Identification of Specific Organic Compounds in a Highly Anoxic

16 Identification of Specific Organic Compounds in a Highly Anoxic Sediment by Gas

Downloaded by UNIV OF TEXAS AT DALLAS on July 12, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0147.ch016

Chromatographic-Mass Spectrometry and High Resolution Mass Spectrometry R O N A L D A . HITES and W I L F R I E D G . B I E M A N N Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Mass. 02139

The sediment of the Charles River Basin (an exclusively anaerobic system) was analyzed by computerized gas chromatographic-mass spectrometry (GC-MS) and high resolution mass spectrometry (HRMS). The organic compounds were separated first by methylene chloride extraction followed by gradient chromatography on alumina. This analysis revealed a large number of aliphatic and olefinic hydrocarbons, elemental sulfur, various and abundant polycyclic aromatic hydrocarbons (PCAH) and their alkyl derivatives, and two phthalate esters. Since the PCAH were the most abundant single class of compounds, their identification was pursued in detail. Possible sources of these compounds in the aqueous environment are petroleum, incomplete combustion, anaerobic biosynthesis, or chemical dehydrogenation of natural products.

' " p h e Charles River i n Boston is not one of the world s most commercially ·*• significant rivers. It is, however, of considerable aesthetic importance to metropolitan Boston. Unfortunately, it is highly polluted, a condition which is aggravated b y the geometry of the river basin and the associated dams. This has caused the irreversible intrusion of salt water back into the river. Because of its high density, this salt water wedge (shown i n Figure 1) prevents the normal seasonal, thermal inversion of the river. As a result very little if any vertical mixing of water occurs. This effect, taken together with the very low flow and with the occasional input of 188 Gibb; Analytical Methods in Oceanography Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

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Organic Compounds by GC-MS and HRMS 189

sanitary sewage, has led to a highly anoxic condition of the water. Thus the Charles River may be considered a typical, but extreme case of most bodies of polluted water. The Charles River is typical of other polluted bodies of water i n still another way, namely, there is little detailed information on the specific chemical nature of the pollutants i n the water. Previous work i n our CHARLES RIVER CROSS SECTION


HIGH CHLORIDE

MILES ABOVE DAM

Figure 1. Diagram of the Charles River Basin in Boston. NYC, Newton Yacht Club; LAB, Lars Ander son Bridge; BUB, Boston University Bridge; HB, Harvard Bridge; LFB, Longfellow Bridge. The sedi ment sampling location is indicated by X . Data from Ref. 14. laboratory ( I ) identified some of the specific organic compounds i n this water. A number of normal alkanes, several alkyl naphthalenes, a few alkyl anthracenes or phenanthrenes, pyrene, fluoranthene, and phthalate esters were found. The most abundant compounds present were di-(2ethylhexyl) phthalate and di-n-butyl phthalate. Detailed analysis of these data indicated that the alkanes (see Figure 2 A ) were probably of natural origin (from algae and bacteria), the phthalate esters were plasticizers

Gibb; Analytical Methods in Oceanography Advances in Chemistry; American Chemical Society: Washington, DC, 1975.


