ANALYTICAL
BIOCHEMISTRY
Angle
Rotor YOICHIRO
Laboratory
of Technical
65, 310-320 (1975)
Countercurrent ITO AND
ROBERT
Chromatography L.
BOWMAN
Development, National Heart Bethesda, Maryland 20014
and Lung
Institute,
Received September 30, 1974; accepted November 22, 1974 A new rotor for performing countercurrent chromatography holds a coiled helix column at 30” from the vertical. Versatility of the angle rotor is demonstrated on high-efficiency separations of biological compounds using a variety of two-phase solvent systems. Advantages of the angle rotor over other centrifugal countercurrent chromatographic schemes are discussed.
Previously two countercurrent chromatographic schemes have been reported, both utilizing a coiled tube in a centrifugal field. One is the flow-through coil planet centrifuge ( 1-5) with a vertical rotor and the other, the elution centrifuge (5-7) with a horizontal rotor. Experiments with various two-phase solvent systems have disclosed specific features inherent to each scheme. The advantage of the vertical rotor is that a high-efficiency partitioning is achieved with a short separation time by providing a vigorous phase mixing. However, it fails to retain the stationary phase of some low inter-facial tension phase systems, such as polymer phase systems, due to intensive emulsification. On the other hand, the horizontal rotor provides a stable centrifugal force field to retain polymer phase systems but a longer separation time is generally required due to increased mass transfer resistance. Since these different features are apparently derived from the difference in rotor angle between two schemes, there may exist an optimum rotor angle between 0” (vertical) and 90” (horizontal) that permits universal application of the solvent systems with a high partition efficiency. Efforts have been made to determine the optimum rotor angle with which capability of the method is demonstrated on separations using a variety of two-phase solvent systems having different physical properties of inter-facial tension, viscosity, and density difference between two phases. APPARATUS
Two angle rotors have been constructed by modifying a conventional refrigerated centrifuge (MSE, Model LR-6). Both rotors were designed, as in the previous schemes, to produce a counterrotation of the column 310 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
COUNTERCURRENT
CHROMATOGRAPHY
311
holder to establish a continuous flow-through system without rotating seals. (1) Forty-jive degree angle rotor. The first rotor was made at a fixed angle of 45” as follows: A cylindrical hollow aluminum column holder was supported by a pair of aluminum blocks equipped with bearings, which were then sandwiched by a pair of aluminum side plates to support the column holder at 45” with an accurate bearing alignment. To prevent twisting the flow tubes counterrotation of the holder was established by coupling a pair of toothed pulleys of equal diameter, one fixed at the middle portion of the holder and the other, on the motor housing at the center of centrifuge drive. A pair of idler pulleys mounted at the bottom plate of the rotor serves for changing direction of the running belt at 45”. The dummy side of the rotor was made similarly to facilitate accurate counterbalancing of the rotor. The revolutional speed was continuously regulated from 400 to 1500 rpm at a mean revolutional radius of 20 cm. The performance of the 45” rotor revealed several items to be improved. At a high revolutional speed, the bearings suffered from loss of lubricant through the seal resulting in overheating and wearing out. Temperature inside the column holder also rose considerably higher than regulated ambient temperature due to heat conduction from the bearing through the holder wall. After a few weeks of operation, the column holder began to rotate against the inner race of each bearing which resulted in wearing out the holder wall at the bearing sites. In the design of the second prototype described, efforts have been made to eliminate these problems. (2) Adjustable angle rotor (30”, 20”, and IO’). Since the 45” angle rotor described above permits a satisfactory phase retention of the polymer phase systems, the critical rotor angle for phase retention must be present between 0” and 45” from the vertical. Further investigation has been performed to determine the optimum rotor angle by constructing an adjustable angle rotor which provides 30”, 20”, or 10” from the vertical by a simple adjustment. Figure 1 shows the design of adjustable angle rotor fixed at the 30” position. A pair of bearing blocks (a), holding the aluminum column holder (b) (32 mm i.d. and 25 cm long) on each side of the rotor, is sandwiched between a pair of rectangular aluminum plates (c) each equipped at the middle with a pin (d) that fits into the pin hole (d) made in the side plate (e) of the rotor. This arrangement allows the column holder blocks to be rotated around the pins without disturbing the bearing alignment and then bolted to the side plates at a selected angle. A toothed pulley (f) mounted on the column holder is coupled to a stationary pulley (g) of equal diameter on the axis of the centrifuge through a toothed belt (h) by the aid of a pair of idler pulleys (i) mounted on the bottom plate (j) of the rotor. This coupling es-
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FIG. 1. Diagram of the adjustable angle rotor fixed at the 30” position. a: bearing block, b: column holder, c: side plate of column holder block, d: pin and pin hole in side plate, e: side plate of rotor, f: toothed pulley on column holder, g: stationary toothed pulley, h: toothed belt, i: idler pulley, j: bottom plate of rotor, k: ball bearing, 1: bakelite pipe, m: side hole in column holder, n: column support carrying a coiled helix column, o: feed and return tubes, Rotor angle is adjusted to 30”, 20”, or 10” from the vertical by rotating each column holder block around the pin hole (d) and bolting to the side plates (e). Counterrotation of the column holder is established by coupling pulleys if) and fg) with a toothed belt (h) to prevent twisting of the flow tubes (0). Idler pulleys (i) change direction of the running belt fh).
