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Other Types of Countercurrent Distribution Apparatus and Continuous Flow Chromatography Techniques
5 Other Types of Countercurrent Distribution Apparatus and Continuous Flow Chromatography Techniques IAN A. SUTHERLAND Department of Engineering NationaJ Institute of Medical Research London, United Kingdom
I. II. III. IV. V. VI.
Introduction Enhanced Gravity Countercurrent Distribution Column Chromatography Countercurrent Chromatography Emerging Techniques Commercial Availability References
149 150 152 154 156 157 157
I. INTRODUCTION
Thin-layer countercurrent distribution (CCD) has now become an accepted method of separating material of similar partition coefficient. However, the high labor content of the technique and the long separating times have led to attempts to develop new methods that are more suited to the modern laboratory and eventual automation in industry. These methods fall into two categories: those based on countercurrent distribution (Section II) making use of the Craig discrete mixingsettling-transfer approach, and those based on continuous flow chromatography. The chromatographic methods are also divided into two groups: those using (Section III) and those not using (Section IV) a solid support. Some of the processes described below use centrifugation to speed up the separation of the phase systems. It should be emphasized that 149
PARTITIONING IN AQUEOUS TWO-PHASE SYSTEMS
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Ian A. Sutherland
this is low-speed centrifugation and will only have a significant sedimentation effect on the sample when specifically stated. Only laboratory-scale equipment will be considered in this section. Industrial-scale processes will be discussed in the chapter on biotechnology (Chapter 15). II. ENHANCED GRAVITY COUNTERCURRENT DISTRIBUTION
Pritchard et al. (1975) describe a miniature version of the original stainless steel CCD machine developed by Craig and Post (1949). The device, illustrated in Fig. 1, consists of an upper and a lower section each with 18 circumferentially arranged chambers. Each chamber is filled with the appropriate amount of phase system (top left of figure) and has a small nickel mixing ball added. The sample is added to chamber 1 and the sections are then assembled together with their appropriate seals. The principle of operation is quite simple. First the whole device is gently inverted a number of times to allow the balls to thoroughly mix the phases. The whole device is then centrifuged in a swinging bucket rotor for 5 min at 300 g. After these mixing and centrifugation steps, the upper section is indexed anticlockwise by one cham-
Fig. 1. Diagram of Pritchard CCD device. From Morris and Peters (1982).
5. Methods and Types of Apparatus
151
ber and the whole process repeated 17 times. This method offers a cheap and simple way of performing a few countercurrent distribution steps but it can be labor-intensive, with each run taking about two hr. Ákerlund (1984), on the other hand, has developed a fully automated enhanced-gravity version of Albertsson's thin-layer CCD apparatus (Chapter 4). The major design difference between the machines is that the Ákerlund one rotates at speeds of up to 3000 revolutions/min to separate the phases centrifugally. The transfer plane is therefore vertical as opposed to horizontal. Ákerlund has achieved a transfer step while the rotor is still rotating and hence significantly reduced the cycle time. Mixing is achieved by stopping or slowing down the rotor and using the conventional Albertsson vortex mixing procedure. The advantage of Ákerlund's system is that it is quick (30 sec mixing, 90 sec centrifugation including acceleration and deceleration, giving a total cycle time of 2 min). Also, the use of enhanced gravity allows separations with viscous phase systems that would not settle out in unit gravity (see Chapter 6). The disadvantage of this device is that it is complicated from the engineering point of view and the plates must be extremely accurately machined to avoid leaks between the chambers when the apparatus is spinning. Another quite different and innovative approach is offered by Nakazawa et al. (1978). The device consists of a rotating column separated into chambers of loculi by up to 20 disks. Each chamber contains a number of steel balls to promote mixing. The column can rotate at two speeds: the lower speed is arranged to just tumble the balls and thoroughly mix the phases, while the higher speed is sufficient to hold the balls stationary against the outer wall of the column and separate the phases. The disks dividing each chamber have axial holes to allow transfer of the lighter phase by pumping in a volume equivalent to one chamber through one end of the column via a rotating seal. The major differences between the three devices are summarized in Table I, where their operating conditions are compared to those of an Albertsson unit gravity machine. All the methods are thin-layer ones apart from the Pritchard method, which accounts for its lengthy separation time even at 300 g. The Nakazawa device works at a relatively low acceleration field (3.3 g) and so is the only one suitable for separating cells. This is reflected in the applications of each technique. Both the Pritchard unit (Pritchard et al., 1975; Morris and Peters, 1982; Sutherland et al., 1984) and the Ákerlund unit (Ákerlund, 1984) have been used to fractionate organelles, while the Nakazawa unit (Nakazawa et al., 1978) was used for cells. Ákerlund also demonstrates that enhanced-gravity CCD units can be used for separations based on multi-
152
Ian A. Sutherland TABLE I Operating Conditions of Various CCD Devices
Unit
No. of chambers
Chamber height (cm)
Enhanced gravity (g)
Settling time (min)
Cycle time (min)
Pritchard et al. (1975) Akerlund (1984) Nakazawa et al. (1978) Albertsson (1971)
18 60 13-20 120
2.5 0.2-0.3 0.35-0.45 0.2-0.3
300 100 3.3 Ia
5 1 2 5-10
7-8 2 4 5.5-10.5
a
Unit gravity.
