examination on the applicability of the phenol sulfuric

The Journal of the Oceanographical Society of Japan Vol.22, No.3, June 1966

Examination on the Applicability of the Phenol Sulfuric

Acid Method to the Determination of Dissolved

Carbohydrate in Sea Water*

Nobuhiko HANDA**

Abstract : The applicability of phenol sulfuric acid method to the determination of total

carbohydrate in sea water is discussed. When treated with phenol and concentrated sulfuric

acid, carbohydrates give an yellow-brown color, the color is strong and the formed chromato-

phore is stable enough to determine the carbohydrate in sea water. The effect of inorganic micro-elements and some organic compounds possibly to be found in sea water on the formation

of chromatophore is examined. Except eugenol, tested agents gave no effect when the sulfuric

acid used contains reducing agents. The eugenol effect can be removed by the extraction

procedure with organic solvent.

1. Introduction

The importance of the studies of the biologi-cally active organic compounds governing the life of marine microbes has been generally re-cognized for understanding the biology and chemistry of the ocean.

It has been also pointed out that dissolved carbohydrates in sea water play an important role in metabolic processes of plankton. The extracellular production of carbohydrates was observed for some marine flagellates in culture studies (GUILLARD and WANGERSKY 1958), while the dual capacity of autotrophy in the light and glucose heterotrophy in the dark was shown in marine littoral diatoms in culture

(LEWIN 1961). Evidences for the carbohydrates decomposition by marine bacteria have been reviewed by ZoBELL (1964) and later by WOOD

(1958). However, biochemical discussion of dissolved

carbohydrate in sea water has rarely been made from the data of direct measurement of carbo-hydrate, vertical and horizontal distribution and temporal change. Quite a few determinations of dissolved carbohydrate in sea water has been made because of lack of a sufficiently sensitive and precise method for that purpose. LEWIS and RAKESTRAW (1955) compared the anthrone

and N-ethylcarbazole methods for the deter-mination of total carbohydrate in sea water and showed that both methods give nearly the same sensitivity. ZEIN-ELDIN and MAY (1958) im-

proved the N-ethylcarbazole method for routine techinique of total carbohydrate determination in sea water. However, MCLAUGHLIN et al.

(1960) reported that N-ethylcarbazole reaction is interfered with non-carbohydrate materials

giving rise to different colors. ANTIA and LEE (1963) examined the applicability of anthrone method to total carbohydrate determination in sea water. The result was that while this method is satisfactory for the determination of free sugars and their glycosides, it is good only under limited conditions for methylated sugars and pentoses.

The reaction of carbohydrate with phenol and sulfuric acid in aqueous solution gives a brown color. DUBOIS et al. (1956) reported that this reaction can be 'used for the quantitative color-imetric microdetermination of monosaccharides and their polymerization products, such as oligosaccharides and polysacchrides.

In the present paper the author reports the examination of the applicability of the phenol sulfuric acid method to sea water on three

points: 1) dependency of color development on the amount of phenol added, 2) establishment of absorption curves of developed color for different sugar, and 3) interference with the color development of D-glucose caused by the

* Received Jan . 24, 1966 ** Water Research Laboratory , Faculty of Science,

Nagoya University

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80 Jour. Uceanogr. Soc. Japan, Vol.22, No.3 (1966)

presence of organic compounds and inorganic

ions.

It was proved that the examined organic

compounds and inorganic ions in concentrations

as found in sea water give no harmful effect on

color development. The color is easily developed

free from a strict control of temperature or

reaction time as compared with the anthrone

or N-ethylcarbazole method. Furthermore, the

developed color is stable at least for 48 hours.

Thus the method as processed in the way

described later can be recommended as the most

simple, rapid and reliable tool for determining

dissolved carbohydrate in sea water.

2. Experiments and Results

Excepting cases otherwise stated, the phenol

sulfuric acid method was processed as follows:

In a test tube 1 ml test solution was placed

followed by immediate addition of 1 ml 5%

phenol solution and 5 ml concentrated sulfuric

acid. The test solutions were prepared by dis-

solving test materials in synthetic sea water of

salinity 38.5•ñ. For interference examination,

however, test solutions were prepared by adding

a suitable amount of test materials and 25 ƒÊg

D-glucose to synthetic sea water per ml.

