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Rapid, sensitive determination of periodate
Carbohjdmfc
Rrrcarch
119
Ekvier Fublirhine Company. Amsterdam Printed in BcIginm
RAPID, SENSITIVE DETERMINATION GAD
OF PERIODATE
AVIGAD*
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (U. S. A.)
(Received April lOth, 1969)
Periodate rapidly oxidizes the violet ferrous-2,4,6-tri-2-pyridyl-s-triazine complex to a colorless compound. This reaction serves as the basis for a simple procedure for the quantitative, calorimetric determination of periodate at the nanomolar level. Use of this assay was exemplified by a study of the course of oxidation of 1- to Zpmolar samples of several representative carbohydrates. INTRODUCTION
Periodic acid is a powerful oxidant that has found many uses in analytical and organic chemistry1-4. Its ability to cleave 1,2-glycols provides a reagent extensively employed in carbohydrate chemistry5- ‘. A selection of methods is available for the determination of periodate in solution1*2*6*‘. Titrimetric procedures10-14, which are relatively slow and sensitive only to pmolar levels of periodate per sample, are the most widely employed. Direct determination of periodate by U.V. spectrophotometry provides a sensitive method”-“, but it often cannot be applied effectively, owing to the presence of other compounds having high absorption in the same spectral region. Another spectrophotometric method, based on the oxidation of 1,2-bis(p-dimethylaminophenyl)-l,Zethanediol, has recently been described’ ‘. Calorimetric assays for periodate that are based on the oxidation of aromatic amines havealso beensuggested’g920. These procedures are relatively slow, or involve the use of organic solvents and the formation of a colored product that is not very stable. In the present article, a simple, rapid, sensitive procedure for the assay of periodate is described. This method is based on the very fast oxidation of the stable, violet complex [Fe(TPTZ)~f] (1)of ferrous ion with 2,4,6-tri-2-pyridyl-s-triazine2 ’ (TPTZ) to yieid a colorless ferric derivative. The amount of residuil violet compound is determined calorimetrically. A convenient range for the assay of 5 to 200 nmoles of periodate is suggested. However, the method can readily be adapted for the determination
of one nmole
of periodate.
Compounds
(other
than
periodate)
usually
*On leave from the Hebrew UniVersity, Terusaiem, Israel. Curbohyd- Res..
11 (1969) 119423
120
G. AVIGAD
found in a standard oxidation system of a carbohydrate do not interfere with the assay. As measurements are made at 593 nm, the presence of such u.v.-absorbing materials as compounds containing unsaturated bonds, aromatic aglycons, purines, pyrimidines, and proteins does not interfere. The method may aiso be suitable for the assay of periodate in certain water-miscible, organic solvents. The present assay-procedure was found to be particularly valuable when only a small quantity (one to two irmoles) of the sugar to be examined was available, but a study of the kinetics of its oxidation by periodate was desired. EXPERIMENTAL
Materials. - 2,4,6-Tri-2-pyridyl-s-triazine was obtained from the G. E. Smith Chemical CO., Columbus, Ohio 43223. The preparations of sodium metaperiodate used were analytical reagents from Fluka AG, Buchs, Switzerland, and Fisher Scientific Co., Pittsburgh, Pa. Spectrophotometry. Readings were made with a Zeiss Model PMQII spectrophotometer, with cuvets having a I-O-cm light-path. A Gilford model 300 spectrophotometer was occasionally employed (for rapid assay of a large series of samples). Reagents. - The violet solution of 1 was prepared as follows. TPTZ (75 mg, 0.24 mmole) was dissolved in acetic acid (46 ml), and M sodium acetate (210 ml) and a freshly prepared solution of Fe(NH,),(SO,),. 