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L5l Everaerts, F. M., Beckers, J. L. and Verheggen, T.P.E.M. Isotachophoresis: Theory, Instrumentation and Applications, Elsevier, Amsterdam 1976. 161 Everaerts, F.M.,in:JorgensenJ. W.andPhillips.M.(Eds.),American Chemical Society, Washington, D.C., 1987, pp. 199-221. 171 Hjerten, S. and Zhu, M.-D.,J. Chromatogr. 1985,346. 265-270. [Sl Hjerten, S., Elenbring, K., Kilar, F. and Liao, J.-L., J. Chromatogr. 1987,403.47-61. [91 Terabe, S., Otsuka, K., Ichikawal, I.,Tsuchiya, A. and Ando,T.AnaL Chem. 1984,56 111-113. 1101 Terabe,S.,Otsuka,K.andAndo.,T.Anal.Chem. 19&5,57,834-841. I 1 I1 Cohen, A. S., Terabe, S., Smith, J. A. and Karger, B. L. Proc. Natl. Acad. Sci. USA 1988,85,9660-9663. 1121 Minard, R. D., Chin-Fatt, D., Curry, J. P. and Ewing; A. G. 36th ASMS Conference on Mass Spectrometry and Allied Topics , June 5-10, 1988, San Francisco, CA, p. 950. [ 131 Moseley, M. A,, Detering, L. J., Tomer, K. B. and Jorgenson, J. W.,

Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlanta, GA, March 6-10, 1989, Abstract No. 713. I141 Moore, W. T., Martin, M., Dague, B., Caprioli, R. M., Wilson, K . J. and Moring, S . E., 37th A S M S Conference on Mass Spectrometry and Allied Topics, May 21-26, 1989, Miami Beach, FL, p. 106. I151 Olivares, J. A., Nguyen, N. T., Yonker, C. R. and Smith, R. D., Anal. Chem. 1987,59, 1230-1232. [ 161 Smith, R. D., Olivares, J. A,, Nguyen, N. T. and Udseth, H. R.,Anal. Chem. 1988,60,436-441. 1171 Smith, R. D., Barinaga, C. J. and Udseth, H. R., Anal. Chem. 1988, 60,1948-1952. I181 Lee, E. D., Muck, W., Henion, J. D. and Covey, T. R., Biomed. Environ. Mass. Spectrom. 1989,18,844-850. 191 Lee,E.D.,Muck, W.,Henion, J. D. andCovey,T. R.,J. Chromatogr. 1988,458,313-321.

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I201 Udseth, H. R., Loo, J. A. and Smith, R. D., Anal. Chem. 1989.61, 228-232. 1211 Meng, C. K., Mann, M. and Fenn, J. B., Z . Phys. D - Aloms, Molecules and CIuslers 1988, 10, 361-368. 1221 Meng. C. K., Mann, M. and Fenn, J. B.. 36th ASMS Conference on Mass Spectrometry and Allied Topics. June 5-10, 1988, San Francisco, C A p. 77 I. I231 Loo, J. A., Udseth, H. R. and Smith, R. D., Anal. Biochem. 1989,179, 404-4 12. 1241 Edmonds, C. G., Loo,J. A,,Barinaga, C . J., Udseth, H. R. and Smith, R. D., J. Chromatogr. 1989,474,21-37. 1251 Dolnik, V., Deml, M. and Bocek, P., in: Holloway, C. J. (Ed.) Analytical and Preparative lsotachophoresis, Walter de Gruyter, Berlin 1984, p. 55-65. 1261 Kodama, H., Yamamoto, M. and Sasaki, K., J . Chromatogr. 1980, 183,226-228. [271 Mikasa, H., Sasaki, K. and Kodama, H., J . Chromatogr. 1984,305, 204-209. [281 Clark, P. M. S., Kricka, L. J. and Whitehead, T. P., J . Chromatogr. 1980,181,347-354. 1291 Driesen, O., Beuckers, H., Belfroid, L. and Emonds. A., J. Chromatogr. 1980, 181.44 1-448. 1301 Smith, R. D., Loo, J. A., Barinaga, C. J.,Edmonds,C.G.,and Udseth, H. R., J . Chromatogr. 1989. 480, 211-232. I 3 11 Smith, R. D., Loo, J. A,, Barinaga, C. J., Edmonds, C. G. and Udseth, H. R., J . Amer. Sac. Mass Spectrorn. 1990, I , 53-65. [321 Barinaga, C. J., Edmonds, C. G., Udseth, H. R. and Smith, R. D., Rapid Comm. Mass Spectrom 1989,3, 160-164. 1331 Loo, J. A., Edmonds, C. G., and Smith, R. D., Science 1990,248, 20 1-204.

