The Science of the Total Environment, 116 (1992) 213-220 Elsevier Science Publishers B.V., Amsterdam
213
Determination of the carboxyl content in humic substances by methylation I. Arsenie, H. Boren and B. Allard Department of Water and Environmental Studies, Link6ping University, S-581 83 LinkOping, Sweden (Received January 24th, 1991; accepted April 25th, 1991) ABSTRACT The carboxyl content of different fulvic acids was estimated by means o f a methylation technique. The procedure involved methylation by diazomethane followed by base catalyzed hydrolysis, esterification with propionyl chloride and gas chromatographic analysis of the methyl ester formed. After optimization of each step, the whole sequence of reactions was performed using only a few milligrams of starting material. The method was applied to two fulvic acid samples of different origin (surface water and groundwater, respectively). The carboxylic content was estimated to about 3.5 meq/g material for the surface water fulvic acid and about 4.5 meq/g material for the groundwater fulvic acid. This estimates approximately 80% of the total content of acidic groups obtained for the same materials by using potentiometric titration.
Key words. humic acid; fulvic acid; carboxylic acid; derivatization; methylation; saponification
INTRODUCTION
Humic materials represent one of the largest sources of natural organic carbon on earth [1]. They are formed by chemical and biological degradation of plant and animal residues and by synthetic activities of microorganisms [2]. The increased interest in the chemistry of the humic compounds in the last decade is based on the understanding of the important role of humic materials in the natural environment. The ability of humics to form both water-soluble and water-insoluble complexes with other chemical species as well as their capacity to reduce some metal ions make them play a crucial role in the speciation, transport and deposition of a variety of compounds ranging from metal ions to lipophilic organics [3,4]. Due to a continuous leaching from the soil and also a possible formation in aqueous systems, humic and fulvic acids may be found in all kinds of fresh 0048-9697/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved
214
i. ARSENIE ET AL.
waters, typically at concentrations from tens of mg/l in bog waters to fractions of mg/1 in deep ground waters [5]. Minor amounts of fresh water humics may also be produced autochthonously [6]. The origin of humic compounds in the marine environment is still a matter of controversy [7]. Structurally, humic substances comprise a heterogeneous group of organic compounds. Their building blocks are believed to be a mixture of aromatic rings and aliphatic chains. The predominant functional groups responsible for reactivity, complexation ability and solubility of humic compounds are carboxyls, alcohols and phenols. Common analytical techniques used to study the structure of humic compounds are various kinds of titration [8,9], spectroscopic techniques such as 1H- and 13C-NMR [10,11] and chemical degradation followed by analysis by GC/MS [12]. Chemical derivatization has been utilized to a less extent in such studies, and only in combination with spectroscopic techniques [13]. This paper describes the quantitative determination of the carboxylic component of two fulvic acids by a method that is more specific than most techniques utilized so far. The stages of the method are methylation, saponification and subsequent gas chromatographic analysis of the methanol formed as a methyl ester. EXPERIMENTAL
Recovery of Humic Substances Surface water fulvic acid (FA-B) was isolated from a bog area at Bersbo, approx. 200 km south of Stockholm. The groundwater fulvic acid (FA-G) was isolated from Gide~, approx. 300 km north of Stockholm (recovered from groundwater sampled in a 107-m deep borehole in granitic rock). The techniques for isolation and purification of these samples (based on adsorption on DEAE-cellulose at neutral pH in the first step) are described in detail elsewhere [14].
