5. It is unlikely that all commercial varieties of the solids studied here would yield precisely the same results. However, it is judged that the rankings would be comparable. 6. The surface chemistry parameters studied here should be important considerations in selecting detergent formulations which may become mixed with oily wastewaters requiring ecological cleanup, and in choosing substrates for the internal structures of coalescer filters. Literature
Cited
(1) McBain, M. E. L., Hutchinson, E., “Solubilization and Related
Phenomena”, Academic Press, New York, N.Y., 1955.
(2) Kaufman, S., in C. A. Hampel and G. G. Hawley, Eds., “The Encyclopedia of Chemistry”, 3d ed., p 1021, Van Nostrand-
Rheinhold, New York, N.Y., 1972.
(3) Kaufman, S., unpublished Navy report, “Fourth Progress Report on Oil-Water Demulsification, Project No. SF53-554-70616469-01, NRL Problem 61CO2-20.203’’ (5 October 1973). (4) Becher, P., “Emulsions, Theory and Practice,” 2d ed., Rheinhold, New York, N.Y., 1966. ( 5 ) McAuliff, C., J . Phys. Chem., 70,1267 (1966). (6) Sniegoski, P., Water Res., 9,421 (1975). (7) Hamilton, W. C., J . Colloid Interface Sci., 40,219 (1972). ( 8 ) Fox, H. W., Zisman, W. A., J. Colloid Sci., 5,514 (1950). (9) Harkins, W. D. and Brown, F. E., J . A m . Chem. SOC.,41, 499 (1919). (10) Strenge, K. H., J. Colloid Interface Sci., 29, 732 (1969). (11) Pontello, A. P., et al., unpublished Navy report, “Oilmater Pollution Program, Phase 11”, NAPTC-PE-46, October 1974. Receiued for reuiew J u n e 30, 1975. Accepted November 11, 1975. Work supported by the Naual Sea Systems Command.
Sensitive Chemical Method for Routine Assay of Cobalamins in Activated Sewage Sludge Robert A. Beck and John J. Brink” Department of Biology, Clark University, Worcester, Mass. 0 16 10
A method for the routine determination of 1.0-10 wg of total extractable cobalamins from activated sewage sludge has been described. The method involves benzyl alcohol extraction of cobalamins; removal of spectrophotometrically interfering substances from cobalamins using a combination of gel filtration and chromatography on alumina; concentration of trace extracts by lyophilization; and direct quantitation of total cobalamins by high-speed liquid chromatographic (HSLC) peak areas compared to cyanocobalamin standards. The HSLC technique utilized a reverse phase column and a detector a t 550 nm. Radioactive tracer recovery studies for the benzyl alcohol extraction step ranged from 80.2-92.3% depending on sample size.
In recent years a number of studies have implicated cobalamin concentration levels to the population dynamics of phytoplankton as well as dinoflagellates responsible for the red tide (1-8). In view of the mounting evidence that cobalamins may have a significant impact on the marine or freshwater environments, a routine analytical procedure for the direct determination of cobalamins in activated sewage sludge was sought, one that avoided the long meticulous techniques presently available in addition to being quantitative in the 1.0-10 pg range. The method described has been developed for the assay of activated sewage sludge since it is a particularly abundant source of cobalamins. Classical methodologies for the qualitative and quantitative determination of cobalamins in activated sludge have relied heavily on microbiological assays (9-13), each of which has its own idiosyncratic interfering response to one or more compounds such as methionine, thymidine, or deoxyribonucleosides that commonly occur in chemically complex environmental samples. Indirect analyses for cobalamins have been reported (14-16), but such methods are not suitable for activated sewage sludge because of its complex chemical composition.
A method for the determination of cobalamins has been described by Rudkin and Taylor (17) based on the differences in the absorption spectra between cyanocobalamin and the dicyanide complex. The major difficulty with the method is its inability to separate and differentiate cobalamins from colored impurities that interfere with spectrophotometric analyses, especially in the case of activated sludge. Moreover, the method requires a relatively large sample size so that accurate cobalamin absorption spectra may be determined. The direct assay of cobalamins reported in this study is partially based on the combined extraction methods of Rudkin and Taylor (17) and Bacher et al. (18); however, it significantly deviates from available methodologies in that trace cobalamin extracts are concentrated by lyophilization of water rather than heated evaporation; spectrophotometrically interfering colored impurities are effectively separated from cobalamins by means of gel filtration prior to chromatography on activated alumina; and total extractable cobalamins are directly quantitated using high-speed liquid chromatographic (HSLC) analysis. Experimental Sodium nitrite and potassium cyanide were respectively mixed with a sample of known volume or slurry having a known weight of solid in a ratio of 0.5 : 0.2 g per 100 ml, and the p H was adjusted to 4.0. The sample was boiled for 5 min and if necessary, octanol was used as a defoaming agent (18). Zinc acetate dihydrate was dissolved in the pH 4.0 solutions or slurry to a final concentration of 10% w/v (to eliminate the formation of emulsions) and the pH adjusted to 8.5 using sodium hydroxide (50%w/w). The zinc hydroxide floc was separated from the clarified liquor using suction filtration, and the filtrate was mixed with sodium sulfate (20% w/v) and then extracted three times with one-tenth volume of benzyl alcohol (17). One-half volume of chloroform was added to the comVolume
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1
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Figure 2. Minimum detectable quantity of cyanocobalamin was set at two times background levels: peak areas were calculated from product of peak height and width of peak at one-half height
.