tablishes counterrotation of the column holder to prevent twisting of flow tubes (0). The positions of the pulley (f) on the coiumn holder and idler pulleys (i) are also adjusted for each angle to provide an adequate tension to the running belt (h). In the light of test results obtained by the 45” angle rotor, several design items were improved. Properly sealed ball bearings (k) (Moffatt Bearings Co., Cat. No. 209-SZZ) were employed, which can hold lubricant much longer and permit easy lubrication with a syringe needle inserted between the inner ring of the bearing and the plastic seal. Between the column holder and the inner race of each bearing, a piece of bakelite tube (1) was tightly placed to minimize heat conduction and also prevent rotation of the column holder against the bearings. In order to cool the column, several side holes (m) were made at the top and bottom of the column holder to allow air flow inside the holder during a run. A cylindrical column support (n) made of delrin rod was tightly secured at the top and bottom of the holder to prevent vibration during operation, Two types of column configurations were employed. The straight helix column was prepared by winding Teflon tubing onto a rigid plastic rod to make a column unit. Multiple column units were interconnected in series and tightly mounted in parallel onto the column support.
COUNTERCURRENT
313
CHROMATOGRAPHY
The coiled helix column (shown in Fig. I) was prepared by winding Teflon tubing onto a flexible core which in turn was tightly coiled around the column support. In order to minimize any adverse effect of the Coriolis force (6) previously described, these columns were placed within 1.3 cm from the central axis of the column holder with a mean revolutional radius of 21 cm. Both feed and return tubes (0) were passed through the center hole of the column support and then held tightly at the center of the upper member of the centrifuge. The flow tubes served several months of operation, when protected with a piece of greased silicone rubber tube at each supported end. Performance of the apparatus has been evaluated at revolutional speeds ranging between 500 and 1300 rpm during a period of 6 mo. Leakage of lubricant from the bearing seal was much reduced and weekly lub~cation seems satisfactory. The column temperature became close to the controlled ambient temperature, and maximum rise no more than a few degrees in centigrade after several hours of operation. DETERMINATION
OF THE OPTIMUM
ROTOR ANGLE
Both straight and coiled helix columns were prepared from two sizes of Teflon tubing (Zeus Industrial Products, Inc., Raritan, NJ), 0.55 mm and 0.3 mm i.d. for examination. The polymer phase system composed of 4% polyethylene glycol 6000, 5% dextran T 500, and 0.1 M sodium phosphate (pH 7) was mainly used, but two other phase systems, set-BuOH/aqueous dichloroacetic acid and n-BuOHIO.01 M NaCl containing 1% cetylpyridinium chloride (CPCl), were also chosen for study because of their specific physical properties. Set-BuOH system has a low interfacial tension but can be retained by a 60” coiled helix column configuration with the vertical rotor. The n-BuOHlaqueous CPCl system shows a high tendency of emulsification and a complete phase separation requires a few hours in a separatory funnel. The polymer phase systems have extremely low inter-facial tension, high viscosity, and small density differences between two phases. Because of these properties, the vertical rotor failed to retain the latter two phase systems even with the 60” flat coiled helix column configuration described earlier (4). In each experiment, the column was filled with the stationary phase and the mobile phase was introduced at various flow rates while the rotor was run at a constant speed at 25°C until the mobile phase was eluted through the column. Percentage of the stationary phase volume retained within the column was calculated from the eluted stationary phase volume and the capacities of the column and the dead space calibrated beforehand. Phase retention for each phase system was examined at various rotor
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angle of lo”, 20”, 30”, and 45” at 1200 and 1500 rpm at maximum flow rates of 2.5 ml/hr for 0.55 mm i.d. columns and 1.0 ml/hr for 0.3 mm i.d. columns. At lo”, all phase systems showed poor phase retention of below 30%. At 20”, the set-BuOH system gave a sufficient phase retention while other two-phase systems exhibited intensive carry-over resulting in poor phase retention. At 30” and 45”, however, all phase systems gave sufficient phase retention ranging from 30 to 50% in both column configurations. The straight helix column was found to retain a greater amount of the stationary phase at a given flow rate and revolutional speed, while the coiled helix column yielded a higher partition efficiency demonstrated by partition of bovine insulin on secBuOH/aqueous dichloroacetic acid systems. APPLICATION
TO VARIOUS
TWO-PHASE
SYSTEMS
Capability of the angle rotor (30”) has been evaluated on separations of biological materials with a variety of two-phase solvent systems of different physical properties. In order to demonstrate analytical potential of the method, a fine coiled helix column, 0.3 mm i.d. and 40 m long with a helix diameter of 1.2 mm, was used throughout unless otherwise specificed. In each separation, the column was filled with the stationary phase and a sample solution was introduced through the feed tube followed by the elution with the mobile phase using a syringe drive (Harvard Apparatus) at the indicated flow rate, while the rotor was run at the indicated revolutional speed. Separations were performed at 25°C unless otherwise indicated. Eluate was monitored through a uv monitor (LKB Uvicord II) at either 276 or 254 nm except for the polymer phase system. In separation of the colony-stimulating factor with the polymer phase system, eluted fractions were added to human bone marrow cell culture for colony count, while the total protein contents were estimated by turbidity measurement at 300 nm in 1 M perchloric acid solution. (1) Chloroform/acetic acid/O.1 N HCI (2 :2 :1) (8). This phase system has a moderately low interfacial tension, low viscosity, and a great density difference between two phases, which is ideal for countercurrent chromatography. Figure 2A shows a chromatogram of nine dinitrophenyl (DNP) amino acids at a flow rate of 0.5 ml/hr at 500 rpm, using the upper aqueous phase as a mobile phase. Sample volume was 10 ~1 containing each component in the lower phase at about 1% where solubility permits. All components were eluted out within 17 hr. Figure 2B shows a similar chromatogram obtained at a higher flow rate of 1.5 ml/hr at 1000 rpm. The separation was completed within 6 hr without serious loss of the resolution. (2) Ethyl acetate/lo% acetic acid, 5% NaCl (1 :l) (3). This phase system has a high interfacial tension and low viscosity and tends to
COUNTERCURRENT
CHROMATOGRAPHY
17 1.0 0.8 i R
0.6 0.5
3 z z 2 6
0.4 0.3 0.2
0
0
5
10 TIME Ihours)
15
20
3 1.0: 0.8 z z
0.60.5-
& 0.4 8 z 0.3 2 8 0.2 2 4
0.1 -
OO
J I, 1
,
I1 2
I 3
I
I 4
I
I 5
I
I
I 6
a
TIME (hours1
FIG. 2. Chromatograms (2 : 2 : 1) at different flow
of DNP amino acids on rates. A: Elution at 0.5 ml/hr.
