Improved Method for Characterizing Environmental Hydrocarbons by Gas Chromatography Oliver C. Zafiriou Woods Hole Oceanographic Institution, Woods Hole, Mass. 02543
Routinely correlating environmental hydrocarbons from natural waters by gas chromatography requires optimai resolution and separation of weathering-resistant components, operational simplicity, and good precision. Methods for comparing results from different columns must precede spectral libraries. A novel method of protecting columns from sample residues involves injecting solutions of oil-rich samples into a capillary injection/splitter with disposable glass liner operated at 175 "C; reproducible results are obtained without sample cleanup. Temperature-programmed O V - 1 01 support-coated open tubular columns and F I D detection yield excellent characterization. Characteristic signal intensity ratios have long-term relative standard deviations of 1.8-4%; 0.7% has been achieved short-term. Ratios from three columns were similar enough for comparison directly or with a standard. The performance attained is adequate to correlate artificially weathered oils with sources and to differentiate most of thirty oils found in a major port. Extensions and improvements of the method are discussed.
Many workers have been attracted by the possibility of utilizing gas chromatographic "fingerprints" of hydrocarbon samples from the sea and natural waters to correlate them with suspected source oils or petroleum products (1-5). Since analyzing such chemically complicated samples is extremely demanding, no simple yet discriminating and practical system for accomplishing correlations has appeared. The great potential of gas chromatography for this purpose is widely recognized and one or another of the cited workers (1-5) has demonstrated most of the separate capabilities such a system requires. For example. Blumer and coworkers (5-7) have followed the fate of a small oil spill by hydrocarbon analyses of sediments and organisms. The distribution, and also the processes responsible for compositional alteration of the oil, were determined. That study demonstrates low-level sample recovery and cleanup methods, as well as generation of geochemically interpretable information by gas chromatography. The oil was not correlated with a variety of similar possible sources, but rather the fate of a known spill was followed. The "fingerprinting" and correlation of relatively fresh oil samples using packed columns has been performed by several workers (1-5, 8, 9) and standard methods have ( 1 ) Petroleum Standardization Committee, J . lnst. Petrol., 56, 107
(1970) (2) E. R . Adlard, L. F. Creaser, and P. H . D. Matthews, Anal. Chem., 44, 64 (1972). (3) E. R. Adlard, J. lnst. Petrol.. 58, 63 (1972). ( 4 ) R. E. Kreider, Proc. Joint Conf. Prevention and Contr. of Oil Spills, Washington, D. C., June 1971, p 119. ( 5 ) M. Blurner. G . Souza, and J. Sass, Mar. Bioi., 5 , 195 (1970). (6) M . Ehrhardt and M . Biumer, Envvon, Pollut.. 3, 179 (1972). ( 7 ) Fvl. Blurner and J. Sass, Science. 176, 1 1 2 0 (1972). (a) C. B. Koons, P. H . Monaghan, and G . S. Bayliss, Esso Production
Research Co., Houston, Texas, 77001: unpublished manuscript. 1972. (9) D . W . Mayo and D. J. Donavon, 4 t h Northeast Regional Meeting, American Chemical Society, Hartford, Conn.. October 1972. 952
ANALYTICAL CHEMISTRY, VOL. 45,
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MAY 1973
been proposed ( I , 4 ) . However, these systems are clearly inadequate for differentiating among similar oils. More specific characterization has been demonstrated using capillary columns (IO, 1I ) and sulfur-specific detectors (2). We have considered how to construct a practical correlation system. Unfortunately, the separate capabilities cannot simply be spliced together, because analyses of complex mixtures such as oils are compromises of many conflicting factors affecting simplicity, analysis time, resolution, separation, and reproducibility. Furthermore, the literature has neither delineated the reproducibility of the analytical characteristics used for discrimination, nor considered the lifetimes of columns and the comparability of data obtained a t different times or with different columns of the same nominal construction. We have constructed an improved system and determined its performance characteristics in these respects. The system was also used to analyze the oil products occurring in several high-volume oil ports, to determine how well the levels of performance achieved can differentiate the products which actually occur. These studies will be reported elsewhere.
