Separation of Porphyrins by Paper Chromatography MAX BLUMER Exploration and Production Research Division, Shell Development Co., Houston, Tex.
A paper chromatographic method has been developed for the resolution of the aggregate of porphyrin pigments occurring in ancient sediments and petroleum. In a two-dimensional chromatogram the nonacidic pigments, the esters, and the metal complexes of both are separated first. The free acids which remain at the origin are then esterified with diazomethane. A second development at a right angle to the first one resolves the newly formed methyl esters of the acidic pigments and their metal complexes. The location of the spots on the completed chromatogram can be correlated with structural features of the porphyrins. As little as 5 X gram of free porphyrin and 4 X 10-8 gram of the nonfluorescent metal complexes can be detected; a rapid semiquantitative determination is possible by comparison with a series of standards or by measurement of the spot areas.
A
LL porphyrin pigments can formally be deduced from one parent compound, the porphin. A very large number of substituted porphins are known today; most of them have been prepared synthetically, and a considerable number have been isolated from biological sources. Very few of these porphyrins have been found in petroleum and ancient sediments. These fossil pigments apparently are the most stable end members formed from the chlorophylls and hemins through a series of transformation reactions. Most abundant in petroleum and ancient sediments are the two chlorophyll derivatives, deoxophyllerythrin ( D P ) and deoxophyllerythroetioporphyrin ( D P E P ) [for the structural formulas see Fischer and Orth ( 7 ) ] . I n coals, two other pigments, mesoporphyrin ( M P ) and mesoetioporphyrin (MEP, also designated as etioporphyrin 111)are more abundant. The porphyrins occur in sediments and petroleum, both free and as complexes with heavy metal ions. The most abundant pigments are the vanadium complexes of deoxophyllerythroetioporphyrin and deoxophyllerythrin (DPEP-V and DP-V). Two other types of complexes have been described as containing nickel (8)and iron (10)as the central atoms. Complexes of other metals have never been identified with certainty. The presence of porphyrins in geological materials was described for the first time in 1934 by Treibs ( 1 3 ) . Historically, this discovery is of great importance because it established for the first time a direct link between constituents of crude oil and their parent compounds in the biological source material. With the finding of porphyrin metal complexes, Treibs also indicated that organic molecules can be the carriers and possibly also the concentrators of heavy metals in petroleum and oil shales. The occurrence of these pigments in petroleum and ancient sediments is still of great interest; it was therefore decided to review the available methods for the determination of porphyrins in geological materials.
substitution on the porphyrin ring and can be used for a t least a tentative structural analysis of the pigments. Unfortunately, the spectra of many porphyrins with similar structure are almost identical. Mesoporphyrin and mesoetioporphyrin, for instance, and also deoxophyllerythrin and deoxophyllerythroetioporphyrin cannot be distinguished by spectroscopy in the ultraviolet and visible regions. The spectra of the metal complexes are determined by the complexing metal ion rather than by the substitution of the porphin ring. Absorption-spectroscopic methods (Q), although they present a very sensitive tool for the detection of porphyrins, cannot provide the necessary detailed information about the relative contribution of individual closely related pigments in a fossil porphyrin mixture. This information can be obtained only by a separation of the mixture by some selective method and a consecutive determination of the individual pigments. Methods for the isolation of individual porphyrins from mixtures have been described by Treibs (14). These separations, based on fractionated chromatography and stepwise solvent extraction, are extremely tedious and cannot be carried out as routine methods. Improved techniques for the isolation of porphyrins from biological materials have been developed recently. With paper chromatography, they effect an excellent and fast resolution of the pigments and require only very small samples.