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Richard A. Mosher Wolfgang Thormann" Center for Separation Science, University of Arizona, Tucson, AZ

Experimental and theoretical dynamics of isoelectric focusing: IV. Cathodic,anodic and symmetrical drifts of the pH gradient The production of anodic, cathodic and symmetrical drifts of a pH 3.5- 10 gradient formed by isoelectric focusing in polyacrylamide gels is demonstrated experimentally by manipulation of the electrolyte concentrations. Experimental behavior is reproduced by computer simulation of a model mixture of 15 hypothetical carrier ampholytes whose PIS span the p H range 3-10. The mechanism which produces the drifts is elucidated and approaches to minimize such drifts are discussed. The data suggest why most experimentally observed drifts are cathodic.

1 Introduction I n the most common variant of isoelectric focusing (IEF), the p H gradient is established using a mixture of synthetic carrier ampholytes to form the pH gradient. An experiment begins ~

Correspondence: Dr. Richard A. Mosher, Center for Separation Science, University of Arizona, Tucson, AZ 85721, USA

Bis, N,N'-methylenebisacrylamide; IEF, isoelectric focusing; ITP, isotachophoresis; PAG, polyacrylamide gel; PI, isoelectric point; TEMED,

N,N,N'.N'-tetramethylethylenediamine 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

with a transient period during which this mixture forms a pH gradient and the amphoteric samples migrate within the gradient to a position where the pH is equal to their isoelectric point (PI). At this position thenet chargeon thesamplesiszero and migration ceases. The end of the experiment is characterized by a unique steady state distribution of carriers and samples. Schumacher I 11, Kauman [21 and Svensson-Rilbe 13,41 developed a mathematical description of this steady state, showing it to result from a balance between mass transports

*

Present address: Department of Clinical Pharmacology, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland 01 73-0835/90/0Y0Y-0717 %3.50+.25/0

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due to diffusion and to electromigration. According to the theory, this steady state, once established, should remain invariant as long as the applied current density is constant. In practice, however, this has been seen not to be the case. At least two types of pH gradient instabilities have been identified, the plateau phenomenon 151 and cathodic drift [61. The first refers to a flattening of the p H gradient in the neutral region, accompanied by a steepening of the gradient at its extremes. The second generally refers to a progressive loss of the basic end of a pH gradient upon prolonged focusing. We have presented a theoretical explanation for the plateau phenomenon in the second ofthis series of papers dealing with the fundamental aspects of IEF 171. In a subsequent paper which explored the impact of the boundary conditions (the manner in which the driving electrodes communicate with the focusing space) on I E F 181, one of the fundamental causes of cathodic drift was identified. In that paper it was shown that cathodic drift can result from an isotachophoretic (ITP) mechanism, and it was clear that anodic and symmetrical drifts were possible as well. Svendsen and Schafer-Nielsen [91have also suggested that pH gradients in I E F can decay by an ITP mechanism. It was further postulated that the nature of the drift can be controlled by appropriate choice of electrolyte type and concentration as well as the volume of the electrode reservoir. That paper presented experimental and computer simulation data derived from simple model systems comprised of 3 ampholytes. The experimental arrangement utilized was afreefluidcapillaryformat. In this paper wepresent anextension of this work'to more complex systems. It is shown theoretically by computer simulation, and experimentally with I E F in polyacrylamide gels (PAG), that when NaOH and H,PO, are used as catholyte and anolyte, respectively, simple changes in their concentrations can produce not only cathodic, but also anodic and symmetrical drifts of the pH gradient.