6H,O (31.4 mg, 0.08 mmole in 100 ml of water) were added. The volume was then made to 1 liter with water. The solution should have pH 4.0-4.2 and E:‘,“, ~1.8. No apparent decrease in absorbance of the solution occurs during one month at room temperature. This reagent may be diluted with M acetate buffer (pH 4.0)to provide solutions of lower absorbance at 593 nm. A stock solution (50 mM) of sodium metaperiodate was prepared by dissolving NaIO,, (1.0695 g) in water (100 ml) in an amber-colored measuring flask or one wrapped in aluminum foil to ensure complete protection from light. This solution was kept no longer than a week at room temperature. Dilute solutions were freshly made daily with water, and these solutions were kept protected from light. Assay of the periodate. In a standard procedure, sampIes containing 5 to 200 nmoles of periodate were added to tubes containing 4.5 ml of the violet reagent (1). The volume was made to 5.0 ml with water, and the solution was well mixed. The absorbance of the solution was read at 593 nm, to determine the amount of residua1, violet 1.As this compound is very stable under the conditions employed, measurement of it can be made at one minute, or many hours, after a sample of periodate has been removed for analysis. This property proved to be especially convenient when several series of samples had to be taken at short time-intervals during a kinetic study of oxidation. Typical standard-curves for periodate are presented in Fig. I. The slope of the lines is the same, irrespective of the initial concentration of the violet 1 present Cuddzyd-
Res., 11
(1969) 119-123
DETJXMINATION
OF PERIODATE
121
in the reagent (see Fig. 1A). Also, the slope can be proportionally increased when a smaller initial volume of the solution of 1 is employed for the assay of similar amounts of periodate (see Fig. 1B). It is apparent that the range and sensitivity of the method for periodate can readily be adjusted for the needs of any particular experiment over a range of concentrations of oxidant. Thus, the sensitivity can be increased to permit determination of one nmole of periodate.
Fig. 1. Oxidation of 1 by periodate. A. Reaction conducted in a volume of 5 ml, but containing various initial concentrations of the violet, ferrous complex 1.B. Different volumes of a solution of 1 used. Numbers near each line indicate number of ml of reagent employed.
The decrease in the molar extinction coefficient of the violet 1 that is ‘caused by oxidation with one mole of periodate was found to be 37,000 under the conditions of assay described; this value corresponds to the oxidation of I.7 g-atoms of ferrous ions2 ’ (the maximum theoretical equivalent expected is 2.0). E#ect of cariotu compounds on the oxidation of 1 by periodate. At a final concentration of 5 mu, formaldehyde, acetaldehyde, NaIO,, NaCIO,, KH,P04, Na,CO,, MgC12, and Tris do not interfere with the assay. Although 2 mM citrate does not decolorize the violet ferrous complex (l),concentrations higher than 0.2 mu lessen the sensitivity of its oxidation by periodate. Addition of ethylene(d&trilotetraacetate) to the ferrous complex 1 at equimolar concentrations causes instantaneous
decolorization”.
The presence of any one of several such ions as Cu2+, Ag+, CN-,
and NO; may interfere with 21 the stability of the reagent 1. The reaction can also be performed in the presence of any one of several water-soluble, organic solvents. Thus, pyridine (4%), acetonitrile (40%) and acetone (40%) do not interfere significantly with the reaction at concentrations lower than those indicated. NJGDimethyIformamide at concentrations higher than 1% lessens the extent of oxidation, whereas p-dioxane at 1% decolorizes the violet, ferrous complex (1). Several aliphatic primary alcohols decolorize the ferrous complex 1 and, CurbohycZ.Res., 11 (1969) 119423
122
G. AVIGAD
at very low concentration, inhibit the periodate reaction. Thus diethylene glycol, diethylene glycol monomethyl ether, methyl Cellosolve, and 2-chloroethanoi significantly interfere with the assay when present in a concentration as low as 0.2%. Methanol, ethanol, and propyl alcohol at final concentrations >0.05% exhibit an inhibitory effect on the extent of oxidation of the ferrous complex 1 by periodate (see refs. 23 and 24). Kinetics of oxidation of mrious substrates. - As an example, a representative group of compounds containing the 1,2-glycol grouping was oxidized by periodate, and the course of the consumption of oxidant was estimated by the method already described (see Fig. 2). Each oxidation was conducted at pH 5.2, to ensure slow hydrolysis of any formyl esters formed 25+26.Similar oxidations at pH 3 to 8 could conveniently be analyzed by the present method, as the reagent was prepared in M
acetate buffer at pH 4.0 to 4.2.
