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A polarographic study of d -glucuronolactone
Talanta, 1961. Vol. 7. pp. 175 to 180. Pergamon Press Ltd. Printed in Northern Ireland
A POLAROGRAPHIC
STUDY OF D-GLUCURONOLACTONE
ROGER J. THIBERT*@ and ALBERT J. BOYLE Department of Chemistry, Wayne State University, Detroit, Michigan, U.S.A. (Received 26 February 1960)
polarographic study of D-glucuronolactone using a dropping mercury electrode is made. The effects on the wave of various supporting electrolytes, buffers, and maximum suppressors are investigated. The diffusion current shows a linear relationship to concentration for lithium chloride and potassium chloride as supporting electrolytes in the range of 10-100 pg/ml. Pyruvate interferes whereas D-glucose does not. INTRODUCTION Summary-A
THE fact that D-glucuronolactone (D-glucurone) is a lactone and is potentially reducible at the dropping mercury electrode suggested the possibility of a polarographic determination. Aldoses and ketoses give kinetic currents and their waves depend on the transformation of the non-reducible cyclic hemi-acetal form to the aldehydoor keto-form at the electrode .ls2 Carbohydrates may also be determined polarographically by forming their hydrazones, then reducing them at the dropping mercury electrode.3,4 A polarographic study of D-glucuronic acid was reported by Ishidate and Shimozawa.5 These investigators were not able to obtain reproducible waves and the results In this communicawere not quantitative for either D-glucuronic acid or D-glucurone. tion, an investigation of the effect of the supporting electrolyte, maximum suppressor, pH, types of buffers, and the interference of glucose is discussed. EXPERIMENTAL Apparatus
A Beckman pH-meter, Model G, was used to measure pH. An E. H. Sargent polarograph, Model XXI, with a Heyrovskjr-type cell was used to obtain the polarograms. A polarimeter with monochromator assembly, Rudolph 503, 0. C. Rudolph and Sons, served to measure the specific rotation of D-glucurone solutions. Reagents The prepared D-glucurone solutions presented a specific rotation [tin*‘] of +20.3”. Polarograms obtained after digestion of D-glucurone solutions with nitric-perchloric acid mixture6s7 gave the same wave as the supporting electrolyte (0.02N potassium chloride). Procedure The solutions to be analysed were prepared by mixing the appropriate concentrations of Dglucurone, supporting electrolyte, and maximum suppressor in a IOO-ml volumetric flask and diluting to volume with distilled water. After proper mixing, the solution to be analysed was transferred to a 25-ml Heyrovskjr-type polarographic cell. Nitrogen was passed through the solution for 10 min and a polarogram was obtained for each solution, using a drop rate of 4 sec. The polarograms were run through the range of 0.0 to -3.0 V versus the mercury pool. * Research Fellow, Michigan Heart Association. Present address: Essex College, Assumption of Windsor, Windsor, Ontario, Canada. 175
University
176
ROGER J. THIBERTand ALBERT J. BOYLE RESULTS
Supporting
AND
DISCUSSION
electrolytes
The relationship between diffusion current and concentration of D-glucurone using 0.2N potassium chloride solution as the supporting electrolyte and 0405% gelatin as the maximum suppressor is given in Table I. TABLE 1. EFFECTOFD-GLIJCURONECONCENTRATION ON DIFFUSION CURRENT D-Glucurone
concentration,
Diffusion current,
mA
f&ml
1.41
100 80
l*OO
80
1.02
60
0.882
40
0.495
20
0.176
20
0.148
20
0.159
10
0.096
10
0.132
The half-wave potential was observed to be - 1.56 V versus the mercury pool. The polarographic wave has a sharp maximum at about - 1.6 to - I.7 V versus the mercury pool using 0*005’~ gelatin as the maximum suppressor. Further work revealed that 0.01 y0 gelatin completely suppresses the maximum but the total wave height is somewhat reduced. Lithium chloride as a supporting electrolyte proved to be superior to potassium chloride if 0405 % gelatin was used as the maximum suppressor. The wave exhibits no maximum and, as is shown in Table II, the relationship of concentration to diffusion current is much more nearly linear than is the case with potassium chloride. TABLE II.
