SEPARATION OF PORPHYRINS BY COUNTER-CURRENT DISTRIBUTION*

BY S. GRANICK AND LAWRENCE BOGORADt

WITH THE TECHNICAL ASSISTANCE OF EILEEN FRANKFORT

(From the Laboratories of The Rockefeller Institute for Medical Research, New York, New York)

(Received for publication, December 10, 1952)

The recent development by Craig (l-3) of an all-glass machine for liquid- liquid counter-current fractionation makes possible the application of his method to the separation of porphyrins which cannot readily be separated by the sensitive Willstatter “HCl number” method. In this paper we have studied the application of the Craig method to the separation of a mixture of three known porphyrins, using the Willstiitter system of aqueous HCl and ether.

The principle of separating porphyrins by liquid-liquid partition was first used in 1906 by Willstatter and Mieg (4). In their method, the porphyrin mixture was dissolved in ether and the ether solution was extracted suc- cessively with equal volumes of increasingly higher concentrations of aque- ous HCl. They defined a partition value called the HCl number as that per cent of HCl (in gm. per 100 cc.) in the aqueous phase which would extract two-thirds of the porphyrin from the ether phase when the volume of the aqueous phase saturated with ether was equal to the volume of the ether phase saturated with water. In 1937 Zeile and Rau (5) attempted to extend the method of Willstatter and Mieg to the separation of a mix- ture of two porphyrins that differed only slightly in their HCl numbers, namely deuteroporphyrin (HCl number = 0.36) and mesoporphyrin (HCl number = 0.60) (Fig. 1). They calculated that, starting with a 1: 1 mix- ture of these porphyrins, it should be possible to obtain one-third of the mesoporphyrin admixed with only 1.3 per cent of deuteroporphyrin after 60 successive extractions. The Craig method, as we shall see below, per- mits a complete separation of these two porphyrins, uncontaminated with each other, and at the same time has made possible the recognition of unsuspected porphyrin impurities in the original crystalline materials.

Technical and Theoretical Considerations

Apparatus, Phases, and Porphyrin Concentration Bands-The Craig ma- chine used in our work consists of 100 glass tubes connected with each

* This is the sixth in a series of papers on porphyrins and related compounds. t Merck Fellow in the Natural Sciences of the National Research Council.

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other in series. With this machine two kinds of manipulations are possible, namely a transfer and an equilibration. Details of the apparatus and its manipulation have been described by Craig and coworkers (l-3).

In the experiments to be described for porphyrin separation the phases used are ether and aqueous HCl. The over-all effect of the progressive transfers in the machine is to cause a series of upper phase portions to pass over a series of lower stationary phases. If a mixture of porphyrins is present, the porphyrin with a greater relative affinity for the ether phase (Le. the porphyrin with a higher partition coefficient K, or the porphyrin with a higher HCI number) will tend to progress more rapidly along the series of tubes, since it is the ether phase that is being transferred to the next successive tubes. If the partition ratios of the porphyrins are suffi- ciently different, each porphyrin may come to occupy a series of tubes and appear as a discrete pink concentration band. In a band region, a plot of the concentration of the porphyrin per tube versus tube number should

FIG. 1. Deuteroporphyrin isomer 9; the lower half of the molecule is not shown. Replacement of both H* atoms by -CHOH-CH3 = hematoporphyrin, -CHs-CH3 = mesoporphyrin.

form a bell-shaped curve, t,he Gaussian curve of error (see Fig. 3). By visual inspection, the number of bands and their degree of separation or overlapping can be readily seen. In the purification of a porphyrin, for example, conditions can be arranged so that at the end of the experiment the purified porphyrin will occupy a series of tubes in the middle of the machine and impurities will occupy tubes on either side. Such a distribu- tion into three band regions would be due to differences in the respective K values of the three porphyrins.