2
3
Time
A- 6
12
24
(hours1
C
V 1
Time (hours)
2
Time
3
A
5
(hours1
Fig. 2. Course of consumption of periodate during oxidation of different substrates- All reactions were conducted in the dark in 40 mM acetate buffer (PH 5.2). Samples (5 to 25 al) were withdrawn with a constriction micropipet and directly added to the violet, ferrous reagent (1) to determine the amount of residual periodate. A. Disaccharides (2 mhf) and tetrasaccharide (1 mhr) were oxidized with 15.4 m&i NaI04: 0, stachyose; 0, lactose; 8, a,a-trehalose; 31, sucrose. B. AIdopyranosides (2 mt.@ were oxidized with 10 mM NaI04: 0, methyl a-D-xylopyranoside: 0, methyl a-D-arabinopyranoside; 0, methyl a-D-ghrcopyranoside; I, methyl a-D-mannopyranoside. C. Substrates (2 mM) were oxidized with 20.2 mM NafO;r for inositol (e)), D-glucitol (o), and D-ghtcose (a); with 10.1 mM for 2-deoxy-n-lyxo_hexose (8) and 3-O-methyl-D-glucose (a; with 7.9 mM for glycerol (A) and r-serine (8); and with 5 mM for ethylene glycol (v)_
in all cases (see Fig. 2), the course of the oxidation and the periodate uptake per mole of substrate correspond to the theoretical value predicted. As expected6*‘, Cmbohyd.
Res., 11 (1969) 119-123
DETERhfJNATIbN
the oxidation
123
OF PERIODATE
of acyclic
glycols
is very fast compared
to that
of reducing
sugars,
involving the intermediary formation of formic esters (see Figs. 2 A, B). Also, cleavage between or-erythro-hydroxyl groups (distorted cis-hydroxyl groups) is faster than that of a-tizreo-hydroxyl group (distorted trans-hydroxyl groups),as is evident from the pattern of oxidation of glycosides (see Fig. 2C). ACKNOWLEDGMENT
I thank Dr. S. Englard for the hospitality of his laboratory. REFERENCES 1 C. A. BUMON, in K. B. WIBERG (Ed.), Oxidation in Organic Chemistry, Part A, Academic Press, New York, 1965, p. 367. B. SKLARZ, Quart. Rev. (London), 21 (1967) 3. A. J. FATIADI, J- Res. Nat. Bur. Stand., 72A (1968) 361_ W. J. BAUhfAN, H. H. 0. ScHhfm, AND H. K. MANGOLD, J. Lipid Res., 10 (1969) 132. R. D. GUTHRIE, Aduan. Carbohyd- Chem., 16 (1961) 105. J. STANEK, M. CERNS, J. KOCOUREK, AND J. PACAK, The Monosaccharides, Academic Press, New York, 1963, p_ 903. 7 I. J. GOLDSTEIN, G. W. HAY, B. A. LEWIS, AND F. SMITH, Mefhods Curbohyd. Chem., 5 (1965) 361. 8 L. HOUGH, Methods Carbohyd. Chent., 5 (1965) 370. 9 L. HOUGH, A. C. RICHARDSON, AND C. H. BOLTON, in Rodd’s Chentistry of Carbon Compounds, Vol. lF, Elsevier, Amsterdam, 2nd edition, 1967, p. 305. IO G. F. SMITH, Analytical Applications of Periodic Acid and Zodic Acid and Their Salts, G.F. Smith Chemical Co., Columbus, Ohio, 5th edition, 1950. 11 J. R. DYER, Methods Biochem. Anal., 3 (1956) 111. 12 I. M. KOLTHOFF AND R. BELCHER. Vohtmetric Analysis. Vol. III, Interscience Publishers, New York, 1957, p. 475. 13 R. D. GUI-HRIE, Methods Carbohyd. Chem., I (1962) 435. 14 G. W. HAY, B. A. LE\VIS, AND F. S?.IITH, Methods Carbohyd. Chent., 5 (1965) 357. 15 J. S. DIXON AND D. LIPKIN,Anal. Chem., 26 (1954) 1092. 