Isolation of Alkaloid Isomers. A 400-mg. sample of a reaction product containing two oxindole alkaloid isomers is applied to a plate coated with silica gel G. The developing solvent is chloroform-ethyl acetate (5:95). At this concentration, overlapping of the bands occurs, and this section is removed and discarded. The isomers are completely separated when a 200mg. sample is applied. The results of the isolation are given in Table I. Isolation of 17a-methyl-17B-hydroxy - 1,4 androstadiene 3 one oxime Isomers. A 400-mg. sample
-
- -
mixture is applied to a plate coated with silica gel G. The plate is first with chloroform-methanol (99:1), removed, and dried. The plate is then redeveloped with chloroform-ethyl acetate (713). The results of the isolation are given in Table 11. ACKNOWLEDGMENT
The authors thank F. F. Anderson for the construction of the apparatus used. LITERATURE CITED
( 1 ) Brockman, H., Schodder, H., Ber. 74,73 (1941).
(2) Dahn, H., Fuchs, H., Hela. Chim. Acta 45,261-70(1962)* (3) HgrmBnek, S., Schwartz, V., Cekan, Z,, Coli. Czeck. Chem. Comm. 26, 3170-3 (1961). (4)-Mickibbins, S. W., Harris, J. F., hoeman, J. F.1 J. Chromatog. 5, 207-16 (1961). (5) Ritter, F. J., Meyer, G. M., A'ature 193,941 (1962). (6) Stahl, E., Pharm. Rundschau 1, 2 (1959), ( 7 ) Wollish, E. G., Schmall, Morto, Hawrylshyn, Mary, ANAL.CHEM.33, 1138-42 (1961). RECEIVED for review Sovember 19, 1962. Accepted A ril 22, 1963. Division of Analytical &hemstry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962.
The Chromatographic Determination of Trace Amounts of Polynuclear Hydrocarbons in Petrolatum, Mineral Oil, and Coal Tar WILLIAM LIJINSKY, IRVING DOMSKY, GLORIA MASON, HUSNI Y. RAMAHI, and TAHER SAFAVI Division o f Oncology, The Chicago Medical School, 2020 West Ogden Ave., Chicago 12, 111.
b Methods of analyzing petrolatums, mineral oils, creosote, and coal tar for polynuclear hydrocarbons ore discussed. Current methods involve absorption chromatography, solvent partition, and paper chromatography. These procedures are fairly quantitative but time consuming. Individual polynuclear hydrocarbons at a concentration of 40 p.p.m. or more have been determined in a 250-mg. creosote sample and those at a concentration of 0.01 p.p.m. or more in a 1 -kg. petrolatum sample. Gas chromatography at moderate temperatures has been used for polynuclear hydrocarbon analysis. Using a column of SE-30 on glass beads, all of the common polynuclear hydrocarbons with boiling points up to that of benzo(9, h, ilperylene can be chromatographed in less than 1 hour at 200' C. The major polynuclear hydrocarbon constituents of petrolatum were observed in a chromatogram of a concentrate and those of coal tar in a dilute solution of the tar. Very small samples were required for gas chromatography (1 pg. of each hydrocarbon constituent).
