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Pulse polarography of nitrosated GPC fractions of humic acids
Talanta, Vol. 31, No. 3, pp. 207-208, 1984 Printed in Great Britain. All rights reserved
Copyright
PULSE POLAROGRAPHY OF NITROSATED FRACTIONS OF HUMIC ACIDS
0
0039-9140/84 $3.00 + 0.00 1984 Pergamon Press Ltd
GPC
MARIA TERESA LIPPOLIS, VITTORIO CONCIALINI and GIUSEPPE CHIAVARI Istituto Chimico G. Ciamician, Universita di Bologna, Via Selmi 2, Bologna 40126, Italy (Received 30 Augusf 1983. Accepted 28 September 1983)
Summary-A pulse polarographic method for the analysis of humic acids fractionated by gel permeation chromatography is described. It is concluded that the pulse polarography of the nitroso-derivatives, already reported as an analytical method for the determination of humic acids, really gives responses due to the presence of iron in the samples. Atomic-absorption measurements confirm this conclusion quite unambiguously.
Humic acids are extremely complex polymeric substances, which have been the subject of extensive research because of their importance in environmental chemistry. I4 Their composition varies according to the origin and nature of the water or soil in which they are found and methods for characterizing them are highly desirable. A variety of techniques have been used for this purpose, such as X-ray scattering,5 gel chromatography, viscosity measurement, dialysis and ultrafiltration,6*7 chemical analysis8 and polarographic determination of the nitroso derivatives.’ We have very recently investigated the gel filtration of humic acids, using an electrochemical detector and comparing its performance with that of the usual ultraviolet detector.” The present work is a continuation of the investigation, with the aim of comparing the pulse polarography of the nitrosoderivatives of the various fractions obtained in the gel filtration, with the responses given by the electrochemical detector. To clarify a discrepancy observed between the two methods, atomic-absorption measurements were also made.
Redirac fraction collector. Molecular weight calibration of the column is reported elsewhere.‘O The pulse polarograph was a Southern Analytical model A 3100. Procedure The nitrosation procedure was essentially the same as that reported by Ederle er ~1.~Each fraction (cu. 3.5 ml) was acidified to pH 4.5 with 1M acetate buffer, 1 ml of 10% sodium nitrite solution was added and the mixture was left at 50-60” for 20 min. After cooling, the solution was diluted to volume in a 25-ml standard flask with water and transferred to a conventional three-electrode polarographic cell, with a platinum counter-electrode and a saturated calomel electrode as reference. After deaeration with pure argon for 10 min, the solution was polarographed with a pulse amplitude of 50 mV and a sweep-rate of 133 mV/min. The peak height (at Ewk = -0.63 V us. SCE) was measured and corrected with respect to that found for a blank solution treated similarly.
RESULTS AND DISCUSSION
Figures 1 and 2 summarize the elution curves obtained by the three techniques, for the gel filtration of the two commercial samples.
EXPERIMENTAL Reagents
Commercial samples of humic acid were purchased from Aldrich and Fluka. The other chemical products were of analytical grade (C. Erba IWE) and used without further purification. The gel filtration columns were made with Ultrogel (LKB) having exclusion limits of 1000-15000. A O.OSM solution of sodium pyrophosphate was used as eluent, at a flow-rate of cu. 0.5 ml/min. Apparatus The electrochemical detector was a Metrohm mode1 656 with a three-electrode detection cell (model EA 1096/2). The working electrode was a carbon-paste electrode, the counter-electrode a glassy-carbon electrode and the reference electrode an Ag/AgCl system. Atomic-absorption measurements were done with a Perkin-Elmer mode1 372 instrument. The gel filtration column was 25 x 1.6 cm, packed according to the maker’s instructions and used with an LKB 2112
IO
20
30
40
50
ml
Fig. 1. Elution curves of Fluka humic acid. Sample volume: 0.5 ml of 0.5% solution in 0.05M Na,P,O,: ~ electrochemical detector response (sensitivity 0.5 PA full-scale deflection); --- ppm of iron from pulse polarographic measurements of the nitrosated fractions; .-.-. ppm of iron from atomic-absorption measurements. 207
208
SHORT COMhtUNlCATIONS
n
1
followed by polarography: these solutions gave a reduction peak at -0.63 V vs. SCE, confirming the attribution to reduction of an iron complex. The peak can be used to evaluate the concentration of iron, as shown in Figs. 1 and 2. Conclusions
ml
Fig. 2. Elution curves of Aldrich humic acid. Conditions and symbols as for Fig. 1.