Characterization of the Humic Samples A sequence of reactions (methylation - - hydrolysis - - esterification) was performed, starting with the natural humic substances (denoted as XCOOH), in the following steps: O
(i)
x
//O
/~/\
+ CI{,N, . . 01t
~'
X
/~x,x
+ N2 0
CH 3
CARBOXYLCONTENTIN HUMICSUBSTANCES
215
O
(ii)
x
+ NaOlt CH3
~x~,,,o
•
X
O (iii)
H3C
+C1t3Ot] O-
Na+
O
~
+ CH30H
I~
H3C~ ~ , , ~
O/CH3
+ HCI
CI
(i) Methylation The methylating agent diazomethane (CH2N2) was obtained by reacting 372 mg potassium hydroxide in 2 ml ethanol with 1.42 g N-methyl-N-nitrosop-toluene-sulfonamide dissolved in 5 ml diethyl ether at 40°C. The diazomethane was trapped in a flask immersed in an ice bath and the ether solution was distilled twice. The fulvic acid sample (4-16 mg), as well as a blank, was dissolved in 5 ml N,N-dimethylformamide (DMF) and treated with 0.8 ml diethyl ether solution of CH2N2 at ambient temperature. After 24 h, when the yellow colour in the blank sample had vanished, the solvent was vacuum-rotary evaporated at 60°C.
(ii) Hydrolysis To the methylated fulvic samples, 300/~1 of 2.5 M sodium hydroxide solution and 5 #1 of buthyl acetate (internal standard) were added. The sample was transferred to a glass ampoule, sealed and heated at 100°C for 12 h.
(iii) Esterification To the hydrolysed fulvic sample in a round bottomed flask fitted with a condenser, 1.5 ml of propionyl chloride was added cautiously, with shaking; the flask was kept in an ice-water bath to prevent a violent reaction. After 24 h at ambient temperature, 10 ml of dichloromethane (CH2C12) was added and the solution was washed twice with 15 ml of 1 M NaOH, and once with 15 ml distilled water. The remaining water was freezed out at -20°C. The same washing procedure was performed on a standard sample consisting of 10 ml CH2C12 mixed with 5 /zl methyl propionate and 5 #1 buthyl propionate.
(iv) Gas Chromatography The organic extract was analysed by gas chromatography using the following equipment: Hewlett Packard 5890 with flame ionization detector (FID), attenuation 5; crosslinked methyl silicone column (25 m × 0.31 mm i.d.)
216
i. A R S E N I E E T AL.
DB1 (J and W) 0.57/~m; helium carrier gas at 35-40 cm/s flow-rate; split injection; injected volume 1.5 /zl; temperature program 30°C for 2 min. and then raised at 10°C/min to 125°C. RESULTS
Optimization of the Reaction Steps The whole sequence of reactions was tested on simple substances. To optimize the conditions of each step using one aromatic compound (2,4-dihydroxy benzoic acid) and one aliphatic compound (stearic acid) were used as model substances for humic materials. The linearity between the methyl propionate formed and different internal standards was tested by gas chromatography. After optimization of the different steps the correlation coefficient for the methyl propionate/internal standard ratio and the initial amount of model acid was 0.999. Furthermore, a few milliequivalents of a methyl ester was quantified by the reaction sequence within 5% of the correct value. Most of the experimental error was due to the uncertainty of the electronic integrator.
Characterization of Natural Humic Samples The possible presence of methyl esters in the native fulvic samples was checked by following the reaction sequence but omitting the methylation step. Under these conditions, by comparing with the results obtained for a blank sample (only solvent, DMF, and no methylation step) the amount of methyl propionate formed was negligible. Consequently the methyl propionate found after the complete sequence and after subtraction of the blank sample was due to methyl esters formed from carboxylic groups of the fulvic sample. In a similar analysis for butyl esters, without added butyl acetate (internal standard) no butyl propionate was found. The results of these analyses justify the interpretation that the methyl propionate obtained in the ordinary analysis corresponds to carboxyl groups of the humic sample and that butyl acetate is an acceptable internal standard. A typical chromatogram is displayed in Fig. 1. The amounts of carboxyl groups in the sample was estimated by comparing the area quotient methyl propionate/butyl propionate, with that of a corresponding reference pair, taking into account that the butyl propionate in the sample is derived from 5 ~1 butyl acetate. The results obtained for the two fulvic samples analyzed are shown in Table 1. These results (the mean value in each serie) correspond to 81-88% of the amount of acidic groups in the same materials, as obtained by potentiometric titration to pH 7-8 (for FA-B: 4.78 meq/g, for FA-G: 5.33 meq/g [15]).
