SciELO - Scientific Electronic Library Online

 
vol.50 número3SYNTHESIS OF CONDENSATION POLYMERS DERIVED FROM SILYLATED MONOMERS UNDER PHASE TRANSFER CATALYSIS índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

Links relacionados

Compartir


Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.50 n.3 Concepción sep. 2005

http://dx.doi.org/10.4067/S0717-97072005000300001 

 

J. Chil. Chem. Soc., 50, N° 3 (2005), págs: 527-533

 

CHARACTERIZATION OF HUMIC ACIDS ORIGINATED FROM ARGENTINEAN COMPOST AND SOIL BY CAPILLARY ELECTROPHORESIS AND MASS SPECTROMETRY.

 

CEPPI, S. B.1*, M. I. VELASCO1, P. A. CAMPITELLI1, E.M. PEÑA-MÉNDEZ2 and J. HAVEL3

1Faculty of Agronomy Science, National University of Cordoba, CC 509 CP 5000 Cordoba, Argentina
2Department of Analytical Chemistry, Nutrition and Food Science, University of La Laguna, Campus de Anchieta, 38071 La Laguna, Tenerife, Spain
3Department of Analytical Chemistry, Faculty of Science, Masaryk University, Kotlárská 2, 611 37 - Brno, Czech Republic

E-mail address: sceppi@agro.uncor.edu


ABSTRACT

Humic acids (HAs) extracted from different organic compost and soil from Argentina have been characterized by capillary electrophoresis (CE) and mass spectrometry. The chemical properties of HAs show changes depending on their origin (compost, soil). Capillary electrophoresis analyses were carried out using a background electrolyte consisting of boric acid, TRIS and EDTA (BTE). BTE was also modified with cyclodextrins. After optimization of BTE, the best results were obtained when b-cyclodextrin was used. Under optimized separation conditions, the characteristics CE fingerprints of compost derived HAs (CHAs) were studied. CE fingerprints show differences in the composition of the different CHAs. Mass spectra obtained by laser desorption ionization time of flight mass spectrometry show differences between the CHAs and HAs extracted from Argentinean soil. A comparison with the soil standard HAs (International Humic Substances Society) was made. CHAs show mostly compounds in the mass spectra with low m/z values. The typical patterns exhibited in soil HAs of different origin are present in the Argentinean soil HA, i.e. groups of peaks around mass-to-charge (m/z) 800.

Keywords: Compost; Humic acids; Capillary electrophoresis; cyclodextrins modified buffer; mass spectrometry; fingerprints


INTRODUCTION

At present, the production of organic and inorganic wastes in the modern world is increasing, while the soils loose organic matter through intensive and extensive agriculture uses and adverse weather conditions (1). Organic matter (OM) content in soils strongly affects their fertility, increasing the plant nutrients, enhancing the soil structure and the water-holding capacity, and acting as a sink phase for xenobiotics (toxic organic compounds and heavy metals) in the soil environment. The recycling of organic wastes is a necessity and matter of high interest in the modern world. The recycling of organic wastes from urban and/or agricultural origin after composting usually gives an organic rich material. It has been proposed that this organic rich material (when composted to maturity) could produce a high quality soil amendment, which can be used for agricultural purposes supplying large quantities of humic substances (HS) and humic-like substances to the soil (2, 3).

Different organic fractions can be distinguished in composting organic materials: extractable humic acids (HAs), the so-called "humic like substances", other compounds that are bound to mineral surfaces, microbial biomass, and among others, non-degradable organic matter. In comparison with HAs extracted from the soil, the compost HA (CHAs) is formed in a relatively short time period. Therefore, it is necessary to carefully study and understand the physico-chemical transformations of the humic substances originated during composting. These substances ("humic-like substances") resemble partially native soil humic acids and are instruments to obtain a reduced environmental impact and successful agronomic performance of the compost in soil (4, 5).

Several methods have been proposed to evaluate the quality of the organic matter during the composting process (5-7). However, the application of such methods has not proved to be reliable, because it is not able to distinguish between a raw and a stabilized organic material (1).