Some description of the reagents, test ma-

terials and apparatus, is thought to be useful.

Reagents: 1) Sulfuric acid of reagent grade

with specific gravity 1.84, supplied from Wako

Pure Chemicals to which hydrazine sulfate

(0.5%) or stannous chloride (2.5%) was added.

Thiourea may be substituted. 2) Phenol, re-

distilled twice at 198•Ž with a glass distiller,

was diluted with water to 5% solution. The

solution was left to stand in a refrigerator for

later use.

Synthetic sea water of the composition of

LYMAN and FLEMMING was made up to a

salinity 192.5•ñ. The stock solution thus pre-

pared was diluted to any desired salinity.

Test materials: Commercial preparations

were used for mono- and oligosaccharide sam-

ples. Among polysaccharides, laminarin, amy-

lose and dextran were isolated respectively from

Eckronia cava and Laminaria angustata, both

brown marine algae, potato and Leuconostoc

mesentrioides. Sugar alcohols, D-glucitol, D-

galactitol, D-mannitol and D-xylitol, were obtained by the reduction of D-glucose, D-

galactose, D-mannose and D-xylose with sodium borohydride. Aldonic acids, D-gluconic acid

and D-galactonic acid were prepared from D.

glucoseand D-galactose by oxidation with bromine in the presence of barium carbonate.

Apparatus: Hitachi photoelectric spectro-

photometer EPU-2 and especially, for obtaining the absorption spectra of monosaccharide chro-

matophores, Perkin-Elmer spectrophotometer 202 were found to be satisfactory. 1 cm cells

were used in both cases.

For quick delivery of sulfuric acid within 5

second, a 5 ml rapid delivery pipet proved to be convenient. The pipet was made by cutting

the tip of an ordinary 5 ml measuring pipet.

In a number of cases the phenol sulfuric acid method was paralleled by anthrone method and

the N-ethylcarbazole method. The latter was

conducted as follows: To 1 ml of a sugar so-lution was added 10 ml N-ethylcarbazole sulfuric

acid solution. The mixture was kept for 30 min

in boiling water. After cooled with running

tap water, developed color was measured by using Hitachi photoelectric spectrometer EPU-2.

The N-ethylcarbazole sulfuric acid solution was

prepared by dissolving 0.2 g N-ethylcarbazole in 100 ml sulfuric acid containing 2.5 g thiourea.

N-ethylcarbazole preparation from Katayama

Fig. 1. Changes of the absorbance with the

amount of phenol added.

1. D-Xylose, 2. D-Mannose, 3. D-Glucose,

4. D-Fructose, 5. D-Galactose.

25 ƒÊg of sugars were used in each of the

experiments.

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Examination On the Applicability of the Phenol Sulfuric Acid Method to the Determination of Dissolved Carbohydrate in Sea Water

81

Chemical Co. was recrystallized twice from

absolute alcohol before use.

1) Color development and the amount of

phenol added.

Different amounts of phenol were tested with

1 ml solution containing 25 ƒÊg of different

sugars.

The results are shown in Fig. 1. The in-

tensity of the developed color varies with the

amount of added phenol for different sugars. In

general the absorbance increases to a maximum

and then tends to decrease with the increase of

the amount of phenol added. For D-xylose the

gradient of absorption curve is steep and the

stable phenol concentration range for color

measurement is narrow. D-mannose and D-

glucose have absorption maxima at 25 mg and

50 mg phenol concentrations, and keep high

values for certain ranges of higher phenol

concentration. The absorption maximum for

D-galactose occurs at a lower concentration of

phenol the absorption decreases at higher phenol

concentrations.

2) Different amounts of various sugars were

proposed following the general procedure stated

above with 1 ml 5% phenol solution. The de-

veloped color was measured by using Perkin-

Elmer spectrophotometer to construct absorption

curves. Results are given in Figs. 2 and 3.

Fig. 2. Absorption curves.