DETERMINATION OF PORPHYRINS IN GEOLOGICAL MATERIALS
The porphyrin pigments possess a typical absorption spectrum with strong bands in both the visible and near-ultraviolet regions. The spectra are sensitive to differences in the type of 1640
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V O L U M E 28, NO. 11, N O V E M B E R 1 9 5 6 Another method, described by Chu, Green, and Chu ( d ) , permits the separation of the porphyrin methyl esters. -4chloroform-kerosine mixture first serves as developer. hfter the chromatogram has been dried, a second development follows in the same direction, this time with a 1-propanol-kerosine mixture. This unidirectional double development appeared necessary for a satisfactory resolution between the adjacent spots and for reduction of trailing. Here again, an inverse relationship is found between the R , values and the number of the (esterified) carboxyl groups on the porphyrin ring. The method has been modified by Bogorad and Granick (s),who use the same developer systems but apply them in a two-directional double development. Their technique permits a better resolution of adjacent spots, especially if they are caused by the presence of very different quantities of pigments. Another modification has recently been described by Rappoport and associates (12),who use the high degree of resolution obtained by a circular development. The method requires relatively large samples, however, and necessitates elution of the pigments for quantitative determination. All these methods show a serious lack of resolution for the etioporphyrins and porphyrin mono- and dicarboxylic acids which are most abundant in geological materials. Attempts were made, therefore, to find developer systems that would spread these pigments more evenly over the paper. The ability to differentiate sharply betneen the free acid group and the ester-etioporphyrin group in Chu's technique is very desirable. Several difficulties, however, prohibit the application of this method t o the separation of fossil porphyrins. I n the first place, mesoetioporphyrin and deoxophyllerythroetioporphyrin are not resolved, because both migrate with the solvent front. The one-dimensional double development is time-consuming and requires close attention during the development. Kerosine is not a desirable component of a developer because of its nonreproducible composition and low volatility. During the slow drying of the chromatograms, an increase of the spot areas has been observed. This prohibits the direct quantitative estimation of the porphyrins by scanning for light absorption or by measurement of the spot areas. I t was hoped that most of the difficulties mentioned above might be overcome by a better choice of solvents and paper. Preliminary experiments indicated that a single development did not necessarily lead to heavy trailing, if the paper was properly selected. Contrary to Chu's report, no loss of sensitivity in the porphyrin detection was found, if a pure hydrocarbon like isooctane was substituted for the kerosine as the paraffin component of the developer system EXPERIMENTAL
The reagents used were iso-octane (2,2,4-trimethylpentane), Phillips Petroleum Co., pure grade; carbon tetrachloride, Mallinckrodt, analytical reagent grade; and fluoranthene, Matheson, Coleman and Bell. Whatman No. 3 filter paper is recommended for general use because of the reduced tendency for trailing. The chromatograms were carried out on the filter paper supported in a horizontal position in a large desiccator, the walls of which were lined with heavy filter paper wetted with solvent in order to keep the atmosphere in the developing tank saturated. Any other development technique (ascending, descending) might be used. The chromatograms are developed in the direction of the grain of the paper; in the two-dimensional separations, the first separation is carried out in this direction. Standard chromatographic techniques were used for spotting, developing, and drying of the sheets and the planimetry of the spots. Esterification of Spots by Diazomethane. The spot of the acidic porphyrins which remains a t the origin after the first development can be esterified with either diazomethane gas or a solution. The reaction of the methylating agent has to be restricted to the immediate area of the spot because excessive methylation of the paper would change the R, values.
1641 I n practice, this is easily achieved by slowly adding a solution of diazomethane from a capillary pipet to the spot, balancing the rate of addition and the rate of evaporation of the solvent. Cyclohexane has been selected as a solvent that dissolves a sufficient amount of diazomethane but will not affect the porphyrin spots. Diazomethane is prepared from either nitrosomethylurea or Xmethyl-N-nitroso-p-toluenesulfonamide (b) (Eastman organic chemicals). Nitrosomethylurea is not available commercially, but small quantities as needed for this procedure are rapidly prepared following Weygand's procedure 1 (15,p. 261). Nitrosomethylurea is unstable a t room temperature and should be kept under refrigeration; because of its toxicity, diazomethane and its solutions should be handled with due care.
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Figure 2. R , values of porphyrins in carbon tetrachlorideiso-octane mixtures
Whatman No. 1 paper. R( values on this paper are very close t o those obtained o n N o . 3 paper.