Different methods have been proposed to study separation, characterization and behavior of HAs of different origin Polyacrylamide Gel Electrophoresis (PAGE) was used by Trubetskoj et al. (8, 9) for the comparative analysis of soils from different origins. Recently, Trubetskaya et al., (1) have used coupling size exclusion chromatography-page (SEC-PAGE) for the evaluation of the transformation of organic matter to humic substances in compost. Special attention is paid to the capillary zone electrophoresis (CZE), mostly with UV­Vis, diode array, or laser-induced fluorescence detection; while as background electrolytes (BGE) usually borate, phosphate or a mixture of both are used (10). An extensive overview of the various background electrolytes can be found in the literature (11, 12). Pokorná et al., (13) and Gajdosová et al., (14) have studied the influence of substances as cyclodextrins (CDs) on the HA separation by CE. CDs consist of cyclic oligosaccharides composed of D-glucose units connected to a ring with glycosidic bonds. This arrangement of the D-glucopyranose units forms a bucket (''cone-shaped''), consisting of 6, 7, or 8 glucose rings fused to form a, b, or g-cyclodextrins, respectively. These torus-shaped molecules have relatively hydrophobic interiors and hydrophilic exteriors with primary hydroxyl groups located on the smaller end of the torus, the secondary hydroxyl groups being located on the larger end (15, 16). CDs have been selected not only for their influence on the HA separation, but also due to the possibility of CDs interactions with different xenobiotics (17, 18). Despite the great effort applied to improve the CZE separation of HAs, there is still a lack of reproducibility and a need for better CZE methods.

Characterization of HA using various soft ionization mass spectrometric techniques has been shown as a complex problem (14, 19,20). Among the soft ionization mass spectrometric techniques, the use of laser desorption ionization mass spectrometry (LDI TOF MS) has not been reported for the characterization of compost derived HA, as far as we know.

The main goal is to characterize humic substances obtained from compost materials using CZE and LDI TOF mass spectroscopy in order to increase knowledge about "humic-like substances" extracted from compost. Results obtained were compared with those found with HAs extracted from an Argentinean soil and standard soil HAs (IHSS).

EXPERIMENTAL

MATERIALS

Compost and Soil Characteristics

The samples of compost used to extract their HAs were taken from composting plants located in Argentina. Table 1 presents the sampling location and the raw materials of each compost sample used to extract the compost humic acids (CHAs). Sampling was made at the end of the stabilization process according to the composting time informed in Table 1.


Soil HAs were extracted from an Entic Haplustol located in the semiarid region (Province of Cordoba, Argentine) with Gramma Rhodes as vegetal coverage.

Humic Acids

The HAs samples used in this work were: HAs extracted from i) different compost, ii) Argentinean soil (HAs Gramma) and iii) HAs from the International Humic Substances Society 1S102 (IHSS) (soil HAs standard).

Humic acids obtained from compost (CHAs) and from soil were extracted with NaOH solution 0.1 M, purified with HCL:HF (1:3) and finally dried at low temperature, according to the procedure recommended by Chen et al.,(21).

All reagents were of analytical grade purity. Sodium hydroxide and phosphoric acid were from Merck (Darmstadt, Germany), hydrochloric acid, monosodium phosphate and sodium acetate were purchased from Lachema (Brno, Czech Republic). Sodium tetraborate salt was purchased from Lachema (Brno, Czech Republic). Boric acid, 1,1-tris-(hydroxymethyl) amino methane (Tris), ethylene-diaminetetraacetic acid (EDTA). Cyclodextrins (a - b and g) were from Sigma­Aldrich (Steinheim, Germany). Deionized water used to prepare all solutions was double-distilled in the quartz apparatus of Heraeus Quartzschmelze (Hanau, Germany).

Methods

Elemental analyses of all HAs studied were determined using the Perkin Elmer 2400 CHNS/O analyzer. The analyses were made in triplicate. The ash content was obtained by burning a certain amount of HA in an oven at 900o C for 6 h.

Optical densities of soil and compost HAs solution (3 mg of HA in 10 ml 0.05N NaHCO3) were measured at wavelengths, 465 and 665 nm., respectively, on a Spectronic 20 Genesys. These values were used for the calculation of the E4/E6 coefficient value (22).

A Beckman CZE (Model P/ACE) System 5500 (Palo Alto, CA, USA) equipped with the diode array detection (DAD) system, an automatic injector, a fluid-cooled column cartridge and the System Gold Data station were utilized for all CZE experiments. A fused-silica capillary tubing of 37 cm (29.7 cm length to the detector), 75 mm I.D. was used. The normal polarity mode of the CZE system (cathodic pole at the side of detection) was applied.