2 Materials and methods 2.1 Chemicals Phosphoric acid, 85 %, was purchased from Fisher Scientific (Fair Lawn, NJ). Sodium hydroxide, analytical reagent grade, was from Mallinckrodt (Paris, KY). Ampholine was a product of LKB Produkter (Bromma, Sweden). A mixture of acrylamide and N,N'-methylenebisacrylamide (Bis), 29 : 1, was purchased from Bio-Rad Laboratories (Richmond, CA). N,N,N',N'-tetramethylethylenediamine(TEMED) was obtained from Eastman Kodak (Rochester, NY) and ammonium persulfate was from Sigma Chemical (St. Louis, MO).

2.2PAG-IEF Electrophoresis was performed in the Bio-Rad Model 155 electrophoresis cell for tubegels. Gels were 12 cminlength and 3 mm in diameter. The bottom electrolyte reservoir of the cell was magnetically stirred, and a coolant (4 "C) was circulated through the cell for the duration of each experiment. Experiments were performed at a constant 1 W/gel. The polymerization mixture contained 6.4 m L acrylamide/Bis (30 % w/v), 80 pL of TEMED, 80 pL of 40 '36 w/v ammonium persulfate, 4.4 mL of Ampholine (pH 3.5- 10) and water to 35 mL. Polymerization was allowed to proceed at ambient temperature for 1 h, after which the gels were either used immediately or sealed in a ziplock bag and stored overnight at 4 "C.

2.3 pH Gradient measurement When the appropriate time was reached the power was turned off, the upper reservoir was removed from the electrophoresis cell and the upper buffer poured into a beaker. Three gels were

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Figure 1. Computer simulation of the focusing of 15 ampholytes with 10 mM phosphoric acid as the anolyte and 90 mM NaOH as catholyte. The latter concentration is out of the range of the figure. The anode is to the left. The ampholytes are initially distributed uniformly, 2 mM each, at time zero (upper left-hand panel) over the center one cm, which is referred to as the focusing space. Time points presented are after 20, 40, 60.80 and 100 min of current flow. The length of the simulation space is 5 cm, with only the center 3 cm being displayed. At this current density the ampholytes are focused by 20 min. The migration of phosphoric acid toward the anode causes the acidic ampholytes to form an isotachophoretic stack which results in those ampholytes being lost from the focusing space. The ampholyte zones are identified by their P I S .

pH Gradient drifls in isoelectric focusing

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removed and the empty sockets plugged with rubber stoppers. The upper reservoir was returned to the electrophoresis cell, the original buffer used to refill the reservoir and the power (appropriately reduced) was reapplied. The gels were extruded from the glass tubes by applying hydrostatic pressure with a hand-held syringe. Each was immediately cut into 1 cm lengths and each segment was placed in 0.5 mL of deionized water. The tubes were capped and the elution allowed to proceed for a minimum of 4 h. The gel fragments were removed and the pH of each solution measured with a Radiometer p H meter.

