Quantitative Determination of the Porphyrin Aggregate in Petroleum SIGURD GROENNINGS Shell Development Co., Emeryville, Calif. To facilitate investigation of porphyrins in petroleum, an expedient method of analysis was developed, based on the original work of Treibs. The porphyrin aggregate is extracted with hydrogen bromide in glacial acetic acid, transferred to chloroform, and the light transmittance is measured at two characteristic wave lengths. The content is read from a graph of the relation between transmittance and concentration obtained from measurements of standard solutions of porphyrins isolated from petroleum. The procedure is of particular interest in the study of the origin of petroleum and is potentially useful in oil exploration where the porphyrins may serve as crude oil “markers.”
0
RGANIC compounds of complex structures definitely a b tributable to characteristic components of plant and animal life-as, for instance, porphyrins (15), chitin (1), anthraquinone dyes ( I d ) , and estrogenic active substances ($)-have been encountered in rocks of various ages as mineral components. In 1933 green colored derivatives of chlorophyll (16) as well m of hematin (in fossil animal excrements) ( 5 ) had been found intact in Tertiary sediments. These discoveries led Treibs to undertake a systematic search for well-defined organic compounds in rocks, and he found that extracts of oil shales as old as the Triassic, and also extracts of petroleum, exhibit spectra characteristic of complex salts of porphyrins with metals (11). Recently the presence of vanadium complexes in petroleum has been confumed by Skinner ( 1 0 ) and they have been isolated by Overberger et al. (9); Dunning et al. ( 4 ) have shown that metalporphyrin complexes are major contributors t o the interfacial activity of crude oil. Trace constituents of petroleum are stimulating growing interest owing to their effect on oil processing. The porphyrins are singularly interesting because, by virtue of their nature, they may be regarded as remnants of prehistoric vegetal or animal life. Hence further study of them may well contribute to the solution of the problem of the origin of petroleum, and the amount remaining throughout the eons of time could conceivably mark the oils and thus be useful in exploration. As an aid in future work, therefore, a procedure is presented for their determination in bituminous materials, which is believed to be quantitative. The name “porphyrins” in a broad sense designates a class of compounds of the following general structure:
solids of high melting point, and, being difficult to crystallize they are usually obtained as reddish brown powders. Dissolved in organic solvents, in which they exhibit beautiful red colors, the porphyrins are readily detected even in the minutest amounts by aid of the spectroscope. Their spectra show distinct absorption bands in the visible region (green to red) and four of these bands are so characteristic for this class of pigments as to leave no doubt about their identification. The positions in the spectrum of these four absorption bands of the main porphyrins from chlorophyll and from hematin, when dissolved in ether, are a t the following wave lengths: from chlorophyll, 4975, 5317, 5675, and 6216 A , ; and from hematin, 4949, 5282, 5676, and 6233 A. Thus they show essentially the same absorption spectrum, but there is a slight difference between the chlorophyll porphyrine and the hematin porphyrins which may be detected in a good spectroscope.
WAVE LENGTH.
Figure 1. Upper.
Lower.
Thus, they consist of four pyrrol rings linked together to form a large ring, and this is the nucleus upon which the complex molecules of both chlorophyll, the green coloring matter in plants, and hematin, the red coloring matter in blood, are constructed. For further information pertaining to this large class of compounds, reference must here be made to the textbooks (6, 8). The porphyrins, of which a great variety occur in nature, are
f
Spectrophotometric Absorption Curves of Porphyrins
Absorption curve of porphyrins isolated from crude oil sample No. 60. Beckman spectrometer; ether aolution Microphdtometric curve of etioporphyrin I (from chlorophyll) in alcohol-ether through 18-mm. test tube (3)
These porphyrins, together with others of less importance, were detected by Treibs in various bitumina, and as a means of further substantiation of these findings, he synthesized some of the porphyrins and found them to be identical with those actually isolated from bitumina. The porphyrins are present in these materials as complex salts with metals; they have also occasionally been found in the free state as well as in their more or less original combination in the chlorophyll molecule, either as chlorophyll as such or as intermediate decomposition products (phyllins, phaeophorbides, chlorins, rhodins. etc.). In view of
938