stage sedimentation. He shows that at 100 g polystyrene latex particles of 2.2 μτη diameter hardly sediment at all in distilled water, while particles of 7.6 μ,πι sediment significantly. Considering the relative densities of the materials used in two-phase partition, it is reasonable to assume that devices employing enhanced gravities in excess of 100 g should be used for particle sizes lower than 1 μ,πι if sedimentation effects are to be avoided. III. COLUMN CHROMATOGRAPHY There are three basic forms of chromatographic columns utilizing polymer systems. The major differences are in the way the stationary phase is retained or supported by the column. The first method, which has been extensively reviewed (Albertsson, 1971; Blomquist and Albertsson, 1972; see also Chapter 15, Section II,G,1), uses unit gravity to retain the stationary phase. This is illustrated schematically in Fig. 2a. The column is first filled with a homogeneous mixture containing 50% upper phase and 50% lower phase. When the phases have settled, each mixing chamber (C) will be filled with lower phase and each settling chamber (E) with upper phase. Sieve plates (B) have holes that are sufficiently small to prevent the lower phase from passing through the plate to the chamber below. Sieve plates (D) are designed to limit the mixing zones to (C) but allow heavy phase to return from the settling zones (E). The sample is injected with the mobile phase at (A) and goes through a series of mixing and settling stages before eluting at (F). Flow rates are relatively low to prevent carryover of the stationary phase. These columns form the basis for large-scale industrial separations, but have found little use in laboratory work, as they are slower than thin-layer CCD and have an
5. Methods and Types of Apparatus
a)
C
~\-^
153
b)
c)
Fig. 2. Various chromatography columns used with two-phase polymer systems.
efficiency of about 88%. Most biological material can be processed, except for cells, which tend to adhere to the sieves. The second method uses a solid support (Fig. 2b) which adsorbs one of the phases. Morris (1963) used Celite and synthetic calcium silicates as a solid support for the dextran-rich lower phase. He tested proteins such as lysozyme, ribonuclease, cytochrome c, ovalbumin, and bovine serum albumin, but he found the range of partition values restricting, despite well-defined symmetrical distributions. Later, Müller et al. (1979) used cellulose or Celite as a solid support for the fractionation of DNA fragments. His distribution profiles were also very broad until he developed a way of coating the support particles with a very thin layer of lower phase. He did this by suspending dry cellulose or Celite in the PEG-rich upper phase. The uptake of water from the phase by the support particles disturbed the phase equilibrium, resulting in a fine deposition of lower-phase droplets on the surface of the particles. With careful selection of flow rates and the use of salt gradients, he was able to produce fractionations of double-stranded DNA fragments of 150 to 22,000 base pairs (see Chapter 7). The major drawback of the technique is that it is time-consuming (50-100 hr per fractionation) and requires elevated temperatures for optimum results.
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Ian A. Sutherland
The final method involves the use of chemically bonded polyethylene glycol) (PEG) as the stationary phase. This method (Fig. 2c) has been extensively developed by Matsumoto and Shibusawa. They use a column packing consisting of chemically bonded PEG 20M on Sepharose 6B and an isotonic buffered solution containing dextran T40 or T500 as the mobile phase. They separate human blood cells (Matsumoto and Shibusawa, 1980) and study the influence of mobile phase composition on the retention behavior (Matsumoto and Shibusawa, 1981). They found that the blood cells were eluted from the column in the following order: erythrocytes, platelets, granulocytes, and lymphocytes. This elution order was found to correlate with the results of hydrophobic affinity partition (Matsumoto et al., 1983, 1984) and was independent of cell size, adhesiveness, and surface negative charge. Recoveries were found to be between 50 and 80% with elution times between 6 and 12 hr. It should be noted that the separations in Fig. 2c are probably not based on cell partition phenomena in the sense discussed in this book, because all the PEG is immobilized and cannot distribute freely between the mobile and stationary phases. Since both phases of a Dx/PEG system must always contain a finite amount of PEG (Chapter 3), no phase system can form in the present case. No free liquid interface will be present, therefore. Presumably the bound PEG is still interacting with the dissolved Dx and the suspended cells in a manner similar to that which occurs in the true phase systems, but the detailed effects of polymer concentration, salt type, and so forth are likely to differ in the absence of a liquid interface with a finite interfacial tension. IV. COUNTERCURRENT CHROMATOGRAPHY Countercurrent chromatography (CCC) can be considered as a continuous form of countercurrent distribution or liquid-liquid chromatography without a solid support. Two types of apparatus exist for countercurrent chromatography with polymer phase systems: the toroidal coil centrifuge and the nonsynchronous coil planet centrifuge. The toroidal coil consists of a helically wound polytetrafluoroethylene coil mounted circumferentially on a rotating disk (Fig. 3). The coil is initially filled, while stationary, with the Dx-rich phase. The plate is then rotated at 1000 rpm while PEG-rich mobile phase is pumped in. Centrifugal force retains the heavier Dx-rich phase in the outer half of each coil unit, while the PEG-rich mobile phase progressively displaces the Dx-rich phase from the inner half of each coil unit. Continuous
5. Methods and Types of Apparatus
155
Fig. 3. Schematic layout of toroidal coil operating system. Reprinted from Sutherland et al. (1984), p. 370, by courtesy of Marcel Dekker, Inc.
