Ind. Eng. Chem. Fundam. 1985, 2 4 , 489-497
489
EXPERIMENTAL TECHNIQUES Semicontinuous Protein Separation via Variable-Field-Strength Electrophoresis Jen-Fu Chao, Helen C. Holleln," and Chlng-Rong Huang Department of Chemical Engineering and Chemistry, New Jersey Institute of Technology, Newark, New Jersey 07102
Variabbfield-strength electrophoresis is proposed as a new semicontinuous separation process for protein mixtures. Conventional preparative-scale polyacrylamide gel electrophoresis equipment is used, but new feed is added in successive cycles of operation and the gel is reused for a number of cycles rather than dismantling the system and polymerizing new gel in situ for each feed sample. Experimental results for the new process are presented, based on the separation of a mixture of human hemoglobin and human serum albumin. Two modes of operation are investigated with the electric field being switched from positive to negative polarity or from low to high voltage during each cycle.
Introduction Biotechnology is currently a focus of intense interest in the chemical and pharmaceutical industries with a large number of genetically engineered products including human insulin, human growth hormone, and CY, P, and y interferon in various stages of commercialization. Since the protein separation and purification steps comprise a significant portion of the production costs for these recombinant DNA products, process development and equipment design are advancing simultaneously with the genetic frontiers. Typical separation methods depend on the scale-up of conventional analytical techniques such as affinity chromatography, high-performance liquid chromatography (HPLC), and electrophoresis. New separation tools such as commercial-scale HPLC equipment and affinity-binding resins fixed with monoclonal antibodies are now available. Electrophoresis is also being developed industrially even though some scientists are reported to be pessimistic about "the lack of versatility" of this process (Graff et al., 1983; Michaels, 1974; Webber, 1984). Electrophoresis. There are many different types of electrophoretic separation methods including moving boundary electrophoresis, continuous flow electrophoresis, molecular sieve zone electrophoresis, and isoelectric focusing (van Oss, 1979). A continuous or free-flow electrophoresis device for the separation of biological materials has been tested successfully on several protein mixtures in the space shuttle flights, and the manufacturer, McDonnell Douglas Astronautics Co., plans to send a 5000-lb, fully-automated production model into space in 1985 (Chem. Eng. News, 1983). In continuous flow electrophoresis, the protein mixtures is injected continuously into a stream of buffer solution which flows between two charged plates. The fluid stream spreads out fan-like with the most highly charged particles moving nearest to the plates. Different product fractions are collected across the *Author to whom correspondence should be addressed a t Manhattan College, Riverdale, NY 10471. 0 196-43 13/85/ 1024-0489$0 1.50/0
width of the fan-like stream with the protein molecules separated according to charge magnitude. McDonnell Douglas reports greatly improved separations under the microgravity conditions of space as compared with Earth-bound operation of the same equipment. The separation principle in all electrophoretic processes is based on the selective migration of charged particles in the presence of an applied electric field. Protein molecules have both acid (COOH) and amine (NH,) functional groups incorporated into their skeletal structures. In solution, these groups interact with hydrogen and hydroxyl groups to give charged protein ions as shown in Figure 1. A t low pH, the protein molecule carries a net positive charge and will be attracted toward the negative electrode or anode in an electric field. At high pH, the protein molecule carries a net negative charge and will be attracted toward the cathode in an electric field. A t some intermediate pH known as the isoelectric point I*, the protein exists as a zwitterion or double ion with a net charge of zero. Each protein species carries a number of these charged functional groups in its molecular structure, the summation of which gives each species a unique value for the isoelectric point. Isoelectric focusing is done in a gel or a solution with a pH gradient so that each protein species migrates to the point where the pH matches its isoelectric point and stops. Continuous flow electrophoresis and zone electrophoresis are generally done a t one pH so that the different protein molecules move at different velocities depending on their net charges. Polyacrylamide gel electrophoresis (PAGE) is in the category known as molecular sieve zone electrophoresis which also includes starch gel, agar gel, and paper electrophoresis. In contrast to chromatography or to continuous flow electrophoresis, there is no bulk flow of solvent in zone electrophoresis processes. Instead, only the protein molecules move through the pores in the gel or the paper with the net movement dependent both on the net charge and on the ionic radius of the protein molecules in the presence of the electric field. Analytical PAGE apparatuses are available with the gel in the shape of vertical 6 1985 American Chemical Society
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Ind. Eng. Chem. Fundam., Vol. 24, No. 4, 1985
N H:
I
,? o t i on
“3 I
isoelectric
NH
I
orion
Doirt
Figure 1. Protein charge as a function of solution pH.
