New Micro Liquid-Liquid Partition Techniques with the Coil Planet Centrifuge Yoichiro Ito,’ Ichiro Aoki, and Eiichi Kimura Department of Physiology, Osaka City University Medical School, Abeno-ku, Osaka, Japan
Kanichi Nunogaki and Yoshiaki Nunogaki Sanki Engineering, Limited, Nagaoka-cho, Kyoto, Japan
Two immiscible liquids of different density, fiiling a closed helical tube, become distributed into a pattern of alternating segments when the tube is rotated on its axis, perpendicular to a gravitational field. A solute loaded at the initial single liquid-liquid interface will become distributed in segments of each liquid along the coil according to its partition coefficient. Using centrifugal force instead of gravity, this principle has been applied to develop a countercurrent system on an unprecedented small scale. With the equipment developed, 12 samples of microgram quantities of material can be subjected to countercurrent separation, each in a total volume of about 0.5 ml of solvents, in about 5 hours. The factors affecting efficiency have been analyzed and improvements in resolution can be expected. The resolving power of this method increases (1) in proportion to the number of turns of the coil (coil units), (2) inversely with the internal diameter of the tube, and (3) in proportion nearly to the square root of time required to complete the segmentation of the two solvents along the length of t.he tube. With the apparatus in use and 6 meters of plastic capillary tubing coiled as 300 turns, the resolving power is equivalent to 300 “theoretical plates.” Preliminary experiments were performed to demonstrate separation of basic dyes, algal proteins, and erythrocytes.
LARGESCALE liquid-liquid partition techniques have been developed, the Craig apparatus (I) and recently the Ronor column ( 2 ) being widely used in laboratories. In contrast, existing micro-scale liquid-liquid partition techniques have employed materials holding one of the two phases, and consequently the results are often disturbed by adsorption of the solute onto the carrier surface. We have devised a carrierfree liquid-liquid partition technique in a micro scale. It involves a simple technique in conjunction with a coil planet centrifuge (3-5) and can be applied on a very small scale. Resolutions equivalent to several hundred theoretical plates have been attained, and methods to further increase the efficiency are suggested. The present paper describes the mechanism of the technique in relation to the exerimental separations presented. Present address, Laboratory of Technical Development, National Heart Institute, National Institutes of Health, Bethesda, Md. (1) L. C. Craig, “Comprehensive Biochemistry,” Elsevier Pub-
lishing Co., Amsterdam, London, and New York, Vol. 4, 1962,
P 1. (2) R. Signer and H. Am, Heh. Chim. Acra, 50, 46 (1967). (3) Y.Ito, Abstracts of 23rd International Congress of Physiological Sciences, Tokyo, Japan, p 66 (1965). (4) Y . Ito, M. A. Weinstein, I. Aoki, R. Harada, E. Kirnura, and K. Nunogaki, Nature, 212, 985 (1966). (5) Y. Ito, I. Aoki, E. Kirnura, and K. Nunogaki, Abstracts of 7th International Congress of Biochemistry, p 953 (1967).
