1350
Anal. Chem. 1991, 63,1350-1354
albumin followed quite closely to the expected variation in volume with temperature. In the case of myoglobin, there was also reasonable correspondence; however, the slope was slightly lower than the calculated volume change. Nevertheless, the results of Figure 8 clearly show a strong temperature dependence. A corollary of this study is that precision in the amount injected will depend strongly on the column temperature being maintained constant. Finally, the precision of the measurements with column temperature controlled is shown in Table I. The relative standard deviation in migration time was found to be 0.5% or less, that of the peak area 2.3% or less, and that of peak height 3.8% or less. It is interesting to note that good precision is obtained even at 50 "C. The values in Table I are comparable to those reported by other authors in HPCE (13).
CONCLUSIONS This study has been shown that column temperature can dramatically affect the electrophoretic pattern of proteins in HPCE. Reduction of iron in the heme protein, myoglobin, has been observed. Moreover, this on-column reduction is kinetically controlled, and column temperature affects the rate significantly. Thus, the specific electrophoretic pattern observed will be dependent on sample handling of myoglobin, column temperature, electric field, and column length, among other factors. In addition, we have observed the electrophoretic consequences of a conformational change in a-lactalbumin. In analogy to the behavior in HPLC, the temperature region where both the folded and conformationally altered species simultaneously exist yields an electrophoretic peak that is significantly broadened and somewhat asymmetric. The results point to the need for good temperature control in order to achieve a reproducible electrophoretic pattern.
Moreover, as in HPLC ( 5 9 ,IO), it must be recognized that broadened or multiple peaks do not necessarily mean that a protein sample is impure. Finally, since other proteins may be far less labile than those studied here, subambient temperature control is a desirable feature of HPCE equipment in the future. Operation at 4 "C is often recommended for protein separations on slab gels as well (14). ACKNOWLEDGMENT We gratefully acknowledge support by Beckman Instruments, Inc., and the James L. Waters chair in analytical chemistry. We further thank Dr. Shiwen Lin and Dr. Peter Oroszlan for helpful discussions.
LITERATURE CITED Hjerten, S., Electrophoresis 1000, 1 7 , 665-690. Privalov, P. L. Adv. in Protein Chem. 1070, 33. 167-241. Nelson, R. J.; Pauius. A.; Cohen, A. S.; Guttman. A,; Karger, B. L. J. Chromatogr. 1080, 480, 111-127. Kronman, M. J. Critical Reviews in Biochemistry 8 Molecular Biology; CRC Press: Boca Ratan, FL, 1969; Voi. 24, pp 566-667. Oroszian, P.; Blanko, I?.; Lu, X.-M., Yarmush, D.; Karger, B. L. J . Chromatogr. 1000, 500, 481-502. Terabe, S.; Otsuka, K.; Ando. T. Ami. Chem. 1980, 6 1 , 251-260. Rothgeb, T. M.; Gurd, F. N. R. Enzymol. 1078, 52. 473-466. Mayo, S. L.; Ellis, W. R., Jr.; Crutchley, R. J.; Gray, H. B. Science 1086, 233, 948-942. Lu, X. M.; Benedek, K.; Karger, B. L. J . Chromatogr. 1086, 359, 19-29. Wu, S . L.; Benedek, K.; Karger, B. L. J . Chromatogr. 1086, 359, 3-17. Oroszlan, P.; Karger, 8. L. Unpublished results. Kim, P. S.; Baidwin, R. L. Ann. Rev. Blochem. 1082, 51, 459-489. Morina, S. E.: Colburn. J. C.: Grossman. P. D.: Lauer. H. K. LC-GC 1900,-8, 34-46. Chrambach, A. The Practice of Quant&thre Gel Electrophoresis; VCH: Deerfield Beach, FL. 1985: Chapter 5.
RECEIVED for review December 20,1990. Accepted March 14, 1991. This is contribution No. 421 from the Barnett Institute of Chemical Analysis and Material Science.
