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Boletín de la Sociedad Chilena de Química

versión impresa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.44 n.4 Concepción dic. 1999 



1 Department of Natural Resources, Faculty of Agricultural Scienes, University National of
Cordoba, CC 509-5000-Cordoba, Argentina.
2Department of Analytical Chemistry, Faculty of Sciences, Masaryk University, Kotlàrskà 2,
61137 Brno. Czech Republic.
(Received: Januanry 19, 1999 - Accepted: July 5, 1999)


Humic substances (HS) have a great importance in regard to the quality and productivity of a soil, and in the retention of the metal ions and pollutants by the environment. The characterization of humic compounds provides important information to evaluate the interaction of HS metal ions and with different classes of organic compounds (e.g., herbicides and pesticides). In order to understand HS behavior, it is necessary to separate this complex organic matter into fractions, and determine the structure of each compound in the fractions obtained.

Numerous studies applying many different analytical techniques have been used for this purpose. In particular, the application of capillary zone electrophoresis (CZE) for the characterization of HS has evoked an increased interest.

The aim of this work is to study the CZE behavior of HA from a different origin. The HA of Argentinean soils and also the HA from compost of urban wastes will be studied and compared to the reference and standard samples of the international Humic Substances Society (IHSS) in DL-alanine as background electrolyte (BGE).

The IHSS HA has electropherograms similar to those of Argentinean HA. Additionally, a CZE method developed here can be used to follow the maturation process in the composting of urban wastes.

KEY WORDS: Humic substances, capillary zone electrophoresis, fingerprints, separation.


Las sustancias húmicas (SH) tienen gran importancia en la calidad y productividad de los suelos, retención de iones metálicos y contaminantes del medio ambiente. La caracterización de las sustancias húmicas (SH) provee importante información para la evaluación de su capacidad de interacción con iones metálicos y diferentes clases de compuestos orgánicos (herbicidas y pesticidas).

Para entender su comportamiento es necesario realizar su separación en fracciones y determinar su posible estructura.

Numerosos estudios aplicando diferentes técnicas analíticas han sido realizados con este propósito. La aplicación de la Electroforesis Capilar para la caracterización de las SH ha cobrado gran interés.

Se trabajó con ácidos húmicos (AH) extraídos de suelos de diferentes zonas climáticas de la Argentina, con AH extraídos de compost de residuos sólidos urbanos (RSU) y con HA standard y referencias de la IHSS.

El principal objetivo de este trabajo es estudiar el comportamiento de AH de diferentes orígenes mediante la Electroforesis Capilar, utilizando DL Alanina como electrolito soporte.

Los patrones electroforéticos de los AH estudiados son similares. Los AH de IHSS utilizados como standard y referencia tienen patrones electroforéticos similares a los HA de los suelos estudiados.

La electroforesis capilar puede ser utilizada para el seguimiento del proceso de maduración de los compost de RSU.

PALABRAS CLAVES: Sustancias húmicas, electroforesis capilar, fingerprints, separación.

*To whom correspondence should be addressed.


Organic matter contributes to plant growth through its effect on the physical, chemical, and biological properties of the soil. They have (i) a nutritional function and serve as a source of N, P, and S for plant growth, (ii) a biological function that deeply affects the activities of microflora and microfaunal organisms, and (iii) a physical function that promotes good soil structure, thereby aeration and retention of soil moisture. Humic substances (HS) play an indirect role in soils through their effect on the uptake of micronutrients by plants and the performance of herbicides and other agricultural chemicals.

HS have a great importance to the quality and productivity of a soil and, at the same time, in the retention of the metal ions and pollutants by the environment. Dissolved humic materials have a tendency to bind and to complex metal ions or pollutants and thus may alter the fate and transfer of them in the soil1,2). Also, humic substances play an important role in the speciation and mobility of the metals in the environment. The abundance of oxygen, nitrogen and sulfur containing functional groups in these substances make HS efficient metal complexing ligands3).

Humic Acids (HA) are organic macromolecules exhibiting a wide variety of molecular-mass (Mr) distribution, substructures and functional groups. These compounds are of high interest for study of environmental systems because they have considerable influence on the bioavailability of toxic elements, which is a result of their high complexation capability4).