pumping of the mobile phase sets up a series of cascades (much like waterfalls) of the PEG-rich phase through the retained segments of the heavier Dx-rich phase in each coil unit. The sample is injected with the mobile phase by using a conventional liquid chromatography sample loop and undergoes a series of mixing and settling steps before it eventually elutes to the fraction collector (Fig. 3). Sample components partitioning toward the PEG-rich mobile phase will elute early while components favoring the Dx-rich phase or interface will be retained in a way similar to the chromatographic processes described in Section III. However, as there is no solid support, either phase, or even a mixture of the two, can be used as the mobile phase. Adding a small proportion of the Dx-rich phase in the
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Ian A. Sutherland
above example would speed up the elution of all the retained components and clear the coil system for another sample loading. Applications of the toroidal coil have included bacteria (Sutherland and Ito, 1978), rat liver organelles (Heywood-Waddington et al., 1984; Sutherland et al., 1984), and the use of affinity partition to separate nicotinic cholinergic receptors (Flanagan et al., 1984; see Chapter 8) with phase systems both near and far from the critical point. Fractionations with a 550 coil-unit device give the equivalent of 60-100 transfers of the Treffry Bioshef unit gravity thin-layer CCD machine in a third of the time (I. A. Sutherland et al, 1985). Sutherland et al. (1984) have concluded that mixing is limited by constraints of the coil geometry and that higher resolution will be achieved only by increasing the number of coils. All toroidal coil rotors have a system which avoids the use of rotating seals. This helps to give recoveries in the order of 70100% (Heywood-Waddington et al, 1984). The toroidal coil system is ideal for fractionating samples with particle diameters less than 1 /xm, but can lead to sedimentation effects if the particles are larger. The nonsynchronous coil planet centrifuge works on exactly the same principle as the toroidal coil except that the coils are not stationary but slowly rotate relative to the acceleration vector. This has the effect of enhancing mixing of the phases in each coil unit (there will be mixing zones on each side of the helical coil due to the rotation) and causing particulate material to move in small circles, thus avoiding sedimentation. Nonsynchronous coil planet centrifuges have been used successfully to separate erythrocytes (Sutherland and Ito, 1980) and Salmonella typhimurium (Ito et al., 1983; Leive et al., 1984). However, these devices are quite complicated to build and results are difficult to reproduce from one machine to another, particularly when different phase systems are used (Harris et aJ., 1984).
V. EMERGING TECHNIQUES
The recent development of the multilayer coil planet centrifuge (Ito et al., 1982) capable of both analytical and preparative countercurrent chromatography with aqueous/organic phase systems with up to three times the resolution in a quarter of the time, has led to speculation concerning similar developments with polymer phase systems. The coils undergo an epicyclic motion, described in detail by Ito (1981), whereby one side of the multilayer coil is at high g, giving phase sepa-
5. Methods and Types of Apparatus
157
ration, and the other side is at low g (the cusp of the epicyclic motion), causing phase mixing. A fractionation of rat liver organelles has already been successfully demonstrated with toroidal coils wound on this device and further developments are in progress (I. A. Sutherland et al., 1985). VI. COMMERCIAL AVAILABILITY
One feature common to all the devices reviewed above is that, with few exceptions, they have not been widely used for a range of different applications and none of them is yet commercially available. Manufacturers are unlikely to take up new ideas unless they are well proven and scientists cannot use these techniques until they become commercially available, unless of course they have a well-equipped engineering facility at hand to make their own. This is the renowned chicken-and-egg situation that frequently delays the development of new technology. However, thanks to the pioneering work of the authors reviewed above, there are a number of new ideas that could potentially benefit the user. There is clearly a need for a simple, low-cost, enhanced-gravity CCD device, much along the lines of the Pritchard one but with a reduced cycle time. With such a device, exploratory CCD could be performed before investing time and effort in using the higher resolution techniques. Countercurrent chromatography has been used for the widest range of applications, but the rotors are complicated to build, particularly the nonsynchronous one used for cell separation. While both chromatographic techniques are easy to use and are suited to automation, CCC has the distinct advantage of having no solid support, minimizing adsorption problems and reducing running costs. If the multilayer coil planet centrifuge (P.C. Inc., Potomac, Maryland), which is commercially available for CCC with aqueous/organic phase systems, proves to be suitable for use with polymer phase systems, there could develop a competition between CCC and column development that could stimulate the commercial interest that is so urgently needed. REFERENCES Akerlund, H.-E. (1984). An apparatus for counter-current distribution in a centrifugal acceleration field. /. Biochem. Biophys. Methods 9, 133-141. Albertsson, P.-A. (1971). "Partition of Cell Particles and Macromolecules," 2nd ed. Almqvist & Wiksell, Stockholm; Wiley (Interscience), New York.
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Blomquist, G., and Albertsson, P.-Á. (1972). A study of extraction columns for aqueous polymer two-phase systems. /. Chromatogr. 73, 125-133. Craig, L. C, and Post, H. O. (1949). Apparatus for countercurrent distribution. Anal. Chem. 21, 500-504. Flanagan, S. D., Johansson, G., Yost, B., Ito, Y., and Sutherland, I. A. (1984). Toroidal coil countercurrent chromatography in the affinity partitioning of nicotinic cholinergic receptor enriched membranes. /. Liq. Chromatogr. 7, 385-402. Harris, J. M., Case, M. G., Snyder, R. S., and Chenault, A. A. (1984). Cell separations on the countercurrent chromatograph. /. Liq. Chromatogr. 7, 419-431. Heywood-Waddington, D., Sutherland, I. A., Morris, W. B., and Peters, T. J. (1984). Subcellular fractionation of rat liver homogenates using two-polymer systems in a toroidal-coil centrifuge. Biochem. /. 217, 751-759. Ito, Y. (1981). Countercurrent chromatography. /. Biochem. Biophys. Methods 5, 105129. Ito, Y., Sandlin, J., and Bowers, W. G. (1982). High-speed preparative counter-current chromatography with a coil planet centrifuge. /. Chromatogr. 244, 247-258. Ito, Y., Bramblett, G. T., Bhatnagar, R., Humberman, M., Leive, L. L., Cullinane, L. M., and Groves, W. (1983). Improved nonsynchronous flow-through coil planet centrifuge without rotating seals: Principle and application. Sep. Sci. Technol. 18, 33-48. Leive, L., Cullinane, M., Ito, Y., and Bramblett, G. T. (1984). Countercurrent chromatographic separation of bacteria with known differences in surface lipopolysaccharide. /. Liq. Chromatogr. 7, 403-418. Matsumoto, U., and Shibusawa, Y. (1980). Surface affinity chromatographic separation of blood cells. I. Separation of human and rabbit peripheral granulocytes, lymphocytes and erythrocytes using polyethylene glycol-bonded column packings. J. Chromatogr. 187, 351-362. Matsumoto, U., and Shibusawa, Y. (1981). Surface affinity chromatographic separation of blood cells. II. Influence of mobile phase composition on the chromatographic behaviour of human peripheral blood cells on polyethylene glycol-bonded Sepharose. J. Chromatogr. 206, 17-25. Matsumoto, U., Shibusawa, Y., and Tanaka, Y. (1983). Surface affinity chromatographic separation of blood cells. III. Effect of molecular weight of polyethylene glycol bonded stationary phases on elution behaviour of human blood cells. /. Chromatogr. 268, 375-386. Matsumoto, U., Ban, M., and Shibusawa, Y. (1984). Surface affinity chromatographic separation of blood cells. IV. Relationship between surface hydrophobicity of human peripheral blood cells and their retention behaviour on polyethylene glycol 20M-bonded Sepharose columns. /. Chromatogr. 285, 69-79. Morris, C. J. O. R. (1963). A new method of protein chromatography. Protides Biol. Fluids 10, 325-328. Morris, W. B., and Peters, T. J. (1982). Micro analytical partition of rat liver homogenates by polyfethylene glycolj-dextran counter-current distribution. Eur. /. Biochem. 121, 421-426. Müller, W., Schuetz, H.-J., Guerrier-Takada, C, Cole, P. E., and Potts, R. (1979). Size fractionation of DNA fragments by liquid-liquid chromatography. NucJeic Acids Res. 7, 2483-2499. Nakazawa, H., Tanimura, T., and Tamura, Z. (1978). A device for countercurrent distribution of particles by an aqueous polymer two-phase system. Sep. Sci. Technol. 13, 745-752.
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Pritchard, G. D., Halpern, R. M., Halpern, J. A., Halpern, B. C, and Smith, R. A. (1975). Fractionation of mucopolysaccharides by countercurrent distribution in aqueous polymer two-phase systems. Biochim. Biophys. Acta 404, 289-299. Sutherland, I. A., and Ito, Y. (1978). Toroidal coil chromatography. A new high-speed, high-resolution method of separating cells and cell organelles on their distribution in two-phase polymer systems. HRC CC, ]. High Resolut. Chromatogr. Chromatogr. Commun. 3, 171-172. Sutherland, I. A., and Ito, Y. (1980). Cell separation using two-phase polymer systems in a nonsynchronous flow through coil planet centrifuge. Anal. Biochem. 108, 367373. Sutherland, I. A., Heywood-Waddington, D., and Peters, T. J. (1984). Toroidal coil countercurrent chromatography: A fast simple alternative to countercurrent distribution using aqueous two phase partition. J. Liq. Chromatogr. 7, 363-384. Sutherland, I. A., Heywood-Waddington, D., and Peters, T. J. (1985). Countercurrent chromatography using a toroidal coil planet centrifuge: A comparative study of the separation of organelles using aqueous two-phase partition. /. Liq. Chromatogr. 8(12), 2315-2335.
I. II. III. IV. V. VI.
Introduction Enhanced Gravity Countercurrent Distribution Column Chromatography Countercurrent Chromatography Emerging Techniques Commercial Availability References
149 150 152 154 156 157 157
I. INTRODUCTION
Thin-layer countercurrent distribution (CCD) has now become an accepted method of separating material of similar partition coefficient. However, the high labor content of the technique and the long separating times have led to attempts to develop new methods that are more suited to the modern laboratory and eventual automation in industry. These methods fall into two categories: those based on countercurrent distribution (Section II) making use of the Craig discrete mixingsettling-transfer approach, and those based on continuous flow chromatography. The chromatographic methods are also divided into two groups: those using (Section III) and those not using (Section IV) a solid support. Some of the processes described below use centrifugation to speed up the separation of the phase systems. It should be emphasized that 149
PARTITIONING IN AQUEOUS TWO-PHASE SYSTEMS
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
150
Ian A. Sutherland
this is low-speed centrifugation and will only have a significant sedimentation effect on the sample when specifically stated. Only laboratory-scale equipment will be considered in this section. Industrial-scale processes will be discussed in the chapter on biotechnology (Chapter 15). II. ENHANCED GRAVITY COUNTERCURRENT DISTRIBUTION
Pritchard et al. (1975) describe a miniature version of the original stainless steel CCD machine developed by Craig and Post (1949). The device, illustrated in Fig. 1, consists of an upper and a lower section each with 18 circumferentially arranged chambers. Each chamber is filled with the appropriate amount of phase system (top left of figure) and has a small nickel mixing ball added. The sample is added to chamber 1 and the sections are then assembled together with their appropriate seals. The principle of operation is quite simple. First the whole device is gently inverted a number of times to allow the balls to thoroughly mix the phases. The whole device is then centrifuged in a swinging bucket rotor for 5 min at 300 g. After these mixing and centrifugation steps, the upper section is indexed anticlockwise by one cham-
Fig. 1. Diagram of Pritchard CCD device. From Morris and Peters (1982).
5. Methods and Types of Apparatus
151
ber and the whole process repeated 17 times. This method offers a cheap and simple way of performing a few countercurrent distribution steps but it can be labor-intensive, with each run taking about two hr. Ákerlund (1984), on the other hand, has developed a fully automated enhanced-gravity version of Albertsson's thin-layer CCD apparatus (Chapter 4). The major design difference between the machines is that the Ákerlund one rotates at speeds of up to 3000 revolutions/min to separate the phases centrifugally. The transfer plane is therefore vertical as opposed to horizontal. Ákerlund has achieved a transfer step while the rotor is still rotating and hence significantly reduced the cycle time. Mixing is achieved by stopping or slowing down the rotor and using the conventional Albertsson vortex mixing procedure. The advantage of Ákerlund's system is that it is quick (30 sec mixing, 90 sec centrifugation including acceleration and deceleration, giving a total cycle time of 2 min). Also, the use of enhanced gravity allows separations with viscous phase systems that would not settle out in unit gravity (see Chapter 6). The disadvantage of this device is that it is complicated from the engineering point of view and the plates must be extremely accurately machined to avoid leaks between the chambers when the apparatus is spinning. Another quite different and innovative approach is offered by Nakazawa et al. (1978). The device consists of a rotating column separated into chambers of loculi by up to 20 disks. Each chamber contains a number of steel balls to promote mixing. The column can rotate at two speeds: the lower speed is arranged to just tumble the balls and thoroughly mix the phases, while the higher speed is sufficient to hold the balls stationary against the outer wall of the column and separate the phases. The disks dividing each chamber have axial holes to allow transfer of the lighter phase by pumping in a volume equivalent to one chamber through one end of the column via a rotating seal. The major differences between the three devices are summarized in Table I, where their operating conditions are compared to those of an Albertsson unit gravity machine. All the methods are thin-layer ones apart from the Pritchard method, which accounts for its lengthy separation time even at 300 g. The Nakazawa device works at a relatively low acceleration field (3.3 g) and so is the only one suitable for separating cells. This is reflected in the applications of each technique. Both the Pritchard unit (Pritchard et al., 1975; Morris and Peters, 1982; Sutherland et al., 1984) and the Ákerlund unit (Ákerlund, 1984) have been used to fractionate organelles, while the Nakazawa unit (Nakazawa et al., 1978) was used for cells. Ákerlund also demonstrates that enhanced-gravity CCD units can be used for separations based on multi-
152
Ian A. Sutherland TABLE I Operating Conditions of Various CCD Devices
Unit
No. of chambers
Chamber height (cm)
Enhanced gravity (g)
Settling time (min)
Cycle time (min)
Pritchard et al. (1975) Akerlund (1984) Nakazawa et al. (1978) Albertsson (1971)
18 60 13-20 120
2.5 0.2-0.3 0.35-0.45 0.2-0.3
300 100 3.3 Ia
5 1 2 5-10
7-8 2 4 5.5-10.5
a
Unit gravity.
stage sedimentation. He shows that at 100 g polystyrene latex particles of 2.2 μτη diameter hardly sediment at all in distilled water, while particles of 7.6 μ,πι sediment significantly. Considering the relative densities of the materials used in two-phase partition, it is reasonable to assume that devices employing enhanced gravities in excess of 100 g should be used for particle sizes lower than 1 μ,πι if sedimentation effects are to be avoided. III. COLUMN CHROMATOGRAPHY There are three basic forms of chromatographic columns utilizing polymer systems. The major differences are in the way the stationary phase is retained or supported by the column. The first method, which has been extensively reviewed (Albertsson, 1971; Blomquist and Albertsson, 1972; see also Chapter 15, Section II,G,1), uses unit gravity to retain the stationary phase. This is illustrated schematically in Fig. 2a. The column is first filled with a homogeneous mixture containing 50% upper phase and 50% lower phase. When the phases have settled, each mixing chamber (C) will be filled with lower phase and each settling chamber (E) with upper phase. Sieve plates (B) have holes that are sufficiently small to prevent the lower phase from passing through the plate to the chamber below. Sieve plates (D) are designed to limit the mixing zones to (C) but allow heavy phase to return from the settling zones (E). The sample is injected with the mobile phase at (A) and goes through a series of mixing and settling stages before eluting at (F). Flow rates are relatively low to prevent carryover of the stationary phase. These columns form the basis for large-scale industrial separations, but have found little use in laboratory work, as they are slower than thin-layer CCD and have an
5. Methods and Types of Apparatus
a)
C
~\-^
153
b)
c)
Fig. 2. Various chromatography columns used with two-phase polymer systems.
efficiency of about 88%. Most biological material can be processed, except for cells, which tend to adhere to the sieves. The second method uses a solid support (Fig. 2b) which adsorbs one of the phases. Morris (1963) used Celite and synthetic calcium silicates as a solid support for the dextran-rich lower phase. He tested proteins such as lysozyme, ribonuclease, cytochrome c, ovalbumin, and bovine serum albumin, but he found the range of partition values restricting, despite well-defined symmetrical distributions. Later, Müller et al. (1979) used cellulose or Celite as a solid support for the fractionation of DNA fragments. His distribution profiles were also very broad until he developed a way of coating the support particles with a very thin layer of lower phase. He did this by suspending dry cellulose or Celite in the PEG-rich upper phase. The uptake of water from the phase by the support particles disturbed the phase equilibrium, resulting in a fine deposition of lower-phase droplets on the surface of the particles. With careful selection of flow rates and the use of salt gradients, he was able to produce fractionations of double-stranded DNA fragments of 150 to 22,000 base pairs (see Chapter 7). The major drawback of the technique is that it is time-consuming (50-100 hr per fractionation) and requires elevated temperatures for optimum results.
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Ian A. Sutherland
The final method involves the use of chemically bonded polyethylene glycol) (PEG) as the stationary phase. This method (Fig. 2c) has been extensively developed by Matsumoto and Shibusawa. They use a column packing consisting of chemically bonded PEG 20M on Sepharose 6B and an isotonic buffered solution containing dextran T40 or T500 as the mobile phase. They separate human blood cells (Matsumoto and Shibusawa, 1980) and study the influence of mobile phase composition on the retention behavior (Matsumoto and Shibusawa, 1981). They found that the blood cells were eluted from the column in the following order: erythrocytes, platelets, granulocytes, and lymphocytes. This elution order was found to correlate with the results of hydrophobic affinity partition (Matsumoto et al., 1983, 1984) and was independent of cell size, adhesiveness, and surface negative charge. Recoveries were found to be between 50 and 80% with elution times between 6 and 12 hr. It should be noted that the separations in Fig. 2c are probably not based on cell partition phenomena in the sense discussed in this book, because all the PEG is immobilized and cannot distribute freely between the mobile and stationary phases. Since both phases of a Dx/PEG system must always contain a finite amount of PEG (Chapter 3), no phase system can form in the present case. No free liquid interface will be present, therefore. Presumably the bound PEG is still interacting with the dissolved Dx and the suspended cells in a manner similar to that which occurs in the true phase systems, but the detailed effects of polymer concentration, salt type, and so forth are likely to differ in the absence of a liquid interface with a finite interfacial tension. IV. COUNTERCURRENT CHROMATOGRAPHY Countercurrent chromatography (CCC) can be considered as a continuous form of countercurrent distribution or liquid-liquid chromatography without a solid support. Two types of apparatus exist for countercurrent chromatography with polymer phase systems: the toroidal coil centrifuge and the nonsynchronous coil planet centrifuge. The toroidal coil consists of a helically wound polytetrafluoroethylene coil mounted circumferentially on a rotating disk (Fig. 3). The coil is initially filled, while stationary, with the Dx-rich phase. The plate is then rotated at 1000 rpm while PEG-rich mobile phase is pumped in. Centrifugal force retains the heavier Dx-rich phase in the outer half of each coil unit, while the PEG-rich mobile phase progressively displaces the Dx-rich phase from the inner half of each coil unit. Continuous
5. Methods and Types of Apparatus
155
Fig. 3. Schematic layout of toroidal coil operating system. Reprinted from Sutherland et al. (1984), p. 370, by courtesy of Marcel Dekker, Inc.
pumping of the mobile phase sets up a series of cascades (much like waterfalls) of the PEG-rich phase through the retained segments of the heavier Dx-rich phase in each coil unit. The sample is injected with the mobile phase by using a conventional liquid chromatography sample loop and undergoes a series of mixing and settling steps before it eventually elutes to the fraction collector (Fig. 3). Sample components partitioning toward the PEG-rich mobile phase will elute early while components favoring the Dx-rich phase or interface will be retained in a way similar to the chromatographic processes described in Section III. However, as there is no solid support, either phase, or even a mixture of the two, can be used as the mobile phase. Adding a small proportion of the Dx-rich phase in the
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above example would speed up the elution of all the retained components and clear the coil system for another sample loading. Applications of the toroidal coil have included bacteria (Sutherland and Ito, 1978), rat liver organelles (Heywood-Waddington et al., 1984; Sutherland et al., 1984), and the use of affinity partition to separate nicotinic cholinergic receptors (Flanagan et al., 1984; see Chapter 8) with phase systems both near and far from the critical point. Fractionations with a 550 coil-unit device give the equivalent of 60-100 transfers of the Treffry Bioshef unit gravity thin-layer CCD machine in a third of the time (I. A. Sutherland et al, 1985). Sutherland et al. (1984) have concluded that mixing is limited by constraints of the coil geometry and that higher resolution will be achieved only by increasing the number of coils. All toroidal coil rotors have a system which avoids the use of rotating seals. This helps to give recoveries in the order of 70100% (Heywood-Waddington et al, 1984). The toroidal coil system is ideal for fractionating samples with particle diameters less than 1 /xm, but can lead to sedimentation effects if the particles are larger. The nonsynchronous coil planet centrifuge works on exactly the same principle as the toroidal coil except that the coils are not stationary but slowly rotate relative to the acceleration vector. This has the effect of enhancing mixing of the phases in each coil unit (there will be mixing zones on each side of the helical coil due to the rotation) and causing particulate material to move in small circles, thus avoiding sedimentation. Nonsynchronous coil planet centrifuges have been used successfully to separate erythrocytes (Sutherland and Ito, 1980) and Salmonella typhimurium (Ito et al., 1983; Leive et al., 1984). However, these devices are quite complicated to build and results are difficult to reproduce from one machine to another, particularly when different phase systems are used (Harris et aJ., 1984).
V. EMERGING TECHNIQUES
The recent development of the multilayer coil planet centrifuge (Ito et al., 1982) capable of both analytical and preparative countercurrent chromatography with aqueous/organic phase systems with up to three times the resolution in a quarter of the time, has led to speculation concerning similar developments with polymer phase systems. The coils undergo an epicyclic motion, described in detail by Ito (1981), whereby one side of the multilayer coil is at high g, giving phase sepa-
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ration, and the other side is at low g (the cusp of the epicyclic motion), causing phase mixing. A fractionation of rat liver organelles has already been successfully demonstrated with toroidal coils wound on this device and further developments are in progress (I. A. Sutherland et al., 1985). VI. COMMERCIAL AVAILABILITY
One feature common to all the devices reviewed above is that, with few exceptions, they have not been widely used for a range of different applications and none of them is yet commercially available. Manufacturers are unlikely to take up new ideas unless they are well proven and scientists cannot use these techniques until they become commercially available, unless of course they have a well-equipped engineering facility at hand to make their own. This is the renowned chicken-and-egg situation that frequently delays the development of new technology. However, thanks to the pioneering work of the authors reviewed above, there are a number of new ideas that could potentially benefit the user. There is clearly a need for a simple, low-cost, enhanced-gravity CCD device, much along the lines of the Pritchard one but with a reduced cycle time. With such a device, exploratory CCD could be performed before investing time and effort in using the higher resolution techniques. Countercurrent chromatography has been used for the widest range of applications, but the rotors are complicated to build, particularly the nonsynchronous one used for cell separation. While both chromatographic techniques are easy to use and are suited to automation, CCC has the distinct advantage of having no solid support, minimizing adsorption problems and reducing running costs. If the multilayer coil planet centrifuge (P.C. Inc., Potomac, Maryland), which is commercially available for CCC with aqueous/organic phase systems, proves to be suitable for use with polymer phase systems, there could develop a competition between CCC and column development that could stimulate the commercial interest that is so urgently needed. REFERENCES Akerlund, H.-E. (1984). An apparatus for counter-current distribution in a centrifugal acceleration field. /. Biochem. Biophys. Methods 9, 133-141. Albertsson, P.-A. (1971). "Partition of Cell Particles and Macromolecules," 2nd ed. Almqvist & Wiksell, Stockholm; Wiley (Interscience), New York.
