(37) Ganley, J. T., Springer, G. S., ibid., 8,340-7 (1974). (38) Mueller, P. K., ibid., 4,248-53 (1970). (39) Huntzicker, J. J., Friedlander, S. K., Davidson, C. I., 166th ACS National Meeting, Chicago, Ill., August 1973; 167th ACS National Meeting, Los Angeles, Calif., March 1974; Enuiron. Sci. Technol., 9,448-56 (1975). (40) Robinson, E., Ludwig, F. L., J. Air Pollut. Control Assoc., 17, 664-9 (1967). (41) Daines, R. H., Motto, H., Chilko, D. M., Enuiron. Sci. Technol., 4,318-22 (1970). (42) Lundgren, D. A., J. Air Pollut. Control Assoc., 20, 603-8 (1970). (43) Gillette, D. A., Winchester, J. W., Atmos. Enuiron., 6, 443-50 (1972). (44) Moyers, J. L., Colovos, G., ibid., 8, 1339-40 (1974). (45) Junge, C. E., “Air Chemistry and Radioactivity,”pp 131-41, 289-98, Academic Press, New York, N.Y., 1963.
(46) Martens, C. S., Wesolowski, J. J., Kaifer, R., John, W., Enuiron. Sci. Technol., 7,817-20 (1973). (47) Lundgren, D. A., J . Colloid Interface Sci., 39,205-10 (1972). (48) Duprey, R. L., “Compilation of Air Pollutant Emission Factors,” Public Health Service Publication No. 999-AP-42,1968. (49) Kircher, D. S., Armstrong, D. P., “An Interim Report on
Motor Vehicle Emission Estimation,” U.S.Environmental Protection Agency, Office of Air Quality Planning and Standards, APTD-1471, October 1972 (Revised January 12, 1973). (50) Roth, P. M., Roberts, P. J. W., Liu, M.-K., Reynolds, S. D., Seinfeld, J. H., Atmos. Enuiron., 8,97-130 (1974). (51) Colucci, J. M., Begeman, C. R., Kumler, K., J. Air Pollut. Control Assoc., 19,255-60 (1969). (52) Rahn, K., Wesolowski, J. J., John, W., Ralston, H. R., ibid., 21,406-9 (1971). Received for review November 15,1974. Accepted July 21, 1975.
Determination of Trace Organics in Municipal Sewage Effluents and Natural Waters by High-Resolution Ion-Exchange Chromatography W. Wilson Pitt, Jr.,* Robert L. Jolley, and Charles D. Scott Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
.Automated, high-resolution ion-exchange chromatographs, previously developed for the analysis of the molecular biochemical constituents in human body fluids, have been applied to the analysis of various polluted waters. Samples of polluted waters have been collected from selected sources, concentrated up to 10,000-fold, and chromatographed on high-pressure ion-exchange columns. Monitoring of the column eluates for ultraviolet absorbance and cerate oxidizability has revealed the presence of numerous organic contaminants, many of which have been subsequently identified by auxiliary techniques such as mass spectrometry. In the primary and secondary effluents from a domestic sewage treatment plant, 56 and 13 organics, respectively, have been identified and quantified. A total of 16 different organics have been identified in samples from five different natural waters. Our continually expanding population and industrialization have greatly increased the need for multiple reuse of surface waters with their concomitant concentrations of trace organic compounds from diverse sources. Regardless of the source, it is imperative that identities, quantities, biological effects, transformation products, and ultimate fate of the individual contaminants in the aquatic systems be established. The impact of these contaminants on the water ecosystems and, indeed, mankind, cannot be accurately assessed without quantitative information concerning the specific compounds and the effects of various treatment processes such as chlorination. Nor can adequate systems for removal or recovery of the organic content be designed without that prior knowledge. The American Chemical Society addressed this problem in 1969 with the recommendation that “an effort should be made to learn more of the specific chemical compounds, particularly organic compounds, that are present in industrial, and other wastes, and in natural waters,” and that “emphasis should be placed on the development of analytical methods for specific organic pollutants” ( I ) . Organic contamination in polluted waters has generally been determined in terms of gross organic content, while 1068
Environmental Science & Technology
only limited work has been done in identifying the individual molecular species (2, 3). In the past few years, however, significant progress has been made in developing methods and instrumentation for the identification and quantification of such species in the effluents of waste treatment plants (4-6). Because of its sensitivity and capability of analyzing for many low-molecular-weight refractory organics in aqueous samples, high-resolution anion-exchange chromatography is a logical choice for adaptation to the problem of analyzing for trace organics (particularly polar and/ or nonvolatile compounds) found in natural waters.
