© by PSP Volume 12 – No 7. 2003
Fresenius Environmental Bulletin
CONFORMATIONAL BEHAVIOR OF LIGNITE HUMIC FRACTIONS
SEPARATED BY SEQUENTIAL pH-EXTRACTIONS
J. Kučerík1, P. Conte2, M. Pekař1, A. Piccolo2*
1
Institute of Physical and Applied Chemistry, Faculty of Chemistry,
Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech Republic
2
Dipartimento di Scienze del Suolo della Pianta e dell’Ambiente,
Università di Napoli Federico II, via Università 100, 80055, Portici (Na), Italy
SUMMARY
A bulk humic acid (HA) extracted from a South Moravian lignite was further fractionated by sequential extraction in buffers at different pHs. All samples were
analyzed by high performance size exclusion chromatography (HPSEC) and elemental analysis. Weight-averaged
molecular weights (Mw) of fractions dissolved at pH 7, 8,
and 9 were larger than for the parent bulk HA, thereby
indicating that Mw of humic matter is related only to
molecular size as affected by sample hydration rather than
its molecular mass. Moreover, hydration radius was a
function of the specific chemical properties of humic
fractions. Addition of small amount of either mineral
(HCl) or organic acids (formic, acetic, propionic) to humic solutions induced significant changes in conformational arrangements of the treated sample with respect to
control. Also the conformational changes among humic
fractions depended on their different molecular composition and suggested a weakly-bound supramolecular association of different humic molecules in solution.
polymers [6], with molecular weights ranging from 500
to more than 10 6 Dalton [1], arranged either in a random
coil [7] or in micellar conformations [8, 9]. Recent findings have lead to an alternative understanding of humic
substances structure that is described as a supramolecular
association of different small and heterogeneous molecules self-assembled by weak forces such as van der
Waals, -, CH- bonds, and hydrogen bonding [4, 1017]. Therefore, the loosely-bound humic conformations
have an apparently large molecular size that can be reversibly altered by treating humic solutions with low
concentrations of mineral or carboxylic acids [12, 13].
Fractional precipitation at different pH is a commonly
applied procedure [18] to separate polydisperse macromolecules according to their molecular sizes. Simpson et
al. [19], have shown that dissolution of humic substances
in water can be achieved by sequential extraction with
buffer solutions at increasing pH values. In the present
work a sequential pH-based extraction was applied on a
humic acid (HA) from a South Moravian lignite in order
to obtain humic fractions of different molecular sizes and
composition. High performance size exclusion chromatography (HPSEC) was used to evaluate the conformational behavior of the pH-extracted humic fractions and
their response to treatments with mineral and organic
acids.
KEYWORDS: HPSEC, humic substances, lignite, supramolecular
associations, molecular weight distribution, sequential extraction.
INTRODUCTION
MATERIALS AND METHODS
Humic substances are the most widely-distributed
natural organic compounds on the Earth’s surface derived
from decomposition of plant tissues and dead animal
bodies [1]. They are present in soils, waters, and sediments
and play important roles in sustaining plant growth [1],
stabilizing soil structure [2], and protecting from environmental contamination [3, 4]. However, there are still
opened questions on their basic chemical structure and
conformational properties [5]. The traditional view depicts
humic substances (HS) as polydisperse macromolecular
Humic samples
A humic acid (HA1) was isolated from a South Moravian lignite collected from the Mír mine in the area of
Mikulčice, nearby Hodonín, Czech Republic. Extraction
of the humic acid was as by established procedures [1].
Briefly, 50 g of previously air-dried and 0.3-mm-sieved
lignite, were added with 500 mL of a 0.1M NaOH solution and shaken for 24 h. After centrifugation, the super-
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© by PSP Volume 12 – No 7. 2003
Fresenius Environmental Bulletin
natant was treated with concentrated HCl until pH 1 in
order to precipitate the humic acid (HA1). The HA1 was
treated overnight with a 0.5% (v/v) HCl-HF solution to
remove residual ashes, dialyzed (Spectrapore 3 dialysis
tubes, 3500 Mw cut-off) against distilled water until chloride-free and freeze-dried.
and subjected to HPSEC analysis. The same solutions
were then added with HCl, acetic, propionic or formic
acid to lower the pH to 3.5 before HPSEC analysis. The
small addition of these acids (<10-2 M) did not change the
ionic strength of the humic solutions [11]. All solutions
were filtered through quartz filters (Glass Microfibre Filterm Whatman International, LTD) before injection. The
HPSEC analyses were conducted in duplicate and no significant differences were observed between measurements.
