Three-Phase Counter-Current Distribution: Theory

and Application to the Study of Strandin*

HEEBERT L. MELTZER

From the Departments of Biochemistry, New York State Psychiatric Institute and College of Physicians and Surgeons, Columbia University, New York, New York

(Received for publication, July 15, 1958)

The technique of discontinuous countercurrent distribution has been applied successfully to the separation of mixtures of biochemical importance. The advantages of this procedure, which requires that each component of the mixture be distrib- uted in constant ratio between two immiscible liquid phases, have been set forth, and the procedure has been used exten- sively, by Craig and coworkers. The scheme of distribution gives curves which are mathematically predictable on the basis of the binomial expansion (1).

Recently, an analogous scheme of discontinuous counter- current distribution which uses three immiscible liquid phases, has been developed and applied to the partial separation of brain lipide mixtures (2). It is the purpose of this paper to set forth the scheme of distribution, the applicable mathematical treatment, the technical features of the automatic apparatus for effecting three-phase distribution, and some experimental results obtained in its application to strandin.

PROCEDURE

General-The solute to be separated is equilibrated among equal volumes of top, middle, and bottom phases contained in one tube which is placed in a position corresponding to that of the top left position of Fig. 1 and assigned the number (0,O). Top and middle phases are transferred to tubes corresponding to positions (0,l) and (1 ,O), respectively in which aliquots of the bottom phase were previously placed. Aliquots of the top phase are added to tubes (0,O) and (1,O) and aliquots of the middle phase are added to tubes (0,O) and (0,l). After equili- bration, the top phase is transferred from (0, l), (0 ,O), and (1 ,O) to (0,2), (0, l), and (I, I), respectively. The middle phase is transferred from (l,O), and (O,O), and (0,l) to (2,0), (l,O), and (I, l), respectively. Aliquots of the appropriate solvents are added to tubes containing fewer than three phases, after which the contents of all tubes are again equilibrated.

This is the basic distribution pattern, illustrated in detail for a two-transfer system. It may be extended to any desirable number of transfers. The pattern is that of an equilateral triangle. Solute is placed at the apex, top phase is added along one adjacent side, and middle phase along the other adjacent side. Fractionated solute is obtained from the opposite side which mill be designated as the output side. Thus, if the system is made to contain 11 equilibration positions along each

* This investigation was supported in part by a research grant (No. B-344) from the National Institute of Neurolocrical Disease and Blindness, United States Public Health Service, and by a research grant from the National Multiple Sclerosis Society.

side, 10 transfers are required to move solute from the apex to the opposite side. On the 11th and each succeeding transfer, output samples are obtained as 11 separate fractions, each containing top and middle phase.

Apparatus-The apparatus necessary to carry out the simul- taneous transfers of top and middle phases from each stage is based on the decantation principle that has been used so success- fully by Craig (3). Each stage consists of an equilibration section and a decantation section. Top and middle phases are transferred as a unit to the decantation section from which they are transferred separately to the appropriate equilibration sections of adjacent stages (Fig. 2). There is a four-point connection between adjacent tubes. In the apparatus now in use, these connections are effected by ball a.nd socket joints. The tubes are mounted in rows parallel to the output side. These rows slide apart through closely fitting guides so that any tube can be made accessible for cleaning or repair when necessary. Each tube is clamped to its own holder which is positioned reproducibly in its place on the mounting row by guide pins. Thus, removal and replacement of a tube can be done without the need for repeated readjustment of its position in the assembly. All tubes are interchangeable.

It will be evident from the foregoing discussion that the con- struction of such an assembly is a very involved undertaking. First a heat-resistant metal form has to be machined to very close tolerance. This is then used to limit each bend and joint of the glassware to the precise position required. The finished unit must then be annealed without distortion, after which it must be mounted on a rack which has been machined so that it is comprised of repeating units whose dimensions are defined by the requirement for four-point attachment between glass units. The time, effort, and unanticipated difficulties involved in the construct,ion of such an assembly have been considerable. It does not seem likely that any laboratory would find it economi- cally feasible to do all the machining necessary to make only one such assembly. For this reason, publication of mechanical drawings and other specific information concerning details of construction are unwarranted. The apparatus has been con- structed by Llr. Joseph Buchler,l who is responsible for the successful calibration and for many of the essential details of design.

Mathematical Analysis-When a unit quantity of a single solute is distributed among three phases, and when the fractions in the top, middle, and bottom phases are designated by T, M,

1 Laboratory Glass Supply Company, 514 West 147 Street, New York 31, N. Y.

1327

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1328 Three-Phase Distribution of Strand& Vol. 233, No. 6

FIG. 1. Schematic diagram of three-phase countercurrent distribution. distribution.

Each large rectangle represents a stage in the The top and the middle phases are moved through

a matrix of the stationary bottom phase in the direction indicated by the arrows. The numbers on the arrows indicate the first transfer at which solute originating from the (0,O) tube can move into the indicated position.