Materials and Methods The preparation of samples for analysis (concentration), the separation of individual contaminants (chromatographic analysis), and the application of analytical methods to separated fractions involve an integrated series of manipulations and investigative techniques. These have been described in detail elsewhere (6-9). Since many of the individual organic substances in polluted waters are present a t microgram-per-liter concentrations, and the detection capability of the chromatography used in this investigation was of the order of 1-10 mg/l., a suitable technique for concentrating aqueous samples by factors of 103-104 was required. A procedure based on lowtemperature vacuum evaporation (Figure 1) was used. In this procedure, the bulk (90-95%) of the water is removed in a low-temperature vacuum evaporator and the remainder by freeze-drying. The product from freeze-drying is redissolved in an acetic acid-ammonium acetate buffer (pH 4.4) and adjusted to the desired final volume. Any undissolved solids a t this point are removed by centrifugation. The sample concentrates are analyzed by high-resolution anion-exchange chromatography (7,8). Column: 316 stainless steel, 1.5 m X 2.2 mm i.d. or 1.5 m X 3.0 mm i.d. packed with strongly basic anion-exchange resin, Bio-Rad Aminex A-27. Eluent: ammonium acetate-acetic acid buffer (pH 4.4) a t 8 ml/hr; concentration gradient; 0.015-6.OM. Detectors: dual-wavelength ultraviolet photometer, 254 and 280 nm; cerate oxidation system ( I O ) . The use of the cerate oxidative monitor in series with the
-
UV-ANALYZER
SAMPLE N A T U R A L WATER 10-100 LITERS
+ CATION-EXCHANGE CHROMATOGRAPHY .L
REMOVE BUFFER
DOC
*
I(
UV SPECTRUM IN METHANOL+ CATION EXCHANGE
L
MASS SPECTROMETRY (MS)
GAS CHROMATOGRAPHY (GC)
4
COMBINED GC-MS Doc‘
Figure 2. Multiple-analytical procedure for identification of individual compounds in high-resolution chromatographic fractions EVAPORATOR PRESSURE
DOC = DISSOLVED ORGANIC CARBON
200 m l
FREEZE
D R Y AT D I S S O L V E IN A C E T I C
ACIO
DISSOLVE IN DILUTE ACETATE BUFFER AND CENTRIFUGE S OC* D
A
W
d
TO CHROMATOGRAPH
Figure 1. Schematic of procedure for concentrating polluted water samDles
UV photometer permits detection of additional compounds and a much greater sensitivity to others such as phenols. It also provides another parameter useful in compound identification, since many compounds that are not UV-absorbing are oxidizable and vice versa. Tentative identification of each individual compound responsible for a chromatographic peak may be made from its elution position, its relative absorbance a t the two ultraviolet wavelengths monitored, and its cerate oxidizability. More positive identification, on the other hand, requires corroborative evidence from one or more analytical techniques. At this laboratory, separated fractions from a preparative-scale chromatograph are collected and subjected to the multiple-analytical procedure shown in Figure 2. A positive identification requires confirmative information from two or more different analytical methods.