EXPERIMENTAL Gas chromatograms are obtained .using Varian 1440 gas chromatographs equipped with Y 8 - h and capillary splitting injection ports. The standard method (12) adopted used glass liners in the capillary injection port (Figure 1). Each instrument's temperature programmer initial and final temperatures and rate, and the injection port pyrometers, were calibrated. Support-coated open tubular (SCOT) columns (13) were 50-ft X 0.02-in., obtained from Perkin-Elmer Corporation, and were stabilized briefly a t 275 "C before use. Salient features of the standard operating conditions adopted are: capillary injector, 195-210 "C, with silicone rubber septum and glass liner; splitter adjusted to 50 ml/min He a t the split exit and 44 second retention time for methane at 75 "C (yielding 4.5-6 ml/min He for the SCOT columns used; split ca. 1O:l); column temperature ranging from 75 to 275 "C at G"/min with the program started a t the elution of the CS2 solvent peak; detector, flame ionization at 300 f 10 "C with 10 ml/min He makeup gas and optimized compressed air and hydrogen flows; electrometer, 1 X 1 O - I Q A/mV driving a 1-second repsonse, 1-mV recorder with 0.5 in./min chart advance and positive left-to-right pen movement. 0.5-5 p1 injections of ca. 10% solutions in purified CS2 were used. Reproducibility and comparability studies utilized base-line drift-corrected signal intensity ratios at selected peaks which occur in virtually all samples. Measurements were made utilizing calibrated scales and hand magnifiers; the defined terms are illustrated in Figure 2. Injector cleaning, septum changes, blank determinations, and other operating procedures were also standardized (12). A commercial No. 2 fuel oil stabilized by passage through a bed of alumina and silica gel was utilized as a standard sample; it is available upon request. (10) R. D. Cole, Nature. 233, 546 (1971). (11) A . G. Douglas,J. Chromatogr. Sci., 9 , 742 (1971). (12) 0. Zafiriou, M . Blumer, and J. Myers, Woods Hole Oceanographic Institution, Tech. Rep. 72-55, J u l y 1972, unpublished manuscript. (13) L. S. Ettre, J. E. Purceil, and K . Billeb, J. Gas Chrornatogr., 3, 584
(1965).
GLASS LINER IN INLET TUBE
{
EXACT DIMENSIONS TO FIT
I
GLASS - LINED INJECTOR ASSEMBLY CARRIER GAS INLET
,
SY PLITTER
INJECTOR BODY
Figure 1. Detail and
’
profile of glass injection port liner
Figure 2.
tative data output of good short and long-term reproducibility, and (4), hopefully, sufficient comparability of results from different columns to make spectral libraries feasible. Existing work has focused almost entirely on the first two capabilities, though the latter two are of great practical importance. For example, the Western Oil and Gas Association (4) recommended comparison of chromatograms run on the same instrument and column, preferably on the same day! This would mean that only four or five samples can be analyzed under highly comparable conditions. In contrast, we have been requested by an enforcement agency to attempt to match each of 35 samples with an array of 17 possible sources-a task which required a substantial fraction of the expected life of a column. Although the reproducibility of results over long periods and the comparability of results from different columns are quite important, they are neither predictable nor easily measurable under a variety of conditions. We therefore specified constraints on system design which we believed would enhance reproducibility, prolong column lifetime, and maximize the similarity of column/instrument combinations. Within these bounds, we explored the feasible trade-offs among resolution, separation, simplicity of operation, and sample preparation, and selected optimized conditions from this work, which was heavily guided by previous experience. Finally, we adopted a fixed set of conditions and set up several instrument/column combinations. Utilizing measurements defined in Figure 2, these were tested to determine their reproducibility and comparability characteristics.