Figure 2. Ruchler Polv-Prep 200 experimental apparatus
cylinders (discs) or in the shape of thin slabs. The disc gels give ”exquisite resolution” and are, therefore, one of the most powerful analytical tools for the separation and characterization of protein mixtures (van Oss, 1979). In analytical operation, the protein mixture is applied at one end of the gel and the proteins are pulled into the gel, separating into sharp bands or zones as they move through the electric field. The gel is then removed from the experimental apparatus and stained to highlight the individual zones. The stained gels are compared with standards produced under similar conditions using known proteins in order to identify the components of the mixture. This paper examines preparative-scale polyacrylamide gel electrophoresis (prep-PAGE), which is currently operated as a rather tedious batch process, and demonstrates that prep-PAGE can be operated in a semicontinuous manner giving good separation with much simpler experimental methods. The Buchler Poly-Prep 200 (Figure 2) is one of the few commercially available prep-PAGE systems. The gel for prep-PAGE is polymerized directly in the electrophoresis column prior to each run. The protein solution is layered on top of the gel and the electric field is applied with the appropriate electrode at the bottom of the column. The protein molecules are pulled downward through the gel pores toward the elution chamber where the individual protein bands are eluted with fresh buffer solution. In conventional operation, the apparatus is completely dismantled after each batch run, the gel is discarded, and the apparatus is reassembled in order to prepare new gel and repeat the procedure. Elimination of the tedious gel preparation step would obviously increase the average hourly production capability of this equipment and make the apparatus more desirable to use. Semicontinuous operation on the same gel for long periods of time offers the potential for taking advantage of the proven separation capability of electrophoretic processing without the expense of going into space. Experimental studies in the Buchler Poly-Prep system on the purification of the enzyme, human placental alkaline phosphatase (HPAP), show that significant improvements in purification can be achieved by optimizing
the operating conditions in the batch process with respect to electric field strength, feed concentration, buffer pH, and buffer ionic strength. Crude HPAP is difficult to purify and typifies the separation problems encountered in most biochemical separations. There are six isomeric forms of the enzyme and the crude enzyme mixture contains protein impurities such as human placental albumin with molecular weights and/or isoelectric points close to the values for HPAP, thus making these impurities difficult to remove (Chao et al., 1983). Furthermore, the enzyme concentration is usually measured by recording its activity compared to the total protein concentration in the sample. Thus, if the enzyme is denatured by the electric field, HPAP will be incorrectly measured as being one of the inactive impurities. In order to better evaluate and develop variable-fieldstrength electrophoresis, i.e., the new semicontinuous process, an arbitrary mixture of two known proteins (human hemoglobin plus human serum albumin) was chosen as the model system for this investigation. Accurate concentrations of these globular proteins are readily measured. Batch studies on a number of proteins including hemoglobin and albumin were previous carried-out in an LKR 7900 Uniphor prep-PAGE system by Righetti and Secchi (1972). These researchers optimized the operating conditions in the batch process with respect to total protein loading, gel height, percent acrylamide in the gel, buffer ionic strength, and elution buffer rate. Their data show total protein eluted with respect to fraction number or time. The operating conditions in the present work are based on the batch studies by Righetti and Secchi (1972) and Chao et al. (1983). The elution profiles for the individual proteins are shown. Cyclic Separations. Pigford and co-workers introduced cycling zone adsorption as a new separation process in 1969. Using this process, conventional batch chromatographic separations can be made continuous by periodically changing a thermodynamic variable such as temperature or pressure while continuously adding fresh feed to the system. The change in variable institutes adsorption or desorption of specific solutes, thereby producing a continuous product stream that is alternately enriched in either the adsorbed or the unadsorbed solute. Separation of mixtures of multiple solutes can be achieved by setting multiple levels of the thermodynamic variable (Nelson et al.. 1978). Appropriate variables for protein separation are solution pH, solution ionic strength, electric field strength, and molecular affinity. We have recently investigated parametric pumping and cycling zone adsorption as cyclic processes for the separation of blood proteins, enzymes, and lectins using these particular intensive variables to institute adsorption and desorption in packed beds of ion exchangers and affinity resins (Chen et al., 1981a,b; Hollein et al., 1982; Chao et al., 1982). Parametric pumping is similar to cycling zone adsorption except that the former process couples flow reversal with the change in variable while the latter process is unidirectional. Both of these cyclic processes eliminate the batch-type adsorbent regeneration step. Thompson and Bass (1974) and Oren and Soffer (1978) have successfully used electric field as the cyclic variable for the adsorptive separation of NaCl and water in cyclic electrodialysis processes. Lee and Kinvan (1975) suggested cycling electrosorption as a potential separation process for proteins based on data for adsorption of ribonuclease A imd glucoamylase on carbon electrodes. They found that the proteins tended to adsorb irreversibly because of de-
Ind. Eng. Chem. Fundam., Vol. 24, No. 4, 1985 491
naturation a t the electrical surface. Based on the ideas gained from our cyclic protein separation research plus the cyclic electrodialysis and cycling electrosorption studies, we decided to investigate the potential application of cyclic variation of electric field strength and/or electric field polarity in a nonadsorptive protein separation process such as electrophoresis. In the new variable-field-strength electrophoresis process, a pulse feed is periodically introduced into the system and the field strength is changed to different levels during each cycle while a continuous product stream is withdrawn from the system. The new process is designated as semicontinuous because the product stream is continuous but the feed stream is discontinuous. Preliminary experiments with a continuous feed stream indicate that the pulse feed is required in this equipment for good separation. The mechanical design of the Buchler Poly-Prep 200 system is such that only unidirectional flow is possible. An alternate apparatus, the LKB 7900 Uniphor, is normally operated with one elution stopper giving unidirectional flow; however, the LKB electrophoresis column can be fitted with two elution stoppers making bidirectional flow possible.