Laser Desorption/Ionization Mass Spectrometry measurements were carried out using the AXIMA CFR of Kratos Analytical (Manchester, United Kingdom) mass spectrometer, equipped with the nitrogen laser wavelength of 337 nm operated in the linear and positive ion mode. Energy of the laser was changed in the range 0-180 relative units. The volume of 1 ml of sample solution (100 mg l-1 HA) was pipetted onto a sample plate and dried at room temperature in an air stream. External calibration with the monomer ions of molecular ions [M+H]+ of a-cyano-4-hydroxycinnamic acid and bradykinin were used. Each mass spectrometric analysis was performed using at least 500 shots and the obtained data were accumulated.

Electrophoretic Procedure

At the beginning of daily work, the capillary was washed for 5 min with 0.1 M NaOH solution, followed by 5 min washing with tri-distilled water and 5 min with background electrolyte (BGE) at 25oC. In this work the BGE used were: 50 mM sodium tetraborate; 50 mM sodium phosphate; 20 mM sodium acetate and a mixed BGE containing 115 mM boric acid, 95 mM Tris, and 0.06 mM EDTA.

In each analysis the capillary was first washed for 5 min with deionized water and for 5 min with BGE. The 25oC temperature was found to be optimal. Hydrodynamic injection of samples was used. The absorbance was monitored at 214 nm and 260 nm. Previous to the analysis, the HA solution was filtered through a 0.45-m filter Spartan®-3 of Schleicher & Schüll (Dassel, Germany) and degassed in an ultrasonic bath from Branson (Shelton, USA) for 10 min.

Mass Spectrometry Procedure

LDI TOF mass spectra were measured in various modes depending on the sample analyzed (linear positive, linear negative or reflectron positive and negative modes), but generally, the spectra obtained with more signals were those done in the linear positive mode.

Results and Discussion

Basic Characteristics of Humic Acids

Elemental analysis for the CHAs was carried out and the data are given in Table 2. Carbon to nitrogen ratio (C/N) for CHAs samples varies in the range from 9.3 to 19.9. In the case of Soil Standard HA (IHSS), the C/N was 16.4, while HAs Gramma presented a C/N value of 22.3, which is higher than that present in the different HAs under study. This is probably due to the vegetal materials that are in the soil from which HAs were extracted. It also follows from Table 2 that the average nitrogen content is slightly high in CHAs samples, with the only exemption of CHAs labeled as Sorghum. From the hydrogen and oxygen content, expressed as percentage, it was observed that for all HAs the values range is: H (%) 3.7-4.9, O (%) 34.1-38.6. The hydrogen, oxygen, carbon, and nitrogen percentage in CHAs samples are similar to those reported by Miiki et al. (23).


The absorbance in the wavelengths range 460 - 480 nm reflects the organic material present in the sample at the beginning of the humification process, while, the absorbance in the range 600 - 670 nm is related to a strongly humified material (24). The ratio E4/E6 was calculated; this ratio is related to the humification characteristics and in this way, to the degree of condensation of the aromatic C network of HAs. Table 2 presents the values obtained for the HAs under study. Zbytniewski et al., (25) suggested that an increase in ratio E4/E6 indicates that the humification process is not complete and matured compost differs from organic matter present in soils. The E4/E6 values suggest that CHAs have a more aliphatic structure and low molecular weight than those HAs extracted from soil. This fact is probably due to the limited and incomplete period of humification that takes place in the composting process, originating the so-called "humic-like substances", because they are humic acids in neoformation.

Capillary Zone Electrophoresis

Several parameters for the characterization of CHAs and soil HAs samples by capillary zone electrophoresis (CZE) were studied. Background electrolytes (BGE) such as sodium acetate (pH 4.7), sodium phosphate (pH 9.6), sodium phosphate (pH 6.5), sodium tetraborate (pH 9.6), and BTE (Boric acid-Tris-EDTA pH 8.6) were examined. When sodium acetate, sodium phosphate and sodium tetraborate were used no good separation was achieved. For either CHAs or soil HAs samples, the most expressive and reproducible electropherograms were obtained under the following conditions: potential 20 kV, temperature 25oC, injection time 3 s and HAs concentration 100 mg l-1 and BTE as BGE. Figure 1 gives an example of the separation pattern obtained during the electrophoretic separation. Each CHAs shows similar patterns and some characteristic peaks. The electropherograms present the typical "unresolved hump", where some individual not sufficiently separated peaks are also observed. For migration time 12-25 min, no significant peaks were observed.