EFFECTOF D-GLUCURONECONCENTRATION ON DIFFUSIONCURRENT
D-Gluckrone
concentration,
tcglml 100
Diffusion current,
mA 1.23
80
0.888
60
0.612
60
0.623
60
0.576
40
0.366
20
0.120
20
0.111
10
0.062
A typical polarogram using lithium chloride as the supporting electrolyte and 0405 % gelatin as the maximum suppressor is shown in Fig. 1. The half-wave potential is -1.60 V versus the mercury pool. Attempts to use ammonium chloride as a
117
A polarographicstudy of o-glucuronolactone
supporting electrolyte were not successful since the electrolyte gave a wave starting at - I.65 V versus the mercury pool. Degree of hydrolysis
The influence of time on the wave height of D-glucurone was investigated. It was observed that a three-weeks old solution had a similar diffusion current (l-41 mA) to a one-day old solution (1.40 mA). The polarogram was run using 100 pg/ml of D-glucurone in 0.2N potassium chloride solution as the supporting electrolyte and
2 VOLTAGE .VERSUS
FIG. I.-Polarogram
MERCURY POOL
of 100 pg/ml of D-glucurone using 0.02iV LiCl as the supporting electrolyte.
0.005% gelatin as the maximum suppressor. If the fresh solution is heated before analysis, the diffusion current is much less (I.02 mA), pointing to some hydrolysis. Standard solutions of D-glucurone were always stored at O”-9, thereby slowing down hydrolysis. Reduction of D-glucurone in the presence of hydrochloric acid
Polarograms of 100 pg/ml of D-glucurone in O-IN potassium chloride solution using 4 drops of methyl orange solution (O-1% in water) as the maximum suppressor, were run at various hydrogen ion concentrations. The effect of hydrogen ion concentration on the diffusion current is shown in Table III. At acid concentrations above 1 x 10-4M, the D-glucurone wave cannot be obtained, but rather a very large wave, beginning at about - l-5 V versus the mercury pool and going off scale rapidly, is observed. Under basic conditions no wave is obtained; upon neutralisation with hydrochloric acid, the wave re-appears. The wave
178
ROGER J. THIBERTand ALBERT J. BOYLE
exhibits a more pronounced maximum using methyl orange as the maximum suppressor, but the wave height is somewhat elevated. TABLE III.
EFFECTOF HYDROGENION CONCENTRATION ON DIFFUSIONCURRENT
Concentration
of HCl,
Diffusion current,
molesllitre
mA
1 x 10-a
1.23
1 x 10-s
1.68
1 x 10-G
1.80
no acid present
1.70
Interference
The use of any of the standard buffer mixtures, completely suppressed the wave. The buffers usually caused the appearance of a gradual wave at about -1.32 to -1.7 V versus the mercury pool. This gradual wave completely eliminated the D-glucurone wave. The buffer systems used were: phthalate buffer,8 pH 4; Coleman buffers, pH 2, 6, 9; K,HPO,-KH,PO, buffer? pH 6; NaC,H,O,-HC,H,O, buffer,g pH 4; (C,H,),NOH + H,PO, buffer, pH 2.1, 4.1, 6.0, 8.0, 9.4; barbiturate buffer, pH 6, Li,HPO,-LiH,PO, buffer, pH 6. Even upon dilution, these buffers inhibited the D-glucurone wave. So extreme is the effect with any form of phosphate buffer, that if, for example, one drop of O*lM potassium dihydrogen phosphate solution is added to the 25-ml polarographic cell containing 100 ,ug/ml of D-glucurone with potassium chloride as the supporting electrolyte containing 0.005 % gelatin as the maximum suppressor, the D-glucurone wave is entirely suppressed. This suggested the possibility of an indirect polarographic determination of phosphate. Present studies indicate that this indirect method for phosphate is feasible. Barbiturate buffer enhanced the wave height because it exhibited a wave of its own in the region of the D-glucurone wave. Pyruvic acid, when added to D-glucurone solutions, interferes with the D-glucurone wave height. The relationship is not linear, nor is it additive. (Pyruvic acid is converted to lactic acid at the dropping mercury electrode.lO) Determination
