The present study evaluates the possibility of eliminating the purification steps involved in the characterization of HA by capillary zone electrophoresis (CZE). The HAs of various sources were analyzed, showing different electropherograms by CZE, which depend on the charge and size of HA. The data suggest that the purification of the sample is not necessary to characterize HAs. Based on the results, CZE showed to be a promising tool to characterize HA of different origins without the purification step of the sample.Neste estudo, avaliou-se a possibilidade de eliminação das etapas de purificação envolvidas na caracterização de HA. HA de diferentes fontes foram analisados por CZE apresentando diferentes eletroferogramas. Essas diferenças observadas mostram que a caracterização do HA por CZE depende da carga e do tamanho do HA. Os resultados apresentados neste trabalho sugerem que não é necessária a purificação da amostra para a caracterização de HA por CZE. Baseados nos resultados, a CZE mostrou-se uma ferramenta promissora para caracterização de HA de diferentes origens sem a etapa de purificação da amostra.CNPqFAPES

MORAES, Solange L. de

REZENDE, Maria Olímpia O.

Biblioteca Digital da Produção Intelectual da Universidade de São Paulo (BDPI/USP)

Ar
ti
cl
e
J. Braz. Chem. Soc., Vol. 19, No. 1, 24-28, 2008.
Printed in Brazil - ©2008  Sociedade Brasileira de Química
0103 - 5053  $6.00+0.00
*e-mail: soso@fcfrp.usp.br
Capillary Electrophoresis (CE): a Powerful Tool to Characterize Humic Acid (HA)
Solange L. de Moraes* and Maria Olímpia O. Rezende
Instituto de Química de São Carlos, Universidade de São Paulo, CP 780, 13560-970 São Carlos-SP, Brazil
Neste estudo, avaliou-se a possibilidade de eliminação das etapas de purificação envolvidas na 
caracterização de HA. HA de diferentes fontes foram analisados por CZE apresentando diferentes 
eletroferogramas. Essas diferenças observadas mostram que a caracterização do HA por CZE 
depende da carga e do tamanho do HA. Os resultados apresentados neste trabalho sugerem que 
não é necessária a purificação da amostra para a caracterização de HA por CZE. Baseados nos 
resultados, a CZE mostrou-se uma ferramenta promissora para caracterização de HA de diferentes 
origens sem a etapa de purificação da amostra.
The present study evaluates the possibility of eliminating the purification steps involved in 
the characterization of HA by capillary zone electrophoresis (CZE). The HAs of various sources 
were analyzed, showing different electropherograms by CZE, which depend on the charge and 
size of HA. The data suggest that the purification of the sample is not necessary to characterize 
HAs. Based on the results, CZE showed to be a promising tool to characterize HA of different 
origins without the purification step of the sample.
Keywords: humic acid, characterization, capillary zone electrophoresis 
Introduction
Humic substances (HS) have an important role in the 
environment. They are related to the fertility of the soils, 
contributing to the increasing the harvest production.1,2
In soil, HS are implicated in water-holding capacity, 
pH buffering, ion exchange, binding of metal ions and 
hydrophobic compounds.3-6 These substances are classified 
into three groups, humic acids (HAs), fulvic acids (FAs) 
and humins, on the basis of their solubility in acidic and 
basic solutions.7-9
HAs present relatively high molecular mass 
polyelectrolytes containing aromatic, aliphatic and 
heterocyclic subunits.10 Their chemical structure is 
less known due to the origin and the natural conditions 
prevailing during their formation.11The degree of ionization 
of these macromolecules is governed by the amount of 
ionized phenolic and carboxylic groups of HAs, which is 
a function of solution pH.12-14
As a modern analytical technique, capillary 
electrophoresis (CE) has become an attractive method 
for the characterization of molecules with high structural 
complexity and wide range of molecular weights, such as 
HA.15-18 The CE, as a method of characterizing a variety 
of HS, is suitable due to its polyelectrolyte properties,
reflecting its charge-to-mass ratio.19-26 The characterization 
of humic compounds provides important information for the 
evaluation of their capacity of interaction with metal ions 
and different classes of organic compounds as pesticides.25
Some valuable features of this analytical technique are short 
analysis time, selectivity for the analytes, simple analytical 
procedure, and micro-quantity consumption of samples. In 
CZE, the introduction of samples is carried out at the anode, 
while the detection is realized at the cathode. Ionic species 
are separated based on their charge and size, which are 
functions of the pH value of the electrolyte solution.27
The aim of this work was to characterize HAs samples 
of different origins by CZE, considering each step of 
purification.
Experimental
Chemicals
All reagents used were of analytical grade acquired from 
Merck or Aldrich. Deionized water obtained by Millipore 
system was used to prepare all solutions, standards and 
electrolytes.
Moraes and Rezende 25Vol. 19, No. 1, 2008
Apparatus
CE experiments were performed on a Hewlett Packard 
3D Capillary Electrophoresis equipped with a diode 
array detector (DAD). Uncoated fused silica capillary 
(50 mm id., 56 cm length from the detector, 64.5 cm total 
length) was obtained from Hewlett Packard. The samples 
were injected by pressure (50 mbar × 10 s), and a voltage 
of 20 kV was applied at a temperature of 30 °C. The signals 
were recorded with UV-Vis detection at 214 nm. The mode 
electrophoresis used for all experiments was CZE. 
Before starting the analyses or between determinations 