V O L U M E 25, NO. 6, J U N E 1 9 5 3 the great complexity of chlorophyll and hematin chemistry it is not possible, a t least not for the time being, to specify the exact mode of occurrence of porphyrins in a given material and to isolate each of these compounds, They can, however, be liberated from their combinations in extracts of the bitumina by digesting the latter with glacial acetic acid saturated with hydrogen bromide, followed by separation from the oily matrix by virtue of their great solubility in aqueous mineral acids. I n this way a p parently all types of porphyrins are released from their combinations and extracted collectively by the acids. The porphyrins are finally collected in ether for spectroscopic examination. Spectroscopic examination was made of porphyrins thus obtained from numerous crude oil samples. T h e red ether solution of the porphyrins was studied in a quartz prism Gaertner spectroscope. This instrument is designed primarily for investigations in the ultraviolet and near-violet region, and the dispersion in the visible region, where the porphyrin bands occur, is rather small; however, a grating spectroscope was not available. A typical spectrophotometric absorption curve is shown in Figure 1 (upper curve); the four absorption bands, situated a t wave lengths of approximately 4975, 5300, 5675, and 6225 il., all of which are characteristic of the chlorophyll porphyrins and the hematin porphyrins, are well recognizable. Further identification is afforded by comparison with the absorption curve for etioporphyrin obtained from chlorophyll with maxima a t these wave lengths (lower curve), according to Conant ( 8 ) . The identification was further substantiated by results of an elemental analysis of the porphyrins isolated from a California crude oil sample in connection with the development of a rapid method for quantitative estimation of porphyrin content, as discussed below. These isolated porphyrins also exhibited the characteristic absorption bands. Actually the band just below 5000 -4. is closer to 4975 A. (chlorophyll porphyrins) than to 4949 A. (hematin porphyrins). It is interesting in this connection that the porphyrins found by Treibs were almost exclusively of chlorophyll origin. The absolute amount of porphyrins in petroleum is small. Treibs made rough estimates on the basis of spectroscopic analyses and found that the porphyrin content of crude oils varies from nil to ea. 400 p.p.m., or 0.04%, and similar results have been observed in these laboratories. In shale oil he found amounts of the same order of magnitude; in coal, little or none; and in peat, none. The greatest amount of porphyrins found in any bituminous material was extracted from an oil shale (marl) of Triassic age; it contained 0.4%. Considering the fact that dry leaves contain only 0.8% chlorophyll, corresponding to 0.6% porphyrins, the amount of porphyrins found in bituminous materials is truly remarkable. Porphyrins, particularly their vanadium and iron complexes, are very stable compounds, provided they are not exposed t o oxidation, which may be the reason why they are not encountered in peats. I n shale oil and petroleum the porphyrins are well protected from oxidation. They are contained in the heavier portion (over 400 molecular %-eight)of petroleum and may be completely extracted by acids, provided that heavy sludges are not allowed to form prior to or during the acid digestion period by precipitation of asphaltenes upon dilution with petroleum ether. This solvent, employed by Treibs, has therefore been replaced by benzene, in which the asphaltenes are almost completely soluble. QUANTITATIVE DETERMINATION OF PORPHYRINS IN CRUDE OIL
Extraction Procedure. The extraction reagent is a saturated solution of hydrogen bromide in glacial acetic acid prepared by introducing anhydrous hydrogen bromide into the acid a t room temperature, Ion-ering the temperature as the absorption proceeds; the saturated solution will have a density of 1.30 a t 0" C.
939
b
The crude oil is first topped by a continuous short-contact vacuum flash distillation to remove light ends (boiling below ea. 250' C. under atmospheric pressure), which might cause separation of the reaction mixture into two phases, impeding the reaction. This distillation may be conveniently conducted a t 3 mm. and 111" C., the refluxing temperature of toluene, employed as constant boiling liquid in the jacket of the column. Topped crude oil equivalent to 100 grams of whole crude oil (dry basis), or more if the porphyrin content is exceptionally low, is diluted with an equal volume of C.P. benzene, placed in a glass ampoule and cooled in solid carbon dioxide. One volume of reagent (based on topped crude) is added, and the ampoule is sealed, wrapped in a cloth for safety, and kept in an oven at 50'-C. for 4 days, with occasional shaking. TOPPED CRUDE OIL, BENZENE, ACETIC ACID+ H b 4 d o y r at 5 0 ' C
[Ti
[A]
I
/
Mixed
OILY PWSE
brownish red
I I
Washing with ether
-20%
-------
HCi e:tractim (black oily pha* dircardcd )
Purified mixed
[O]
I
Addition af ether and saturated sodium a etate .solution
(dark ether phase discarded)
f
Washing k b wafer and I wRh 7% HCI extroctions
.
(faintly ycllaw odd phase discarded 1
I Addion of ether and saturated sodium acetate solution. and step [C] to [HI repeated.