1. D-Mannose, 2. D-Glucose, 3. Sucrose,

4. D-Galactose, 4. D-Fructose.

Fig. 3. Absorption curves.

1. D-Xylose, 2. D-Rhamnose,

3. D-Arabinose, 4. L-Fucose.

Different groups of monosaccharides are charac-

terized by different patterns of curve. Stepping

further, for a number of sugars absorbance was

observed at wave lengths 480 mp and 490 mp

respectively for aldopentoses and hexoses. The

results are listed in Table 1, based upon which

standard curves were constructed as given in

Fig. 4. Standard curves.

1. D-Xylose, 2. D-Mannose, 3. D-Rhamnose,

4. D-Alabinose, 5. D-Glucose, 6. Sucrose,

7. D-Galactose, 8. D-Fructose, 9. L-Fucose.

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82 Jour. Oceanogr. Soc. Japan, Vol.22, No.3 (1966)

Table 1. Absorbance index of monosaccharides.

Fig. 4. Standard curves for oligo- and poly-saccharides are shown in Fig. 5.

Examination of the interferering effects on the phenol sulfuric acid method caused by same chemical constituents of sea water.

(1) Salinity To different volumes of the synthetic sea

water was added 1 ml D-glucose solution (25

Fig. 5. Standard curves.

1. Laminarin from Eckronia cava,

2. Laminarin from Laminaria angustata,

3. Amylose from potato,

4. Dextran from Leuconostoc mesentrioides,

5. Lactose.

mg/l) and evaporated using a rotary evaporator to dryness. The dry sample was dissolved in 1 ml distilled water to be subjected to further steps of color development. The result given in Fig. 6 proved that in the possible range of salinity in sea water, color development is little affected.

(2) Inorganic components

Fig. 6. Absorbance vs. salinity.

25 ƒÊg of D-glucose were used in each of the

experiments.

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Examination on the Applicability of the Phenol Sulfuric Acid Method to the Determination of Dissolved Carbohydrate in Sea Water

83

NaNO3, NaNO2, NH4Cl, FeCl3, NaH2PO4, CuSO4, (NH4)6Mo7O24, Na2SiO3, MnSO4 and ZnSO4 were examined for testing the effects of PO34-, SO24-, Mo7O62-4, Fe3+, Cu2+, Zn2+, Mn2+, NO-3, NO-2; NH+4, SiO23-. By adding appropriate

amounts of these substances to the synthetic

sea water to obtain those solution in which the

tested elements were contained in concentrations

20, 60 and 100 times as found in natural sea

water. To 0.5 ml of the solution thus obtained,

0.5 ml D-glucose solution containing 25 ƒÊg of

the sugar was added and subjected to further

processes. The results in Table 2, show that

the tested substances give no significant effect

in their concentrations in which they usually

occur in sea water.

Table 2. Inorganic compounds interference

in glucose determination.

* The abundance of elements in sea water after

GOLDBERG.

** In Column a, b and c, inorganic compounds

were tested in 20, 60 and 100 times concetrations

respectively as much as their abundances in

sea water.

*** In blank experiment, 25 ƒÊg of D-glucose gave

0.2366 in optical density.

(3) Organic compounds A series of organic compounds from the

presence of which might cause in sea water any intereference with the application of the

present carbohydrate determination method were selected. They included 19 amino acids, one

protein, gammaglobuline, seven carboxylic acids, (acetic acid, Krebs cycle member acids, such as citric, succinic and malic acids and aldonic acids such as D-gluconic, D-galactonic and

D-mannonic acids), four alkyl alcohols, methyl,

ethyl, n-propyl and iso-propyl alcohols, four sugar

alcohols, D-glucitol, D-galactitol D-mannitol and

D-xylitol, and some aromatic compounds such as

benzoic acid, gallic acid, catechol, veratoric acid,

protocatechuic acid, eugenol, o-,m-, and p-cresols

and vanillin. In general, as in the case of testing

the effect of inorganic substances, to 5 ml syn-

thetic sea water sample containing different

amounts of the test material, 0.5 ml D-glucose

solution containing 25 ƒÊg of the sugar was

added to be processed further. A special care

was taken for some aromatic compounds not

easily soluble in synthetic sea water.