S
of the 1958 hmendment to the Food, Drug, and Cosmetic Act of the U. S. A., with its specific reference to carcinogenic compounds, interest in the development of rapid analytical methods for the detection of such compounds has intensified. Attention has focused particularly on INCE PASSAGE
952
rn
ANALYTICAL CHEMISTRY
the polynuclear hydrocarbons, firstly because among this group of compounds is the largest number of known chemical carcinogens and, secondly, because of their ubiquitous occurrence, a consequence of the widespread use of coal, petroleum, and products derived from them. Methods of detecting polynuclear hydrocarbons have improved as new analytical tools have been developed, since the pioneer work of Cook, Hewett, and Hieger led to the isolation of benzo(a)pyrene from coal tar in 1933 ( I ) . Analysis of coal tar which once took months can now be completed in 7 to 10 days and, using gas chromatography, conceivably can be accomplished in 1 day. Parallel with improvements in resolution of mixtures of polynuclear hydrocarbons, the sensitivity of the analytical methods has been increased and the detection of 0.01 p.p.m. of a polynuclear hydrocarbon in a mixture is currently attainable, although only by rather lengthy procedures (8). A detectable limit of 0.5 p.p.m., on the other hand, can be realized with a 1-day procedure. The essence of all of the analytical methods currently used is the concentration of the aromatic material in the mixture, with elimination of most nonaromatic compounds. This is achieved by adsorption chromatography or by partition between two suitable solvents. The aromatic concentrate thus prepared is resolved into its components by paper chromatography and
the compounds are identified from their ultraviolet spectra and, if possible, their fluorescence emission spectra. In some cases, examination of the ultraviolet absorption spectrum of the aromatic concentrate is adequate for the identification of polynuclear hydrocarbons. The use of gas liquid partition chromatography offers the prospect that paper chromatographic resolution of the aromatic concentrates can be dispensed with and the concentrate analyzed directly. Gas chromatography has already been used for analysis of lower boiling polynuclear hydrocarbons (6, I S ) and for tars and heavy oils (3-6), in which high column temperatures were employed for detection of 4and 5-fused-ring hydrocarbons, among which group almost all of the carcinogenic hydrocarbons are found. Unfortunately, a t these high temperatures (approaching 400' C.) bleeding of many stationary phases drastically reduces the sensitivity. Gas chromatography of individual polynuclear hydrocarbons and synthetic mixtures at lon-er temperatures (230' to 260" C.) has been described ( 2 ) , but here again large sample sizes were employed. Our concern has been to resolve complex mixtures of polynuclear hydrocarbons a t moderate temperatures (180' to 210" C.) and with high sensitivity. Improvements in the procedure leading possibly to quantitative determination of the components can be expected. Such improvements include temperature
programming and collection of fractions from the gas chromatograph for absorption or fluorescence spectrometric analysis; the latter would reduce the sensitivity of the procedure, which would be a serious disadvantage when, as with concentrates from petrolatums, waxes, etc., only minute quantities of sample are available. EXPERIMENTAL
T h e mineral oils and petrolatums (U.S.P. grade) were obtained commercially, a,s was the creosote. Medicinal coal tar was obtained from Gazzolo Drug and Chemical Co., Chicago. All hydrocarbon solvents were chromatographed (9) and distilled before use. One-kilogram samples of petrolatum, dissolved in 1 liter of hot isooctane (2,2,4-trimethylpentane), were chromatographed on silica gel (grade 922, Davison Chemical Co.: as described in (11). After washing the column with hot isooctane the arclmatic material was eluted with 1 liter cf benzene. The residue after evaporation of the benzene was dissolved in 25 ml. of cyclohexane and partitioned with nitromethane (11 ) . After distillation of nitromethane under reduced pressure, the residue was dissolved in a minimum volume of benzene and chromatographed on 15 X 50 cm. sheets of Whatman No. 1 filter paper (20 mg. of residue per sheet). The sample was applied with a micropipet to a line 10 cm. from the end of the sheet. Chromatography was carried out in a N,N-dimethylformamide-isooctane system (8). The fluorescent bands were cut out, eluted with benzene-ethanol ( 3 : l ) in a Soxhlet extractor, the solvent was evaporated under nitrogen, and the absorption spectrum taken from 2.50 to 430 mp in isooctane. The fractions were rechromatographed unt 1 spectra corresponding to those of known polynuclear hydrocarbons were obtained. The identification was ( onfirmed by the fluorescence emission spectra where possible. The concen ;ration of each identified compound in the starting material was calculated from the absorption spectra. The method used for analysis of mineral oil was essentially similar, except that 18 liters of mineral oil was mixed with an equal volume of hot isooctane and chromatog-aphed on silica gel: the subsequent steps were the same as for petrolatum. In the analysis of materials relatively rich in polynuclear h,ydrocarbons (as compared with waxes and mineral oils), such as coal tar and creosote, much smaller quantities of material could be used. T o remove pheiols and similar compounds, 100 to 500 mg. of tar or creosote was dissolvec! in 25 ml. of cyclohexane and shakm with 25 ml. of 90% methanol ( 7 ) . The methanol extract was shaken with three 25-ml. portions of cyclohexan? and the combined Cyclohexane solulions were evaporated to dryness. The residue was dissolved in 25 ml. of cyclohexane and Procedure.
