Because FFE uses no organic solvents, salts, or supporting media, it can be used to solve the problems associated with the purification of biological molecules and cells Mark C. Roman Phyllis R. Brown Univenify of Rhode lsland
he tremendous growth of biotechnology over the past decade has necessitated the development of purification methods for isolating a single compound from a complex biological matrix. Compounds of interest include biologically important molecules such as amino acids, peptides, and proteins. Traditional$. the preparative purification of proteins and peptides has been accomplished by methods such as precipitation and dialysis techniques (which are not very selective), LC methods (which denature proteins and peptides), and gel electrophoresis (which is limited by small sample loads and the difficultyof separating the compound of interest from the gel matrix). With the advent of genetically engineered cells, cell purification has become increasingly important Ultracentrifugation and isopycnic centrifugation, which separate cells on the basis of density differences, are the most common methods
80 A Analflical Chemistry, Vol. 66, No. 2, January 15, 1994
of purification. These techniques, however, are not very selective, because the densities of different cell types are often similar. New preparative separation techniques are needed to address the problems associated with the purification of peptides, proteins, and cells. Free-flow electrophoresis W),frst introduced by Barrolier, Watzke, and Gibian (I)in 1958and Hannig (2) in 1961,has evolved i n b one of the most promising techniques for preparativescale separation. In FFE, a curtain of carrier buffer flows between two narrowly spaced vertical plates. An electric field is applied perpendicular to the carrier flow while a sample solution is injected continuously into the camer buffer as a MITOW band. Sample components are then separated laterally in the electric field according to differences in their electrophoretic mobilities, and the separated components are collected continuously at the outlets
w i 1).
OW3-270OB4~0366-86A604SW @ 1994 American Cnemical Society
As a Preparative Separation Technique The primary advantage of FFE over other separation techniques is that it is a continuous separationprocess as opposed to a batch process. Unlike LC and gel electrophoresis,in which repeated sample loadings are required for puriryins large quantities of compound, FFE can be used for continuous separation until the entire sample has been treated. Therefore, using FFE saves a great deal of time and labor. Because it does not use organic solvents. high salt concentrations, or supporting media such as silica or gels, FFE is gentle enough for use with proteins and cells. Unfortunately, problems such as distortions of the sample stream and difficulty with scale-up have limited the use of FFE.Nevertheless, because it has shown a great deal of promise for the separation of hiomolecules; work is proceeding on developing FFE as a preparative technique for biotechnology. Migration of ions in an electric tieid Electrophoretic separations are possible with any ionic or ionizable species. When an ion in solution is placed in an electric field, it will move in a direction parallel to the electric field with a velocity
pi E (1) where & is the electrophoreticmobility of the ion and E is the potential gradient. The ionic mobility of a particular ion can be described by
ui
pi
=
= lil
go /6xqri
(2)
where 4 is the charge on the ion, qo is the elementary charge (1.6022 x l(r” C), is the viscosity of the solution,and 5 is the radius of the ion. The electrophoretic mobility of an ion depends largely on its
charge-to-size ratio. Because different ions have different charge-to-size ratios, each ionic species has a unique mobility and thus a unique velocity when placed in an electric field. Therefore, separations of ions based on different ionic mobilities are possible. If a compound is amphoteric, its charge is dependent on the pH of the surrounding carrier solution. An amphoteric compound migrates until the surrounding solution has a p y equal to its isoelectric point, where it will have no net charge and thus will not migrate any farther in the electric field. This phenomenon, known as isoelectric focusing, has been used as a powerful technique for pdfyhg proteins.