using different electrolytes, the capillary was flushed with 
1 mol L-1 NaOH for 5 min, then with deionized water for 
10 min and with the used electrolyte for 15 min. Between 
each run, the capillary was flushed with water for 2 min,
followed by a flush with the running electrolyte for 
3 min.
The electrolytes utilized were 20 mmol L-1 borate 
buffer pH 9.3.
Humic acids
The samples evaluated in this work were: HAs extracted 
from peat (PHA), 10-25 cm depth from Mogi-Guaçu 
River margin, São Paulo State, Brazil, HAs obtained from 
vermicompost (VHA) originated from animal manure and 
treated with Eisenia foetida or Lumbriculus rubellus, and 
a commercial HA obtained from Aldrich (AHA), lot N0 
05924 KN.
Extraction and purification of the sample
The extraction and purification of HA samples from 
peat and vermicompost were performed according to the 
International Humic Substance Society (IHSS) adapted 
method,24,28 based on acid and basic extractions followed 
by purification with successive elution through cationic and 
anionic resins. The collected fractions were: non-purified 
HA (fraction 1), HA after Cl– removal (fraction 2) and 
HA after purification through cationic and anionic resins 
(fraction 3). Each collected fraction was liofilized and 
stored for further analysis by CZE. Figure 1 illustrates the 
described procedure.
To perform CZE analyses the HA-containing solutions 
were prepared by dissolving 1.0 mg of HA in a small 
volume of 0.1mol L-1 NaOH solution (approximately two 
drops) and diluting up to the fixed volume of 1 mL with 
deionized water (Millipore system). An ultrasonic bath 
was applied for 3-5 min to enhance dispersion (Branson 
ultrasonic).
The effect of organic matter was determined through 
the values of ash for each fraction 1, 2 and 3 (Figure 1). 
The values of ash were obtained by ashing the sample at 
600 °C for 5h. 
Results and Discussion
In this work, all the purification steps involving HA 
samples were evaluated. Firstly, the extraction of sample 
with NaOH was performed. Next, the HA samples was 
flushed several times to eliminate chloride ions originated 
from the HCl solution used to separate humic and 
fulvic acids. Then, HA was exhaustively eluted through 
exchange columns to remove cations and anions present
in the samples. This step is very long and depending on 
the sample, it may last several weeks. In this context, the 
possibility of working with HA without purification was 
investigated, considering that the distribution patterns for 
HA of different origins could be compared if the separation 
were carried out under the same conditions. Figure 2 shows 
the electropherograms of PHA in borate buffer considering 
the purification steps of the sample.
If two HA samples exhibit the same behavior in an 
electric field, then they should have a comparable charge-
to-mass ratio and the migration time will be a distinctive 
criterion if the molecular weight is unknown.19 However, 
we tried to compare the electrophoretic behavior of different 
fractions of HA samples (non-purified, after Cl– removal 
and purified) by evaluating the migration times. 
Figure 1. Scheme of the fractions of HA collected: (fraction 1) 
non-purified, (fraction 2) after Cl– removal, and (fraction 3) purified. 
Capillary Electrophoresis (CE): a Powerful Tool to Characterize Humic Acid (HA) J. Braz. Chem. Soc.26
The degree of ionization of these macromolecules is 
known to be governed by the number of ionized phenolic 
and carboxylic groups of the humic core, which is a 
function of the pH solution. In this case, we observed 
superimposed peaks as an unresolved broad shoulder in all 
electropherograms, which is assigned to the anionic region 
of the HA. No individual component can be distinguished 
under the present experimental conditions. At pH 9.3, the 
negative charges carried by the analytes are maximized and 
the acid groups carry all the negative charges. According to 
several authors,18-26 this region is attributed to homogeneous 
“humic hump” in the anionic region with a distribution 
around an average electrophoretic mobility (AEM). 
The shapes of the broad bands that appear for PHA 
are quite similar, Figure 2, but the most intense peak was 
attributed to the purified PHA (Figure 2c). It is reasonable 
to assume that for more purified samples, the number 
of negative charges that they carry is larger. However, 
it is useful to ratify that the region of interest of the 
eletropherogram (broad band known as “humic hump”) 
presents basically the same behavior for the purified HA 
(Figure 2c), non-purified case (Figure 2a) and after Cl–
removal (Figure 2b). It is worthwhile to notice that after 
the Cl– removal, the “humic hump” region increased in 
intensity, in comparison to the non-purified sample. The 
same procedure was repeated for other HA samples, as 
shown in Figures 3 and 4.
Figure 3 presents the electropherograms obtained for 
purified and non-purified AHA. It is important to point out 
that AHA was obtained commercially and the purified AHA 
was made only by ionic resins. Again, the same behavior 
was observed in the PHA case: the broad bands are similar. 
However, the most intense peak is observed in the purified 
sample. The broad forms of the peaks and the CE profiles 
are quite similar, denoting that these shapes are due to the 
acid groups with a similar structure. 
These lower peaks of the “humic hump” of the non-
purified PHA sample can be attributed to the presence of 
several components, metals for instance, associated with 
the humic structure. In this case, the molecule of HA would 
not be completely dissociated, resulting in a minor amount 