Preparation and Evaluation of a Bimodal Size-Exclusion Chromatography Column Containing a Mixture of Two Silicas of Different Pore Diameter David M. Northrop, R. P. W. Scott, and Daniel E. Martire* Chemistry Department, Georgetown University, Washington, D.C. 20057
Size-exclusion results from four dlfferent pore-sized silicas were used to select two of these silicas for use In a bimodal SlZe-excJudon cokmn. A mbttwe of 80- and 500-A poresized slllcas provided an excluslon curve of elutlon volume versus log molecular welght with a linear range from 5 X lo2 to 2 X 10' MW. The excluslon propertles were retained when a reversed-phase packlng was prepared by uslng the mlxed pore-slzed a k a . Column efficiency was found to be virtually Independent of pore slze.
INTRODUCTION Size-exclusion chromatography (SEC) is a liquid chromatographic method that provides molecular weight discrimination of samples. Conditions are chosen such that interactions between the solute and the stationary phase are mini-
mized. As a result, separations occur based on a solute's ability to migrate into and out of the pores of the stationary phase. This is largely determined by the solute's size, specifically the solute's hydrodynamic radius (the molecule and associated solvent) or the radius of gyration for macromolecules (the coiled molecule plus associated and entrained solvent) ( I ) , which are related to a solute's molecular weight and shape. Because there is essentially no interaction of solutes with the stationary phase, there is no retention; thus, each solute's elution volume is no greater than the total solvent volume of the column, V,. As described by Alhedai et al. (2), V , can be divided into several different regions including contributions from the various parts of the interstitial, Vi, and pore volumes, V,, where V , is the sum of Vi and V,. Nonionic molecules may sample as much of the total solvent volume as their size will permit. As the effective size of a solute increases, it is excluded
0003-2700/91/0363-1350$02.50/00 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991
from pores that are smaller. If a solute is completely excluded from the pores, it may only sample the accessible interstitial volume. Thus,in SEC, all solutes elute between the accessible interstitial volume and the total solvent volume. Because of this limited range, the analysis is rapid and complete, but this is at the expense of selectivity. It has been shown (I) that columns with different pore-size stationary phases provide separations over different molecular weight ranges; thus, it is possible to select a column to provide information about solutes in many different ranges. SEC has been applied to a variety of separation problems, particularly those involving macromolecules. Polymers with a narrow molecular weight distribution can be separated into narrow bands (3), while the characteristic molecular weight distribution curve of broad molecular weight polymers can also be obtained (4). SEC of biopolymers has been of particular interest with the development of many procedures for the molecular weight separation of peptides and proteins (5). The narrow molecular weight range that can be separated by a single pore-sized column is one of the limitations of SEC. Ambler e t al. (6) demonstrated the use of several columns, containing silicas of varying pore sizes, in series to provide a wide range of molecular weight separations. Yau e t al. (7) found that using a bimodal column set could produce the same result as long as the two columns were picked such that their individual linear exclusion ranges did not overlap. The resulting columns in series provided exclusion over a molecular weight range equal to the combination of both individual ranges. It was also found that the same result could be achieved by manufacturing a single silica particle containing both pore sizes. This was accomplished by layering microspheres of one pore size over a core of microspheres of another pore size. Thus, it is possible to obtain exclusion of solutes over a 4-5-decade molecular weight range by using either two columns in series or a single column containing bimodal pore-sized silica particles. The purpose of this paper is to describe the characterization of silica packing materials, to be used in SEC, and to describe a simple procedure for the formulation of a single mixed-bed column containing bimodal pore-size silica, using the resulting information. The effect of pore size on efficiency was also studied to determine if mixing silica particles would have any effect on efficiency and, in particular, on the resistance to mass-transfer term. One of the primary reasons for constructing such a column was to study the retention behavior of macromolecules, under reversed-phase conditions, in a system