The characterization of HA has been the focus of intense research for many years because soil orgainic matter contributes to the quality of soil more than the other soil constituents. Nowadays, one of the main goal in humus science is the separation of humic substances into fractions. These fractions can then be studied independently with the aim to elucidate HS structure, is a problem that still remains unresolved in spite of the enormous efforts that have been devoted to and the remarkable progress has been achieved recently in this field.

The characterization of humic compounds provides important information to evaluate the interaction of HS with metal ions and with different classes of organic compounds (e.g., herbicides and pesticides)5). The high structural complexity and the wide range of molecular masses of HS has lead to the application of various techniques and methods to obtain more information about these compounds6).

HS show structural complexity and polyelectrolyte properties. For more fully understand HS behavior, it is necessary to first separate the complex organic matter into fractions, and then to determine the structure of each compound in the fractions obtained. Numerous studies applying many different analytical techniques have been used for this purpose and much valuable knowledge has been gained3,7). However, there arestill a significant amount of problems to be solved.

Recently, the application of capillary zone electrophoresis (CZE) for the characterization of HS has evoked an incresed interest8-13). The potential of CZE as a method to characterize/fingerprint variety of humic substances (fulvic and humic acids), reflecting their charge-to-mass ratios and their state of dispersity, has been reported10,11). The application of the CZE to study the intraction between HS and metal ions has also recently received some attention12,13).

The fractionation and characterization of HA have been studied in recent years using chromatography14), spectroscopy15), electrophoretic techniques such as isoelectric focusing in a polyacrylamide gel16), and capillary isotachophoresis8). Electrophoretic methods were applied for the purpose of humic substances already in the 1960's 17-19) without much success.

For the determination of HA directly following the extraction process, only two new methods have been applied recently. The first is based on high performance liquid chromatography (HPLC) with fluorimetric detection14), while the second uses capillary zone electrophoresis (CZE) with UV/vis detection of the HA content in soil samples after a single extraction-precipitation-solubilization steps4,9).

Rigol et al.4) observed that a commercial and natural HA (the latter having been obtained by extraction from a soil sample) may be separated into a set of Mr ranges with a given membrane cut-off. All the fractions had the same electropherogram patterns when applying a CZE separation method. The lack of dependence of the electropherogram on the Mr of the HA and the equal electrophoretic response for all the Mr fractions permits each fraction to be quantified by an ultra-filtration-CZE method. The non-ultrafiltrated HA samples from Fluka is used as a reference.

Nordén et al.5) observed differences in migration behavior between FA and HA using the same buffer composition. Fetsch et al.20) showed that CZE is the most powerful tool applied so far for the separation and characterization of humic substances. They successfully obtained the ionic and/or polyelectrolyte properties, as well as an excellent separation of up to 25-30 fractions.

It has been known from the literature for a long time that humic substances undergo aggregation in solution6,18,19). This property was described by Von Wandruszka et al.21) as pseudomicelles, by

Wershaw 22,23) and Chien et al.24) as humic membrane-micelle, by Dachs et al.25) as fractal aggregates, by Schvchenko et al.26) as polymers and finally as oligomers27-29). Buffle et al.27) noted that humates are observed to be very heterogeneous and they are charged polymers.

Recently, aggregation properties of HS were studied by CZE by Fetsch et al.17,28-30). An oligomerization process of HS was observed during their CZE separation when the effect of the HS concentration on CZE separation patterns was studied28).

The aim of this work is to study the CZE behavior of HA from a different origin. The HA of Argentinean soils and also the HA from compost of urban wastes will be studied and compared to the reference and standard sample of the International Humic Substances Society. As carboxylic31) or amino acids based9,32) BGE (alanine buffer, especially) were found to be suitable for obtaining fingerprints of HA and for oligomerization studies, we have selected DL-alanine as the background electrolyte for the present study.



All reagents were of analytical grade purity. Mesityl oxide (MSO) used as a neutral marker for electro-osmotic flow (EOF) determination was from Fluka (Buchs, Switzerland). NaOH and HCl were from Carlo Erba (Roma, Italy) and Merck (Darmstadt, Germany), respectively. Deionized water used to prepare all solutions was double-distilled from a quartz apparatus made by Heraeus Quartzschmelze (Hanau, Germany).