 
Figure 1: Electropherograms of HAs from different origin (compost and soil HAs) obtained with BTE buffer (115 mM boric acid + 95 mM TRIS + 0.6 mM EDTA pH=9.6). CZE conditions: separation voltage: 20 kV; hydrodynamic injection 3 sec; detection at 214 nm; Total length: 37 cm; length to the detector: 29.7 cm; 75 µm ID

Great differences between electropherograms corresponding to CHAs and soil HAs are observed in Figure 1. The obtained electropherograms may be considered as fingerprint of the humic substances. Different fingerprints are shown in their patterns, being an evidence of a difference in their structures. These differences may be explained considering the compost "like-humic acid" or HAs in neoformation as a mixture of several single compounds, with low degree of condensation, high content of aliphatic fractions and low content of aromatic rings. Humic substances originated during the composting process (CHAs) show colloidal properties and may probably form supra-molecular aggregates (25). The formation of supra-molecular aggregates implies the transformation of low molecular organic compounds to humic-like substances. These humic-like substances present a high molecular weight, and are responsible of the "humps", which can be observed in the eletrophoretic patterns (fingerprints) of CHAs from Argentina (26).

Effects of cyclodextrins (CDs) on the separation.

The effects of cyclodextrins for improving the separation of HAs during the electrophoretic procedure were also studied. The HAs solutions were spiked with different levels of a, b, or g-cyclodextrins. Then, the mixture of HAs + CDs were injected into the capillary and analyzed.

Figure 2 shows an example of the electropherograms obtained when different additions of a-cyclodextrins were applied to CHAs. After the addition of CDs to HAs solutions, a substantial effect was found. It can be seen in Figure 2, that even the addition of low concentration of a-CDs produces a rather high shift (for migration times 10-25 min, no significant peaks were observed).


 

Figure 2: Electropherograms of CHAs (Sorghum) obtained with CHAs solution spiked with a-CD. Buffer used, as BGE was BTE. Separation conditions are the same as given in Fig 1

The main group of peaks (tm from 5 to 10 min) shift to lower migration time (tm) (3 to 5 min). When b-CDs or g-CDs were added to the HAs solutions, a similar behavior was found out for soil HAs (Gramma and Soil standard) and CHAs (INTA, Tarsu, La Para).

The decrease of the tm values can be explained by the formation of a supra-molecular complex, an inclusion complex, between CDs and HAs components (low-molecular-mass fractions):

HAs + CD Û {HAs, CD}

It leads to increase the molecular weight of HAs and, thus, to decrease of anion mobility. Because CE separation is counterelectroosmotic (EOF gives to negative electrode at the side of the detector), a decrease in the mobility of HAs charged negatively leads to shorter migration time.

However, the classical unresolved "hump" is still present in the electropherograms. Therefore, we modified the BGE with a-, b- and g-cyclodextrins. The anionic CDs were used in the range 10 to 80 mM. The results for CDs modified BTE show that addition of b-CDs to the BGE improves the separation patterns, while no substantial effect has been found out when a- or g-CDs were used. Figure 3 shows the electropherograms obtained for CHAs samples (Sorghum, Tarsu, and INTA) and Soil standards HA (IHSS) with BTE modified with 10 mM b-CDs. The effect of the modified BGE on the separation of HAs was followed. A better resolution of the "humps" in the electropherograms of soil standard HAs (IHSS) was observed in comparison with those presented by CHAs samples. Also, CHAs present a lower number of fractions than soil standard HAs (IHSS). This difference is probably due to the short and incomplete period of humification of the composted materials. In addition, the components present in CHAs structure are aggregating because of the aliphatic chains and, thus, they form a supramolecule that migrates independently, without separation of fraction as in soil standard HAs (26).


 
Figure 3: Electropherograms of AHs from different origin (compost and IHSS AHs) in BTE modified with 10 mM b-CD. Separation conditions are the same as given in Fig 1 (for curves INTA, Tarsu and Sorghum absorbance values were multiplied by the factor of 7) (for migration time 5-25 min, no significant peaks were observed).

Mass Spectrometry

On the basis of the studies conducted by Gajdosová et al., (14); Peña-Méndez et al., (27), the laser desoption/ionization time of flight mass spectrometry (LDI TOF MS) was applied to characterize the CHA after a carefully optimization of the parameters affecting the LDI measurements. A comparison of the mass spectra obtained for CHAs (INTA and Sorghum) and soil HAs (IHSS) is present in Figure 4 a,b. From the mass spectra it can be observed that there are some common peaks (features) present in CHAs samples and soil HA (IHSS).