MECHANISM
We consider a tube coiled in a cylindrical helix sealed at both ends rotating slowly around its long axis placed horizontally and wish to study the motion of fluids and/or particles confined in the tube (Figure la). First one can observe the asymmetric nature of the extremities of the rotating coiled tube. When the tube is filled with water, a glass bead or air droplet introduced into the tube moves toward one end of the tube which is then called the head; the other end of the tube is defined as the tail. The direction of movement is determined by both the “handness” of the coil and the direction of rotation. In this system a particle with a density either higher or lower than that of the medium has a tendency to advance toward the head of the tube. When the rotation is slow enough, the heavier particle stays exclusively near the bottom and the lighter particle near the top of the coiled tube, both advancing toward the head at a constant speed of one coil unit per one rotation. This also occurs with two mutually immiscible phases of an equilibrated countercurrent system. When the tube is filled with one phase and a small amount of the other phase is introduced at the tail, a slow rotation permits the latter phase to move through the former at a rate of one coil unit per one rotation. One “coil unit” is the length of tube in a complete turn (Le., 360”) of the helix. Accordingly, when the sealed tube contains a two-phase system, the light phase in one half-end and the heavy phase in the other half-end, each slow rotation of the coil induces a countercurrent between these media starting at the interface. In this case, the rate of rotation of the coil about its axis determines the ratio of the volumes of each of the two media in each coil unit. The volume of the medium which advances depends on the rate of rotation of the coil; the volume approaches 50 of the total volume of the coil unit at slow rotation rates, but is less at higher rates of rotation. Figure l b shows schematically this exchange process in a tube of 4 coiled units. The coiled tube at the top is filled with a white medium (light phase) at the head-end and a grey medium (heavy phase) in the tail-end, and the interface is shown at the bottom of the coiled tube. When the tube is subjected to a slow rotation under the gravitational field as indicated, the media begin to interchange through the interface. In each half rotation of the coil, a new interface is formed at the bottom of the coil and an interchange of the media takes place. This process occurs four times during two complete turns when these media complete what is here called a direct countercurrent process. At this stage each coil unit is occupied by approximately equal volumes of the two media as illustrated, and the whole length of the tube contains alternating segments, each about half-a-coil unit in length, of the two phases. Further rotation results in an “alternating” countercurrent process in which the pattern of segments remains the VOL. 41, NO. 12, OCTOBER 1969
1579
-Air
Bubble
Glarr Bead c-
Figure la. Motion of particles in a rotating helix Turn
Rotating Coil& Tube
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314
1
I
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Fimrc 2. l h e coil planet cenlrifugc Simultaneou~rotalion of the fmmc (3, 4, and 5) and the ccntrill shaft (1) at a diRerenl angular tclncily results in a rewlulion and a rotation of the coil holders ( 6 ) as n plinct
Figure lh. Mechanism of phase exchange in a rotating helix same, but in which the segments of one of the two phases pass alternately backward and forward through segments of the other This can be visualized bv insnection of ~i~~~ lb. If initially the direction of rotation had been in the opposite sense, the original head becomes the tail and the .. counrercurrenr process procews rnrougn me mreriaces appearing at the top of the coiled tube. Consequently, samples of a solute introduced beforehand at the initial liquid-liquid interface of the two media are subjected to a continuous partition process and finally distribute throughout the length of alternating segments of the two media in the tube according to the partition coefficient. It is apparent that the relation of the area of the interface to the volume of the media plays an important role in the efficiency of partitioning, and therefore we expect to obtain better results with a fine tube; in practice, good resolution bas been obtained with a tube measuring less than 1 mm in internal diameter. To enable the countercurrent process to take place inside such a tube, however, necessitates enhancement of the gravitational field, this being achieved by the use of the coil planet centrifuge (3-5).
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EXPERJMENTAL
Apparatus. The coil planet centrifuge (Figure 2) consists of a coil-bolder subjected to a centrifugal force of up to 350 g acting perpendicularly to the axis of the coil-holder. The 1580
ANALYTICAL CHEMISTRY