190

ANALYTICAL METHODS IN OCEANOGRAPHY

of ubiquitous urban origin, and the various polycyclic aromatic hydro carbons were probably of petrochemical origin. The hypothesis was ad vanced (J) that the aromatic hydrocarbons entered the water by the urban runoff system and originated i n gasoline and other petroleum fuels used i n the urban area. This paper reports further on the analysis of the Charles River system by a detailed qualitative examination of the organic compounds which are present i n the river sediment. This investigation of the sedi ment was undertaken from two points of view. First, the sediment is an obvious sink for pollutants which enter the river, and it was of interest to determine the identities of these compounds and, ultimately, their primary sources. Second, the sediment is a possible pollutant source i n itself, i.e., organic compounds may be generated i n the sediment by chemical or biological reactions and these would, i n turn, pollute the water. In both of these cases, a comparison of the organic compounds found i n the water and i n the sediment would provide information on the natural processes which tend to modify the pollutant load of a body of water. Experimental The sediment sample was obtained from the site indicated i n Figure 1 on September 28,1973. The sampling device was a weighted thin-walled brass tube with a Teflon flap closing the bottom, suspended on a cotton rope. It was allowed to fall into the water where it hit the bottom ver tically and penetrated into the soft m u d about 7 cm. W h e n pulled up slowly, the flap retained the m u d core and some supernatant water. The sample was transferred to a glass beaker, covered with aluminum foil, and immediately taken to the nearby laboratory. The sample consisted of a grey-to-olive, fine grained mud, smelling of hydrogen sulfide. In the laboratory it was immediately transferred to a large Buchner funnel, and the water was removed. The filter cake was dried i n a desiccator for two days. The dried mud was extracted with methylene chloride ( Nanograde) i n a pre-extracted Soxhlet apparatus for 18 hr. The solvent was evaporated under vacuum at 25°C, and the extract was weighed. T w o batches of the sediment were extracted. The first batch ( 14.97 g) was broken into pieces 2-10 m m i n diameter, and the resulting methyl ene chloride-extractable matter was 1.92% (0.287 g ) . The second batch (19.41 g) was powdered with a mortar and pestle, and the resulting methylene chloride-extractable matter was 1.96% (0.380 g ) . There was no increase i n the extractable organic fraction after pulverization, so it was assumed that the extraction was complete. The first batch of extract was dissolved i n a small volume of methyl ene chloride and subjected to gradient chromatography on alumina. W o e l m alumina was washed with methylene chloride several times and then activated at about 95°C for several hours. A 26-ml column (9 m m i d ) was prepared with a slurry of this alumina i n pentane, and 122 frac tions were eluted with a succession of solvents as follows: pentane



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191

(fractions 1-30), 4:1 pentane-benzene (fractions 31—40), 7:3 pentanebenzene (fractions 41-60), 1:1 pentane-benzene (fractions 61-80), ben zene (fractions 81-102), 1:1 benzene-methanol (fractions 103-112), and methanol (fractions 113-122). A l l solvents were nanograde and were shown to be pure enough for this application by G C analysis. E a c h fraction was evaporated to dryness i n 10-ml pear-shaped flasks, redissolved in a small amount of methylene chloride, and gas chromatographed. G C conditions were as follows: column, 6 ft X 0.125 in. od stain less steel packed with 3 % OV-17 on 100-120 mesh Gas-Chrom Q ; tem perature program, 7 0 - 3 3 0 ° C at 12°/min, holding at the final temperature for up to 20 min; and carrier gas flow rate, 28 m l / m i n . Fractions contain ing very little material or with G C patterns very much like adjoining fractions were combined. These fractions were then analyzed on the G C - M S computer system to obtain mass spectral data on the individual components. F o r certain fractions additional information was gained from ultraviolet spectra. The second batch of extract was fractionated into pentane-soluble and pentane-insoluble components; 31% of the methylene chloride ex tract was soluble i n pentane. This pentane-soluble fraction was then analyzed by H R M S by directly inserting the sample into the ion source. The G C - M S computer system consists of a Perkin-Elmer 990 gas chromatograph interfaced to a Hitachi R M U - 6 L mass spectrometer which is i n turn interfaced to an I B M 1802 computer. The details of this hardware and associated software have been published previously (2). The H R M S system consists of a DuPont Instruments 21-110B mass spectrometer and a D . W . M a n n comparator interfaced to the I B M 1802 computer. Details on this system are available elsewhere (3). Both mass spectrometers were operated at 70 eV ionizing energy. Results and Discussion In general, the compounds which were identified i n the sediment of the Charles River Basin can be divided into three groups. The first group comprises fractions 1-30 from the alumina chromatography and contains aliphatic and olefinic hydrocarbons and elemental sulfur. The second group is made up of fractions 31-91 and contains polycyclic aromatic hydrocarbons. The third group consists of fractions 92-122 and contains phthalate esters. Aliphatic and Olefinic Hydrocarbons. Of the first 30 fractions, only fractions 4-7 contained significant quantities of material. The explora tory gas chromatographic analysis of these four fractions showed them to be very similar; they were pooled and analyzed by G C - M S . Figure 2 shows the total ionization plot (trace B ) and the mass chromatogram of m/e 57 (trace C ) for these combined fractions. The peak at scan 165 is elemental sulfur. Its mass spectrum shows peaks separated by 32 mass units and a molecular ion at m/e 256 corresponding to eight sulfur atoms. The presence of sulfur is not unexpected i n such highly anoxic sediment (4).