Results and Discussion By the methods described above, samples of effluents from primary and secondary stages of a municipal sewage treatment plant and samples of five natural waters were analyzed for trace organic contaminants. More than 150 UV-absorbing and/or cerate oxidizable compounds were separated from samples of effluent from the primary stage of a municipal sewage treatment plant. T o date, 56 of these compounds have been identified. The identifications, along with approximate concentrations of the compounds in the original effluent and the methods for confirming identifications, are listed in Table I. Figure 3 is a typical chromatogram of primary sewage treatment plant effluents, showing the elution positions of the identified compounds. In addition to the identified compounds, 103 unknown constitu-
ents have been characterized with respect to gas chromatographic and mass spectrometric properties. Similarly, 13 of the approximately 50 compounds separated from samples of effluent from a secondary sewage treatment plant have been identified, and an additional 20 characterized with respect to gas chromatographic and mass spectrometric properties. The identified compounds are listed in Table 11. Although natural waters have only recently been analyzed using this technique, 16 organic compounds have been identified among the many found in samples from five different natural waters (see Table 111).Typical chromatograms of natural water samples are shown in Figure 4. Use of the cerate oxidative monitor for natural water samples is particularly advantageous because of its 1-10 Wgh. sensitivity to phenols and carbohydrates. The compounds listed in Tables I, 11, and I11 represent many different classes of organic compounds. Because of the hydrophilic nature of the compounds in several of these classes-i.e., carbohydrates (glucose), polyols (inositol), glycosides (methyl-0-D-glucopyranoside), sulfate conjugates (indican), and nucleosides (inosine),they would not have been detected by the usual extraction-gas chromatographic methods. In addition, purine and pyrimidine bases (uracil) and amino acids (glycine) would have been quite difficult to detect. On the other hand, the absence from the lists of volatile hydrocarbons and chlorinated hydrocarbons, quite likely present in the samples and which would readily have been detected by extraction-gas chromatographic techniques, illustrates the complementary nature of HRLC and GC for analysis of dissolved organic contaminants. One of the most fruitful applications of high-resolution ion-exchange chromatography to an aquatic pollution problem is its use in studying the effects of chlorination of various waters. Since little definitive information was available concerning the potential hazards resulting from the chlorination of sewage plant effluents and other polluted waters, a series of experiments was initiated to determine whether chlorinated organic compounds are produced during chlorination of various waters. These ongoing experiments combined high-resolution anion-exchange chromatography with the use of chlorinating agents tagged with radioactive 36Cl. Earlier results had indicated that relatively stable, chlorinated organics are formed during chlorination under conditions similar to those existing at sewage treatment plants (11, 12). Present investigations with two different condenser cooling waters are yielding similar results. Material balances of the 36Cl activity show that approximately 1%of the chlorine is incorporated into chloroorganic compounds by addition andlor substitution reactions. These compounds are of great significance in terms of possible ecological effects, both in the short and long term ( 1 3 ) . Volume 9, Number 12, November 1975
