. These changes result capillary modified with a monomeric hydrophilic bonded phase in variation of the elution time of the proteins in separations with the putative structure -Si(i-Pr),[(CHZ),OCH2CH(OH)- that depend on flow for transport through the capillary; in CH20CH2CH(OH)CH2(OH)]. Because of the bulky nature of the contrast, separations that depend solely on the electrophoretic diisopropyl protecting groups, bonded phases prepared with silanes mobility of the proteins for transport through the capillary containing these shielding groups have been demonstrated to are not subject to these flow variations. exhibit superior stability relative to bonded phases prepared with The reduction in electroosmotic flow in Figure 2 could also silanes containing methyl or methoxy groups (14). result in part from the stabilization of the silica through the Phosphate Binding Experiments. The level of phosphate binding to the silica capillaries was measured with [S2P]orthoformation of a complex of silanol and phosphate groups on phosphoric acid. Lengths of capillary (35 cm) were first sethe surface of the capillary wall (19). Experiments revealed quentially flushed with 1 M KOH and water for 10 min each to that phosphate binds to 50-pm capillaries a t a level of -30 prepare the silica surface. The capillaries were then flushed for nmol/m (assuming, based on a simple cylindrical capillary 15 min with 150 mM H3P04containing 0.067%of 37 GBq/mmol geometry, a surface area of 1.6 X m2/m, and a silanol orthophosphoric acid. Unbound phosphoric acid was washed population of 8 pmol/m2, this corresponds to -23 phosphates from the capillary with 150 mM H3P04until no radioactivity was per silanol). These phosphate groups appear to bind strongly detected in the effluent. Bound phosphate was then desorbed to the silica surface and are not removed by flushing the from the capillary by flushing with 1M KOH until radioactivity capillary with water or buffer solutions. The presence of these could not be detected in fractions of the capillary effluent. bound phosphate moieties on the surface of the capillary wall Collected fractions containingthe desorbed phosphate were added I
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Flgure 4. Separation of anglotensln I1 octapeptides: capillary, 1 10 cm of 53 pm i.d. fused silica, 75 cm separation distance; buffer, 150 mM NaH,PO,, pH 3.0; detection, 190 nm; lnjectlon, 5 s at 2.5 kV; separation voltage, 2.5-30 kV linear program In 300 s; sample, angiotensln I1 homologues: (A) S a r Arg Val Tyr Ile His Pro Gly, (B) S a r Arg Val Tyr Ile His Pro Leu, (C) Sar Arg Val Tyr Ile His Pro Thr, (D)
60 -
Sar Arg Val Tyr lie His Pro phe, (E) Asp Arg Val Tyr Val His Pro Phe, (F) Asp Arg Val Tyr Ile His Pro Phe. Conventional abbreviations for amino acid are as follows: Sar, sarcosine; Arg, arginine; Val, valine; Tyr, tyrosine; Ile, isoleuclne; HIS, histidine; Pro, prollne; Gly, glycine; Leu, leuclne; Thr, threonine; Phe, phenylalanine; Asp, aspartic acid. I
I
3 .O 4.5 BUFFER pH
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Flgure 2. Electroosmotlc flow as a functlon of buffer pH. Flow rate was measured In a 110 cm length of 53 pm Ld. fused silica filled with 150 mM phosphate buffer at the specified pH. The applied potential was 30 kV; the separation was monitored 75 cm from the anode. Phenol and acetone dissolved in the separation buffers were used as
C
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Figure 3. Separation of dipeptldes in low pH phosphate buffer: capillary, 110 cm of 50 pm i.d. fused silica, 75 cm separatlon distance; buffer, 150 mM HaPo,, pH 1.5; detection, 190 nm; injectbn, 5 s at 500 V; separatlon voltage, 0.5-25.0 kV linear program over 15 mln; sample: tyrosyCx dipeptides, where x Is (a) glycine, (b) alanine, (c)valine, (d) leucine, (e) glutamic acid, (f) tyrosine.