Experimental Section A Buchler Poly-Prep 200 experimental electrophoresis system was set up as shown in Figure 2. The prep-PAGE system was maintained at 278 K by circulation of cooling water through the inner and outer cooling jackets. A Buchler 3-1500 power supply was used for a direct current source. The power supply was set at constant wattage or constant voltage in the individual experimental runs as indicated in Table I, and the voltage, amperage, and wattage were recorded throughout the runs. A volt-hour integrator is available for electrophoretic systems, but was not used in the present work. A multistaltic pump (manufactured by Buchler) was used to circulate the buffer solution between the upper and lower electrode compartments and the external buffer reservoir (1L volume). The buffer was titrated to maintain the desired pH. During runs of more than 10 h duration, the buffer solution was periodically replaced in order to maintain the correct buffer concentration. A reversible peristaltic pump (manufactured by Pharmacia Fine Chemicals) was used to pump pre-cooled elution buffer in through the elution chamber and out through the central elution capillary tube to the product stream. Prior to the start of each experimental run, the polyacrylamide gel was polymerized directly in the prep-PAGE system. The composition of the gel was 5 wt % acrylamide, 0.13 wt 70N,N’-methylenebisacrylamide, 0.15 wt % ammonium persulfate, plus 0.07 volume % TEMED (Rodbard and Chrambach, 1971). The gel solution was titrated to the desired pH with Tris for the Tris/glycine runs and with NaOH for the Tris-maleate/NaOH runs. The gel heights are listed in Table I. The gel has an annular cross-sectional area of 18 cm2. After about 30 min was allowed for polymerization, the prep-PAGE system was assembled and filled with buffer solution. The power supply was set at constant voltage (200 V), and the system was run for 6 h in order to remove ultraviolet detectable materials from the gel. For the experimental runs, the buffer solutions were either a mixture of Tris plus glycine (0.05 M each) at a pH of 8.6 or a mixture of Tris-maleate plus NaOH (0.05 M each) a t various pH’s as listed in Table I. The feed was made-up by adding 0.03-0.05 w t % total proteins plus 3-10 wt % sucrose to the buffer solution. The added sucrose makes the feed denser than the buffer solution. The feed
32r
o
w
LIB 7900 UNIPHOR
A LUC BUCHLER 2 8 L
A
POLY-PREP 2 0 0
I I
2 4-
t 0 4 t
0
0
AA 2
4
6
\A
\
II 87 8
,
IO
2
14
I6
hours
Figure 3. Comparison of hemoglobin elution curves from the Buchler and LKB prep-PAGE systems.
is pumped into the upper electrode compartment and forms a separate layer directly above the gel and directly below the circulating buffer. The dense feed layer remains in place throughout an experimental run except as explained below for Figures 5 and 6. In semicontinuous operation, the spent feed is periodically removed from the system and fresh feed solution is pumped into the column. The gel has been reused for up to seven cycles or 68 h with no apparent degradation of physical properties. The feed proteins were human hemoglobin purchased from Sigma Chemical Co. and human serum albumin purchased from Worthington Biochemical Corp. The product samples from the elution chamber were collected at either 15- or 30-min time intervals at an elution rate of 20 cm3/h. The feed and product samples were analyzed with a Bausch and Lomb spectrophotometer. Hemoglobin concentration was determined at a wavelength of 403 nm and total protein concentration was determined using Bio-Rad protein assay at a wavelength of 595 nm. Albumin concentration was then determined by difference. A preliminary experiment was done with hemoglobin feed at a pH of 8.6 in both the Buchler Poly-Prep 200 and the LKB 7900 Uniphor as shown in Figure 3. Hemoglobin has an isoelectric point of 6.7 and thus has a net negative charge a t a pH of 8.6, so the cathode is placed a t the bottom of the column. The Buchler column has a larger cross-sectional area than the LKB column (18 cm2 vs. 5 cm2),so the production capacity of the Buchler column is higher. As seen in Figure 3, the peak position and peak shape are similar under similar operating conditions in the two columns. The larger area under the Buchler peak is expected since the total feed was 15 mg of hemoglobin to the Buchler column in run 2 as compared with 8 mg of