2.4 Computer simulation The generalized model for transient electrophoretic processes developed by Bier et nl. 1 10)was used for the simulation work. This model is one-dimensional and isothermal and assumes the absence of fluid flows. It i s capable of treating biprotic ampholytes, weak and strong rnonovalent acids and bases and monovalent strong electrolytes. Component fluxes result from electromigration and diffusion. The model predicts the evolution of concentration, p H and conductivity profiles as a function of time. Inputs required include the pK and mobility values which describe each component, the length of the separation space and its segmentation, the current density, amount of electrophoresis time, and the initial distribution of each component. The permeabilities of the ends ofthe separation space are also specified. Simulations were run on a Cyber 205 supercomputer at the John von Neumann National Supercomputer Center at Princeton, NJ. The 15 biprotic car rier ampholytes used for the sinIdation work were assigned equally spaced pfs (0.5 pH unit:;), from 3-10. The difference between pK, and pK2 for each was 2 units, and each ionic mobility was 3 x 10 'm2/Vs. The ionic mobility ofthe sodium ion was 5.19 x 10 '/Vs. Phosphoric acid was modelled as a univalent acid with a pK of 2 and an ionic mobility of 3.67 x 10-*m2/Vs. All simulations were performed with a 5 cm separation space overlaid with a grid which defined 400 segments of equal length. Current density was a constant 20 A/m2 for every simulation. The boundary conditions employed allowed free transport of mass into and out of the separation space.

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mM. The catholyte is N a O H at a concentration of 90 mM. The 5 remaining panels present the concentration distributions of all components at 20 min intervals to 100 min. In the subsequent discussion, a specific ampholyte will be referred to by using its pl, thus ampholyte 4.5 refers to that ampholyte whose PI=4.5. The second panel shows that the ampholytes have focused by 20 min. There has also been adistinct migration of the H , P 0 4 boundary toward the anode. The electrophoretic velocity of the Na'boundary under these conditions is much less. There is actually some diffusion of the catholyte into the focusing space. The 40 min profile shows ampholyte 3, the

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COLUMN LENGTH (cm) Figure 2 . The pH gradients which correspond to the 20 (solid line) and 100 min time points from Fig. 1. Only the center 1 cm is shown. Diffusion of NaOH into the focusing space produces a very high pH at the cathodic end at 20 min, which has nearly vanished by 100 min. The loss of acidic ampholytesfrom the focusing space has resulted in an anodicdrift ofthepH gradient.

3 Results and discussion

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The term anodic drift, as used here, is analogous to cathodic drift, and means a progressive loss of the acidic end of a pH gradient during anexperiment. Extending this terminology, an experiment which displays symmetrical drift shows both anodic and cathodic drifts.

3.1 Anodic drift Computer simulation data which illustrate anodic drift are presented in Fig. I . Each ofthe six panels presents thedistribution of the ampholytes after a specified duration of current flow which is indicated in minul-es in the upper left-hand corner. The initial distribution of all components is shown in the first panel (T = 0). The ampholytes are uniformly distributed at a concentration of 2 m M acrom the central 1 cm of the separation axis (from 2-3 cm) which is herein referred to as the focusing space. The phosphoric acid occupies the anodic portion (left side) of the separation space at a concentration of 10

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is difficult for several reasons: (i) The composition of the Ampholine preparation is unknown, (ii) there is a lack of information concerning the impact of the PAG on the process, (iii) the temperature assumed for the simulation is 25 O C whereas the experiment was performed as 4 "C, (iv) the experimental drift rate varies because the experiment was performed at constant wattage rather than constant current. With these limitations in mind, a number can be extracted for comparison purposes if it is assumed that the average current density for the experiment is 1 mA/gel (it is greater than thisinitially, ca. 2 mA, and less at the end of the experiment, ca. 0.1 mA) and the simulation data is corrected for current density and column length. A value of 0.45 pH units/h is obtained. Thus, the simulation represents a reasonable approximation of the experimental system.