not only may reduce the electroosmotic flow by converting the residual highly acidic silanols to more easily protonated silica-phosphate complexes (and thus reducing the magnitude of the negative charge on the capillary wall) but may also screen the silica surface (19),thereby reducing the sorption of peptides and proteins in much the same way that phosphate deactivates the surface of GC capillaries for fatty acid analyses (20). Phosphate has also been reported to reduce the interaction of proteins with polysilicic acid (21). With these low pH phosphate buffers, dipeptides differing by one amino acid can easily be resolved (Figure 3) in 150 mM
F l g m 5. CZE separation of protein " u e in low pH buffer: capillary, 110 cm of 53 pm Ld. fused silica, 75 cm separation distance; buffer, 150 mM H,PO,, pH 1.50; detection, 190 nm; Injection, 10 s at 2.5 kV; separation voltage, 2.5-30 kV linear program in 300 s;sample, protein mixture; (A) j3-lactoglobulin A, (B) cytochrome c (horse),(C) lysozyme (chicken),(D) myoglobin (horse heart), (E) parvalbumin (rabbit).
H3P04at pH 1.5. In this separation, the first four peaks (A-D) correspond to dipeptides that differ by addition of methyl or methylene groups to the side chain in the second amino acid of the dipeptide. These four dipeptides elute in order of increasing molecular weight. Changes in the chemical nature of the side chain in the amino acids (e.g., change of a neutral to an acidic or basic amino acid) can also have a significant effect on the mobility of the dipeptides. For example, peaks E and F in Figure 3, where the side chains in one of the amino acids of the dipeptide contain acidic and aromatic groups, respectively, have different mobilities than the dipeptides that contain amino acids that differ by neutral methylene groups in the side chain (peaks A-D). The power of CZE for resolving closely related molecules is further demonstrated in Figure 4, which shows the separation of a mixture of synthetic octapeptides at a pH of 3.0 in a silica capillary filled with phosphate buffer. These peptides are all similar in sequence, differing in most cases by single amino acid substitutions. Separation of peptides E and F, which differ by only a methylene group, is possible because substitutions on the side chains of amino acids alter the pK values of the acidic and basic functionalities in the amino acids, and these changes alter the electrophoretic mobility of the peptides. By use of these low pH buffers, it is often possible to obtain sharp, symmetrical bands for proteins in CZE separations. Figure 5 shows the separation of a mixture of proteins in pH 1.5 phosphate buffer in a fused silica capillary. The protein
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
six proteins in Figurej 6 are given A 0,b. c . d, e. f r B m
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in Figure 7. Cytochrome
c proteins from the following pairs differ by three amino acids
in a total sequence of 104: horse-pig, dog-pig, and rat-pig. Yet, these proteins are easily resolved in the separation shown. I' In determining differences in the mobilities of the proteins, f' 1' f' a variation in the identity of the amino acids is more important than is the number of amino acids changed. For example, 9 00 13 75 1850 900 13 75 18 5 0 horse and chicken cytochrome c differ by 11amino acids but C have greater mobility differences than do the cytochrome c proteins from horse and tuna, which vary by 20 amino acids. Likewise, the six amino acid variation between horse and dog cytochrome c does not impart as great a mobility change as does the three amino acid difference between the horse and pig cytochromes. This effect is understandable since sub9 00 I3 75 1850 900 13 75 18 50 stitution of an acidic or basic amino acid for a neutral one Figwe 6. Effect of buffer pH on separation of cytochrome c proteins: should change the mobility of the protein more than a simple capillary, 125 cm of 53 pm i.d. fused silica, 75 cm separation distance; interchange of one or more neutral amino acids. buffer, 150 mM phosphate, pH as indicated; detection, 190 nm; injection, 5 s at 2.5 kV; separation voltage, 2.5 to 30 kV linear program The subtle changes in protein sequences illustrated in in 300 s; sample, cytochrome c proteins: (a) horse, (b) dog, (c) rat, Figures 6 and 7 may not be detectable by conventional in(d) pig, (e) chicken, (f) tuna. Sequence data are shown in Figure 7. teractive chromatographicseparations such as reversed-phase, ion-exchange, or hydrophobic-interaction chromatography, particularly if the changes occur in the interior of the proteins bands are fairly symmetricalbut elute together in a 3-min time period. The major limitation of performing CZE separations or are located away from the contact region responsible for at such a low pH is that the peak capacity of the separations protein retention (22). In this regard, CZE may represent a is low because the acidic buffer fully protonates the proteins, unique separation technique that will permit resolution of thereby diminishing charge differences between the species. species that is not achievable by other methods. However, it is necessary to operate a t a pH well below the The high-resolving power of CZE is further illustrated in lowest-pIprotein in the mixture (e.g., in the separation shown, the separation of @-lactoglobulinsA and B (Figure 81, which the PI of parvalbumin is -4.5) to attain efficient band shapes; differ by substitution of two amino acids in a total sequence if the buffer pH is close to the PI of a protein, that protein of 162. &Lactoglobulin B has a glycine at position 64 and an tends to interact with the silica surface at the capillary wall alanine at position 118, whereas 0-lactoglobulin A contains and will not elute or will elute with a badly tailed peak shape. aspartic acid and valine at the two respective positions. Such The effect of pH on the efficiency of CZE separations is subtle changes in amino acid sequence can substantially alter further illustrated in Figure 6, which shows the separation at the mobility of proteins, either by changing the net charge four pH values of a group of similar cytochrome c proteins on the protein at a given pH or possibly by altering the tertiary (PI -10.5) from various species. The identity, amino acid structure due to differences in protein folding. sequence, and molecular weight of these six cytochrome c The preceding examples illustrate the ability of CZE to proteins are shown in Figure 7. At pH 1.5, the differences separate peptides and proteins that differ only slightly in in mobilities of these six proteins are m i n i and the proteins sequence and/or structure. Actually, CZE might best be coelute as shown in Figure 6A. As the pH is raised, mobility suited for separationsof this type,(i.e., for separation of closely differences become greater and partial separation is achieved related proteins), since, with current technology, it is difficult at pH 4.0 (Figure 6B). An optimal separation of these proteins to achieve the separation of proteins with a range of PI values in 150 mM phosphate buffer is achieved at pH 5.0 (Figure 6C). and molecular weights in a single run due to the limited peak As the pH is raised further (pH 5.25), the separation begins capacity of the technique. Since small changes in buffer pH can have a substantial effect on the mobility of proteins in to deteriorate (Figure 6D). This loss of resolution presumably CZE (in some cases, reversal of the elution order of select arises because the proteins begin to adsorb onto the silica proteins has been observed with changes in buffer pH), procapillary, resulting in tailed band shapes. Also evident in Figure 6 is the reduction in elution times of t h e protein bands gramming the buffer pH during the course of a separation would extend the peak capacity and utility of CZE for sepain the pH 5.25 buffer relative to the separations at pH 4 and rating a broad range of proteins. Deactivating the walls of 5; this effect is due to an increase in electroosmotic flow through the capillary at the higher pH and may possibly the capillary so that proteins do not irreversibly adsorb in contribute to the observed deterioration in the quality of the higher pH buffers is another method of achieving this goal separation. and has been partly successful in recent studies. The pH 5.0 separation in Figure 6C also illustrates the Silica modified with poly(vinylpyrro1idinone) (PVP) has power of CZE to separate charged macromolecules that differ been used for separation of proteins by both size-exclusion and hydrophobic-interaction chromatography (23);little or only slightly in structure. The amino acid sequences of the Cytochrome c protein from horse consists of 104 amino acids'in the sequence shown. Cytochrome c proteins from other species are identical in sequence except for the amino acid substitutions indicated. 15
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GDV EKG KKI FVQ KCA OCH TVE KGG KHK TGP NLH GLF GRK TGO APG FTY TDA NKN KGI TWK EET LME YLE NPK KYI PGT KMI FAG IKK KTE RED LIA YLK K A T NE'
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Identity, amino acid sequence, and molecular weights of cytochrome c proteins. Conventional one-letter abbreviations for amino acids
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 B
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Flgure 8. CZE separation of @-lactoglobulinsA and B capillary, 110 cm of 53 pm i.d. fused silica, 75 cm separation distance; buffer, 150 mM NaH,PO,, pH 4.5; detection, 190 nm; injection, 5 s at 2.5 kV; separation voltage, 2.5 to 30 kV linear program in 300 s.
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Flgure 11. Effect of applied potential on quality of CZE separation: capillary, 100 cm of 49 pm 1.d. fused silica, derivatized with glycerogiycidoxypropylsilane(seetext), 60 cm separation distance; buffer, 150 mM NaH,P04, pH 4.5; detection, 190 nm; Injection, 8 s hydrostatic injection, 15 cm head; sample, protein mixture contalning (a) lysozyme (turkey), (b) lysozyme (chicken), (c) cytochrome c (horse), (d) ribonuclease A, (e) trypsinogen, (f) a-chymotrypsin; separatlon voltage, (A) 2.5 to 25 kV linear program in 300 s, (B) 2.5 to 30 kV linear program in 300 s.
A. Voltaae: 2-25 KV steD at 0 min
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Flgure 9. CZE separation of protein mixture in PVP-modified capillary: capillary, 110 cm of 52 p m i.d. fused silica derivatized wkh [(3methacryloyloxy)propyl] trimethoxysilane and l-vinyC2-pyrroliinone, 75 cm separation distance: buffer, 38.5 mM H3P04,20 mM NaH2P0,, pH 2.0; detection, 190 nm; injection, 5 s at 5 kV; separation, 5 to 25 kV linear program in 150 s. Sample: (A) @-lactoglobulinB, (B) &lactoglobulin A, (C) lysozyme (ovine egg), (D) albumin (human serum), (E) albumin (bovine serum), (F) cytochrome c (horse), (G) trypsinogen (bovine), (H) myoglobin (whale), (I) transferrin, (J) conalbumin, (K) myoglobin (horse), (L) carbonic anhydrase B (bovine), (M) carbonic anhydrase A (bovine), (N) hemoglobin (human), (0)parvalbumin (rabbk).
0.11
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Flgure 12. Effect of voltage program time on separation: capillary, 110 cm of 50 p m i.d. fused silica, derivatized with [(methacryioyloxy)propyi]trimethoxysllane/1-vinyl-2-pyrrollinone (see text): buffer, 150 mM H3P04,pH 1.45; detection, 190 nm; injection, 5 s at 2 kV; separation voltage, (A) 2 to 25 kV step program at 0 s, (B) 2 to 25 kV linear program in 300 s;sample, protein mixture of @-lactoglobulin A, lysozyme, cytochrome c , myoglobin, and parvalbumin.
'
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Flgwe 10. Elution time of proteins as a function of applied potential: capillary, 110 cm of 55 @mi.d. fused silica, 75 cm separation distance; buffer, 150 mM H3P04,pH 1.5; detectlon, 190 nm; injection, 5 s at 1.25 kV; separation voltage, 30 kV; sample, proteins; (0)/3-lactoglobulin B. (A)cytochrome c , (0)lysozyme; (A)parvalbumln.
no interaction with the protein samples was found. Figure 9 shows the separation of a broad range of proteins in a capillary modified with 1-vinyl-2-pyrrolidinoneat a pH of 2.0. Fifteen proteins with molecular weights ranging from 12K to 77K and PI values of 4.5-11 were separated in less than 25
min. Two bands were obtained for both human serum albumin and hemoglobin, presumably because of denaturation of these proteins to constituent subunits in the low pH buffer. The separation in Figure 9 exhibited peaks with plate numbers exceeding 700 000. However, not all proteins (e.g., insulin, ovalbumin, chymotrypsinogen) could be separated with this capillary because of protein adsorption onto the capillary walls. Other attempts to modify capillaries by substituting acrylamide and acrylic acid for 1-vinyl-2-pyrrolidinone were not successful in yielding capillaries that produced high-efficiency separations. Strong interaction between the proteins and capillary walls was apparent, as evidenced by the long elution times and asymmetrical peak shapes of the protein bands. Further work on silica capillary deactivation to expand the peak capacity and separation efficiency of CZE is currently under way. Effect of Applied Voltage on Separations. According to the theory of CZE as derived by Jorgenson (24),the elution time for a solute band should be inversely and linearly proportional to the applied voltage ( t 0: 1/V) and the plate
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MINUTES
Figure 13. Effect of voltage program on elution time, peak width, and apparent plate number for 8-lactoglobulin A: capillary, 110 cm of 50 Pm i.d. fused sllica, 75 cm separation distance; buffer, 150 mM H,PO,, pH 1.45; detection, 190 nm; injection, '5 s at 2.5 kV; separation voltage, 2.5 to 30 kV log program in 300 s.
number should be directly proportional to the applied voltage. This conclusion is based on the assumption that higher applied voltages reduce elution times and thus reduce the time available for the bands to broaden by diffusion. However, in practice, the elution time of a protein band is not a linear function of voltage (%), as illustrated by the data in Figure 10. As the applied voltage is increased, elution times decrease much more rapidly than the predicted linear behavior, presumably because the increased temperature in the capillary at the higher applied voltages reduces the buffer viscosity and thus increases protein mobilities. Increased thermal effects resulting from higher applied potentials can also alter the mobility of specific proteins more than others; proteins that are well-resolved under one set of experimental conditions can coelute at a higher applied potential (Figure 11, peaks E and F). In some cases, proteins can become denatured by the increased capillary temperatures accompanying higher applied potentials, causing deterioration of band shapes of selected proteins (Figure 11,peak C). Thus, the contention that higher applied voltages (9)w ill yield more efficient separations should be viewed as a general but not universal rule. In some cases, better separations can be achieved at lower applied potentials. Another factor that strongly influences the quality of CZE separations is the rate at which the voltage is applied across the capillary at the start of the separation. Figure 12A shows a separation of a protein mixture in which the voltage was instantaneously increased from 2 to 25 kV and held at 25 kV for the duration of the separation. In contrast, in Figure 12B, the voltage was programmed from 2 kV to the separation voltage of 25 kV in a linear fashion over a 5-min period at the start of the separation. The components in Figure 12B are much better resolved, not only because the bands are more symmetrical but also because the peaks are sharper (smaller band widths). This significant improvement in resolution is realized even though the elution times in the voltage-programmed separation (Figure 12B) are slightly longer because of the 5-min delay in applying the full running voltage; the peaks in Figure 12B should be broader if diffusion was the primary band-broadening mechanism contributing to separation efficiency. The effect of the length of the voltage program on protein separations has also been investigated. Figure 13A shows an essentially linear increase in the elution time of a protein band (@-lactoglobulinA) with increased length of the voltage program. In contrast, in Figure 13B, the half-height width of the protein band passes through a minimum (at between 100 and 300 s for the proteins studied) and then increases at longer
program times. These trends translate to an increase in the apparent plate number of bands in the separation, as shown in Figure 13C. At very long program times (not shown), the apparent plate number passes through a maximum ( 2000 s) and then decreases slowly up to the longest program times studied (SO00 s). These results suggest that, for very long program times (>2000 s), band broadening due to diffusion outweights the improvement in performance arising from the uge of the voltage program. Though these apparent plate numbers do not have the same significance as plate numbers measured in linear (nongradient) separations, they do show that sharper band shapes can be attained by voltage programming and, at very long separation times, diffusion becomes a significant band-broadeningmechanism,as expected. The underlying cause of this improvement in separation efficiency is not currently understood, though it is suspected that it derives from the establishment of equilibrium conditions (thermal, ionic, etc.) in the capillary as the voltage is imposed at the start of the separation. Unlike chromatographic separations in which sample injection is made into a column under equilibrium operating conditions, injection of the sample in CZE is normally made at low or no applied voltage; gradual rather than instantaneous imposition of the running voltage apparently results in reduced broadening of the sample zone by allowing the electroosmotic flow, capillary temperature, double-layer characteristics, etc., to slowly attain equilibrium conditions. Further investigations into this phenomenon, as well as into improvements in capillary deactivation in particular and capillary zone electrophoresis of proteins in general, are currently under way. N
ACKNOWLEDGMENT The author wishes to thank Professor J. W. Jorgenson of the University of North Carolina, Chapel Hill, for several valuable discussions at the inception of this investigation. LITERATURE CITED Robey, F. A. 7th International Symposium on HPLC of Proteins, Peptides, and Polynucleoths, Nov 2-4, 1987, Washington, DC, paper 701. Mortensen, H. B.; Cristophersen, C. Blochim. Biophys . Acte 1982, .707. - . , 15A-163. .- . . - - . SiragElden, E.; Gercken, G.; Herm. K.; Voight, K. D. J . Ciln. Chem. Clin. Biochem. lS86. 2 4 . 283-292. Tsuda, T.; Nomura, K.; Nakagawa, 0. J . Chromatcgr. 1983, 264, 385-392. Gassmann, E.; Kuo, J. E.; Zare, R. N. Science (Washington, D . C . ) 1085, 230, 813-814. Tsuda, T.; Nomura, K.; Nakagawa, G. J . Chromatogr. 1982, 248, 241-247. Jorgenson, J. W.; Lukacs, K. D. J . Chromatcgr. 1981, 218, 209-216. Cohen, A. S.; Terabe, S.; Smith, J. A.; Karger, B. L. Anal. Chem. 1987, 59, 1021-1027.
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(9) HjertGn, S. Chromatogr. Rev. 1967, 9 , 122-219. (10) Herren, B. J.; Shafer, S.0.;Alstine, J. V.; Harris, J. M.; Snyder, R. S. J. ColloM Inferface Sci. 1987, 115, 46-55. (11) Jorgenson, J. W.; Lukacs, K. D. Science (Washington, D.C.) 1983, 222, 266-272. (12) HjertGn, S. J. Chromafogr. 1985, 347, 191-198. (13) Lauer, H. H.; McManigHI, D. Anal. Chem. 1986, 58, 166-170. (14) Glajch, J. L.; Kirkiand, J. J. U S . Patent 4705725, 1987. (15) Regnier, F. E. J. Chromafogr. 1987, 418, 115-143. (16) Iler, R. K. The Chemistry of Silica; Wlley: New York, 1979; p 42. (17) Lukacs, K. D.;Jorgenson, J. W. HRC CC,J . High Res. Chromafogr. Chromafogr. Commun. 1985, 8 , 407-411.
Parks, G. A. Chem. Rev. 1985, 65,177-198. Mltsyuk, B. M. Russ. J. Inorg. Chem. 1972, 17, 471-473. Metcalfe, L. D. Nature (London) 1960, 188, 142-143. Emerick, R. J. J. Nutr. 1987, 117, 1924-1928. Fausnaugh, J. L.; Regnler, F. E. J. Chromarogr.1986, 418, 131-146. , Kohler, J. Chromafographis 1966, 2 1 , 573-582. i24) Jorgenson, J. W. TrAC, Trends Anal. Chem. (Pers. Ed.) 1984, 3 , 51-54. (25) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302.
RECEIVED for review April 29, 1988. Accepted August 2,1988.