where the exclusion effects were well characterized and where polymers as large as 2 X lo6 MW would not be completely excluded. That study is the subject of a subsequent paper. It should be mentioned that mixed beds of polymer gels have been prepared and utilized in the separation of hydrophilic (8)and hydrophobic (9) polymers using gel-permeation chromatography (GPC). Columns for this type of work are commercially available. However, silica-based columns are not as expensive as gel columns, they are not as sensitive to solvent changes, they are not as prone to damage if the solvent dries out of the column, and they can, in general, withstand higher operating pressures. The idea of mixed-bed silicas was suggested in a patent by Kirkland and Yau (US. Patent 4160728) and was a t one time marketed by Waters Associates, Inc. (Milford, MA), under the trade name rBondage1, E-linear. Specific details as to the procedures for the formulation of these types of columns are not available and, therefore, are presented here. The effect of pore size on efficiency has been examined by de Vries e t al. (IO) for silica particles and by Copper and Johnson (11)for porous glass. However, neither study looked
1351
Table I. Solutes Used for the SEC Study solute benzene naphthalene anthracene sodium iodide polystyrene (PS) PS 517 PS 1024 PS 4000 PS 9000 PS 17500 PS 30740 PS 5oooo PS 100000 PS 233000
PS 390000 PS 600000 PS 1800000 PS 2800000 PS 15000000
PS 30000000
MW, g/mol 78.12 128.19 178.24 149.89 517 1024 4000 9000 17 500 30 740 50 000 100000 233 000 390 000 600 000 1800000 2800000 15000000 30000000
a t the specific effect of pore size on the various terms of the van Deemter equation and, in particular, the resistance to the mass-transfer term. Careful characterization of mixed-bed silica columns with respect to efficiency also has not been reported.
EXPERIMENTAL SECTION Four standard high-performance liquid chromatography (HPLC) columns, 25.0 cm X 0.46 cm, each packed with a different pore-sized (80-, 120-, 300-, and 500-A average pore diameter) 5 r m particle-size silica, were received from Advanced Separation Technologies, Inc. (ASTEC). AU solvents were either HPLC grade or Spectra-analyzed grade from Fisher Scientific (FairLawn,NJ). Probe solutes (see Table I) included reagent-grade benzene, naphthalene, anthracene, sodium iodide, and narrow distribution molecular weight standard polystyrenes obtained from Scientific Polymer Products, Inc. (Ontario,NY). 4-(Dimethylamino)pyridine was purchased from Sigma (St. Louis, MO) and n-butyldimethylchlorosilane from Petrarch Systems, Inc. (Bristol, PA). The HPLC system consisted of a Beckman Model 112 pump, a Valco injector with a 0.5-pL internal sample loop, a specially constructed ultraviolet absorbance detector operating at 254 nm with a specially designed 2-pL flow cell, and a constant-temperature circulating bath, which maintained the column temperature at 30.00 i 0.02 O C throughout the experiments. Elution volumes and flow rates were measured by using a 5.00-mL graduated buret (1 division = 0.01 mL f 0.001 mL). A PerkinElmer Model 56 chart recorder was used for data collection. All solvents were degassed by stirring under vacuum. Prooedures. AU experiments described herein were performed in triplicate unless otherwise noted. The size-exclusion properties of each column used in this project were determined by the following procedure. Tetrahydrofuran (THF) was pumped through the column at a flow rate of 0.5 mL/min (the actual flow rate was accurately determined). Injections (0.5 pL) of the polystyrene standards, at approximately 0.05 mg/mL, were made. The retention volume of each solute was measured by using the calibrated buret to collect the effluent. Efficiency studies were conducted with a 90% THF, 10% HzO mobile phase with NaI as a pore-excluded marker, to determine the mobile-phase linear velocity, and benzene as the pore probe. Flow rates of 0.1,0.2,0.4,0.8,1.6, and 3.2 mL/min were used with the linear velocity determined by l i= L / ( V , , / F ) (1) where ti is the linear velocity in cm/s, L is the column length in cm, Vexis the retention volume of the pore-excluded NaI in mL, and F is the volumetric flow rate in mL/s. The efficiency ( N = theoretical plates) was determined by N = 4(tR/Wi)' (2) where tR is the retention time of benzene and wi is the peak width
1352
ANALYTICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991
Table 11. Results of the Total Column Volume Studies
column, A
V,, mL
80 120 300 500
3.031 3.207 3.415 3.338 3.189 2.999
V,
mL
%
3
1
diff
2 j c n q s t r o m pores
A
3CC angstrom Dores 530 anas'rcm pores
0
Mix-NPa Mix-RPb
2.969 3.104 3.363 3.273 3.158 2.913
3 3 angstrom pores
o
2.09 3.32 1.55 1.99 0.98 2.95
a Mix-NP = mixed silica, normal phase. * Mix-RP = mixed silica, reversed phase.
