Improved Chroma tog raphic Ana lysis of Petroleum Porphyrin Aggregates and Quantitative Measurement by Integral Absorption W. WARREN HOWE Colorado School o f Mines Research Foundation, Inc., Golden, Colo. b By using silica gel as the surface, and chloroform as the eluting agent, the petroporphyrins are separated into groups according to the spectral types of rhodo, etio, phyllo, etc. The free porphyrin aggregates from four petroleums are analyzed quantitatively, showing the proportions of the spectral types. The efficiency of the chromatographic separations i s checked b y an hydrochloric acid extraction of an ether solution of each fraction, using a quantitative procedure. A significant amount of a rhodo-type porphyrin was found in one Wyoming crude. Chlorin-like porphyrins were found in two of the crudes. Improved methods of purifying certain fractions are discussed.
T
IKVESTIGATION explores the possibilities of analyzing the demetallized petroporphyrin aggregate M ith respect t o its spectral types. The method, with some developments, should become precise. An abundance of data is developed here n-hich should be of use to those studying the origins and migration of petroleum. The method shon s success in separating interesting traces pure enough to give a clear cut absorption spectrum. Methods proposed heretofore (1, 5, 7 , 12) have not been too successful in isolating the rhodo-type porphyrins or in separating the etio-type from the phyllo-type porphyrins. Lucas and Orten (9) have a procedure for separating the esterified porphyrin acids which is very similar to the procedure outlined here. HIS
EXPERIMENTAL
Reagents and Apparatus. Chloroform, acetic acid, and all other liquid reagents were freshly distilled t o avoid contamination or degradation of t h e porphyrins. The porphyrins were protected from daylight by putting a collar of black paper on t h e chroniatographic column. The column was observed in a dark room, using t h e ultraviolet lamp, t o detect t h e fluorescence of t h e thinner bands which form. The danger of using chloroform with porphyrins hasxbeen pointed out pre-
viously (2, 6); in this work chloroform was used freely, but the porphyrins IT-ere in contact M ith this solvent for a maximum of 1 or 2 hours. The products of chromatography were tested carefully for chlorine, which might have come from chloroform (4). Xone was found. Solvents other than chloroform were tried on the silica gel column; among these TT ere ethylene chloride, benzene, and nitromethane. S o n e performed as well as chloroform, the univer-a1 solvent for the porphyrins. Davison silica1 gel S o . 923 is recommended (IT. R. Grace and Co., Baltimore, Md.). Other silica gels were tested but the porphyrins adhered too strongly. Howe.c.er, the Xo. 923 gel must be extracted u i t h acid t o remove traces of copper and iron and possibly other metals ( I O ) , otherwise copper porphyrins will contaminate the products of chromatography. About 50 grams of silica gel in a 1 X 8 inch column n ill handle u p to 50 pmoles of porphyrin. An acid-n ashed, chromatographic grade alumina was used, deactivated according t o recommendations of Fisher and Dunning ( 7 ) . ilbout 60 grams of alumina in a 1 X 6 inch column will handle up to 50 pmoles of porphyrin. Since materials move quite slowly over the alumina column, a n air pressure of 3 to 5 p.6.i. was applied intermittently to the top of the column. The use of air to hasten the evaporation of solutions of porphyrins may be objected to because of the danger of oxidation. HOTT ever, when dealing with a large number of fractions which should be evaporated in a hurry, some comproniise n ith ideal conditions must be admitted. I n the last stages of purification, certainly the evaporation should be made in a stream of nitrogen or carbon dioxide. The solvent was removed from a fen- samples by distilling at reduced pressure. The same spectra were obtained repeatedly for the various fractions, evidence that little degradation of the porphyrin structure occurred. Production of F r e e Porphyrin Aggregate. A weighed sample of t h e crude oil is treated with glacial acetic acid containing 25 t o 30% hydrobromic acid by weight in t h e proportions recommended by Costantinides et al. (S), at 50" C. for a period of from 2 t o 4 days. The sample is then poured into a separatory funnel containing from 3 t o 10 times its volume of 337, acetic acid. The resultant oil layer is washed with 50% acetic acid to remove
last traces of porphyrins. The acid layer is washed three times n i t h benzene to remove oily constituents. The free porphyrins, now in about 50% acetic acid, are extracted with three portions of chloroform. This removes all traces of porphyrin from the acid layer. The chloroform solution is washed four or five times n-ith a n equal volume of water and finally dried over anhydrous S a n S 0 4 , for 15 minutes or more. The solution is then decanted and concentrated on a water bath. Chromatographic Procedure. FIRST STAGE(Table I). The concentrated chloroform solution of t h e porphyrin is poured onto t h e silica gel column previously wet with chloroform. T h e eluted fractions are evaporated n i t h a gentle blast of clean d r y air on a \T ater bath as fast as they are obtained and dissolved in benzene. Ordinarily, there are enough impurities present to render the porphyrins soluble in benzene. If not, they are dissolved in benzene containing 2 to 4y0 isopropyl alcohol, .Irhich also serves as a n antioxidant. The extent of separation of one class of porphyrin from another must be judged from the abqorption spectra. The mixture of rhodos and etios shows a double band head a t 568 and 57-1 mp in benzene. Similarly the mixtures of etios and phyllos show double band heads. The mixtures come intermediate to the main bands and constitute a small fraction of the 11 hole.