217
C A R B O X Y L C O N T E N T IN H U M I C S U B S T A N C E S
1
2
Fig. 1. Analysis of methyl propionate generated from a methylated humic sample. 1: Methyl propionate; 2: Butyl propionate (internal standard).
DISCUSSION
The techniques most frequently used for characterization of acidic functional groups are potentiometric titrations and NMR-spectroscopy. Although the various methods all claim to analyse carboxylic groups they use in fact different and sometimes indirect characteristics for their analysis. Only groups that will undergo methylation by diazomethane and subsequent base catalyzed hydrolysis will be identified as carboxyl groups by the present method. Very few non-carboxylic groups will be able to react in this manner and consequently the technique will give a more specific estimate of the carboxyl content.
218
i. A R S E N I E ET AL,
TABLE 1 Estimated amounts of carboxyl groups in the fulvic samples Fulvic acids sample
Butyl prop. (mg)
meq COOH/g
FA-Ba
4.1 7.3 10.2 15.0
4.57 3.69 3.35 3.66
4.4 7.5 15.5
3.83 4.96 3.82
4.20
8.0 11. l 14.6
3.73 4.23 3.74
3.90
7.4 9.5 12.1
4.52 4.14 4.56
4.38
FA-B~
FA-B
a
FA-G
Mean value
3.81
aThree different runs
The carboxyl content was calculated with respect to the internal standard, butyl propionate (formed from butyl acetate). The values obtained are somewhat spread (S.D. 0.485, for FA-B) and this could be due to the fact that buthyl propionate (which generate buthyl acetate) is participating in many steps of the procedure and the risk for losses is high. The results obtained for F A - G and FA-B are similar in the respect that the carboxyl content was only approximately 80-90°/,, of the total acidity calculated from the potentiometric titration. The deviation between the derivatization and titration results could, in principle, be due to incomplete reaction steps in the derivatization sequence, alternatives which will be discussed below.
(i) Methylation with Diazomethane Incomplete methylation of the fulvic samples could be a possible source of error for the estimation. However, repeated methylations did not increase the amount of methyl propionate analysed, thus nullifying the assumption that methylation was not complete.
CARBOXYL CONTENT IN HUMIC SUBSTANCES
219
(ii) Saponification The saponification of the methyl esters represents the most fundamental step of the reaction sequence. The hydrolysis of the internal standard (buthyl acetate) was not affected by the presence of humic compounds. The same reaction tested on model substances always ran quantitatively. Consequently, this step should not be incomplete.
(iii) Quantification of the Liberated Methanol The methanol formed in the saponification step was analysed as the corresponding ester of propionic acid. This reaction was quantitative in the absence of humics. There is no reason to believe that methanol or methyl propionate would complex with the humics present since the corresponding compounds of the internal standard, butanol or butyl propionate, were adequately quantified from the same solutions. The liberated methanol was correctly quantified. Another explanation to the difference in results from the two methods might be that the titration includes not only carboxylic groups but also phenolic or other acidic groups with pKa-values within the pH range of the titration. Thus the derivatization technique offers a possibility to distinguish a minor fraction of acidic groups other than the carboxylic ones.
CONCLUSION
The development of more specific methods for the characterization of humic substances is essential for a better understanding of their structural elements. The present methylation-saponification sequence constitutes a method to quantify carboxylic groups in humic substances based on a specific chemical reaction. Therefore the technique would give apparent values for the carboxyl content in humic substances that are below the levels indicated by less specific techniques, which set an upper limit. However, the reason for the deviation between results from potentiometric titration and the present method should be further studied and the origin of the additional acidity obtained from the titration should be identified. ACKNOWLEDGEMENT
We are indebted to the Swedish Nuclear Fuel and Waste Management Co for their financial support.
