Chromatographic Analysis of Hydrocarbons in Marine Sediments and Sea water** W.E. May, S.N. Chesler, S.P. Cram*, B.H. Gump, H.S. Hertz, D.P. Enagonio and S.M. Dyszel, Bioorganic Standards Section, Analytical Chemistry Division, National Bureau of Standards, Washington, DC 20234
Abstract Sediment or Water Sample The low concentration of hydrocarbons anticipated in pollution baseline studies necessitates the development of analytical techniques sensitive at the sub-microgram per kilogram concentration level. The method of analysis developed in this laboratory involves dynamic headspace sampling for volatile hydrocarbon components of the sample, followed by coupled-column liquid chromatography for the non-volatile components. These techniques require minimal sample handling, reducing the risk of sample component loss and/ or sample contamination. Volatile sample components are separated from the matrix in a closed system and concentrated on a TENAX-6C packed pre-column, free from large amounts of solvent and ready for GC/GC-MS analysis. Non-volatile compounds, such as the benzpyrenes, may be extracted from large volumes of water and concentrated on a Bondapak C18 packed pre-column for coupled-column liquid chromatographic separation and analysis. Results of the application of these techniques to the analysis of samples from sites of known low level hydrocarbon contamination are presented and discussed.
TENAX-GC Pre column
C 1 8 - Pellicular Pre-column
Introduction
GC (GC-MS]
LC
The low concentration of hydrocarbons anticipated in pollution baseline studies necessitates the development of analytical techniques sensitive at the sub-microgram per kilogram level. At this low level, the problems of analytical blanks and component recoveries become paramount. Furthermore, analytical methods that ultimately permit the identification of individual hydrocarbon components are desired. Several authors have developed methods for the analysis of hydrocarbons in marine samples (1-6). These methods are of three basic types: screening for total extractable hydrocarbons (1), static headspace analysis, specific for low molecular weight hydrocarbons (3,5), and digestion or extraction of the sample followed by clean-up, concentration and gas chromatographic (GC) analysis of the hydrocarbon components (2,4,6). These methods are subject to certain limitations. The screening technique for total extractable hydrocarbons is not sufficiently specific, i.e., it does not afford single-compound identification. Static headspace analysis methods are suitable only for compounds of relatively high volatility. Extraction/ digestion procedures are generally lengthy, involve considerable sample handling and usually result in considerable losses of hydrocarbons having molecular weights of 200 and below. An alternative scheme for the analysis of hydrocarbons in marine sediments and in water has been developed in this laboratory (Figure 1). It involves dynamie headspace sampling and the trapping of volatile components on a TENAX-GC packed pre-column. The trapped components are subsequently analyzed by GC or gas chromatography-mass spectrometry (GC-MS). The non-volatile components are then analyzed by coupled-column liquid chromatography (LC). Samples which have been collected and frozen in the field, are thawed and transferred to a headspace sampling flask in a class 100 JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 13
Clean Air at 4 C Add Water to Sediment Closed System *2 H to 70 C t4h Vapors
Liquid
Figure 1. Scheme for analysis of hydrocarbons in marine sediments and water.
laminar flow, clean air hood maintained in a cold (4°C) room. They are then headspace-sampled for a period of four hours using scrubbed nitrogen gas to sweep volatile components into the TENAX-GC packed pre-columns. After headspace sampling is completed, the liquid remaining in the flask is pumped through an LC pre-column packed with Bondapak C18. Subsequent GC or GC-MS (utilizing capillary columns) and analytical LC are used to separate, identify and quantitate the volatile and non-volatile components of the sample. Various headspace sampling and coupled-column LC techniques have been applied in other analysis systems (e.g. 5,7-15). Grob (8) used charcoal as an adsorbent for headspace sampling of drinking water. Trapped material was desorbed with carbon disulfide and analyzed by GC and GC-MS. Zlatkis and co-workers used Porapak P, Carbosieve and TENAX-GC as adsorbents in air pollution studies (12) and in the headspace-analysis of gases and biological fluids (9,11). Trapped material was desorbed by heating and transferred to a pre-column for subsequent GC and GC-MS analysis. Snyder (15) has discussed the use of coupled-column LC techniques with Corasil-II and Porasil A for the separation of non-polar, intermediately polar, and polar antioxidants. •Present Address: Varian Aerograph, Walnut Creek, CA 94598. * * Identification of any commercial product does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose.