Sample chromatogram of No. 2 fuel oil with terms defined
RESULTS AND DISCUSSION Even the best capillary columns fail to separate oils completely in the region of interest in correlation (retention indices >low). Clearly, gas chromatographic analyses with other goals in addition to highest resolutionsuch as simplicity, reproducibility, and extended column life-are run under conditions which challenge all phases of the system and are optimal for none. Therefore, we attempted to formulate the essential requirements of a useful correlation system, and to optimize the marriage of existing and easily developed capabilities to meet these demands. Problem Analysis a n d Approach. A practical GC correlation system should provide: (1) adequate resolution and separation of key components in the retention index region 1500-3500 encompassing the most environmentally persistent and geochemically variable components, (2) simplicity of sample preparation and analysis, (3) quanti-
Aside from the obvious standardizing of instrumental and operating parameters to enhance reproducibility, we judged that comparability and reproducibility required minimizing irreversible changes in the columns themselves. High operating temperatures and excessive inputs of semi-volatile material to the column were considered to be the principal causes of avoidable column ageing. Choice of Column a n d Operating Parameters. The low flow resistance per plate of open tubular columns recommended them over packed columns, as this asset can be flexibly apportioned among characteristics such as resolution, shortened analysis time, and lowered operating temperatures. Such flexibility facilitates optimizing systems with severe requirements. Furthermore, oils and environmental samples have been analyzed with superior resolution on such columns (2, 10, 14). (14) M . Ehrhardt, lnstitut fur Meereskunde, Kiel, West Germany, personal communication, 1971
ANALYTICAL CHEMISTRY, VOL. 45, NO. 6, MAY 1973
953
r’
I-. .-
*
-
r-T’
Figure 4. SCOT OV-101 column comparability Figure 3. Analysis of
No. 2 Fuel Oil on various columns
Top to bottom 7’h-ft Apiezon L, 3% on Chromosorb W, nitrogen, 50-ft Dexsil 300 SCOT, He, 50-ft OV-101 SCOT, nitrogen, same, helium
Furthermore, we have encountered difficulty in obtaining the same oil “fingerprint” on nominally identical packed columns, and have traced this problem to the difficulty of making columns with simultaneously identical plate counts and flow resistances; efficiency-resistance relationships are expected to be more uniform for open tubular columns. We therefore experimented initially with short capillary columns (10 meters) of OV-101 (15) similar to those utilized by Adlard and coworkers (2). Although we obtained an outstanding column, our next dozen coating efforts (with the same apparatus, stationary phase batch, and tubing roll) were complete failures, even with coating aids (16), and we therefore abandoned this column type. Next, we experimented with commercially available support-coated open tubular (SCOT) columns (50-ft X 0.02-in.), which have lengths and plate-counts similar to short, well-coated capillaries, but bear more stationary phase. The higher loading increases elution temperatures, (15) T. H. Gouw, I . M. Whittemore, and R. E. Jentoft, Anal Chem., 42, 1394 (1970) (16) E. J. Malec, J. Chromatogr. Sci.. 9,318 (1971).
954
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 6 , M A Y 1973
Top to bottom: separation of a 2: 1, 2: 1 heptadecane/pristane, octadecane/phytane test mixture: chromatograms of No. 2 fuel oil on columns 216, 218, 219
but also durability, relative to wall-coated columns. Figure 3 shows chromatograms of a No. 2 fuel oil obtained on a good packed column (Apiezon L) and on two SCOT columns (OV-101 and Dexsil 300); an Apiezon L SCOT column offered similar resolution but no separation of key peak pairs. The SCOT column runs are slightly faster and terminate a t a lower temperature, and elution temperatures are lower. The marked improvement in performance obtained by using He carrier gas is also clear. The precise temperature program and flow variables are not critical in oil correlation analyses, so long as they are exactly reproducible. Programmers and pyrometers must be calibrated, and flow rates carefully standardized and monitored; the latter is easily accomplished by determining the retention time of methane at a standard temperature. The chromatograms of Figure 3 on SCOT columns were determined under the standard conditions of operation. We selected the silicone phase OV-101 for further study, because it is thermally stable, and columns have good resolution and yield particularly favorable separations of key pairs heptadecane/pristane and octadecanel phytane (Figure 4).
~~~~~
Table I . Variability of Consecutive Analyses of Crude Oils for Several SCOT OV-101 Columns Relative Standard Deviation of Ratios, %a
Conditions Columnb
No. of runs
Crude oil
Pres/Phy
21 6c 218 218 219
6 6 6 6
TJ It. (crude) Agha Jari Agha Jari Agha Jar1
3.94 2.20 2.10 5.83d
a Ratios measured as defined in Figure 1 and related discussion. Columns used had been utilized previously for 80-160 analyses. After period