of HA by UV detection. 
Figure 4 presents the electropherograms for the VHA 
fractions. It is interesting to observe that after chloride 
removal (Figure 4b), the VHA presents almost the same 
profile of the “humic hump” (Figure 4c), but with a minor 
intensity. 
We evaluated the influence of inorganic matter (ash) on 
each fraction analysed by CZE. Table 1 presents the ash 
content obtained for different HA samples.
Figure 2. Electropherogram of PHA (1.0 mg mL-1): (a) non-purified, 
(b) after Cl– removal (c) purified. Conditions: 20 mmol L-1 borate buffer 
pH 9.3; 20 kV, 10 s, 214 nm.
Figure 3. Electropherogram of AHA (1.0 mg mL-1): (a) non-purified, 
(b) purified. Conditions: 20 mmol L-1 borate buffer pH 9.3; 20 kV, 10 s,
214 nm.
Figure 4. Electropherogram of VHA (1.0 mg mL-1): (a) non-purified, 
(b) after Cl– removal (c) purified. Conditions: 20 mmol L-1 borate buffer 
pH 9.3; 20 kV, 10 s, 214 nm. 
Moraes and Rezende 27Vol. 19, No. 1, 2008
The ash values indicate the purity of the material. The 
lower the value, the purer the material. The values of ash 
(Table 1) were compared with the results obtained by CZE 
(Figures 2, 3 and 4). We observed that samples with a low 
value of ashes presented higher “humic hump” bands. It 
is reasonable to suppose that in the non-purified HA there 
are more complex groups that hamper the dissociation of 
the HA molecule by UV detection. 
In all CZE analyses, the non-purified HA sample 
presents minor intensity of peaks probably due to the 
smaller amount of HA available with UV detection. 
The result is a loss of signal. This fact reinforces the 
possibility of eliminating the several steps of purification 
related to elution through the ion exchange columns, 
therefore various samples of HAs can be rapidly 
analyzed by CZE in a short time with the advantage 
of reducing costs and submitting the sample to fewer 
manipulations. 
The average apparent mobility (M
ap, cm
2
 Vs-1) in Table 2 
was calculated for each fraction of HAs studied, according 
to the equation: M
ap = v/E = (Ld/tm)/V/Lt), where M is the
velocity, E is the electric field strength, Ld is the length 
from the capillary to the detector, Lt is the total length 
of the capillary, V is the applied voltage and tm is the 
migration time. 
The data of average apparent mobility as a normalized 
parameter permits, in principle, the comparisons between 
the HA fractions. These values showed that the mobility of 
the non-purified HA (fraction 1) is similar for the purified 
HA (fraction 3) and minor for the HA after Cl– removal 
(fraction 2), considering the same experimental conditions. 
As the values of mobility presented for fractions 1 and 3 
are similar, the characterization of the HAs samples can be 
made directly in CZE after the extraction of the sample. 
Characterization of humic acids by CZE
It is well-known that the migration of analytes in 
CZE depends on their charge-to-mass ratio. Therefore, 
the characterization of HAs by CZE is due to their 
charge and size, which depend on the HA origin and 
natural condition during their formation. Since the 
HAs present polyelectrolyte properties, they can be 
easily characterized by CZE, depending on the pH. 
Consequently, the choice of the separation buffer plays 
a key role in the interpretation of the electrophoretic 
patterns. It is obligatory that the very same analytical 
steps be followed in order to characterize different 
samples of HA. Considering the fraction purified for 
all HAs, Figures 2c, 3c and 4c, the shortest migration 
time was obtained for AHA, suggesting that the AHA 
possesses a major charge-to-size ration and a minor 
apparent mobility compared with other HAs. On the 
other hand, the PHA and VHA indicate an approximately 
similar number of dissociated groups (minor migration 
time), suggesting that they present similar charge-to-
size ration. These characteristics are due to different 
charge-to-size rations and various structural properties 
of the individual components of HA caused by their 
different origin.
Conclusions
The characterization of HAs by CZE is due to their 
charge and size, which is a function of different structural, 
physical and chemical properties. The present study 
shows the potential of CE for the characterization of HAs 
of different origins, without purifying them through the 
ionic exchange columns. Therefore, a greater number 
of HA samples could be analyzed in a short time, with 
minor modifications in the molecular structure of the HA. 
According to our results, the step of extraction is sufficient 
to characterize HA by CZE with the advantages of rapid 
separation, small amounts and fewer manipulations of the 
samples.
Acknowledgments
The authors would like to acknowledge CNPq and 
FAPESP (fellowship to S. L. M., 00/02368-5), for the 
financial support given to this research.
References 
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Table 2. Values of average apparent mobility obtained from different 
fractions of HA samples
HA samplesvalue of average apparent mobility / (cm2 Vs-1)
fraction 1 fraction 2 fraction 3
THA 2.96 r 10-4 2.74 r 10-4 2.98 r 10-4
AHA 2.06 r 10-4 2.04 r 10-4
VHA 2.97 r 10-4 2.88 r 10-4 2.95 r 10-4
Table 1. Ash content obtained from different fractions of HA samples
HA samples Values of ash / (%)
fraction 1 fraction 2 fraction 3
THA 72.30 53.20 4.80
AHA 48.90 2.60
VHA 71.20 42.60 1.40
Capillary Electrophoresis (CE): a Powerful Tool to Characterize Humic Acid (HA) J. Braz. Chem. Soc.28
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Received: October 25, 2006
Published on the web: January 14, 2008
FAPESP helped in meeting the publication costs of this article.