NOVEMBER 1975* 535
Table III Benzene
Butanol
Pentanone
Nitropropane
Pyridine
682:»0.6 683±1.0 +0.1 702:0.6 700:0.3 -0.3 733:0.3 732dO.6 -0.1
672:0.3 706:0.3 708:0.5 +0.3 738:0.9 735±1.3 -0.4 767d0.3 769:0.6 +0.3
716±1.7 778+1.3 782:0.9 +0.5 818:0.9 821±1.7 +0.4 866dO.3 872±1.5 +0.7
738^0.3 783dO.9 793±1.5 +1.3
OV-11 OV-11* %Dif
663:0.5 689:0.3 692:0.6 +0.4 712:0.6 716:0.6 +0.6 742:0.3 745:0.3 +0.4
OV-17
765:0.0
747±1.0
788±1.3
894:0.9
898:0.9
OV-101 OV-3 OV-3* %Dif OV-7 OV-7* %Dif
816:0.9 827:0.9 +1.3 863:0.6 871:0.9 +0.9
* : mixed bed
Columns to be compared, for example, OV-7 and OV-7* (* = mixed bed), were conditioned together from 30°C to 250°C at 8°C/min and allowed to bake at 250°C for 1 hour. All experiments were performed with the column oven set at 100°C. Furthermore, all data were obtained simultaneously for each set of columns being compared. Column comparisons were, therefore, uninfluenced by minor temperature variations. Corrected retention times were derived using methane to approximate time in the mobile phase. It was assumed that methane spends no time in the liquid phase. Injection size was on the order of 0.05 y. 1 per solute plus 5 \k 1 of methane. Flow rates were adjusted to optimize peak shapes.
mixed bed silicone polymer columns certainly increases mono tonically as a function of the number of phenyl groups. Th« approach described in this paper, basing the polarity on the average weight percent of phenyl groups within the column, is a valuable modification of the proposal by the latter authors making quantitative correlations of column selectivity possible. Manuscript received February 18, 1975
Acknowledgment
Results and Discussion Table III lists the Kovats' Indices for the five McReynolds' test solutes (benzene, 1-butanol, 2-pentanone, 1-nitropropane and pyridine) on all columns, as well as the standard deviation of the mean for each value reported. The peak maxima were used for the measurement of all retention times from which the Kovats' Indices were derived. The reported indices are the average for three replicate determinations. Also included in Table III are the percentage differences between the index values on the copolymer columns and the mixed bed columns. It should be noted that tail suppressing compounds were not used in this study because they could camouflage the effects of the phenyl groups. The data in Table III clearly show the similar retention properties of the copolymer columns and the mixed bed columns containing the same number of phenyl groups (same average weight percent of phenyl groups within the column). Mann and Preston (6) suggested that the retention behavior of a 1:1 mixture of dimethylsilicone polymer and 50% phenyl substituted methylphenylsilicone polymer should give equivalent results to a methylphenylsilicone-dimethylsilicone copolymer with 25% phenyl substitution. This concept seems practical and qualitatively correct because the polarity of
534-NOVEMBER 1975
The authors gratefully acknowledge the assistance of Dr. Shin Tsuge (Department of Chemistry, Faculty of Engineering, Nagoya University, Nagoya, Japan) for his help during the planning stages of this project.
Literature Cited 1. Rohrschneider, L., Z. Anal. Chem. 170, 256 (1959). 2. Hildebrand, G.P. and Reilley, C.N., Anal. Chem. 36, 47 (1964). 3. Keller, R.A. and Stewart, G.H., Anal. Chem. 36, 1186 (1964). 4. Molera, M.J., Dominguez, J.A.G. and Biarge, J.F., J. Chromatog. Sci. 7, 305 (1969). 5. Pilgrim, G.W. and Keller, R.A., J. Chromatog. Sci. 77,206(1973). 6. Mann, J.R. and Preston, S.T., J. Chromatog. Sci. 77, 216(1973).
JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 13
The use of dynamic headspace sampling and coupledcolumn LC alone and in combination offers a number of advantages over most of the current techniques for analysis of marine sediment and water samples. Both techniques require minimal sample handling, thereby reducing the risks of loss of sample components and of possible contamination. Volatile components of the sample are efficiently separated from the matrix in a closed system and collected in a concentrated form that is free from large amounts of solvents and ready for GC/GC-MS analysis. The headspace-sampling technique is well-suited for naphthalene and substituted naphthalenes, compounds of special interest due to their suggested immediate toxicity to marine organisms (16,17). Analysis at the sub-microgram per kilogram level is readily attainable by GC. High molecular weight non-volatile compounds, such as the benzpyrenes, may be readily analyzed by the coupledcolumn LC technique. This latter method can be very sensitive, since trace amounts of hydrocarbons can be concentrated from very large volumes of water. The analysis is nondestructive and separated compounds may be detected by UV photometry (254 nm) or collected and identified by other techniques. A significant advantage derives from the fact that water is the only solvent needed for sample preparation. The use of hydrocarbon-free water reduces opportunities for contamination of the sample by fossil hydrocarbons.
Experimental Preparation of Hydrocarbon-free Water Hydrocarbon-free water (18) is prepared by distillation from alkaline permanganate followed by passage through a column packed with XAD-2 copolymer (Rohm and Haas, Philadelphia, PA) Preparation of Sediments and Water for Gas Chromatographic Analysis Due to the low levels of hydrocarbons present in the samples, special caution was excerised at all stages during the collection and handling of water and sediment samples (18). An internal standard is added to the water samples at the time of collection. All samples were quick-frozen in the field for return to the laboratory. Until analyzed, samples were stored in a freezer at -10°C. For analysis, the samples were allowed to thaw overnight in a laminar flow hood in a cold (4°C) room. They were then transferred into 2-liter tared flasks for headspace sampling. Approximately 100 g of sediment or 750 ml of water were taken for analysis. Approximately 600 ml of hydrocarbon-free water and 1 iA of the internal standard solution were added to the sediment samples. The internal standard consisted of a solution of aromatic hydrocarbons, each present at a known concentration (approximately 2 y% per MOThe inlet of each headspace sampling flask was connected to a supply of scrubbed nitrogen gas. The exit of each flask was connected to a 6.5 x 0.6 cm (1/4 in.) o.d. stainless steel column packed with TENAX-GC (Applied Science Laboratories, State College, PA) which had been heated previously to 35O°C for 30 minutes to remove any contaminants. Details of the sampling apparatus are shown in Figure 2. Experiments were carried out on six samples simultaneously, one of the six being a system blank that consisted of hydrocarbon-free water plus the internal standard. 536 •NOVEMBER 1975
Figure 2. Headspace sampling vessel, (a) N2 gas inlet, (b) copper tubinc (1/8 in.), (c) glass tubing (1/4 in.) (d) tapered joint 24/40, (e) heatinc mantle, (f) magnetic stirrer, (g) dispersion tube with coarse frit, (h) taperec joint 45/50, (i) glass transfer line (1/4 in.), (j) TENAX trap, (k) glass coolinc jacket maintained at 15°C with air, ( I ) 2-liter round-bottom flask, (m) Teflor tubing (1/8 in.), (n) to rotometer, (0) copper coil, (p) ice bath, and (q; compressed air inlet.
The headspace sampling procedure was as follows: the cool CW5°C) air flow around the TENAX-GC column was begun, the magnetic stirrer was started, and a flow of approximately 150 ml/min of pre-purified nitrogen was established through each sampling vessel. The headspace was sampled at room temperature for two hours. Then the flasks were heated tc 70°C and the headspace sampling was continued for an addi tional two hours. At the end of the four-hour sampling period, each cooled TENAX-GC column was connected for two hours directly tc the nitrogen line to remove trapped water from the column (flow rate of nitrogen = 150 ml/min). Each TENAX-GC column was then capped tightly and stored at 4°C until analyzed by GC/GC-MS. Preparation of Water for Liquid Chromatographic Analysis Water remaining in the flask following headspace sampling was decanted into a clean one-liter beaker which was then covered with cleaned aluminum foil. The water was then pumped through a 6.5 x 0.6 cm (1/4 in.) o.d. stainless steel liquid chromatographic pre-column at the rate of 10 ml/min by use of a Milton-Roy Minipump. This column was packed with a 37-50 yon pellicular (superficially porous) support with a bonded Cjg-stationary phase (Bondapak C18 — Waters Associates, Milford, MA), and was fitted on both ends with Swagelok stainless steel reducing unions with 2 pan snubbers. Liquid Chromatography After the headspace sampled water was pumped through the LC pre-column, the pre-column was attached to a liquid chromatograph capable of gradient elution (Figure 3). The two reservoirs of the gradient pumping system contained degassed, redistilled, reagent grade methanol and hydrocarbon-free water, respectively. Ten ml of water were pumped through the pre-column to remove entrapped air. Then a 30 cm x 0.6 cm (1/4 in.) o.d. stainless steel analytical column packed with a 10 pan micro-particulate (totally porous) support with a bonded Cjg-stationary phase (/iBondapak C18 — Waters Associates, Milford, MA) was connected to the outlet end of the pre-column. Elution of adsorbed compounds from the coupled columns was begun with a 30:70 (v/v) JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 13
methanol-water mobile phase pumped at 3 ml/min. The gradient was programmed to increase the percentage of methanol in the mobile phase to 100% in 40 minutes. The effluent from the analytical column was passed through a UV photometer (245 nm) and the chromatogram was recorded. Individual fractions were collected for subsequent analysis of the individual compounds (by UV, fluorescence and mass spectrometry). Methanol Reservoir
Figure 4. Schematic of gas chromatographic flow system showing location of TENAX-GC pre-column.