16 G. V. MARINETTI AND G. ROUSER, J: Amer. Chem. Sot., 77 (1955) 5345. 17 G. 0. ASPINALL AND R. J. FERRIER, C/tern. Ittd. (London), (1957) 1216. 18 R. FIELDSAND H. B. F. DIXON, Biochem. J., 108 (1968) 883. 19 M. GUERNET, Bail. Sot. Chim. France, (1964) 478. 20 J. ARES PONS, PubI. Inst. Invest. Microquim., Unit. Nacl. Litoral (Rosario, Arg.), 26 (1965) 175, 199. 21 P. F. COLLINS, H. DIEHL, AND G. F. SMITH, Anal. C/tent., 31 (1959) 1862. 22 B. KRATOCHVILAND M.C. WHITE, Anal. Chem.,37(1965) 111. 23 J. F. TAYLOR, B. SOLDANO, AND G. A. HALL, J. Amer. Chem. Sot., 77 (1955) 2656. 24 R. D. GUTHRIE, Chem. Ind. (London), (1960) 691. 25 L. HOUGH AND M. B. PERRY, Chem. Znd. (London), (1956) 768. 26 L. HOUGH, T. J. TAYLOR, G. H. S. THOMAS, AND B. M. WOODS, J. C/tern. Sac., (1958) 1212. Carbohyd- Res., 11 (1969) 119-123
Rrrcarch
119
Ekvier Fublirhine Company. Amsterdam Printed in BcIginm
RAPID, SENSITIVE DETERMINATION GAD
OF PERIODATE
AVIGAD*
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (U. S. A.)
(Received April lOth, 1969)
Periodate rapidly oxidizes the violet ferrous-2,4,6-tri-2-pyridyl-s-triazine complex to a colorless compound. This reaction serves as the basis for a simple procedure for the quantitative, calorimetric determination of periodate at the nanomolar level. Use of this assay was exemplified by a study of the course of oxidation of 1- to Zpmolar samples of several representative carbohydrates. INTRODUCTION
Periodic acid is a powerful oxidant that has found many uses in analytical and organic chemistry1-4. Its ability to cleave 1,2-glycols provides a reagent extensively employed in carbohydrate chemistry5- ‘. A selection of methods is available for the determination of periodate in solution1*2*6*‘. Titrimetric procedures10-14, which are relatively slow and sensitive only to pmolar levels of periodate per sample, are the most widely employed. Direct determination of periodate by U.V. spectrophotometry provides a sensitive method”-“, but it often cannot be applied effectively, owing to the presence of other compounds having high absorption in the same spectral region. Another spectrophotometric method, based on the oxidation of 1,2-bis(p-dimethylaminophenyl)-l,Zethanediol, has recently been described’ ‘. Calorimetric assays for periodate that are based on the oxidation of aromatic amines havealso beensuggested’g920. These procedures are relatively slow, or involve the use of organic solvents and the formation of a colored product that is not very stable. In the present article, a simple, rapid, sensitive procedure for the assay of periodate is described. This method is based on the very fast oxidation of the stable, violet complex [Fe(TPTZ)~f] (1)of ferrous ion with 2,4,6-tri-2-pyridyl-s-triazine2 ’ (TPTZ) to yieid a colorless ferric derivative. The amount of residuil violet compound is determined calorimetrically. A convenient range for the assay of 5 to 200 nmoles of periodate is suggested. However, the method can readily be adapted for the determination
of one nmole
of periodate.