The results as shown in Tables 3, 4 and 5

proved that the tested organic compounds except

eugenol do not interfere with the application

of the method in question in the ranges of

concentration tested. Really, the concentrations

of some of the tested compounds in sea water

have been reported rarely to exceed the tested

Table 3. Amino acids interference in

glucose determination.

* Without D -glucose.

** In blank experiment, 25 ƒÊg of D-glucose gave


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Table 4. Carboxylic acids, alkyl alcohols and

their related compounds interference in glucose

determination.

* In blank experiments, 25 ƒÊg of D-glucose gave


Table 5. Aromatic compounds interference

in glucose determination.

* Without phenol from complete system which is

consisted of phenol, D-glucose, sulfric acid and

aromatic compound.

** Complete system and 50 ƒÊg of each aromatic

compounds are used.

*** In blank experiment, 25 ƒÊg of D-glucose gave


Table 6. Extraction of the aromatic compounds with chloroform.

* Aromatic compounds mixture is consisted of

gallic acid, vanilin, benzoic acid, catechol and

eugenol.

** Recovery of D-glucose is calculated on the base

of 0.2321 of optical density which is obtained in

25 ƒÊg of D-glucose without any treatment.

ranges. Thus citric acid, malic acid, acetic

and formic acids, and amino acid have been

determined as 0.025-0.145 me/ by CREAC'H

(1955), 0.028-0.277 mg/l by KOYAMA et al.

(1959), 0.1 mg/l by KOYAMA et al. (1959) and

13 mg/l by TATSUMOTO et al. The problem

of eugenol was solved by the extraction of

eugenol as shown in Table 6.

From the results, some basic knowledge was

given upon which conditions can be found to

permit the present method of gross carbohydrate

determination to run with a high sensitivity and

reproducible results. Thus as for the amount

of phenol added 50 mg/ml is recommended to

produce a high stable coloration. Secondly, as

far the wave-length of color determination 480

or 490 mƒÊ is suggested as the best where the

absorption maximum found for the sugars tested.

The final problem concerns with the difference

in magnitude of absorption index between dif-

ferent sugar species. On Table 7, eight different

kinds of sugars are compared for relative ab-

sorbance for three proposed methods for sugar

determination in water, the phenol sulfuric acid

Table 7. Relative absorbances of the sugar chro-

matophores in phenol, N-ethylcarbazole and

anthrone reactions.

* After ANTIA and LEE .

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Examination on the Applicability of the Phenol Sulfuric Acid Method to the Determination of Dissolved Carbohydrate in Sea Water

85

method, N-ethylcarbazole method and anthrone method.

The data for anthrone method are quoted from ANTIA and LEE and those for phenol sulfuric acid method are from the author's data listed in Table 1, while the values for the N-ethylcarbazole method are from an experiment which the author purposely carried out. Table 6 shows that the absorbance widely diverges from 100% of D-glucose, standard, to 3% of D-arabinose and D-xylose for the anthrone method. The divergece is less for the method of N-ethylcarbazole in which it varies from 100% of D-xylose to 7% of L-fucose. The

phenol sulfuric acid method proved to be best for which the values are distributed in the range of 100% for D-xylose to 35% of L-fucose. The conclusion is that among these three current methods the phenol sulfuric acid method is recommendable for the gross estimation of gross carbohydrate amounts in sea water.

3. Summary

1) The applicability of phenol sulfuric acid method to the determination of total dissolved carbohydrate in sea water was examined.

2) It was shown that the intensity of de-veloped color varies with different amounts of

phenol added and for different monosaccharides. 3) Absorption curves were constructed for

eight different monosaccharides. Aldohexoses, ketohexose and aldopentoses showed a absorption maximum respectively at 490, 488 and 480 mp.

4) Several inorganic microelements and some organic compounds, possibly contained in sea water were examined for their interference with the method. Excepting eugenol, no tested elements and compounds cause interference in the range of tested concentrations so long as the sulfuric acid contains reducing agents such as hydrazine sulfate, stannous chloride or thiourea. The effect of eugenol can be cancelled by extracting this compound with chloroform.