hemoglobin to the LKB column in run 1. Calculation of the hemoglobin recoveries in the two runs, however, shows a recovery of only 49% in the LKB column as compared with 84% in the Buchler column (Table I). Righetti and Secchi (1972) carried out recovery studies in the LKB column because of what they described as “the peculiar design of the elution stopper in the Uniphor column”. They reported 80% recovery for cytochrome C and 91-97% recovery for bovine serum albumin, ovalbumin, and lysozyme, but for some unknown reason they did not report the hemoglobin recovery even though they did experimental work on this protein. The Buchler Poly-Prep 200 was chosen for the experimental studies below because of its higher capacity and
492
Ind. Eng. Chem. Fundam., Vol. 24, No. 4, 1985
Table I. ExDerimental Conditionsaand Results feed wt, % A1 Hb
feed, cycle cm3 Buffer. 0.05 M Tris-maleate 20 50
_ _ _ l _ _ _ _ l
run
0.04 0.03 0.03
1
0.03
5
0.03
ti
0.03
0.03
Buffer: 0.05 M Tris-maleate 1 30 2 30 3 30 1 30 2 30 3 30 1 30
0.03
2
30
1
15 15 15 15 15 15 15 15
2 3 4
0.05 0.05
7
n 9 10 11
0.05 0.05 0.025
0.025
12 13
0.025 0.025
0.025 0.025
14
0.025 0.025
0.025 0.025
1,i
16 17
Buffer: 0.05 M Tris 30 30 30 30 1 30 2 30 3 30 10 I 10 2 30 10 1 10 2 10
Buffer: 0.05 M Tris-maleate 0.025 0.025 0.025 0.025
3
10
4
10
5
10
6
10
n
10
W
V
+ 0.05 M NaOH a t pH 8.6 10 10
354 176
recovery, YO A1 Hb
S.F.h -
-
49 84
90 92 112
52 61 69 -
99
21
39.0
100 84 14 47 25 95 15 62 24
20 5 26 7 16 8 31 10 44
5.5
-
91 75
-
+ 0.05 M NaOH a t pH 6.0 17.8 17.8 17.8 18.2 18.2 18.2 +27.6 - 27.6 +27.6 -27.6 +30.3 -30.3 +30.3 -30.3 +30.3 -30.3 f30.3 -.30.3
-120 -120 -120 +120 +120 +120 +150 -150 +150 -150 +150 -150 +150 -150 + 150 -150 +150 -150
-
-
-
30.5 4.1
23.9 11.6
+ 0 05 M Glycine a t pH 8.6 5 10 5 10 10 10 10 10 5 5 3 5 5 10 7
10 5 10 3 10 3 10 3 10
233 281 234 347 298 298 298 331 234 234 170 230 230 321 230 321 230 321 201 321 201 321 201 321
87 73 56 67 96 100 100 100 100 95
64 91 108 100 89 94 100 100
9.4 8.3 3.4 5.5 12.6 6.6 11.3 2.1
106
100
9.4
99
100
6.9
100
92
6.3
-
90
-
100
92
3.7
100
100
3.5
-
-
+ 0.05 M NaOH a t pH 5.0 and 0.05 M Tris + 0.05 M Glycine at pH 8.6 -
10 30
10 10
257 262
100 98
94 82
10.6 8.0
"Elution rate (run): 20 cm3/h (1, 3-17), 24 cm3/h (2); sucrose concentration in feed (run): 3 wt O7o (1-2), 5 wt % (3-5), 10 wt YO (6-17); gel height (run): 4.0 cm (I),5.0 cm (Z), 2.3 cm (3-5), 3.1 cm (6), 8.3 cm (7-17); power (run): constant wattage (1-2, 7-17), constant voltage (3-6). bHigh separation factors are not considered meaningful unless the numbers are reproducible and the recoveries are also high.
because of the apparently superior mechanical design of the cooling system and of the elution membrane. The wattage in the electrophoresis process generates heat which must be removed in the cooling jackets in order to prevent gel degradation and protein denaturation. The Buchler system has both an inner and an outer cooling jacket (Figure 2), while the LKB column has only an outer cooling jacket. The elution chamber and the lower electrode chamber in the Buchler system are separated by a rigid glass semipermeable membrane. The LKB column has a thin polymeric membrane which is held to the elution stopper by a brittle nonmetallic ring. Both the membrane and the securing ring were easily ruptured during normal operation in the LKB column, while the membrane in the Buchler column was quite sturdy. The recovery was also bett,er in the Buchler column in the preliminary runs,
Table 11. Selected Physical Properties
hemoglobin albumin
isoelectric Roint 6.7 4.7
mol w t 64500 69000
equiv spherical radius. A 31.0 36.1
diffusivity a t 25 "C, lo7 cm*/s 7.7 7.1
although additional hands-on experience with the LKB column would probably result in improved recovery for this system. Results and Discussion Hemoglobin and albumin are both globular proteins with similar physical properties except for their isoelectric points (Table 11). Elution profiles for the electrophoretic separation of mixtures of hemoglobin and albumin via
Ind. Eng. Chem. Fundam., Vol. 24, No. 4, 1985
v o+l t s 0
l
5
O
493
O
1
Pulse Feed
(cc) I
0
-
Albumin HemoqIobin---
c-.
hours
-+ CYCLE3 + Hemoglobin and albumin elution profiles a t ZB < pH < +C Y C L E I -+ CYCLE
Figure 4.
2
0
I-CYCLE
----'----
10
hours
I +-CYCLE
30
40
50 2 __I(
Figure 5. Experimental results for alternating-field-polarity mode of operation (run 5 ) .