Compounds
(other
than
periodate)
usually
*On leave from the Hebrew UniVersity, Terusaiem, Israel. Curbohyd- Res..
11 (1969) 119423
120
G. AVIGAD
found in a standard oxidation system of a carbohydrate do not interfere with the assay. As measurements are made at 593 nm, the presence of such u.v.-absorbing materials as compounds containing unsaturated bonds, aromatic aglycons, purines, pyrimidines, and proteins does not interfere. The method may aiso be suitable for the assay of periodate in certain water-miscible, organic solvents. The present assay-procedure was found to be particularly valuable when only a small quantity (one to two irmoles) of the sugar to be examined was available, but a study of the kinetics of its oxidation by periodate was desired. EXPERIMENTAL
Materials. - 2,4,6-Tri-2-pyridyl-s-triazine was obtained from the G. E. Smith Chemical CO., Columbus, Ohio 43223. The preparations of sodium metaperiodate used were analytical reagents from Fluka AG, Buchs, Switzerland, and Fisher Scientific Co., Pittsburgh, Pa. Spectrophotometry. Readings were made with a Zeiss Model PMQII spectrophotometer, with cuvets having a I-O-cm light-path. A Gilford model 300 spectrophotometer was occasionally employed (for rapid assay of a large series of samples). Reagents. - The violet solution of 1 was prepared as follows. TPTZ (75 mg, 0.24 mmole) was dissolved in acetic acid (46 ml), and M sodium acetate (210 ml) and a freshly prepared solution of Fe(NH,),(SO,),. 6H,O (31.4 mg, 0.08 mmole in 100 ml of water) were added. The volume was then made to 1 liter with water. The solution should have pH 4.0-4.2 and E:‘,“, ~1.8. No apparent decrease in absorbance of the solution occurs during one month at room temperature. This reagent may be diluted with M acetate buffer (pH 4.0)to provide solutions of lower absorbance at 593 nm. A stock solution (50 mM) of sodium metaperiodate was prepared by dissolving NaIO,, (1.0695 g) in water (100 ml) in an amber-colored measuring flask or one wrapped in aluminum foil to ensure complete protection from light. This solution was kept no longer than a week at room temperature. Dilute solutions were freshly made daily with water, and these solutions were kept protected from light. Assay of the periodate. In a standard procedure, sampIes containing 5 to 200 nmoles of periodate were added to tubes containing 4.5 ml of the violet reagent (1). The volume was made to 5.0 ml with water, and the solution was well mixed. The absorbance of the solution was read at 593 nm, to determine the amount of residua1, violet 1.As this compound is very stable under the conditions employed, measurement of it can be made at one minute, or many hours, after a sample of periodate has been removed for analysis. This property proved to be especially convenient when several series of samples had to be taken at short time-intervals during a kinetic study of oxidation. Typical standard-curves for periodate are presented in Fig. I. The slope of the lines is the same, irrespective of the initial concentration of the violet 1 present Cuddzyd-
Res., 11
(1969) 119-123
DETJXMINATION
OF PERIODATE
121
in the reagent (see Fig. 1A). Also, the slope can be proportionally increased when a smaller initial volume of the solution of 1 is employed for the assay of similar amounts of periodate (see Fig. 1B). It is apparent that the range and sensitivity of the method for periodate can readily be adjusted for the needs of any particular experiment over a range of concentrations of oxidant. Thus, the sensitivity can be increased to permit determination of one nmole of periodate.
Fig. 1. Oxidation of 1 by periodate. A. Reaction conducted in a volume of 5 ml, but containing various initial concentrations of the violet, ferrous complex 1.B. Different volumes of a solution of 1 used. Numbers near each line indicate number of ml of reagent employed.