Table I. Fractionations Using Silica Gel Column and Chloroform Eluent
Fraction
Same
Yellow-green imDurities Rhodo-t ype porphyrins Rhodos plus etios Etio-type porphyrins Etios plus phyllos Phyllo-type porphyrins Diffuse brown immrities Impdrity plug" a
Remarks ITauy pigments Small fraction Small fraction
...
Waxy pigments Extrude if necessary
Isopropyl alcohol added to chloroform
eluent.
VOL 33, NO. 2, FEBRUARY 1961
255
The various classes of porphyrins can be seen by looking through the translucent column, where the rhodos are obberved to be magenta, the etios a ruby red, and the phyllos a darker red. The colors on the column do not necessarily match the color of the fraction after further purification. The materials proceed down the column at a rapid rate. The whole operation can be carried out in a little over 2 hours. SECOND STAGE. Each fraction from the silica gel column is further purified by chromatography over deactivated alumina eluting with benzene (Y), Table 11. The alumina column will not resolve mixtures of rhodos and etios or primary mixtures of etios and phyllos, but i t will remove the pigmented waxes always found associated with porphyrins. After the main band (etio or phyllo) is eluted from the column, one or more smaller bands will be developed by adding chloroform to the benzene. These are referred to as secondary polar traces, and may be etios, phyllos, or chlorins. .Idditional chromatographic steps must be taken at the discretion of the experimenter. A single chromatograph does not give a satisfactory separation of rhodos from etios. Sometimes one must repeat the chromatograph to clean out the colored impurities. Integral Absorption Method. The original acetic acid solution containing all the porphyrins was adjusted as to volume and an aliquot taken for analysis by t h e integral absorption method proposed by Costantinides et al. ( 3 ) where absorptivity = 65.2 X 108 mole-l sq. em. mp, using a spectropure sample of etioporphyrin I for a standard. In our work and using the same spectropure standard, absorptivity = 57.2 X 108 mole-1 sq. em. mp, which is considered t o be a satisfactory check on this
Table II. A Further Purification of Fractions Using Deactivated Alumina
Fraction
Name Tellox-green impurities 2 Main band 3 Polar traces of etios, phyllos. or chlorins 4 Brown impurities" 5 Impurity plug* a Partially separated. * Will not elute. 1
Eluent Benzene Benzene Benzene, chloroform (1: 1) Benzene, chloroform ( 1 : l )
...
c
,
IV
I1
Ill
I
0.2
02.