..,...,
~
I V L
v11 /Sl
Figure 1. Chromatogram on SE-30 of silica gel adsorbate from 100 grams of Petrolatum 10, after cyclohexane-nitromethane partition; residue dissolved in 0.5 ml. of benzene, 1 -pi. sample 1. 2.
3. 4.
Phenanthrene Fluoranthene/pyrene Methylpyrene (?) Triphenylene (a)
extracted with 25-ml. portions of nitromethane, as previously described. The residue, after evaporation of nitromethane, was diluted to 5 ml. with benzene and aliquots of the solution were chromatographed on paper to enable both major and minor components t o be determined. Since the concentration of tricyclic and tetracyclic polynuclear hydrocarbons in coal tar and creosote is a t least 10 times that of the pentacyclic hydrocarbons, it was sufficient to chromatograph 0.05 or 0.1 ml. of the benzene solution to determine the former compounds. For the determination of the pentacyclic compounds, 1 ml. or more of the benzene solution was chromatographed on a suitable number of sheets of paper (0.2 ml. on each 15 x 50 cm. sheet), and the four or five zones below and including that containing benz(a)anthracene were discarded, the compounds in these zones having been determined by chromatography of the small volume of benzene solution already described. The upper zones (numbering 6 or 7 ) were cut out, analogous ones from the several chromatograms were combined, and extracted with benzene-ethanol in a Soxhlet apparatus. The residues after evaporation of the solvent were rechromatographed on single sheets of paper in the dimethylformamide-isooctane system. Adequate separation of the components in this case required 12 to 20 hours development. Each fraction was examined spectrometrically as previously described. All of these analyses required approximately 1 meek (35 to 40 hours) t o complete, the limiting factor being the time required to take the spectra, which again depended on the amount of dilution of the fractions necessary. GAS CHROMATOGRSPHY. A BarberColman Model 20 instrument with a strontium-90 ionization detector, using argon.as carrier gas, was used. Before applying this method to the analysis of unknown mixtures, it was necessary to establish the operating parameters for detection and separation of polynuclear
hydrocarbons. A series of solutions pf several polynuclear hydrocarbons in benzene, differing in concentration by factors of 10, was prepared. X standard sample size of 1 pl. was used and the lowest detectable concentration of each hydrocarbon was determined. The parameters varied were column temperature and argon pressure. The column was 8 ft. long, l/q inch in diameter, and was packed with glass beads 60-80 mesh coated with 0.25% of SE-30 Silicone (prepared by Chemical Research Services, Inc., Addison, 111.). The aromatic concentrate from the cyclohexane-nitromethane partition of the adsorbate from a petrolatum was dissolved in 0.1 or 0.5 ml. of benzene, and 1 pl. of this solution was used for gas chromatography. The results of this analysis applied to petrolatum sample 10 are given in Figure I . Similarly, in Figure 2 are shown the chromatograms obtained from 5 PI. of the solution of the aromatic concentrate from coal tar. RESULTS
The analysis of five samples of mineral oil revealed the absence of identifiable polynuclear hydrocarbons, even at a concentration of 1 part in lo9. Similar analyses of hydrocarbon solvents led to the detection of some polynuclear hydrocarbons in most samples a t this concentration (9). The petrolatums analyzed by the procedure described all contained identifiable polynuclear hydrocarbons a t concentrations of 1 part in IO8 or higher. No carcinogenic compounds were detected. Of the 10 samples examined (Table I), there was a quantitative difference in polynuclear hydrocarbon content between the white petrolatums (samples 1, 2, and 3) and the amber petrolatums (samples 4 to 10). The total polynuclear hydrocarbon content of several petrolatum samples was of VOL. 35, NO. 8, JULY 1963
953
ences in polynuclear hydrocarbon content (Table 11), although in general the same compounds were identified in both (two samples of different size are shown for each). The concentrations of tricyclic and tetracyclic hydrocarbons
the order of 10 p,p.m., whereas the highest value for any petroleum wax examined was approximately 1 p.p.m, (14). The creosote and coal tar samples examined showed quantitative differ-
Table 1.