IA.
prep-PAGE are shown in the figures below. The concentrations of the individual proteins are given rather than showing only the total protein eluted as is commonly done in PAGE experiments (Righetti and Secchi, 1972). The protein concentrations are used to calculate the hemoglobin and albumin recoveries as well as the overall separation factors in Table I. Experimental operating conditions are also listed in Table I. The new separation process can be extended to any mixtures of proteins A and B with different isoelectric points I A and IB. Two modes of separation are considered below. In the first set of experiments, the operating pH is set so that I B < pH < I A and the electric field is switched from positive to negative polarity during each cycle of operation. In the second mode of separation, the operating pH is set so that I B < I A < pH and the electric field is changed from low voltage to high voltage during each cycle of operation. The second mode can also be used to separate mixtures of proteins which have similar isoelectric points but different charge magnitudes due to differences in molecular structures. Either of these two modes of separation can be repeated for a number of cycles by removing the spent feed solution and adding fresh feed to the column, then repeating the positive-negative or the low-high field strength variation. The eluted product from each cycle is divided into two product fractions a t the crossover point of the elution profiles of the individual proteins. The separation factor (S.F.) for each cycle or batch is defined as
S.F. =
(mg B),/(mg B), (mg A),/(mg
(1)
where fraction 1 is richer in protein B or albumin and fraction 2 is richer in protein A or hemoglobin. Alternating-Field-Polarity Experiments. First consider operation a t a pH of 6.0 which makes IB< pH < IA. This pH makes protein A or hemoglobin positively charged and protein B or albumin negatively charged. If the cathode is placed a t the bottom of the column, protein B will move through the gel to the elution chamber while protein A will remain in the feed layer. Alternately, if the anode is placed a t the bottom of the column, protein A will move through the gel and protein B will remain in the feed layer. Thus, alternation of the field polarity will institute separation of proteins with unlike charges. Separate experiments were run on albumin and hemoglobin a t a pH of 6.0 as shown in Figure 4. Three cycles of operation are shown for each protein with fresh feed at 10-h intervals. A gel height of only 2.3 cm was used be-
cause the operating pH is close to the isoelectric points of the two proteins making the charge magnitudes small and the migration velocities low. The anode was placed at the bottom of the column for the hemoglobin run-this electrode placement is designated as negative voltage in the following experiments. Hemoglobin gives broad peaks under these operating conditions with maxima occurring a t 8.5-10.5 h after the feed pulses. The cathode was placed at the bottom of the column for the albumin run in Figure 4. As expected, albumin travels slightly faster through the gel than hemoglobin since the difference between the operating pH and the isoelectric point is greater for albumin than hemoglobin or IpH - IBI > IpH - IAI. Actually, albumin has a greater charge magnitude even if JpH - IBI = IpH - IAl, because it has more charged functional groups or a greater { potential than hemoglobin. Albumin gives sharper peaks than hemoglobin with maxima occurring a t 6.5-7.5 h after the feed pulses. The albumin recovery in Figure 4 was 98% but the hemoglobin recovery was only 61% (Table I). In experiments with cytochrome C, Righetti and Secchi (1972) reported that recovery losses were due to reddish protein material remaining in the gel after electrophoresis. In the present hemoglobin experiments at a pH of 6.0, the gel and the feed layer contained yellow material or hemoglobin at the completion of the runs. This phenomenon did not occur in later hemoglobin experiments at a pH of 8.6, which suggests that the lower pH is too close to the isoelectric point of hemoglobin for good elution of this protein. Figure 5 shows the separation of the protein mixture using alternating polarity for two complete cycles of operation. The mixture was pumped into the column at a pH of 6.0 and the cathode was first applied to the bottom of the column in order to collect the albumin-rich product. After 28 h of operation, fresh feed was added and the complete cycle was repeated. The albumin moved a little faster in this experiment than in the previous experiment because the field strength was increased to 150 V. The shape of the albumin peaks is similar in Figures 4 and 5. The hemoglobin peaks in Figure 5 , which supposedly occur at about 19 and 47 h, are almost nonexistent due to recoveries of only 20% in this experiment as compared with 61% in the previous experiment (Table I). During the first part of each cycle when the field was placed in the positive position for elution of negatively charged albumin, the feed layer containing the positively charged hemoglobin expanded with the upper boundary moving toward the negative electrode a t the top of the column.
494
Ind. Eng. Chem. Fundam., Vol. 24, No. 4, 1985 Exp run watts --
Exp r u n w o l f s ---
12:
-0-9 -..-IO
I Ob
F-CYC-E
-
CYCLEZ-
i CY;,E
3
L
C CLEa
5
IO
--
Figure 6. Alternating-field-polarityexperiment with two feed pulses per cycle (run 6).