The curves obtained by use of the electrochemical detector and by pulse polarography of the nitrosated fractions differ noticeably in shape and in the position of the maximum. In the earlier communicationi it was pointed out that the response of the electrochemical detector is mainly due to the oxidation of phenolic and amino groups present in the humic acids. The comparison therefore suggests that the pulse polarographic response is not attributable to nitrosated phenolic compounds. It is known that the humic acids contain variable amounts of several metals such as Al, Fe, Cu, etc., so we have made atomic-absorption (AA) measurements on the chromatographic fractions, to try to elucidate the discrepancy. The elution curves obtained by AA by determination of iron, show excellent agreement with those obtained by pulse polarography, so it seems reasonable to attribute the polarographic response to the reduction of some nitroso-complex of iron. This assumption has been confirmed by nitrosation of solutions containing known quantities of Fe(III),
From the results reported we can conclude that though phenolic and amino groups are certainly present in the humic acids, they are not all easily converted into nitroso derivatives, so the nitrosation technique9 may give unreliable results. However, since under our chromatographic conditions the iron is practically concentrated in one or two fractions, the nitrosation technique, coupled with pulse polarography, can be used for the determination of iron traces in natural humic acids. REFERENCES
1. M. M. Kononova, Soil Organic Matter, 2nd Ed., pp. 183-228. Pergamon Press, Oxford, 1966. 2. M. Schnitxer and S. U. Khan, Humic Substances in the Environment, pp. 281-302. Dekker, New York, 1966. 3. E. T. Gjessing, Physical and Chemical Characteristics of Aquatic Humus, pp. 56-73. Ann Arbor Science, Ann Arbor, 1976. 4. G. T. Felback, in Soil Biochemistry, A. D. McLaren and J. Skujins (eds.), Vol. 2, pp. 36-59. Dekker, New York, 1971. 5. R. L. Wershaw, S. L. Heller and D. J. Pinckney, Adv. X-Ray Anal., 1970, 13, 609. 6. J. H. Reuter, Geol. Sot. America, Abstracts, 9 (1977). 7. M. Schnitzer and S. I. M. Skinner, Isotopes and Radiation in Soil Organic Matter Studies, p. 41. International Atomic Energy Agency, Vienna, 1968. 8. S. A. Visser, J. Environ. Sci. Health, 1982, A17, 767. 9. S. H. Ederle, C. Hoesle and C. Krtickeberg, IRCh, KFK-1969 UF Kemforschungszentrum Karlsruhe, 1974. 10. G. Chiavari, V. Concialini, M. T. Lippolis and F. Scarponi, J. Chromatog., in press.
Copyright
PULSE POLAROGRAPHY OF NITROSATED FRACTIONS OF HUMIC ACIDS
0
0039-9140/84 $3.00 + 0.00 1984 Pergamon Press Ltd
GPC
MARIA TERESA LIPPOLIS, VITTORIO CONCIALINI and GIUSEPPE CHIAVARI Istituto Chimico G. Ciamician, Universita di Bologna, Via Selmi 2, Bologna 40126, Italy (Received 30 Augusf 1983. Accepted 28 September 1983)
Summary-A pulse polarographic method for the analysis of humic acids fractionated by gel permeation chromatography is described. It is concluded that the pulse polarography of the nitroso-derivatives, already reported as an analytical method for the determination of humic acids, really gives responses due to the presence of iron in the samples. Atomic-absorption measurements confirm this conclusion quite unambiguously.
Humic acids are extremely complex polymeric substances, which have been the subject of extensive research because of their importance in environmental chemistry. I4 Their composition varies according to the origin and nature of the water or soil in which they are found and methods for characterizing them are highly desirable. A variety of techniques have been used for this purpose, such as X-ray scattering,5 gel chromatography, viscosity measurement, dialysis and ultrafiltration,6*7 chemical analysis8 and polarographic determination of the nitroso derivatives.’ We have very recently investigated the gel filtration of humic acids, using an electrochemical detector and comparing its performance with that of the usual ultraviolet detector.” The present work is a continuation of the investigation, with the aim of comparing the pulse polarography of the nitrosoderivatives of the various fractions obtained in the gel filtration, with the responses given by the electrochemical detector. To clarify a discrepancy observed between the two methods, atomic-absorption measurements were also made.
Redirac fraction collector. Molecular weight calibration of the column is reported elsewhere.‘O The pulse polarograph was a Southern Analytical model A 3100. Procedure The nitrosation procedure was essentially the same as that reported by Ederle er ~1.~Each fraction (cu. 3.5 ml) was acidified to pH 4.5 with 1M acetate buffer, 1 ml of 10% sodium nitrite solution was added and the mixture was left at 50-60” for 20 min. After cooling, the solution was diluted to volume in a 25-ml standard flask with water and transferred to a conventional three-electrode polarographic cell, with a platinum counter-electrode and a saturated calomel electrode as reference. After deaeration with pure argon for 10 min, the solution was polarographed with a pulse amplitude of 50 mV and a sweep-rate of 133 mV/min. The peak height (at Ewk = -0.63 V us. SCE) was measured and corrected with respect to that found for a blank solution treated similarly.