most acidic ampholyte, clearly forming an isotachophoretic zone, with a characteristic plateau shape, behind the phosphate boundary. This is fully established by 60 min, as the H,P04 boundary continues to move toward the anode. The 80 rnin profiles show that the H,P04 boundary has nearly left the region presented in the figure. (It is approximately 1.1 cm from the anodic end of the simulation space). A classical isotachophoretic stack forms behind this boundary, which consists of the fully formed plateaus of the acidic ampholytes 3 and 3.5, migrating in order of their pls. At 100 rnin the plateau consisting of ampholyte 4.0 has completely formed. The adjusted concentrations (in the Kohlrausch sense) of ampholytes 3,3.5 and 4 successively decrease. At this current density and time interval, the NaOH boundary has hardly moved. No ampholytes are lost from the cathodic end of the gradient. The impact this behavior has on the pH gradient is shown in Fig. 2; the profiles correspond to 20 and 100 rnin of current flow, and present the gradients which exist in the region initially occupied by the ampholytes, from 2-3 cm of the simulation space. The 20 min profile shows a zone of high pH caused by the diffusion of the NaOH into the focusing space. This zone has nearly vanished by 100 min. The 100 rnin profile clearly shows the progressive loss of the acidic end ofthe pH gradient, or anodic drift. The limited number of ampholytes used in the simulation produces ITP zones whose length is significant compared to the length of the focusing space. Thus, the drift occurs in steps rather than as a continuous process.

3.2 Cathodic drift Cathodic drift is illustrated by computer simulation data in Fig. 4. The anolyte for this simulation was 100 mMH,PO, and the catholyte was l O m NaOH. ~ The mixture of ampholytes used was the same as in the simulation presented in Fig. 1. Each ampholyte was uniformly distributed at a concentration of 2 mM across the center 1 cm ofthe simulation space. This is shown in the upper left-hand panel (T = 0) in Fig. 4, as are the initial distribution of H,PO, and NaOH. The ampholytes have focused by 20 rnin and theNa+boundary has migrated a substantial distance toward the cathode. This boundary acts as leader for ampholyte 10 which is forming an isotachophoretic zone immediately behind. There is a clear adjustment predicted in the concentration of ampholyte 10. After40 min of current flow the boundary between Na+ and ampholyte 10 is 4 cm from the anode. Ampholyte 9.5 is forming an adjusted isotachophoretic zone behind ampholyte 10. The successive loss of basic ampholytes continues, with the 100 rnin profile depicting a long zone of ampholyte 9 due to the pre-

An experiment in which the same anolyte and catholyte were used for IEF of a pH 3.5-10 Ampholine preparation in polyacrylamide gels is shown in Fig. 3. The data presented show the pH profiles in the gels after 1,3 and 6 h of focusing. Each data point represents the average of 3 measurements. The progressive loss of the anodic end of the pH gradient is obvious. There is also a small loss of the cathodic end of the gradient. The anodic drift rate from hour 1 to hour 5 is 0.26 pH units/h. A quantitative comparison of this value to the simulation data I

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Figure 4 . Computer simulation of the focusing of 15 ampholytes with 100 mM H3P0, and 10 mM NaOH as anolyte and catholyte, respectively. The former concentration is out of the range of the figure. The anode is to the left. All other conditions are identical to those used in Fig. 1. The NaOH acts as a leader in the isotachophoretic sense for the basic ampholytes, causing their progressive loss from the focusing space (the central 1 cm of the column). The ampholyte zones areidentified by their PIS. None of the acidic ampholytes have been lost from the focusing space by 100 rnin.

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pH Gradient drifts in isoelectric focusing

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phosphoric acid has diffused into this region by 20 min and significantly lowered the pH at the anodic end. This situation has hardly changed by 100 min. At the cathodic end, the loss of the basic ampholytes has resulted in a progressive loss of the basic end of the pH gradient or cathodic drift. As with the anodic drift experiment presented in Fig. 2, in the simulation this loss occurs in steps, rather than continuously, because of the significant length of the adjusted ampholyte zones in comparison to the length of the simulation space.

dicted low adjusted concentration. The predicted adjusted concentrations for the basic ampholytes in this simulation are much less than those predicted for the acidic ampholytes in Fig. 1. Figure 5 shows the pH gradients which correspond to the component distributions at T = 20 and 100 min in that space originally occupied by the ampholytes, i. e. 2-3 cm. The 11.c