The decrease in the molar extinction coefficient of the violet 1 that is ‘caused by oxidation with one mole of periodate was found to be 37,000 under the conditions of assay described; this value corresponds to the oxidation of I.7 g-atoms of ferrous ions2 ’ (the maximum theoretical equivalent expected is 2.0). E#ect of cariotu compounds on the oxidation of 1 by periodate. At a final concentration of 5 mu, formaldehyde, acetaldehyde, NaIO,, NaCIO,, KH,P04, Na,CO,, MgC12, and Tris do not interfere with the assay. Although 2 mM citrate does not decolorize the violet ferrous complex (l),concentrations higher than 0.2 mu lessen the sensitivity of its oxidation by periodate. Addition of ethylene(d&trilotetraacetate) to the ferrous complex 1 at equimolar concentrations causes instantaneous
decolorization”.
The presence of any one of several such ions as Cu2+, Ag+, CN-,
and NO; may interfere with 21 the stability of the reagent 1. The reaction can also be performed in the presence of any one of several water-soluble, organic solvents. Thus, pyridine (4%), acetonitrile (40%) and acetone (40%) do not interfere significantly with the reaction at concentrations lower than those indicated. NJGDimethyIformamide at concentrations higher than 1% lessens the extent of oxidation, whereas p-dioxane at 1% decolorizes the violet, ferrous complex (1). Several aliphatic primary alcohols decolorize the ferrous complex 1 and, CurbohycZ.Res., 11 (1969) 119423
122
G. AVIGAD
at very low concentration, inhibit the periodate reaction. Thus diethylene glycol, diethylene glycol monomethyl ether, methyl Cellosolve, and 2-chloroethanoi significantly interfere with the assay when present in a concentration as low as 0.2%. Methanol, ethanol, and propyl alcohol at final concentrations >0.05% exhibit an inhibitory effect on the extent of oxidation of the ferrous complex 1 by periodate (see refs. 23 and 24). Kinetics of oxidation of mrious substrates. - As an example, a representative group of compounds containing the 1,2-glycol grouping was oxidized by periodate, and the course of the consumption of oxidant was estimated by the method already described (see Fig. 2). Each oxidation was conducted at pH 5.2, to ensure slow hydrolysis of any formyl esters formed 25+26.Similar oxidations at pH 3 to 8 could conveniently be analyzed by the present method, as the reagent was prepared in M
acetate buffer at pH 4.0 to 4.2.
2
3
Time
A- 6
12
24
(hours1
C
V 1
Time (hours)
2
Time
3
A
5
(hours1
Fig. 2. Course of consumption of periodate during oxidation of different substrates- All reactions were conducted in the dark in 40 mM acetate buffer (PH 5.2). Samples (5 to 25 al) were withdrawn with a constriction micropipet and directly added to the violet, ferrous reagent (1) to determine the amount of residual periodate. A. Disaccharides (2 mhf) and tetrasaccharide (1 mhr) were oxidized with 15.4 m&i NaI04: 0, stachyose; 0, lactose; 8, a,a-trehalose; 31, sucrose. B. AIdopyranosides (2 mt.@ were oxidized with 10 mM NaI04: 0, methyl a-D-xylopyranoside: 0, methyl a-D-arabinopyranoside; 0, methyl a-D-ghrcopyranoside; I, methyl a-D-mannopyranoside. C. Substrates (2 mM) were oxidized with 20.2 mM NafO;r for inositol (e)), D-glucitol (o), and D-ghtcose (a); with 10.1 mM for 2-deoxy-n-lyxo_hexose (8) and 3-O-methyl-D-glucose (a; with 7.9 mM for glycerol (A) and r-serine (8); and with 5 mM for ethylene glycol (v)_
in all cases (see Fig. 2), the course of the oxidation and the periodate uptake per mole of substrate correspond to the theoretical value predicted. As expected6*‘, Cmbohyd.