Humic acids

The HA samples used in this work were:

1) HA extracted from Argentinean soils from the Province of Cordoba (Argentine) located in two different climatic zones: semiarid zone and arid zone.

HA were extracted from soils of different culture and are denoted as: a) "Grama", b) "Pastizal", c) "Algarrobo" and d) "Desmonte". Grama and Pastizal samples from a semiarid zone, while Algarrobo and Desmonte were from an arid zone.

2) HA extracted from compost waste urban residual of two cities from the Province of Cordoba, Argentina: La Para and Oncativo.

Composting was performed in piles, mixing solid urban waste and a low content of vegetal materials, in the open air with occasional turning every 10 or 15 days. In La Para the composting procedure was the same but mixing solid urban waste, vegetal materials, manure and a high content of soil (@ 40-50%).

The Oncativo sample presents the particularity to have been extracted after two periods of compostage: 6 or 14 months. Moreover, fractions of HA obtained in the compost of 6 months of compostage were collected as: a) first extraction, b) second extraction (performed on the sample after the first extraction of HA).

The HA obtained are denominated: HA "La Para", HA "Oncativo 14", HA "Oncativo 6(1st)" (first extraction) and HA "Oncativo 6 (2nd)" (second extraction).

The extraction of the humic acid (HA) fraction from soil and compost was performed with NaOH 0.1 M, purified with HCl:HF (1:3) and finally dried at low temperature, according to the procedure recommended by Chen et al., 1987.

Characteristics of these Argentinean HA are described elsewhere33,34).

3) Samples of the International Humic Substances Society (IHSS) denoted as "Leonardite standard", "Peat reference" and "Peat standard".

Procedure of humic acid dissolution

Stock solutions of HA were prepared by weighing a given amount, dissolving it in a small volume of 0.1 M NaOH, and diluting to a fixed volume using double-distilled water.


The pH value of the background electrolyte (BGE) was adjusted with diluted aqueous solutions of NaOH and/or HCl. Solution of BGE were prepared daily. BGE solutions were kept in a freezer in order to prevent their decomposition by the action of fungus. Because HA are interacting strongly with most of othe organic compounds35-37), antifungal compounds were not added either to BGE or HA solutions.


A Spectaphoresis 2000 of Thermo Bioanalysis Corporation (San José, CA, USA) unit, equipped with a fast scanning detector with deuterium and tungsten lamps working from 190 to 700 nm, was used. Untreated fused-silica capillaries with an inner diameter (I.D.) 75 µm (Avery Dennison, MA, USA), total capillary length L 43.6 cm, (length to the detector l 35.6 cm) were housed in a cartridge. Samples were injected using a hydrodynamic injection by applying a vacuum (10342.14 Pa).

The electropherograms were recorded and data reprocessed by PC1000 software of Thermo Bioanalysis Corporation (San José, CA, USA) to obtain peak parameters namely: migration time, peak height, peak area, theoretical number of plates, symmetry factor, and others.

The pH was measured using a glass G202C electrode, standard calomel electrode K401 of Radiometer (Copenhagen, Denmark) and a Precision Digital pH-meter OP-208/1 of Radelkis (Budapest, Hungary) while standard buffer solutions of Radiometer (Copenhagen, Denmark) and/or Radelkis (Budapest, Hungary) were used for the calibration.


The capillary was washed every day first with the BGE solution which was degassed for 10 min. Furthermore, it was washed 5 min with 0.1 M NaOH at 25°C, 10 min with deionized water at a temperature gradient from 25 to 40°C and 5 min with BGE at 40°C. The capillary inlet vials were replenished after each injection. In each analysis a pre-wash for 2 min with 0.1 M NaOH, 2 min with deionized water, 1 min with 0.1 M NaOH and 1 min with deionized water at 40°C was applied. Separation of HA was done at 40°C and hydrodynamic injection time was 30 s if not otherwise mentioned. The separation potential value was optimized for BGE.