main bod! of the atmaratus consislo of ihree rims. each capable of rotation a i a unit: a pair of coil-holders (Figure 2, 6) and interchangeable gears (Figure 2, 7) with shaft (Part I); frame or a pair of arms (Figure 2, 4) and discs (Figure 2, 3) bridged with links (Figure 2, 5) (Part 11); central shaft (Figure 2, 1) and central gear (Figure 2, 2) which interlocks with the gear (Figure 2, 7) of Part I (Part 111). Simultaneous rotation of Parts I1 and I11 at a different angular velocity results in a revolution and a rotation of Part I as a nlanet. The relative rotation of Part I is determined bv the Gifference in angular velocity between Parts I1 and I11 and also by the gear ratio between the central (Figure 2, 2) and peripheral (Figure 2, 7) gears. In this apparatus, the rotation revolution ratio is adjustable into four steps-i.e., one rotation of Part I and 1000 revolutions of Part 11, 1/2000, 1/3000, and lj5000. The rate of revolution of Part I1 may be regulated up to 1600 rpm, which is equivalent to 350 X g on the axis of the coil-holder. Each coil-holder, measuring 24 cm in length and 3 cm in diameter, has six grooves on its periphery into which 6 coiled tubes can be loaded-a thick strong vinyl cylinder is then slipped over the holder to hold the coiled tubes in place. Another coil planet centrifuge, developed for the separation of particles, has a similar capacity except that the rotation-revolution ratio can be adjusted to ljl00, 1/200, 11300, and lj500. PREPARATION OF COILED TUBES. Polyethylene tubes with internal diameters of 1.0 mm, 0.7 mm, 0.5 mm, and 0.35 mm were employed. The thickness of the wall varied from 0.2 mm to 0.3 mm. When calibration of the volume or internal diameter of the tube was required, the tube was marked with ink at 1-meter intervals. The tube was coiled uniformly and tightly onto a glass rod measuring 24 cm in length and 6 mm in diameter, making as many turns as possible into a length of about 22 cm. Each end of the capillary tube coil was
fixed to the core glass rod with a rubber band, leaving 20 to 25 cm of free tubing at each end. The capacity of the tube was determined by measuring the volume of water with a precisely graduated syringe between two of the previously marked points. Then the water was sucked out, the tube washed by sucking alcohol and ether in turn several times, and finally dried by sucking air through it for several minutes. Reagents. The reagents employed were mostly of analytical grade and the products of Takeda Chemical Industries Ltd., Osaka, Japan, or E. Merck A.G., Darmstadt, Germany. Dextran 500 was obtained from Pharmacia, Uppsala, Sweden, and polyethylene glycol 6000 from Nihon Yushi Kogyo, Ltd., Osaka, Japan. Human albumin (25%;) was obtained from the Green Cross Corporation, Osaka, Japan. Phase Systems. The following phase systems were used: A. Solvent system for separation of basic dyes: A mixture of isoamyl alcohol (4 volumes), ethanol (2 volumes), acetic acid (1 volume), and distilled water ( 5 volumes) was chosen specifically for methylene blue distribution studies as it gives a centrosymmetric distribution for methylene blue. This mixture is compatible with the polyethylene tubing, as it does not permeate the wall easily (polyethylene, although inexpensive and readily available in various sizes, may not be compatible with some solvent systems, and Teflon or other materials could be used). The mixture was allowed to equilibrate at room temperature and separate with a separatory funnel. The two phases were separated and used in the experiments to be described. A two-phase system composed of isopropyl alcohol (3 volumes), n-butanol(6 volumes), ethanol (1 volume), distilled water (12 volumes), and various amounts of o-phosphoric acid was used to study the effect of droplet size on efficiency. This system, in addition to providing a suitable medium for studying the distribution of methylene blue, tended to form an emulsion as the concentration of phosphoric acid was reduced. B. System for algal protein separation: Table I lists the composition of the mixture used to separate algal proteins; this is based on the work of Albertsson (6). The mixture was allowed to equilibrate and separate as in A, above. C. System for separation of erythrocytes: Two different phase systems, based on Albertsson’s work (7), were used for separation of erythrocytes. Table II lists the components of each system, which were equilibrated and separated as described above. Preparation of Samples and Separation Procedure. A. Basic dyes: Stock solutions of methylene blue (0.5% w/v), neutral red (l.Ox),basic fuchsin (l.O%), and methyl green (2.073 were prepared. These stock solutions were diluted with the solvents used to make A, above. The lower phases were used to carry the samples. The final concentration and the dose of each dye was determined by preliminary experiments. The final concentration of each dye in the phase system was 0.03x for methylene blue, 0.05% for neutral red, 0.08% for basic fuchsin, and 0.17% for methyl green, respectively. B. Algal proteins: Phycoerythrin and phycocyanin were extracted from dried Asakusa-nori (Porphyru teneru) by adding it to 20 to 30 times its volume of water containing 0.5% toluene and 0.01 % mycillin and then leaving it in the dark at 4 “C for 7 to 10 days. The mixture was filtered and the filtrate was salted out with a 60z saturated (NH4)*S04 solution, followed by centrifugation at 4000 rpm for 10 minutes. The sediment was diluted with twice the volume of distilled water, stirred vigorously, and centrifuged again
A. Albertsson, “Partition of Cell Particles and Macromolecules,” Almqvist and Wiksell, Stockholm, and John Wiley and Sons, New York, 1960, p 164. (7) P. A. Albertsson and G. D. Baird, Experimental Cell Research, 28, 296 (1962). (6) P.