Sample Preparation and Introduction. As previously indicated, the usefulness of an oil correlation system depends heavily on the longer-term stability of columns and intercomparability of results, as well as on simplicity of operation. We judged that relatively oil-rich samples should not require treatment more complex than dissolving and filtering or centrifuging to remove insoluble matter; distillation or liquid chromatographic cleanup is both time-consuming and a potential source of fractionation and additional variability. Dilute samples, such as sediments and organisms lacking a macroscopic oil phase, will require more elaborate extraction and cleanup for any gas chromatographic method. Ruling out clean-up operations for routine samples eliminates the most common method of protecting columns, and places the entire burden on the injection and operating parameters. Columns could be protected by using short packed precolumns and/or backflushing routines. However, even packed columns tend to change flow resistance at the front end (14) with oil residues, and capillary backflushing presents technical problems. We have found that injection of 0.5-5 p1 of ca. 10% solutions of sample in CS2 into a glass-lined injector a t 195-210 "C, with subsequent 1 O : l splitting and column initially a t 75 "C, yields reproducible sharp-peaked traces, with long column lifetimes as well. The low temperature minimizes pyrolysis and introduction of semi-volatiles to the column, and the liner permits residue removal and avoids metal-catalyzed decomposition. Thus, the column can be effectively protected from residues by the use of a lowered injection port temperature without concurrent peak broadening; Grob and Grob ( I 7) have discussed the importance and manipulation of the injector temperature relative to the column temperature. With similar programs and the injector at 175 "C, nearly all the volatile material from residue-bearing oil samples is recovered (6). The drastic injector temperatures (300350 "C) favored by other workers (1, 3, 4, 8) are unnecessary for programmed oil analyses. Indeed, for unsplit gas flows and 300 "C injectors followed by columns kept at low temperature, the load to the front of the column arising from bleed of septa-even conditioned ones-can easily exceed the load from injection. We see no significant bleed peaks a t operating sensitivity even after overnight standing with a cold column. The relatively nonretentive SCOT column is also maintained a t a temperature above that of the injector for about 30-45 minutes per analysis, spreading residues over a larger portion of the column. Even after 100-300 injections, the column bleed signal a t 275 "C is still dropping slightly, suggesting that the column is not loaded with high-boilers. Finally, infrequent solvent cleaning of the injector/splitter lines minimizes variability. Reproducibility and Intercomparability. Three SCOT OV-101 columns in different instruments were utilized to (17) K. Grob and G. Grob, J. Chrornatogr. Sci., 7,590 (1969)
C1 ,/Pris
Cis/Phy
C17ICl8
2.65 1.07 0.91 1.72
4.16 2.87 1.75 6.10d
3.00 0.70 0.82 1.30
Ci7/BG
9.59 3.41 1.85 2.68 of disuse; variability may be caused in part by "atrophy effect." Variability decreased after injection port cleaning.
-
4.5 4.3
83
A
#219 I- (COLUMN 442 EO. STD. I
4.4
-
3.9
A
A A
-
L
8 ;
A
A
0
h
3.7
0
L-
1.5
g
c3
n-Cv/BACKGROUND n-CWPHYTANE PRISTANE/PHYTANE n -CWPRISTANE n - Ci7/ n -Ci 8
0
a
x
0
0 l.i
0
I
I
20
I
I
40
I
I
60
I
I
80
RELAUVE SAMPLE SIZE Effect of sample size on signal intensity ratios: see Figure 2 for term definitions Figure 5.
analyze a variety of oils, oil products, and environmental samples. About half the oil samples were residue-bearing; each column received from 1'75-285 injections during the study. Periodic analysis of the standard No. 2 fuel oil or of Agha Jari crude yielded variability and intercomparability data. Table I shows consecutive-analysis variabilities of ratios (see Figure 2) at about the midpoint of the study. Relative standard deviations (RSD) for most ratios are in the region 0.7-37'0, except for a system with dirty injection splitter (RSD's improved after cleaning) and one which had not been used regularly. The data in Table I contain systematic variability caused by the small sample-size dependence of ratios, illustrated in Figure 5. This effect is small for ratios involving signal intensities a t peaks, but the 17lbackground ratio is much more variable; one would expect it to change with changing resolution. Table 11 shows RSD's of ratios for the same three columns over a longer period, using data from No. 2 fuel oil analyses. These relative standard deviations are surprisingly uniform; almost all lie in the 1.5-3% range, though again the 17/BG ratios are the most variable. I t is quite clear that analyses run a t widely separated intervals are closely comparable, with standard errors not much greater than those obtained in consecutive analyses. The longterm stability of ratios of partially resolved peaks is extremely stable. Comparison of Tables I and I1 suggests that operating irregularities (dirty injector, column disuse) may be more important in limiting comparison than simple column ageing. Also, the data of Table I are deANALYTICAL C H E M I S T R Y , VOL. 45, NO. 6, M A Y 1973 * 955
Table II. Long-Term Variability of Analytical Ratios of
No. 2 Fuel Oil Standard
Conditions Relative Standard Deviations of Ratios, %a Column
Total No. of runs
Injection NO.^
Pris/Phy
CIT/Pris
Cie/Phy
C17/C18
Ci7JBG
21 6 21 a 21 9
13 11 11
121-136 33-1 75 34-207
2.15 2.44 3.43
3.1 1 2.30 2.60
2.85 1 .a0 1.59
2.56 2.86 1.56
4.52 2.98 6.21
a Ratios measured as defined in Figure 2. Each sample injection is numbered; tabulated analyses of standards occurred during the interval
given,
Table 111. lntercolumn Comparison of Ratio Means Conditions Signal Intensity Ratiosa
No. runs Column No.