chloroform/acetic acid/O.1 B: Elution at 1.5 ml/hr.
N
HCI
produce a plug flow in the fine bore column in the vertical rotor (4). However, the present method overcomes the problem of plug flow and five catecholamine metabolites were well separated and eluted out in 13 hr (Fig. 3A). Applied sample volume was 20 ~1, containing each component at 0.5 g%/ 100 ml in the lower phase. The lower phase was used as the mobile phase at a flow rate of 1 ml/hr at 1200 rpm. The same solvent system was applied to detect urinary catecholamine metabolites from patients (Fig. 3B). Each urine sample was prepared by adding NaCl (5%) and acetic acid (10%) according to the phase composition and 100 ~1
316
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TIME ihours)
metabolites on ethyl acetate/lo% FIG. 3A. Chromatogram of catecholamine CH,COOH, 5% NaCl (1: 1). B. Chromatogram of urinary catecholamine metabolites. Abnormal levels of VMA (4-hydroxy-3-methoxymandelic acid) and HVA (4-hydroxy-3methoxyphenylacetic acid) were detected from 100 ~1 of urine sample.
loaded, while the pure sample was dissolved in the lower phase at 0.5 g/100 ml for each component and 20 ,ul charged. Abnormal levels of VMA (4-hydroxy-3-methoxymandelic acid) and HVA (4-hydroxy-3methoxyphenylacetic acid) were detected from 100 ,ul of mine. VMA was cochromatographed with several other compounds but none of them interfered with any of the calorimetric assays for VMA. HVA was completely separated from other compounds. (3) N-BuOHlaqueous systems. The two-phase systems have moderate interfacial tension and relatively high viscosity, and are extremely useful for partition of various biological compounds. Figure 4A shows a chromatogram of purines and pyrimidines on a two-phase system composed of n-BuOH/l M potassium phosphate (pH 6.5) at 1: 1 volume ratio (9), obtained at a flow rate of 0.5 ml/hr and at 1200 rpm. Sample was prepared by dissolving each component in the upper phase at 0.1 g/ 100 ml and 10 ~1 charged. Since partition coefficients of these compounds favor to the lower phase, the upper organic phase was used as a mobile phase for better resolution of peaks. Thus, the present method permits a choice of the mobile phase according to partition coefficients of the solutes of interest. Figure 4B shows a chromatogram of a set of dipeptides using a gradient elution technique. A linear gradient was applied between 1% and 0% dichloroacetic acid on the base of n-BuOH and 0.1 M am-
COUNTERCURRENT
CHROMATOGRAPHY
317
Chemodectoma
0.2~ I\ I.:-
0.8 0.6
Normal Urine
0.4
TIME (hours1
FIG.
3B
monium formate at 1: 1 volume ratio (4), using the lower phase as a mobile phase. Sample size was 100 ~1 containing each component at 0.2 g/l00 ml in the lower phase. All components were separated and eluted out in 6 hr at a flow rate of 1 ml/hr at 1200 rpm. Two pairs of isomers, L-valyl-L-tyrosine and L-tyrosyl-L-valine, L-leucyl-L-tyrosine, and Ltyrosyl-L-leucine, were completely separated in this method. (4) ~ec.-~uO~~3% aqueous ~ic~f~r~uce~ic acid (1 :I) (5). This twophase system is characterized by low interfacial tension and high viscosity with a high tendency of emulsification, Figure 5 shows a countercurrent chromatogram of bovine insulin compared with the partition data by countercurrent distribution method on a similar phase system reported by Harfenist and Craig (10). The CCD revealed a second component, identified as a deaminated form of insulin which was not separable by
318
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17 1.0 1 0.8 $ 0.6 $ 0.5 5 0.4 8 z 0.3 $ 6 0.2 3 a 0.1 -
0-
1; 1.0 -
0.6
g 0.6 R
0.5
2
0.4
8 z
0.3
&% g 0.2
0.1
0
0
01234567 TIME
Ihours)
FIG. 4A. Chromatogram of purines and pyrimidines on n-BuOH/l M potassium phosphate (1: 1). The upper organic phase was used as the mobile phase. B. Chromatogram of a set of dipeptides using a gradient elution. A linear gradient of 1% to 0% dichloroacetic acid was applied on the base of n-BuOH/. 1 M ammonium formate using the lower phase as a mobile phase.
either electrophoresis or ultracentrifugation. Countercurrent chromatography completely separated the second component from the main peak. Applied sample size was 1 mg dissolved in 50 ~1 of the lower phase and the separation was performed at a flow rate of 1 ml/hr at 1200 rpm.