w
Same
Z
510
550
530
111
IV
630
670
I
I1
tenes
9 0 3.7 24 0 Lloydminster, Canada 16 6 Venezuela No. 1 a American Petroleum Institute gravity 79-C, Wyoming B. C. Prescott No. 3, Wyomiiig
256
ANALYTICAL CHEMISTRY
II
111
ia
I
490
I
538 502 631 576
0
nI1
m m oet
0A
"
L 490
530
650
610
Ill
II
I
500 618 566 536
0.6
n
A
02
570
530
IV
0et
04
/I
\
-
Q 2 t
I
w
I \
I
_L_LL
570
610
650
WAVE
430
530
570
610
650
LENGTH, rnN
Figure 1 . Examples of petroporphyrin absorption spectra in benzene-2-propanol solvent
value. The advantage of the integral absorption method seems t o be that all porphyrins and chlorins give a similar Soret absorption band with a maximum near 400;mp. The porphyrin content of all fractions reported here was determined by this method assuming that the absorptivity used would apply to all kinds of porphyrins. Data pertaining to the four crude oils analyzed are given in Table 111. Spectral Types of Porphyrins. &lost porphyrins show four principal absorption bands occurring around 500. 530, 565, and 625 mp, These bands are numbered I-11-111-IV from long to short wave lengths (Figure 1). The order of the bands is used here to indicate the order of the intensity of the absorption peaks. The order IV-III11-1 indicates the 500 peak to be the strongest and the 625 peak t o be the weakest. Two etio-type spectra were found shon ing the order IV-111-11-1 (ordinary etio) and IV-111-11-I-Ia (special). The Ia peak is a minor one between I and I1 (coming near 595 mp, benzene solvent). The Ia peak is supposed to show that the vinyl substituents (at positions 2 and 4) on the porphyrin nucleus have been reduced
Crude Oils Analyzed
yo Asphal-
IV
498 535 568 622 595
.
u
(11).
Table 111.
,
652 500 532 566 620
V, P P.11. 70 0 21 0 105 0 157 0
XI, P P 11 28 7 51 99
4 1 5 0
.I.P.I Grav a 24 5
32 9 17 0 22 1
Tn o phyllo-tj pe spectra showed the orders IV-11-111-1 (the ordinary type) and IV-1-11-111 (special type). In both of these types, peak 111 is weaker than peak 11. They are both referred to, in this invwtigation. as of the phyllotype spectra. Two rhodo-type spectra are referred to in the literature ( 2 1 ) shoning the orders IILIV-11-1 (the ordinary type) and 111-11'-1-11 (special type). Peak 111is accentuated in the rhodo types. TKO types believed to be closely related to the chlorins (dihydroporphins) were encountered. This structure is present in the chlorophylls and their immediate decomposition product. The chlorins shon a very strong absorption peak in the neighborhood of 660 to 680 mp. Apparently this same peak shon ed in some of the spectra found in this investigation a t from 638 to 653 mp. The chlorin peak will be designated here as the C peak. The order C-IV111-11-1would be called a n etiochlorin; the C-111-IV-11-1, a rhodochlorin, the C-IV-11-111-1,a phyllochlorin, etc. -1rhodo-type porphyrin was found in the S o . 79-C Wyoming crude. One part of this rhodoporphyrin underwent a reversion as to type, while evaporating solvent on the water bath. The change \vas from the 111-IV-1-11type to the 111-ITr-11-1type. The fraction is referred to as a reverted rhodo. What is believed to be a n etiochlorin n a s found in crude S o . 79-C. This fraction underwent a reversion on standing for 3 months in benzene-2propanol solvent. The change was from the C-ITr-111-11-1type to the IVC-111-11-1. The ratio of the intensities of the 653-mp peak to that of the 500-mp
peak was originally 1.23. After 3 months the ratio dropped to 0.86. The so-called chlorins here are referred to as being pseudochlorins because their spectra are imperfect, Certain of the trace materials have been designated as polar etios or polar phj-110s because they stick on the chromatographic column more firmly. I n the experimental results shown in Tables IV to VI, wave lengths are assigned to the principal absorption peaks, as measured in benzene-2-propan01 solvent. These wave lengths must be considered as being accurate to within 1 and 2 mp. All fractions are impure; however, they do fluoresce with a satisfactory red color. Figure 1 s h o w several of the spectra found in this investigation.
Table
IV.