of D-glucurone in the presence of D-glucose
A D-glucose solution (1000 ,ug/ml) was analysed in O*lN potassium chloride solution using 0.005 ‘A gelatin as the maximum suppressor. At sensitivities of 0.003, O$Kl4,0~006,and 0.01 mA/mm, the polarograms showed a break at - 1.80 V versus the mercury pool in each case (i.e. no characteristic wave was observed). A solution of 50 ,ug/ml of D-glucurone and 1000 lug/ml of D-glucose in O*lN potassium chloride solution using OGO5‘A gelatin as the maximum suppressor was analysed in the usual manner. The usual D-glucurone wave was obtained with a diffusion current of 0.60 mA and half-wave potential of - 1.56 V versus the mercury pool (Fig. 2). CONCLUSION
D-Glucurone exhibits a polarographic wave in potassium chloride or lithium chloride as the supporting electrolyte using gelatin as the maximum suppressor. It is suggested that this wave is due to the lactone since it disappears at high pH levels
A polarograpbic
study of ~~~u~no~a~one
179
(hydrolysis occurs) and decreases in magnitude upon heating solutions of D-glucurone before analysis. The wave obtained with D-glucurone is not due to metal ions, since digestion to dryness of D-glucurone solutions with nitric-perchloric acid mixture causes the wave to disappear. The complete inability of the system to function in the presence of any type of buffer sets it apart from many types of organic reductions which require well defined media with respect to pH .lx The type of reduction occurring does not appear to be of the reversible type with controlled pH, but rather ofthe catalytic type in which the overvoltage of hydrogen is decreased by the la&one, and the diffusion current obtained is ~ro~~~ona~ to the concentration of the reducible species in sdution.
/” OOO/ 00
04
08
1.2
1.6
2.0
VOLThGE VERSUS MERCURYFOOL
~~~a~-Ejne ~l~~~a~~isc~~ &die mit ~~lu~on~lacto~ an der tropfen&n Der EinRuss verschiedener GrundBsungen, Elektrolyte, Q~~~~l~elek~ode wird mitgeteilt. Puffer und Maximasuppressoren auf die WeHe wurde studiert. fn Lithium- oder Kaiiumchlorid als Grundliisungen ist die Beziehung zwischen Diffusionsstrom und Konzentration linear im Bereich 10-100 ,ug per ml. Pyruvate stBren wahrend D-Glucose ohne Eitiuss ist. R&m&-Les auteurs ont fait une Etude palarographique de la D-glucuronolactone en utilisant une &&rode B gouttes de mercure. Les actions de diffkrents 6lectrolytes supports, des tampons et des Le courant de diffusion varie linhirement suppresseurs de maximum sur la vague ant Ct&&dikes. avec la concentration dans le domaine 50 rg-1OQ yg par ml pour les Clectrolytes supports: chlorure de lithium et chlorure de potassium. Le pyruvate gene, mais non le n-glucose. REFERENCES 1 i. M, KoIthoB and 3. I_ Lingane, ~~~~r~~ra~~~. Interscience Publishers, New York> 2nd Ed., 1952. Vol. 2, p. 77%. 3 S. M. Cantor and Q, P. Peniston, & Amer. Chem. SW., 1940,6Z, 2113.
180
RICER J. THIBERTand ALBERTJ. BOYLE
* L. Mester and A. Major, ibid., 1955, 77,4297. 4 J. W. Haas and C. C. Lynch, Analyt. Chem., 1957,29,479. 5 M. Ishidate and T. Shimozawa, J. Pharm. Sot. Japan., 1944, 64, 53. 6 G. Frederick Smith, Mixed Perchloric, Sulphuric and Phosphoric Acid and their Applications in Analysis. The G. Frederick Smith Chemical Co., 1942. p. 10. ’ G. Frederick Smith, Analyt. Chim. Acta, 1953, 8, 397. 8 H. H. Willard, L. L. Merritt and J. A. Dean, Znstrumental Methods of Analysis. D. Van Nostrand Company, Inc., New York, 1951. p. 182. p F. C. Koch and M. E. Hanke, Practical Methods in Biochemistry. The Williams and Wilkins Company, Baltimore, 6th. Ed., 1953. p. 522. lo I. M. Kolthoff and J. J. Lingane, Polarography. Interscience Publishers, New York, 2nd. Ed., 1952. Vol. 2, p. 730. l1 0, H. Muller, Polarography, in A. Weissberger, Technique of Organic Chemistry. Interscience Publishers, New York, 1949. Vol. 1, Part 2, p. 1833.