1069
Table I. Identification of Molecular Constituents in Primary Domestic Sewage Plant Effluents Concentrati0n.b Compound
Ethylene glycol Maltose Galactose Glucose GIycer ol Galacitol Erythritol Urea "-Met h y l-4-p yr i done-3-car boxa m i de Phenylalanine Uracil 5-Acetylamino-6-amino-3-methyl uracil N' Met hy I-2-p yr i don e- 5-car bo x a m ide Tyrosine Thymine Theobromine 7-Methylxanthine Inosine Hypoxanthine Xanthine Adenosine 1,7-Dimethylxanthine 3-Methylxanthine Caffeine Guanosine 2-Deoxyglyceric acid 4-Hydroxybutyric acid 3-Deoxyarabinohexonic acid Quinic acid 1-Methylxanthine 2-Deoxytetronic acid Glyceric acid 4-Deoxytetronic acid 3-Deoxyerythropentonic acid 2,5-Dideoxypentonic acid 3,4-Dideoxypentonic acid Ribonic acid Oxalic acid 2-Hydroxyisobutyric acid Uric acid Palmitic acid Orotic acid Succirlic acid Phenol 3-Hydroxyphenylhydracrylic acid Phenylacetic acid 4-Hydroxyphenylacetic acid Benzoic acid 2-Hydroxybenzoic acid 4-hydroxy benzoic acid 3-Hydroxybenzoic acid 3-Hydroxyphenylpropionic acid Indican 3-Hydroxyindole @Phthalic acid p-Cresol
-
Identification methoda
AC, AC, AC, AC, AC, AC, AC, AC, AC, AC, AC,
GC, MS GC GC GC GC, MS GC, MS GC, MS GC, MS UV, GC GC, MS CC, UV, GC, MS
AC, CC, U V , GC AC, CC, UV, GC AC, GC, MS AC, CC, GC, MS AC, CC AC, CC AC, CC, UV, GC, MS AC, GC, MS AC, CC, U V , GC, MS AC, CC, U V , GC, MS AC, CC AC, CC AC, CC, UV, MS AC, CC, UV, GC, MS MS GC, MS MS MS AC, CC, U V MS MS MS MS MS MS MS AC, GC, MS GC, MS AC, GC, MS GC, MS AC, U V , GC, MS AC, GC, MS AC, GC, MS AC, UV, GC, MS AC, GC AC, UV, GC, MS AC, GC, MS AC, GC, MS AC, GC AC, GC, MS AC, GC, MS AC, GC, F MS AC, UV, MS AC, GC, MS
PgA
3 0.5
-
15-19 2 5 16-43 I O ; 14 9 0 ; 50 4 0 ; 16-58
140 2 0 ; 25 34 7; 9-28 90; 2 50; 11-23 25; 12-42 70; 2-7 13 - 1 0 ; 29-46 50; 4-28 7 7 50 70 6 5 6 4 6 13 4 2 4 20 6; 12 5; 2 24 6; 12 10-22 -1 0 1 9 0 ; 16- .52 3 7; 2 1 -40; 7 -20; 6 -2; 1 2 200 20; 29
Anion-exchange elution position, ml
8 8 8 8 10 10 10 10 14 16 18 20 21 23 29 29 29 29 30 55 64 64 64 67 76 80 80 80 80 82 85 90 90 90 90 90 90 95 95 106 128 145 145 205 205 230 235 260 290 295 295 295 325 340 400 400
a A C anion exchange chromatography; CC, cation excha ge chromatography. U V ultraviolet spectrum. G C gas chromatography on t w o colum&; MS, mass spectroscopy; F, fluorescent spectrum. Based on ultraviolkt abdorbance during A C (i:e., vhlues in italics), or on flame Ionization detector response during GC.
a
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Environmental Science & Technology
E LUTlOY VOLUME. rn C
25
50
75
I50
2w
21T--*
ELUTION MLLm,rnl
2yI
Figure 3. Chromatogram of 1000-fold concentrate of effluent from the primary stage of a domestic sewage treatment plant
Table I I . Identification of Molecular Constituents in Secondary Domestic Sewage Plant Effluents Approximate anionexchange elution position, ml
Cornpou nd
I dent if icat io n methodn
Concentration,b PdI.
Glycerine Uracil 5-Acetylamino-6-amino-3-methyI uracil 1-Methylinosine Inosine 7-Methylxanthine 1-Methylxanthine 1,7-Dimethylxanthine Succinic acid Catechol I ndole-3-acetic acid 3-Hydroxy indo le p-Cresol
AC, GC, MS AC, CC, U V , MS AC, CC, U V
4 3 0 ; 16 30
10 18 20
AC, AC, AC, AC, AC, AC, MS MS MS AC,
80 20 5 6 -6 4 1 13 2 9 0 ; 20
29 29
CC, U V
cc, uv CC, U V GC CC, U V MS
GC, MS
29
-
82 64 135 235 235 320 300
a AC, anion-exchange c h r o m a t o g [ a p h y ; C C ,cation-exchange c h r o m a t o g r a p h y ; p V , ultraviolet s p e c t r u m ; M S , mass spectroscopy: G C , gas c h r o m a t o g r a p h y . b Based o n ultraviolet absorbance during A C ( [ . e . ,values in italics), or o n f l a m e l o n l z a t i o n detector response d u r i n g G C .