at 0.6065h where h is the peak height. The height equivalent to was calculated by a theoretical plate (H) H =L/N (3) Values of H and ii were used to construct plots of H versus ii and Hii versus ii, with the latter plot being fitted to a second-order polynomial in order to determine the multipath term, A , the longitudinal diffusion term, B , and the resistance to mass-transfer term, C, according to the van Deemter equation H = A + B / i i + Cii (4) or Hii= A a + B + C a 2 (44 Total solvent volume was determined by the small probe solute method and by the column weighing method (2). Benzene was used as the small probe solute and ita retention volume measured with neat THF as the mobile phase. The columns were weighed after equilibration with each of four solvents with different densities. The total solvent volume, V, (mL),was calculated by Vt = (W1- Wz)/ (dl - 4) (5) where W1 and W, are the weights of the column plus solvents 1 and 2 and d l and d2 are the densities of solventa 1and 2 in g/mL. A mixed ore-sized silica was prepared by thorough mixing of 4.80 g of pore-sized silica and 5.20 g of 500-Apore-sized silica. This mixed silica (3.68 g) was reacted following the procedure of Sentell et al. (12) using n-butyldimethylchlorosilaneto form a C4-bonded phase for future reversed-phase studies. Phase loading was determined as per Sander (13): NpL = 106Pc/S[1200nc - P,(M - 111 (6) where NpLis the surface coverage in bmol/m2; P, is the percent carbon, as determined by elemental analysis from Galbraith Laboratories, Inc. (Knoxville, TN); S is the surface area of the silica substrate in m2/g, as determined by the BET method at ASTEC; n, is the number of carbons in the bonded phase chain; and M is the molecular weight of the bonded phase chain in g/mol. Two columns were packed with mixed silica (one normal phase and one reversed phase) by using pentane as a slurry solvent, at 6300 and 9OOO psi, respectively,for vertical slurry packing. Each of these columns was examined by using the procedures described above. Exclusion work on the reversed-phase column was done with methylene chloride and THF as the mobile phases so that comparisons to other work could be made.
80-1
RESULTS AND DISCUSSION Molecular weight calibration of the four single pore-sized silica columns was done by measuring the exclusion volume of each of the aromatic and polystyrene solutes listed in Table I. A plot of exclusion volume versus log of molecular weight
15
21
27
33
3 9 C 5 51 57 icg M W of 3nlystlrenes
63
69
Flgure 1. Size-exclusion curves for four different pore-sized silica columns. Conditions: solvent, THF; flow, 0.5 mL/min.; injection of polystyrene solutions, 0.5 bL; detection, 254 nm.