 

Figure 4 a, b: Comparison of LDI TOF MS of AHs extracted from compost (INTA, Sorghum) with IHSS derived HAs (Soil IHSS) in the region 100-500 Dalton (a) and 500-1000 Dalton (b)

The mass spectra of soil HAs (IHSS) and Gramma HAs show a relative higher number of peaks in comparison with CHAs samples (Figure 5 a,b). Peaks at m/z values 104, 160, 368.4 are present in the mass spectra of CHAs and, also, in the soil HAs (IHSS) mass spectra. The mass spectra of two different HAs extracted from soil, soil HAs (IHSS) and soil HAs (Gramma), not only present common peaks in the mass spectra (m/z 105.0, 160.1, 165.1, 278.9, 305.0, 368.4, 396.4) but also the pattern exhibit in the m/z range between 800 and 900 is present for both (Figure 5b). The presence of such patterns has also been described by Havel et al. (19) Gajdosová et al. (14) and Peña-Méndez et al. (27) in the mass spectra of HAs extracted from different soils of different origin. The greatest differences between CHAs and HAs extracted from soil (soil standard HAs and Gramma HAs) were found in the region 500<m/z<1000.


 
Figure 5 a, b: Comparison of LDI TOF MS of HAs derived from the Argentinean soil (Gramma) and IHSS derived HAs (Soil IHSS) in the region 100-500 Dalton (a) and 500-1000 Dalton (b)

The characteristics commented above suggest that CHAs are a mixture of very simple compounds with low molecular weight, as we can see through their high E4/E6 values (Table 2). CHAs are formed within a short period of time with an incomplete and inefficient humification process. In this way, probably, low molecular weight compounds are present in these HAs in neoformation, as shown by mass spectra. These characteristics of CHAs should explain their higher possibility to form supramolecular aggregates than soil HAs and, in this way, understand the differences observed in the electropherograms.

CONCLUSIONS

Capillary zone electrophoresis is a practical method for the separation and characterization of humic acids extracted from compost. In this paper several BGE for the separation of CHAs by CZE were studied. It was found that the best separation conditions were obtained when a BGE (Borate, Tris and EDTA, BTE) was modified with b-CD. In this experimental condition well reproducible electropherograms were recorded and it showed better performance for the characterization of the CHAs by CZE. The CHAs showed lower values of molecular weight components than those in soil HAs (IHSS) or extracted from Argentinean soil (HAs Gramma).

LDI mass spectrometry has proved to be a suitable technique to characterize and observe the differences and similarities between soil HAs and CHAs. The structure of humic acids is very complex and it is reflected in the mass spectra. Even if CHAs and HAs extracted from soil represent different stages in the humification process, some common features are present in them. This fact is clearly shown in the mass spectra of the HAs studied and may be indicative of the presence of common structural facts. Argentinean soil HAs (Gramma) present in their mass spectra patterns in the region of m/z values between 700 and 800 described in humic acids from different origin (peat, coal derived) and country and/or continent (Central America, Europe and Antarctica). Such pattern was not found in CHAs.

 

References

1 Trubetskaya, O.E., Trubetskoj, O.A., Ciavatta, C. Biores. Technol. 77 (2001) 51-56.         [ Links ]

2 Atiyeh, R.M., Lee, S., Edwards, C.A., Arancon, N.Q., Metzger, J.D.,. Technology 84 (2002) 7-14.         [ Links ]

3 Campitelli, P. A., Velasco, M. I., Ceppi, S. B. J. Chl. Chem. Soc 48 (3) (2003) 91-96         [ Links ]

4 Romero, L., Velasco, M. and Ceppi, S. Bol. Soc. Chil. Quim. 44 (1999)139-146         [ Links ]

5 Veeken, A., Nierop, K., de Wilde, V., Hamelers, B. Biores. Technol. 72 (2000) 33-41         [ Links ]

6 Senesi, N., Brunetti, G. Chemical and Physico-chemical Parameters for Quality Evaluation of Humic Substances Produced during Composting. In: The Science of Composting. London - Glasgow - Weinheim - New York - Tokyo ­ Melbourn ­ Madras. 195-212, 1996.         [ Links ]