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Blomquist, G., and Albertsson, P.-Á. (1972). A study of extraction columns for aqueous polymer two-phase systems. /. Chromatogr. 73, 125-133. Craig, L. C, and Post, H. O. (1949). Apparatus for countercurrent distribution. Anal. Chem. 21, 500-504. Flanagan, S. D., Johansson, G., Yost, B., Ito, Y., and Sutherland, I. A. (1984). Toroidal coil countercurrent chromatography in the affinity partitioning of nicotinic cholinergic receptor enriched membranes. /. Liq. Chromatogr. 7, 385-402. Harris, J. M., Case, M. G., Snyder, R. S., and Chenault, A. A. (1984). Cell separations on the countercurrent chromatograph. /. Liq. Chromatogr. 7, 419-431. Heywood-Waddington, D., Sutherland, I. A., Morris, W. B., and Peters, T. J. (1984). Subcellular fractionation of rat liver homogenates using two-polymer systems in a toroidal-coil centrifuge. Biochem. /. 217, 751-759. Ito, Y. (1981). Countercurrent chromatography. /. Biochem. Biophys. Methods 5, 105129. Ito, Y., Sandlin, J., and Bowers, W. G. (1982). High-speed preparative counter-current chromatography with a coil planet centrifuge. /. Chromatogr. 244, 247-258. Ito, Y., Bramblett, G. T., Bhatnagar, R., Humberman, M., Leive, L. L., Cullinane, L. M., and Groves, W. (1983). Improved nonsynchronous flow-through coil planet centrifuge without rotating seals: Principle and application. Sep. Sci. Technol. 18, 33-48. Leive, L., Cullinane, M., Ito, Y., and Bramblett, G. T. (1984). Countercurrent chromatographic separation of bacteria with known differences in surface lipopolysaccharide. /. Liq. Chromatogr. 7, 403-418. Matsumoto, U., and Shibusawa, Y. (1980). Surface affinity chromatographic separation of blood cells. I. Separation of human and rabbit peripheral granulocytes, lymphocytes and erythrocytes using polyethylene glycol-bonded column packings. J. Chromatogr. 187, 351-362. Matsumoto, U., and Shibusawa, Y. (1981). Surface affinity chromatographic separation of blood cells. II. Influence of mobile phase composition on the chromatographic behaviour of human peripheral blood cells on polyethylene glycol-bonded Sepharose. J. Chromatogr. 206, 17-25. Matsumoto, U., Shibusawa, Y., and Tanaka, Y. (1983). Surface affinity chromatographic separation of blood cells. III. Effect of molecular weight of polyethylene glycol bonded stationary phases on elution behaviour of human blood cells. /. Chromatogr. 268, 375-386. Matsumoto, U., Ban, M., and Shibusawa, Y. (1984). Surface affinity chromatographic separation of blood cells. IV. Relationship between surface hydrophobicity of human peripheral blood cells and their retention behaviour on polyethylene glycol 20M-bonded Sepharose columns. /. Chromatogr. 285, 69-79. Morris, C. J. O. R. (1963). A new method of protein chromatography. Protides Biol. Fluids 10, 325-328. Morris, W. B., and Peters, T. J. (1982). Micro analytical partition of rat liver homogenates by polyfethylene glycolj-dextran counter-current distribution. Eur. /. Biochem. 121, 421-426. Müller, W., Schuetz, H.-J., Guerrier-Takada, C, Cole, P. E., and Potts, R. (1979). Size fractionation of DNA fragments by liquid-liquid chromatography. NucJeic Acids Res. 7, 2483-2499. Nakazawa, H., Tanimura, T., and Tamura, Z. (1978). A device for countercurrent distribution of particles by an aqueous polymer two-phase system. Sep. Sci. Technol. 13, 745-752.
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Pritchard, G. D., Halpern, R. M., Halpern, J. A., Halpern, B. C, and Smith, R. A. (1975). Fractionation of mucopolysaccharides by countercurrent distribution in aqueous polymer two-phase systems. Biochim. Biophys. Acta 404, 289-299. Sutherland, I. A., and Ito, Y. (1978). Toroidal coil chromatography. A new high-speed, high-resolution method of separating cells and cell organelles on their distribution in two-phase polymer systems. HRC CC, ]. High Resolut. Chromatogr. Chromatogr. Commun. 3, 171-172. Sutherland, I. A., and Ito, Y. (1980). Cell separation using two-phase polymer systems in a nonsynchronous flow through coil planet centrifuge. Anal. Biochem. 108, 367373. Sutherland, I. A., Heywood-Waddington, D., and Peters, T. J. (1984). Toroidal coil countercurrent chromatography: A fast simple alternative to countercurrent distribution using aqueous two phase partition. J. Liq. Chromatogr. 7, 363-384. Sutherland, I. A., Heywood-Waddington, D., and Peters, T. J. (1985). Countercurrent chromatography using a toroidal coil planet centrifuge: A comparative study of the separation of organelles using aqueous two-phase partition. /. Liq. Chromatogr. 8(12), 2315-2335.