5) Three different methods for sugar deter-mination, phenol sulfuric acid method, N-ethyl-carbazole method and anthrone method, were compared with phenol sulfuric acid method as standard for their relative absorbances using

eight different monosaccharides. The divergence

of relative absorbance proved to be the least for phenol sulfuric acid method.

6) The conclusion is that the phenol sulfuric acid method is the most recommendable for determining total carbohydrate in sea water.

Acknowledgements

The author would like to express his thanks to Dr. T. KOYAMA and Dr. Y. SAIJO of Nagoya University for their suggestions and encourage-ments in the course of this study. He is also indepted to Dr. K. SUGAWARA for his invaluable discussion and advice given during the pre-

paration of this manuscript.

References

ANTIA, A. L. and C. Y. LEE (1963) : Studies on the determination and differential analysis of of dissolved carbohydrate in sea water. Manu-script report series No. 168, Fisheries Research Board of Canada.

CREAC'H, P. (1955) : Sur la presence des citrique et malique dans les eaux marines littorales. C. R. Acad. Sci. Paris, 240, 2551-2553.

DUBOIS, M., K. A. GILLES, J. K. HAMILTON, P. A. REBERS and F. SMITH (1956) : Colorimetric method for determination of sugars and related substances. Anal. Chem., 28(3), 350-356.

GUILL ARD, R. R. L. and P. J. WANGERSKY (1958) : The production of extracellular carbohydrates by some marine flagellate. Limonl. Oceanogr., 3, 449-454.

KOYAMA, T. and T. G. THOMPSON (1959) : Organic acids in sea water. Reprints Intern. Oceanogr. Cong. A. A. A. S., 925-926.

LEWIN, J. C. (1961) : Heterotrophy in marine diatoms. Bacteriol. Proc., Soc. Am. Bacteriol., Abstr. Symposium on Marine Micro-biol., Chi- cago, Illinois, April, 20-22.

LEWIS JR., G. J. and N. W. RAKESTROW (1955) : Carbohydrate in sea water. Jour. Mar. Res., 14, 253-258.

MCLAUGHLIN, J. J. A., R. A. ZAHL, A. NOWAK, J. MARCHISOTTO and J. PRAGER (1960) : Mass cultivation of some phytoplanktons. Ann. N. Y.

Acad. Sci., 90, 856-865. TATSUMOTO, M., M. T. WILLIAMS, J. M. PRESCOTT

and D. W. HOOD (1961) : On the amino acids in samples of surface sea water. Jour. Mar. Res., 19, 89-96.

WOOD, E. J. F. (1958). The significance of marine microbiology. Bacteriol. Rev., 22, 1-19.

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ZEIN-ELDIN, Z. P. and B. Z. MAY (1958) : Improved N-ethylcarbazole determination of carbohydrates with emphasis on sea water samples. Anal. Chem., 30, 1935-1941.

ZOBELL, C. E. (1946) : Marine microbiology. Chro-nica Botanica Co., Waltham, Massachusetts, 138- 141.

フェノール硫酸法による溶存炭水化物の定量について

半田暢彦

要旨従来海水中の溶存炭水化物の定量にはN-ethyl-

carbazole法もしくはanthrone法が用いられていたが,

これ等の方法は共存する有機化合物によりcolorationを

受けたり,また単糖類に対するreactivityの相違が大き

いなどの欠点があった。比較的これ等の欠点を持たない

フェノール硫酸法の海洋への応用を検討した。

本法は海水中の主要無機化合物の何れによっても妨害

されなかった。また,有機化合物についてはアミノ酸・

蛋白質,アルキルアルコール,糖アルコール,カルボン

酸および数種の芳香族化合物等の影響が検討されたが,

オイゲノール以外は何れも本法による炭水化物の定量に

影響を及ぼさなかった.オイゲノールについては有機溶

媒による抽出で容易に海水からとりのぞかれることを示

した。これ等のことは本法が海水中の炭水化物の定量に

用いられるべく可能であることを示していると思われ

る。

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examination on the applicability of the phenol sulfuric

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