Figure 2. Molecular weight range over which linear size exclusion is provided by different pore-sized silicas. Data extracted from Figure 1 results.
is seen in Figure 1. The log-linear region on each curve is the region where separation based on size-exclusion occurs. Columns with different pore-sized silicas provide exclusion separations over different molecular weight ranges as seen in Figure 2. It is also interesting to note the larger slope of the linear region for the 300-Acolumn as compared to the other three columns, indicating that the relatiue pore-size distribution for the 300-A column is skewed more toward the smaller pore diameters. Information from ASTEC indicated that the silica for the 300-Acolumn was obtained from one source while the silica for the other three columns was obtained from another source. Total solvent volume and efficiency studies were also conducted on each of these columns. The results of the column weighing and small probe solute procedures are shown in Table 11. Note that there is consistently a slightly larger total solvent volume obtained by the weighing method. The efficiency study results are listed in Table 111. The values of Fop,were found by Fopt = a o p t V i / L (7)
Table 111. Results of the Column Efficiency Studies
column, A 80 120 300 500
Mix-NP Mix-RP
Hmim
1-05 x 0.95 x 1.36 x 1.06 x 1.15 x 1.42 x
N,, = theoretical plates.
cm
"ax"
10-3 10-3 10-3 10-3 10-3 10-3
23 800 26 300 18 300 23 600 21 800 17 600
aopt,cm/s 0.184 0.206 0.222 0.210 0.181 0.229
'5
A , cm 2.36 X 3.15 x 2.81 x 4.50 X 2.41 X 2.11 x
lo-" 10-4 10-4 lo4 lo4 10"'
B, cm2/s 7.47 x 6.18 x 12.00 x 6.38 x 8.21 X 13.88 X
10" 10-5 10-5 10-5
c, 9 2.21 x 1.45 x 2.44 x 1.45 x 2.51 x 2.64 x
10-3 10-3 10-3 10-3 10-3 10-3
1353
ANALYTICAL CHEMISTRY, VOL. 83, NO. 14, JULY 15, 1991 Multi-path Term A-term of van Deemter equation
1 6
- 4 x 2
e
T
I
I . * I.
P-edicted Exclusion ExDerimental Exclusion
0
3.3
u
0 t both RP R NP mixed lilicos
0 ' 0
60
120
180
240
300
360
420
480
540
600
Longitudinal D i f f u s i o n
.1-
T
m 12
4 x
n
NP silica mixed
8
._ 2.3
4
VI
$x 2 1 w1 9
4
15' 1 5
C-term of van Deemter equation
4 -
b 3 ;; 12
;I 2.5
E-term of van Deemier equation
-
0
L
-
both RP k NP mixed silicas
0
I
21
27
33
39
45
51
log MW of Polystyrenes
57
63
69
75
Figwe 5. Comparison of the predicted optimiied exclusion c w e from Figure 4 with excluslon results from the normal-phase mixed-silica column. Condtions are the same as Figure 1.
0
35r
0
Normal Phase: Soldent - THF
35r
15
1 5 '
1 5
21
27
3 3 39 4 5 51 5 7 log MW of Polystyrenes
6 3
69
75
Flgure 4. Exclusion results for the 80- and 500-A pore-sired silicas and the predicted, optimized exclusion curve using 38% of the 80-A exclusion volumes and 82% of the 500-A exclusion volumes. Condltions are the same as in Figure 1.
where Ciopt is found by taking the partial derivative of eq 4a and solving for ii, which gives iiopt
=
(B/C)0.5
(8)
Substitution of eq 8 into eq 4 allows one to calculate the minimum plate height, Hh,and thus from eq 3 the maximum efficiency, N-. The purpose of the efficiency study was to determine if changes in pore size had any significant effect on the efficiency or any of the terms in the van Deemter equation. Figure 3 shows plots of the A, B, and C terms, of eq 4 and 4a, versus average pore diameter. The results indicate that these terms are largely independent of pore size. As can be seen, the efficiency data for the mixed-silica columns, which will be discussed later, also follow this trend. The 80- and 500-A pore-sized silicas were selected to be used in a mixture based on the nonoverlap of their linear exclusion regions. The optimum exclusion curve was determined by calculating the contributions from each of the silicas by using the lever rule. The result was a mixture in which 38% of the exclusion volume was contributed by the 80-A silica and 62% by the 500-A silica. The resulting optimum exclusion volume versus log MW curve demonstrates a linear region from 5 X lo2 to 2 X lo6 M W as seen in Figure 4. It was found that a limited range of volume percentages around 4060 would also provide the desired results, giving rise to the idea that an exact
21
27
33
3 9 4 5 51 5 7 log MW of Polystyrenes
63
69
75
Flgure 6. Exclusion results for the normal-phase mixed-silica column using THF as the eluting solvent and exclusion results for the reversed-phase mlxed-dlca column using M F as the eluting solvent and CH,CI, as the eluting solvent.