Partition Coeficient-K, the partition coefficient, or distribution coefli- cient, is the ratio of the concentration C1 of porphyrin in the upper phase to the concentration C2 in the lower phase; i.e., K = CJC,. For fractiona- tion in the Craig machine it is desirable to select a two-phase system in which the porphyrin, whose purification is desired, has K = 1. This means that after 100 transfers the porphyrin will appear as a concentration band occupying the middle thirty tubes of the apparatus (see Equations 2 and 3). Such a K value can be obtained for any particular porphyrin by se- lecting an appropriate concentration of HCl as the aqueous phase.

If the K of a porphyrin at one particular concentration of HCI is known,

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S. GRANICK AND L. BOGORAD 783

then one may calculate approximately the K of the porphyrin at another concentration of HCl from the following equation,

(1) Ka tHCl.]* = Kb [HCl$

where K, is the partition coefficient of a porphyrin at a given HCl concen- tration a of the aqueous phase, and Kb is the partition coefficient of the porphyrin at another KC1 concentration b of the aqueous phase. This approximate equation may be applied if the concentration of HCl does not greatly exceed 0.1 N (5); otherwise, corrections for activities must be ap- plied. For example, with this formula, one may calculate the HCl con- centration at which a porphyrin has a K of 1.0, if the HCl number of the porphyrin is known, since by definition the HCl number of a porphyrin represents a concentration of HCl in per cent (gm. per 100 cc.) at which the partition coefficient of the porphyrin is 0.5.

If the HCl number of a porphyrin is not known, an estimate of the value of the partition coefficient may be obtained from a comparison of the struc- ture of this porphyrin with similar porphyrins and from a consideration of the following rules. (1) Porphyrins with resonating side chains (vinyl, for- myl, etc.) at the /3, /3’ positions of the pyrroles will tend to have a higher partition coefficient than porphyrins lacking such side chains. The reason for this effect is that the resonating side chains depress the pK of the ring nitrogens. Since the diacid form of the porphyrin, which carries 2 protons at the ring nitrogens, is insoluble in ether and since the neutral form of the porphyrin is insoluble in the aqueous acid, a depression of the pK of the ring nitrogens will tend to retain more of the porphyrin in the upper ether phase. (2) The partition coefficient of a porphyrin will also be influenced by the number and kinds of side chains. The presence of hydrophilic side chains, such as those containing COOH or OH, will tend to decrease the partition coefficient, whereas aliphatic side chains will tend to increase the partition coefficient.

The K of a porphyrin may be estimated experimentally by distributing the porphyrin between the two solvents in a separatory funnel and measur- ing the amount of pigment in each phase after equilibrium is attained. This measurement can be made readily spectrophotometrically at appro- priate wave-lengths. An accurate value for K is obtained by use of Equa- tion 2 after a fractionation has been made in the Craig machine (2, 3).

Rapid Method for Determining Partition Coeficieflt in Each Tube of Band Region-When the porphyrins have been separated by counter-current dis- tribution, it is often desirable to determine not only the total concentration of porphyrin in each tube of the machine, but the K value per tube as well. These determinations can be made rapidly by using a modified Beckman test-tube holder attached to the spectrophotometer in place of the cus-

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784 SEPARATION OF PORPHYRINS

tomary cell holder. The Beckman test-tube holder has been modified by having a hole cut through its bottom plate (Fig. 2). The test-tubes, 20 X 150 mm., used are selected for optical uniformity and for a light path deviating no more than f 1 per cent from the mean. When the test-tube is filled with 10 cc. of each of the two liquid phases and placed in the tube holder so that the lip of the tube touches the first or upper stop, the lower phase will be in the light path; by pushing the tube down so that the lip of the tube touches the second stop, the upper phase will now be in the light path. By selecting an isosbestic point (in our apparatus it was 579

FIG. 2. Modified test-tube holder for the Beckman spectrophotometer. In the position shown, the absorption of the upper phase can be determined. By raising the tube to the upper stop, the absorption of the lower phase can be determined.

rnp for deuteroporphyrin), the extinction of the upper and lower phases can be determined at the same wave-length. It was found best to deter- mine the extinction against an air blank and then to correct for the water blank. In this way the extinctions of both the upper and lower phase of each of 100 tubes could be determined in less than 2 hours.

Equations Used in Two-Phase Counter-Current Fractionation and Their Application to Porphyrin Separations-The theoretical basis for the distri- bution of solutes in the Craig apparatus and appropriate equations for the application of the theory are discussed by Craig (1, 3). A few equations which are immediately pertinent to this paper and which were used in selecting conditions for the separation of porphyrins and for the analysis of data are given below. These equations are applicable if the number of transfers exceeds twenty and if the K values are not too far from unity.

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The number of the tube N in the Craig apparatus which will contain the maximal concentration of a porphyrin is given by Equation 2

where n is the total number of transfers and K is the partition coefficient of this porphyrin.

The number of tubes over which 99 per cent of the solute is distributed after n transfers can be calculated from Equation 3.

(3) W = GdnK/(K + 1)2

Thus W is the width of a concentration band. Given the K values of two porphyrins in a particular two-phase system, Equations 2 and 3 permit the calculation of the extent to which the bands of the porphyrins would over- lap each other or be separated from each other in n transfers. An example of such a calculation is given in Experiment 3 below.

A curve for the theoretical distribution of each porphyrin in the tubes of the apparatus can be constructed by applying Equation 4.

Yo Yz = ~

antilog where 2 =

0.217 (K + 1)2

2 nK 1 2t

Yo is the total concentration of porphyrin in both phases of tube N. (For example, for the central band of Fig. 3, Yo is 1.13 mg.) Yz equals the total concentration of porphyrin in tube x. (For these calculations tube N is numbered 0 and the tubes on either side of N are numbered +l, +2, +3, etc., and -1, -2, -3, etc. The theoretical curve is symmetrical about N, so the total concentration of solute in tube x = +l is the same asthatinx = -1.)

Overloading-Because the porphyrins are relatively insoluble compounds, a limit is set to the quantity of porphyrins that can be separated in the Craig machine at any one time. One way to overcome this concentration effect is to scatter the initial porphyrin solution in the first six to seven tubes of the apparatus (2). This procedure does not result in any appreci- able distortion of the shape of the distribution curve from that to be expected from theory (see Experiment 4 below). Another way to over- come the overloading is to add some additional component to the two- phase system to increase solubility. This has been investigated in Experi- ments 2 and 3 and its advantages and disadvantages are discussed.

EXPERIMENTAL

Experiment 1 demonstrates that the porphyrins used behave as solutes which conform to the partition law and that marked deviations from the theoretical curves (Fig. 3) represent porphyrin impurities.

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The phases used were prepared by equilibrating 1500 cc. of 0.085 N

aqueous HCI with 1500 cc. of ethyl ether free from peroxides. A mixture of 19 mg. of hematoporphyrin.2HC1, 25.2 mg. of deuteroporphyrin*2HCl, and 25.5 mg. of mesoporphyrin (Fig. 1) was dissolved in a solvent consisting of 7 cc. of aqueous phase plus 3 cc. of tetrahydrofuran. The tetrahydro- furan was added to increase the solubility of the porphyrins. This solution was placed in Tube 0 of the counter-current apparatus. After several transfers had been made, thus diluting the tetrahydrofuran, a sludge was formed at the interphase, indicating oversaturation or overloading with

Tube No. FIG. 3. Counter-current fractionation in a Craig machine of a mixture of hema-

toporphyrin, deuteroporphyrin, and mesoporphyrin, with ether and 0.1 N HCl. The concentration of porphyrin contained in each tube of the machine is plotted against the tube number as abscissa. The first concentration band at the left represents hematoporphyrin, the second represents deuteroporphyrin, and the third represents mesoporphyrin. The dash curve is the distribution to be expected from partition theory.

porphyrins. This sludge gradually disappeared during the next ten trans- fers. During a period of 6 hours a total of 118 transfers was made. The room temperature at the start was 22’ and rose to 25.5’ by the end of the experiment.

At the end of the experiment the three main porphyrin components had separated into three broad “concentration bands,” each band being pink in color and occupying a group of tubes (Fig. 3). The first band, repre- senting hematoporphyrin, extended from Tubes 0 to 17. The second band, representing deuteroporphyrin, extended from Tubes 40 to 70, and the third band extended from Tubes 75 to 101. Tubes 100 to 118 represent porphyrins withdrawn from the end of the machine. Fig. 3 is a plot of porphyrin concentration as ordinate versus tube number as abscissa.

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s. GRANIcK AND L. BOGORAD 787

Deviations from theory are evident. A minor deviation is to be ex- pected because of the gradual increase in temperature during the experi- ment. A more serious deviation is caused by porphyrin overloading at the start of the experiment, which would result in a broadening of the concentration band. A broadening is observed in the second, i.e. deutero- porphyrin band, suggesting that it was this porphyrin which formed a major part of the sludge.

Greater deviations from theory are probably to be ascribed to porphyrin impurities. For example, the second or deuteroporphyrin band is unsym- metrical, indicating impurities in Tubes 59 to 71. Evidence that this deviation can be accounted for by at least one porphyrin impurity has been obtained by a paper chromatography method to be published later. By this method, Tubes 62, 65, and 67 have been found to contain besides deuteroporphyrin one additional porphyrin which has an RF value between that of deuteroporphyrin and mesoporphyrin. Considering the method of preparation of deuteroporphyrin, its distribution in the counter-current machine, and its RF value, the unknown porphyrin may well be a monoethyl deuteroporphyrin (Le., in which an H at the 2 or 4 position (Fig. 1) is replaced by an ethyl group). From a comparison of the areas under the experimental and theoretical curves, it may be estimated that the original crystalline deuteroporphyrin might have contained as much as 10 to 15 per cent of this impurity.

Another impurity is evident in Tubes 110 to 118. Observation by a hand spectroscope revealed a sharp band at 645 rnp, suggesting that one of the porphyrins present here was a porphyrin oxidation product. Other impurities are suggested by deviations of the curves from theory. For example, some impurities are possibly present in Tubes 20 to 35. Also an impurity is suggested by the region of the curve representing Tubes 94 to 106.

In practice, these minor impurities may be eliminated by discarding the contents of certain tubes. Thus, Tubes 9 to 44, Tubes 60 to 80, and Tubes 95 to 119 may be discarded without appreciable sacrifice of the major components.

In Experiment 2 the influence of tetrahydrofuran as a component of the two-phase system on the solubility and separation of the porphyrins was studied. The two phases were made by equilibrating 1200 cc. of 0.100 N

aqueous HCl plus 300 cc. of tetrahydrofuran with 1500 cc. of ether. The acidity of the aqueous phase was 0.089 N. The tetrahydrofuran used was freshly distilled over Na to free it of peroxides. The following amounts of porphyrin were used in the experiment: 6.5 mg. of crystalline hematopor- phyrin.2HCl which had undergone a purification by the counter-current procedure described in Experiment 1, 22 mg. of deuteroporphyrin.2HC1, and 19 mg. of mesoporphyrin. A total of 120 transfers was made. The

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results of the experiment are presented in Fig. 4, at the top of which are plotted the K values or partition coefficients of the porphyrins in the respective tubes.

The solubility characteristics of the porphyrins in the two phases were found to be appreciably enhanced by the presence of tetrahydrofuran, no sludge being formed even during the first transfers. The partition coe& cients or K values of deuteroporphyrin and mesoporphyrin were 0.875 and 2.63, respectively, as compared to values obtained in the aqueous HCI-ether

1.4 K-0875 X= 2.63

12

0 10 20 30 40 50 60 70 80 90 0 Tube No.

FIG. 4. Fractionation of the mixture of three porphyrins in an aqueous HCl- tetrahydrofuran-ether system. The K values (partition coefficients) per tube in a band region are plotted at the top of the figure; deviations of K from the mean indi- cate the presence of porphyrin impurities.

of Experiment 1 (0.816 and 2.93). Using Equations 2 and 3 to calculate the tube numbers of the band maxima and the number of tubes over which the band is spread, one may calculate that for 100 transfers there would have been, in the presence of tetrahydrofuran, a separation of two tubes between the extreme ends of the bands and a separation of one tube in the absence of tetrahydrofuran. Thus, the property of the aqueous HCl-ether phases to separate deuteroporphyrin from mesoporphyrin was not appre- ciably affected by the presence of tetrahydrofuran.

Comparison of the plotted values of Fig. 4 with the theoretical curves shows that the second or deuteroporphyrin band fits the theoretical curve rather well, whereas the third or mesoporphyrin band does not. To deter- mine whether these deviations were due to some physical defect or to

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impurities, the partition coefficients of the porphyrins in the tubes were determined. As will be seen at the top of Fig. 4 the K value for deutero- porphyrin is constant from Tube 47 to Tube 63. Tubes 47 to 63 may be considered to contain pure deuteroporphyrin (i.e. pure as far as this par- ticular fractionation will permit). Discarding the other tubes of the band which contain impurities would entail a loss of only 15 per cent of the total deuteroporphyrin.

Considering the third or mesoporphyrin band, the K values suggest that impurities are present on either side of the band maximum. Evidently the effect of the tetrahydrofuran has been to change the K values of certain

Tube No. FIG. 5. Fractionation of the mixture of the three porphyrins in an aqueous HCI-

acetone-ether system. The dash curve is that to be expected from partition theory.

impurities so that the impurities now lie in the region of the mesoporphyrin band.

Although the addition of tetrahydrofuran to the aqueous HCl-ether phases has the desirable effect of making the porphyrins soluble and of not diminishing the spread between the partition coefficients, it has been found to form peroxides during the course of the experiment. This undesirable property led us to investigate the use of acetone.

In Experiment S the influence of acetone as a component of the two- phase aqueous HCl-ether system on the solubility and separation of the porphyrins was studied (Fig. 5). It was found that although acetone in- creased the solubility of the porphyrins it had the undesirable effect of diminishing the difference between the partition coefficients of deutero- porphyrin and mesoporphyrin; i.e., of diminishing the distance between the two bands.

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The phases were prepared by equilibrating 1200 cc. of 0.100 N HCl plus 300 cc. of c.p. acetone with 1500 cc. of ether. The acidity of the aqueous phase was 0.081 N. The crystalline porphyrin mixture contained 25 mg. of hematoporphyrin.2HC1, 25 mg. of deuteroporphyrin.2HC1, and 22 mg. of mesoporphyrin. The presence of the acetone diminished the difference between the partition coefficients of deuteroporphyrin (K = 0.576) and mesoporphyrin (K = 1.33). It was therefore necessary to increase the total number of transfers to 156 in order to obtain a separation of the two bands. The decrease in separating properties of the phases containing acetone, compared to the phases lacking acetone, is made evident by a calculation similar to the one described in Experiment 2. Assuming 100 transfers in the case of the phases containing the acetone, the extremes of the bands of deuteroporphyrin and mesoporphyrin would overlap by seven tubes, whereas in the phase system lacking acetone the extremes of the two bands would be separated by one tube.

To determine how well the impurities in the porphyrin mixture could be removed, the contents of the Tubes 1 to 15, Tubes 40 to 65, and Tubes 80 to 110 were collected and used for Experiment 4. The porphyrins were transferred to ether, and the ether phase was washed with water and evaporated to dryness.

In Experiment 4 the purified porphyrins of Experiment 3 were used and the purity of the separated porphyrins was examined. The two-phase system was prepared by equilibrating 1500 cc. of aqueous 0.100 N HCI with 1500 cc. of ether. The acidity of the aqueous phase was 0.090 N. The porphyrins to be separated were obtained from the selected tubes of Ex- periment 3. This porphyrin mixture was dissolved in 3 cc. of acetone, to which were added 47 cc. of aqueous phase. (This small amount of acetone does not affect the phase distribution, since it is rapidly diluted during successive transfers.) 10 cc. of this aqueous solution were added to each of the first five tubes, together with 10 cc. of ether. In this way the por- phyrin mixture was distributed equally over the first five tubes. This initial distribution of the porphyrins was made in order to avoid overload- ing. Such an initial distribution causes the final shape of the curve of a concentration band to be only slightly broadened towards its base. Prac- tically, the number of initial tubes in which one may distribute the por- phyrins is found to be about 20 per cent of the expected band width (2). The band width can be calculated from Equation 3. A total of 128 trans- fers was made. The room temperature was 20.0’ at the start and rose to 22’ by the end of the experiment.