Volume 9, Number 12, November 1975
1071
Table Ill. Molecular Constituents Identified in Natural Water Samples Identification methodb
ldentif ication Constituent
met hodb
Constituent
AC, AC, AC, AC, AC, AC, AC, AC,
p-Cresol Diethylene glycol Glycerin Glycine Mannitol Methyl-a-D-glucopyranoside Methyl-p-D-glucopyranoside Sucrose
CC, MS MS GC, MS GC, MS GC, MS GC, MS GC, MS GC, MS
5'.
il I\ I
10
'
'
,
I
;
....- - - CERIUM
'\\
/
/I
$\,
(111)
FLUORESCENCE
-254
om P B S O R B A N C E
-
nm P B S O R B A N C E
_ _2 8 0
9,
_/--
,,A-
//--
_/--,----I
0
1
2
3
4
5
6
7
8
9
IO
TIME (hr)
I/
I2
I3
14
I5
O
16
I7
18
Y 1 0
0
0 18
19
20
21
22
23
24
25
26
27
2a
29
TiME (hr)
Figure 4. High-resolution chromatogram of trace organics in natural water Top, sample from Watts Bar Lake: bottom, sample from Lake Marion, S.C 1072
Environmental Science & Technology
GC, MS GC, MS MS MS
rograpny; CC. cation-excnange c n r o m a t o g r a p n y : J V , J traviolet soectrkm; G C , gas c n r o v a r o g r a p n y : MS, mass sDec:roscoDY.
a l L a k e M a r i o n . 2 F o r t L o u d o n a k e ; 3, Holston River: 4, Miss;ssippi River; Watts Bar Lake.$ AC, anion-exchange c h r o m a -
I
AC, AC, GC, GC, MS MS MS MS
Xy I itol Urea Inositol o-Methylinositol Linoleic acid Oleic acid Palmitic acid Stearic acid
30
31
32
33
34
35
36
In addition to the above results, some interesting preliminary data have been obtained by chromatographing some of the samples of concentrated polluted water on high-resolution amino acid analyzers and carbohydrate analyzers. Several chromatographic peaks were obtained on both analyzers, and indicate an additional area for research.
Literature Cited (1) ACS Subcommittee on Environmental Improvement, “Cleaning Our Environment, The Chemical Basis for Action,” pp 105-55, American Chemical Society, 1969. (2) Skrinde, R. T., “Analytical Methods-Chemistry. Organics,” J . Water Pollut. Control Fed., 44,911 (1972). (3) Tyckman, D. W., Irvin, F. W., Young, R. H. F., “Trace Organics in Surface Waters,” ibid., 39,458 (1967). (4) Garrison, A. W., Keith, L. H., Walker, M. M., “The Use of Mass Spectrometry in the Identification of Organic Contaminants in Water from the Kraft Paper Mill Industry,” Proc. 18th Annual Conference on Mass Spectrometry and Allied Topics, pp B205-13, San Francisco, Calif., June 14-19, 1970. (5) Jolley, R. L., Pitt, W. W., Jr., Scott, C. D., “High-Resolution Analyses of Refractory Organic Constituents in Aqueous Waste Effluents,” Proc. 19th Annual Technical Meeting of the Znstitute of Environmental Sciences,. DD 247-52, Anaheim, Calif., April 2-5, 1973. (6) Katz. S., Pitt. W. W., Jr.. Scott, C. D., Rosen. A. A., “The Determination of Stable Organic Compounds in Waste Effluents a t
__
Microgram per Liter Levels by Automatic High-Resolution Ion Exchange Chromatography,” Water Res., 6,1029 (1972). (7) Scott, Charles D., “Analysis of Urine for Its Ultraviolet-Absorbing Constituents by High-pressure Anion Exchange Chromatography,” Clin. Chem., 14,521, (1968). (8) Scott, C. D., Jolley, R. L., Pitt, W. W., Johnson, W. F., “Prototype Systems for the Automated, High-Resolution Analyses of UV-Absorbing Constituents and Carbohydrates in Body Fluids,” Am. J . Clin. Pathol., 53,701 (1970). (9) Mrochek, J. E., Butts, W. C., Rainey, W. T., Jr., Burtis, C. A,, “The Separation and Identification of Urinary Constituents Using Multiple-Analytical Techniques,” Clin. Chem., 17, 72 (1971). (10) Katz, S., Pitt, W. W., Jr., “A New Versatile and Sensitive Monitoring System for Liquid Chromatography: Cerate Oxidation and Fluorescence Measurement,” Anal. Lett., 5 (31, 177-85 (1972). (11) Jolley, R. L., “Chlorination Effects on Organic Constituents in Effluents from Domestic Sanitary Sewage Treatment Plants,” PhD Dissertation, University of Tennessee, Knoxville, Tenn., 1973. (12) Jolley, R. L., “Determination of Chlorine-Containing Organics in Chlorinated Sewage Effluents by Coupled 36Cl TracerHigh-Resolution Chromatography,” Enuiron. Lett., 7 (41,32140 (1974). (13) Gehrs, C. W., Eyman, L. D., Jolley, R. L., Thompson, J. E., “Effects of Stable Chlorine-Containing Organics on Aquatic Biota,” Nature, 249,675 (1974).