Concentration of Polynuclear Hydrocarbons in Petrolatum (p.p.m.1
Benz(a)BenzoSam- anthra(e)ple cene pyrene 0 1 0 2 0 0 0 3 0 0.23 4 0.19 5
6 7
8
9 10
0.02 0.01 0 0.90
0
0.03
0.01
0
0.025 0.01 0
0
Table II.
0 0 0
0.07
0 0 0
0.03 benzo(g, h, i) perylene
0.36
2.79 2.56
0.03 0.17 3.52
0.59 6.70
0.31 2.32
0.15 4.83
0.04
0.7
0.15 0.93
0.04 0.04
0.59
0.08
0
Other
0 0 0
0.06 0.01 0.18 4.71
0.03 0.01 0.02 0.33
0.01
Triphenylene
0.11 benzo(g, h, i) perylene 0.02 anthracene 0 0
Polynuclear Hydrocarbons in Creosote and Coal Tar
Anthracene Benz(a)anthracene Benzo(b)chrysene Benzo(j )fluoranthene Benzo(k)fluoranthene Benzo(g, h, i)perylene Benzo(a)pyrene Benzo(e)pyreno Carbazole Chrysene Dibenz(a, h)anthracene Fluoranthene Perylene Phenanthrene Pyrene
Table 111.
Pvrene -finil.methylChry- Fluor- Phenan- pysene anthene threne rene) 0 0.01 0.01 0 0.01 0 0.015 0 0.015 0 0.01 0 0 0 0.44 0
Concen-
Concentration in creosote (g./kg.) (1) (2) 12.1 12 .o 2.77 2.94 0.03 0.06 0.29 0.29 0.30 0.11
...
given In (19) g.A. 6.2 2.75
... ... ...
...
...
0.14 0.18 2.20 1.34
0.22 0.15 1.42 0.94
0.12 ... 2.75 1.27
24.8 0.04 39.9 9.1
22.2 0.04 33.3 6.8
7.8 0.04 47.9 4.2
...
...
...
Concentration in coal tar (g./kg.) (1) (2) 2.88 4.35 6.24 6.98 0.93 0.80 0.63 0.45 1.08 1.07 1.23 1.89 2.08 1.76 1.85 1.88 1.32 1.27 2.13 2.86 0.30 0.23 17.7 17.8 0.70 0.76 13.6 17.5 7.95 10.5
Gas Chromatography of Polynuclear Hydrocarbons on SE-30
Hydrocarbon Phenanthrene Fluoranthene Pyrene 4-Methylpyrene 1-Methylpyrene 3-Methylpyrene Chrysene Benz(a)anthracene 7,12-Dimethylbenz(a)anthracene Triphenylene Benzo(a)pyrene Dibenz(a, h)anthracene Benzo(g, h, i)perylene
Sample wt., fig. 0.01" 0.05" 0.02 1 1
Retention time (min.) at 200° c. 210'
1 0,2a 0.1" 0.5"
6.0 11.5 11.5 30
14 10
38
la
3"
c.
180" C.
11
..
3
7 6.5 13
5.5 16 40 44
I
.
.
4.5 4.5 9 4 12 26 28
These were minimum quantities giving a good response with relative gain 30 and attenuation 1. Operatin parameters were: argon pressure 15 p.s.i. (32 ml./min.), detector voltage 1250, flash geater temperature 285' C., detector cell temperature 250" C., sample volume 5
1 pl.