Diazomethane in Cyclohexane. I n a well-ventilated hood, 10 ml. of cyclohexane and 10 ml. of 20% aqueous potassium hydroxide are warmed to about 36' C. in a 50-ml. Erlenmeyer flask. One hundred milligrams of nitrosomethylurea is dropped into the hydroxide layer. As soon as all of it has reacted, the flask is cooled in ice. The diazomethane solution is withdrawn for use with a capillary pipet. Porphyrins. The porphyrins used for the separations described here were repared in this laboratory. Hemin (Eastman organic chemicalsr served as a starting material for the preparation of protoporphyrin ( P P ) (removal of iron by treatment with iron powder and formic acid), of mesoporphyrin (catalytic hydrogenation of protoporphyrin), and of mesoetioporphyrin (decarboxylation of mesoporphyrin). The vanadium complexes of deoxophyllerythrin and its etioporphyrin were isolated from a porphyrin-rich Triassic oil shale from Serpiano, Switzerland ( 1 ). Free deoxophyllerythrin and deoxophyllerythroetioporphyrin were prepared from the vanadium complexes and the nickel and copper complexes were synthesized from nickel or copper acetate and a solution of the porphyrins in acetic acid. The methyl esters of the porphyrins were prepared by esterification of the pigments with diaxomethane. Selection of Solvent Systems. As a consequence of the preliminary experiments, a series of chromatograms was prepared in which chloroform-iso-octane mixtures of varying compositions were used as developers (Figures 1 and 2). No trailing was observed, and a good resolution between mesoetioporphyrin and deoxophyllerythroetioporphyrin was obtained, with a maximum a t about 30% chloroform in the developer mixture. Replacement of the polar component, chloroform, by the unpolar solvent, carbon tetrachloride, should decrease the R , values
1642
ANALYTICAL CHEMISTRY
of the porphyrins in a developer with comparable concentration of chlorinated hydrocarbons. This effect was confirmed experimentally (Figure 3). Again, an excellent resolution of mesoetioporphyrin from deoxophyllerythroetioporphyrin and DP-Me (Me is used to characterize a methyl group) from MP-Me2 took place, The relative merits of the two different developer types were compared in a series of chromatograms using two developers giving an equal R/ value for one standard porphyrin (DP-Me) and giving also the highest degree of resolution possible within the chloroform and carbon tetrachloride developer series. The R f values for several porphyrins were then determined in the two systems, and the resolution was calculated as the difference in the R f values (Table I).
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Relationship between Rf Value and Constitution of Porphyrins. I n normal phase chromatography, the solutes are distributed between the more polar stationary phase and the less polar mobile one. The distribution coefficients are influenced by the constitution of the solutes. Relatively small differences in the polarity of similar compounds can cause a change of the partition coefficient and R, value of sufficient magnitude to permit a separation. Systematic changes of the Ri values are frequently found between the members of homologous series of compounds and can be used in the determination of the structure of unknowns.
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A square sheet of filter paper is spotted with the initial mixture in one corner and then developed as described above. The solvents are removed by evaporation, and the unresolved spot a t the origin is then made to react with diazomethane. After a few seconds, the acidic porphyrins are completely esterified. The paper is then developed a second time with the same solvent but a t a right angle to the first development. The pigments that migrated during the first development migrate again with the same Rf value and reach a position on a diagonal line over the paper. The esters that were formed by the reaction with the diazomethane now migrate away from the origin and are found along one edge of the sheet (Figure 4).
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amount of trailing found on Whatman S o . 1 filter paper. With the Whatman KO.3 paper, a single development is satisfactory. Elimination of the second development results in a considerable saving of time as compared to the original method. Separation of Acidic from Nonacidic Porphyrins by TwoDimensional Development. When mixtures of porphyrin carboxylic acids with etioporphyrins and esters are chromatographed, an unresolved spot representing the acids remains a t the origin. A simple method for their subsequent resolution has been found.
