938 IMPROVED TECHNIQUE

NOTES

FOR ANALYSIS IN SEDIMENTS~

OF CARBOHYDRATES

ABSTRACT The phenol-sulfuric acid method for determining total carbohydrates was applied to surface sediment samples from a brackish water lake, using an improved technique in which the order of addition of reagents proved to be essential for reproducibility. Corrections are made for noncarbohydrate materials that may be present in the samples,

Methods of analysis of carbohydrates in sediments usually involve extraction, separation and, finally, quantitation of the sugars. These procedures are generally used in cases where resolution of individual sugars is necessary. In many cases where an estimate of total carbohydrates is more meaningful, especially when mass balance of organic matter, or a correlation with total organic matter is sought: direct analysis with a nonspecific calorimetric test relating to a glucose standard is sufficient (Rogers 1965; Swain 1966, 1967). Three major methods presently exist for estimating total carbohydrates : anthrone, N-ethylcarbazole, and phenol-sulfuric acid, The anthrone method was demonstrated as a qualitative test for carbohydrates (Dreywood 1946); later it was used quantitatively (Morris 1948). Starch, cellulose, glucose, fructose, and other naturally occurring carbohydrates vary greatly in their molar absorptivities with the anthrone method (Koehler 1952; Handa 1966). It has proven satisfactory for free sugars in seawater but less sensitive to methylated sugars and pentoses (Antia and Lee 1963). Nevertheless, it has been used extensively for the determination of carbohydrates in seawater and sediments (Strickland and Parsons 1968; Biggs and Wetzel 1968). The N-ethylcarbazole method was introduced for carbohydrate analysis in sea1 Contribution No. 1544 from the Rosenstiel School of Marine and Atmospheric Science, UniThis work was supported in versity of Miami. part by National Science Foundation Grant GA 14473 and by U.S. Atomic Energy Commission Contract AT ( 40-l ) -3801-3 sub 2.

water in the mid-50s (Lewis and Rakestraw 1955). Various sugars exhibited about the same molar absorptivities with the N-ethylcarbazole as with the anthrone method (Zein-Eldin and May 1958). However, noncarbohydrate materials in seawater interfere in the N-ethylcarbazole analysis of total carbohydrates (McLaughlin et al. 1960 ) . The phenol-sulfuric acid method was originally described as a nonspecific quantitative test for carbohydrates (Dubois et al. 1956). Later it was compared with the anthrone and N-ethylcarbazole methods (Handa 1966) and found to be best suited for total carbohydrate analysis because its molar absorptivities for most sugars fall within a short range, while large variations in molar absorptivities for various sugars exist with the other two methods. Subsequently, the phenol-sulfuric acid method was used for the analysis of total carbohydrates in sediments ( Artemyev 1969, 1970) and in particulate matter (Handa 1967). However, it suffers from extraneous color development and from lack of reproducibility; an improved method is therefore proposed.
METHODS

Collection

of samples

Surface sediment samples were collected from a rowboat on Mangrove Lake, Bermuda, with a hand corer. A cylinder extended from the surface of the water into the sediment, its top end was capped, and it was then withdrawn slowly. The cylinder, filled with water and sediment, was brought up near the surface of the water, and the bottom end was capped. The top cap was removed and the water inside drained. The bottom cap was then removed and the extruded sediment collected in a plastic bag and deep frozen until analysis. The frozen samples were placed in beakers and freeze-dried, disaggregated with a spatula, and sieved through a 2-mm

NOTES

939
1.2

sieve to remove large shells and bark. The sediment < 2 mm was crushed to achieve homogeneity, stored dry, and used as needed. We would like to acknowledge the assistance of A. C. Neumann in obtaining the samples. Materials Phenol solution-25 g of phenol (Mallinckrodt, A.R.) was dissolved in 400 ml of water and diluted to make 500 ml of solution. Glucose stock solution-10 mg of P-Dglucose (Pierce Chem. Co.) was dissolved in 80 ml of water and diluted to make 100 ml of stock solution (100 pg/ml). Analytical procedure Glucose standards-To eight 30-ml beakers were added 1, 2, 3, 4, 5, 8, 10, and 20 ml of glucose stock solution, with enough water to make a final solution volume of 20 ml. Three 2-ml aliquots were taken from each beaker with a 5-ml glass hypodermic syringe and placed in three series of 20- X 150-mm test tubes labeled Ai, Bi, and Ci. To the A series were added consecutively 2 ml of phenol solution and 10 ml of concentrated HZSOA. To the B series were added consecutively 2 ml of water and 10 ml of concentrated H$O+ To the C series were added 2 ml of phenol solution and 10 ml of water. All additions to the test tubes were made with good mixing, from rapid delivery pipettes. The solutions were transferred into l-cm path length cuvettes and their absorbances were measured at 485 nm with a spectrophotometer (Perkin Elmer model 356) at a slit setting of 4 mm. Absorbances of the solutions from the A tubes were measured against the corresponding solutions from the B tubes. Phenol correction for each sample was determined by measuring the absorbance of the solution from the Ci tube against a solution of 2 ml of water, 2 ml of phenol solution, and 10 ml of water added consecutively to a test tube as described above. A standard curve of corrected absorbance vs. glucose concentration was drawn (Fig. 1).