averaged
Sample
Pris/Phy
C17/Pris
Cie/Phy
Ci7ICia
Ci7IBG
21ac 21 gC 21 6d 21 ad 21gd
11 11 13
No. 2 Fuel oilb No. 2 Fuel oil* No. 2 Fuel oil*
1 .ai 1 .aa 1 .a3 0.93 0.89
1.41 1.41 1.29 1.97 1.93
1.90 1.90
1.34 1.40 1.44 1.17 1.11
4.36 4.34 3.42 5.56 5.49
a
6 6
Agha Jari Agha Jari
1.65 1.59 1.55
Ratios measured as defined in Figure 2. Standard sample. Runs made during period of -150 injections.
rived from samples bearing a residue; those in Table I1 are nok. The smaller RSD values for some ratios in Table I suggest that the presence of a residue does not increase variability. Of the columns a t hand, 216 (175 injections) has lost considerable resolution and is no longer suitable for correlations. Column 218 (220 analyses) retains excellent properties; however, even week-ends of disuse now require half a dozen programs to be run before stable values are obtained. Column 219, after 285 injections, is still excellent. These columns thus have useful lifetimes in the 150-300+ injection range. One of our goals was to determine how closely results from different columns would agree; we felt that the construction of open tubular columns was much more likely to be uniform. Figure 4 shows gas chromatograms of standard No. 2 fuel oil obtained on the three columns. They appear quite similar. In order to obtain a quantitative comparison of the differences, we calculated the ratio means; these are presented in Table 111. The means are quite close; in most instances the spread is about 2 standard errors. Column 216 was always of lower resolution than the other two, and this shows in the trends of the ratio values-especially the heptadecane/background ratio. However, even in this case the differences in mean values are so small that they can be corrected by reference to a standard sample. In short, the columns are so similar that only by the use of replicate analyses and statistical tests can the differences be clearly discerned. We have also found that the system is capable of distinguishing uniquely most of the 30 oils collected in a 2month ship sampling program in Greater New York Harbor, and of distinguishing many of the crudes occurring in the oil port of Portland, Maine. The correlation of 35 artificially weathered oil samples with seventeen possible source oils on a “blind” basis was also accomplished with a high degree of success, as will be discussed elsewhere (18).
CONCLUSIONS A gas chromatographic correlation system which meets practical reouirements can be realized. Geochemically characteristic and weathering-resistant sample parameters (18) 0 Zafiriou and F Freestone, submitted to 1973 Conference on Prevention and Control of Oil Spills, Washington, D C , March 1973
956
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 6, MAY 1973
Consecutive analyses.
have been measured with relative standard deviations ranging from a few per cent to fractions of a per cent. Results may be compared over long time periods or from different (but calibrated) apparatus, so that spectral libraries or data banks are now feasible. The necessary reproducibility and comparability was obtainable without resort to complex cleanup procedures for oil-rich samples. The demonstrated system could be improved and extended. For example, the S-sensitive detector could be used in conjunction with SCOT columns to overcome the sensitivity limitations found (2) with wall-coated capillaries and to provide highly reproducible data. OV-101 SCOT columns, with their low bleed, are also highly suited for use in GC-MS systems. Several measures might improve reproducibility. The best RSD values obtained-ca. 0.7%-are approaching the precision of analog recording and visual measurement; digital methods might be fruitfully employed. The larger standard errors appear to be caused by such factors as injector cleanliness and column hysteresis effects. We strongly suspect that data obtained in long continuous runs (that is, with the column in regular use) is markedly better than occasional-use data. Basic work on the nature of the hysteresis effects obtained in programmed-temperature GC may be required to lower all RSD values to the 0.5% range, A t these levels, data will merit correcting for sample-size variations. For very close comparisons, sample pre-treatment to obtain selected fractions-isolation of aromatics or molecular-sieve treatments-may enhance correlations. ACKNOWLEDGMENT I am indebted to M. Blumer and M. Ehrhardt for helpful suggestions, and to J. Myers for technical assistance.
Received for review November 17, 1972. Accepted January 22, 1973. This work was supported by Contract 15080 HEC of the Office of Research and Monitoring of the Environmental Protection Agency. Oil samples were furnished by the Edison, N.J., laboratory of EPA, by the U.S. Bureau of Mines, by the U.S. Coast Guard Port Captain’s Office, Governor’s Island, by the Maine Environmental Improvement Commission, and by M. Blumer of the Woods Hole Oceanographic Institution. Contribution Number 2978 from the Woods Hole Oceanographic Institution.