FIG. 2. Schematic diagram of 1 unit of the apparatus. The lower section is the equilibration tube, from which the top and middle phases are decanted through the side arm to the decanta- tion section, Equilibration is effected when the unit is rocked so that lines A and B are alternately made horizontal. After phase separation in Position B, the top and middle phase decanta- tions occur when a clockwise rotation brings line C to the hori- zontal position. Top phase transfer to another unit in the lower left position occurs upon counterclockwise rotation to Position B and middle phase transfer to a third unit in the lower right position occurs upon further counterclockwise rotation to Position A.

TABLE I

Pattern of fraction

Tube No. Total fraction

(O,O) B (l,O) M (O,l) 1’

ation after one transfer

Fraction

TOP

T.B T.&l T2

Middle

M.B 1w M.T

Bottom

B2 B.iU’ B.1’

and B, respectively, the relation, (T + M + B) = 1, expresses the distribution. When the transfer pattern described above is applied, the conditions applying after the first transfer are as indicated in Table I. The conditions applying after the second

transfer are shown in Table II. When this analysis is continued for three- and four-transfer systems, and the total content of each tube is displayed in a pattern corresponding to that of Fig. 1, Figs. 3 and 4 are obtained.

A general term, expressing the total fraction of solute present in any tube after n transfers through a pattern containing (n + 1) tubes on each side may be derived from inspection of Figs. 3 and 4. If each horizontal row is designated by j and each vertical column by i, the contents of each tube, (i, j), are defined by the expression

CTjjj&iBn-i-j.

The coefficient C is derived as follows. The terms of Row 0 are identical with those of the binomial expansion (M + B)n. (Similarly for Column 0, the expansion of (T + B)n describes each term.) The general term for the binomial coefficient is

n!

i!(n - i)!

I f the coefficients of Row 1 are divided by the coefficient of tube (0, 1), which is obtained from the expression

n!

j!(n - j)!

where j = 1, the resulting coefficients are identical with those of the corresponding terms of the binomial expansion

(a + b)n-I.

For this expansion, the general term for the coefficient is

(n - l)!

i!(n - i - 1) ! .

Thus, when the latter term is multiplied by

n ! l!(n - I)!

the coefficients of Row 1 are obtained. Similarly, for any row, the coefficient C is given by

(j!(n)L! j,!) (;!&“!,),). The total expression is then

QG = jrjy, “!i _ j)! TjWBVt-C-j, (1)

. .

This gives the total fraction of solute present in the combined phases of any tube. If the trinomial expansion of (T + M + B)” is performed for the case n = 3, the individual terms can be tabulated to correspond exactly with those of Fig. 3. Three phase countercurrent distribution is described by the trinomial expansion, in contrast to the two-phase system which is described by the binomial expansion.

It was indicated in the general description of the procedure that samples of top and middle phases from each tube of the output side would be obtained by performing more than n transfers. The fraction of solute present in each output tube may be derived from Equation 1. From inspection of Fig. 1, it is apparent that for the output side, i + j = n. The first output set of samples is obtained by transferring the fraction

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December 1958 H. L. Meltxer 1329

(T + M) from each tube on this side. Each tube in this set therefore contains the fraction

n! i! j!(n - i - j)!

limb-i-i(T + M)

This set is placed in the first column of a matrix (p,j) where j has the same value as the corresponding tube on the output side of the apparatus, and p designates the sequence of sample sets (Fig. 5). I f the fraction of solute in each tube of this matrix is to bear a simple relation to the total number of transfers performed, the numbering of p must start with zero. Thus, where QP represents the fraction of solute present in any tube of the pth set of samples,

(n + p)! Qp*i = i!j!(n + p - i - j) Ti~Bn+?ri-i(T + M)

and since i = n - j,

Q ,= ,(n+p)! PI J!p!(n - j)!

TW"-~BP(T + M) w

It is to be expected that a single solute will appear in more than one Row j, that the distribution curve in each row will contain a maximum, and that, since the number of transfers is the same for all rows at any value of p, the maxima will have the same value of p. It is also to be expected that one Row j will have a maximum which is greater than all other maxima, that this will bear some relation to the fraction T/M.

The problem of locating the maxima mathematically mill be greatly simplified if we assume that the distribution curves will be symmetrical in the region of the maximum. This, of course, is an approximation where n is a small number, but we may expect it to become a better approximation as n becomes larger. Thus, if we assume a maximum at p, for j = j, so that

h + p, - 1) !

(n + p, + 1) !(p, - 1) 1 Bpm-' =- (n + p,, - 1) !(p, + 1) ! Bm+l

(n+pJ(n+p,+l) 1 Pm(Pm + 1) = Bz

(3)

Thus, the position of the maximum is a function only of n and B, i.e. of the size of the apparatus and the relative pref- erence of the solute for bottom phase.

The row containing the principal maximum may be located mathematically if we assume symmetry in the region of the maximum of a curve drawn through all values of p, for all

2 This equation may also be expressed in terms of the two distribution ratios: T/M = K, and (T + M)/B = D. After some algebraic manipulation, the equivalent form of Equation 2 be- comes :

TABLE II Pattern of fractionation after two transfers

Tube No. Contributing tubes

(O,O)

(1,O); (O,O)

(1,O)

@,l); @,O)

@,I)

(l,O); @,I)

-

/ Total fraction

B2 M.B -j- M.B

M2 T.B + T.B T2 T.M + T.M

0 1 2 3

0 T0M”B3 3 TOMB2 3 TOM2B T0M3B0 j=g 3 3 T2MoB TMoB2 3 6 TMB T2MB0 3 TM2BO

3 T3MOBQ

FIG. 3. Display of fractional distribution of solute after three transfers according to the scheme of Fig. 1.

i= 0 1 2 3 4 ~_____ 0 ToMOB 4 TOMB3 6 T”M2B2 4 T”M3B TOM4Bo

j=; 4 6 T2M”B2 TMOB3 12 12 T2MB TMB2 6 12 T2M2B0 TM2B 4 TM3Bo

3 4 T3M”B 4 T3MBo 4 T4MoBo

FIG. 4. Display of fractional distribution of solute after four transfers according to the scheme of Fig. 1.

(l=N 1 (J=o) P 0

\ \ I \ \ @ I + J=N

v&L----~ FIG. 5. Schematic relation of output matrix to distribution

matrix. The dashed lines indicate that the distribution scheme and the number of sample sets collected can be ext.ended indef- initely. The first sample set is placed in the vertical row p = 0.

TABLE III Relation of&* to Kt for a IO-transfer apparatus

* j, is the output row containing the principal maximum. j K = T/M.

values of j. This would be a vertical curve in the rectangular array which contained the output sa,mples. Then,

Q Pm.i,+l = Qp,.i,-I

and after substituting these values of j in the general expression for the output samples, the equation may be simplified to

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1330 Three-Phase Distribution of Strandin Vol. 233, No. 6

T2 jm(jm + 1) -= M2 (n-j~)(n-jm+l)

(4)

I f T/M = K, the row containing the greatest concentration of solute is determined only by the size of the apparatus and the affinity of solute for the top phase relative to that for the

TABLE IV

Relation of RQm * to jk for a IO-transfer system

The principal maximum is assumed at j = 8 from which K = 3.464 by substitution in Equation 4.

je RQ,

0 1.07 x 10-B

1 3.72 X 1O-6 2 5.79 x 10-d

3 5.35 x 10-a

4 0.0322 5 0.135

6 0.389

7 0.7698 8 1.00

9 0.7698

10 0.267

* RQ~ is the ratio obtained by dividing the fraction of solute

present in a tube originating from any Row j, by the fraction pres- ent in a tube originating in Row j, and having the same location p in the output matrix.

TABLE V

Relation of RQ,* to pm? and pk$. The tabulated numbers are

values of RQ~ for a IO-transfer system

9m BI Pk = 1 Bk = 5 Pk = 9 $k = 15

5 0.354 0.233 1.00 0.454 0.0096 9 0.487 0.038 0.59 1.00 0.0439

* RQ~ is the ratio obtained by dividing the fraction of solute

present in tube pk at any one value of j by that present in tube

Pm. t pm is the tube number in the output matrix at which the out-

put has a maximal value. $ pk is any other tube in the same Row j as pm.

0 By substitution in Equation 3.

TABLE VI

Theoretical fractionation of a pure substance within a five-transfer apparatus after jive transfers

Distribution coefficients are assumed to be T = 0.1, M = 0.3, B = 0.6. The location of each fraction corresponds to the posi- tion of each stage in Fig. 1.

i

j 0 1 2 3 4 5

0 0.07776 0.1994 0.1994 0.0972 0.0243 0.00243 1 0.0648 0.1296 0.0972 0.0324 0.00405 2 0.0216 0.0324 0.0162 0.0027

3 0.0036 0.0036 0.0009 4 0.0003 0.00015 5 0.00001

middle phase. It is to be expected that where these affinities are equal, the principal maxima will occur in the central row. Table III shows the values of K that lead to maxima in each row of a lo-transfer apparatus.

Since the curves are assumed to be symmetrical in both dimensions, the quantity of solute in any given Row j, should always be in fixed ratio to the quantity in any or all tubes of the row containing the principal maximum. The ratio may be designated RQ,. Then,

Qn.ik RQm = ~ =

ss! TGMn-ikB~(T + M)

QPd, (n ‘” f;;;!p! Ti7nMn-imB~(T + M)

(a- j,)!j ’ RQ, = (n - j,) ! jz!

(5)

when T/M = K.

From Equation 5 it is possible to estimate the fraction of solute present in any Row j, after Row j, has been determined. As an illustration, some values of this ratio are calculated in Table IV. Thus, there is no appreciable quantity of this solute within the area bounded by j = 0, j = 3.

A similar ratio may be derived for the horizontal distribution within one row, such that

(6)

As an illustration, this ratio is calculated for a few values of pk in Table V.

Discussion of Mathematical Treatment-The theoretical fractionation of a pure substance within the apparatus can be visualized by assigning specific numbers to the distribution coefficients. The pattern of such a fractionation is presented in Table VI, in a form comparable to that of Fig. 1. Since the distribution has been carried out over five transfers in an ap- paratus containing only five transfer positions on each side, the sum of all the fractions in the apparatus is equal to unity. A continuation of the transfer process will result in output curves whose sizes and shapes are predictable by Equation 2. The curve containing the principal maximum will originate from the row j = 1, as may be seen by inspection of the output diagonal, where the fraction of solute is greater in the position (4,l) than in any other position on this diagonal. (Equation 4 predicts that the principal maximum will originate from Row j = 0.97 for the case under consideration.)