FIG. 5. Partition of bovine insulin by countercurrent distribution method and countercurrent chromatography. Countercurrent distribution (top) demonstrates a second component which is hardly separable by other separation methods. Countercurrent chromatography (bottom) completely separates the second component from the main peak.
COUNTERCURRENT
319
CHROMATOGRAPHY
(5) Polymer phase system (I I, 12). In general, polymer phase systems have extremely low interfacial tension, high viscosity, and small density difference between the phases and require a considerable time for phase separation. Because of the aqueous/aqueous phase composition, application to the conventional chromatography becomes difficult and, therefore, the partition relies upon a time-consuming countercurrent distribution method. In order to demonstrate applicability of polymer phase systems to the present method, preliminary studies were performed on separation of colony-stimulating factor (CSF) (13) from human embryonic kidney supernatant using a charged poly(ethylene glycol) phase system (12) composed of 6% trimethylaminopolyethylene glycol (supplied by courtesy of Dr. G. Johansson), 6% dextran T 500, and 0.05 M sodium phosphate.’ The pH range of the polymer phase system suitable for partition of CSF was determined on test tube experiments and the toxicity of the upper and lower phases was ruled out on bone marrow cell culture. Subsequently, the separation was performed by applying an exponential gradient elution between the starting (pH 4.4) and ending (pH 6.3) media, using the upper phase as a mobile phase. Two-milliliter human embryonic kidney supernatant (Flow Laboratories) was lyophilized and dissolved in 2 ml of the upper phase of the starting medium. A coiled helix separation column, 0.55 mm i.d., 18 m long, and 4 ml capacity, was first filled with the lower phase of the starting medium and then 1.2 ml of the above sample solution was introduced at a rate of 0.6 ml/hr at 1300 rpm, followed by gradient elution for 15 hr at 18°C. Thirty fractions, each 0.3 ml, were obtained during run and then 10 fractions were collected from the column. These fractions were added to human bone marrow cell culture for evaluation of CSF activity by colony count,
“r
Prote,n
FRACTION NUMBER
FIG. 6. Chromatogram of CSF (colony-stimulating factor) from human embryonic kidney supernatant using a charged poly(ethylene glycol) polymer phase system. CSF (solid line) is fairly well separated from the total protein (dotted line). ’ This Stashick,
work has been carried out Medical Oncology, National
in collaboration Cancer Institute,
with Dr. Bethesda,
J. M. MD.
Bull
and
Ms.
E.
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while the total protein was measured by turbidity in I M perchloric acid solution at 300 nm with a Beckman spectrophotometer. Figure 6 shows the results of separation. CSF was fairly well separated from the rest of the proteins. The CSF activity at the peak was more than twice that of the original sample solution. Minor activity found in the later fraction may not be significant. DISCUSSION Versatility of the 30” angle rotor has been demonstrated on separations using a variety of two-phase systems including a high interfacial tension ethyl acetate system and a low interfacial tension polymer phase system. These separations are performed with a single-column configuration of coiled helix which is easily prepared from a piece of Teflon tubing and the column can be used for many weeks without damage. Once a suitable phase system is found, a proper operating condition is easily found by adjusting a flow rate and revolutional speed, since the method permits a wide range of applicability for phase retention and solute partitioning. Though a fine separation is usually attained with a slow flow rate by an overnight run, a relatively high resolution can be achieved in several hours with a higher flow rate and revolutional speed. Partition efficiency may be further increased by using a smaller bore and/or longer tubing while scaling up can be attained by a large-bore column. ACKNOWLEDGMENT We are deeply indebted to Mr. H. Chapman and Dr. E. K. Achter for the design and fabrication of the instruments and to Ms. E. Stashick for analysis of CSF. We also thank Mr. P. Carmeci, and Drs. J. M. Bull and J. Mabry for their kind cooperation and helpful suggestions for separation and analysis of CSF.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
lto, Y., and Bowman, R. L. (1971) Science 173, 420. Ito, Y., and Bowman, R. L. (1971) Anal. Chem. 43, 69A. Hurst, R. E., and Ito, Y. (1972) Clin. Chem. 18, 814. Ito, Y., and Bowman, R. L. (1973) J. Chromatogr. Sci. 11, 284. Ito, Y., Hurst, R. E., Bowman, R. L., and Achter, E. K. (1974) Separation and Purification Methods 3, 133. Ito, Y., Bowman, R. L., and Noble, F. W. (1972) Anal. ~~~c~em. 49, 1. Ito, Y., and Bowman, R. L. (1973) Science 182, 391. Hausmann, W., Weisiger, J. R., and Craig, L. C. (1955)J. Amer. Chem. SW. 77, 723. Tinker, J. F., and Bown, G. B. (1948) J. Biol. Chem. 173, 585. Harfenist, E. J., and Craig, L. C. (1952) J. Amer. Chem. Sot. 74, 3083. Albertsson, P. A. (1971) Partition of Cell Particles and Macromolecules, 2nd ed., Wiley-Interscience, New York. Johansson, G.. Hartman, A., and Albertsson, P. A. (1973) Eur. J. Biochem. 33, 379. Met&f, D., and Moor, M. A. S. (1971) in Haemopoietic Cells (Neuberger, A., and Tatum, E. L., eds.). p. 383, Noah-Holland, Amsterdam.