954
ANALYTICAL CHEMISTRY
were higher in creosote than in coa1 tar, the reverse being true for the pentacyclic hydrocarbons. The variation in concentration given by analysis of two different aliquots of the same material is due to the greater difficulty in some cases of separating two particular components-e.g., benzo(j)fluoranthene and benzo(k)fluoranthene, anthracene and phenanthrene, benzo (a) pyrene and benzo(g, h, i)perylene. The results of the creosote analysis may be compared with the analysis of a similar sample by a much longer method, which has been reported previously (1.2). By using gas liquid partition chromatography it was possible to separate and detect polynuclear hydrocarbons at moderate column temperature (Table 111). The retention times given are relative and varied somewhat from one column to another. Even the high boiling point hydrocarbon dibenz(a, h)anthracene was easily detectable. The sensitivity of the system varied considerably between different compounds, being approximately 100 times greater for pyrene than for dibenz(a, h)anthracene. At the column temperature adequate for the detection of tricyclic and tetracyclic hydrocarbons (180' C.), most of the pentacyclic hydrocarbons were not detected. On the other hand, at 220' C., at which temperature benzo(a)pyrene and dibenz(a, h)anthracene were detectable, the retention times of phenanthrene, fluoranthene, and pyrene were so short as to make these compounds difficult to distinguish from the solvent. A mixture of pyrene, benz(a) anthracene, benzo (a) pyrene, and dibenz (a, h) anthracene was easily resolved. It proved possible to separate pyrene homologs from pyrene (although not from one another) and 7,la-dimethylbenz(a)anthracene from benz(a)anthracene in this system. Benao(a)pyrene could be separated from benzo(g, h, i)perylene by gas chromatography, whereas their separation was difficult by paper chromatography, ultraviolet absorption spectrometry, and fluorescence spectrometry (IO). By submitting a concentrate p r e pared from petrolatum sample 10 to gas chromatography (Figure I), several polynuclear hydrocarbons could be identified including fluoranthene and pyrene, phenanthrene, triphenylene, and methylpyrene. The gas chromatogram of the coal tar sample (Figures 2a and b) showed that many of the polynuclear hydrocarbons detected by paper chromatography could be detected by gas chromatography. It would seem desirable to collect the fractions from the gas chromatograph and to examine them spectrometrically. This has not yet been done because the low capacity of the column would make the collection of sufficient material difficult. Secondly, collection
\,
-
--.
6
P
,
IC
\fi
--+-
. ,-I---io
IT.*/
10
7
-
-
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35
-
-====-&=
TIME I C I N U T E S I
Fiaures 2a and 2b. at"l80"C.
Chromatoaram on SE-30 of coal tar, 250 mg. in 5 ml. of benzene, 5-pL sample; 20 at 160" C.,
2b
1. Phenanthrenejanthracene Carbazole Fluoranthene Pyrene 1 -Methyl or 3-methylpyrene
2. 3. 4. 5. 6.
Benz(a)anthracene/chrysene
7. Benzo(a)pyrene
of fractions would increase the sample size required and, therefore, greatly reduce the sensitivity of the procedure. DISCUSSION
I n the analysis of mixtures containing polynuclear hydrocarbons, the factors limiting the sensitivity of the method used (adsorption chromatography, cyclohexane-nitromethane partition, and paper chromatography) are: (a) the quantity of starting material to produce fractione containing 5 to 10 pg. of any polynuclear hydrocarbon (this being the miniinum quantity required for accurate spectrophotometric determination) and (tl) the time allotted to paper chromatography, this being dependent on the amount of background ultraviolet absorbing material present. Removal of background absorption is necessary to obtain ispectra which can be compared with those of pure polynuclear hydrocarbons. A practical limit is set by the rapid increase in the number of paper chroma1,ograms needed as the proportion of bac iground absorbing material to polynuc [ear hydrocarbons increases. This limit, has been, in our experience, 0.01 p.p.m. in waxes and petrolatums, and 01.0003 p.p.m. in mineral oils and solvents (9). The identification of pc lynuclear hydrocarbons is facilitated by the well de-