The freeze-dried HA1 was further shaken in aqueous
buffer solutions to sequentially extract fractions soluble at
different pHs. First, 1 liter of phosphate buffer at pH 7
(0.062M NaH2PO4·H2O+0.038M Na2HPO4·12H2O, ionic
strength 0.176M) was added to 2 g of HA1, and the suspension shaken for 24 hours. The supernatant was separated by centrifugation, added with concentrated HCl to
pH 1, and freeze-dried as humic fraction HA2. The solid
residue was again added with 1L of a phosphate buffer at
pH 8 (0.014M NaH2PO4·H2O+0.086M Na2HPO4·12H2O,
ionic strength 0.272M). The resulting humic extract, HA3,
was obtained as for HA2. The solid remaining after extraction of HA3, was further added with 1L of a buffer solution
at
pH
9
(0.002M
NaH2PO4·H2O+0.098M
Na2HPO4·12H2O, ionic strength 0.296M) and the extracted humic acid, HA4, was isolated as for HA2. Finally, the
solid residue from the HA4 extraction was treated with 1
L of a pyrophosphate buffer solution (0.10M, ionic
strength 1M) at pH 10. A humic acid sample, HA5, was
obtained as above. All samples were characterized for
their elemental content using a Fisons EA 1108 Elemental
Analyzer, whereas the ash content was obtained by muffle
burning 50-100 mg of each material at 750C for 8 hours.
Elemental and ash contents are reported in Table 1. All
humic samples (50 mg) were suspended in distilled water
(60 ml) and titrated to pH 7 with a CO2 free 0.1M NaOH
solution by an automatic titrator (VIT 90, Videotitrator,
Radiometer, Copenhagen) as described by Conte and
Piccolo [10]. The resulting sodium humates were freezedried and homogenized in an agate mortar for further
HPSEC analyses.
Weight-average molecular weight (MW)
Weight-average molecular weight (Mw) was calculated using the PE-TC-SEC 4.01 software and the following
relationship:
N
MW 
 (h M
i
i 1
i
)
N
h
i 1
i
where Mi and hi are the molecular weight and the
height of each ith fraction in the chromatogram, respectively [20]. All data were processed using a SEC noise
threshold of 100, and a Savitzky-Golay smoothing with a
filter size of 5.
Standards of known MW, such as polysaccharides
(PSC) of 186, 100, 23.7 and 12.2 kD (Polymer Sciences
Laboratories, UK) and sodium polystyrenesulphonates
(PSS) of 169, 123, 30.9 and 6.78 kD (Polymer Standard
Service, Germany) were used for column calibration.
Water was used to determine the total volume of the column (21.40 mL), whereas blue dextran (2000 kD) was
used to measure the void volume (11.04 mL). Calibration
curves were semi-log linear over the range defined by
standards and were used to obtain the molecular weights of
humic samples. Standard samples were measured several
times and relative standard deviations never exceeded 5%.
HPSEC analysis
The HPSEC system consisted of a Perkin–Elmer
LC200 pump equipped with two detectors in series: a
Perkin–Elmer LC295 UV/VIS detector set at 280 nm for
humic analyses, and a refractive index detector from
Fisons Instrument (RefractoMonitor 4.0) for calibration
with polysaccharides. A rheodyne rotary injector, with a
100 μl sample loop, was used to load HPSEC solutions
and a Phenomenex Biosep S2000 (600 x 7.5 mm) column
was used for size exclusion separations. The column was
preceded by a Biosep Guard column and a 0.2 m stainless-steel inlet filter. Flow rate was set at 0.6 ml min-1,
while the HPSEC eluent was a 50 mM NaH2PO4.H2O
solution adjusted at pH 7 with 1M NaOH. The salt concentration was chosen to have a constant ionic strength of
50 mM in order to minimize ionic exclusion or hydrophobic interactions with the column [10, 11].
RESULTS AND DISCUSSION
The elemental composition of the bulk humic acid
(HA1) and the pH-separated humic fractions (HA2-HA5)
is reported in Table 1. HA1 showed a C/H ratio of 12.4,
whereas the C/H ratios of the humic fractions were in the
order: HA2>HA3>HA4=HA5. Decreasing C/H ratios
appears to be related to decreasing aromaticity or degree
of unsaturation [19]. The HA2 sample, with the largest
carbon amount and the lowest hydrogen content, was
extracted from HA1 by a pH 7.0 buffer solution. This
fraction may then be composed by aromatic systems possessing acidic groups with pKa values <7.0. In fact, Simpson et al. [19] found that fractions extracted with buffer
solution at pH 7.0 was aromatic and had a large content of
highly acidic groups. Fractions HA3 and HA4, were extracted with buffer solutions at pH 8.0 and pH 9.0, respectively. Literature [1, 19] reports that decreasing C/H ratios
Freeze-dried sodium humates were dissolved in the
HPSEC eluent to achieve a 0.6 mg mL-1 humic solution
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Fresenius Environmental Bulletin
TABLE 1 - Ash content and ash-free based elemental analyses of HAs.