a separation, the only variable from solute to solute is the ionic mobility. Therefore ions with different mobilities are deflected at different angles (8)and thus travel along different paths V i 1). The separated components are then collected continuously in multiple tubes at the outlet The current i can be expressed as
i
=
-Afldt/clz)x lzd pici
(4) where.4 is the area of the electrode sur-
CCE theory
In FFE, an ionic solute is injected at a point in a continuouscurtain of buffer. The solute travels in the z direction as it is carried hy the buffer flow (perpendicular to the electric field) and in they duection as it migrates parallel to the electric field. The angle of deflection of the solute in the electric field increases with the electric field strength and the ion mobility and decreases with increasing flow rate. The ratio of the electrophoretic velocity to the buffer velocity is tan 0 and is given hy tan0
=
pii/qxw
(3)
where 8 is the angle of deflection, i is the electric current, q is the cross section of the separation chamber, xis the specific conductance of the buffer solution, and o is the linear velocity of the buffer solution. Because the current, the cross section of the chamber, the conductance of the buffer solution, and the linear velocity of the buffer are held constant during Analyikal Chemistry, Vd. 66, No.2,January 15, 1994 01 A
face, Fis Faraday’s constant, dt/dr equals the potential gradient E, and c, is the concentration of the species. The conductivityK however, is given by IC = F ~ l Z i l ~ c i and Equation 2 can be simplified to
(5)
i = -AEK (6) By substitution, Equation 1becomes
tan 0 = -p, EA / qw
(7)
At constant E, the angle of deflection of a solute is independent of the buffer solution conductivity if the conductivity is uniform across the buffer solution. If the conductivity is not uniform, however, the potential gradient will not be uniform and the angle of deflection will vary. Thermal instabilities. Whenever a current passes through a conductor, the temperature of the conducting medium increases. This phenomenon is known as Joule heating. In a narrow FFE separation chamber (0.5-1.0 mm thick) the Joule heat is efficiently dissipated to the surroundings. As the bed thickness increases, however, the buffer solution in the center of the bed cannot dissipate the heat effectivelyand the buffer temperature increases.The amount of Joule heat H generated is H
=
iZRt
=
~/AK
FFE can be performed in four differentmodes7 which are determined by the buffercomposition and can be combined to form “hyphenated modes.
-
.~
”
(8)
where R is the resistance and t is the time during which the current is applied, or (in FFE)the residence time of the buffer in the separation chamber. The resistance R can be related to K by R
is proportional to the residence time in the separation chamber. High flow rates help prevent overheating of the carrier buffer hy m i n i the time that the buffer spends in the electric field. Finally, the heat generated is proportional to the area of the electrodes, which is directly related to the chamber thickness. Narrow cross sections reduce the heat generated and facilitate heat dissipation.
(9)
where h i s the distance between the electrodes. Therefore, the Joule heat generated in a FFE experiment is
H = AE’Kth (10) Four things are evident from this equation. First, because the heat generated is proportional to the square of the voltage gradient, there is a limit on the voltage that can be used in an experiment. Second, the heat generated is proportional to the specific conductance of the buffer solution. Buffer solutions with low conductivities are preferred for ensuring minimal heat generation. Third, the heat absorbed by the buffer
Once the voltage is applied, the buffer solution will enter the chamber with a temperature Toand exit with a temperature TpThe value of TImay be signiificantly greater than Tobecause of the heat absorbed by the buffer. In addition, the buffer near the walls will be cooler than the buffer in the center of the chamber because the walls can transfer heat to the surroundings.The temperature differential in the chamber creates density gradients within the buffer and may cause thermal convection, which can severely distort or destroy any separation. According to the equations derived by Ostrach (3),for a separation unit measuring 50 cm long, 10 cm wide, and 1mm deep, the maximumallowable temperature differencebetween the cooler carrier buffer near the walls and the wanner buffer in the center of the chamber is about 10 “C if the residence time of the buffer is 10 min. Alarger temperature
88 A Ana/ytica/ Chemistry, Vo/. 66.No. 2,January 15, 1994
gradient will cause flow instabilities from thermal convection. Increasing the thickness of the chamber not only will decrease the allowable critical temperature difference but also will decrease the heat dissipation efficiency of the unit. For this reason, narrow separation chambers must be used to allow efficient heat transfer throughout the entire buffer solution (4-7).In addition, buffer solutions are often cooled by passing them through a refrigeration unit, or by cooling the walls of the separation unit. Laminar flow pmfiie. In addition to these thermal convection problems, another problem inherent in FFE is the laminar flow profile of the carrier fluid. When a fluid flows between two parallel plates it develops a parabolic flow profile in which the maximum flow velocity is midway between the two plates, and the flow at the surface of the plates is effectively zero. The equation describing the flow is given by
u
-(l/Zp)(dP/dr)(BY-y’,) (11) where u is the velocity of the buffer at any distance y from one of the plates, is the dynamic viscosity of the buffer, dpldr is the pressure gradient along the axis of flow, and B is the distance between the plates. This buffer velocity gradient across the thickness of the chamber will cause the ions in the sample stream to travel at different velocities, depending on their positions relative to the wall. Solute ions in the center of the sample stream, and hence in the center of the chamber, will move the fastest; they will spend the least amount of time in the electric field. Solute ions near the wall will travel slowly and thus spend much more time in the electric field. The net result is band broadening of the sample because of differences in the migration times; the sample band assumes the shape of a crescent instead of a straight ribbon ( 4 9 ) . This “crescent” effect, caused by the laminar flow profile, can be referred to as hydrodynamic distortion. This effect can be minimized by either reducing the diameter of the sample band at the point of injection or increasing the distance between the walls of the separation chamber. Reducing the diameter of the sample band decreases sample throughput; an =
increase in the separation chamber thickness causes heat dissipation and thermal convection problems. Electroosmosis.Electroosmotic flow, caused by a charged double layer near the walls of the unit, can also distort the sample stream and result in a net flow at the walls toward the cathode. In FFE,electroosmotic flow distorts the sample stream into a crescent-the solute ions near the wall are dragged toward the cathode, whereas the solute ions in the center of the chamber are relatively unaffected. Therefore the sample stream develops a crescent shape similar to the results caused by a parabolic flow profile. The band spreadingcaused by electroosmotic flow can be referred to as electrodynamic distortion (10).Ifthe electmasmotic flow opposes the direction of solute ion migration, it can actually offset the band spreadingcaused by hydrodynamic distortion. EIectrohydmdymmic distortion. In addition to hydrodynamicand electrodynamic distortion of the sample band, Rhodes et al. described a third type of sample distortion called electrohydrodynamic distortion (11, 12).This distortion results from shear stresses caused by differences in the conductivity between the sample and the surrounding buffer and is independent of electrodynamic and hydrodynamicdistortions. When the sample stream is less conductivethan the surrounding buffer, it will flatten in the transverse direction, toward the wall; when the sample stream is more conductive than the surrounding buffer, it will distort in the lateral direction,toward the electrode ( F i2). Both types of electrohydrodynamic distortion cause band broadening. Rhodes et aL have suggested that elearnhydrodynamic distortion can be reduced or e l i t e d if the buffer is chosen carefullyso as to reduce conductivity differences. Mod.. ot WE
FFE can be performed in four different modes, which are determined by the buffer composition and can be combined to form ‘%hyphenated modes.”They are zone electrophoresis, stepfield electrophoresis, isotachophoresis, and isoelectric focusing. In zone electrophoresis, the carrier
bufferpossesses uniform pH and conduc- between throughput and resolution must be made by adjusting the voltage and the tivity across the chamber. Separation is flow rate. based on the charge-to-size ratios of the The step-field mode is similar to zone solute ions. Buffers that can maintain staelectrophoresis; however, the buffer conble pH and conductivityin the electric field must be chosen because protein mo- ductivity is not uniform across the separation chamber. In this mode, “walk” of bility is highly influenced by pH. The pH of the buffer is chosen according to the PI high-conductivitybuffer at the edges of the separation chamber, next to the elecof the compound of interest, the PISof trodes, are used. Because the voltage grathe contaminating compounds, and the pH dependence of the sample’s solubility. dient is inversely proportional to the conductivity, the voltage gradient in these Often the pH of the buffer is chosen so that the pH is between the PI of the com- high-conductivity walls is low compared with that in the low-conductivitycenter pound of interest and the PI of the major zone. When solute ions migrate to these contaminant. Alternatively,the pH of the walls, they will slow as they enter the buffer can be adjusted to the PI of the high-conductivity zones This retardation compound of interest; at this pH, the in their migration narrows the solute compound will not be deflected in the bands at the junction of the high- and electric field and the contaminants will be deflected away from the component of low-conductivitybuffers; in this way, the separated components can be focused interest. The importance of the voltage and the into sharp bands. In isotachophoresis.three zones are flow rate used in a zone electrophoresis created. The first, or leading, zone conseparation can be seen from Equation 4. tains an electrolyte with a high ionic moIncreasing the voltage or decreasing the bility. The middle zone is composed of flow rate will increase the separation disthe sample; the thud, or trailing, zone is tance between compounds. Avoltage incomposed of an electrolyte with a low crease, however, will also increase the ionic mobility. When the voltage is apamount of Joule heat generated and inplied, the ions arrange themselves in orcrease the electroosmotic distortion. Deder of ionic mobility with no clear separacreasing the flow rate will increase the tion between the species; the sample chance of thermal convection while also ionic species are “stack& between the decreasing throughput A compromise leading and the trailing electrolytes.Although this technique is limited because of low resolution between adjacent species, it has been used for applications such as the purification of ovalbumin and lysozyme (13.14). In isoelectric focusing a pH gradient is used in the separation chamber. This pH gradient is formed through the use of either commerciallyavailable ampholytes (nahually formed pH gradients) or buffers of different pH across the chamber (artificially preformed gradients). Roteins and peptides migrate in the electric field until they reach a buffer pH equal to their PIS, at which point they will stop Flgum 2 Eloctmhwhdymrnlc migrating and become focused. In this dktortlon technique, there is no voltage depenSample stream is the shaded area, and chamber wails are the hatched areas in this dence; the solute ions are immobile when cross section lwking down from the top of an they have reached their isoelectric points. FFE unit. (a) No voltage: sample is CirCuiW. (b) Voltage is applied: sample conductivity is less than the conductivityof the surrounding buffer. (c) Voltage b applied; sample conductivity is greater Man the conductivity 01 the surrounding buffer.
Instnnnontatlon Instrumentation has come a long way since Tiselius’ f i s t U-tube apparatus
Analyiical Chemistry, Vol. 66.No. 2, January 15, 1994 OS A
(15). Several methods have been developed to overcome convection and sample stream distortion problems and many different types of instruments are commercially available. Atypical FFE instrument consists of four components: pumps for the carrier buffer, sample, and electrode buffer washing solutions; a power supply: a fraction collector: and the separation unit as shown in F I3. The pump for the carrier buffer is usu aUy a multichannel peristaltic pump that can be positioned either in front of the carrier inlet (pushingthe buffer through the separation chamber) or after the outlets (pulling the carrier buffer through the separation chamber and the outlet tubes). The sample injection pumps and the electrode buffer pump are also urnaUy peristaltic pumps; the carrier buffer pump and the electrode buffer pump are sometimes the same pump. The separation unit consists of inlets for the buffer and the sample, a separation chamber, and electrodes. The number of carrier inlets can vary; anywhere from 5 to 90 have been used (1s). The purpose of having multiple carrier inlets
is to allow pH and/or conductivity gradients to be created across the separation chamber by varying the pH or the conductivity of the carrier buffer entering each inlet port The separation chamber is usuaUy made of two plates of glass, polycarbonate, Plexiglas or h c i t e separated by spacers that act as gaskets or seals. The electrodes, a l i i e d on opposite sides behveen the two plates, are usually composed of a metal such as platinum, which will not easily be oxidized in the electric field. The electrodes are separated from the separation chamber by ion-exchange, nylon, or cellulose acetate membranes. The electrodes are washed constantly by a salt or a buffer solution to remove the electrolysis products that are mated during the process. The carrier buffer and the separated sample leave the separation chamber through a fraction collector consisting of a multitude of outlet tubes. The outlet tubes then dispense the carrier buffer and the separated components into test tubes or vials. Although some units monitor the W absorption of each fraction, only the total protein concentration in
00 A Analflical Chemistry, Vol. 66, No. 2,January 15, 1994
each fraction can be determined from the W absorption. Fraction purity must be determined after the separation by LC, CE, or gel electrophoresis. The power source must supply a dc voltage of up to 3OOV/m and sustain a current of up to 200 mk One of the f i s t commercially available instruments was the Elphor VaP 21, manufaclured by Bender and Hobeln (Munich, Germany). It has since been succeeded by the Elphor VaP 22, which can regulate the buffer temperature to within 0.20 "C by cooling one of the chamber walls with a cooling module. A 90-channe1 peristaltic pump pulls the carrier buffer through five carrier inlets and 90 outlet tubes. The chamber thickness can be either 0.5 or 1.0 mm. A W-vis detector monitors the 90 fractions for proteins or cells to indicate the relative concentration in each fraction. A miniature FFE system, the ACE 710, has been developed by Huschmann Wnterhaching, Germany).The cross-sectional area of the ACE 710 is only 60 mm2 and the separation length is 4 cm; the area of the Elphor VaP 22 is 250 mm2 (for the 0.5-mm-thickchamber) and the length is 10 cm. The advantages of the ACE 710 are very fast residence times for the sample (30-90 s). direct W monitoring of the fractions at 225 nm,and an autosampler that holds 60 samples. The drawbacks of both instruments are the hydrodynamic and electrodynamicflow distortions of the sample bands because of narrow separation chambers and the inability to scale up the units to allow haher throughput Milan Bier of the University of AriZOMflhcson) has dweloped two systems designed to avoid problems such as thermal convection, electroosmotic flow, and hydrodynamic distortion: the Rotofor R e p IEF Cell (marketed by Bio-Rad Laboratories) and the RFJ Protein Fractionator (built by ProteinTechnologies and marketed by Rainii). The Rotofor unit is a horizontal cylindricalcellwith the electrodes placed at opposite ends. The unit is divided into 19 evenly spaced circular polyester screens with a pore size of 10 pm. The sample is intrcduced along with amphoiytes to generate a pH gradient The cell then rotates about its horizontal axis at 1 rpm, and the
combinationof the rotation and the screens prevents thermal convection.After several hours the proteins are focused in the pH gradient, and the 20 compartments created by the screens are quickly drained by vacuum into a hction collector. The RF3 unit is similar to the Elphor VaP 22 and the ACE 710; instead of continuous fraction collection, however, the RF3 recycles the eluent from the outlets hy reinjecting it into the inlets. This recycling technique is known as r e c y c l i isoelectric focusing (RIEF). Ahigh flow rate ensures short residence time and minimizes thermal convection. In addition, dter exiting the unit, the carrier and the sample are cooled in a heat exchanger before b e i returned to the inlet Ampholytes create pH gradients as in the Rotofor. By recycling the ampholyte buffer and sample several hundred times over a period of 2 h, high resolution of proteins is achieved. After 2 h, a manifold directs the sample and the buffer into a fraction collector. The disadvantage of both the Rotofor and the RF3 is that neither are continuous. Because both instruments are batch processors, one of the major advantages of FFE is lost. In addition, both systems are dependent on amphoiytes, which are chemically ill defined, costly, and can be difficult to remove from the purified proteins and peptides. Nevertheless, these instruments can be used for high-resolution separations that often are not possible by using conventionalFFE units. Eby (17) has reviewed many of the commercial FFE instruments. Other unique innovations have been tested to solve thermal convection and electroosmotic flow distortion problems. One of the most ambitious endeavors was taking an electrophoresis unit into space (microgravity electrophoresis) (18-24). The microgravity environmentof space prevents thermal convection, which is gravity dependent The lack of thermal convection allows the chamber thickness to be increased,thus increasing throughput while decreasing electrodynamic and hydrodynamic distortions. Scale-up of the FFE apparatus in space has limitations, however; the buffer temperahre must be kept below the denaturation temperahre of proteins.
The fluid endless belt system (25,26) developed hy Kolin and Luner showed much promise as a possible solution to thermal convection and sedimentation problems. In this system, a curtain of electrolyte is circulated in a flattened elliptical cylinder with a water-cooled core by a magnetic field generated by four bar magnets. Electrodes are on opposite ends of the flattened cylinder. A sample stream is injected near one end of the cylinder, and sample components are deflected at different velocities toward the outlets at the other end. The periodic reversal of the direction of the buffer and the solutes eliminates mixing caused by
Figurn 4. FluM d k u b d l dactrophomis
. .
N: north poles of magnets; w window in iron core: E electric field: B: maanetic field: J . wrrent density: F. electrimagnet c force: 1' and 2': hist ana Second ascending strea6o (bnina core): I. 2, and 3: fmt. SBcono. ana thim descending streaks (m front 01 core). (Adapted with perm sslon from Reference25.)