RESULTS AND DISCUSSION
Figures 1 and 2 summarize the elution curves obtained by the three techniques, for the gel filtration of the two commercial samples.
EXPERIMENTAL Reagents
Commercial samples of humic acid were purchased from Aldrich and Fluka. The other chemical products were of analytical grade (C. Erba IWE) and used without further purification. The gel filtration columns were made with Ultrogel (LKB) having exclusion limits of 1000-15000. A O.OSM solution of sodium pyrophosphate was used as eluent, at a flow-rate of cu. 0.5 ml/min. Apparatus The electrochemical detector was a Metrohm mode1 656 with a three-electrode detection cell (model EA 1096/2). The working electrode was a carbon-paste electrode, the counter-electrode a glassy-carbon electrode and the reference electrode an Ag/AgCl system. Atomic-absorption measurements were done with a Perkin-Elmer mode1 372 instrument. The gel filtration column was 25 x 1.6 cm, packed according to the maker’s instructions and used with an LKB 2112
IO
20
30
40
50
ml
Fig. 1. Elution curves of Fluka humic acid. Sample volume: 0.5 ml of 0.5% solution in 0.05M Na,P,O,: ~ electrochemical detector response (sensitivity 0.5 PA full-scale deflection); --- ppm of iron from pulse polarographic measurements of the nitrosated fractions; .-.-. ppm of iron from atomic-absorption measurements. 207
208
SHORT COMhtUNlCATIONS
n
1
followed by polarography: these solutions gave a reduction peak at -0.63 V vs. SCE, confirming the attribution to reduction of an iron complex. The peak can be used to evaluate the concentration of iron, as shown in Figs. 1 and 2. Conclusions
ml
Fig. 2. Elution curves of Aldrich humic acid. Conditions and symbols as for Fig. 1.
The curves obtained by use of the electrochemical detector and by pulse polarography of the nitrosated fractions differ noticeably in shape and in the position of the maximum. In the earlier communicationi it was pointed out that the response of the electrochemical detector is mainly due to the oxidation of phenolic and amino groups present in the humic acids. The comparison therefore suggests that the pulse polarographic response is not attributable to nitrosated phenolic compounds. It is known that the humic acids contain variable amounts of several metals such as Al, Fe, Cu, etc., so we have made atomic-absorption (AA) measurements on the chromatographic fractions, to try to elucidate the discrepancy. The elution curves obtained by AA by determination of iron, show excellent agreement with those obtained by pulse polarography, so it seems reasonable to attribute the polarographic response to the reduction of some nitroso-complex of iron. This assumption has been confirmed by nitrosation of solutions containing known quantities of Fe(III),
From the results reported we can conclude that though phenolic and amino groups are certainly present in the humic acids, they are not all easily converted into nitroso derivatives, so the nitrosation technique9 may give unreliable results. However, since under our chromatographic conditions the iron is practically concentrated in one or two fractions, the nitrosation technique, coupled with pulse polarography, can be used for the determination of iron traces in natural humic acids. REFERENCES
1. M. M. Kononova, Soil Organic Matter, 2nd Ed., pp. 183-228. Pergamon Press, Oxford, 1966. 2. M. Schnitxer and S. U. Khan, Humic Substances in the Environment, pp. 281-302. Dekker, New York, 1966. 3. E. T. Gjessing, Physical and Chemical Characteristics of Aquatic Humus, pp. 56-73. Ann Arbor Science, Ann Arbor, 1976. 4. G. T. Felback, in Soil Biochemistry, A. D. McLaren and J. Skujins (eds.), Vol. 2, pp. 36-59. Dekker, New York, 1971. 5. R. L. Wershaw, S. L. Heller and D. J. Pinckney, Adv. X-Ray Anal., 1970, 13, 609. 6. J. H. Reuter, Geol. Sot. America, Abstracts, 9 (1977). 7. M. Schnitzer and S. I. M. Skinner, Isotopes and Radiation in Soil Organic Matter Studies, p. 41. International Atomic Energy Agency, Vienna, 1968. 8. S. A. Visser, J. Environ. Sci. Health, 1982, A17, 767. 9. S. H. Ederle, C. Hoesle and C. Krtickeberg, IRCh, KFK-1969 UF Kemforschungszentrum Karlsruhe, 1974. 10. G. Chiavari, V. Concialini, M. T. Lippolis and F. Scarponi, J. Chromatog., in press.