The mathematical analysis makes it possible to apply rigid criteria to the purity of any solute for which the distribution among top, middle, and bottom phases is constant. Thus a pure solute must distribute so that the row containing the maximal output is defined by the constant T/M and the value of n; the concentration in any other row must be in a certain fixed ratio to it; the maxima of the output curves of all rows must exist at the same value of p which is defined by the values of n and B. The method of withdrawing samples makes pos- sible a ready check on the constant T/M. Substances whose affinity for top phase is greater than that for middle phase will be found principally in the regions having high values of j.

The scheme of partition depicted in Fig. 1 is capable of con- siderable variation. The composition of top and middle phases

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December 1958 H. L. Meltxer 1331

applied along the input sides does not necessarily have to be constant. For example, if the middle phase is partly aqueous, it is feasible to vary its pH within certain limits, and to apply to each tube of the middle phase input side a reservoir of middle phase differing from the preceding reservoir with respect to pH. Thus the virtues of gradient elution analysis can be combined with the precision of countercurrent distribution.

More extensive variations are possible. Fig. 6 shows one extreme variation. Top and middle phases are returned to the

two input sides, so that only two sets of output samples are obtained. The effect here is analogous to the reflux principle. Such variations involve a loss of generality and resolving power. However, it is conceivable that they would be preferable for special problems. Of course, each variation in distribution pattern requires a separate mathematical treatment. Each also requires a separate apparatus, but it is possible to build an apparatus that would perform a large variety of such patterns.

EXPERIMENTAL

Xolvent Systems-The factors that limit the selection of com- ponents of a solvent system are related to the nature of the substances under investigation, the analytical methods avail- able for detection of the various fractions, and the physical facilities of the laboratory. Although some exceptions will be apparent for specific cases, it is generally desirable that all components of a solvent system be relatively nonreactive with the solute, easily removed by evaporation at low temperatures, free of excessive odor or toxicity (substances such as carbon disulfide and tetrachloroethane are undesirable), and readily obtainable in a reproducible state of purity. A three-phase solvent system formulated from such components should separate readily after equilibration (this means in practice that each phase should differ from its neighbor by at least 0.1 density unit), have a constant composition under the range of tempera- ture variation expected, and should show little or no tendency to form emulsions with the solute.

Despite these general limitations, it has been possible to formulate a large number of solvent systems that appear to be useful for the separation of lipides. Most of these are based

FIG. 6. Schematic diagram of modified three-phase counter- current distribution. The symbols have the same meaning as in Fig. 1. A single middle phase output is obtained from the posi- tion at the top right and a corresponding top phase output from the bottom left. The depleted phases are supplied to the top and left side positions as needed.

TABLE VII

Some useful three-phase solvent systems

I<:ach vertical array gives the code number and the proportional parts of t.he components of a particular solvent system

Solvent system number

XIV xv

25 25

6 10 15 25

19 25

20 25

5

YXIX E P

fi

46

18 48 34

48

6 1

Component

I II IV XIII

25

20

10 25

XXI

13

5 14 13 12

4

XVIII

20 12.2

2.5 14

12.5 20

LVI

-.-

9t

1%

41‘

8t

4t

- Hcpt nne Water.

Nitromethane. Methanol

1, 1, LTrichloroethane 1.2.Dichloroethane.. Methglene chloride.

Chlorocyclohexane LChlorobutane 1,1,2-Trichloroethylene

LKitropropane.. 4.Methyl-2.pentanone Ethylene glycol

Ethylene diformate Methyl acetate.. Ethyl acetate.

Diethyl carbonate. l’yridirre. Acetic acid..

50 50 25 25 50 50 25 25

10 10

13

7 14 13 12

4

17t 6*

19.51

let 16t

5:

205

38 116 154

216

* Redistilled from permnnganute.

t Fractionally distilled. : Distilled a few days before use.

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1332 Three-Phase Distribution of Strandin Vol. 233, No. 6

TABLE VIII

Effect of structure of chlorinated hydrocarbons on partition between heptane and nitromethane

5 ml. of each component was mixed with the indicated volume of the chlorinated compound.

component Phase volumes after equilibration with:

1 ml. 2 ml. 4 ml. 6 ml.

Ccl, .___.................... 5.715.3 6.5/5.5 8.4/5.6 10.2/5.8 CH&H&H&H&l 5.8/5.2 6.6/5.4 8.4/5.6 11/5 CH&Cl, 5.6/5.4 6.2/5.8 7.6/6.4 9.6/6.4 CHCl=CClz. 5.5/5.5 t-v6 7.5/6.5 9.516.5 C6H&l............... 5.3/5.7 5.8/6.2 6.6/7.4 1 phase CH&HClz 5.4/5.6 5.5/6.5 5.5/8.5 1 phase CHC13. 5.3/5.7 5.4/6.6 5.2/8.8 1 phase CH2C12..................... 5.1/5.9 5.1/6.9 4.6/9.4 1 phase CH&lCHClz . 516 5/7 4.6/9.4 3.2112.8

CHzClCHzCl 4.8/6.2 4.6/7.4 4/10 2.5113.5

TABLE IX

Initial compositions of the strandin preparatiolzs used in the

distribution studies; all values are expressed in percentages, except where indicated otherwise

9 2 K d

%

s-2 s-5 S-6 5

-

upon the immiscible system: heptane, water, nitromethane. It is possible, however, to omit either water or nitromethane from a three-phase solvent system. (See Systems XIII and XVIII in Table VII.) Addition of various chlorinated aliphatics, alcohols, acetic acid, or other components produces substantial alterations in the properties of each phase. Generally the top phase is nonaqueous and nonpolar, the middle phase is aqueous and polar and the bottom phase is nonaqueous and polar.3

The choice of a particular solvent system depends upon obtaining favorable distribution coefficients for the mixture of solutes. Where the mixture must be regarded as an unknown it is desirable that initially it distribute equally among the three phases. To obtain such a distribution of brain lipides it has been found necessary to formulate solvent systems of low water content. This is equally true for cholesterol, which has a high affinity for nonaqueous solvents, and for strandin, which has a high affinity for water and a limited solubility in other solvents. In order to obtain a system of low water content it was neces- sary to be able to add to the aqueous phase a large proportion of a water soluble organic solvent, such as methanol or acetic acid. However, there is a percentage point beyond which further addition of the latter component causes the aqueous phase to become miscible with the nitromethane phase. Addi- tion of a water-insoluble chlorinated aliphatic solvent to the

3 Polarity was inferred from the instrument readings obtained with the Sargent Oscillometer. The reading increases with the dielectric constant.

nitromethane increases the allowable percentage of the water soluble organic component in the aqueous phase. By this reasoning, then, the problem of obtaining systems of low water content can be equated with the problem of increasing the chlorinated aliphatic solvent content of heptane-water-nitro- methane solvent systems. Since the chlorinated compound is partitioned between heptane and nitromethane, it is desirable to choose a substance whose affinity for nitromethane is high. It was noticed that when increasing volumes of carbon tetra- chloride were shaken with equal volumes of heptane and nitro- methane, the increase in volume of the upper (heptane) phase was almost equal to the volume of carbon tetrachloride added, indicating that the nitromethane had no affinity for carbon tetrachloride. It seemed that the occurrence of hydrogen bonding might be necessary for retention in the nitromethane phase. Since the presence of neighboring chloride groups could conceivably influence the tendency to hydrogen bond forma- tion, a survey was made of the relation of increment in phase volume of the lower (nitromethane) phase to the volume of various chlorinated aliphatic compounds added to different portions of heptane-nitromethane mixtures. The results (Table VIII) appear to bear out the hypothesis.4 On the basis of these results 1,2-dichloroethane was chosen as a component of a three-phase solvent system. The system so formulated (System LVI, Table VII) did have a lower water content than that obtainable when 1 , 1, I-trichloroethane was used instead, and it was markedly more satisfactory as a solvent system for strandin.

Application to Study of Strandin-Strandin was isolated from brain cortex by Folch (4), who reported that it contains a main component with a minimal molecular weight of about 250,000. From similarities in composition of constituent groups, strandin is now considered to be closely related to the gangliosides, first isolated by Klenk (5).

For the first and second distributions, S-2 and S-5, strandin was prepared by the partition dialysis method (4). The third distribution, S-6, was performed on a purified fraction for which I am indebted to Dr. Folch and which is estimated by him to be 95 per cent pure. 193 mg. of X-2 was dissolved in 6 ml. of water followed by proportionate volumes of the other components of solvent System XXXIV (Table VII). Appropriate quantities of each phase were added from reservoirs so that, after equilibra- tion, the volume of each phase was 80 ml. Then 25-ml. aliquots of each phase were placed in tubes (0 ,O), (0,l) and (1 ,O) of a IO-transfer apparatus. The distribution was carried out at 22-24” until 11 sample sets were obtained.

198 mg. of S-5 was dissolved in 12 ml. of acetic acid and 1.5 ml. of water followed by proportionate volumes of other com- ponents of solvent System LVI (Table VII). The additional quantity of water needed to prepare this solvent system was added last. After the phase volumes were adjusted to 30 ml. for each phase, 25-ml. aliquots were placed in tube (0,O) of a five-transfer apparatus and 29 sample sets were collected at 20-21”. 190 mg. of S-6 were distributed in the same manner until 35 sample sets were obtained.

4 Supporting evidence for an interaction between nitromethane and 1,2-dichloroethane was obtained by plotting a curve of Sargent Oscillometer inst.rument readings of mixtures of these two substances. Every experimental point on this curve was above the theoretical line predictted for mixtures of noninter- acting substances.

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Analytical Methods-Nitrogen was determined by digestion with HzS04 in sealed tubes at 380”, followed by nesslerization. Phosphorus was determined calorimetrically by the Sperry method (6). Sialic acid was determined calorimetrically at 530 rnE.1 by reaction with diphenylamine (7). The color produced was compared with a sample of authentic sialic acid obtained as a gift from Dr. K. Meyer. Hexosamine was determined by a modification of the Elson and Morgan procedure (8). Hexose was determined by reaction with anthrone (9). Sphingosine was determined by a slight modification of the method of Robins et al. (10). After color development, the dinitrophenyl sphingosine was extracted into chloroform. An aliquot of the extract was evaporated almost to dryness and then dissolved in water, HCl and propionic acid in the proportions stated by Robins et al. (10). The color was compared with sphingosine and dihydrosphingosine standards (obtained through the courtesy of Dr. B. Weiss). Each of these substances had a molar extinction coefficient of 5200, either with or without chloroform extraction. The extraction procedure is necessary to eliminate a turbid material that results when strandin is put through the procedure. Fatty acid ester groups were deter- mined as the ferric hydroxamate acid complex (ll), with the modification that anhydrous ethyl ether was refluxed with hydroxylamine and distilled to insure low blanks. The total amount of fatty acids was determined as follows. A sample containing 0.1 to 1.0 mg. of fatty acid was placed in a 180 x 25mm. tube with a standard taper joint; 5 ml. of methanolic HCl were added and the tube was closed by inserting a standard taper cold finger which was secured in position by taping the outside of the joint. After the tube was heated for 2 hours at 70”, the solvent was removed in a stream of nitrogen at 4&50”. The methyl esters thus formed from free fatty acids and amide groups were analyzed by the ester reaction.

RESULTS

Analytical data on the three preparations before fractionation are given in Table IX. Distribution of S-2 gives clear evidence of the inhomogeneity of the starting material. The analytical values for selected samples obtained from the output samples and from samples obtained from the apparatus are shown in Fig. 7. Some output curves obtained from the region favored by a substance having a T/M ratio less than unity are shown in greater detail in Fig. 8. A small fraction remained in the apparatus, another small fraction moved principally with the top phase, and the major fraction which moved principally with the middle phase, has at least two components as may be seen from the dissimilarities in curves of weight and content of sialic acid. Consideration of the data of Table X supports and extends these conclusions. For example, sialic acid components of the initial strandin were resolved into at least two groups, one moving with the middle phase and associated with substantial amounts of hexose and sphingosine, and the other remaining in the apparatus, associated with substantial amounts of fatty acid.

Output samples obtained by distributing S-5 were pooled in 13 groups on the basis of peaks, valleys, plateaus, and inflections in the weight curves (Fig. 9). It should be emphasized that some of these weights were so low, in some cases less than 0.1 mg., that the shape of the curves is not reliable. However, samples grouped according to these weights were evidentlv

I IOMG

0 -

FIG. 7 Distribution of strandin, Preparation S-2. The analyti- cal values for the output curves were obtained from tubes on the output side of the apparatus corresponding to the intersection points of the 11 horizontal base lines with the large diagonal. The weight of material remaining in certain positions in the apparatus is represented by the height of the vertical lines in the triangular region of the figure. The abscissa is the sample set number. The ordinate is weight.

FIG. 8. Detailed study of three output curves obtained from s-2.

TABLE X

Some components of pooled fractions obtained from S-2

Pooled fractions

j oto3 pot05

j oto3 p6to8

j 5to7 p 0 to 10

j 8 to 10 p 1 to 10

Apparatus diagonal 0 to 8

leight arbo- ,drate

exos- mine

mg.

88.9

%

17.4

%

4.0

13.8 10.3 2.8

11.9 3.6

8.2

25.9

3.4

3.5

a& cid -

%

24

13

2

0

3

-

#phin- :osine

%

24

16.5

17

3.4

Ester

mole/ mg.

1.83

0.58

0.85

2.65

2.05

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1334 Three-Phase Distribution of Strandin Vol. 233, No. 6

25 20 15 IO 5 0 TABLE XI

J=O

>J=,

d J= ;

?

;

FIG. 9. Some output curves obtained by distributing strandin, Preparation S-5. The weights were obtained by analyzing the top phase portion of samples derived from j = 4 and 5 and the middle phase portion of samples obtained from j = 0, 1, and 2.

j 3to5 p 25 to 29

14 1 5 12

j Oto2 pOto6

29 2 3 34

distinct fractions (Table XI). For example, pooled samples (j = 3 to 5, p = 15 to 21) and (j = 3 to 5, p = 25 to 29) differed with respect to sphingosine and hexose content, both of which were low or absent in the former and of appreciable magnitude in the latter. Samples in the group (j = 0 to 2, p = 0 to 6) seem to be a relatively pure fraction. The shapes of the curves (Fig. 9) from rows j = 1, 2 are somewhat broader than the theoretical curve calculated from p, = 2, B = 0.30. The curve fromj = 0 differs still more. A curve obtained by plotting the values of the maxima of each of these curves as ordinate and the value of j as abscissa is also different from the theoretical curve calculated from j, = 1, T/M = 0.33. Thus in both dimensions, points in this sample group do not bear the strict mathematical relationship to each other required of a pure substance. However, this group does represent the most clear-cut fraction among the minor components and as such its composition is of some interest. The major fraction, com- prising about 50 per cent of the weight of starting material, was found as expected in the samples remaining with the ap- paratus (Fig. 10). Samples from apparatus positions (1 ,O) to (5,0) represent that portion of the slowly moving component having a greater aflinity for the middle phase than for the top phase. This group of samples is free of phosphorus, in contrast to the sample from the starting position (O,O), which contains 0.5 per cent of phosphorus. It thus appears that more than one component is present in this portion of the slowly moving component. The other analytical data, as well as the shape of the weight curve of samples (0,O) to (5,0) (Fig. lo), confirm this conclusion. The samples remaining in position (0,l) to (0,5), which are symmetrically related to the (1 ,O) to (5,O) group, differing with respect to their higher affinity for the top phase than for the middle phase, also appear to form a distinct group as is evidenced by their lower hexosamine and sialic acid content.