Reproducibility of the CZE analysis (migration time, peak area, etc.) was determined repeating each measurement 3 to 5 times and estimating the standard deviation (RSD) of the parameters using standard formula30).


Argentinean soil HA

Figures 1a and 1b show the electropherograms of Pastizal and Desmonte HA at 10, 100, 200, 400 and 650 ppm. For both samples the process of oligomerization of HA when increasing HA concentration injected at constant injection time can be observed as proposed previously by Fetsch et al.28). It shows that the electropherogram pattern of HA can be obtained only after a critical micellar concentration is reached. This strongly suggests that the successful separation patterns are due to the oligomers formed in solution.

FIG. 1a. Capillary zone electrophoresis separation of oligomers of Pastizal HA observed when increasing HA concentration. HA concentration; 10; 100; 200; 400 and 650 ppm. Fused-silica capillary: L = 43.5 cm (l = 35.5 cm) x 75 µ m I.D. BGE: 60 mM DL-Alanine adjusted with HCl, pH = 3.20. Separation conditions: +15 kV, 40°C, 220 nm, 30 s hydrodynamic injection.

FIG. 1b. Capillary zone electrophoresis separation of oligomers of Desmonte HA observed when increasing HA concentration. HA concentration; 10; 100; 200; 400 and 650 ppm. Fused-silica capillary: L = 43.5 cm (l = 35.5 cm) x 75 µ m I.D. BGE: 60 mM DL-Alanine adjusted with HCl, pH = 3.20. Separation conditions: +15 kV, 40°C, 220 nm, 30 s hydrodynamic injection

Figure 2 shows the electropherograms patterns of Argentinean soil HAs, where some similarities and differences can be observed. All electropherograms show three fractions or peaks (A, B and C), and sometimes some small peaks between the last two peaks (B and C) can be observed. The shape of peak A is quite similar for Grama and Desmonte HA (Fig. 2d and 2b), and for Pastizal and Algarrobo (Fig. 2c and 2a), but some differences in the shape (namely, a shoulder) of the A peaks between both groups (Grama-Desmonte and Pastizal-Algarrobo) are observed.

Desmonte and Algarrobo HA (arid zone extracts) present a peak C that is more intensive than that observed for Pastizal and Grama HA (semiarid zone extracts). This, indicates that the former two have more quantity of this fraction than the latter two. The electropherograms of Pastizal and Grama HA (Figure 2c and 2d) show differences in peak C intensity and in migration times. Similar results were obtained for Algarrobo and Desmonte HA (Figure 2a and 2b) . These differences observed in the electropherograms indicate some differences in their charge/mass ratio. Furthermore, the absorbance of peak C is much lower for Algarrobo than for Desmonte HA, indicting differences in this fraction concentration.

FIG. 2. Electrophoregram fingerprints of different Argentinean soil HA. Humic Acids: a) Algarrobo, b) Desmonte, c) Pastizal, d) Grama, HA concentration: 650 ppm. Capillary, BGE and condition are the same as in Figure 1.

We suggest that these differences are due to either the climatic zone of the origin of these HA studied or to the different vegetal materials that are present in these regions or the soil characteristics. This is in agreement with properties studied for these HA by Ceppi et al.33).

Compost HA

The electropherograms of HA extracted from compost of urban waste are shown in Figure 3. The primary organic materials (wastes) from which HA La Para and Oncativo were produced are completely different. Accordingly, some similarities and differences are observed in the electrophoretic patterns, of their HA extracts.

The first peak present in La Para HA is of a lower intensity than in Oncativo HA (Figures 3a and 3b), which indicates a lower concentration of this fraction. The second peak is similar for both HA. Furthermore, the observed electropherograms (Figure 3) show a third peak taht differ significantly in migration time. This value of migration time is higher for HA Oncativo (Fig. 3b), indicating the presence of compounds with higher charge or lower molecular weight than those presented in La Para HA (Fig. 3a). Finally, between the second and the third peak, small peaks are observed in Oncativo HA (Fig. 3b), which probably represent other compounds not present in La Para HA (Fig. 3a).

FIG. 3. Electrophoregram fingerprint of HA extracted from compost. Humic Acids: La Para, Oncativo 14 month, Oncativo 6 month (1st), Oncativo 6 month (2nd). HA concentration: 650 ppm. Capillary, BGE and condition are the same as Figure 1.