Receiued for reuiew September 20,1974. Accepted July 28,1975
NOTES
Comparative Atomic Absorption Spectroscopic Study of Trace Metals in Lake Water Terry Surles,’ John R. Tuschall, Jr., and Theodore T. Collins Limnetics, Inc., 6132 W. Fond du Lac Ave., Milwaukee, Wis. 53218
Two methods of atomic absorption spectroscopic analysis of lake water were used in this study, flameless atomization, using a Massmann-type graphite furnace, and flame atomization preceded by a chelation/solvent extraction concentration procedure. Analyses were performed for seven metals, Cu, Cr, Cd, Mn, Pb, Ni, and Zn. The sensitivities and detection limits for many metals using a graphite furnace are known to be better than those using any flame methods. Results from this study indicate that, by using proper procedures, accuracy and precision of flameless atomization methods are equal to those for chelation/solvent extraction flame methods.
In recent years, a considerable amount of water analysis has been performed by atomic absorption spectroscopy. A recent review article by Ediger ( 1 ) discussed recent trace metal analyses of water samples by atomic absorption spectroscopy. Walsh ( 2 ) has recently discussed the present state-of-the-art of atomic absorption spectroscopy with an emphasis on the continually increasing potential on new techniques, such as flameless atomic absorption spectroscopy. The flameless methods have a number of advantages over the conventional flame techniques. These include: lower detection limits than can normally be obtained by conventional flame methods; less sample pretreatment; much smaller sample size is necessary; more rapid method
of analysis; a greater variety of samples is possible, such as solvents not normally used in flames and, in some cases, solids applied directly into the sample chamber. There can be interferences with both methods of analysis, although corrections for lake water matrix interferences are relatively simple in flameless atomic absorption spectroscopy. The primary interference encountered in the flameless technique is nonatomic background absorption, attributab!e to molecular absorption and/or light scattering. These interferences and corrective procedures have been discussed in a previous publication ( 3 ) . Despite the advantages of the flameless method over the flame technique, there is still controversy regarding the precision and accuracy of the flameless technique ( 4 ) . Many still prefer the chelation/solvent extraction method. The chelation/solvent extraction technique has received mixed reviews. Although excellent results are achievable on any given water system, some problem areas should be recogn iz ed : a. No one chelation/solvent extraction system produces optimum results for all metals or water systems. By varying the pH and the nonaqueous solvent, the.detection limit for some metals will be enhanced, while, simultaneously, for other metals, it will become poorer. b. Only select groups of metals can be simultaneously extracted. c. An increased probability for sample contamination arises from increased sample handling. d. Stability varies among the various chelates. Volume 9, Number 12, November 1975
1073