192

SCAN MDMBER Figure 2. (A) Mass chromatogram of m/e 99 representing the distribution of normal alkanes in Charles River water. The numbered peaks represent normal alkanes of the indicated chain length. Data are from Réf. 1. (B) Total ionization plot of fractions 4-7 from the extract of Charles River sediment. (C) Mass chromatogram of m/e 57 representing the distribution of alkanes in Charles River sediment.



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The other components of this mixture are not resolved from one another and form a wide continuum i n the gas chromatogram centered at scan 220 which corresponds i n retention time to octacosane. The general nature of these compounds can be determined by inspection of a l l the mass chromatograms from this mixture. F r o m these data it can be con cluded that these compounds are alkanes, cycloalkanes, and alkenes (containing up to three double bonds) w i t h considerable branching. A mass chromatogram indicative of the alkanes found i n the river water ( at 2 m depth) is included for comparison (Figure 2 A ) ; the G C peaks caused by the individual normal alkanes are readily apparent. In the sediment, however, the normal alkanes are missing as noted b y the lack of regularly spaced predominant peaks i n the mass chromatogram of m/e 57 (Figure 2 C ) . The lack of normal alkanes i n the sediment is believed to result from biodégradation. The facile microbiological decomposition of normal alkanes (as opposed to branched species) is w e l l known (5). Polycyclic Aromatic Hydrocarbons. Gas chromatographio-mass spectrometric analysis of fractions 31-91 indicated the presence of vari ous polycyclic aromatic hydrocarbons ( P C A H ) with molecular weights of 178-316. Because it is impossible to discuss i n detail all the chromatoTable I. Polycyclic Aromatic Hydrocarbons Found in Sediment of the Charles River Basin by GC-MS . Unsubstituted Compounds Maximum — No. of No. of Alkyl Rings + Carbon Elemental Double MW Atoms Composition Bonds

Compound Found Anthracene or phenanthrene 4,5-Methylenephenanthrene Pyrene ( U V confirmation ) Fluoranthene ( U V confirmation ) Benzofluorenes (isomers unknown) Benzo[a]anthracene / Chrysene J Benzo[6 or fc]fluoranthene Benzo[a]pyrene Benzo[e]pyrene Perylene (trace quantities) C22H12 (isomer unknown) Dibenzopyrenes or dibenzofluoranthenes (isomers unknown)

12 9

C14H10

CuHio

10 11

178 190

15

CuHio

12

202

C17H12

12

216

10

CieHl2

13

228

5

C20H12

15

252

3

C22H12

17

276

3

C24H14

18

302

0

a

A UV spectrum of fraction 43 showed peaks characteristic of a 1-1 mixture of pyrene and flouroanthene. β


194



36-40

0

5

Py,45mp ,ap< cc

10

15

20

25

TIME , MIN.

Figure 3. Gas chromatogram of fractions 36-40 from the extract of Charles River sediment, ap, anthracene or phenanthrene; 45mp, 4,5methylenephenanihrene; f, fluoranthene; py, pyrene; chry, chrysene. The number of superscript cs indicates the number of alkyl substituted carbon atoms. graphic and spectrometric evidence, only a few examples w i l l be pre sented. A complete summary of a l l compounds found i n these fractions is presented i n Table I. F o r each compound listed, the mass spectrum and gas chromatographic retention times agreed with those of authentic material. The maximum number of alkyl carbon atoms found for the various polycyclic systems is also given i n Table I. The exact substitution of these alkyl carbon atoms on the ring system is, of course, not known. It seems likely that many isomers are present simultaneously. The data on alkyl substitution are based on the mass spectra and relative G C retention times of the methyl and most dimethyl or ethyl derivatives and on peaks i n the mass chromatograms corresponding to the higher alkyl homologs. As an example of the chromatographic and mass spectrometric data for these aromatic fractions, Figure 3 shows the gas chromatogram of combined fractions 36-40. Examination of the mass spectra correspond ing to the various chromatographic peaks, the gas chromatographic retention times, and the liquid chromatographic behavior allows one to make the structural assignments shown i n Figure 3. Anthracenes or phenanthrenes containing one, two, and three carbon atoms as alkyl substituents are present. T h e two major components are fluoranthene