A POLAROGRAPHIC
STUDY OF D-GLUCURONOLACTONE
ROGER J. THIBERT*@ and ALBERT J. BOYLE Department of Chemistry, Wayne State University, Detroit, Michigan, U.S.A. (Received 26 February 1960)
polarographic study of D-glucuronolactone using a dropping mercury electrode is made. The effects on the wave of various supporting electrolytes, buffers, and maximum suppressors are investigated. The diffusion current shows a linear relationship to concentration for lithium chloride and potassium chloride as supporting electrolytes in the range of 10-100 pg/ml. Pyruvate interferes whereas D-glucose does not. INTRODUCTION Summary-A
THE fact that D-glucuronolactone (D-glucurone) is a lactone and is potentially reducible at the dropping mercury electrode suggested the possibility of a polarographic determination. Aldoses and ketoses give kinetic currents and their waves depend on the transformation of the non-reducible cyclic hemi-acetal form to the aldehydoor keto-form at the electrode .ls2 Carbohydrates may also be determined polarographically by forming their hydrazones, then reducing them at the dropping mercury electrode.3,4 A polarographic study of D-glucuronic acid was reported by Ishidate and Shimozawa.5 These investigators were not able to obtain reproducible waves and the results In this communicawere not quantitative for either D-glucuronic acid or D-glucurone. tion, an investigation of the effect of the supporting electrolyte, maximum suppressor, pH, types of buffers, and the interference of glucose is discussed. EXPERIMENTAL Apparatus
A Beckman pH-meter, Model G, was used to measure pH. An E. H. Sargent polarograph, Model XXI, with a Heyrovskjr-type cell was used to obtain the polarograms. A polarimeter with monochromator assembly, Rudolph 503, 0. C. Rudolph and Sons, served to measure the specific rotation of D-glucurone solutions. Reagents The prepared D-glucurone solutions presented a specific rotation [tin*‘] of +20.3”. Polarograms obtained after digestion of D-glucurone solutions with nitric-perchloric acid mixture6s7 gave the same wave as the supporting electrolyte (0.02N potassium chloride). Procedure The solutions to be analysed were prepared by mixing the appropriate concentrations of Dglucurone, supporting electrolyte, and maximum suppressor in a IOO-ml volumetric flask and diluting to volume with distilled water. After proper mixing, the solution to be analysed was transferred to a 25-ml Heyrovskjr-type polarographic cell. Nitrogen was passed through the solution for 10 min and a polarogram was obtained for each solution, using a drop rate of 4 sec. The polarograms were run through the range of 0.0 to -3.0 V versus the mercury pool. * Research Fellow, Michigan Heart Association. Present address: Essex College, Assumption of Windsor, Windsor, Ontario, Canada. 175
University
176
ROGER J. THIBERTand ALBERT J. BOYLE RESULTS
Supporting
AND
DISCUSSION
electrolytes
The relationship between diffusion current and concentration of D-glucurone using 0.2N potassium chloride solution as the supporting electrolyte and 0405% gelatin as the maximum suppressor is given in Table I. TABLE 1. EFFECTOFD-GLIJCURONECONCENTRATION ON DIFFUSION CURRENT D-Glucurone
concentration,
Diffusion current,
mA
f&ml
1.41
100 80
l*OO
80
1.02
60
0.882
40
0.495
20
0.176
20
0.148
20
0.159
10
0.096
10
0.132
The half-wave potential was observed to be - 1.56 V versus the mercury pool. The polarographic wave has a sharp maximum at about - 1.6 to - I.7 V versus the mercury pool using 0*005’~ gelatin as the maximum suppressor. Further work revealed that 0.01 y0 gelatin completely suppresses the maximum but the total wave height is somewhat reduced. Lithium chloride as a supporting electrolyte proved to be superior to potassium chloride if 0405 % gelatin was used as the maximum suppressor. The wave exhibits no maximum and, as is shown in Table II, the relationship of concentration to diffusion current is much more nearly linear than is the case with potassium chloride. TABLE II.
EFFECTOF D-GLUCURONECONCENTRATION ON DIFFUSIONCURRENT
D-Gluckrone
concentration,
tcglml 100
Diffusion current,
mA 1.23
80
0.888
60
0.612
60
0.623
60
0.576
40
0.366
20
0.120
20
0.111
10
0.062
A typical polarogram using lithium chloride as the supporting electrolyte and 0405 % gelatin as the maximum suppressor is shown in Fig. 1. The half-wave potential is -1.60 V versus the mercury pool. Attempts to use ammonium chloride as a
117
A polarographicstudy of o-glucuronolactone
supporting electrolyte were not successful since the electrolyte gave a wave starting at - I.65 V versus the mercury pool. Degree of hydrolysis
The influence of time on the wave height of D-glucurone was investigated. It was observed that a three-weeks old solution had a similar diffusion current (l-41 mA) to a one-day old solution (1.40 mA). The polarogram was run using 100 pg/ml of D-glucurone in 0.2N potassium chloride solution as the supporting electrolyte and
2 VOLTAGE .VERSUS
FIG. I.-Polarogram
MERCURY POOL
of 100 pg/ml of D-glucurone using 0.02iV LiCl as the supporting electrolyte.
0.005% gelatin as the maximum suppressor. If the fresh solution is heated before analysis, the diffusion current is much less (I.02 mA), pointing to some hydrolysis. Standard solutions of D-glucurone were always stored at O”-9, thereby slowing down hydrolysis. Reduction of D-glucurone in the presence of hydrochloric acid
Polarograms of 100 pg/ml of D-glucurone in O-IN potassium chloride solution using 4 drops of methyl orange solution (O-1% in water) as the maximum suppressor, were run at various hydrogen ion concentrations. The effect of hydrogen ion concentration on the diffusion current is shown in Table III. At acid concentrations above 1 x 10-4M, the D-glucurone wave cannot be obtained, but rather a very large wave, beginning at about - l-5 V versus the mercury pool and going off scale rapidly, is observed. Under basic conditions no wave is obtained; upon neutralisation with hydrochloric acid, the wave re-appears. The wave
178
ROGER J. THIBERTand ALBERT J. BOYLE
exhibits a more pronounced maximum using methyl orange as the maximum suppressor, but the wave height is somewhat elevated. TABLE III.
EFFECTOF HYDROGENION CONCENTRATION ON DIFFUSIONCURRENT
Concentration
of HCl,
Diffusion current,
molesllitre
mA
1 x 10-a
1.23
1 x 10-s
1.68
1 x 10-G
1.80
no acid present
1.70
Interference
The use of any of the standard buffer mixtures, completely suppressed the wave. The buffers usually caused the appearance of a gradual wave at about -1.32 to -1.7 V versus the mercury pool. This gradual wave completely eliminated the D-glucurone wave. The buffer systems used were: phthalate buffer,8 pH 4; Coleman buffers, pH 2, 6, 9; K,HPO,-KH,PO, buffer? pH 6; NaC,H,O,-HC,H,O, buffer,g pH 4; (C,H,),NOH + H,PO, buffer, pH 2.1, 4.1, 6.0, 8.0, 9.4; barbiturate buffer, pH 6, Li,HPO,-LiH,PO, buffer, pH 6. Even upon dilution, these buffers inhibited the D-glucurone wave. So extreme is the effect with any form of phosphate buffer, that if, for example, one drop of O*lM potassium dihydrogen phosphate solution is added to the 25-ml polarographic cell containing 100 ,ug/ml of D-glucurone with potassium chloride as the supporting electrolyte containing 0.005 % gelatin as the maximum suppressor, the D-glucurone wave is entirely suppressed. This suggested the possibility of an indirect polarographic determination of phosphate. Present studies indicate that this indirect method for phosphate is feasible. Barbiturate buffer enhanced the wave height because it exhibited a wave of its own in the region of the D-glucurone wave. Pyruvic acid, when added to D-glucurone solutions, interferes with the D-glucurone wave height. The relationship is not linear, nor is it additive. (Pyruvic acid is converted to lactic acid at the dropping mercury electrode.lO) Determination