BIOCHEMISTRY
Angle
Rotor YOICHIRO
Laboratory
of Technical
65, 310-320 (1975)
Countercurrent ITO AND
ROBERT
Chromatography L.
BOWMAN
Development, National Heart Bethesda, Maryland 20014
and Lung
Institute,
Received September 30, 1974; accepted November 22, 1974 A new rotor for performing countercurrent chromatography holds a coiled helix column at 30” from the vertical. Versatility of the angle rotor is demonstrated on high-efficiency separations of biological compounds using a variety of two-phase solvent systems. Advantages of the angle rotor over other centrifugal countercurrent chromatographic schemes are discussed.
Previously two countercurrent chromatographic schemes have been reported, both utilizing a coiled tube in a centrifugal field. One is the flow-through coil planet centrifuge ( 1-5) with a vertical rotor and the other, the elution centrifuge (5-7) with a horizontal rotor. Experiments with various two-phase solvent systems have disclosed specific features inherent to each scheme. The advantage of the vertical rotor is that a high-efficiency partitioning is achieved with a short separation time by providing a vigorous phase mixing. However, it fails to retain the stationary phase of some low inter-facial tension phase systems, such as polymer phase systems, due to intensive emulsification. On the other hand, the horizontal rotor provides a stable centrifugal force field to retain polymer phase systems but a longer separation time is generally required due to increased mass transfer resistance. Since these different features are apparently derived from the difference in rotor angle between two schemes, there may exist an optimum rotor angle between 0” (vertical) and 90” (horizontal) that permits universal application of the solvent systems with a high partition efficiency. Efforts have been made to determine the optimum rotor angle with which capability of the method is demonstrated on separations using a variety of two-phase solvent systems having different physical properties of inter-facial tension, viscosity, and density difference between two phases. APPARATUS
Two angle rotors have been constructed by modifying a conventional refrigerated centrifuge (MSE, Model LR-6). Both rotors were designed, as in the previous schemes, to produce a counterrotation of the column 310 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
COUNTERCURRENT
CHROMATOGRAPHY
311
holder to establish a continuous flow-through system without rotating seals. (1) Forty-jive degree angle rotor. The first rotor was made at a fixed angle of 45” as follows: A cylindrical hollow aluminum column holder was supported by a pair of aluminum blocks equipped with bearings, which were then sandwiched by a pair of aluminum side plates to support the column holder at 45” with an accurate bearing alignment. To prevent twisting the flow tubes counterrotation of the holder was established by coupling a pair of toothed pulleys of equal diameter, one fixed at the middle portion of the holder and the other, on the motor housing at the center of centrifuge drive. A pair of idler pulleys mounted at the bottom plate of the rotor serves for changing direction of the running belt at 45”. The dummy side of the rotor was made similarly to facilitate accurate counterbalancing of the rotor. The revolutional speed was continuously regulated from 400 to 1500 rpm at a mean revolutional radius of 20 cm. The performance of the 45” rotor revealed several items to be improved. At a high revolutional speed, the bearings suffered from loss of lubricant through the seal resulting in overheating and wearing out. Temperature inside the column holder also rose considerably higher than regulated ambient temperature due to heat conduction from the bearing through the holder wall. After a few weeks of operation, the column holder began to rotate against the inner race of each bearing which resulted in wearing out the holder wall at the bearing sites. In the design of the second prototype described, efforts have been made to eliminate these problems. (2) Adjustable angle rotor (30”, 20”, and IO’). Since the 45” angle rotor described above permits a satisfactory phase retention of the polymer phase systems, the critical rotor angle for phase retention must be present between 0” and 45” from the vertical. Further investigation has been performed to determine the optimum rotor angle by constructing an adjustable angle rotor which provides 30”, 20”, or 10” from the vertical by a simple adjustment. Figure 1 shows the design of adjustable angle rotor fixed at the 30” position. A pair of bearing blocks (a), holding the aluminum column holder (b) (32 mm i.d. and 25 cm long) on each side of the rotor, is sandwiched between a pair of rectangular aluminum plates (c) each equipped at the middle with a pin (d) that fits into the pin hole (d) made in the side plate (e) of the rotor. This arrangement allows the column holder blocks to be rotated around the pins without disturbing the bearing alignment and then bolted to the side plates at a selected angle. A toothed pulley (f) mounted on the column holder is coupled to a stationary pulley (g) of equal diameter on the axis of the centrifuge through a toothed belt (h) by the aid of a pair of idler pulleys (i) mounted on the bottom plate (j) of the rotor. This coupling es-
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FIG. 1. Diagram of the adjustable angle rotor fixed at the 30” position. a: bearing block, b: column holder, c: side plate of column holder block, d: pin and pin hole in side plate, e: side plate of rotor, f: toothed pulley on column holder, g: stationary toothed pulley, h: toothed belt, i: idler pulley, j: bottom plate of rotor, k: ball bearing, 1: bakelite pipe, m: side hole in column holder, n: column support carrying a coiled helix column, o: feed and return tubes, Rotor angle is adjusted to 30”, 20”, or 10” from the vertical by rotating each column holder block around the pin hole (d) and bolting to the side plates (e). Counterrotation of the column holder is established by coupling pulleys if) and fg) with a toothed belt (h) to prevent twisting of the flow tubes (0). Idler pulleys (i) change direction of the running belt fh).