Results of the Chromatographic Separation of No. Crude
Spectral Type Rhodo Reverted rhodo
+
Rhodo etio Red etio Phyllo Etiochlorin Pseudochlorin Pseudochlorin
Rhodo
+
etio Rhodo Red etios Brown etios
+
Etio phyllo Phyllos
Total, pmoles
5OO-GRAM SAMPLE^ 111-IV-1-11 538-502-631-576 111-IV-11- I 538-502-574-632
Brown etio
RESULTS
Porphyrin aggregates from four petroleums were fractionated according to the chromatographic scheme described above. One crude, 79-C, contained a rhodo-type porphyrin n-hich was eluted as the first fraction. Two crudes, 79-C and the Venezuela crude, showed chlorins or pseudochlorin-type porphyrins. Otherwise the porphyrin aggregates were separated into etios, phyllos, and polar traces of either type, the bulk of the etios coming off the column before the bulk of the phyllos. Most of the impurities associated 1% ith the petroporphyrins fluoresce blue. The yellow-green impurities which come off the column first fluoresce blue-white. The dark brown impurities which come off the column last fluoresce a darker blue. Toward the end of the elution, the impurity plug left a t the top of the column became nonfluorescent in the case of the Lloydminister crude, and fluoresced green in the case of the B. C. Prescott crude. The impurities seem to be complexed with the various porphyrin fractions, especially in the case of the so-called brown etios or bronm phyllos. An analysis was made on the highly refined impurities from the 79-C ll7yoming crude. The yellow-green impurity from this crude analyzed 0, 0.8%; N, 2.8%; S, 2.53%, by %%-eight. The brown impurities from this crude analyzed 0. 2.4%; K,3.42%; S, 2.94%. These impurities contain no metals. It is significant t h a t they are not just hydrocarbons. The fact that the b r o a n impurities contain more oxygen than the yellow-green impurities may explain n h y the browns are more polar (come off the column toward the end of the elution). Tables IV t o VI show the analytical results. The fractions are listed in the order in which they come off the chromatographic column. Each fraction 1%as analyzed by the integral absorption
Wave Length Order, ;\lp
79-C Wyoming
5 4
0.4088
0 565
0.4279 48.15
0 GOO 66 0
16 l A b
22 4h
2 6Sh
3 7b
0.0432
IV-1-11-111 1 .i445b 5OG618-566-536 Total moles 17.93 =
Values are corrected, see below. Total pmoles of porphyrin in eample
=
method applied to the Soret band (3). The errors in the chromatographic procedure cause losses of from 18 to 25%. This type of error is systematic and difficult t o avoid. One should understand that the analyses are by spectral types. Rhodos refers to the fraction showing the rhodotype spectrum (the . porphyrin here might be one of the pyrroporphyrins). Moreover, the fractions listed are a mixture of molecular structures, all showing approximately the same spectrum. The position of the porphyrin fraction in the tables indicates its polarity. The less polar fractions appear a t the top, the more polar fractions appear a t the bottom. The red etios show the spectral order of IV-111-11-I-Ia, whereas the polar etios have the order IV-III11-1. This may mean t h a t the red etios lack the vinyl group in positions 2 and 4 of the tetrapyrrole ring and t h a t the polar etios have a substituent causing polarity. Hence the polar etios stick to the chromatographic column more firmly. If this reasoning is correct, the red phyllos with the order IV1-11-111lack a polar substituent which is present in the polar phyllos \%-iththe older IV-11-111-1. The rhodo-type porphyrin found in
5
3.9
500-566-534-618 C I V - 111-11-1 0.245 652-500-532-566-620 IV-c-111-11-1 0 1852 500-650-534-568-620 IV-c-111-11-1 0 1285 500-645-536-568-620 Total moles 72.2ti 100-GRAMSAMPLEC 111-IV-1-11 0.569 538-502-631-576 0.618 IV-11131-I-Ia 12,75 498-530-565-622495 IV-111-11-I-Ia 2.21b 498-530-568-622-595
Total pmoles of porphyrin in sample
Mole
110.5; Loss 110.5 - 72.26 x 100 110.5
0 34 0,254 0.18
3.17 3.45 71.0
12.P 0,024 9.75b
= 25.4%,
22.1; Loss 22.1 - 17.93 x 100 = 18.9?$. 22.1
Wyoming crude 79-C is of interebt. Its spectrum is shown in Figure 1. So far as known this is the first time a rhodotype porphyrin has been reported as occuiring in crude oil. Treibs ( I d ) makes brief reference to such a porph! rin in the mother liquor obtained from oil shale. Special Treatment For Certain Fractions. Chlorin-like porphyrins were found in two of t h e crudes. T h e Venezuela crude (Table V) showed red psuedochlorins in significant amount and t h e more polar brown psuedochlorins in small amount. T h e red psuedochlorins were discovered mixed with red phylloq. To separate them, the complex between the two types of porphyrin had to be broken up. The original fraction containing the mixtures of phyllos and psuedochlorins was extracted \\ith 33% acetic acid (by volume). The soluble part and the insoluble part were then chromatographed separately, first on alumina, then on silica gel, and then on alumina again. By analysis the results showed 23 mole YGred psuedochlorins, 2 mole yG brown psuedochlorins, T4 mole % red phyllos, 1 mole % polar phyllos, and 0.23 mole % vanadyl chlorin complex. The original fraction conVOL. 33, NO. 2, FEBRUARY 1961
* 257
Results of the Chromatographic Separation of Venezuela Crude, Pederanallis Field
Table V.