01

, 20

, 60

, pg

, , , , 100 GLCOS:4/02rnl

, 180

, 220

FIG.

Calibration

curve for glucose standards.

Sediment analysis--Total organic carbon content of the samples was determined by dry combustion at 650C in a stream of oxygen and absorption of the evolved CO2 on Ascarite. At this temperature CaC03 is not decomposed (Konrad et al. 1970). Interfering gases (i.e. Cl2 and SOP) were removed in the combustion tube packing. Water and nitrogen oxides were removed by absorption tubes of magnesium perchlorate and manganese dioxide respectively (Gustin and Tefft 1966). Carbohydrates in sediments comprise about 10% of the total organic carbon ( Artemyev 1969). A sediment sample containing about 1,000 ,ug of carbohydrates was weighed into a 30-ml beaker, 20 ml of water added, and the mixture ultrasonically homogenized. With a 5-ml hypodermic syringe, three 2-ml aliquots of the homogenate were immediately taken and treated as above. Centrifugation at 4,000-5,000 rpm for 30 min resulted in flotation of sedimentary debris; to prevent erroneous results in the spectrophotometric analysis, the solutions were transferred from the centrifuge tubes to the cuvettes with disposable Pasteur pipettes, thus avoiding the floating particles. Carbohydrate contents of the sediment samples, expressed in terms of glucose,

940
TABLE

NOTES

1.

method)

Total organic carbon ( TOC) and carbohydrates (A-our in Mangroce Lake, Bermuda, surface sediments
Carbohydrate Artem'yev method (%> 1.33

method; B-modified

Artemyev

Sample No. ML1 1a 2 IiL 3 EL 4 ML5 ML6 ML7 ML8 ML9 ML 10 Ia 11 FL 12 ML 13 m 14 ML 15 ML 16 ML 17 ML 18 ML 19 ML 20

TOC (%> 3.06 10.30 1.20 17.50 15.44 14.76 14.77 17.77 14.49 15.42 9.52 15.24 15.19 15.80 17.30 6.23 19.40 6.27 10.44 6.93
0.99

A B-A 0.15 0.05

0.00

A% x 100

1.14 0.51 0.01 13.55 12.78 8.58 9.25 10.22 8.50 12.06 6.76 12.38 6.88 12.65 13.75 2.51 8.06 2.04 7.16 5.12

15.0 10.9
0.0

0.46 0.01 12.20 12.06 7.55 8.62 7.60 8.12 11.58 6.43 11.70 4.98 11.64 13.18 2.14 6.22 1.74 5.28 4.10

8.80

1.35 0.72 l,C3 0.63

11.1 6.0 14.6 7.3 34.5 4.7 8.5 5.1 5.8 38.2 8.7 4.3 17.3 29.6 17.2 35.6 24.9

7.42

2.62 0.38
0.98

0.33 0.68 1.90 7.18 1.01 0.57 2.05 0.37 1.84 1.71 0.30 1.88
1.02

were calculated from the corrected absorbantes for the treated sediments and the standard curve in Fig. 1.
RESULTS AND DISCUSSION

When surface sediment samples were analyzed by the Artemyev ( 1969) method, the sedimentary particles were charred. Subsequently, we dispersed the samples ultrasonically before adding the reagents; the modified Artemyev method yielded higher values for carbohydrate than the original Artemyev method (Table 1). The interaction of sulfuric acid with

noncarbohydrate organic constituents in sedimentary samples may result in a finite absorbance that can interfere with carbohydrate determination; in fact, Llarsh and Weinstein ( 1966) determined lipids by the charring reaction with suIfuric acid and the subsequent measurement of absorbance at 330 nm. Tailing from this and other charring reactions may significantly contribute to absorption at 485 nm. Correction for interferences of this kind may be made appropriate with the sample/reagent blanks. The analytical procedure described above makes the correction for absorbances

NOTES

941

0-c 550

I 530 510 490 470 450 430

I
! I oj-550 I 510 5x, 490 470 450

/r3 -4 430

WAVELENGTH

(nm)

Duplicate runs for the absorption spectra of 2 ml of glucose solution (50.5 ,~g/ml) to which were added: 1 and 2-2 ml of 5% phenol solution and 10 ml of concentrated sulfuric acid in that orcler; 3 and 4-10 ml of concentrated sulfuric acid and 2 ml of 5% phenol solution in that order. All solutions were scanned against water in a l-cm cuvette.
FIG. 2.