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Cyrnocobilimin concentralion (ut)
Figure 1. Relationship between HSLC peak areas and various concentration levels of cyanocobalamin
bined benzyl alcohol extracts and the resulting solvent phase was extracted three times with one-tenth volume of water (17). The combined aqueous extracts were lyophilized to dryness and reconstituted to a known volume with water. The resulting amber-colored extract was passed through a 0.9 X 30-cm column packed with activated alumina (20 cm), overlayed with Sephadex G-10 (8 cm), and eluted with water. One-half milliliter fractions were collected and the concentration of the combined pink cobalamin fractions were quantitated by injecting a 10-20 pl sample into a Tracor 3200 Liquid Chromatograph. A 1220 X 2.1-mm RPV-reverse phase column was employed with 0.01-M potassium dihydrogen phosphate-methanol (3:l) as a carrier solvent having a flow rate of 0.5 ml/min. Cobalamins were detected a t 550 nm using a Beckman K-24/25 spectrophotometer equipped with an 18-bl flow cell. T o determine the extraction efficiency for the procedure, C ~ ~ ~ - l a b e cyanocobalamin led was added to samples prior to benzyl alcohol extraction (19). The radioactivity of the sample extracts was assayed a t the 0.122-Mev energy level on a 1600-channel Nuclear Chicago Gamma Spectrometer calibrated at 5 Kev per channel. The spectrometer was equipped with a 3-in. sodium iodide crystal.
Results The linearity of HSLC detector response for various cobalamin concentrations was determined using a standard cyanocobalamin solution (1.0 mg/ml). A plot of concentration levels ranging from 1.0-10 bg vs. chromatogram peak areas produced a straight line with a calculated correlation coefficient of 0.99 (Figure 1).The concentration range for the standards was chosen to cover the cobalamin concentration levels in activated sewage sludge samples. The precision of HSLC detector response was determined from 10 replicate analyses at the 2.O-bg concentration level. Peaks with an average area of 70 mm2 were obtained, having a standard deviation of 2.13 and a coefficient of variation of 3.04%. Figure 2 demonstrates the minimum detectable quantity (MDQ) of cobalamins using the cyanocobalamin standard, while Figure 3 illustrates the reproducibility of HSLC detector response on a cyanocobalamin standard as well as an activated sewage sludge extract. 174
Environmental Science & Technology
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Flgure 3. Typical set of analyses demonstrating reproducibility of HSLC peak areas for a cyanocobalamin standard and a sample from dried, activated sewage sludge calculated to have 980 ng/g (dry weight) total extractable cobalamins.
The extraction recoveries for 89 ng of Co5' cyanocobalamin were 80.2% for 500-ml samples and 92.3% for 100-ml samples having a solid content of 1.08% (dry weight). The respective standard deviations were f1.7% and f 4 . 5 % for triplicate assays.
Discussion The cobalamins in activated sewage sludge show absorption maxima at 278, 361, and 550 nm (Figure 4 ) (20); however, substances co-extracted with the cobalamins may interfere with their quantitation a t any wavelength. Alumina has been reported to remove colored substances from cobalamin extracts (21-23) but in the present study a sample of sufficient purity could not be obtained using alumina as the only method of purification. Therefore, Sephadex G-10 was used in conjunction with alumina to successfully minimize spectrophotometric interference from yellow-colored substances. Sephadex G-10 permitted rapid elution of one yellow band prior to a cobalamin fraction, while retarding elution of a number of lighter-molecular-weight yellow compounds. The yellow substances were not identified. The yellow band eluted prior to the cobalamin fraction remained a t the interface of the Sephadex G-10 and alumina column, and the pink cobalamin fraction was eluted from the column prior to any other colored substances. The calculated ratio for the cobalamin fraction a t A361/ A550 was 3.21 (18,24). The acceptability of the method rests on the following five points: The chromatographic procedure outlined permits selection of an appropriate variety of Sephadex to achieve removal of interfering substances on the basis of their molecular weights; HSLC analysis provides excellent correlation between cobalamin concentrations and chroma-
l !
of analysis for cobalamins are less sensitive than biological and microbiological assay methods, but are generally faster, more reproducible, and discriminative (25).