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F i g u r e 3. Two-dimensional s e p a r a t i o n of p o r p h y r i n s
The resolving poner of the carbon tetrachloride system is better than that of the chloroform system. Carbon tetrachlorideiso-octane mixtures are therefore recommended for general use. The relative concentration of the two components of the developer can be adjusted to conform to the type of porphyrin mixture to be separated. If only etioporphyrins are present, a low R j developer containing 50 to 70% carbon tetrachloride is used; if esters have to be resolved, a developer containing between 70 and 85% of the same solvent is preferred. Selection of Filter Papers. The choice of the most suitable type of filter paper is one of the many factors which influence the success of a paper chromatographic separation. On its texture and composition depend, among other factors, the rate of migration of the mobile phase, the degree of resolution, and the amount of trailing of the spots. I n order to find the best conditions for the separation of porphyrins, chromatograms were carried out using Whatman filter papers 1, 2, 3, and 4. The R f values were determined and the shape of the spots and the amount of trailing were compared. The R f values obtained on the four papers are identical within =kO.O5 unit, but a great difference exists in the appearance of the spots. U n i m u m trailing, maximum resolution between adjacent spots, and the sharpest definition of the spots are obtained on the thick filter paper, Whatman No. 3. The quality of the separation decreases from this paper to Whatman No. 1 and is insufficient on No. 2 and S o . 4. The technique of unidirectional double development had been devised by Chu, Green, and Chu in order to overcome the serious
T a b l e I.
Resolution of Porphyrins by Different Developer Solvents
Development. Horizontal in saturated atmosphere of the solvent vapor Paper. Whatman A-0. 3 Developers. Iso-octane containing 30 rolume 70 chloroform, and isooctane containing 70 rolume yo carbon tetrachloride Difference in R/ Value in System Carbon Porphyrins tetrachloride C hlorof a r m RIEP hIP-Me2 0 42 0 52 DPEP DP-Me 0 22 0 32 PP-Me2 0 05 DP-Me 0 OS DP-hle MP-hlel 0 02 0.06 DPEP RlEP 0 18 0 18
+ + + + +
The present method for paper chromatography of porphyrins permits a direct correlation of the R f values with structural properties of the pigments (Figure 5 and Table 11). The high R, values of nonacidic porphyrins-e.q., D P E P and MEP-are reduced to zero upon the introduction of one or more carboxyl groups. Upon esterification, the R f values increase again, but remain considerably loiver than those of the etio pigments. In the series of the esters, the R , values decrease from the monoto the dicarboxylic ester (from DP-Me to MP-Men and PP-Me*). Esters of polycarboxylic acids would have a very low R fvalue or would not migrate at all, but they could be resolved with a more polar eluent-e.g., a chloroform-iso-octane mixture containing a high percentage of chloroform. Upon complexation m-ith a metal like copper or nickel, the porphyrin loses tlvo strongly polar sites (the --NH groups of the pyrrole rings), and the R , values increase slightly. Complexation with vanadium introduces the oxygen atoms of the vanadyl ion, increasing the polarity of the molecule and lowering the R , value.
1643
V O L U M E 28, N O . 1 1 , N O V E M B E R 1 9 5 6 Even very *mall differences in the substitution of the porphin system show up clearly. DPEP with the isocyclic five-membered ring is well separated from M E P with an open side chain and the same number of carbon atoms. LlP-Me2, which does not have any double bonds in the side chains, moves slightly faster than PP-1\Ie2 with two vinyl groups, and can be separated from it on a sufficiently large sheet of paper.
MEP
statement that the use of pure alkanes in place of kerosine reduces the fluorescent intensity of the porphyrin spots. Phorphyrin metal complexes lack the sensitive fluorescence of the uncomplexed pigments, and their characteristic color can be detected only a t relatively high levels of concentration, where the danger of trailing exists. An indirect method for their detection, making use of their intense ultraviolet light absorption, has been developed. For this purpose, the completed and dried chromatogram is briefly sprayed with or dipped in a saturated solution of fluoranthene in n-pentane. I t is then rapidly airdried and observed over a source of long-m-ave ultraviolet (366 mfi). The sheet exhibits an even bright fluorescence except for dark spots where porphyrin-metal complexes or free porphyrins absorb the ultraviolet radiation and prohibit the fluorescence of the fluoranthene.