Wave Length Order, Mp
Spectral Type
Total, pmoles
Mole yo
~OO-GRAM SAMPLE, SHORTPROCEDURE^ 117-111-11-I-Ia 21.35b 498-530-568-622-595 IV-1-11-111 50.04b 500-618-566-536 IV-11-111-1 2.926 -_ 500-568-536-620 IV-111-11-1 0.95 500-534-568-620 Total moles 75,265
Red etios Red phyllos Polar phyllos Polar etios
28. 3b 66. 5b 3.9 1.26
~OO-GRAM SAMPLE, LONGPROCEDURE” Red etios
IV-111-11-I-Ia 22.2 498-530-568422-595 568-536-635 0.07b IV-(3-11-111-1 6. 75b 502-638-568-534-620 IV-1-11-111 21.7b 500-6 18-566-536 IV-11-111-1 0.55’ 500-568-536-620 IV-c-11-111-1 1.164b 504- 638-568-534-620 IV-111-11-1 0.154 500-534-568-620 IV-11-111-1 4.8 502-566-536-618 Total moles 57.38
Vanadyl chlorins Red psuedochiorins Red phyllos Polar phyllos Brown psuedochlorins Polar etio Phyllo acid
a
*
Total pmoles of porphyrin in sample = 91.0; Loss Values have been corrected, see below. Total pmoles of porphyrin in sample = 72.5; Loss
91.0 - 75.26 91.0
X
38.6 0.01’
11.7b 38. Ob
0.96b
2 . OOb 0.027 8.37
100 = 17.470.
72’57i.j57’38 100 20.8%.
Table VI.
=
X
Results of the Chromatographic Separation of Two Crudes, 1 00-Gram Samples of Each
Kave Length
Spectral Type
Order,
Total, pmoles
Mp
Mole %
LLOYDMINSTER~ Red etios Red phyllos Brown phyllos
Polar etios
IV-111-11-I-Ia 6. 073b 500-532-568- 622-595 IV-1-11-111 8.4b 500-618-566-536 IV-1-11-111 0.23 500-618-566-536 IV-111-11-1 0.04 502-536-566-620 Total moles 14.743
41 .2b 57.Ob 1.56 0.27
B.C. PRESCOW Etios Phyllos
I\’-111-11-I-Ia 498-530-568-622-595 IV-11-111-1 500-566-532-620 Total moles
Total pmoles of porphyrin in sample
=
* Values have been corrected, see below. Total pmoles of porphyrin in sample
258
0
ANALYTICAL CHEMISTRY
=
4.6
96.5
0.1845
3.5
4.7845
19.0; Loss 19‘0 - 14’74 x 100 = 22.4%. 19.0 6.25; Loss
6.25 - 4.78 6.25
X
100 = 23.5%.
tained 29.4 pmoles of porphyrin. Corrections were applied t o Table V. The red psuedochlorins were less polar than the red phyllos. The brown phyllos originating in Table V were given the same treatment as that given the red phyllos, outlined above. The analysis showed 87 mole % red phyllos, 8.6 mole yo brown pseudochlorins, and 4 mole % polar phyllos and rejected brown impurities. This fraction contained 6.55 pmoles originally. Corrections were made for each of these constituents in Table V, the brown psuedochlorins being combined. The brown etios from Table IV (Wyoming crude) were given the treatment with 33% acetic acid as outlined above for the phyllos. The operation divided the brown etios into 63.5 mole % ’ red etios and 36.5 mole % red phyllos and rejected brown impurities. The phyllos were soluble and the etios mostly insoluble in 33% acetic acid. The original fraction contained 3.475 pmoles. Corrections Rere made in Table IV. The brown psuedochlorins in Table I V were listed just as they came off of the alumina column in the second stage of the chromatography, without further treatment. A porphyrin acid was found (Table V) which was removed from the silica gel column by using alcoholic ammonia followed by overnight soaking in strong ammonia. The ammonia solution upon dilution appeared to be a true water solution. It was acidified with acetic acid and the acid solution extracted with chloroform to recover the porphyrins, after which i t mas transferred to benzene-2-propanol solution for spectral examination. No other fraction reported in these tables could be proved to be definitely a porphyrin acid. Interesting Traces. T h e vanadyl chlorin complex listed in Table V came from the red psuedochlorins while chromatographing on silica gel. It is listed as a vanadyl chlorin complex on t h e basis of its absorption spectrum, showing one vanadyl peak a t 536 mp and a more intense peak a t 568 mp. The third weak peak at 635 mp should identify it as a chlorin. The corresponding red psuedochlorin peak came a t 638 mp. This was the only trace of metalloporphyrin found in any of the fractions. It is of interest because it suggests that a vanadyl chlorin complex must be quite stable to resist the action of the glacial acetic acidhydrobromic acid reagent. A chlorophyll-like substance was found in the 79-C crude, 100-gram sample, Table IV, which came off the silica gel column in front of the rhodoporphyrins. It forms a blue-green solution in benzene-2-propanol solvent. It shows a very strong band, quite sharp, at 418 mp, which would not be a Soret band. The spectrum is very simple. The sub-