Capillary electrophoresis (CE): a powerful tool to characterize humic acid (HA)

Solange L. de Moraes

Maria Olímpia O. Rezende

Crossref

English

The present study evaluates the possibility of eliminating the purification steps involved in the characterization of HA by capillary zone electrophoresis (CZE). The HAs of various sources were analyzed, showing different electropherograms by CZE, which depend on the charge and size of HA. The data suggest that the purification of the sample is not necessary to characterize HAs. Based on the results, CZE showed to be a promising tool to characterize HA of different origins without the purification step of the sample.Neste estudo, avaliou-se a possibilidade de eliminação das etapas de purificação envolvidas na caracterização de HA. HA de diferentes fontes foram analisados por CZE apresentando diferentes eletroferogramas. Essas diferenças observadas mostram que a caracterização do HA por CZE depende da carga e do tamanho do HA. Os resultados apresentados neste trabalho sugerem que não é necessária a purificação da amostra para a caracterização de HA por CZE. Baseados nos resultados, a CZE mostrou-se uma ferramenta promissora para caracterização de HA de diferentes origens sem a etapa de purificação da amostra

RCAAP - Repositório Científico de Acesso Aberto de Portugal

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Journal of the Brazilian Chemical Society

http://www.scielo.br/pdf/jbchs/v19n1/a05v19n1.pdf

Capillary electrophoresis (CE): a powerful tool to characterize humic acid (HA)

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