1 discarded 1
1
[I]
Purified l7%HCl Z T C T
Addition of c h k o f m and saturated sodium acetate sdution
1
[ J ]
I
PORPHYRINS IN CHLOROFORM deep red
f X H C l PHASE calorless
(discarded 1
940
ANALYTICAL CHEMISTRY
0
shaken a i t h (usually three) portions of 25 ml. of ether until the acid phase becomes very nearly colorless, whereupon it is discarded. The ether washings are combined mith the main ether phase, E. The latter is washed once with an equal volume of distilled water and the porphyrins are extracted from the ether with 25-ml. portions of iyo hydrochloric acid until the color of the ether phase, H , has been reduced to a faint yellow or possibly faint green. The iyohydrochloric acid extract, G , is red. Transference of the porphyrins to ether with the aid of sodium acetate, and extraction with 7y0hydrochloric acid, are repeated, I . (Complete removal of porphyrins from a phase may be checked with the aid of a pocket spectroscope.) Identification tests (qualitative spectroscopic examination) were carried out u ith this ether solution. For quantitative tests (colorimetric measurements), it R as found preferable to transfer the porphyrins to chloroform owing to the greater solubility of the porphyrins in this solvent. The transfer is made mith the aid of sodium acetate as described abovr. The deep red chloroform solution, J , is washed twice with distillr I r a t e r and filtered through cotton to remove moisture. COLORIXIETRIC MEASUREMENTS AhD STANDARDIZATION
The volume of chloroform solution is adjusted so that the porphrrin eoncentration falls within the range of the most conveniently observed color intensities (20 to 50 % transmittance) and the light transmittance is determined using the two principal absorption hands, near 5000 and 5300 A . Other bands may be used in addition if desired. I n the present study a Pulfrich photometer (Zeisx) R as used, u ith a 1-cm. cell, and filters S-50 (trans nittance maximum a t 4900, approximate range 4600 to .il-tO'i _-.., and S-53 (transmittance maximum a t 5300. aooroximate range 5070 to 5600).
\
\
- -
\
5
0
IS
20
25
35
30
40
P O R P H Y R I N C O N C E N T R A T I O N , MG. PER LITER
Figure 3.
acetate) arid re-extraction p i t h 770 hydrochloric acid were repeated 10 times; the final chloroform solution was washed well with Rater and the chloroform was distilled off, leaving about 4 grams of a dark reddish-brown mass of semicrystalline (*onsistency. Sumerous attempts a t crystallizing the porphyrins proved futile, but a nonsticky powder could be obtained by re-solution in chloroform and precipitation with five parts of 95% methanol. After 10 such precipitations, involving considerable loss of material, 385 mg. of a purified powder were obtained which, under the microscope, appeared as tiny dark reddish brown broken prisms of a violet outer tone.
Table I.
Elemental rinalysis of Petroleum Porphyrins
Composltion, YG
C32H36N4
Carbon Hydrogen Xitrogen Total
80 62 7.62 11 76 ___ 100 00
Porphyrins Isolated from Trinidad Crude (Treibs) 79 8 7.3 98 9
Porphyrins Isolated froiii Crude Oil 60 7 9 . 5 (micro) 7 . 7 (micro) 1 1 . 5 (macro) ~. 98.7
S o definite melting point could be observed, but a t about 368" C. some sintering took place (copper block method), \vhilr heating to 430' C. did not result in definite melting. Treibs reported a melting point of 358" C. for synthetic desoxophyllerythro-etioporphyrin (a chlorophyll porphyrin) as well as for porphyrins isolated from shale oil and from Trinidad crude oil (12). Elemental analysis by combustion gave results as shown in Table I, including for comparison those obtained by Treibs 011 porphyrins isolated from the Trinidad oil as well as those ralculated for desoxophyllerythro-etioporphyrin,Ca2H38S4. Since these isolated porphyrins were intended for use as standard solutions for colorimetric measurements, it was necrssary to establish their identity wit,h the porphyrin aggregate observed iii numerous crude oil samples; spectroscopic examination$ ievealeti identical absorption peaks in all cases. During the course of extraction of this crude oil, a very interesting observation was made. The ether phase remaining froin the first extraction of porphyrins with iY0 hydrochloric acid ( H in Figure 2) contained traces of material extractable hy 20y0 hydrochloric acid. After purification by two cycles of transference to ether and re-extraction with 20% hydrochloric acid, the resulting ether extract, exhibited