Acknowledgment Thanks are expressed to Dr. John T. Reynolds, Clark University; to Dr. Frank J. Wolf and Merck Sharp & Dohme for supplying C ~ ~ ~ - l a b ecyanocobalamin; led and to Mr. John Sieckarski of the United States Army Natick Laboratories for technical assistance. Special thanks are extended to Dr. Charles Zapsalis of Framingham State College. ........... -
.-...-
Literature Cited (1) Guillard, R. R., Cassie, V., Limnol. Oceanogr. 8,161-5 (1963). (2) Guillard, R. R., Ryther, J. H., Can J . Microbiol., 8, 229-39 (1962). (3) Menzel, D. W., Spaeth, J. P., Limnol. Oceanogr., 7, 151-4 (1962). (4) Parker, B. C., Nature, 219,617-8 (1968). (5) Parker, B. C., Wodehouse, E. B., “Ecology and Water Quality Criteria”, 15th Water for Texas Conference, p 15, 1970. (6) Provasoli, L. “Algal nutrition and eutrophication”, i n “Eutro-
Figure 4. Typical absorption spectra for cobalamins extracted from
activated sewage sludge; absorption maxima were observed at 550, 361, and 278 nm (not indicated in Figure) tographic peak areas; very dilute cobalamin extracts can be effectively concentrated by lyophilization without significant loss of trace cobalamin concentrations; concentrated cobalamin extracts as small as 20 pl can be subjected to HSLC analysis; and the method outlined is a direct method of quantitative analysis that easily lends itself to routine analyses of complex samples such as activated sewage sludge. The MDQ of cobalamins in the present study was limited by an 18-pl flow cell (1.0-cm light path). An 8.O-pl flow cell may appreciably increase the MDQ. Moreover, if the sample under investigation is not chemically complex, and a high-purity cobalamin extract can be assured, the MDQ may be increased by setting the HSLC detector a t 361 nm, thereby taking advantage of a higher extinction coefficient. In the event that colored substances should interfere with quantitative HSLC analysis, the polarity of the carrier solvent may be adjusted to obtain improved resolution of the cobalamins although this was not necessary in the present study. In the case of chemically complex samples such as activated sewage sludge, there can be no substitute for the isolation of cobalamins prior to their assay. Chemical methods
phication: Causes, Consequences, Correctives”, National Academy of Sciences, Washington, D.C., 574-93,1969. (7) Robbins, W. J., Hervey, A,, Stevens, M. E., Bull. Torrey Bot.
Club, 77,423-41 (1950). ( 8 ) Sweeney, B. G., Am. J. Bot., 38,669-77 (1951). (9) Hoover, S. R., Jasewicz, L. B., Porges, N., Science, 114, 213 (1951). (10) Sebrell, W. H., Harris, R. S. “The Vitamins”, Vol. 11, pp 120-258, Academic Press, New York, N.Y., 0000. (11) Shorb, M. S., Science, 107,397 (1948). (12) Smith, E. L. “Vitamin B-12”, Wiley & Sons, New York, N.Y., 1960. (13) Strohecker, R., Henning, H. M., “Vitamin Assay-Tested Methods”, p 360, Chemical Rubber Co. Press, Cleveland, Ohio, 1965. (14) Boxer, G. E., Rickards, J. C., Arch. Biochem. Biophys., 39, 281 (1952). (15) Boxer, G. E., Rickards, J. C., Arch. Biochem., 29,75 (1950). (16) Wide, L., Killander, A., Scand. J . Clin. Lab. Inuest., 27, 151 (1971). (17) Rudkin, G. O.,Taylor, R. J.,Anal. Chem., 24,1155 (1952). (18) Bacher, F. A., Boley, A. E., Shonk, C. E., ibid., 7,1146 (1954). (19) Chaiet, L., Miller, T. W., Boley, A. E., J . Agri. Food Chem., 2, 784 (1954). (20) Lewis, V. J., Tappan, D. V., Elvehjem, C. A., J . Biol. Chem., 194,539 (1952). (21) Brink, N. G., Wood, T. R., US.Patent 2,609,325,1952. (22) Fricke, H. H., ibid., 2,582,589 (1952). (23) Rickes, E. L., Wood, T. R., ibid., 2,563,794 (1951). (24) US.Pharmacopeia XVIII, p 153. (25) Freed, M., “Methods of Vitamin Assay”, 257-85, Wiley & Sons, New York, N.Y., 1966. Received for review May 8,1975. Accepted October 14,1975.
Volume 10, Number 2,February 1976 175