7 Sánchez-Cortés, S., Francioso, O., Ciavatta, C., García-Ramos J. V., Gessa , C. J. Col. Int. Sci. 198(2) (1998) 308-318.         [ Links ]8 Trubetskoj, O.A., Trubtskaya, O.E., Afanas'eva, V., Reznikova, O.I., Sainz-Jimenez, C., J of Chromatography A 767 (1-2) (1997) 285-292         [ Links ]

9 Trubtskoj, O.A., Trubtskaya, O.E., Reznikova, O.I., Afanas'eva, V. Geoderma 93. (1999) 277-286.         [ Links ]

10 Fetsch, D., Albrecht-Gary, A.M., Peña-Méndez, E.M., Havel, J. A review. Scripta Fac. Sci. Nat. Univ. Masaryk Brun. 27 (1998a) 3-26.         [ Links ]

11 Havel, J., Fetsch, D. Humic Substances/Capillary Zone Electrophoresis. Monograph Separation Science. 3018-3025. London, UK, 2000         [ Links ]

12 Schmitt-Kopplin, P., Junkers, J. 2003. J. Chromatogr. A 998 (2003) 1­20.         [ Links ]

13 Pokorná, L., Pacheco, M.L., Havel, J. J. Chromatogr. A 895 (2000), 345-350.         [ Links ]

14 Gajdosová, D., Novotná, K., Prosek, P., Havel, J. J. Cromatogr. A 1014 (2003), 117-127.         [ Links ]

15 Lamparczyk, H., Zarzycki, P., Ochocka, R.J., Sybilska, D. J. Chromatogr. 30 (1990) 91­94.         [ Links ]

16 Wang, X. and Brusseau, M.L. Environ. Sci. Technol. 29 (1995), 2632­2635.         [ Links ]

17 Ishiwata, S., Kimiya, M. Chemosphere 38 (1999), 2219-2226.         [ Links ]

18 Lindsey, M.E., Xu, G., Lu, J., Tarr, M.A. Sci. Total Environm. 307 (2003) 215 ­229.         [ Links ]

19 Havel J., Fetsch D., Peña-Méndez E. M., Lubal P., Havlis J. Recent Developments in Humic Acid Characterization by CE and MALDI-TOF Mass spectrometry. In Understanding and Managing Organic Matter in Soils, Sediments and Waters (Swift R.S. and Spark K.M., Eds.) IHSS, Australia, 77-82, 2001.         [ Links ]

20 Mugo S.M and C.S. Bottaro. Rapid. Commun. Mass Spectrom. 18 (2003) 2375-2382.         [ Links ]

21 Chen, V., Senesi, N. F., Schnitzer, M. Geoderma 20 (1978) 87-104.         [ Links ]

22 Schnitzer, M. and Khan S.U. Humic Substances in the Environment. Marcel Dekker, New York, 1972         [ Links ]

23 Miiki, V., Senesi, N., Hänninen, K. Chemosphere 34 (1997) 1639-1651.         [ Links ]

24 Gieguzynska, E., Kocmit, A., Golebiewska, D. Studies on Humic Acids in Eroded Soils of Western Pomerania. In: Zaujec, A., Bielek, P., Gonet, S.S. (Eds), Humic Substances in Ecosystems, Slovak Agricultural University, Nitra, pp. 35-41,1998.         [ Links ]

25 Zbytniewski, R., Kosobucki, P., Kowalkowski, T., Buszewski, B. Environ. Sci. Pollut. Res. 1 (2002) 68-74.         [ Links ]

26 Fetsch, D., Hradilová, M., Peña-Méndez, E.M., Havel, J. J. Chromatogr. A, 817 (1998b) 313-323.         [ Links ]

27 Peña-Méndez, E.M., Gajdosová, D., Novotná, K., Prosek, P., Havel, J., Talanta (2005) (In print).         [ Links ]

 

ACKNOWLEDGEMENTS

This work was supported by The Ministry of Education, Youth and Sports, MSM 143100007 and by the Grant Agency of the Czech Republic, Project No. 203/02/1103, Czech Republic.

Ceppi and Velasco thank SeCyT UNC, Cordoba Science Agency and CONICET for the financial support.

Peña-Méndez would like to thank to Dirección General de Universidades of Canarian Government (Canary Islands, Spain) and Masaryk University (Czech Republic) for the financial support .

 

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons