Anal. Cham. 1991, 63, 1126-1132
1126
(19) LochmuHer, C. H.; Hamzavl-Abedl, M. A.; Ou, C.-X. J. Chromatogr. 1987, 387, 105. (20) Qluckman, J. C.; Hlrose, A.; McQuffln, V. L.; Novotny, M. Chromatographs 1983, 17, 303. (21) Dorsey, J. G. Personal communication. (22) Gilpin, R. K. J. Chromatogr. Set. 1984, 22, 371. (23) Gilpin, R. K. Anal. Cham. 1985, 57, 1465A. (24) Scott, R. P. W.; Simpson, C. F. J. Chromatogr. 1980, 197, 11.
(25) Carr. J. W.; Harris, J. M. Anal. Cham. 1987, 59, 2546. (26) Cole, L. A.; Dorsey, J. G. Anal. Cham. 1990, 82, 16.
Received for review November 21,1990. Accepted March 11,1991. This research was supported by the Michigan State University Foundation and the Dow Chemical Company.
Capillary Electrophoretic Separations of Proteins Using Nonionic Surfactant Coatings John K. Towns and Fred E. Regnier* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
well suited for the analysis of positively charged solutes where they are repelled from the surface, but severe wall interactions result when anionic proteins are analyzed. Stable, reproducible deactivation of the capillary is needed before capillary zone electrophoresis (CZE) will be of general utility in protein separation. The goal of this study was to develop a stable, reproducible coating that would reduce protein adsorption on capillaries and provide good recovery while some eiectroosmotic flow was maintained. The use of ionic surfactants has been used to control eiectroosmotic flow in both CZE (25, 26) and open tubular chromatography (27). Nonionic surfactants have been used for quite a different reason. It has been determined, in the case of chromatography, that nonionic surfactants can be hydrophobically adsorbed onto an alkylsilane-derivatized surface to create a hydrophilic layer that will exclude proteins from the surface (28-35). Borgerding and Hinze (29) examined the chromatographic effects of polyoxyethylene[23]-dodecanol (BRIJ 35) on an octadecylsilane (C18) column and found that unlike ionic surfactants, BRIJ 35 adsorbs in substantial amounts onto the reversed-phase surface. Other papers demonstrated that after the modification of reversed-phase columns by nonionic surfactant adsorption, proteins could be eluted with aqueous mobile phase either when the surfactant was in the mobile phase (32) or when it was deleted (33). Deschampe (32) found that surfactants in the mobile phase improved efficiency by reducing the denaturation of proteins. Chang (33) recognized that the hydrophilic layer established by the surfactants over a wide-pore reversed-phase sorbent creates a nearly permanent and ideal surface for the size-exclusion separation of proteins. The most definitive work on the effects of nonionic surfactants in exclusion media for large molecules was contributed by Desilets et al. (28). This study examined the chromatographic effects of polyoxyethylene surfactant size and structure on protein exclusion and small analyte separations. Adsorption of polyoxyethylene-based surfactants apparently creates a semipermeable, hydrophilic layer of adsorbed surfactant on an alkylsilane-derivatized surface that prevents the adsorption of proteins. A hydrophilic network is apparently created by the polyoxyethylene portion of the surfactant that may include polyoxyethylene loops, trains, and tails. It is suggested that this hydrophilic “forest” would keep proteins at a sufficient distance from either the reversed-phase surface or the residual silanol groups on the capillary wall to prevent adsorption and denaturation (28). The TWEEN and BRIJ series surfactants
Capillary zone electrophoretic separations of proteins have been achieved by using nonionic surfactant coated capMarles. Capillaries were prepared by derlvatlzatkm of the silica surface with octadecyMane Mowed by the deposition of a layer of non ionic surfactant from an aqueous solution above the critical micelle concentration. This coating Is of sufficient thickness and hydrophMdty to reduce both protein adsorption and electroosmotlc pumping. This hydrophWc coating reduces electroosmotlc pumping 5-8-fold while resolving proteins quickly and efficiently with good recovery. The coating provides a stable and reproducible means of deactivation, while the rate of electroosmotlc pumping stays relatively constant throughout the pH range 4-11. This allows the pH to be varied to enhance selectivity without adversely affecting the flow rate.
are
INTRODUCTION High-resolution capillary electrophoresis is proving to be of great utility in the separation of small molecules such as inorganic ions (1, 2), amino acids (3-6), small organic ions (7-10), peptides (11-13), and oligonucleotides (14,15). Unfortunately, the enormous resolving power of capillary electrophoresis has been of minimal value in the separation of proteins. The difficulty in applying this technique to proteins arises from silanol groups on the surface of fused-silica capillaries. Silanols ionize above pH 4 and greatly increase band spreading and peak tailing through adsorption or denaturation of many proteins on the capillary walls. Although acidic pH may be used to repress the ionization of silanols (16), or basic pH to produce a net negative charge on the protein, which is then repelled by the negatively charged capillary wall (17,18), these approaches introduce several new problems. Many proteins are denatured by extremes in pH, and the full pH range is necessary to discriminate between proteins on the basis of charge. Attempts to deactivate capillary walls by either silane derivatization (19, 20) or physically coating the silica surface (21,22) have been reported. These approaches, however, have been of limited success due to a lack of consistency in intercolumn performance, rapid deterioration of column efficiency, and limited utility at neutral pH. An alternative strategy to reduce protein adsorption has been to reverse the charge on the capillary wall from negative to positive through coating (23) or the addition of an amine polymer to the buffer (24). These two approaches 0003-2700/91/0363-1126802.50/0
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 11, JUNE 1, 1991
particularly useful due to their commercial availability, high purity, low cost, low toxicity, high cloud temperature, and low background absorbance compared to the other types of surfactants. This paper describes methods used to coat capillaries with an alkylsilane followed by water-soluble TWEEN and BRIJ series surfactants in such a manner that they are acceptable for capillary electrophoresis of proteins. Surfactant headgroup structure and size and hydrocarbon chain length are evaluated in terms of electroosmotic pumping, separation efficiency, peak capacity, and peak symmetry. BRIJ 35-coated capillaries were evaluated in terms of migration reproducibility, coating reproducibility, protein recovery, and stability and compared to uncoated capillaries. The results demonstrate the simplicity and viability of nonionic surfactant coatings in the separation of proteins for capillary electroare
phoresis.
EXPERIMENTAL SECTION Instrumentation. Capillary electrophoresis was performed instrument based on an in-house design. All high-voltage components of the system were contained in a Lucite cabinet fitted with a safety interlock that would interrupt the line voltage to the transformer in the power supply when the cabinet door was opened. A Spellman Model FHR 30P 60/El (Spellman High Voltage Electronics Corp., Plain view, NY) power supply was used on an
to apply the electric field across the capillary. The power supply output was connected to 22-gauge platinum wire electrodes immersed in 3-mL buffer reservoirs along with the capillary ends. Polyimide-coated fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) of 50 and 75 µ i.d., 200 µ o.d., were used with the total length varying between 50 and 100 cm and the separation length from 35 to 85 cm. On-line detection was performed with a variable-wavelength UV absorbance detector (Model V4 Isco Inc., Lincoln, NE). When two detectors were employed, the BAS UV-8 detector (Bioanalytical Systems, West Lafayette, IN) with an in-house capillary cell design served as the second detector. Detection was monitored at either 200 or 214 nm for proteins and peptides and 254 nm for mesityl oxide. The signal from the detector was fed to a Linear 2000 (Linear, Reno, NV) strip chart recorder. Electrophoresis. Protein solutions of 1.0 mg/mL were introduced into the capillary by syphoning for a fixed time (1-3 s) at a fixed height (10-15 cm). Mesityl oxide was used as the neutral marker. In order to help prevent gradual loss of surfactant from the reversed-phase surface, 0.001% by weight of the surfactant was dissolved in the buffer. Since 0.001% of surfactant is 10-100 times less than the critical micelle concentration of TWEEN and BRIJ series surfactants, no significant amount of micelles was ever present in these buffers. Several buffer solutions were used to operate capillaries over the pH range 3-11: 0.01 M acetate at pH 3 and 5, 0.01 M phosphate at pH 7, 0.01 M diami nopropane at pH 9 and 11. Salt was added to each buffer to give comparable ionic strengths and currents. During electrophoresis, current through the capillary never exceeded 50 µ , with all analyses being run at ambient temperature without temperature control. Between analyses, the capillary was flushed with double-distilled water and the separation buffer for 1 min. Capillary Coating. Capillaries were first treated with 1.0 M NaOH for 15 min followed by 15 min of washing with deionized (DI) water. Residual water was evaporated from the capillaries by connecting them to a gas chromatography (GC) oven at 100 °C for 2 h under a nitrogen pressure of 400 kPa. Octadecyltrichlorosilane with 5% methylene chloride was then pulled through the capillary by syringe. The capillary was next placed into an oil bath at 90 °C for 3 h, with new solution being pulled through the capillary every 15 min. After the 3-h silylation, the capillary was removed from the bath and wiped clean of oil, and the residual octadecyltrichlorosilane was removed from the capillary by pushing nitrogen through the capillary. The capillary was washed with several capillary volumes of methanol followed by a 30-min wash with filtered (0.2 µ ), double-deionized water. Micellar solutions of surfactant were made by dissolving 0.5% by weight of the desired surfactant in filtered (0.2 µ ), double-
·
1127
deionized water. This solution was pulled through the capillary for 2 h to ensure complete coating of the capillary. The capillary was then washed with the appropriate running buffer to remove
all
excess
surfactant.
Percent Protein Recovery. Protein recoveries were determined with a dual detector. Placing detectors at 20 and 70 cm from the capillary inlet allowed the percent recovery to be measured over the 50 cm of capillary between the detectors. Recovery of approximately 10 ng of protein ranging in pi from
11.0 for lysozyme to 3.2 for pepsin was determined by using an unabsorbed internal standard to account for discrepancies between the detectors and changes in flow rate between detectors. Detectors were also switched from front to back to take into account any differences in detector response. All capillaries were nm with a 0.01 M phosphate buffer at pH 7, with the capillaries coated with BRIJ 35 surfactant. For the protein recovery study, both coated and uncoated capillaries were discarded after each protein run to prevent any error due to protein adsorption from an earlier run. Percent recoveries were determined by subtracting peak areas from each detector after taking into account differences in detector responses. Reagents. Protein samples were purchased from Sigma Chemical Co. (St. Louis, MO) except for ovalbumin, which was
purchased from Pharmacia Fine Chemicals (Piscataway, NY). Nonionic surfactants, octadecyltrichlorosilane, mesityl oxide, and methylene chloride were purchased from Aldrich (Milwaukee, WI), as were all buffer reagents. Electrophoresis buffers were prepared from in-house double-distilled-deionized water.
RESULTS AND DISCUSSION In the deactivation of silanol groups by coating chemistry, it was a concern that an attempt to reduce protein adsorption would come at the expense of electroosmotic pumping. Electroosmotic pumping is needed so that proteins of positive, neutral and negative charge could all be swept through the capillary to the detection end. It was therefore necessary to reduce the negative silanol charges on the capillary wall that cause protein adsorption yet still allow for sufficient electroosmotic pumping. The rate of electroosmotic flow is governed by the potential drop across the surface double layer, which in turn is a function of the charge density on the capillary wall (36, 37). It has been noted above that one approach to controlling electroosmotic flow is to reduce the charge density at the capillary surface through the silylation of surface silanols. Because electrical potential decreases much more slowly than charged density (37), it is very difficult to eliminate electroosmotic flow by sequestering surface silanols. The influence of charge density on protein adsorption is quite different. Adsorption of macromolecules at a surface is generally a cooperative process. A decrease in charge density on the surface of either the molecule or the sorbent produces an exponential decrease in the force of interaction (38). When these two phenomena are considered together, it is seen that electrostatic adsorption of proteins to surfaces is expected to decrease more rapidly than the reduction of electroosmotic flow by derivatizing surface silanols and applying the surfactant coating. Thus there should be a surface charge and coating density where there is no protein adsorption to the capillary surface but adequate electroosmotic flow. In an attempt to prepare such a surface, nonionic surfactants of the TWEEN or BRIJ series were adsorbed onto alkylsilane-derivatized surfaces and their utility was examined in increasing protein recovery while electroosmotic flow was still maintained. The use of octadecylsilane phase alone was found to cut electroosmotic flow in half, but due to this phase’s strong hydrophobicity, proteins readily adsorbed to this surface and go undetected. Figure la shows the chemical structure of the TWEEN series of polyoxyethylene sorbitan monoalkylates. Table I shows the structural characteristics of the three selected TWEENs. Figure lb and Table II contain the analogous
1128 (a)
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HOfCHzCHgOl^CHz^CHa
Table III. Performance Parameters for the Five Selected Surfactant Molecules Adsorbed onto Alkylsilane-Coated
(OCH2CH2)jPH
(b) H2C
Capillary
A CHA CH2(OCH2CH2)/OOC(CH2)„^CH3
electroosmo-
surfactant
H0(CH2CH20)„HC—CH(OCH2CH2),OH z= no. of OEs
m=w+x+y+ OE
=
(OCH2CH2)
=
oxyethylene unit
Figure 1. (a) Chemical structure of TWEEN series of polyoxyethylene sorbitan monoalkylates, (b) Chemical structure of BRIJ series of polyethylene alkyl ethers.
TWEEN 20 TWEEN 40 TWEEN 80 BRIJ 35 BRIJ 78 1
n
surfactant
name
m
TWEEN 20 TWEEN 40 TWEEN 80
(oxyethylene units)
(alkyl
chain length)
20 20 20
12 16 18
Table II. Selected Water-Soluble BRIJ-Series Surfactants n
surfactant
BRIJ BRIJ
name
35 78
m
(oxyethylene units) 23
20
(alkyl
chain length) 12 18
information for two of the BRIJ series polyoxyethylene alkyl ethers selected. The TWEENs selected have the same hydrophilic head group (20 polyoxyethylenes) but differ in the alkyl chain length (12,16, and 18 carbons). The BRIJ surfactants differ in alkyl chain length (12 and 18 carbons) and differ slightly in hydrophilic head-group size (20 and 23 polyoxyethylenes). Several factors were considered in the selection of surfactants for this study. It will be shown below that the type of surfactant is very important in terms of both the chemical stability and ability to form a hydrophilic network above the alkylsilane surface. BRIJ series surfactants have a stable ether group linking the hydrophilic head group to the hydrophobic chain, while the TWEEN series has ester group linkages, which may be less stable. Although both types of surfactants create a hydrophilic network of “trees" or “branches” that can keep proteins at a sufficient distance from the surface to diminish adsorption, the size of the polyoxyethylene head group and degree of branching may play a role. In addition to chemical type, the critical micelle concentration (CMC) is important because the surfactant concentration must exceed the CMC to ensure complete surface coverage. For TWEEN and BRIJ series surfactants, the CMCs are very low, on the order of 10"4 to 10"6 M (28). It is also important that the surfactant be reproducibly adsorbed onto the alkylsilane surface. Less soluble members of the TWEEN and BRIJ series must be dissolved with organic solvents and might not be deposited on the alkylsilane surface reproducibly. The five surfactants listed above were examined to determine how parameters such as head-group structure and length of the hydrocarbon tail affected capillary performance. The influence of each surfactant was evaluated in terms of electroosmotic flow, separation efficiency, peak capacity, and peak symmetry (Table III). It was found from examining the TWEEN series, where the head-group size is held constant against different hydrocarbon chain lengths, that the length of the hydrocarbon chain over the range from 12 to 18 had little influence on the capillary performance. In contrast, there was a large change in capillary performance when head-group size and structure were varied by coating the capillary with
plates0
peak capacity6
peak skew6
2.03 2.48 2.27 1.50 1.26
170000 135000 150000 240000 115000
35 30 30 60 25
0.945 0.948 0.945 0.946 0.858
no.
“N/m = 5.54(t./uj,,,)8 for myoglobin on Interval between lysozyme and myoglobin c Ratio of a/b 15 10% peak height. 6
Table I. Selected Water-Soluble TWEEN-Series Surfactants
of
tic flow x m2/V s
108,
an
80-cm capillary.
on 80-cm
capillary.
the BRIJ series of surfactants. Comparing data for the branched TWEEN surfactants with that of the linear BRIJ series shows that head-group structure had a large impact on capillary performance. Capillaries coated with the linear BRIJ series showed a marked decrease in electroosmotic flow. Since the electroosmotic flow is coupled to both charge density and double-layer thickness, the decrease in electroosmotic flow resulting from the various surfactants probably reflects differences in coating thickness. This is further indicated by the increase in efficiency on the BRIJ 35 capillary compared to the TWEEN series. This increase in efficiency and decrease in electroosmotic flow may be due to the smaller BRIJ surfactant head groups being able to cover the alkylsilane surface more efficiently and mask the silanol groups. The decrease in electroosmotic flow is continued for BRIJ 78-coated capillaries where the head group is smaller than it is for BRIJ 35 but the alkyl chain length is increased from 12 to 18. However, decreasing electroosmotic flow does not correspond to an increase in efficiency. The BRU 78-coated capillary suffers from reduced efficiency, peak capacity, and peak symmetry compared to BRIJ 35. This could be due to the smaller head group of BRIJ 78 not sufficiently masking the surface, which would allow the proteins to interact with the hydrophobic C18 surface. Thus the nature of the oxyethylene head group, either head-group structure or length, is a critical parameter in determining capillary performance. A sample of small hydrophobic compounds similar to those separated by chromatographic exclusion media (28) were run to determine the small-analyte separation capability of these capillaries. These compounds could not be separated presumably due to insufficient solute partitioning between the mobile and stationary phases as a result of the large inner diameter of the capillary (39), the inner diameter of 75 µ being far from the ideal diameter of 2 µ for open tubular chromatography (27). An interesting feature of the BRIJ 35 capillary is the increased peak capacity compared to the TWEEN series. The peak capacity, which approximates the maximum number of peaks that can be separated into the available separation space, is taken in this case as the interval from lysozyme to myoglobin. This increase in peak capacity for TWEEN 20 compared to BRIJ 35 is shown for a 50-cm capillary in Figure 2 for proteins ranging in pi from 11.0 for lysozyme to 7.3 for myoglobin. The electropherograms for the two capillaries show that although elution times for lysozyme, the most positive protein, increase only slightly, the elution times increase dramatically for proteins of lower p7 values. This occurs without a significant increase in the peak widths. The separation of acidic proteins is shown in Figure 3. These proteins are negatively charged at neutral pH and therefore move against the electroosmotic flow toward the positive electrode. This decrease in net velocity allows shorter capillaries to be used (30-cm length as compared to 50 cm for the basic pro-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 11, JUNE
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Figure 3. Electropherogram of acidic proteins at pH 7.0: (1) myoglobin, (2) conalbumin, (3) transferrin, (4) /3-lactoglobulln B, (5) d-lactoglobulln A, and (6) ovalbumin. Detection was at 200 nm, 300 V/cm, and 26 µ on a 75 µ X 30 cm modified capillary using 0.01 M phosphate/0.001 % (w/w) BRIJ 35 buffer.
Figure 4. Dependence of electroosmotic flow (m2/V s) X 10® on pH for ( ) uncoated and (A) BRIJ 35/alkylsilane-coated capillary (75 µ X 50 cm).
Figure 2. Electropherograms showing the separation of five basic proteins [(1) lysozyme, (2) cytochrome c, (3) rlbonudease A, (4) -chymotry psinogen, and (5) myoglobin] using a 0.01 M phosphate buffer (pH 7.0) on a 75 #tm X 50 cm (A, top) TWEEN 20/alkylsilane capillary and (B, bottom) BRIJ 35/alkylsilane capillary at 300 V/cm.
teins). The speed and resolving power of CZE is well illustrated by the separation of d-lactoglobulins A and B in the acidic protein separation. These two proteins differ by only two amino acids in a total sequence of 162. Through substitutions at position 64 (glycine for an aspartic acid) and position 118 (alanine for a valine) the net charge of d-lactoglobulin B compared to that of A is sufficiently altered to induce a significant change in electromigration rate. The result is base line resolution of the two species in less that 6 min. Due to the success of the BRIJ 35-coated capillary, this coating was used for the remainder of the study. The rate of electroosmotic flow is dependent on the magnitude of the f potential across the solution-solid interface, which in turn is dependent on the charge density at the capillary wall. Thus, an increase in silanol ionization at higher pH would result in an increase in electroosmotic pumping. The effect of pH on electroosmotic flow for both coated and uncoated capillaries was examined over the pH range from
Figure 4 shows the dramatic difference between the two capillaries over this range. There is a 10-fold increase in electroosmotic flow when an uncoated capillary is used over the pH range from 3 to 11 with the largest increase being from 4 to 8. This increase can greatly affect solute residence time in the capillary and therefore separation efficiency and resolution. This dramatic pH sensitivity to electroosmotic flow in uncoated capillaries is in sharp contrast to that found with the BRIJ 35-coated capillaries. Because there is little increase between pH 4 and 11, it is possible to run a BRIJ 35-coated capillary between pH 4 and 11 without a significant change in capillary-dependent separation variables. The best pH can be employed to give optimum selectivity without adversely affecting the analysis time. It was observed in both chromatographic systems (28) and CZE that surfactant gradually leaches from coated surfaces. Thus, a small amount of the surfactant was added to the running buffer to maintain the coating. Although the amount of surfactant should be below the CMC to prevent any micelle-solute interaction, a study was performed to determine the effects of micelle formation on capillary performance. BRIJ 35 surfactant was dissolved in 0.01 M phosphate buffer (pH 7.0) to give the following surfactant concentrations: 1.0 3 to 11.
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Figure 5. Electropherograms for five proteins [(1) lysozyme, (2) cytochrome c, (3) ribonuclease A, (4) a-chymotrypsinogen, and (5) myoglobin] using a 0.01 M phosphate buffer (pH 7.0) on a 75 µ X 80 cm capillary with detectors at (A) 20 cm and (B) 65 cm from the Injection end.
Table V. Protein Recovery on a 75 µ X 50 cm Section of BRIJ 35/Alkylsilane-Coated and Uncoated Capillaries
Table IV. Performance Parameters as a Function of Surfactant Concentration in Separation Buffer concn,M 1.0 1.0 1.0 1.0
X 10"6 X "6 X 10"4 X 10'3
electroosmotic flow X 10®, m2/V 1.781 1.496 1.295 1.028
s
no.
of plates” 80000 110000 90000 55000
% recovery” uncoated BRIJ 35
peak capacity6 25 35 30 25
"N = 5.54(t,/ui1/2)2 for myoglobin on a 50-cm capillary. 6 Interval between lysozyme and myoglobin on 50-cm capillary. X "6 (0.1 times (0.01 times CMC value), 1.0 X "6 CMC value), 1.0 X "4 M (CMC value), and 1.0 X 10"3 M (10 times CMC value). There was little effect on efficiency, peak capacity, or peak symmetry for capillaries run with surfactant concentrations up to the CMC. The electroosmotic flow, however, was proportional to surfactant concentration. Table IV shows the decrease in electroosmotic flow as the surfactant concentration is increased. This decrease in flow is probably the result of an increase in surfactant coating density which would in-tum provide better masking of the negatively charged wall. Above the CMC, the electroosmotic flow continues to decrease. This may be the result of multilayer adsorption with primary and secondary monolayers being formed (29). As the coating thickness is increased, the plane of shear is moved further away from the capillary surface, which would in turn lower the electroosmotic flow. The reduction in electroosmotic flow as a function of surfactant concentration in the buffer is also affected by the reduction in electrical potential. The potential may fall as much as 100 mV in a distance of 20 A from the capillary surface (37). Electrical potential would decrease with increasing layer thickness and result in a reduction in electroosmotic flow. A reduction in efficiency for capillaries run with surfactant buffer concentrations above the CMC was also apparent. Above the CMC, BRIJ 35 monomers cooperatively associate to form micellar aggregates of about 40 molecules (40). It is possible that the presence of these micelles in the buffer solution interact with proteins, causing the observed 2-fold decrease in efficiency. This decreased efficiency could also be attributed to multiple surfactant layers on the capillary wall, as described above.
PROTEIN RECOVERY Due to the small amount of protein injected into the capillary, it was difficult to collect enough sample from the capillary for chemical quantitation. The percent recovery therefore was determined on column. Using two detectors 50 cm apart on a 100-cm capillary, it was possible to determine
protein lysozyme cytochrome c ribonuclease
a-chymotrypsinogen myoglobin conalbumin carbonic anhydrase 3-lactoglobulin B 3-lactoglobulin A ovalbumin pepsin
pi value
capillary
capillary
11.0 10.2 9.3 9.2 7.3 6.3 6.2 5.2 5.1 4.7 3.2
0 0
94 91 92 95 94 96 95 96 97 96 98
0
0 73 76 80 85 86 85 91
“Determined by using two detectors 50 cm apart on a 75-µ capillary. Conditions: 0.01 M phosphate buffer/0.001 % (w/w) BRIJ 35 (pH 7.0); detection at 214 nm, 300 V/cm, 30 µ .
the loss of protein in the 50-cm section between detectors. Percent recoveries were determined by subtracting peak areas. An internal standard was used along with switching the detectors front to back to compensate for any response differences between the detectors. Table V lists the percent recovery of 11 proteins for both a BRIJ 35-coated and uncoated capillary with a 0.01 M phosphate buffer at pH 7.0. The dramatic difference in recoveries between coated and uncoated capillaries is most noticeable for proteins with high pi values. Positively charged proteins with pi values above 8, such as lysozyme (pi 11), cytochrome c (pi 10.2), ribonuclease A (pi 9.3), and chymotrypsinogen A (pi 9.2) were readily adsorbed onto the silica wall. As the pi values of proteins decrease, recovery increased. For example, myoglobin with a pi of 7.3 was the first protein recovered. The increase in recovery with decreasing pi continues until it reaches 91% for pepsin (pi 3.2). The increase in recovery for proteins with low p/ values is due to coulombic repulsion between the negatively charged protein and the negative wall of the capillary. However, there is still significant protein loss for most negatively charged proteins. Recovery from the coated capillaries is in sharp contrast to that found for the uncoated capillaries. The recovery of all the proteins with p7 values 11.0-3.2 on the BRIJ 35-coated capillary was greater than 90%. The surfactant network on the reversed phase is able to keep the proteins at a sufficient distance from the wall to greatly decrease protein adsorption. Although the goal of the two-detector system was to quantitate recovery, detectors at two points along the capillary provided information on the contribution of capillary length
ANALYTICAL CHEMISTRY, VOL. 63, NO. 11, JUNE 1, 1991
Table VI. Capillary Efficiency Length
as a
Function of Separation
Table VII. Stability of BRIJ 35/Alkyleilane-Coated
20-cm separation
66-cm separation
length
length
protein
plates'*
lysozyme cytochrome c ribonuclease A a-chymotrypsinogen myoglobin
8.0 10.0 5.0 9.1 3.6
25000 20000 40000 22000 55000
=
1131
Capillary
plate height, µ
“N
·
no.
plate height,
of 1
no.
protein elution time, min electrophoretic running time, h
of
µ
plates"
protein
pi value
0
40
80
6.5 7.2 3.0 5.4 2.7
100000 90000 220000 120000 240000
lysozyme cytochrome c ribonuclease A a-chymotrypsinogen myoglobin conalbumin carbonic anhydrase /3-lactoglobulin B 0-lactoglobulin A ovalbumin pepsin
11.0 10.2 9.3 9.2 7.3 6.3 6.2 5.2 5.1 4.7 3.2
3.9 4.2 6.8 7.3 9.7 12.5 13.2 16.3 17.0 22.8 31.2
4.0 4.3 6.9 7.4 9.9 12.8 13.5 16.6 17.3 23.2 32.3
4.1 4.4 7.1 7.6 10.2 13.1 13.9 17.0 17.9 24.1
5.54(t,/ui1/2)2 on a 75 Aim x 80 cm total length capillary.
to performance. The electropherograms in Figure 5 show the effect of two detectors placed 20 and 65 cm from the inlet end of an 80-cm capillary using a 0.01 M phosphate buffer (pH 7.0) at 300 V/cm. Results from the first detector show that the five proteins are well separated in 10 min by using a separation length of 20 cm. However, the plate heights are high relative to 35-cm separation length and 50-cm total length capillaries. Although elution times are proportional to capillary length, the total number of plates in the column is not. This is unexpected. The total number of plates and zone width variance should be proportional to separation path length (41). Table VI shows that there is a substantial decrease in plate height and increase in efficiency for zones passing at 65 cm from the inlet compared to 20 cm. The decrease in plate height in turn results in over a 2-fold increase in peak capacity from 30 to 65. The fact that peaks are wider than expected with short capillaries may be due to some band-spreading effect at the capillary entrance (16). For example, a finite period of time may be required to establish equilibrium in flow rate, temperature, and double-layer characteristics after voltage is applied to the capillary. This would become less of a factor as capillary length is increased. A more likely cause is too large of an injection volume, which is due in a large part to limited detector sensitivity (42). As has been observed by others (43), capillary lengths above a certain length showed little improvement in efficiency. The plate numbers in capillary electrophoresis are not proportional to column length as would be the case in chromatography. It was found that column lengths above 80 cm showed no increase in efficiency, and therefore these numbers were not reported in plates per meter. To further evaluate the capillary coating, a study was undertaken to determine the long-term stability of the modified phase. A 50-cm BRIJ 35-coated capillary was run over a 3-week period for 80 h at 300 V/cm with a 0.01 M phosphate/0.001% (w/w) BRIJ 35 buffer ranging in pH from 5 to 8. During these 80 h, over 120 lysozyme samples (p/11.0) were injected. It was found that the elution time did not change significantly over this period (Table VII). However, protein elution times did increase slightly during the test, possibly due to protein accumulation. The elution times, however, were returned to initial values by washing with 0.5% BRIJ in DI water for 15 min followed by a 15-min wash with running buffer. The adsorption/desorption of nonionic surfactant provides a continuously regenerating coating surface that easily removes protein accumulation. The migration reproducibility was studied by using the BRIJ 35/alkylsilane coating on 75 µ X 50 cm capillaries. It was found that the run to run reproducibility was 0.8% relative standard deviation (RSD) ( = 6), while the day to day reproducibility was 1.3% RSD ( = 6). A section to section reproducibility of 1.8% RSD (n 6) was determined by cutting a 6 m long coated capillary into 12 segments and running every other segment. The column to column mi-
gration reproducibility was examined over and found to be 2.4% RSD ( = 10).
a
33.8
3-month period
CONCLUSION Oxyethylene-based surfactants can be readily adsorbed onto alkylsilane derivatized surface to produce a coating that effectively excludes proteins from the negatively charged capillary wall. It is proposed that the hydrophilic head group of the surfactant creates a hydrophilic layer that masks the underlying alkylsilanes and more residual silanol groups. Head-group structure (branched vs linear) is an important performance determinant whereas hydrophobic chain length has less of an effect. Surfactant-coated capillaries give high protein recovery. The masking of negatively charged silanol groups reduces electroosmotic flow, producing a flow rate that increases only slightly from pH 4 to 11. This makes it possible to vary pH to optimize selectivity without significantly afan
fecting the rate of electroosmotic flow. Note Added in Proof. Further research on the capillary electrophoretic separation of macromolecules using nonionic surfactant coatings has found that, in the separation of hydrophobic peptides, an increase in the amount of surfactant in the running buffer from 0.001% to 0.01% drastically improves separation performance. The increased surfactant concentration may provide a thicker and/or more complete coverage of the adsorbed hydrophilic coating layer that better excludes small hydrophobic peptides from the underlying alkylsilane surface.
ACKNOWLEDGMENT We thank Peter T. Kissinger for the use of the additional UV detectors. Funds for this research were provided by the National Institute of Health (Grant GM 25431) and Pfizer, Inc. Registry No. TWEEN 20,9005-64-5; TWEEN 40,9005-66-7;
TWEEN 80,9005-65-6; BRIJ
35, 9002-92-0;
BRIJ 78,9005-00-9.
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Gross, L.; Yeung, E. J. Chromatogr. 1988, 480, 189-178. Gassmann, E.; Kuo, J. E.; Zare, R. N. Science 1885, 230, 813-814. Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1885, 332 , 219. Gozel, P.; Gassmann, E.; Mlchelson, H.; Zare, R. N. Anal. Chem. 1887, 59, 44. (8) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1888, 60, 1832-1834. (7) Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1882, 248, (2) (3) (4) (5)
241-247.
(8) Tsuda, A.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1883, 264, 385. (9) Demi, M.; Foret, F.; Bocek, P. J. Chromatogr. 1985, 320, 159. (10) Fujlwara, S.; Honda, S.; Anal. Chem. 1987, 59, 487. (11) Jorgenson, J. W.; Lukács, K. D. J. Chromatogr. 1981, 218, 209-218. (12) Firestone, . A.; Michaud, J. P.; Carter, R. N.; Thormann, W. J. Chromatogr. 1987, 407, 363. (13) Deyl, Z.; Rohlicek, V.; Adam, M. J. Chromatogr. 1989, 480, 371-378.
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(33) Chang, J. P. J. Chromatogr. 1984, 317, 157-163. (34) Shlhabe, Z. K.; Dyer, R. D. J. Uq. Chromatogr. 1987,
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10 (11),
(35) DeLuccIa, F. J.; Arunyanart, M.; Yarmchuk, P.; Weinberger, R.; Clime Love, L. J. LC Magazine 1985, 3 (9), 794. (36) Pertorlus, V.; Hopkins, B. J.; Schleke, J. D. J. Chromatogr. 1974, 264, 385. (37) Davies, J. T.; Rleal, E. K. Interfacial Phenomena; Academic Press: New York, 1961. (38) Regnler, F. E. Science 1987, 238, 319. (39) Novotny, M. Anal. Chem. 1988, 60, 500A-510A. (40) Kalyanasundaran, K.; Thomas, J. K. In Mlcelllzatlon, Solubilization, and Microemulsions; Mittal, K. L., Ed.; Plenum Press: New York, 1977; Vol. 2, pp 569-588. (41) Jorgenson, J. W.; Lukács, K. D. HRC &CC,J. High Resotut. Chromatogr. Chromatogr. Commun. 1981, 4, 230-231. (42) Huang, X.; Gordon, M. J.; Zare, R. N. J. Chromatogr. 1989, 480,
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Received for review November 21, 1990. Accepted March 11, 1991.
Experimental Evaluation of Conflicting Models for Size Exclusion Chromatography Syed Hussain,1 2Mamta S. Mehta,1,2 Jerome I. Kaplan,3 and Paul L. Dubin*1
Departments of Chemistry and Physics, Indiana University-Purdue University, Indianapolis, Indiana 46205-2820
and it is not always clear what constitutes an appropriate calibration. The requirement for calibration arises in part from the absence of a complete theory for SEC, which would exactly define the relationship between the macromolecular dimensions of the solute and the experimentally measurable chromatographic partition coefficient, KSEC;
Comparisons were made among the predicted dependences of Knc, the chromatographic partition coefficient, on the solute radius R, for three widely acknowledged theories of size exclusion chromatography. It was found that data which appear to agree with the general form of Zf(fl) for any one theory will also appear to support the others, because of the mathematical relationships among the three K(R) functions, over the range of K most usually reported. It must be concluded that the shape of the functional dependence of Kwc on R alone does not provide a meaningful test of theory. To test these theoretical relations, data were obtained by aqueous SEC on Superóse columns for molecular weight fractions of Flcoll. This solute may be shown to closely approximate a rather Inflexible sphere, and hydrophobic and electrostatic Interactions can be neglected for this polymer/statlonary phase pair. The selection of conditions thus reduces uncertainties related to the definition of R and minimizes the influence of enthalplc effects (l.e., "nonideal SEC”). The measured dependence of Kxc on R (with corrections made for polydlsperslty of the samples) is found to agree remarkably well with predictions from a treatment In which the stationary phase Is modeled as a Gaussian distribution of cylindrical cavities.
V0
(1)
where Ve is the retention volume of the solute, V0 the interstitial volume (obtained experimentally as the elution volume of a solute too big to enter the pores), and Vt the total volume of liquid in the column, equal to the sum of V0 and the pore volume. KSBC thus represents the fraction of the pore volume accessible to the solute and ranges from zero to unity. Despite decades of investigation, considerable debate still exists about the proper form of the theoretical relationship among Ksec. pore size, and macromolecular dimensions. Therefore, conversion of SEC elution volumes to some welldefined dimension is not possible in the sense that say the measured diffusivity leads to an equivalent Stokes radius. It is clear that the retention time in SEC—barring nonsteric interactions between the solute and the stationary phase— must depend uniquely on the dimensions of the solute and the stationary-phase pore. If these objects possessed simple geometry, the dependence of K on pore and solute sizes would take a very simple form; i.e., K = (1 R/rp)X (2)
INTRODUCTION
-
Size exclusion chromatography (SEC) is sometimes viewed
where R is the radius of a spherical solute and rp the pore radius and where = 1,2, or 3 for slab, cylindrical, or spherical pores (1). (We designate K as the theoretical equilibrium constant equivalent to the relative solute concentration within the pore, C/C0, and so distinguish it from the measured quantity Ksec· It is important to note that KSBC is equivalent to what others have called KD, but different from K„, which early workers defined as the fraction of the swollen gel ac-
liquid chromatographic separation technique, sometimes as a molecular weight method. It fulfills the latter role only when the column is calibrated with appropriate standards, as a
Department of Chemistry. Current address: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61821. 3 Department of Physics. 1
2
0003-2700/91Z0363-1132502.50/0
Ve-
=
©
1991 American Chemical Society