thermal density differences and sediientation. The separated solutes are then collected at the outlets F w r e 4). The drawbacks are that the system is highly complex and all solutes must be deflected toward the same electrode. Thus very high or very low pH must be used for the carrier electrolyte, which can either precipitate some sample components or denature the sample. Capillary free-flowelectrophoresis (CFE) (27-29) is another method that bas been used to overcome thermal convection and electrodynamicdistortion problems. The CFFE unit resembles a conventional FFE unit except that it has a heat exchanger made of layers of closely spaced, a l i i e d Teflon fluoropolymer capillary tubes placed in the separation chamber. When cooling mter is passed through the interstitial spaces between the capillary tubes (capillary spaces, essentially), efficient heat transfer takes place. F i e 5 shows a schematic diagram of the CFFE unit By increasing the number of layers of capillary cooling tubes, the thickness of the separation chamber can be increased without any thermal convection problems. Because the unit has a 12-nun-thick chamber, the sample stream can be kept away from the chamber walls. Electrodynamicdistortion caused by electroosmosis is greatly reduced because electroosmoticflow occurs only at a d l pohon ofthe surface OftheTeflon fluoroPober capillary Nk.
..
,,
~
,,
~ ; '!$ j #,[;It
Figurn5. CCCE system.
Despite the problems of hydrodynamic, electrodynamic,and electmhydrody~ m i distortion, c FFE is a useful tool for separating complex protein mixtures, peptides, and cells to isolate one or two components at fairly high purity. The separation of proteins by electrophoresis can be traced back to 'Ilselius, who separated three different types of human hemoglobin in a U-tube apparatus. Since then; protein purificationhas become a major focus in FFE because of the tremendous needs of the biotecbnology and the pharmaceutical industries. Much of the work has been experimental and exploratory,yet the results have been successful enough to maintain a high
Analytical Chemistry, Val. 66, No. 2, January 15, 1994 S i A
level of interest. Many of the theoretical and experimental considerations regarding protein purification have been developed by C l i i n et al. (30). Knisely and Rodkey (31) separated proteins by continuous FFE and compared the results with those obtained hy using RIEF. Although the FFE results showed excellent resolution of myoglobin, cytochrome c, and ovalbumin, the proteins ovalbumin and blactoglobulin (which possess PISwith only a 0.5 difference in pH) could not be rU$ resolved from each other. The RIEF unit, on the other hand, dearly resolved these two proteins. It was noted, however, that the use of zwitterionicbuffers may improve the separation of these two proteins in the FFE unit The effects of buffer pH, conductivity, voltage, canier flow rate, sample load, and electrophoresis mode on the purification of alcohol dehydrogenase (ADH) from yeast extract have been studied (3234). By optimizing these conditions and simultaneously injecting three sample streams, a throughput of 2.7 g/h was achieved with a purifcation factor of 4.7
--k.
for the ADH F i e 6).Although complete purification of the ADH was not attained, the high throughput and significant purification factor (the ratio of pmtein purity after separation to the puritybefore separation) demonstrates the potential of FFE for l a r g e . d e separations. Several practical applications of protein and peptide purification have been demonstrated. The acidic protein a-amylase, artificially doped into E. coli, was purified from the E. coli extract, along with the basic protein lysozyme (35). OmpF porin, a membrane fragment from E. coli, has been purified to 9996by using a surfactant in the carrier buffer above its critical micelle concentration: throughput, however, was only 1mg/h (36). By using FFE in the isotachopboresis mode it was possible to purify and characterize high-density lipoproteins, which are believed to play an important role in the transport of cholesterol to the liver (37,38).FFE was demonstrated as a viable means for puriryinglentil lectins. with a potential throughput of 40 mg/h using the Elphor VaP 22. Although complete
purity of the lectins was not achieved with FFE alone, the collected fractions could be further purified by using RIEF; thus, all remainii traces of acidic contaminants were removed. These two preparative electrophoresis steps could replace a chromatographicprocedure in which two ion-exchange steps for the purification of lectins from lentil seeds are used (39). Tissue plasminogen activator @-PA)is an enzyme that has generated a great deal of interest in the biotechnology field because of its role in dissolvingblood clots (40).Ruification of t-PAfrom g e n e t i d y engineered organisms by LC is difficult, however, because t-PA can adsorb to stationary phases and metal parts. FFE was used to pu@ t-PA from yeast extract. The nonionic surfactant 'nveen 80 [sorhitan mono-9-octadecenoatepoly(oxy-1.2ethanediyl) derivatives, better known as Polysorbate 801 was used in the carrier buffer to prevent adsorption onto the walls of the separation chamber. It was found that %% of all foreign protein matter could be removed, and t-PA recovery was 80% (41). The purification of synthetic growth hormone-releasing peptide (GHRP), a short peptide with the structure His-TrpAla-Trp-F'he-Lys-NH,, was used for a correlation study between CE and FFE (42).The results of the anaiytical CE separations were considered in modeling the preparative FFE system used to remove several minor contaminants present after the GHRP was synthesized. The proteins ribonucleaseA and hovine serum alhumin @SA) have been separated hy using CFFE at a throughput of 440 mg/h. Glycine/acetate buffer at pH 6.9, midway between the isoelectric points of BSA and ribonucleaseA, was used as the carrier buffer. F i i 7 shows the results of the LC analysis of the collected fractions after separation. Good resolution was obtained between the two proteins: the slight overlap of =with ribonucleaseAwas most likely caused by mixing immediitely prior to the outlets. An improved design of the outlets should e l i i a t e this prohlem. Cell purification is a challengingprohlem, one for which FFE is particularly well suited. Cell separations can be done hy ultracentrifugation; however, complete resolution is often difficult because of
I
Arrows indicate points of sample injection. Black trace indicates ADH concentration; blue trace indicates total protein concentration. Sample is dialyzed yeast extract, containing 45.8 @L total pmtein and 1.53 ADH. Voltage: 1000 v; residence time: 5 min; flow rate: 20 mUh; electmde bufler: 0.2 M th(hydmxvmethyl)aminomeihaneadjusted to pH 8.0 with HCI. Apparatus is a pmduclion-scale instrument by Bender and Hobein with dimensions 100 cm x 15 cm x 0.05 em; unit has 75 inlet pork and 136 outlet pork. (Adapted with permission from Reference 34.)
92 A Analyiical Chemistry, Vol. 66, No. 2, January 15, 1994
simi!arities in cell densities. Unfortunateiy, LC and gel electrophoresisare not suited to cell separationsbecause the pores in the solid supportsare too smallfor the cells to penetrate. Because solid supports or denaturing conditions are not used in FFE, and because diffemt types of cells usually pos sess different surface charges, FFE is an attractivealternative for cell separations.A great deal of effort has been spent on studyingthe use of FFE for cell separations, both on Earth and in the micropravity environment of space. T and B lymphocytescan be partially separated from each other by using zone FFE (43,44), but complete resolution is very difficult because of their similar mobilities. The lymphocytescan be separated more easily, however, by a d d w polyacrolein microsphereshound with B-cell specific antibodies to the cell mixture. The B lymphocytesbind to the antibody-coated microspheres;thus, their mobility is greatJy reduced, and the two cell types can be resolved (45). In addition to separating the enzyme t - P A h m crude cell extracts, it is desirable to separate cells that produce high levels of t-PAfrom cells that do not producet-PAFFEhasbeenusedtopurify cultured human embryonic kidney cells, which produce urokinase plasminogen activator (u-PA).Some of these cells producet-PAinadditiontou-PAItwas found that passing the human kidney
cells through an FFE unit yields a large number of fractions that contain u-PAproducing cells. A small number of these fractionscontained cells that produced two to three times more u-PA than cells in the other fractions. In addition, cells that produced t-PA showed lower mobdities than non-t-PA-producingcells and thus were concentrated toward one end of the cell fractions (46).Therefore, cells could be concentrated on the basis of their relative abilities to produce u-PA and t-PA FFE experiments conducted in space showed similar results for the separation of human kidney cells (47).
I HERE‘snothingreallynewabouttheEGM;
Princeton Applied Research Model 263.
1 Unlesyoucalltheanaloglookandkltoits
ComlUSion
FFE has the potential to develop into an extremely powerful preparative-de separation technique for cells and biomoled e s such as amino acids, peptides, and proteins. The continuous separation Mture and gentle separation conditions of FFE make it a very attractive alternative to chromatographyand gel electrophoresis. Much of the theoretical framework for FFE has been developed; recent development of new methods can help overcome some of the technicalproblems assoeiated with scaling up FFE. As these new methods overcome the problems of t h e d convection and other sample stream distortions, FFE should become a method of choice for the largescale purification of biological compounds. As with any new technique, however, the acceptance of FFE will come only with exhaustive testing and the demonstration of its selectivity, reproducibility, versatility, and ruggedness.
inputknobnew...Or, ifyoucallitsd