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E S T E R S OF MONOCARBOXYLIC ACIDS
E S T E R S OF DICARBOXYLIC ACIDS
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R Jvalues and constitution of porphyrins 0.2
Limit of Detection and Usable Range of Method. After the separation, the porphyrin spots can be very easily located on the paper b y observing their fluorescence in long-wave ultraviolet radiation. Wetting the sheet with iso-octane during the observation helps in obtaining a higher sensitivity and better visual resolution of adjacent spots. Impregnation with iso-octane makes the paper translucent and increases the amount of fluorescent radiation that reaches the eye by decreasing scattering and absorption. Iso-octane does not dissolve the porphyrins and therefore leaves the spots unaltered for a subsequent quantitative determination. A very low limit of detection can be realized in this manner; for mesoporphyrin dimethyl ester it was found a t 0.005 y This limit is distincly lon-er than the one reported h5- Chu, Green, and Chu, in disagreement n-ith their
Table 11.
R f Yalues of Free Porphyrins, Their .\letal Complexes, and Methyl Esters
Development. Horizontal in saturated atmosphere of solvent vapor Paper. Whatman No. 3 Dereloper. Carbon tetrachloride containing 30 volume R iso-octane Porphyrin Rf Value 0.63 .\IEP DPEP-Ni 0.54 DPEP 0.47 DPEP-T 0 20 DP-Xi-.\le 0.23 DP-Me O,l5 DP-V-Me 0.08 LIP-Xi-hIez 0.12 XlP-hIea 0.10 PP-Cu-JIe2 0.13 PP-Ni-Ale? 0.10 PP-hlez 0.07
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JVith this technique, very small quantities of porphyrin metal complexes can be detected: for DPEP-T' the limit of detection lies at 0.04 y. Compounds that have a strong ultraviolet absorption and are not separated from the porphyrins during the development will interfere with this method of detecting the metal complexes. The usable range of a paper chromatographic method is limited by tx-o concentrations, beyond which separation and accurate determination are impossible. The loTTer one of these values is identical with the limit of detection. The upper limit is determined b j the maximum capacity of the mobile and immobile phases for the compounds. -4t higher concentration, the initial solid or liquid phase is dissolved only gradually upon development causing the formation of trails. The limit a t which this trailing appears depends on the initial spot area, the thickness of the paper, the solubility of the solute in the developers, and other factors that affect the capacity of the mobile and immobile phases Under the experimental conditions described above, trailing of a porphyrin takes place when its concentration in the porphyrin mixture is equal to or higher than 0.5 y . The two limits discussed above restrict the useful range of the method to about 0.5 to 0.005 y for the free porphyrins and 0.5 to 0.04 y for the metal complexes.
A N A L Y TI C A L C 3 E M I S T 2 Y
1644 Table 111. Estimation of Porphyrins by Comparison w i t h Standards Development. Horizontal in saturated atmosphere of developer vapor Paper. Whatman No. 3 Developer. Carbon tetrachloride containing 30 volume 70iso-octane Porphyrin. hfesoporphyrin dimethyl ester Deviation froiii Porphyrin Porphyrin True Valur, Estimated Present, X10-6 G. x10-6 G. % 0.04 0.07 0 10 0.40
0 05 0 OG 0 08
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SEMIQUANTITATIVE DETERMISATION OF PORPHYRINS
From the point of view of speed and simplicity, methods for the quantitative determination of chromatographically separated compounds can be grouped in two classes. Into one class fall methods for a direct evaluation of the spots on the filter paper (comparison with a series of standards, measurement of spot area or diameter, scanning for light absorbance, fluorescence, or radioactivity). Into the other class fall methods that necessitate the recovery of the separated components from the paper before the determination. Methods in the first gro:ip are us.ially very rapid, but often not so accurate as those in the second group. Estimation of Porphyrins by Comparison with Standards or Measurement of Spot Area. By far the most simple, but not a very accurate, method of estimating chromatographically separated compounds consits in visual comparison of the spots with a series of standards. Results falling within the limit of Z ! Z ~ Oerror ~ ~ can be obtained by such an estimation (see Table 111). The main advantage of this method is the speed