fined and distinctive ultraviolet absorption spectra of most of these compounds; they also have high absorptivities, ranging up to 239,000 for dibenz(a, h)anthracene (8). In this laboratory, the best paper chromatographic system for resolving mixtures of polynuclear hydrocarbons was N,N-dimethylformamide-isooctane on Whatman No. 1 paper, run by the descending technique. Some mixtures were difficult to resolve in this system. Benz(a)anthracene and chrysene could be resolved on partially acetylated paper, using toluene-methanol-water as the mobile phase (8). Pyrene and fluoranthene could be estimated in the single zone containing them using the 334mp absorption maximum of pyrene and the 286-mp maximum of fluoranthene. Absorbance of the second compound a t these wavelengths was low. Similarly, anthracene and phenanthrene could be estimated in a mixture of the two using the 375-mp band of anthracene and the 294-mp band of phenanthrene. The results of analysis of the petrolatum samples shows that all contained essentially the same polynuclear hydrocarbons. As expected, the white petrolatums (samples 1 to 3) contained much smaller concentrations of polynuclear hydrocarbons than the amber
petrolatums. This is probably a reflection of the more thorough refining of the white petrolatums. Although no known carcinogenic polynuclear hydrocarbons were detected in these materials, no conclusions can be drawn regarding their biological activity, since no biological testing of them has yet been carried out. One of the difficulties in such analyses is the differentiation of the hydrocarbons from their homologs. Differences in absorption spectrum betwem a polynuclear hydrocarbon and its homologs are often slight (the fluorescence emission spectra usually differ more), and gas chromatography might prove useful in separating a polynuclear hydrocarbon from its homologs. The analysis of the coal tar and creosote samples revealed a higher concentration of the more volatile polynuclear hydrocarbons in the creosote and, conversely, a higher concentration of the less volatile (pentacyclic) hydrocarbons in the coal tar. These analyses, with a sensitivity approximating 10 p.p.m. of individual polynuclear hydrocarbons using 500 mg. or less of starting material, could be completed in about 1 week (40 hours), and could probably be applied to similar materials such as liquid smoke. Estimation of those polynuclear hydroVOL. 35, NO. 8, JULY 1963
* 955
carbons occurring at lower concentrations could be made using larger quantities of starting material, although this would increase the time of analysis considerably. The results of the gas chromatographic investigation showed that most of the polynuclear hydrocarbons studied could be separated at column temperatures between 160' and 210' C. and argon pressure of 15 p s i . , within 1 hour. Of special interest was the fact that it seemed possible to separate polynuclear hydrocarbons from their homologs by gas chromatography. This aspect of the subject is being explored further. Some compounds which were difficult to separate by other means [such as benzo(a)pyrene and benzo(g, h, i)perylene] could be separated by gas chromatography. Since it was impossible to select a single column temperature at which separation of all polynuclear hydrocarbons would be optimal, temperature programming would be excellent for this purpose. This modification of the analytical procedure is now being investigated.
The sensitivity of the gas chromatographic method is superior to that of most other analytical methods for polynuclear hydrocarbons. Thus, since 5 pl. of the solution of coal tar (250 mg. in 5 ml.) contains 250 pg. of tar and this contains 0.5 pg. of benzo(a)pyrene, which is easily detected, the advantage over the spectrometric method is apparent. Although not all of the polynuclear hydrocarbon components of commercial mixtures can be identified, it seems that the presence of known carcinogenic hydrocarbons in materials such as petrolatum above a level of 0.1 p.p.m. can probably be established using a gas chromatographic method. ACKNOWLEDGMENT
The authors are grateful to Philippe Shubik for his interest and encouragement. LITERATURE CITED
( 1 ) Cook, J. W., Hewett, C. L., Hieger, I., J . Chem. SOC.1933,396.
(2) Carugno, N., Tubacco 63, 285 (1959); C.A. 54, 5345a (1960). (3) Dupire, F., NCI Monogr. N o . 9, 183 /,nco\
(lVUL).