220
~ ARSENIE ET AL.
REFERENCES 1 G.M. Woodwell, R.H. Whittaker, W.A. Reiners, G.E. Likens, C.C. Delwiche and D.B. Botkin, The biota and world carbon budget. Science, 199 (1987) 141-146. 2 M. Schnitzer, The chemistry of humic substances, in J. Nriagu (Ed.), Environmental Biochemistry, Second Int. Symp. Environ. Biogeochem., Canada, 1976, pp. 89-107. 3 R.F.C. Mantoura, A. Dickson and J.P. Riley, The complexation of metals with humic materials in natural waters. Estuar. Coast. Mar. Sci., 6(4) (1978) 88-107. 4 G.E. Carlberg and K. Martinsen, Adsorption/Complexation of organic micropollutants to aquatic humus. Influence of aquatic humus with time on organic micropollutants and comparison of two analytical methods for analysing organic pollutants in humus water. Sci. Total Environ., 25 (1982) 387-408. 5 E.M. Thurman, Organic Geochemistry of Natural Water. M. Nijhoffand W. Junk Publ., Dordrecht, 1985, pp. 273-280. 6 C. Steinberg and U. Muenster, Geochemistry and ecological role of humic substances in lakewater, in G.R. Aiken, D.M. McKnight, R.L. Wershaw and P. MacCarthy (Eds.), Humic Substances in Soil, Sediment and Water. John Wiley and Sons Inc., New York, 1985, pp. 105-130. 7 G.R. Harvey and D.A. Boran, Geochemistry of humic substances in seawater, in G.R. Aiken, D.M. McKnight, R.L. Wershaw and P. MacCarthy (Eds.), Humic Substances in Soil, Sediment and Water. John Wiley and Sons Inc., New York, 1985, pp. 233-247. 8 B.A. Dempsey and C.R. O'Melia, Proton and calcium complexation of four fulvic acid fractions, in R.F. Christman and E.T. Gjessing (Eds.), Aquatic and Terrestrial Humic Materials. Ann Arbor Science, Michigan, 1983, pp. 239-272. 9 D.S. Gamble, Potentiometric titration of fulvic acid. Equivalence point calculations and acidic functional groups. Can. J. Chem., 50(16) (1972) 2680-2690. 10 P.G. Hatcher, R. Rowan and M.A. Mattingly, IH and ~3C-NMR of marine humic acids. Org. Geochem., 2(2) (1980) 77-85. 11 P.G. Hatcher, I.A. Breger, L.W. Dennis and G.E. Maciel, Solid state L3C-NMR of sedimentary humic substances: new revelations of their chemical composition, in R.F. Christman and E.T. Gjessing (Eds.), Aquatic and Terrestrial Humic Materials. Ann Arbor Science, Michigan, 1983, pp. 37-81. 12 Y. Chen, N. Senesi and M. Schnitzer, Information provided on humic substances by E4/E 6 ratio. Soil Sci. Soc. Am. J., 41 (1977) 352-358. 13 T.I. Noyes and J.A. Leenheer, Proton Nuclear-Magnetic-Resonance of Fulvic Acid from Suwannee River, in Humic Substances in the Suwannee River, Georgia: Interactions, Properties, and Proposed Structure; U.S. Geological Survey, Open-File Report 87-557, Denver, Colorado, 1989, pp. 231-250. 14 C. Pettersson, I. Arsenie, J. Ephraim, H. Boren and B. Allard, Properties of fulvic acids from deep groundwaters. Sci. Total Environ., 81/82 (1989) 287-296. 15 J. Ephraim, H. Bor6n, I. Arsenie, C. Pettersson and B. Allard, A combination of acidbase titrations and derivatization for functional group determinations of an aquatic fulvic acid. Sci. Total Environ., 81/82 (1989) 615-624.