WAVELENGTH

(nm)

due to sample/sulfuric acid, sample/phenol, and also correction for phenol-sulfuric acid interactions. Three identical portions of the sample are required for these corrections. There are two ways to obtain them. Either the sample can be treated with sulfuric acid to extract the hydrolyzed carbohydrates (or the resulting furfural) and three aliquots of the extract used, or three aliquots of the solid-sample suspension can be appropriately treated. Both approaches were examined. The basic difference between the two is the order of addition of reagents. Artemyev ( 1969) added the sulfuric acid after the phenol solution; Swain (1970) added the phenol solution after the sulfuric acid. The spectra in Fig. 2 clearly show the higher sensitivity and reproducibility obtained when sulfuric acid was added after the phenol solution. This phenomenon may be attributed to the relative instability of the furfural which is formed on treatment of sugars with strong acid. If phenol is al-

FIG. 3. The absorbance spectra of 2-ml aliquots of 20.32 mg of secliments (AIL 13) in 20 ml of water to which were acldecl: l-2 ml of 5% phenol solution and 10 ml of concentratecl sulfuric acicl; 2-2 ml of water and 10 ml of concentrated sulfuric acicl: 3-2 ml of 5% phenol solution and 10 ml of water. 4 represents 2 ml of water to which were added 2 ml of 5% phenol solution and 10 ml of concentrated sulfuric acid. All solutions were scanned against water in a l-cm cuI-ette.

ready present when the furfural is formed, coupling will occur before any competing reaction or decomposition of the furfural. The absorbance due to sediment/sulfuric acid interaction is significant and will result in erroneously high .carbohydrate values, Figure 3 depicts a contribution of about 20% to the total absorbance at 485 nm of sediment/phenol/sulfuric acid. That this contribution is due to components other than carbohydrates is apparent from Fig. 4, showing a contribution of glucose/ sulfuric acid of only 7.4% to the total absorbance at 485 nm. Absorbance due to sample/phenol or glucose/phenol interactions in this study did not contribute significantly to the total absorbance at 485 nm; however, this contribution must be considered inasmuch as it may be significant in sediments from other sources. When sediments of high

942

NOTES

0 0

.5-

30.0

W ii 2 E $ .32 a 2 : .40

-I 20.0

2-

&gigi
550 530 510 490 470 450 430

10.0
WAVELENGTH (nm)

FIG. 4. The absorbance spectra of 2-ml aliquots of a glucose solution (50.5 pg/ml) to which were added: l-2 ml of 5% phenol solution and 10 mI of concentrated sulfuric acid; 2-2 ml of water and 10 ml of concentrated sulfuric acid; 3-2 ml of 5% phenol solution and 10 ml of water. 4 represents 2 ml of water to which were added 2 ml of 5% phenol solution and 10 ml of concentrated sulfuric acid. All spectra were scanned against water in a l-cm cuvette.

FIG. 5. A plot of delta (see Table carbohydrate content by our method.

1)

vs.

carbonate content were subjected to analysis in this laboratory, high turbidities not easily cleared by centrifugation were observed; this artifact must be considered when correction for the contribution of sample/phenol interaction to the absorbance is necessary. Phenol/sulfuric acid interaction makes a minor contribution to the total absorbance and can be generally neglected. The results of total organic carbon, carbohydrate ( Artemyev), carbohydrate (modified ( our method ) , carbohydrate Artemyev) analyses, and the percent difference between the last two (delta) are given in Table 1. As expected, our method yielded results consistently lower than those obtained by the modified Artemyev method. A plot of delta values vs. carbohydrate content as determined by our method (Fig. 5) clearly showed no correlation between the error due to sediment /sulfuric acid interaction and carbohydrate

concentration in the sediments. We propose that correction for absorbance due to sediment/sulfuric acid interaction must be applied whenever the phenol-sulfuric acid method is used to determine total carbohydrates in sediments and must also be considered for biological samples analyzed for total carbohydrates by the phenol-sulfuric acid method. SOL M. GERCHAKOV~ PATRICK G. HATCHERY Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida 33149.
REFERENCES
AKTIA, A. J., AND C. Y. LEE. 1963. Studies on the determination and differential analysis of dissolved carbohydrates in seawater. Fish. Res. Bd. Can., Manuscript Ser. 168. 75 p. 2 Present address: Dep. Microbial., Univ. Miami, Fla. 33152. 3 Present address: Mar. Geol. Geophys. Lab., NOAA, Atl. Oceanogr. Meteorol. Lab., Miami, Fla. 33149.