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Figure 5. The pH gradients corresponding to the 20 min (solid line) and the 100 min time points from Fig. 4. Only the center 1 cm initially occupied by the ampholytes (the focusing space) is shown. The diffusion of phosphoric acid into the focusing space has produced very low pH values at the anodic end ofthe gradient.Thelossofbasic ampholytes has caused the pHgradient to drift toward the cathode by 100 min. 3.0 k

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Figure 6 . The behavior of the p H gradient in tube PAG-IEF with 100 mM H,PO, and 10 mM NaOH as anolyte and catholyte, respectively, is presented. Theprogressivelossofthebasicendofthe pH gradient isobvious (cathodic drift). There is also some residual anodic drift.

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Figure 7 . Computer simulation of the focusing of 15 ampholytes with 10 mM H,PO, and 22.5 mM NaOH as anolyte and catholyte, respectively. The anode is to the left. All other conditions are identical to those used in Fig. 1. The rates of migration of the N a + and phosphate boundaries are approximately equal at these concentrations, which should produce a symmetrical drift (i. e. anodic and cathodic drifts) of the pH gradient. In this idealized system the rate of loss of acidic ampholytes is actually greater than that of basic ampholytes because their adjusted concentration (in the Kohlrausch sense) is higher. The ampholyte zones are identified by their PIS.

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The same anolyte and catholyte were used for an experiment in PAG in which pH 3.5-10 Ampholine was focused. The results are presented in Fig. 6, which shows the pH gradients in the gels after 1 , 3 and 6 h of focusing. The only difference between this experiment and the one reported in Fig. 3 is the concentration of the electrolytes. Each data point in the 1 and 3 h gradients represents an average of 3 measurements. The 6 h data are the average of 2 measurements. The rate of loss of the basic end of the pH gradient over a period of 6 h is much greater than the corresponding rate observed in Fig. 3 . Anodic drift was not eliminated under these circumstances but its extent is less than that present in Fig. 3.

3.3 Symmetrical drift It was postulated in an earlier paper 181that when NaOH and H 3 P 0 4were used as electrolytes, equal rates of drift would be observed if the concentration of the former was 2.25-fold higher than the latter. This concentration ratio produces equal velocities for the Na+and phosphate boundaries. Figure 7 presents the results of a simulation in which the same hypothetical mixture of ampholytes was focused with 22.5 mM NaOH as catholyte and 10 mM H3P0, as anolyte. Simulation conditions were the same as those used to produce the datain Figs. 1 and 4, except for the concentrations of the electrolytes. The rate of migration of the Na' and phosphate boundaries is effectively the same and loss of ampholytes from both the anodic and cathodic ends is obvious, although the rate of loss is not symmetrical. Acidic ampholytes are lost at agreater rate than basic ampholytes because the adjusted concentrations (in the Kohlrausch sense) of the acidic species are greater than those of the basic species. This produces shorter zones when equivalent amounts are present and thus more species will be lost per unit time from the anodic end. At 100 min, ampholytes 3 , 3.5,4, 9.5 and 10 are present as fully formed ITP zones. The impact of this behavior on the pH gradient in the focusing space is shown in Fig. 8. The 100 min profile (dashed line)

shows that both of the extremes of the gradient present at 20 min have been lost from the focusing space. The experimental counterpart of this simulation is shown in Fig. 9 which presents the pH gradients produced in PAG-IEF when 10 mM H,PO, and 22.5 mM NaOH are used as anolyte and catholyte, respectively. There is a drift toward neutrality for both ends of the gradient with the extent of the drift of each end being approximately equal after 6 h.