j Oto2 p 7 to 11 10.36 21 3 9 22 0.89

j 1to2 p 12 to 17 3.24 15 2.6 7 11 0.34

j0 p 12 to 20 15.82 35 5.3 19 20 0.56

j lto2 p 20 to 29

8.19 20 2.8 12 11 0.47

j0 p 22 to 26

j0 p 27 to 29

010

170

2,0 to 5,0

co,1 to 0,5) +

Cl,2 to 1,4) +

(3,2)

1.37

4.06

13.5

15.0

33.3:

4.5:

%

I.5

3

0

0

1.3

1.2

0.3

0.3

0.1

0

0.4

0.1

0

0.16

27 4.7 16 21

40 6.2 28 11

13 3.8 25 19

20 5.4 33 11

32 7.5 27 20

0.37

0.27

7 2.0 I 13 26 1.18

from this preparation, whereas the others were much reduced in quantity (Fig. 11). Again, the principal minor component occurred in the group j = 0 to 2, p = 0 to 4. However, because of the low weight, the shapes of the curves cannot be used to estimate purity. The major slow-moving component was again recovered from the apparatus (Fig. 12). It comprised about 75 per cent of the starting material.

Distribution of S-6 revealed that some of the minor compo- The data (Table XII) indicate that sample X-6 contained at nents observed in the distribution of X-5 were entirely absent least eight components, of which five have been transferred out

Some components of pooled fractions obtained from S-5

Pooled fractions

-

j 3 to 5

pot02

j5 p3to6

j5 p 7 tjo 12

j 3to4 p5to11

j 3to5 p 15 to 21

-

v Veight

1.76

C h: -

:arbo- ydrat’

%

0

16

22

8

6

:xos- nine

%

I

)

1

1

ialic lcid

3

0

Sphin- gosine

6

0

Tatty acids

mole/ mg.

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December 1958 H. L. Meltzer 1335

20MG

5.0 OS

FIG. 10. A contour representation of the weight of 5-5 re- maining in the apparatus after collecting 29 sample sets. The large triangle represents the apparatus, with the apex tube at the top; the middle and top phase input is shown along the right and left side, respectively; and output samples are taken from the bottom. Every intersection of diagonal lines, or of diagonal and horizontal lines, represents the location of a unit of the apparatus. The vertical lines represent the weight of solute remaining in each of these units, with the weight scale given at the right side of the figure. The inset is a two-dimensional representation of the weights in the units along the left and right side of the apparatus.

“5

FIG. 11. Some output curves obtained by distributing strandin, Preparation S-6, in the same manner as for S-5.

5 >o

FIG. 12. A contour representation of the weight of S-6 remain- ing in the apparatus after collecting 35 sample sets.

of the apparatus during the course of the distribution. There is one minor component that moved so slowly with the top phase that it remained in the apparatus, as was the case when S-5 was distributed. Again, the shape of the curve and the analytical values (Table XII) for the contents of tube (0,O) as contrasted with those of tubes (1,O) to (5,0) clearly indicate that there is more than one substance present in the major component.

DISCUSSION

The results of the distribution of X-2 strongly suggested that more complete resolution was possible, since the major com-

TABLE XII Some components of pooled fractions obtained from S-6

Pooled fractions Weight

j 3to4 p2to6

WLR.

0.78

j oto2 pOto6

10.2

j0 p 7 to 16 0.44

j0 p 17 to 25 7.07

j0 p 26 to 35

9.48

(5,O) (4,O) (3,O) G40) O,O)

@,O)

(O,W@,5) + (1,2)-

(1,4)

13.3 16.44 27.70 31.39 19.13 17.86 5.04

P

%

0

0.9

1.2

(0.08)

0

(0.2) 0 0 0 0 0 0

ialil rcid

: $

'2 :

.-

%

0

b ii2 1

C” 1 -

% P

0

8 2 (3) (

15

25 5.9 25 17

25 6.2 23 15

29 7.2 27 23 6.3 28 26 6.9 34 26 6.7 30 25 5.9 33 24 7.2 33 6 1.0 7

16 13 10 13 14 14 13

i

“,%

tmole/ +m.

(0)

0.9

0.85

0.7

0.5

ponents had moved rapidly out of the apparatus in an obviously impure form, as evidenced by the nontheoretical shapes of the output curves and by the analytical data. The occurrence of 0.25 per cent of phosphorus in this fraction was regarded as further evidence of this, since such a small percentage would not be expected to be a part of any repeating unit of a pure substance. Solvent System LVI was set up with the intention that the major components would partition preferentially in its bottom phase (which in this case is the aqueous phase), affording an opportunity for a more extensive separation of the minor components. As the data indicate, such was the result of the use of this solvent system.