stance shows one other band, fairly prominent, a t 592 mp. The 418-mp peak is 7 times as intense as the 592-mp peak. The spectrum was examined over the range of 330 to 850 mp. Only the two peaks mentioned were found. This blue-green substance does not fluoresce under the ultraviolet lamp, and, of course, it contains no metals. Its spectrum resembles that of pheophytin a. Methods of Extraction of Porphyrin Aggregate from Crude Oil. The method for capturing the porphyrin aggregate as used in this research may be referred to as the short procrdure and is described a t the begir,ning of tLis report. Here the por1,tlyrins are extracted from the acid layer immediately with chloroform. The method used by Groennings (8) and others may be referred to as the long procedure, in which the acid layer containing the porphyrins is saturated with sodium acetate, anhydrous, extracted with ether, and then back into acid, the cycle being repeated, finally ending up in a chloroform solution of the porphyrins. The short procedure will capture a larger quantity of porphyrins from the acid layer than the long procedure. The yield from a 100-gram sample of Venezuela crude by the short method = 91.0 pmoles and by the long method = 72.5 pmoles. Table V shows the results of the chromatographic analysis of two samples of porphyrin aggregate from the Venezuela crude, the first (short procedure) based on 91.0 pmoles and the second (long procedure) based on 72.5 pmoles. Fractionation of Porphyrins (13). An ether solution of a mixture of free porphyrins was extracted with increasing concentrations of hydrochloric acid. Willstiitter and U e g (13) assigned an acid number to several of the porphyrins. Table VI1 s h o m the acid numbers of several porphyrins thought to occur in petroleum as furnished by Treibs (12). The analysis by hydrochloric acid extraction was used to test the completeness of the chromatographic separations. It demonstrated clearly, in one instance, that the column had been overloaded. The separation into etios, phyllos, etc. is really brought about by the silica gel. This separation is shown to be good. Table VI11 shows that the red etios listed in Table IV were 100% etios. It would be a duplication of very similar data to include all of these analyses. Suffice it to say that the red etios from Table VI were analyzed by this method and found to be 1007c etios. The red phyllos from Table VI contained 4Oj, etios. Table VI11 shows that the phyllos were poorly separated from the etios in the case of the red etios from Table V.
Table
VII.
Acid Numbers of Petroporphyrins
hcid
Rlesoporphyrin Coproporphyrin Desoxyphyllerythrin Pyrroporphyrin Desoxyphyllerythroetioporphyrin Mesoetioporphyrin
0.09 Etio
0.5 1.2 1.3
Etio Phyllo Etio
2.5 3.0
Phyllo Etio
The acid extract a t from 1.2% to 1.3% HC1 (by weight) shows a mixture of etios and phyllos. This particular sample was put back on a silica gel column and eluted with chloroform. A very satisfactory separation of the etios from the phyllos was accomplished. The experiment means that the column was overloaded in the first place. To conclude, this analysis showed that the red etios from Table V contained 33% phyllos, 65% etios. A correction was made to adjust for this error. Hence, the data are annotated for the red etios and red phyllos in Table V. Similarly the red phyllos from Table VI were shown to contain 4 mole of etios, so a correction was made here in the data of Table VI. I n the same way the brown etios from Table IV were shoxn to contain 14Yc phyllos, hence a correction was made in Table IV. For a summary of these analyses by acid extraction see Table I X . Only those fractions listed in Table I X were analyzed with hydrochloric acid. Finally, the extraction of an ether
Table VIII.