a distinctly green coloi, suggestive of chlorophyll. On removal of the solvent, a hlxkish green paste remained that yielded green solutions in typical chlorophyll solvents (80% alcohol or 80% acetone). However, thesc solutions lacked the red fluorescence characteristic of chlorophyll : henre the pigment was probably a phaeophorbide (chlorophyll deprived of magnesium and phytol). Treibs likewise observed this green solution during the purification of porphyrins from an American crude oil as well as from a shale oil and, in the latter case, was able to identify absorption bands characteristic of magnesium-free chlorophyll derivatives (13). More recently green-colored extracts of bitumina exhibiting absorption spectra of the chlorophyll series have been observed by Russian workers, who, however, were inclined to suspect contamination with coii-
Light Transmittance of Porphyrin Solutions in Chloroform Table 11. Precision of Porphyrin Determination
Pulfrich photometer, 1-om. cell with Zeiss filters
S-50 and S-53 Porphyrins isolated from crude oil No. 60
These pairs of transmittance values are then referred to standard curves of transmittance L I S . porphyrin concentration (Figure 3 ) for the same two absorption bands, and the average of the two values is taken as the porphyrin content of the sample un,ier examination. (The two values differ by about 670, prohahly owing to the presence of impurities.) The standard curve of transmittance us. concentration was obtained by isolating the norphyrins from 2 kg. of a California crude oil sample ( S o . 60) and preparing standard solutions in chloroform. The isolation of petroleum porphyrins from this crude oil was carried out by an elaboration of the above extraction scheme: the purification by transference to ether (using sodium
Approximate Wave Length, A. ' 5000 ,5300
I1
5000 5300
Porphyrins in 16 Vols. of Chloroform Calcd. Porphyyo transmittance, rin Concentration i n Concn., 11'01. of Chloroform, x 100 mg./liter hIg./I.iter 2 1 , 2 2 , 2 2 , 22 Av. 2 1 . 8 4 3 , 4 3 , 4 3 , 44 A,.. 4 3 . 2 2 4 , 2 5 , 2 5 , 25 Av. 2 4 . 7 4 5 , 4 6 , 4 6 , 47 Av. 46 0
28.1
43,:
26.3 Av. 2 7 . 2 25.7
397
23.8
A v . 24 8
A r 416
V O L U M E 25, NO. 6, J U N E 1 9 5 3 temporary organisms because their bituniina were obtained from surface beds ( 7 ) . The possibility of interference by simple nitrogen bases in the porphyrin det,ermination was investigated. The absorption of typical compounds of this kind (pyridine and quinoline) was found t o be negligible at the wave lengths used for porphj-rin determination : moreover! the characteristic absorption spectra of these hiws rould not be detected in any of the porphyrin solutions c~s:iniined. PRECISION
The following shows the precision to he expected. The sample is the same crude oil from which the porphyrins were isolated for
1)rc.paration of the standard transmittance curves. Duplicate s:Lniple8 of topped crude corresponding to 100 grams of crude oil sample 60 were digested with the reagent and the 7 7 0 hydrorliloric acid-extractable porphyrins were transferred to 100 ml. of c*hloroform. This crude oil has a very high porphyrin content, so, in order to bring the concentration within the desired range, t,hr chloroform solution was diluted sixteen fold before optical me:tsurements were carried out. The results are shown in Table 11. Since the porphyrins in 1 volume or 100 ml. of chloroform w r e extracted from 100 grams of crude oil, the porphyrin content ~ i this ‘ oil is 416 mg. per kilogram or 416 p.p.m. hj- weight. From the fi ures, it is seen that the final average results from duplicate runs farid I1 deviate by =!=4.5%from the over-all average value; I)ut taking into consideration the largest differences in results (from measurements made a t the two wave lengths which yielded :L maximum of 28.1 and a minimum of 23.9 mg. per liter in the dilute chloroform solution), the total experimental deviations inwy he taken to be i870 of the average. no more porphyrins cwuld be obtained hy re-treatment of the extracted oil, this figure may represent the expected accuracy.
94 1 ACKNOWLEDGMENT
The author is indebted to M.W. Tamele and A. E. Smith for advice and aid in matters pertaining to spectrophotometry, and to D. C. Crowell for technical assistance. LITERATURE CITED (1) (2)
Abderhalden, E., and Heyns, K., Biochem. Z . , 259, 320 (1933). .Ischheim, S., and IIohln-eg, W., Deut. med. Wochschr., 1, 12 (1933).