This moving boundary was measured in two separate experiments and the hemoglobin retention in the feed layer was calculated based on the measured concentrations and volumes before and after electrophoresis. The amount of hemoglobin in the feed layer dropped to 65% after 10 h and 43% after 15 h. The circulating buffer in the upper electrode compartment was also measured in these experiments and was found to increase slightly in hemoglobin concentration with time, which suggests that some of the positively charged protein migrated out of the dense feed layer into the upper compartment in the direction of the anode. A cloudy, foamy substance was observed in the region of the anode after a number of hours of operation, indicating the presence of denatured proteins in this region. A final experiment on the mixture a t a pH of 6.0 is shown in Figure 6. Instead of adding one 30-cm3pulse of feed at the beginning of each cycle, a 15-cm3pulse was added in each half-cycle in conjunction with the change in electric field polarity. Feed was initially pumped into the column and the cathode was placed a t the bottom of the column in order to elute the negatively charged albumin. After the albumin peak exited the system, fresh feed was added and the anode was placed at the bottom of the column to elute the positively charged hemoglobin. Fresh feed addition and polarity changes follow each peak for four complete cycles. The albumin peaks are sharp and the hemoglobin peaks are slightly more pronounced than in the previous figure, but the recovery is still poor (Table 1). The experiments with the alternating field polarity give good recovery and sharp peaks for albumin but poor recovery and nondescript peaks for hemoglobin when this mixture is separated a t I , < pH < I A . Equipment modification to incorporate a semipermeable membrane between the feed layer and the upper electrode buffer should improve protein recovery. This separation mode generally does not work well for the model protein mixture considered in this study, but it may be more suitable for other mixtures. Variable-Field-StrengthExperiments. In the second mode of separation, the pH is set a t 8.6, which makes IB < 1, < pH. In this case, both proteins carry a net negative charge and migrate through the gel towards the cathode. Since (pH - IB) > (pH - IA) and since the radii and molecular weights are similar for proteins A and B, protein B or albumin will carry a greater net negative charge than protein A or hemoglobin. Thus, protein B will move at a greater velocity through the gel in the presence of an electric field. The electrophoretic migration velocity for a given protein increases in direct proportion to the
hours
Figure 7. Hemoglobin and albumin elution profiles at IB < IA< pH.
strength of the electric field. If the velocities of the solutes in a mixture are too high, the bands will not be completely separated as the mixture passes through the gel. On the other hand, if the migration velocities are too low, the bands will remix due to molecular diffusion. The separation in a mixture may be optimized and the processing time may be reduced by collecting protein B or the faster-moving solute at a low voltage, then increasing the field strength for the slower-moving solute. Multiple levels of field strength can be used for the separation of mixtures of multiple solutes. Separate experiments were run on albumin and hemoglobin at a pH of 8.6 and a gel height of 8.3 cm with constant power at 5 W and also a t 10 W as shown in Figure 7. Electrophoresis of either pure hemoglobin or pure albumin yields a relatively simple elution profile. The albumin peaks move faster than the hemoglobin peaks at both 5 and 10 W, as expected. Also, the shapes of the elution curves for a given protein are identical at 5 and 10 W except that the bands travel faster at the higher field strength. The hemoglobin peaks obtained at a pH of 8.6 are more pronounced than the peaks obtained for this protein at the lower pH in the earlier experiments. Also, the hemoglobin recoveries are significantly improved a t the higher pH (Table I). Hemoglobin clearly exhibits a single peak for the pure component in Figure 7 , but multiple peaks are observed for hemoglobin in the mixture in the figures below. Multiple peaks are observed for pure albumin in Figure 7 with the main peak being the albumin monomer and the shoulder on the main peak being the albumin dimer (Righetti and Secchi, 1972). The small peaks in the albumin elution profiles a t 5-6 h are probably globulin impurities from the purchased albumin sample. The peak concentrations for albumin in runs 9-10 are greater than the feed concentration and can be made even more concentrated by decreasing the elution buffer rate. The elution buffer rate must be kept high enough, however, to provide adequate protein elution. Excess buffer solution can be easily removed from the product via ultrafiltration. The electrophoresis process like the HPLC process usually dilutes the protein mixture as needed in order to separate the solutes in the feed solution from each other. Figure 8 shows the separation of the protein mixture at high pH with 30-cm3feed pulses a t 3.75-h time intervals for three complete cycles. Separation factors of 8-9 were obtained in the first two cycles, and the total recoveries
Ind. Eng. Chem. Fundam., Vol. 24, No. 4, 1985 495
---
4lburnin Hernoglob8n
r I
A
hem CYCLi I
4
CYCLE 3
CYCLE 2
_c/
Figure 8. Cyclic separation experiment on the mixture at high pH and constant field strength (run 11). run 16 ( I O c c I Albumln -0Hemoglobin d-
i
I
t
I\
or
08-
YF yP i; 04-
run I7 ( 3 0 c c )
Albumin -0 Hemoglobin -A-
A
I
02n
2
3
4
5
6
7
8
9
IO
hours
Figure 9. Effect of feed volume on elution profiles.