(4) Dupire, F., Z. Anal. Chem. 170, 317 (1959). (5) Dupire, F., Botquin, G., Anal. Chim. Acta 18.282 f 19581. (6) Hishta, C.,'Mes$erly, J. P., Reschke, R. F., Fredericks, D. H., Cooke, W. D., ANAL.CHEM.32,880 (1960). (7) E. L.. Ibid., . . Hoffmann, D.,. Wvnder. 32, 295 (1960). (81 Liiinskv. W.. Ibid.. 32. 684 11960). (9) Lijinsk?; W.', Raha, C. R.,' To&co~. Appl. Pharmacol. 3, 469 (1961). (10) Lijinsky, W., Raha, C. R., Chestnut, A., ANAL.-CHEM. 33, 1448 (1961). (11) Lijinsky, W., Raha, C. R., Keeling, J.. Ibid.. 33. 810 (1961). (12)' Lijinsky,' W.,'Saffiotti, U., Shubik, P., J . Nut. Cancer Inst. 18, 687 (1957). (13) Pinchin, F. J., Pritchard, E., Chem. & Ind. 1962, 1753. (14) Rhubik, P., e l al. Toxicol. Appl. Pharrnacol. 4, Supplement (Nov. 1962).
RECEIVEDfor review May 28, 1962. Resubmitted February 18, 1963. Accepted April 22, 1963. In part, Division of Analytical Chemistry, 141st Meeting, ACS, Washington, D. C., March 1962. Work supported by grants (3-5170 and CS-9212 from the National Institutes of Health, U. S. Public Health Service.
Detection and Identification of Mercaptans by Gas Liquid Chromatography K. F. SPOREK and M. D. DANYI Owens-Illinois Technical Center, Toledo 7, Ohio
b Gas liquid chromatography is employed in conjunction with an iodine oxidation reaction, which converts mercaptans to disulfides, for separation, identification, and, if necessary, quantitative determination of mercaptans in admixtures with each other or with other substances. The procedure is particularly useful where the mercaptans cannot b e isolated from the sample in a sufficiently pure form to b e directly suitable for separation and identification b y gas liquid chromatography. Thus the crude mercaptan sample is treated with iodine to form the corresponding disulfide; a second portion of the sample i s then spiked with a known mercaptan and this is converted to a mixture of disulfides. In this way a series of disulfides can b e formed from the original mercaptan with predictable retention times and relative abundances, thus providing an almost specific test for the unknown compound.
T
and identification of mercaptans are required in many instances when present either as major components or as traces. Because of HE DETECTIOX
956
ANALYTICAL CHEMISTRY
the difficulties encountered with conventional methods of analysis, several attempts have been made recently to determine mercaptans, alkyl sulfides, and disulfides by gas chromatography. Adams and Koppe (1) tested kraft pulp digester blow gas and black liquor combustion products by a gas chromatographic procedure using Triton X-305 as the column filling. I n their procedure, water interfered and had to be separated prior to the gas chromatographic analysis. Carson and Wong (2) tested Carbowax-1540, Reoplex 400, and Apiezon M for the separation of disulfides. Ryce and Bryce (4) used Celite coated with tricresyl phosphate on which they separated some of the lower mercaptans and dimethyl disulfide. Spencer, Baumann, and Johnson (6) used a column containing Johns--Manville firebrick C-22 coated with dinonyl phthalate; they obtained satisfactory separation of mercaptans used in several commercial odorants, which include methyl, ethyl, etc., thiols. More recently Farrugia and Jarreau (3) applied (2-22 firebrick coated with di-n-butyl phthalate, tritolyl phosphate, and Silicone 200 as column filling. Their work involved
separations of a series of thiols present in odorizing agents for odorless natural gases which also contained isopropyl alcohol as a cloud point suppressant. I n all these procedures the only means of identification for individual mercaptans were the retention times under the specific conditions of the test. It was therefore assumed that an unknown substance would contain no substances other than thiols which would have identical retention times as the calibrating materials. Because of the uncertainty of methods based on the identification by means of retention times alone, attempts were made in presently reported work to combine gas chromatography with a chemical reaction for the mercaptans to give a more specific test. As a result it was possible to convert mercaptans to a series of disulfides by a simple and fast oxidation with iodine in ether; this solvent was also used for extraction and concentration of thiols from aqueous and other samples. The so formed disulfides mere separated and determined by gas liquid chromatography using silicone oil and silicone gum rubber coated firebrick as the fillings for the columns.