0.05 0.09
0.5
1.0
1 2-1.3 2.5 4.0
7.0 12.0
... ...
Etio Etio Etio Etio Etio Etio Etio
Loss = 50 mole yo Table IX.
Crude 79-C 79-c Venezuela Lloydminster Lloydminster B. C. Prescott
solution with hydrochloric acid will ordinarily cause considerable loss in porphyrins. The losses indicated in Table VI11 approximate 50%. Part of this loss results from the precipitation of porphyrin a t the interface between ether and acid. The 50% loss reported is no doubt higher than need be. Moreover, the fractionation by means of this acid is not clear cut. In one case the extraction was repeated 15 times a t the concentration of 1% and the color continued to bleed off. The poor separation of the petroporphyrins by this means is probably due to the complexity of the mixture of porphyrins. CONCLUSIONS
This investigation gives a quantitative analysis of demetallized porphyrins originating from petroleum, aocording to the spectral groups of rhodos, etios, psuedochlorins, and phyllos. The next logical step will be to find a way of resolving each of the above groups into their components with the expectation of obtaining each in crystalline form. Duplicate samples of one Wyoming crude (79-C), and the Venezuela crude were analyzed. The analyses do not check. Single analyses nere made of the Canadian crude and of the B. C. Prescott crude, The purpose here was to investigate the possibilities of the method. It is evident that the procedures must be standardized thoroughly before duplication of the analyses can be attained. ACKNOWLEDGMENT
Thanks are extended to George B. Lucas for many helpful suggestions and
Hydrochloric Acid Extraction of an Ether Solution
...
...
26.8
40.0
6.6 5.68
9.2
8.1 2.87
0.05
0.09
... ...
... ...
Etio Etio 1.2-1.3 Etio phyllo 2.5 Phyllo 4.0 Etio 7.0 Etio 12.0 Etio Loss = 53.5 mole yo 0.5
1.0
13.4 26.5 17.9 25.4 7.6 7.6 2.5
+
Fractionation b y Acid Extraction of Ether Solution (Summary)
Fraction Red etios, Table I V Brown etios, Table IV Red etios, Table V Red etios, Table VI Red phvllos, Table V I Table IX
Analytical Results Table VI11 Not shown Table VI11 Not shown Not shown
Remarks 100% etios
14% phyllos 3370 phyllos 100% etios 4% etios Not treated
VOL 33, NO. 2, FEBRUARY 1961
0
259
for a study of the stability of a copper porphyrin. The sample of etioporphyrin I was furnished through the courtesy of J. Gordon Erdman of the Mellon Institute. The Ohio Oil Co. furnished the crude oil sample from Mill Iron Well No. 79-C, Curtis formation, Wyo., and the Skelly Oil Co., the B. C. Prescott No. 3 oil from the Ten Sleep sand, Wyo. The Canadian oil, Lloydminster, was furnished through the courtesy of G. W. Hodgson of the Research Council of Slberta, Canada, and the Venezuela No. 1 oil, Pederanallis field, was furnished by the Creole Corp.
LITERATURE CITED
(1)Blumer, Max, AXAL.CHEM.28, 1640 (1956). (2) Corwin, Alsoph H., Fifth World Petroleum Congress, Section V, Paper 10,p. 6,1959. (3) Costantinides, G.,Irich, G., Lomi, C.,
Fifth World Petroleum Congress, Section V. PaDer 11. 1959. (4)Erdman,* J. G., Ramsey, V. G., Kalenda, S. W., Hanson, UT.E., J. Am.
Chem. SOC.78,5844 (1956). (5) Dunning, H. N., Carleton, J. K., ANAL.CHEM. 28, 1362 (1956). (6) Dunning, H. N.,Moore, J. W., Myers, A. T., Ind. Eng. Chem. 46,2005 (1954). (7) Fisher, L. R., Dunning, H. K., ASAL. CHEM. 31, 1194 (1059).
(8)Groennings, Sigurd, Ibid., 25, 938 (1953). (9) Lucas, J., Orten, J. M., J. Biol Chem. 191,287 (1951). (IO) Resnick, F. E , Lee, L. A., Powell, A,, -4XAL. CHEM.27,9288 (1955). (11) Stern, A.,Wenderlein, H., 2. physik. Chem. 175A,405 (1936). (12) Treibs, Alfred, rlnn. Chem. Liebigs 410,42(1934). (13) Willstatter, R., Mieg, W., Ibid., 350, I (1906).
w.