tablishes counterrotation of the column holder to prevent twisting of flow tubes (0). The positions of the pulley (f) on the coiumn holder and idler pulleys (i) are also adjusted for each angle to provide an adequate tension to the running belt (h). In the light of test results obtained by the 45” angle rotor, several design items were improved. Properly sealed ball bearings (k) (Moffatt Bearings Co., Cat. No. 209-SZZ) were employed, which can hold lubricant much longer and permit easy lubrication with a syringe needle inserted between the inner ring of the bearing and the plastic seal. Between the column holder and the inner race of each bearing, a piece of bakelite tube (1) was tightly placed to minimize heat conduction and also prevent rotation of the column holder against the bearings. In order to cool the column, several side holes (m) were made at the top and bottom of the column holder to allow air flow inside the holder during a run. A cylindrical column support (n) made of delrin rod was tightly secured at the top and bottom of the holder to prevent vibration during operation, Two types of column configurations were employed. The straight helix column was prepared by winding Teflon tubing onto a rigid plastic rod to make a column unit. Multiple column units were interconnected in series and tightly mounted in parallel onto the column support.
COUNTERCURRENT
313
CHROMATOGRAPHY
The coiled helix column (shown in Fig. I) was prepared by winding Teflon tubing onto a flexible core which in turn was tightly coiled around the column support. In order to minimize any adverse effect of the Coriolis force (6) previously described, these columns were placed within 1.3 cm from the central axis of the column holder with a mean revolutional radius of 21 cm. Both feed and return tubes (0) were passed through the center hole of the column support and then held tightly at the center of the upper member of the centrifuge. The flow tubes served several months of operation, when protected with a piece of greased silicone rubber tube at each supported end. Performance of the apparatus has been evaluated at revolutional speeds ranging between 500 and 1300 rpm during a period of 6 mo. Leakage of lubricant from the bearing seal was much reduced and weekly lub~cation seems satisfactory. The column temperature became close to the controlled ambient temperature, and maximum rise no more than a few degrees in centigrade after several hours of operation. DETERMINATION
OF THE OPTIMUM
ROTOR ANGLE
Both straight and coiled helix columns were prepared from two sizes of Teflon tubing (Zeus Industrial Products, Inc., Raritan, NJ), 0.55 mm and 0.3 mm i.d. for examination. The polymer phase system composed of 4% polyethylene glycol 6000, 5% dextran T 500, and 0.1 M sodium phosphate (pH 7) was mainly used, but two other phase systems, set-BuOH/aqueous dichloroacetic acid and n-BuOHIO.01 M NaCl containing 1% cetylpyridinium chloride (CPCl), were also chosen for study because of their specific physical properties. Set-BuOH system has a low interfacial tension but can be retained by a 60” coiled helix column configuration with the vertical rotor. The n-BuOHlaqueous CPCl system shows a high tendency of emulsification and a complete phase separation requires a few hours in a separatory funnel. The polymer phase systems have extremely low inter-facial tension, high viscosity, and small density differences between two phases. Because of these properties, the vertical rotor failed to retain the latter two phase systems even with the 60” flat coiled helix column configuration described earlier (4). In each experiment, the column was filled with the stationary phase and the mobile phase was introduced at various flow rates while the rotor was run at a constant speed at 25°C until the mobile phase was eluted through the column. Percentage of the stationary phase volume retained within the column was calculated from the eluted stationary phase volume and the capacities of the column and the dead space calibrated beforehand. Phase retention for each phase system was examined at various rotor
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angle of lo”, 20”, 30”, and 45” at 1200 and 1500 rpm at maximum flow rates of 2.5 ml/hr for 0.55 mm i.d. columns and 1.0 ml/hr for 0.3 mm i.d. columns. At lo”, all phase systems showed poor phase retention of below 30%. At 20”, the set-BuOH system gave a sufficient phase retention while other two-phase systems exhibited intensive carry-over resulting in poor phase retention. At 30” and 45”, however, all phase systems gave sufficient phase retention ranging from 30 to 50% in both column configurations. The straight helix column was found to retain a greater amount of the stationary phase at a given flow rate and revolutional speed, while the coiled helix column yielded a higher partition efficiency demonstrated by partition of bovine insulin on secBuOH/aqueous dichloroacetic acid systems. APPLICATION
TO VARIOUS
TWO-PHASE
SYSTEMS
Capability of the angle rotor (30”) has been evaluated on separations of biological materials with a variety of two-phase solvent systems of different physical properties. In order to demonstrate analytical potential of the method, a fine coiled helix column, 0.3 mm i.d. and 40 m long with a helix diameter of 1.2 mm, was used throughout unless otherwise specificed. In each separation, the column was filled with the stationary phase and a sample solution was introduced through the feed tube followed by the elution with the mobile phase using a syringe drive (Harvard Apparatus) at the indicated flow rate, while the rotor was run at the indicated revolutional speed. Separations were performed at 25°C unless otherwise indicated. Eluate was monitored through a uv monitor (LKB Uvicord II) at either 276 or 254 nm except for the polymer phase system. In separation of the colony-stimulating factor with the polymer phase system, eluted fractions were added to human bone marrow cell culture for colony count, while the total protein contents were estimated by turbidity measurement at 300 nm in 1 M perchloric acid solution. (1) Chloroform/acetic acid/O.1 N HCI (2 :2 :1) (8). This phase system has a moderately low interfacial tension, low viscosity, and a great density difference between two phases, which is ideal for countercurrent chromatography. Figure 2A shows a chromatogram of nine dinitrophenyl (DNP) amino acids at a flow rate of 0.5 ml/hr at 500 rpm, using the upper aqueous phase as a mobile phase. Sample volume was 10 ~1 containing each component in the lower phase at about 1% where solubility permits. All components were eluted out within 17 hr. Figure 2B shows a similar chromatogram obtained at a higher flow rate of 1.5 ml/hr at 1000 rpm. The separation was completed within 6 hr without serious loss of the resolution. (2) Ethyl acetate/lo% acetic acid, 5% NaCl (1 :l) (3). This phase system has a high interfacial tension and low viscosity and tends to
COUNTERCURRENT
CHROMATOGRAPHY
17 1.0 0.8 i R
0.6 0.5
3 z z 2 6
0.4 0.3 0.2
0
0
5
10 TIME Ihours)
15
20
3 1.0: 0.8 z z
0.60.5-
& 0.4 8 z 0.3 2 8 0.2 2 4
0.1 -
OO
J I, 1
,
I1 2
I 3
I
I 4
I
I 5
I
I
I 6
a
TIME (hours1
FIG. 2. Chromatograms (2 : 2 : 1) at different flow
of DNP amino acids on rates. A: Elution at 0.5 ml/hr.