were 88% for hemoglobin and 73% for albumin (Table I). Sharp peaks were observed for both proteins, although there is considerable overlapping of the peaks for the two proteins. The albumin peak in the third cycle is malformed, resulting in poor separation for that cycle. The irregular shape of the third albumin peak in this figure as well as the second albumin peak in Figures 5 and 6 is probably due to the experiment being shut off temporarily so that the investigators could attend classes or sleep. These breaks in the continuous operation definitely occurred in the lengthy cyclic experiments, but the times that the breaks occurred were not recorded. Comparison of two batch runs on the mixture in Figure 9 indicates that the separation is increased from 8 to 11 when the feed volume is decreased from 30 to 10 cm3 or when the total protein in the feed is decreased from 15 to 5 mg. The reddish-brown color of the hemoglobin allowed visual confirmation of the experimental data. Two hemoglobin bands were observed moving through the polyacrylamide gel and were also measured experimentally-a faint narrow leading band and a dark broader trailing band. Some of the hemoglobin apparently “sticks to” the albumin. This protein-protein interaction is not well understood and will be studied in more detail in future research. A small hemoglobin peak was eluted with the albumin peak in all of the experiments on the mixture, but this additional peak was more pronounced in the experiments at high pH. Since the extra hemoglobin peak was smaller
in the low pH experiments (runs 5 and 6), elution with two different buffers was attempted in runs 16 and 17 (Figure 9) in an effort to reduce the magnitude of the additional hemoglobin peak. Tris-maleate/NaOH buffer a t a pH of 5.0 was pumped into the prep-PAGE system for the first 4 h of the batch runs in order to elute the albumin peaks; then the eluiion buffer was switched to Tris/glycine at a pH of 8.6. The average product pH in runs 16 and 17 was 7.1 and the elution profiles were similar to those obtained in the experiments with only the high pH buffer (runs 11-15). Evidently, the pH OC the gel and the feed is more important than the pH of the elution buffer. In our pre. vious experiments on HPAP, an optimum pH for the PIJrification was observed (Chao et al., 1983). The separation of the mixture is compared at several different field strengths in Figure 10. As the power increases from 3 to 10 W, the cycle or batch time decreases from 13 to 8 h. The field strengths corresponding to these wattages are 170 and 331 V, resp. Comparison of thew runs in Table I shows that the separation factor increases from 6 to 13 when the field strength decreases from 331 to 231 V, but that the separation factor decreases from 13 to 11 when the field strength decreases from 231 to 1’70 V. Thus, the observed separation goes through a maximum a t about 231 V or 5 W. The optimum wattage for maximum separation was also found to be 5 W in our previous experiments on HPAP (Chao et al., 1983), which is reasonable because the leading peak is albumin in both the crude HPAP mixture and the hemoglobin-albumin mixture. A higher field strength would presumably he required in order to obtain sharp peaks and good separation if HPAP or hemoglobin were to be separated from another protein with lower electrophoretic mobility. Variable-field-strength electrophoresis is demonstrated in Figure 11. In order to minimize the batch or cycle time while still maintaining good separation of the hemaglobin-albumin mixture, the albumin-rich fraction was collected at 5 W to maximize separation. The power was then increased to 10 W to remove the hemoglobin-rich fraction in the minimum time. After the majority of the trailing peak or hemoglobin had exited the prep-PAGE system. the spent feed solution was pumped out of the column and a 10-cm3pulse of fresh feed solution was pumped into the column. The power was simultaneously reduced back to 5 W. Each cycle yields two product fractions in a continuous manner and includes the feed step plus periods of operation at low and high fields. The cycle or the first product begins a t the time when the product stream becomes richer in albumin. The second product fraction begins at the time when the product stream becomes richer in hemoglobin. Three cycles of operation, representing cycles 2-4 of a seven-cycle experiment, are graphed in Figure 11 for run 15. The separation factors and the recoveries for cycles 1-7 are listed in Table I. Complete data for the fifth cycle were not recorded. Cycle 1 was used to determine the appropriate time for shifting the power, cycles 2-4 were run at 5 and 10 W variable field, and cycles 5-7 were run a t 3 and 10 W variable field. The same gel was used for the entire run with no signs of degradation. The separation factor was 9 in cycle 2 which is comparable to the batch experiment at 5 W. The separation factors in cycles 3 and 4 are slightly lower (6-7 vs. 9), because the hemoglobin-rich peak has a long tail which extends into the succeeding albumin-rich peak of the next cycle. Note that the columri was run for a period of time after cycle 1with the power on, thereby removing this left-over hemoglobin from the gel before starting cycle 2. The separation factor is n n h
496
Ind. Eng. Chem. Fundam., Vol. 24, No. 4, 1985
r run 12 ( l O w a t r r ) Albumin
- --
Hemoqlobln
n
run
13 ( 5 w O t t s )
I-
/’
0.2
\\
A
r u n 14
( 3watts)
hours
Figure 10. Effect of electric field strength on elution profiles.
Albumln
P
04-
-
Memoglobin
---
YP YF
A
0.2
I ’
-
0.
I
4 for cycles 6 and 7 , since the hemoglobin tail is even broader and longer a t 3 W than at 5 W. The data suggest that a separation factor of approximately 7 can be maintained with operation a t 5 and 10 W variable field from cycle 4 ad infinitum. The recoveries are close to 100% for both proteins in this experiment. Conclusions Semicontinuous operation of a preparative-scale polyacrylamide gel electrophoresis system has been successfully demonstrated. The gel has been reused up to seven times
or for seven cycles of operation with no visible decline in its physical properties. Two modes of operation were investigated for the experimental separation of a mixture of two proteins with different isoelectric points. Either the operating pH is set between the isoelectric points of the two proteins and the electric field strength is cycled between positive and negative values, or else the operating pH is set above the isoelectric points of the two proteins and the electric field strength is cycled between low and high positive values. A pulse feed is added in each cycle and a continuous product is generated. Variable-field-
Ind. Eng. Chem. Fundam. 1985, 2 4 , 497-499
strength electrophoresis appears to be a promising technique for protein separation and purification with the low-high mode of operation giving better protein recoveries in the present studies. The low-high mode may also be applied to mixtures of multiple solutes by setting the o p erating pH above the isoelectric points of all of the solutes and increasing the field strength in multiple steps in each cycle.