of D-glucurone in the presence of D-glucose
A D-glucose solution (1000 ,ug/ml) was analysed in O*lN potassium chloride solution using 0.005 ‘A gelatin as the maximum suppressor. At sensitivities of 0.003, O$Kl4,0~006,and 0.01 mA/mm, the polarograms showed a break at - 1.80 V versus the mercury pool in each case (i.e. no characteristic wave was observed). A solution of 50 ,ug/ml of D-glucurone and 1000 lug/ml of D-glucose in O*lN potassium chloride solution using OGO5‘A gelatin as the maximum suppressor was analysed in the usual manner. The usual D-glucurone wave was obtained with a diffusion current of 0.60 mA and half-wave potential of - 1.56 V versus the mercury pool (Fig. 2). CONCLUSION
D-Glucurone exhibits a polarographic wave in potassium chloride or lithium chloride as the supporting electrolyte using gelatin as the maximum suppressor. It is suggested that this wave is due to the lactone since it disappears at high pH levels
A polarograpbic
study of ~~~u~no~a~one
179
(hydrolysis occurs) and decreases in magnitude upon heating solutions of D-glucurone before analysis. The wave obtained with D-glucurone is not due to metal ions, since digestion to dryness of D-glucurone solutions with nitric-perchloric acid mixture causes the wave to disappear. The complete inability of the system to function in the presence of any type of buffer sets it apart from many types of organic reductions which require well defined media with respect to pH .lx The type of reduction occurring does not appear to be of the reversible type with controlled pH, but rather ofthe catalytic type in which the overvoltage of hydrogen is decreased by the la&one, and the diffusion current obtained is ~ro~~~ona~ to the concentration of the reducible species in sdution.
/” OOO/ 00
04
08
1.2
1.6
2.0
VOLThGE VERSUS MERCURYFOOL
~~~a~-Ejne ~l~~~a~~isc~~ &die mit ~~lu~on~lacto~ an der tropfen&n Der EinRuss verschiedener GrundBsungen, Elektrolyte, Q~~~~l~elek~ode wird mitgeteilt. Puffer und Maximasuppressoren auf die WeHe wurde studiert. fn Lithium- oder Kaiiumchlorid als Grundliisungen ist die Beziehung zwischen Diffusionsstrom und Konzentration linear im Bereich 10-100 ,ug per ml. Pyruvate stBren wahrend D-Glucose ohne Eitiuss ist. R&m&-Les auteurs ont fait une Etude palarographique de la D-glucuronolactone en utilisant une &&rode B gouttes de mercure. Les actions de diffkrents 6lectrolytes supports, des tampons et des Le courant de diffusion varie linhirement suppresseurs de maximum sur la vague ant Ct&&dikes. avec la concentration dans le domaine 50 rg-1OQ yg par ml pour les Clectrolytes supports: chlorure de lithium et chlorure de potassium. Le pyruvate gene, mais non le n-glucose. REFERENCES 1 i. M, KoIthoB and 3. I_ Lingane, ~~~~r~~ra~~~. Interscience Publishers, New York> 2nd Ed., 1952. Vol. 2, p. 77%. 3 S. M. Cantor and Q, P. Peniston, & Amer. Chem. SW., 1940,6Z, 2113.
180
RICER J. THIBERTand ALBERTJ. BOYLE
* L. Mester and A. Major, ibid., 1955, 77,4297. 4 J. W. Haas and C. C. Lynch, Analyt. Chem., 1957,29,479. 5 M. Ishidate and T. Shimozawa, J. Pharm. Sot. Japan., 1944, 64, 53. 6 G. Frederick Smith, Mixed Perchloric, Sulphuric and Phosphoric Acid and their Applications in Analysis. The G. Frederick Smith Chemical Co., 1942. p. 10. ’ G. Frederick Smith, Analyt. Chim. Acta, 1953, 8, 397. 8 H. H. Willard, L. L. Merritt and J. A. Dean, Znstrumental Methods of Analysis. D. Van Nostrand Company, Inc., New York, 1951. p. 182. p F. C. Koch and M. E. Hanke, Practical Methods in Biochemistry. The Williams and Wilkins Company, Baltimore, 6th. Ed., 1953. p. 522. lo I. M. Kolthoff and J. J. Lingane, Polarography. Interscience Publishers, New York, 2nd. Ed., 1952. Vol. 2, p. 730. l1 0, H. Muller, Polarography, in A. Weissberger, Technique of Organic Chemistry. Interscience Publishers, New York, 1949. Vol. 1, Part 2, p. 1833.