chloroform/acetic acid/O.1 B: Elution at 1.5 ml/hr.
N
HCI
produce a plug flow in the fine bore column in the vertical rotor (4). However, the present method overcomes the problem of plug flow and five catecholamine metabolites were well separated and eluted out in 13 hr (Fig. 3A). Applied sample volume was 20 ~1, containing each component at 0.5 g%/ 100 ml in the lower phase. The lower phase was used as the mobile phase at a flow rate of 1 ml/hr at 1200 rpm. The same solvent system was applied to detect urinary catecholamine metabolites from patients (Fig. 3B). Each urine sample was prepared by adding NaCl (5%) and acetic acid (10%) according to the phase composition and 100 ~1
316
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TIME ihours)
metabolites on ethyl acetate/lo% FIG. 3A. Chromatogram of catecholamine CH,COOH, 5% NaCl (1: 1). B. Chromatogram of urinary catecholamine metabolites. Abnormal levels of VMA (4-hydroxy-3-methoxymandelic acid) and HVA (4-hydroxy-3methoxyphenylacetic acid) were detected from 100 ~1 of urine sample.
loaded, while the pure sample was dissolved in the lower phase at 0.5 g/100 ml for each component and 20 ,ul charged. Abnormal levels of VMA (4-hydroxy-3-methoxymandelic acid) and HVA (4-hydroxy-3methoxyphenylacetic acid) were detected from 100 ,ul of mine. VMA was cochromatographed with several other compounds but none of them interfered with any of the calorimetric assays for VMA. HVA was completely separated from other compounds. (3) N-BuOHlaqueous systems. The two-phase systems have moderate interfacial tension and relatively high viscosity, and are extremely useful for partition of various biological compounds. Figure 4A shows a chromatogram of purines and pyrimidines on a two-phase system composed of n-BuOH/l M potassium phosphate (pH 6.5) at 1: 1 volume ratio (9), obtained at a flow rate of 0.5 ml/hr and at 1200 rpm. Sample was prepared by dissolving each component in the upper phase at 0.1 g/ 100 ml and 10 ~1 charged. Since partition coefficients of these compounds favor to the lower phase, the upper organic phase was used as a mobile phase for better resolution of peaks. Thus, the present method permits a choice of the mobile phase according to partition coefficients of the solutes of interest. Figure 4B shows a chromatogram of a set of dipeptides using a gradient elution technique. A linear gradient was applied between 1% and 0% dichloroacetic acid on the base of n-BuOH and 0.1 M am-
COUNTERCURRENT
CHROMATOGRAPHY
317
Chemodectoma
0.2~ I\ I.:-
0.8 0.6
Normal Urine
0.4
TIME (hours1
FIG.
3B
monium formate at 1: 1 volume ratio (4), using the lower phase as a mobile phase. Sample size was 100 ~1 containing each component at 0.2 g/l00 ml in the lower phase. All components were separated and eluted out in 6 hr at a flow rate of 1 ml/hr at 1200 rpm. Two pairs of isomers, L-valyl-L-tyrosine and L-tyrosyl-L-valine, L-leucyl-L-tyrosine, and Ltyrosyl-L-leucine, were completely separated in this method. (4) ~ec.-~uO~~3% aqueous ~ic~f~r~uce~ic acid (1 :I) (5). This twophase system is characterized by low interfacial tension and high viscosity with a high tendency of emulsification, Figure 5 shows a countercurrent chromatogram of bovine insulin compared with the partition data by countercurrent distribution method on a similar phase system reported by Harfenist and Craig (10). The CCD revealed a second component, identified as a deaminated form of insulin which was not separable by
318
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17 1.0 1 0.8 $ 0.6 $ 0.5 5 0.4 8 z 0.3 $ 6 0.2 3 a 0.1 -
0-
1; 1.0 -
0.6
g 0.6 R
0.5
2
0.4
8 z
0.3
&% g 0.2
0.1
0
0
01234567 TIME
Ihours)
FIG. 4A. Chromatogram of purines and pyrimidines on n-BuOH/l M potassium phosphate (1: 1). The upper organic phase was used as the mobile phase. B. Chromatogram of a set of dipeptides using a gradient elution. A linear gradient of 1% to 0% dichloroacetic acid was applied on the base of n-BuOH/. 1 M ammonium formate using the lower phase as a mobile phase.
either electrophoresis or ultracentrifugation. Countercurrent chromatography completely separated the second component from the main peak. Applied sample size was 1 mg dissolved in 50 ~1 of the lower phase and the separation was performed at a flow rate of 1 ml/hr at 1200 rpm.