C18 - Pellicular Pre-column
CIS - Micro-particulate Analytical Column
UV Photometer 2 5 4 nm
to Fraction Collector
Figure 3. Analysis scheme for non-volatile hydrocarbons by cojjpled-column liquid chromatography.
Gas Chromatography
The gas chromatographic system used for separating the constituents that are trapped on the TENAX-GC pre-column is presented schematically in Figure 4. The helium carrier gas was filtered through a molecular sieve and particle filter before entering the unheated injection port of a Varian Model 2100 gas chromatograph. The TENAX-GC packed pre-column was installed between the injection port and a coiled, glass SE-30 coated, SCOT analytical column (100 m x 0.65 mm i.d. drawn and coated [19] in this laboratory). A removable aluminum heating block, powered by a high intensity cartridge heater and Variac, was placed around the TENAX-GC column. The first eight coils of the capillary column were isolated from the remaining coils for cryogenic cooling to provide thermal focusing of components released by heating the TENAX-GC column. A makeup carrier gas was introduced directly into the detector to reduce the effective detector dead volume. Figure 5 is a photograph of the inside of the gas chromatographic oven with a pre-column installed. The first eight coils of the analytical column were sprayed with liquid nitrogen as the TENAX-GC pre-column containing trapped hydrocarbons was heated to 375 °C for 8 minutes. The flow rate of carrier gas through the system was 20 ml/min during this operation. Then the flow rate was reduced to 6 ml/min, the cryogenic cooling was terminated, and the gas chromatographic oven temperature was raised to 50°C and maintained at 50°C for 8 minutes. For the chromatographic analysis, the oven temperature was programmed to rise at a rate of 6°C/min and hold at 275°C. A flame ionization detector was used. JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 13
Figure 5. Gas chromatographic oven with aluminum heating block, cryogenic cooler and capillary column.
A gas chromatograph-mass spectrometer computer system (Model 5930, Hewlett-Packard, Palo Alto, CA) was used for GC-MS analysis of the samples. These analyses were performed in an analogous manner to the GC analyses. Mass spectra were accumulated every 4 seconds during the course of the chromatogram. Results and Discussion
In designing an analytical method for a hydrocarbon baseline study requiring determinations of single components at the Mg/kg (ppb) level, the criterion of prime importance is the development of a contamination-free procedure that minimizes possible losses of sample. Such a procedure would entail as few sample transfers, as few solvents, and as few concentration steps as possible. The headspace sampling and LC techNOVEMBER 1 9 7 5 * 537
niques described in this paper meet this criterion and in addition offer the following advantages: a) the time involved in the work-up of samples is reduced from days to hours; b) compounds having a wide range of molecular weights can be analyzed; c) samples are handled in a closed system, which provides a hydrocarbon-free nitrogen atmosphere from the time of transfer to the headspace sampling flask until the volatile components are trapped on the TENAX-GC precolumn. The non-volatile components are trapped on an LC pre-column, a concentration step involving neither solvent extractions nor solvents other than hydrocarbon-free water. The early introduction of an internal standard is a prerequisite for good quantitation. By use of a mixture of several aromatic compounds it is possible to correct for the varying recoveries over the range of molecular weights analyzed. The internal standard is added to water samples in the field at the time of collection and to sediment samples at the earliest practical stage, that is, when these samples are in the laminar flow hood being prepared for sample work-up. The timing of this addition is in direct contrast with some earlier procedures (2,4) where the internal standard is added after sample work-up, just prior to final analysis. In sample analysis, the use of recoveries relative to an internal standard enables one to correct for losses that may be due to adsorption on glass as well as losses which arise during the analytical scheme. The recoveries from water of the various internal standard components by headspace