The results of Experiment 4 are presented in Fig. 6. They indicate that the bands of deuteroporphyrin and mesoporphyrin closely approximate, with minor deviations, the curves calculated from theory. To determine

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whether these minor deviations represent impurities, the K values were determined and have been plotted in the upper part of Fig. 6. The K values of deuteroporphyrin deviate from a straight line at Tube 53. This deviation of the K values is a sensitive test for the presence of an impurity. By paper chromatography, contents of Tubes 50 to 60 were examined for impurities; only in Tube 58 was there evidence of a trace of a porphyrin other than deuteroporphyrin. Tube 62 was found to contain deuteropor- phyrin, but no mesoporphyrin, and Tubes 68 and 70 contained mesopor- phyrin, but no deuteroporphyrin. An estimate of the maximal possible

10 20 30 40 50 60 70 80 90 100 110 120 0 Tube No

FIG. 6. Refractionation of the mixture of the three porphyrins purified by a previous counter-current fractionation. The K values per tube in a band region are plotted at the top of the figure. The dash curve is that calculated from par- tition theory. The dotted curve represents the difference between the experi- mental and theoretical curves.

impurities in the deuteroporphyrin and mesoporphyrin curves may be ob- tained by subtracting the experimental from the theoretical curve. From comparison of these areas with the total areas under the curves, the max- imal impurity is calculated to be about 4.3 per cent in the case of deutero- porphyrin and 4.2 per cent in the case of mesoporphyrin. By selecting the contents of Tubes 40 to 52 and Tubes 80 to 95 impurities in these com- pounds may be eliminated.

Tubes 105 to 128 have been plotted in Fig. 6 on the assumption that the porphyrins in these tubes have an extinction coefficient similar to that of mesoporphyrin. Paper chromatography has revealed the presence only of mesoporphyrin in Tube 105. In Tube 115 there are present two uniden- tified porphyrins, neither of which is mesoporphyrin. In Tube 120 three

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unidentified porphyrins are present. These porphyrin impurities are of course small in amount. They have a high K value, and thus they would tend to move more rapidly than the main porphyrin constituents.

SUMMARY

The Craig counter-current method has been applied to the fractionation of a known mixture of three porphyrins. It has been found that the porphyrins behave like ideal solutes in the two-phase system and hence the degree to which they can be separated can be predicted from theory. Certain deviations from theory have been found, but it is shown by paper chromatography that these deviations are due to unsuspected impurities which were present in the original crystalline porphyrins.

Practical manipulative procedures in the application of the Craig ma- chine are considered. Because of the low solubility of porphyrins, the effect of adding tetrahydrofuran or acetone to the two-phase system to increase the capacity of the machine was examined, and the advantages and disadvantages of this procedure are discussed. A modification of a Beckman test-tube holder is described by means of which rapid determina- tions may be made of the concentration of porphyrin in both the upper and lower phases. Methods are discussed for the determination or estima- tion of the partition coefficients of the porphyrins. The application of specific equations for the selection of ideal conditions to carry out a separa- tion of certain porphyrins is described.

We desire to express our gratitude to Dr. Lyman C. Craig for his advice and interest in this study.

BIBLIOGRAPHY

1. Craig, L. C., and Craig, D., Physical methods of organic chemistry, New York, 2nd edition, 171 (1950).

2. Gregory, J. D., and Craig, L. C., Ann. New York Acad. SC., 53, 1015 (1951). 3. Craig, I,. C., in Corcoran, A. C., Methods in medical research, Chicago, 6,3 (1952). 4. Willstitter, R.., and Mieg, W., Ann. Chem., 350, 1 (1906). 5. Zeile, K., and ltau, B., 2. physiol. Chem., 250, 197 (1937).

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S. Granick and Lawrence BogoradCOUNTER-CURRENT DISTRIBUTION

SEPARATION OF PORPHYRINS BY

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