213
Determination of the carboxyl content in humic substances by methylation I. Arsenie, H. Boren and B. Allard Department of Water and Environmental Studies, Link6ping University, S-581 83 LinkOping, Sweden (Received January 24th, 1991; accepted April 25th, 1991) ABSTRACT The carboxyl content of different fulvic acids was estimated by means o f a methylation technique. The procedure involved methylation by diazomethane followed by base catalyzed hydrolysis, esterification with propionyl chloride and gas chromatographic analysis of the methyl ester formed. After optimization of each step, the whole sequence of reactions was performed using only a few milligrams of starting material. The method was applied to two fulvic acid samples of different origin (surface water and groundwater, respectively). The carboxylic content was estimated to about 3.5 meq/g material for the surface water fulvic acid and about 4.5 meq/g material for the groundwater fulvic acid. This estimates approximately 80% of the total content of acidic groups obtained for the same materials by using potentiometric titration.
Key words. humic acid; fulvic acid; carboxylic acid; derivatization; methylation; saponification
INTRODUCTION
Humic materials represent one of the largest sources of natural organic carbon on earth [1]. They are formed by chemical and biological degradation of plant and animal residues and by synthetic activities of microorganisms [2]. The increased interest in the chemistry of the humic compounds in the last decade is based on the understanding of the important role of humic materials in the natural environment. The ability of humics to form both water-soluble and water-insoluble complexes with other chemical species as well as their capacity to reduce some metal ions make them play a crucial role in the speciation, transport and deposition of a variety of compounds ranging from metal ions to lipophilic organics [3,4]. Due to a continuous leaching from the soil and also a possible formation in aqueous systems, humic and fulvic acids may be found in all kinds of fresh 0048-9697/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved
214
i. ARSENIE ET AL.
waters, typically at concentrations from tens of mg/l in bog waters to fractions of mg/1 in deep ground waters [5]. Minor amounts of fresh water humics may also be produced autochthonously [6]. The origin of humic compounds in the marine environment is still a matter of controversy [7]. Structurally, humic substances comprise a heterogeneous group of organic compounds. Their building blocks are believed to be a mixture of aromatic rings and aliphatic chains. The predominant functional groups responsible for reactivity, complexation ability and solubility of humic compounds are carboxyls, alcohols and phenols. Common analytical techniques used to study the structure of humic compounds are various kinds of titration [8,9], spectroscopic techniques such as 1H- and 13C-NMR [10,11] and chemical degradation followed by analysis by GC/MS [12]. Chemical derivatization has been utilized to a less extent in such studies, and only in combination with spectroscopic techniques [13]. This paper describes the quantitative determination of the carboxylic component of two fulvic acids by a method that is more specific than most techniques utilized so far. The stages of the method are methylation, saponification and subsequent gas chromatographic analysis of the methanol formed as a methyl ester. EXPERIMENTAL
Recovery of Humic Substances Surface water fulvic acid (FA-B) was isolated from a bog area at Bersbo, approx. 200 km south of Stockholm. The groundwater fulvic acid (FA-G) was isolated from Gide~, approx. 300 km north of Stockholm (recovered from groundwater sampled in a 107-m deep borehole in granitic rock). The techniques for isolation and purification of these samples (based on adsorption on DEAE-cellulose at neutral pH in the first step) are described in detail elsewhere [14].