Comparing Figures 3b and 3c, the effect of compostage time can be observed on the electropherograms. Both figures are similar to each other with respect to the first two peaks, also shown in Figures 3a and 3b. Concerning the other peaks, however, changes in migration times were observed. Specifically, increasing the time of compostage, decreased the migration times of some of the peaks. This is specially important for the last most intensive peak whose migration time decreased of about two minutes (Figures 3b and 3c).

The effect of extraction can also be followed. Figure 3d shows the electropherogram of HA obtained after a new extraction of the sample of 6 months compostage age and from which HA were already once extracted before (Figure 3c). For this sample significant differences can be observed, (i.e., in the number and the absorbance intensities of the peaks). The decrease of the absorbance is mainly observed for the first two peaks at 1.9 and 2.3 min, which means that these two fractions are in lower concentration than those observed for the first extraction (Fig. 3c). It is particularly occurring for the previously most intensive peak (peak C) observed in the electropherogram, which completely disappeared in Figure 3d. Thus, it means that the procedure of HA extraction used is very efficient with some of its constituents and only one extraction might be enough to reach a high recovery. The recovery with one extraction is not perfect, as can be seen with the two new small peaks with migration times 7.8 and 9.1 min (Fig. 3d).

Comparing the electrophoregrams in Figure 3 with Figure 2, it appears that the HA extracted from compost have similar structure to that extracted from soil, but with a different and limited humification process.

International Humic Substances Society HA

Figure 4 shows the electropherograms of the IHSS HA studied. All electropherograms also present a pattern with three peaks or fractions (A, B, C). Comparing Leonardite standard (Fig. 4a) with Peat reference HA (Fig. 4b) diferences are agian observed in the intensities of the peaks. The area below peaks A and C is more significant in the case of the Peat reference (Fig. 4b), which is indicating a major concentration of those fractions. Peak B is the main fraction, indicating a high concentration of this fraction in Peat reference HA (Fig. 4b). Peat standard HA (Fig. 4c) presents a similar electropherogram as the Peat reference HA but at higher values for the migration times. Finally, the Leonardite standard HA (Fig. 4a) shows sharper peaks with several small negative peaks, whose formation is presently difficult to explain.

FIG. 4. Electrophoregrams fingerprint of International Humic Substances Society HA. Humic Acids: Leonardite Standard, Peat References and Peat Standard. HA concentration: 650 ppm. Capillary, BGE and condition are the same as Figure 1.


It was shown that the electrophoretic patterns of soil HA are similar because they all had three major peaks or fractions. The differences in intensities are due presumably to the diferent humification conditions (e.g. climate, vegetal materials, soil characteristics). The IHSS HA reference or standard materials have electropherograms similar to those HAs extracted from Argentinean soils with an organic matter content lower than 3% and HA content in the organic matter higher than 10%.

The differences in absorbance of the peaks and migration times show evidence of a different composition in the fractions studied. This method can be used for obtaining fingerprints of HA where evidently different oligomers are being separated.

The electrophoretic patterns of HA extracted from compost show some similarities with those extracted from soil indicating similar composition, however its humification processes are different.

Furthermore, the capillary zone electrophoresis method developed can be used successfully to follow the maturation process of urban wastes compost for further agricultural utilization.


This work is a part of a research program within BARRANDE Czech-French (Czech Ministry of Education - French Ministry of Foreign Affairs) bilateral scientific co-operation project N 96006. Czech Ministry of Education (Prague, Czech Republic), French Ministry of Foreign Affairs (Paris, France) and the General Council of Haut-Rhin (Colmar, France) are thanked for their support to D. Fetsch. Masaryk University (Brno, Czech Republic) is thanked for a post-doctoral sojourn award to S.B. Ceppi and M.I. Velasco. The secretary of the Science and Technology of the National University of Cordoba (SeCyT-UNC, Argentina), the Cordoba Provincial Council of the Science and Techology (CONICOR, Argentina) and the National Council of Scientific and Technological Investigations (CONICET-Argentina) are also acknowledged.


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