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and pyrene. Methyl fluoranthene and methyl pyrene were also identified. Mass chromatograms indicated the presence of higher alkyl substituted anthracenes or phenanthrenes, fluoranthenes, and pyrenes. The mass spectrum of an interesting component i n this mixture (see Figure 4) was identified as that of 4,5-methylenephenanthrene, the pres ence of the methylene group causing a characteristically abundant M — 1 ion at m/e 189. A l k y l substituted derivatives of this compound are also present i n the combined fractions 36-40. I n fact, the mass spectrum taken during the emergence of the largest chromatographic peak i n this fraction (see Figure 5) clearly shows the C substituted 4,5-methylenephenan threne. Its molecular weight is 218 amu, and it exhibits fragment ions at m/e 203 and 189. I n addition, this mass spectrum (Figure 5) shows the presence of pyrene (m/e 202) and the three-carbon atom substituted anthracene or phenanthrene, giving ions at m/e 220 and 205. This mass spectrum is an example of the emergence of three different ring systems in the same chromatographic peak; the coincidence of differing levels of alkyl substitution causes them to coelute. In a similar manner, other poly cyclic aromatic ring systems and their alkyl substituted derivatives were identified i n other fractions. Table I summarizes this information. W h e n taken together w i t h the previous data on the aromatic hydro carbons present i n the water ( J ) , it is now apparent that P C A H ranging from naphthalene to dibenzopyrene are present i n the Charles River Basin system. The more water-soluble of these P C A H , the naphthalenes, are found in the water only; those of intermediate solubility, anthracenes or phenanthrenes, are found i n both water and sediment; and those of 2

Figure 4. Mass spectrum corresponding to the peak labeled 45 mp in Figure 3. It has been interpreted as that of 4,5-methylenephenanthrene.


196


202


189

218

1203 220

109

illlllllllliiin)iilllLi^

, i|i,..,J..iilll

M

Figure 5. Mass spectrum corresponding to the most intense peak in Figure 3 (retention time = 18.5 min). See text for interpretation. low solubility, molecular weights above 228, are found only i n the sedi ment. T h e sources of these compounds w i l l be discussed below. Phthalate Esters. Fractions 92-122 contained large amounts of di-(2-ethylhexyl) phthalate and much smaller amounts of di-n-butyl phtha late and dioctyl adipate. The ubiquity of phthalate esters as laboratory contaminants is well known. I n this case, however, the very high abun dance of phthalates i n these later fractions and their absence i n previous Table II.

Nonlinear Abundances of Polycyclic of the Charles River Basin

Total Carbon Number

u

15

16

17

18

19

20

21

22

13600 6792 2492 0 0 0

9676 6172 1248 76 532 0 0

7944 7028 16720 6320 1748 0 0 0

5208 5044 10468 640 2164 88 460 0 0

3132 3676 7020 12444 9140 2388 2g8 0 0 0

1920 2724 3932 7036 5404 556 168 1356 0 0 0

1460 2132 2236 4232 4524 14408 5908 3188 0 0 0 0

1732 2124 1500 2608 2996 7576 1568 1768 92 1072 0 0 0

2132 1984 1588 1316 1640 3744 8152 12232 3016 928 0 0 0

23 1812 1860 1104 696 1484 1868 3528 4344 436 88 624 60 0

These numbers are all divisible by four because a 14-bit A / D converter feeds a 16-bit computer word. α