Conant, J. B., J . A m . Cham. Soc., 53, 3526 (1931). Dunning, H. S . ,Moore, J. W., and Denekas, h l . O., paper presented a t 8th Southwest Regional Meeting of the AMERICAS CHEMICAL SOCIETY, Little Rock, Ark., Dec. 4-6, 1952. ( 5 ) Fikentscher, R.. 2002.A m . , 103, 289 (1933). (6)Gilman, H., “Organic Chemistry, An Advanced Treatise,” 2nd ed., Vol. 11, Xew l o r k , John Wiley & Sons, 1943. ( 7 ) Glebovskaya, E. A , , and Vol’kenshtein, PI. V., J . Gen. Chein. 1C.S.S.R.). , . 18. 1440 11948). ( 8 ) Ka’rrer, P., “Organic Chemistry,” 2nd ed., New Tork, Elsevier Puhlishing Co., 1946. ( 9 ) Overberger. C. G.. and Danishefsky. I.. Polytechnic Inst. Brooklyn, Tech. RcBpt. 2, Technical Information Pilot, 6,4224 (3) (4)
I
~I
I -
(1952).
(10) Skinner, D.
I d . Eng. Chem., 44, 1159 (1952).
Treibs, Alfred, Ann., 509, 103 (1934); 510, 4 2 (1934); 517,172 (1935); 520, 144 (1935). (12) Ibid., 510,62 (1934); 517, 191 (1935). (13) Ibid., 517, 175, 194 (1935). (14) Treibs, Alfred, and Steinmeti, H., Ibid., 506, 171 (1933). (15) Weigelt, J., Forsehungen u . Fortschr., 8 , 300, 357 (1932). (16) Weigelt., J., and Noack, K., .\'ana Acta, Neue Folge, 1, 87 (1932). (11)
RECEIVED for review
?‘;orember 7 , 1952. Accepted March 18, 19.53.
Sodium Carboxymethylcellulose Determination of Degree of Substitution Active Agent C.
V. FRANCIS, Wyandotte Chemicals Corp., Wyandotte, Mich.
The use of available methods of analysis for sodium carboxymethylcellulose for active agent content and degree of substitution has shown that no single method can be universally applied to all commercially available materials, too much time is required for routine control work, and some methods required complicated and expensive equipment not usually found in a control laboratory. .A search for a new method to correct these difficulties resulted in a procedure using the uranyl ion as a precipitating agent, forming insoluble uranyl earboxymethylcellulose. The use of this procedure on a variety of sodium carboxymethylcellulose samples has shown a minimum recovery of 98Yo sodium carboxymethylcellulose and a precision within 1 0 . 0 1 on the degree of substitution. Comparison of degree of substitution obtained by this method and other available methods has shown a maximum difference of 0.04. This method has been found particularly useful in routine control analysis, as a total time of only 2 to 4 hours is required, and no special equipment or experience is necessary.
S
ICVI.~RhT,methods have been proposed for the analysis of sodium carboxymethylcellulose for active agent (sodium carl~oxyr~iethylcellulosc) content and degree of substitution. These methods include the copper salt precipitation method of Conner and Eyler ( 1 ) and the three methods suggested earlier by Eyler, Klug, and Diephuis: an acid wash method, a conductometric method, and a colorimetric method ( 2 ) . .4n alcohol-insoluble method ifi also used in the industry, in which all material insoluble in a specified concentration of alcohol in water is called active agent. Through work in this laboratory on a wide variety of sodium c,arhoxymeth?-lcellulose samples, several objections were r;iised to the chemical methods mentioned above: The methods could not be universally applied t,o all types af materials, the time r,ecluired for analysis was too long for routine ‘control work, and iri some ciises considerable expensive equipment was required, which is tiot normally found in a control laboratory. Csing the
alcohol-insoluble method, various results are obtained b y different laboratories depending on the technique emploj ed, the type of sample, and concentration of alcohol. After unsuccessful attempts to improve existing methods, a new method \vas sought. It was believed that of the approaches tried by the various investigators, a salt precipitation method would be most likely to give the desired results. A study v a s made of the insoluble salts of carboxymethylcellulose, such as copper, zirconium, aluminum, and lead. The main difficulty encountered n a s to obtain a precipitate which, for all types of samples, could be easily filtered and nashed without extensive treatment and preliminary preparation. The uranyl salt appeared to answer these requisites best. Reid and Daul ( 3 ) have briefly investigated the uranyl salt of carboxymethylcellulose and reported that the amount of metal precipitated was higher than the theoretical. This may be due to the use of a 2y0sodium cubox y-