Experimental Observation of Steric Transition Phenomena in Sedimentation Field-Flow Fractionation Seungho Lee and J. Calvin Giddings* Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
The steric transMan region of field-flow fractionation (FFF) Is described as that part of a fractogram, found at very high retention volumes, in which m a l FFF undergoes a tradlon to steric FFF by virtue of increasing particle diameter. The steric transition region Is treated theoretically by assuming first that the steric factor y is constant and second that It Is related by a -le power law to particle diameter. We then report the first experiments in which fractograms dtsplay the characteristic “signature” predicted by theory for the steric transltion: a narrow terminal peak fobwed by a rapid dropoff to base line. I t is shown that the steric transinon polnt, which coincides with the dropoff, Is displaced to higher retention volumes with Increasing field strength, approximately as expected. The expected steric transhion phenomena are fwther confirmed by collecting narrow fractions of a polydisperse po@(vlnylchloride) sample near the steric tradion point and sublectlng them to electron microscopy. The particle size distrlbution of the fractions is found to be sharply bimodal, in accordance with steric transition theory. However, satlsfactory agreement between the measured partlcle diameters and the theoretical expressions Is found only by application of the more complicated equations in which y is assumed to be size dependent.
The methodology of field-flow fractionation (FFF) is based on the action of an external field or gradient whose direction is perpendicular to the axis of flow in a thin channel (1-3). The field forces particulate and macromolecular species to accumulate in narrow zones such that each is intercepted by different flow laminae and thus displaced at different velocities down the flow channel. The technique divides into a number of categories depending on the nature and distribution of the narrow zone and upon the field applied ( 4 ) . Normal FFF is defined as that group of techniques in which species are forced to one wall (the accumulation wall) by the field. Their mean distance from the wall is determined by the force exerted on the particles by the field and by diffusion (Brownian motion), which counteracts the buildup of particles at the wall. For components having the same density, those with the highest molecular mass or size have the greatest force exerted on them and they equilibrate closest to the wall. Here
the downstream fluid motion is highly retarded by the frictional drag of the wall and component particles are displaced only slowly along the flow axis. Species of smaller size occupy laminae positioned further from the wall where the flow displacement is more rapid. A trend is thus established in which particle velocity decreaseswith increasing particle size. This is illustrated by the leftihand branch of Figure 1in which velocity is expressed in terms of retention ratio R, the velocity of the component particles relative to the mean velocity ( u ) of the carrier fluid. In steric FFF the species are also pushed toward the wall. However, in steric FFF the nearness of approach to the wall is determined more by the size of the particles than by the competition between the applied force and Brownian motion. In the simplest model we imagine that the field-induced transverse motion of a particle is halted once it touches the wall, leaving the particle to protrude out into the flow stream by a distance equal to its diameter. Since large particles, by virtue of their size, extend more deeply into the flow channel than small, they are swept more rapidly downstream than the small particles. Thus particle velocity tends to increase with size, as shown by the right-hand branch of Figure 1. The opposing trends shown by normal FFF and steric FFF in Figure 1 are joined smoothly in a transition region. Thus, as particle size increases from ita most miniscule level, particle velocity decreases (retention time t, or volume V , increases) until one reaches a size such that the mean distance of the particle from the wall is little more than the particle diameter. A t this stage the particle size begins to exert a significant influence on displacement velocity by virtue of its physical extension in space. This occurs a t the beginning of the transition region. As the particle size increases further, the size-based effects increase and the particle velocity goes through a minimum (called the inversion point) and begins increasing. For still larger particles the steric effect is fully dominant, with the resultant velocity closely linked to particle diameter. The transition noted above has been characterized theoretically for both sedimentation FFF and flow FFF (5). The transition is generally expected to occur for particles of diameter from 0.1 to 1.0 hm, depending on the field strength. The retention ratio R at the inversion point is very small, of the order of which means that the inversion particles (those eluting at the inversion point) are retained roughly 100
0003-2700/88/0380-2328$01.50/0 0 1988 American Chemical Society