and ease with n-hich the estimation is carried out and the independence of the error from concentration. Most other direct or indirect determinations lose their accuracy in the neighborhood of the limit of detection. Higher accuracy, especially a t higher levels of concentration, can be obtained if the area of the spots is measured and compared nith the area of standard spots. Some personal judgment is involved in marking the edges of the spots; calibration and measurement should therefore be carried out by the same person. The area of the initial spots should be kept as small and constant as possible by forced evaporation of the solution during spotting. Large deviations in the initial spot area are reflected in variations of the final spot size. For best accuracy, calibration spots should be carried along on the same sheet that is used for an actual determination. If all these points are carefully controlled, the error of the determination can be kept within the limit of &IO%. A typical calibration curve for the determination of hlP-i\le:! is plotted in Figure 5 . Certain polar compounds which might contaminate a porphyrin concentrated from natural sources have an eluting effect on the porphyrins. The resulting increase in the R l value causes an increase in the spot area, which mill interfere with a quantitative determination by the above method. To avoid this type of interference, a porphyrin determination from the spot area should
not be attempted if the value is increased more than 0.03 R , unit over the value of a pure standard. The two techniques described are of sufficient accuracy for many purposes; for a more accurate determination, the use of a scanning technique for light absorption or fluorescence or the elution of the porphyrins and a consecutive microspectrophotometric determination of the pigments should be attempted. CONCLUSIOKS
A method developed for the separation of porphyrins that occur in petroleum and sedimentary rocks increases the resolving power from earlier paper chromatographic methods to the point where metal complexes can be separated from each other and from the corresponding free porphyrins. The technical difficulties encountered by Chu, Green, and Chu in the separation of porphyrin methyl esters have been overcome by a careful selection of filter papers and developer solvents, and the need for a onedirectional double development has been eliminated. Twcdirectional development results in a sharp separation of nonacidic and acidic porphyrins and permits the separation of both groups into individual components. The limits of detection of the uncomplexed pigments are lower than previously reported and a sensitive method has been developed for detecting the porphyrin metal complexes which lack the fluorescence of the uncomplexed pigments. A semiquantitative determination of the pigments is possible by comparison with a series of standards; for better results, measurement of the spot areas is recommended. The R , values of the pigments can be used for their identification and also for structural evaluation of porphyrins with unknown structure. They are used in much the same way as the p H and acid values of Fischer and are applicable also to metal complexes and acid-sensitive pigments. ACKNOWLEDGMENT
The author wishes t o express his appreciation to the Shell Development Co. for permission to publish this article. LITERATURE CITED
Blumer, If.,Helu. Chim. Acta 33, 1627 (1950). Boer, Th. J. de, Backer, H. J., Rec. trav. chim. Pays-Bas 73, 229 (1954).
Bogorad, L., Granick, S., J . B i d . Chem. 202, 793 (1953). Chu, T. C., Green, A . A . , Chu, E. J.-H., Ibid., 190, 643 (1951).
Corwin, L. M., Orten, J. M., AN.~L.CHEM.26, 608 (1954). Eriksen, L., Scand. J . Clin. Lab. Inzest. 5, 155 (1953). Fischer, H., Orth, H., “Die Chemie des Pyrrols,” vol. 11, Akademische Verlagsgesellschaft, Leipzig, 1937. Glebovsksya, E. A., Vol’kenshtein, hZ. V., Zhur. Obshchei K h i m . ( J . Gen. Chem. U.S.S.R.) 18, 1440 (1948). Groennings, S., ANAL.CHEM.25, 938 (1953). Moore, J. W., Dunning, H. N., I n d . Eng. Chem. 47, 1440 (1955). Kicholas, R. E. H., Rimington, C., Biochem. J . 48, 306 (1951). Rappoport, D. A., Calvert, C. R., Loeffler, R. K., Gast, J. H., ANAL. CHEM. 27, 820 (1955). Treibs, A., Ann. Chem. 509, 103 (1934). Ibid., 517, 172 (1935).
Weygand, C., ”Organic Preparations,” Interscience, New York, 1946. RECEIVED for review March 23, 1956. Accepted July 2, 1956. Publication 85, Shell Development Co.