It could be argued that the multitude of small fractions isolated was largely the result of degradation of a pure starting material. (When the solvents were removed promptly from samples of the major component of S-6 at low temperature, the residue consisted of pure white strands. When the solution was allowed to stand at room temperature a week or more and then dried, and also when it was dried at or above room tempera- ture, the residue was pale yellow to yellow-brown). This degradation could have occurred either as a continuous process during the course of countercurrent distribution or it could have occurred immediately upon dissolution and could have been substantially complete before the distribution was started. The first possibility can be rejected immediately; if the various fractions were being produced continuously, the appearance of distinct regions of separation in the weight curves would be most unlikely. The second possibility cannot be dismissed so completely, although it does seem improbable in view of the qualitative and quantitative differences between S-5 and S-6.

A consideration of the data resulting from the distribution of

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1336 Three-Phase Distribution of Strandin Vol. 233, No. 6

X-5 and X-6 suggests that almost all of the many minor com- ponents of the starting materials are substances composed of carbohydrate and sphingosine, to which are bound additional moieties of carbohydrate, hexosamine, and neuraminic acid derivatives in variable quantities. From this point of view, the major components appear to be similar substances differing from the minor components only in the greater proportions of the moieties added to the carbohydrate-sphingosine units. Since no component has been obtained in pure form, speculations more extensive than the foregoing concerning the structure of strandin appear to be unwarranted at the present time.

An answer to the question, “Could not two-phase counter- current distribution have accomplished the same results?” is possible if it is assumed that the solvent systems used in this study would also be used in a two-phase distribution. Thus, if the top phase of solvent System LVI was used as the upper phase, and the middle and bottom combined were used as the lower phase in a two-phase system,5 it is evident from the data obtained by distributing X-5 and X-6 that the fast-moving components favored by the middle phase would not have been detected. Similarly, if the top and middle phases were used as upper phase and the bottom phase was used as lower phase, then the fast-moving components favored by the top phase would not have been distinguished from the fast-moving components favored by the middle phase, and the resolution of the material remaining in the apparatus into slow-moving top and middle phase-favored components would not have been accomplished. Similar considerations apply to any sequential arrangement, such as two-phase distribution between top and bottom phase, followed by two phase distribution between middle and bottom

phase. In the latter case, the slow-moving mixture would not have been resolved.

It is, therefore, evident that the application of three-phase countercurrent distribution to this complex lipide mixture has produced information that was otherwise unobtainable.

SUMMARY

1. A technique has been devised for the distribution of solutes between three immiscible liquid phases in a pattern analogous to countercurrent distribution.

2. The design and construction of an apparatus capable of simultaneously transferring the moving phases according to this pattern is described.

3. A mathematical analysis of the distribution pattern has revealed that the contents of any tube within the transfer pattern can be described in terms of a trinomial expansion. Other useful equations have been developed from this relation- ship.

4. The method has been applied to a study of purity of the brain lipide, strandin. At least 15 components were demon- strated in a sample prepared according to a published method. Another, more highly purified sample was shown to have at least 8 components.

Acknozuledgment-The author wishes to express his gratitude to Dr. Warren M. Sperry for the many useful comments which have helped to guide this work, and particularly for the op- portunity to work in a laboratory where such extensive and uncertain endeavors are possible and enjoyable.

REFERENCES

1. CRAIG, L. C., J. Biol. Chem., 165, 519 (1944). 5. KLENH, E., 2. physiol. Chem. Hoppe-Seyler’s, 273, 76 (1942). 2. MELTZER, H. L., Federation PI-oc., 16, 128 (1956). 6. SPERRY, W. M., Ind. Eng. Chem. Anal. Ed., 14, 88 (1942). 3. CRAIG, L. C., HAUSMAN, W., AHRENS, E. H., JR., AND 7. WINZLER, R. J., in D. GLICK (Editor), Methods of biochemical

HARFENIST, E. J., Anal. Chem., 23, 1326 (1951). analysis, Vol. II, Interscience Publishers, Inc., New York, 4. FOLCH, J., ASCOLI, I., ARSOVE, S., AND MEATH, J. A., J. Biol. 1955, p. 297.

Chem., 191, 819 (1951). 8. NEUHAUS, O., AND LETZRING, M., Anal. Chem.,29, 1230 (1957). 9. RADIN, N. S., LAVIN, F. B., AND BROWN, J. R., J. Biol. Chem.,

5 For example, if 5 ml. each of the middle and the bottom phases 217, 789 (1955). were to be added to each equilibration tube of a two-phase 10. ROBINS, E., LOWRY, 0. H., EYDT, K. M., AND MCCAMAN, apparatus calibrated to retain 10 ml. of lower phase, these two R. E., J. Biol. Chem., 220, 661 (1956). phases would remain stationary and function as a lower phase 11. RAPPORT, M. M., AND ALONZO, N., J. Biol. Chem., 217, 193 while the top phase was transferred through the apparatus. (1955).

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Herbert L. MeltzerStudy of Strandin

Three-Phase Counter-Current Distribution: Theory and Application to the

1958, 233:1327-1336.J. Biol. Chem. 

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