Characterization of the Humic Samples A sequence of reactions (methylation - - hydrolysis - - esterification) was performed, starting with the natural humic substances (denoted as XCOOH), in the following steps: O
(i)
x
//O
/~/\
+ CI{,N, . . 01t
~'
X
/~x,x
+ N2 0
CH 3
CARBOXYLCONTENTIN HUMICSUBSTANCES
215
O
(ii)
x
+ NaOlt CH3
~x~,,,o
•
X
O (iii)
H3C
+C1t3Ot] O-
Na+
O
~
+ CH30H
I~
H3C~ ~ , , ~
O/CH3
+ HCI
CI
(i) Methylation The methylating agent diazomethane (CH2N2) was obtained by reacting 372 mg potassium hydroxide in 2 ml ethanol with 1.42 g N-methyl-N-nitrosop-toluene-sulfonamide dissolved in 5 ml diethyl ether at 40°C. The diazomethane was trapped in a flask immersed in an ice bath and the ether solution was distilled twice. The fulvic acid sample (4-16 mg), as well as a blank, was dissolved in 5 ml N,N-dimethylformamide (DMF) and treated with 0.8 ml diethyl ether solution of CH2N2 at ambient temperature. After 24 h, when the yellow colour in the blank sample had vanished, the solvent was vacuum-rotary evaporated at 60°C.
(ii) Hydrolysis To the methylated fulvic samples, 300/~1 of 2.5 M sodium hydroxide solution and 5 #1 of buthyl acetate (internal standard) were added. The sample was transferred to a glass ampoule, sealed and heated at 100°C for 12 h.
(iii) Esterification To the hydrolysed fulvic sample in a round bottomed flask fitted with a condenser, 1.5 ml of propionyl chloride was added cautiously, with shaking; the flask was kept in an ice-water bath to prevent a violent reaction. After 24 h at ambient temperature, 10 ml of dichloromethane (CH2C12) was added and the solution was washed twice with 15 ml of 1 M NaOH, and once with 15 ml distilled water. The remaining water was freezed out at -20°C. The same washing procedure was performed on a standard sample consisting of 10 ml CH2C12 mixed with 5 /zl methyl propionate and 5 #1 buthyl propionate.
(iv) Gas Chromatography The organic extract was analysed by gas chromatography using the following equipment: Hewlett Packard 5890 with flame ionization detector (FID), attenuation 5; crosslinked methyl silicone column (25 m × 0.31 mm i.d.)
216
i. A R S E N I E E T AL.
DB1 (J and W) 0.57/~m; helium carrier gas at 35-40 cm/s flow-rate; split injection; injected volume 1.5 /zl; temperature program 30°C for 2 min. and then raised at 10°C/min to 125°C. RESULTS
Optimization of the Reaction Steps The whole sequence of reactions was tested on simple substances. To optimize the conditions of each step using one aromatic compound (2,4-dihydroxy benzoic acid) and one aliphatic compound (stearic acid) were used as model substances for humic materials. The linearity between the methyl propionate formed and different internal standards was tested by gas chromatography. After optimization of the different steps the correlation coefficient for the methyl propionate/internal standard ratio and the initial amount of model acid was 0.999. Furthermore, a few milliequivalents of a methyl ester was quantified by the reaction sequence within 5% of the correct value. Most of the experimental error was due to the uncertainty of the electronic integrator.
Characterization of Natural Humic Samples The possible presence of methyl esters in the native fulvic samples was checked by following the reaction sequence but omitting the methylation step. Under these conditions, by comparing with the results obtained for a blank sample (only solvent, DMF, and no methylation step) the amount of methyl propionate formed was negligible. Consequently the methyl propionate found after the complete sequence and after subtraction of the blank sample was due to methyl esters formed from carboxylic groups of the fulvic sample. In a similar analysis for butyl esters, without added butyl acetate (internal standard) no butyl propionate was found. The results of these analyses justify the interpretation that the methyl propionate obtained in the ordinary analysis corresponds to carboxyl groups of the humic sample and that butyl acetate is an acceptable internal standard. A typical chromatogram is displayed in Fig. 1. The amounts of carboxyl groups in the sample was estimated by comparing the area quotient methyl propionate/butyl propionate, with that of a corresponding reference pair, taking into account that the butyl propionate in the sample is derived from 5 ~1 butyl acetate. The results obtained for the two fulvic samples analyzed are shown in Table 1. These results (the mean value in each serie) correspond to 81-88% of the amount of acidic groups in the same materials, as obtained by potentiometric titration to pH 7-8 (for FA-B: 4.78 meq/g, for FA-G: 5.33 meq/g [15]).