3.4 Equal concentrations of NaOH and H,PO, Many IEF experiments are carried out with equal concentrations of H,PO, and NaOH as anolyte and catholyte, respectively. The simulated behavior of the mixture of 15 hypothetical ampholytes when 30 mM each of NaOH and H,PO, are used as electrolytes is depicted in Fig. 10. Under these conditions, there is some drift toward both electrodes, but the rate of migration of the Na' boundary is clearly greater than that of the phosphoric acid boundary. When equal concentrations of these electrolytes are used the drift is faster in the cathodic direction.

4 Conclusions The experimental PAG-IEF data clearly show that manipulation of the concentrations of NaOH and H,PO, can produce pH gradient drifts which are predominately anodic, cathodic or symmetrical. The corresponding simulation data present the mechanism which is reponsible for the observed behavior. The progressive loss of one or both ends of a pH gradient can occur as a result of the electrophoretic migration of the terminal carrier ampholytes out of the separation space. This migration occurs when the anolyte and/or catholyte acts as a leader in the isotachophoretic sense. Other weak and strong acids and bases, when used as electrolytes, will cause similar behavior. The rate of loss of carrier ampholytes, i. e.

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COLUMN LENGTH (cm) Figure 8. The pH gradients corresponding to the 20 rnin (solid line) and 100 min time points from Fig. 7. Only the center 1 cm (the focusing space) is shown. The 100 min gradient shows the loss of both the acidic and the basic ampholytes.

Figure 9. The behavior of the pH gradient in tube PAG-IEF with 10 mM H,PO, and 22.5 m M NaOH as anolyte and catholyte, respectively. Each data point is the average of 3 measurements. With these anolyte and catholvte concentrations there is loss of both the acidic and the basic ends of the pH gradient.

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Figure 10. Computer simulation of the focusing of 15 ampholytes with equal concentrations (30 mM) of H,PO, and NaOH as anolyte and catholyte, respectively. All other conditions are identical to those used in Fig. 1. Under these conditions the rate of migration of the Na+ boundary is greater than that of the phosphate boundary, producing a more rapid loss of the basic ampholytes.

the rate of p H gradient drift, will depend on the velocities of the boundaries established by the electrolytes and the adjusted concentration (in the Kohlraiusch sense) of the carrier ampholytes behind the boundary. The boundary velocities will depend on the concentrations and net mobilities of the compounds chosen as electrolytes and on the current density [ 1 1I. It is obviously impossible to reduce this velocity to zero. The residual cathodic drift present in Fig. 3 and the residual anodic drift present in Fig. 6 may reflect this impossibility.

positively charged gel will pump fluid toward the anode. This phenomena and other potential caused of cathodic drift have been reviewed by Righetti ([6]p. 299).

Electrolyte concentration and current density will have an impact on the stability of p H gradients formed with carrier ampholytes in all experimental arrangements which d o not have a physical barrier (such as an ion exchange membrane) to ampholyte migration. This includes recycling free fluid instruments which employ dialysis membranes to isolate the electrolyte reservoirs, gel apparatuses of the type used here and capillary devices. The configurations most susceptible to drifts are those in which the electrode reservoir volume is large in comparison to the volume of the separation space IS]. The ratio of electrolyte concentration to current density should be maximized to reduce the rate of drift. Equal concentrations of NaOH and H,PO, are the most common electrolyte configuration for IEF. The fact that this configuration results in a more rapid drift toward the cathode than toward the anode (Fig. 10) is a likely reason why anodic drifts have not been more commonly observed. Other factors have been identified as contributing to cathodic drift. One ofthe most important of these in PAG-IEF is a net charge on the gel matrix. This is most often due to acrylic acid being a contaminant of the acrylamide. A negative charge on the gel will cause an electroosmotic pumping of fluid in the direction of the cathode. A

Received January 4, 1990

The authors would like to acknowledge theexcellent technical assistance of Douglas Dewey, Michael Zirkle and Roya Tooloian. This work was supported by N A S A grant NAG W693 andgrant NAC-1339from the John von Neurnann Supercomputer Center.

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