RECEIVEDfor review June 14, 19GO. Accepted October 24,1960. This research is being administered by the Colorado School of Mines Research Foundation, Inc., Golden, Colo., under a grant from the Colorado School of hlines Foundation, Inc.
Titrimetric Determination of Hexahydro-lI3,5-trinitros-triazine (RDX) and Octahydro-I,3,5,7-tetranitros-tetrazine (HMX) with Ferrous Sulfate JAROMiR SIMEtEK Military Academy A. ZGpotocki, Brno, Czechoslovakia b The reactions which take place when RDX or HMX are dissolved in concentrated sulfuric acid are explained and also the factors which affect the amount of nitric acid liberated from these compounds. The determination of RDX and HMX in admixiure i s based on the determination of nitric acid liberated from them in sulfuric acid. A colorimetric estimalion can b e used and a titrimetric determination using ferrous sulfate is described.
S
(3) based the determination of R D X and H h I X in admixture on a determination of HK03which resulted from the hydrolysis of RDX and HXX in concentrated H2SOr. They determined the HNOI colorimetrically after reduction with FeS04, as the complex Fe(KO)+2. This reaction has also been used for the determination of HSO3 and HKOZ ( 2 ) and some organic nitrates (1, 6). Semel et al. showed that in Concentrated H2S04, R D X liberated a little more than two thirds and H h I X more than one third of the total HKOs. The reaction proceeds according to the equation EMEL, LACCETTI, AND ROTH
+ +
(-CHZXSO~)~ 22H20 H?S_OI zNH3 zCHzO zHN03 (1) 1: = 3 for R D X and 4 for HMX. This phenomenon was also observed by the author when attempting to determine RDX and H M X by titration with FeS04. Furthermore, i t was found that 3,7-dinitro-1,3,5,7-tetrazobicyclo-
260
+
ANALYTICAL CHEMISTRY
[3.3.l]nonane did not liberate HXO, under these conditions ( 5 ) . I n another study made for nonanalytical purposes, the author quantitatively evaluated the decomposition of RDX in concentrated HzSO4 (4). Results of this study, with other more recent unpublished information, explain the lower yield of "03 in the titrimetric and colorimetric determinations of R D X and H M X and also indicate some of the problems in these determinations. The dissolution of both R D X and HNX in concentrated H2S04involves two reactions, the first being the rapid reversible cleavage of HS03. leading to the equilibrium state
+ +
+
(CHZXNOZ), nH+ nHlSOa 3 (CHSH~),, nSOz+ ~ H S O - ~ (2)
n
+
3 for R D X and 4 for HlIX. The equilibrium state, which depends on the concentration of H2S04as solvent, is characterized by the amount of cleaved " 0 3 . The dependence of the amount of HKoa on the concentration of HzSO4 in a 0.005BI solution is showi in Figure 1. The next reaction is the irreversible destructive decomposition of partly denitrated R D X or HJIX due to Reaction 2 and is, for example,
Because of Reaction 3, Reaction 2 is reversible only temporarily, within the period of existence of hexahydro-striazine or octahydro-s-tetrazine heterocycle. Reaction 3 is also rapid; the rate of destructive decomposition of a n 0.3M solution of RDX in 97.5% is shown in Figure 2 which illustrates that approximately 12% of the RDX decomposed irreversibly during the dissolution. The beharior of H M X is similar and the decomposition of RDX and H h I X can be written as follows:
+
(CHzNKOz), 2rH+ = ( n - z)CH20 ( n - z)SZO
+
zNOz+
+
+ xCHFXH~
(4)
and a t the dilution of the reaction mixture Kith water
z ~ ~ 2 = h %~C zH ~ O+ r
=
n
~
~
= 3 for R D X and 4 for H X X ; z depends on the concentration of and equals approximately 0 to 2.7 for RDX and 0 to 2 for HMX; (see Figure 1). The mechanism of the reaction was demonstrated by the determination of all products of decomposition and by proving the presence of partly denitrated R D X in H&04solution. I n the equilibrium state of Reaction 2, a t a concentration of 85 to 100% H&04 the amount of "03 cleaved is approximately linearly proportional t o the concentration of H2S04. Because of this, the possibility of using this reaction for the indirect determination
a
+