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fractions and i n various blank analyses indicate that the phthalates are indeed present i n the Charles River sediment and are not artifacts. This should not be surprising since phthalate esters were among the more abundant components i n the river water ( I ) . It seems likely, therefore, that the river sediment accumulates these compounds from the water. Degradation by anaerobic bacteria, expected to be present i n the sedi ment, could be slow and, thus, over a period of time high abundances of phthalates could accumulate. High Resolution Mass Spectrometric Quantitation of Polycyclic Aromatic Hydrocarbons. Because of the striking abundance and variety of the P C A H found i n the sediment by G C - M S , it was of interest to measure the relative abundance of these hydrocarbons. This was accom plished by HRMS. A n aliquot of the total pentane-soluble fraction from the second batch of extracted sediment was introduced into a high reso lution mass spectrometer through the direct introduction probe system and vaporized at a continually increasing temperature while seven exposures on a photographic plate were made. After development, the plate was read on the computerized comparator, and the exact masses with their intensities ( i n nonlinear arbitrary units) were stored on magnetic tape. Later these exact masses were converted to elemental compositions (3), and the corresponding intensities were then arranged as tables of carbon number vs. number of double bonds and rings. One such table for each of the seven exposures was generated, and then a composite Aromatic Hydrocarbons Found in the Sediment as Determined by HRMS" Total Carbon Number

1580 1444 1148 588 960 976 1536 2652 8208 4236 1420 100 0

25

26

27

28

1276 1052 764 328 564 352 424 1032 2384 524 128 32 324

1032 924 616 176 84 68 132 268 940 2432 6596 1104 88

916 716 420 144 20 44 24 44 188 536 1520 88 0

292 292 116 76 20 0 0 28 0 20 524 2376 2240

29

80

256 152 0 0 0 0 0 0 0 0 24 100 0

64 100 40 24 0 0 0 0 0 0 0 0 36

Rings + Double Bonds 10 11 12 13 14 15 16 17 18 19 20 21 22



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table was formed by adding the corresponding entries i n each of these tables. This final set of data is shown i n Table II. Because P C A H display mass spectra with extremely abundant molecular ions, this table can be thought of as a relative abundance display of the molecular ions for those P C A H present in the sediment sample. F o r example, the entry at 16 carbon atoms and 12 double bonds or rings corresponds to a mass of 202.0783 and represents the combined abundance of pyrene and fluoranthene (16,720 units). The combined abundance of all of the methyl derivatives of both these compounds and of the benzofluorenes is given by the entry immediately adjacent on the right, namely 10,468. In a similar fashion, all of the benzofluroanthrenes and benzopyrenes are lumped together i n one intensity measure ment of 14,408 units (20 carbon atoms, 15 double bonds and rings). Unfortunately, the entries i n this table are not a linear function of com pound abundance; i n fact, the exact functional relationship is not known. It is known, however, that the higher the entry i n Table II, the more abundant are the compound or compounds represented by that entry. Thus, such a table is a crude semiquantitative indication of the relative abundance of the P C A H and their alkyl-substituted derivatives. Despite this limitation one notices a number of features about the relative distribution of P C A H i n Charles River sediment: 1. Pyrene and fluoranthene (m/e 202) are the most abundant group of isomers present; the C o H i group is the second most abundant. 2

2

2. Coronene ( C , 19 double bonds and rings) is present (4236 units), but it is less abundant than the dibenzopyrenes (8202 units). 2 4

3. A n additional series of P C A H based on the elemental composi tion C H i 4 and including the methyl and ethyl or dimethyl derivatives is present. 26

4. W i t h i n most series of homologs, the abundance monotonically decreases as substitution increases. In fact, a semilogarithmic plot of certain of these data (see Figure 6) shows a linear decrease in abundance as alkyl substitution increases within a given ring series. Indeed, the C i H i series (molecular weight 228) displays a linearly decreasing abundance over 10 atoms. 8

2

The cause of this linearity is not yet clear. Nevertheless, displays of this sort are a particularly powerful, graphic means of semiquantitatively characterizing P C A H mixtures, and, very importantly, their alkyl substituted homologs. Conclusions The single most abundant class of compounds identified i n the sedi ment of the Charles River Basin is the polycyclic hydrocarbons. This


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finding is unusual and surprising, and one immediately wonders about their source. [Concurrent work has shown that mixtures of P C A H of considerable complexity and which contain many of the same compounds listed i n Table I are also present i n a recent near-shore marine sediment (6)]. There are four possible sources of P C A H i n the aqueous environment.


20,000

10,000

3,000

Lu Ζ < Ω Ζ m 1000

Identification of Specific Organic Compounds in a Highly Anoxic

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