Table I. Polymer Phase System for Separation of Algal Proteins 35.0 g
20% (w/w) Dextran 500 30% (w/w) PEG 6OOO 0. O5M KHzPO4 0.05M KzHPOi
14.7 g
10.0 ml 10.0 ml 10.0 ml 20.3 ml
0.22M KCl Hz0
Table 11. Polymer Phase Systems for Separation of Erythrocytes System B
System A 20% (w/w) Dextran 25.0g 500 30% (w/w) PEG 6000 1 3 . 3 g 0.55M NaH2P04 5 . 0 ml 0.5524 Na2HP04 5 . 0 ml
25 human albumin solution
20z (w/w) Dextran 25.0 g 500 30% (w/w) PEG 6000 13.3 g 0.05M NaH2P04 5.0 ml 0.05M NazHPO4 5 . 0 ml 1 . 0 ml 1.0 ml 25 % human albumin
50.7 ml
solution 1.5M NaCl
10.0 ml
HzO
40.7 ml
at 4000 rpm for 10 minutes. The supernatant thus obtained usually contained both proteins in suitable concentration. When further increase of the relative concentration of phycocyanin was necessary, a phycocyanin-rich solution was prepared from the protein mixture by adsorbing phycoerythrin with BaS04. Then the supernatant was eluted through a column of Sephadex G-25 and the protein fraction obtained was concentrated by evaporation under a vacuum. Finally, 0.5 ml of the upper phase of the countercurrent media (B above) was mixed with a suitable volume of the above protein mixture and the total volume was reduced to the original volume of 0.5 ml by evaporation under a vacuum. C. Erythrocytes : Approximately 0.5 ml of heparinized blood from a human and a rabbit was separately delivered into centrifuge tubes and washed twice by mixing with 3 ml of physiological saline solution followed by centrifugation at 3000 rpm for 10 minutes. The sediments were added separately to over five times their volume of the upper phase of the phase system B (Table 11), stirred and centrifuged at 3000 rpm for 20 minutes. The supernatants were discarded and a mixture of equal volumes of each sediment was used as the sample. PROCEDURE.The same procedure was used to load the basic dyes and the algal proteins into the coils. A samplefree portion of the lower phase (A, above, for the dyes, and B, above, for the proteins) was injected into the head-end of the helix to just beyond the half-way point. Then the sample, dissolved in the appropriate lower phase, was injected from the head-end, after admitting an air bubble 2 to 5 mm long between the heavy phase and sample to prevent their mixing. Typically, the sample volume was 30 111; three or four coil units were filled with sample and the actual amount depended on the tubing size used. Next, the lighter phase was slowly injected introducing similar small air bubbles between each centimeter of the first 10 cm of the medium. The liquids were injected slowly (at a speed of about 10 coil units per minute) until the sample reached a position a few turns before the desired starting place and the tube at the tail-end was sealed by tying two knots in it. Then pressure was gradually applied by injecting more medium until the sample reached the desired portion of the tube and the air bubbles were compressed and finally absorbed. The end of the tube on the head-side was clamped and tied. Both ends of the tube were taped onto the core with a piece of vinyl tape. The tube was then inserted into a groove of VOL. 41, NO. 12, OCTOBER 1969
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Figure 3. Separation of basic dyes with a low molecular phase system M.G.: Me1:hyl green; M.B.: Methylene blue; N.R.: Neutral red; F.B.: Basic fuchsin
the coil holder of the ap‘paratus, balanced, and centrifuged under the selected condit:ions of rotation and revolution for various periods of time. A different approach w as used to separate the erythrocyte mixture. The upper pha se of A and the lower phase of B (Table 11) were used as tlle separating media. We used the lower phase of A and ui,per phase of B also. The lighter phase was injected first, f(,llowed by the heavier phase. The coils were loaded into the c:oilplanet centrifuge and centrifuged until the two phases disti:ihuted themselves along the helix. We used a ljl00 relative rotation at 1500 rpm for 30 minutes. This produced a gradient along the length of the helix, and the coils were ready for th e “alternating” partitioning process described earlier. About 5 pl of the mixture of cells in the upper phase of A (Table 11) was injected into a point about 10 coil units from midpoint of the coil toward the head with a syringe and fine needle; the hole was sealed with heat. Centrifugation was resunled until the described separation was achieved which was u sually 1 hour. Estimation of Efficiency (Resolving Power). Careful spectrophotometric analysis of t.he distribution of methylene blue, coil unit by coil unit, showed that the distribution was normal, and a plot of cumulative I>ercentageus. coil unit number was linear when plotted on normal probability graph paper. Because the sample in the Ihelix was quite small, it was difficult to make these analyses routinely. Therefore, a simple method was devised to assess the efficiency in terms of “theoretical plates” (numbers of tubes) of the Craig apparatus, although our method is niIt, strictly speaking, an equilibrium process. First, we took a piece of normal probability graph paper and drew a straight line between the ordinates, 0.15% and 99.85 %, corresponding to +31r (standard deviation units) from the mean of a normal distribution. At the completion ofa centrifuge run, we divided the distributed indicator into three parts noting the> number of coil units in the whole coil and in the central part s. The amount of dye in each part was measured with a spectrophotometer, and the percentage of the total was computf :d for each part. The cumulated percentage contained in t he segment between the head and the first dividing point wiIS noted on the line drawn on the graph paper, as was the :umulated percentage between the head and second dividing point. The remainder of the dye was in the third part. ‘I ‘he difference in abscissa between the two points (head-to-first division and head-to-second division) represented the number of coil units between the dividing points. Then thi:number of turns in which 99.7% of the indicator was distribmuted, was calculated by the number of turns for the central fI,action multiplied by the full scale in abscissa divided by the difference between the two points 1582
ANALYTICAL CH EMISTRY
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Fieure 4. Algal orotein seoaration with a oolvmer ohase . ” !iystem
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in abscissa. ny dividing tnls number by tne total number of coil units and consulting a table of the binomial distrihution, we could obtain a value corresponding to the “theoretical plate” number of the Craig apparatus. In order to apply this method, indicator and media should be selected to give a symmetrical distribution near the middle portion of the coiled tube. By trial and error, media were found which were fairly satisfactory for the distribution of methylene blue so that we could study quantitatively the factors which might influence the efficiency of the method. When the efliciency exceeded the equivalent of 200 “theoretical plates” of the Craig apparatus, however, a slight technical failure or a little aberration of the distribution curve from the normal one became critical, producing a large error in the estimation of efficiency. In this connection, a densitometer, which can record oreciselv the solute distribution inside the coiled tube. i
01a mixture 01 metnyi green (M. u.), mernyiene niue (M. a,), neutral red (N. R.), and basic fuchsin (F. B.). The first tube displays the separation of the mixture and the other tubes show the distribution of the individual components to demonstrate the reproducibility. This separation was performed with 6 meters of 0.35-mm i.d. tubing centrifuged at 300 X g, with a relative coil rotation of 0.25 rpm, for 10 hours. The total volume was 0.6 ml, and the samples contained 2 &gof methylene blue, 3 pg of neutral red, 3 pg of basic . ~. . . . .. . . - . and 20 p g of methyl green. Appearance ofthe bands fuchsin, narkedly varies with the sample size and may be broadened by I heterogeneity of the sample: F. B. is a mixture of three :omponents and M. G. has a minor component. The asym-
from linear isotherm at low concentrations. Similarly, the separation of about 100 fig of a mixture of phycoerythrin and phycocyanin with 0.4-mm i.d. coil is shown in Figure 4. These patterns were obtained in 6 hours under the same rotation-centrifugal forces used to separate the basic dyes. Using the simplified method of calculating efficiency on another experiment, we estimated the efficiencyto he 100 “theoretical plates.’’ An attempt was made to separate erythrocytes of various animals with the present method. When the countercurrent separation was performed in the usual way-Le., loading one phase, then the sample, and then the second phase-erythrocytes showed a tendency to move with the dextran-rich lower
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