Res., 11 (1969) 119-123
DETERhfJNATIbN
the oxidation
123
OF PERIODATE
of acyclic
glycols
is very fast compared
to that
of reducing
sugars,
involving the intermediary formation of formic esters (see Figs. 2 A, B). Also, cleavage between or-erythro-hydroxyl groups (distorted cis-hydroxyl groups) is faster than that of a-tizreo-hydroxyl group (distorted trans-hydroxyl groups),as is evident from the pattern of oxidation of glycosides (see Fig. 2C). ACKNOWLEDGMENT
I thank Dr. S. Englard for the hospitality of his laboratory. REFERENCES 1 C. A. BUMON, in K. B. WIBERG (Ed.), Oxidation in Organic Chemistry, Part A, Academic Press, New York, 1965, p. 367. B. SKLARZ, Quart. Rev. (London), 21 (1967) 3. A. J. FATIADI, J- Res. Nat. Bur. Stand., 72A (1968) 361_ W. J. BAUhfAN, H. H. 0. ScHhfm, AND H. K. MANGOLD, J. Lipid Res., 10 (1969) 132. R. D. GUTHRIE, Aduan. Carbohyd- Chem., 16 (1961) 105. J. STANEK, M. CERNS, J. KOCOUREK, AND J. PACAK, The Monosaccharides, Academic Press, New York, 1963, p_ 903. 7 I. J. GOLDSTEIN, G. W. HAY, B. A. LEWIS, AND F. SMITH, Mefhods Curbohyd. Chem., 5 (1965) 361. 8 L. HOUGH, Methods Carbohyd. Chent., 5 (1965) 370. 9 L. HOUGH, A. C. RICHARDSON, AND C. H. BOLTON, in Rodd’s Chentistry of Carbon Compounds, Vol. lF, Elsevier, Amsterdam, 2nd edition, 1967, p. 305. IO G. F. SMITH, Analytical Applications of Periodic Acid and Zodic Acid and Their Salts, G.F. Smith Chemical Co., Columbus, Ohio, 5th edition, 1950. 11 J. R. DYER, Methods Biochem. Anal., 3 (1956) 111. 12 I. M. KOLTHOFF AND R. BELCHER. Vohtmetric Analysis. Vol. III, Interscience Publishers, New York, 1957, p. 475. 13 R. D. GUI-HRIE, Methods Carbohyd. Chem., I (1962) 435. 14 G. W. HAY, B. A. LE\VIS, AND F. S?.IITH, Methods Carbohyd. Chent., 5 (1965) 357. 15 J. S. DIXON AND D. LIPKIN,Anal. Chem., 26 (1954) 1092. 16 G. V. MARINETTI AND G. ROUSER, J: Amer. Chem. Sot., 77 (1955) 5345. 17 G. 0. ASPINALL AND R. J. FERRIER, C/tern. Ittd. (London), (1957) 1216. 18 R. FIELDSAND H. B. F. DIXON, Biochem. J., 108 (1968) 883. 19 M. GUERNET, Bail. Sot. Chim. France, (1964) 478. 20 J. ARES PONS, PubI. Inst. Invest. Microquim., Unit. Nacl. Litoral (Rosario, Arg.), 26 (1965) 175, 199. 21 P. F. COLLINS, H. DIEHL, AND G. F. SMITH, Anal. C/tent., 31 (1959) 1862. 22 B. KRATOCHVILAND M.C. WHITE, Anal. Chem.,37(1965) 111. 23 J. F. TAYLOR, B. SOLDANO, AND G. A. HALL, J. Amer. Chem. Sot., 77 (1955) 2656. 24 R. D. GUTHRIE, Chem. Ind. (London), (1960) 691. 25 L. HOUGH AND M. B. PERRY, Chem. Znd. (London), (1956) 768. 26 L. HOUGH, T. J. TAYLOR, G. H. S. THOMAS, AND B. M. WOODS, J. C/tern. Sac., (1958) 1212. Carbohyd- Res., 11 (1969) 119-123