sampling/gas chromatography and by coupled-column LC are shown in Tables I and II. By comparing these data on recoveries, it is evident that the LC technique becomes more effective as the headspace sampling becomes less efficient. Thus the two procedures complement each other very well. Capillary column GC was chosen for the analyses since the single compound separation it provides is superior to that obtained using packed columns. Use of thermal focusing with liquid nitrogen during the transfer of volatile components to the capillary column further improves the chromatographic resolution. High resolution GC with a low blank is of extreme importance for GC-MS. Initially XAD-2 resin was used as the trapping agent, but its thermal stability was found to be inferior to that of TENAX-GC for thermal desorption of trace organics. Figure 6 shows chromatograms obtained by the flash heating of XAD-2 to 275°C and of TENAX-GC to 400°C. The minor blank levels observed on flash heating TENAX-GC at 400°C do not appear when it is heated to 375°C. Reverse phase coupled-column LC was chosen as the analytical method for the less volatile compounds since it presents an efficient concentration technique that does not use organic solvents. Use of elution focusing from the pre-column onto the analytical column allows one to extract large volumes of sample with the relatively inexpensive pre-column and then rely on the more expensive analytical column only for the analysis of the compounds of interest. Reverse phase LC affords an advantageous isolation of the non-polar hydrocarbons onto the pre-column with freedom from polar compound interferences. Finally, coupled-column analysis is nondestructive and the eluted compounds are available for spectrometric examination. Efficient elution focusing requires that the pre-column retain hydrocarbons and yet have an affinity such that the hydrocarbons can be subsequently transferred to and focused on the head of the analytical column. Insight into a suitable pair of columns for the coupled-column approach can be gained by examining data on retention volumes of the uncoupled columns. The adjusted retention volumes per gram for phenanthrene on various column packing materials are
538'NOVEMBER 1975
Table I. Recovery from water [600 ml] of internal standart compounds using headspace sampling and gas chromatography Concentration (/ug/kg) Mesitylene 2.7 Naphthalene 3.5 n-Propylnaphthalene 3.3 Phenanthrene 3.3
% Recovery 8±5 38±7
51±10 19±12
Table II. Recovery from water [2 I] of internal standard compounds using coupled-column liquid chromatography Naphthalene Phenanthrene Pyrene Benzo[a]pyrene Dibenzanthracene
Concentration (/ug/k g) 28 1.5 2 1.5 3
% Recovery 19±2 92±12 78±17 58±12 14±8
TENAX BLANK
w XAD-2
BLANK
Figure 6. Gas chromatograms obtained by flash heating XAD-2 to 275°C and TENAX-GC to 400°C.
Table III. Phenanthrene elution from columns of various packing materials — adjusted retention volumes per gram of packing as a function of the composition of methanol-water elution mixtures Elution Mixture % Methanol (in Water)
0 20 40 60 80 100
v
R/Q(ml/g)
Firebrick
BondapakC18
/xBondapakC18
TENAX-GC
200 22 1.6
930 140 21 3.4
23,000 2,400
24,000 3,300
250 26 2.8
460 75 8.1 1.4
0.29 0.03 0.00
0.44 0.07
0.29
listed in Table III. The use of/iBondapak C18 as the analytical column precludes the use of TENAX-GC as the packing material for the pre-column, since the latter has the greater adjusted retention volume per gram. Firebrick has low affinity
JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 13
for hydrocarbons and thus is not an efficient extractant for them. Bondapak C18 pre-columns have sufficient hydrocarbon affinity, and are also well-matched to /jBondapak C18 for use as coupled columns. Separations of a hydrocarbon mixture on Bondapak C18, /iBondapak C18 and coupled columns of these two materials in sequence are shown in Figures 7A-C, respectively. Resolution is essentially the same on the coupled columns as it is on the analytical column alone. On the basis of these chromatograms, resolution of the phenanthrene-pyrene pair was calculated to be 1.4, 3.6 and 3.5 respectively.
Table IV. Trace organic levels [MQ] determined in water, system and chromatographic blanks Headspace Sampling Coupled-column System f/xg] Liquid Chromatography [/ug] Water blank System blank Chromatographic blank.