formulation is not critical. The tap densities for the 80- and 5 0 0 4 silicas are 0.60and 0.40 g/mL, respectively. Thus,the weight ratio for the optimum mixture was 48% of the 80-A silica and 52% of the 500-Asilica. Two mixed-silica columns, one normal phase and one reversed phase, were fabricated as described in the Experimental Section, and these columns were examined for their total solvent volume, efficiency, and exclusion properties. The results of the total solvent volume and efficiency studies are listed in Tables I1 and I11 and are s i m i i to those obtained for the single pore-sized silica. Figure 5 shows a comparison of the exclusion results obtained experimentally on the normal-phase, mixed-silica column versus the predicted optimum exclusion curve. The two curves are in close agreement with each other and provide linear exclusion ranges from 5 X lo2 to 2 X lo6 MW, with correlation coefficients of 0.9971 and 0.9975, respectively. Since adsorption of biopolymers on silica is a problem, a nonpolar stationary phase was made. Kirkland et al. (14) suggested the use of short-chain reversed phases such as the n-butyldimethylchlorosilanephase for use in the analysis of biopolymers. Figure 6 shows the SEC results on both the normal-phase column and the reversed-phase column, with the reversed-phase column being examined with two different mobile phases. It is interesting to note that the curves exactly parallel each other, but the reversed-phase curves are shifted to lower exclusion volumes. This suggests that the exclusion volume is lowered by the volume taken up by the added reversed phase. Observations by Vivileechia et al. (3),with polymeric organosilane surface modifiers, and Sander et al. (15),on monomeric and polymeric C18reversed-phase columns,
1354
Anal. Chem. lQQl, 63,1354-1361
support this. The volume of the stationary phase, V,,, can be estimated by va, = ga,/da, (9) where d,, is the density of the reversed-phase silane and g,, is the grams of stationary phase, which is determined by gap = g$cMWbp / (10) where g, is the grams of packing used, P,is the percent carbon, MWc is the molecular weight of carbon, n, is the number of carbons in the bonded phase, and MWb, is the molecular weight of the bonded-phase chain. The reversed-phase column was found to have a bonded-phase volume of 0.133 mL by this method. The average volume difference between the exclusion results for the normal-phase and reversed-phase columns, using THF, was 0.185 mL. This would suggest that the addition of the bonded phase may have reduced access to or within some of the pores, since the decrease in the exclusion volume is somewhat larger than would be expected due to the volume taken up by the bonded phase.
CONCLUSION We have demonstrated in this study that bimodal pore-sized mixtures can be easily prepared from two different pore-sized silicas with the resulting column exhibiting an exclusion volume versus log molecular weight plot with a linear exclusion range that encompasses the linear exclusion ranges of both silicas. By careful selection of the two silicas, a wide range of molecular weights may be examined by SEC in a short period of time. It was also found that efficiency is not significantly affected by pore size, allowing for the combination of various silicas without a loss in efficiency. Finally, the addition of a reversed phase does not alter the exclusion properties of the column other than to decrease uniformly the total solvent volume in the column. However, the volume taken up by the bonded phase is larger than would be predicted, which suggesta that some restriction of the pores may be occurring. In a subsequent paper, the specific retention behavior of polymers on the mixed-silica, reversed-phase column will be
considered using various mixed-solvent mobile phases. The exclusion properties and column efficiency of this optimized blend have been carefully evaluated, thus permitting a more definitive examination of retention mechanisms (16).