217
C A R B O X Y L C O N T E N T IN H U M I C S U B S T A N C E S
1
2
Fig. 1. Analysis of methyl propionate generated from a methylated humic sample. 1: Methyl propionate; 2: Butyl propionate (internal standard).
DISCUSSION
The techniques most frequently used for characterization of acidic functional groups are potentiometric titrations and NMR-spectroscopy. Although the various methods all claim to analyse carboxylic groups they use in fact different and sometimes indirect characteristics for their analysis. Only groups that will undergo methylation by diazomethane and subsequent base catalyzed hydrolysis will be identified as carboxyl groups by the present method. Very few non-carboxylic groups will be able to react in this manner and consequently the technique will give a more specific estimate of the carboxyl content.
218
i. A R S E N I E ET AL,
TABLE 1 Estimated amounts of carboxyl groups in the fulvic samples Fulvic acids sample
Butyl prop. (mg)
meq COOH/g
FA-Ba
4.1 7.3 10.2 15.0
4.57 3.69 3.35 3.66
4.4 7.5 15.5
3.83 4.96 3.82
4.20
8.0 11. l 14.6
3.73 4.23 3.74
3.90
7.4 9.5 12.1
4.52 4.14 4.56
4.38
FA-B~
FA-B
a
FA-G
Mean value
3.81
aThree different runs
The carboxyl content was calculated with respect to the internal standard, butyl propionate (formed from butyl acetate). The values obtained are somewhat spread (S.D. 0.485, for FA-B) and this could be due to the fact that buthyl propionate (which generate buthyl acetate) is participating in many steps of the procedure and the risk for losses is high. The results obtained for F A - G and FA-B are similar in the respect that the carboxyl content was only approximately 80-90°/,, of the total acidity calculated from the potentiometric titration. The deviation between the derivatization and titration results could, in principle, be due to incomplete reaction steps in the derivatization sequence, alternatives which will be discussed below.
(i) Methylation with Diazomethane Incomplete methylation of the fulvic samples could be a possible source of error for the estimation. However, repeated methylations did not increase the amount of methyl propionate analysed, thus nullifying the assumption that methylation was not complete.
CARBOXYL CONTENT IN HUMIC SUBSTANCES
219
(ii) Saponification The saponification of the methyl esters represents the most fundamental step of the reaction sequence. The hydrolysis of the internal standard (buthyl acetate) was not affected by the presence of humic compounds. The same reaction tested on model substances always ran quantitatively. Consequently, this step should not be incomplete.
(iii) Quantification of the Liberated Methanol The methanol formed in the saponification step was analysed as the corresponding ester of propionic acid. This reaction was quantitative in the absence of humics. There is no reason to believe that methanol or methyl propionate would complex with the humics present since the corresponding compounds of the internal standard, butanol or butyl propionate, were adequately quantified from the same solutions. The liberated methanol was correctly quantified. Another explanation to the difference in results from the two methods might be that the titration includes not only carboxylic groups but also phenolic or other acidic groups with pKa-values within the pH range of the titration. Thus the derivatization technique offers a possibility to distinguish a minor fraction of acidic groups other than the carboxylic ones.
CONCLUSION
The development of more specific methods for the characterization of humic substances is essential for a better understanding of their structural elements. The present methylation-saponification sequence constitutes a method to quantify carboxylic groups in humic substances based on a specific chemical reaction. Therefore the technique would give apparent values for the carboxyl content in humic substances that are below the levels indicated by less specific techniques, which set an upper limit. However, the reason for the deviation between results from potentiometric titration and the present method should be further studied and the origin of the additional acidity obtained from the titration should be identified. ACKNOWLEDGEMENT
We are indebted to the Swedish Nuclear Fuel and Waste Management Co for their financial support.
220
~ ARSENIE ET AL.
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