497
Greek Letters $ = % protein recovered Subscripts 1 = fraction 1 2 = fraction 2
Literature Cited Chao, J. F.; Huang, J. J.; Huang, C. R. AIChE Symp. Ser. No. 219 1982, 7 8 , 39.
Acknowledgment The National Science Foundation (CPE 79-10540) provided financial support for this research. The authors are grateful to Dr. Burke Baker, 111, for his many pertinent remarks in reviewing this paper. Nomenclature AI = albumin Hb = hemoglobin I* = isoelectric point of protein A or hemoglobin IB= isoelectric point of protein B or albumin HPAP = human placental alkaline phosphatase HPLC = high-performance liquid chromatography prep-PAGE = preparative-scale polyacrylamide gel electrophoresis S.F. = separation factor as defined by eq 1 YF = concentration of solute in feed, kg-mol/m3 yp = concentration of solute in product, kg-mol/m3
Chao, J. F.; Rollan, V. P.; Hollein. H. C.; Huang, C. R. Sep. Sci. Techno/. 1983, 78, 999. Chem. Eng. News May 18, 1983, 7. Chen, H. T.: Ahmed, 2. M.; Rollan, V. P. Ind. Eng. Chem. Fundam. I 9 8 l a . 20, 171.
Chen, H. T.; Kerobo, C. 0.: Hollein, H. C.; Huang, C. R. Chem. f n g . Educ. 198lb, 15, 166. Graff, G . M.; Short, H.: Keene, J. Chem. Eng. June 13, 1983, 22. Hollein, H. C.; Ma, H. C.; Huang, C. R.; Chen, H. T. Ind. Eng. Chem. Fundam. 1982, 21, 205. Lee, K. C.; Kirwan, D. J. Ind. Eng. Chem. Fundam. 1975, 14, 279. Michaels, A. S. Chem. Eng. Prog. April 1984, 9; June 1984, 19. Nelson, W. C.; Silarski, D. F.; Wanltat, P. C . Ind. Eng. Chem. Fundam. 1978, 17, 32.
Oren, Y.: Soffer, A. J. Electrochem. SOC.1978, 125, 869. Pigford, R. L.; Baker, B.; Blum, D. E. Ind. Eng. Chem. Fundam. 1969, 8 ,
848. Righetti, P.; Secchi, C. J. Chromatogr. 1972, 7 2 , 165. Rodbard, D.; Chrambach, A. Anal. Biochem. 1971, 4 0 , 95. Thompson, D. W.; Bass, D. Can. J. Chem. Eng. 1974, 5 2 , 345. van Oss,C. J. Sep. Purif. Methods 1979, 8, 119. Webber, D. Chem. Eng. News Aprll 18, 1984, 11.
Received for review December 21, Revised manuscript received August 29, Accepted September 24,
1982 1984 1984
COMMUNICATIONS Nonlinear Sorption Effects on the Determination of Diffusion/Sorption Parameters A comparison is made of sorbate uptake by single particles which exhibit both linear and nonlinear equilibrium sorption. Parameters are identified which may be used to estimate when nonlinear sorption will result in significant error in the determination of kinetic parameters using transient experiments.
Introduction The most popular method for the study of diffusion and sorption kinetics in porous materials is to introduce a change in the concentration of gas or liquid surrounding the solid and measure the solid uptake as a function of time. Numerous experimental techniques exist based on this principle which range from using single particles to packed beds and may use porous solids of either slab, cylindrical, or spherical geometries. In order to extract diffusion and sorption coefficients from these experiments, one must match a theoretical response curve to the experimental uptake curve. For single-particle nonflowing experiments, transport parameters are determined either by using moment analysis (e.g. Kocirik and Zikanova, 1974) or by plotting M J M , vs. t 1 / 2 . For small times, this plot is linear with slope proportional to D1jZ(see Crank, 1975). For complex experimental designs that include flow coupled with diffusion/sorption, more sophisticated moment or Fourier analysis techniques are used. Although the model used to generate the theoretical response curve is dependent on the actual experiment 0196-4313/85/1024-0497$01.50/0
design (i.e. single particle, chromatograph, etc.), an assumption common to all of these transient methods is that of a linear sorption isotherm. For diffusion experiments conducted in this laboratory using beds of monodisperse silica spheres and an experimental approach similar to that reported by Eberly (1968), significant deviations between theoretical and experimental uptake curves were noted (Drake, 1984). Since these deviations are a function of concentration, it was suspected that errors associated with the linear sorption assumption used in our analysis were the cause. A search of the literature indicates that a systematic study of the effects of nonlinear sorption on the determination of diffusion/sorption parameters with transient techniques has not been conducted. Aris (1983) recognized the potential problems associated with nonlinear isotherms in diffusion measurements. The case of concentration-dependent diffusivity, due to both sorption and surface diffusion as related to steady-state measurements, was considered. However, that work is not directly applicable to the more commonly used transient methods. The aim of this communication is to model sorbate uptake by porous particles which exhibit nonlinear equi0 1985 American Chemical Society