ACKNOWLEDGMENT We thank Tom Beesley (ASTEC) for providing the normal-phase silica columns and silica for the mixed-, normal-, and reversed-phase columns. We also acknowledge Lane Sander (NIST) for assistance in preparation and packing of the mixed-silica columns. Registry No. SOz, 7631-86-9;PS, 9003-53-6. LITERATURE CITED Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; Wlley: New York, 1979. Alhedai, A.; Martire, D. E.; Scott, R. P. W. Analyst 1080, 714, 869. Vivileechia, R. V.; Lightbcdy, B. G.; Thinot, N. 2.; Quinn, H. M. J. Chromatogr. Sci. 1977, 15, 434. Buytenhuys, F. A.; van der Maeden, F. P. B. J. Chromatogr. 1078, 749, 489. Welling, G. W.; Welling-Webster, S. I n HPLC of Macromolecules: A Practical Amfoach; Janson, J. C., Rvden, L., Eds.; VCH Inc.: New York, 1989.' Ambler, M. R.; Fetters, L. J.: Kester, Y. J. Appl. Polym. Sci. 1977, 21. 2439. -.. _.
Yau, W. W.: Ginnard. C. R.; Kirkland, J. J. J. Chromatogr. 1978, 149,
465. Kato, Y.; Matsuda, T.: Hashimoto, T. J. Chromatogr. 1085, 332, 39. Schultz, H. S.;Alden, P. G.; Ekmanis. J. L. ACS Symp. Ser. 1084, 245, 145. de Vries, A. J.: LePage, M.; Beau, R.: Guillemin, C. L. Anal. Chem. 1087, 3 9 , 935. Copper, A. R.: Johnson, J. F. J. Appl. Polym. Scl. 1071, 15, 2293. Sentell, K. B.; Barnes, K. W.; Dorsey, J. G. J. Chromatogr. 1088, 455. 95. Sander, L. C. CRC Grit. Rev. Anal. Chem. 1987. 18, 299. Kirkland, J. J.; Glajch, J. L.; Farlee, R. P. Anal. Chem. 1989, 67, 2. Sander, L. C.; Gllnka, C. J.; Wise, S. A. Anal. Chem. 1000, 62, 1099. Alhedai, A.; Bcehm, R. E.; Martire, D. E. Chromatograph& 1000, 2 9 , 313 and references cited within.
RECE~VED for review September 17,1990. Revised manuscript received March 28, 1991. Accepted April 5, 1991. This material is based upon work supported by the National Science Foundation under Grant CHE-8902735.
Electroosmotic Properties and Peak Broadening in Field-Amplified Capillary Electrophoresis Ring-Ling Chien* and John C. Helmer
Varian Research Center, 611 Hansen W a y , Palo Alto, California 94303
Electroosmotic fkw In a fusedgillca capillary column, partially fllled wlth a buffer of one concentratlon and contalnlng a second buffer of the same composttion but dlfferent concentratlon, Is studled. The bulk electroosmotlc veloclty In thls klnd of mixed buffer system Is derlved and shown to be a welghted average of the electroosmotic veloclties of the pure buffers. The theory of lamlnar flow caused by the mismatch between electroosmotk velocttles Is developed and shown to cause extra peak broadenlng for samples Inside the column. Good agreement wlth experlmental results Is found. The length of new buffer Introduced by electrolnjectlon can be determined from the variation In electrophoretic current durlng Injection.
INTRO DUCT10 N High-performance capillary electrophoresis (HPCE) is a major analytical technique for fast and efficient separation of charged species in solutions (1). In HPCE, a high voltage is applied across a fused-silica capillary column filled with an electrolytic buffer. Charged species introduced a t one end of the column migrate under the influence of the electric field to the other end of the column. The migration velocity of a particular ion species is a combination of the electrophoretic velocity of that species and the bulk electroosmotic velocity of the buffer. Under ideal circumstances, where molecular diffusion is the primary source of zone broadening, the separation efficiency is proportional to the field strength times
0003-2700/91/0363-1354$02.50/00 1991 American Chemical Society