FIG. 5. Partition of bovine insulin by countercurrent distribution method and countercurrent chromatography. Countercurrent distribution (top) demonstrates a second component which is hardly separable by other separation methods. Countercurrent chromatography (bottom) completely separates the second component from the main peak.
COUNTERCURRENT
319
CHROMATOGRAPHY
(5) Polymer phase system (I I, 12). In general, polymer phase systems have extremely low interfacial tension, high viscosity, and small density difference between the phases and require a considerable time for phase separation. Because of the aqueous/aqueous phase composition, application to the conventional chromatography becomes difficult and, therefore, the partition relies upon a time-consuming countercurrent distribution method. In order to demonstrate applicability of polymer phase systems to the present method, preliminary studies were performed on separation of colony-stimulating factor (CSF) (13) from human embryonic kidney supernatant using a charged poly(ethylene glycol) phase system (12) composed of 6% trimethylaminopolyethylene glycol (supplied by courtesy of Dr. G. Johansson), 6% dextran T 500, and 0.05 M sodium phosphate.’ The pH range of the polymer phase system suitable for partition of CSF was determined on test tube experiments and the toxicity of the upper and lower phases was ruled out on bone marrow cell culture. Subsequently, the separation was performed by applying an exponential gradient elution between the starting (pH 4.4) and ending (pH 6.3) media, using the upper phase as a mobile phase. Two-milliliter human embryonic kidney supernatant (Flow Laboratories) was lyophilized and dissolved in 2 ml of the upper phase of the starting medium. A coiled helix separation column, 0.55 mm i.d., 18 m long, and 4 ml capacity, was first filled with the lower phase of the starting medium and then 1.2 ml of the above sample solution was introduced at a rate of 0.6 ml/hr at 1300 rpm, followed by gradient elution for 15 hr at 18°C. Thirty fractions, each 0.3 ml, were obtained during run and then 10 fractions were collected from the column. These fractions were added to human bone marrow cell culture for evaluation of CSF activity by colony count,
“r
Prote,n
FRACTION NUMBER
FIG. 6. Chromatogram of CSF (colony-stimulating factor) from human embryonic kidney supernatant using a charged poly(ethylene glycol) polymer phase system. CSF (solid line) is fairly well separated from the total protein (dotted line). ’ This Stashick,
work has been carried out Medical Oncology, National
in collaboration Cancer Institute,
with Dr. Bethesda,
J. M. MD.
Bull
and
Ms.
E.
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while the total protein was measured by turbidity in I M perchloric acid solution at 300 nm with a Beckman spectrophotometer. Figure 6 shows the results of separation. CSF was fairly well separated from the rest of the proteins. The CSF activity at the peak was more than twice that of the original sample solution. Minor activity found in the later fraction may not be significant. DISCUSSION Versatility of the 30” angle rotor has been demonstrated on separations using a variety of two-phase systems including a high interfacial tension ethyl acetate system and a low interfacial tension polymer phase system. These separations are performed with a single-column configuration of coiled helix which is easily prepared from a piece of Teflon tubing and the column can be used for many weeks without damage. Once a suitable phase system is found, a proper operating condition is easily found by adjusting a flow rate and revolutional speed, since the method permits a wide range of applicability for phase retention and solute partitioning. Though a fine separation is usually attained with a slow flow rate by an overnight run, a relatively high resolution can be achieved in several hours with a higher flow rate and revolutional speed. Partition efficiency may be further increased by using a smaller bore and/or longer tubing while scaling up can be attained by a large-bore column. ACKNOWLEDGMENT We are deeply indebted to Mr. H. Chapman and Dr. E. K. Achter for the design and fabrication of the instruments and to Ms. E. Stashick for analysis of CSF. We also thank Mr. P. Carmeci, and Drs. J. M. Bull and J. Mabry for their kind cooperation and helpful suggestions for separation and analysis of CSF.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
lto, Y., and Bowman, R. L. (1971) Science 173, 420. Ito, Y., and Bowman, R. L. (1971) Anal. Chem. 43, 69A. Hurst, R. E., and Ito, Y. (1972) Clin. Chem. 18, 814. Ito, Y., and Bowman, R. L. (1973) J. Chromatogr. Sci. 11, 284. Ito, Y., Hurst, R. E., Bowman, R. L., and Achter, E. K. (1974) Separation and Purification Methods 3, 133. Ito, Y., Bowman, R. L., and Noble, F. W. (1972) Anal. ~~~c~em. 49, 1. Ito, Y., and Bowman, R. L. (1973) Science 182, 391. Hausmann, W., Weisiger, J. R., and Craig, L. C. (1955)J. Amer. Chem. SW. 77, 723. Tinker, J. F., and Bown, G. B. (1948) J. Biol. Chem. 173, 585. Harfenist, E. J., and Craig, L. C. (1952) J. Amer. Chem. Sot. 74, 3083. Albertsson, P. A. (1971) Partition of Cell Particles and Macromolecules, 2nd ed., Wiley-Interscience, New York. Johansson, G.. Hartman, A., and Albertsson, P. A. (1973) Eur. J. Biochem. 33, 379. Met&f, D., and Moor, M. A. S. (1971) in Haemopoietic Cells (Neuberger, A., and Tatum, E. L., eds.). p. 383, Noah-Holland, Amsterdam.