NOTES Carbohydrates in bottom sediments of the Kuril-Kamchatka Trench. Oceanology 9 : 203-207. -. 1970. Carbohydrates in the bottom sediments of the central Pacific. Oceanology 10: 508-513. BIGGS, R. B., AND C. D. WETZEL. 1968. Concentration of particulate carbohydrate at the halocline in Chesapeake Bay. Limnol. Oceanogr. 13 : 169-171. DREY~OOD, R. 1946. Qualitative tests for carbohydrate materials. Ind. Eng. Chem., ,4nal. Ed. 18: 499. DUBOIS, M., K. A. GILLES, J. K. HAMILTON, P. A. REBERS, AND F. S~~ITH. 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350356. GUSTIN, G. M., AND M. L. TEFFT. 1966. Improved accuracy of rapid microcarbon and hydrogen method by modified combustionabsorption techniques. hlicrochem. J. 10: 236-243. on the appliHANDA, N. 1966. Examination cability of the phenol sulfuric acid method to the determination of dissolved carbohydrate in sea water. J. Oceanogr. Sot. Jap. 22 (3): l-8. -. 1967. Identification of carbohydrates in marine particulate matter and their vertical distribution. Rec. Oceanogr. Works Jap. 9 ( 1) : 65-73. KOEHLER, H. L. 1952. Differentiation of carbohydrates by anthrone reaction rate and color Anal. Chem. 24: 1577-1579. intensity. KOSRAD, J. G., G. CHESTERS, AXD D. R. KEESEY. 1970. Determination of organic and carbonate carbon in freshwater lake sediments

943

ARTEMYEV, 17. YE. 1969.

by a microcombustion procedure. J, Therm. Anal. 2: 299-208. LEWIS, G. J., AND N. W. RAKESTRAW. 1955. Carbohydrates in sea water. J. Mar. Res. 14 : 253-258. MCLAUGHLIN, J. J. A., R. A. ZAHB, A. NOWAK, J. MARCHISOTTO, AXD J. PRAGER. 1960. Mass Ann. N. cultivation of some phytoplanktons. Y. Acad. Sci. 90: 89-96. MARSE-X, J. B., AND D. B. WEINSTEIN. 1966. Simple charring method for determination of lipids. J. Lipid Res. 7: 574-576. determination MORRIS, L. D. 1948. Quantitative of carbohydrates with Dreywoods anthrone reagent. Science 107 : 254-255. in aquatic ROGERS, M. A. 1965. Carbohydrates plants and associated sediments from two lakes. Geochim. Cosmochim. Minnesota Acta 29: 183-200. STRICKLAND, J. D. H., AXD T. R. PARSONS. 1968. A practical handbook of seawater analysis. Bull. Fish. Res. Bd. Can. 167. 311 p. 1966. Bottom sediments of Lake SWAIN, F. M. Nicaragua and Lake Managua, western NicaJ. Sediment. Petrol. 36: 522-540. ragua. 1967. Stratigraphy and biochemical -. paleontology of Rossburg Peat ( recent ), North-Central Minnesota, p. 445-475. In C. Teichert and E. Yochelson [eds.], Essays in paleontology and stratigraphy. Univ. Kansas. 626 p. 1970. Non-marine organic geochemis-. try. Cambridge Univ. 445 p. ZEIN-ELDIN,A. P., AND B. 2. MAY. 1958. Improved N-ethylcarbazole determination of carbohydrates with emphasis on sea water samples. Anal. Chem. 30: 1934-1941.

CARBON DIOXIDE CONTENT OF THE INTERSTITIAL WATER IN THE SEDIMENT OF GRANE LANG@, A DANISH LOBELIA LAKE
ABSTRACT The CO2 content of the interstitial water of a Danish Lobelia lake was between 1 and 5 mmole liter-l. hleasurements showed that the sediment must liberate CO, to the lake water in amounts sufficient for optimal photosynthesis of Lobelia. Zonation of Is&es, Littore&, and Lobelia in the lake cannot be correlated with the COs content of the sediment.

Lobelia dortmannu is able to absorb the CO2 necessary for photosynthesis from the sediment through its roots, not, as most aquatic plants, from the water through its leaves ( Wium-Andersen 1971) . For photo-

synthesis to be optimal the amount of free CO? around the roots must be > 1 mmole liter-l. An examination was therefore carried out to determine the amount of free CO2 in the interstitial water of the sediment at one of the localities where Lobelia grows. We chose Lake Grane Lang.@ (surface area 11.8 ha, maximum depth ca. 11 m) because of the extensive background information available. The postglacial development of the lake has been described by Hansen (1965). Nygaard (1965) described the CO2 system of the lake and