Sample
HA1
HA2
HA3
HA4
HA5
Ash
2.27
2.11
1.17
3.11
2.44
C (%)
57.2
53.7
50.1
47.6
50.9
H (%)
4.6
4.7
6.4
6.8
7.2
N (%)
1.0
1.0
1.3
1.3
1.1
are attributed to increasing aliphatic contents. Therefore,
the aliphaticity of HA3 and HA4 can be considered larger
than for HA2 (Table 1). The oxygen content of HA2,
HA3, and HA4 varies in the order HA2<HA3<HA4 (Table 1). Increasing amount of oxygen may be attributed to
an increasing polyacidic nature of the humic systems [21].
Fractions HA3 and HA4 are thus not only more aliphatic
than HA2, but also more polyacidic. The HA5 fraction
revealed the largest amount of hydrogens, whereas carbon
and oxygen content was comparable to that of other humic fractions (Table 1). Simpson et al. [19] reported that
humic fractions extracted at pH 10.0 showed the largest
amount of hydrogen and related the elemental content of
their alkaline-extracted fractions to a large degree of aromaticity. Furthermore, Piccolo et al. [22] reported that
12.8
O (%)
37.2
40.6
42.2
44.3
40.8
C/H
12.4
11.5
7.9
7.0
7.1
C/O
1.5
1.3
1.2
1.1
1.3
phenols were extracted from humic matter more by alkaline pyrophosphate solutions than by sodium hydroxide
solutions. We may then assume that the HA5 fraction was
selectively enriched in polyphenols as compared to fractions extracted in phosphate buffers at lower pHs.
The molecular size distribution of the bulk humic acid
(HA1) and the fractions sequentially extracted from HA1
are shown in Figure 1 as HPSEC chromatograms. The
profile of size distribution for HA1 shows two peaks (Figure 1). The first one is centered at 12.8 mL, whereas the
second peak is at around 17.3 mL of elution volume. The
weight-averaged molecular weight (Mw) corresponding to
that molecular size profile is 9,961 D based on PSS calibration and 18,878 D based on PSC standards (Table 2).
17.3
FIGURE 1 - UV-detected HPSEC chromatograms of humic samples.
HA1: isolated from lignite with NaOH;
HA2: isolated from HA1 by phosphate buffer at pH 7;
HA3: isolated from HA1 by phosphate buffer at pH 8 after extraction of HA2;
HA4: isolated from HA1 by phosphate buffer at pH 9 after extraction of HA3;
HA5: isolated with 0.1 M pyrophosphate at pH 10 after extraction of HA4.
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TABLE 2 - Weight-averaged molecular weight (Mw), in Dalton, of humic samples based on the
calibration by polystirenesulphonates and polysaccharides (PSS/PSC) after treatment with different acids.
Mw
Sample
Control
HCl
Formic
HA1
9,961/18,878
10,592/19,475
8,108/14,648
5,133/8,838
3,144/5,317
HA2
11,829/21,437
14,143/26,616
11,820/21,813
9,337/16,650
7,176/12,671
HA3
11,363/20,588
14,467/27,198
13,470/25,235
9,048/16,243
5,402/9,361
HA4
13,515/26,204
13,394/24,944
14,436/27,147
9,444/16,971
6,693/11,758
HA5
4,768/8,036
2,581/4,302
2,663/4,438
2,485/4,030
3,248/5,419
Although HA2 and HA3 fractions revealed different
molecular-size profiles (Figure 1), being that of HA2 larger
than that of HA3 and both larger than the HA1 profile, they
showed similar Mw values. In fact, Mw values for HA2
were 11,829 D with PSS and 21,437 D with PSC, whereas
those for HA3 were 11,363 D with PSS and 20,588 D with
PSC (Table 2). The HA4 fraction that showed the first peak
at the void volume comparable to that of HA1 but a considerably lower absorbance for the second peak, revealed the
largest Mw values, being 13,515 D with PSS and 26,204 D
with PSC (Table 2). Finally, HA5 showed a molecular sizeprofile with the lowest absorbance and a predominant peak
at larger elution volumes with consequent lowest Mw values: 4,768 D with PSS and 8,036 D with PSC (Table 2).
Acetic
Propionic
ular size smaller than that of HA2, HA3, and HA4, but
larger than that of HA5. This because the parent HA1 was a
mixture of all fractions and showed a molecular size that
was a weighed average of those components selectively
extracted at different pHs.
The molecular sizes of HA1 and its pH-extracted fractions were altered after addition of small amount of either
mineral (HCl) or organic (formic, acetic and propionic)
acids (Table 2). As a general trend, addition of HCl and
HCOOH to reach pH 3.5 increased the Mw values of HA1,
HA2, HA3, and HA4, whereas HA5 showed a strong diminution of its Mw (Figure 2). The acid groups present in both
HA1 and most acidic fractions (HA2, HA3, and HA4) are
deprotonated partly (HA2) or fully (HA3 and HA4) at pH
value 7.0 of the control solutions. When a strong acid such
as HCl or HCOOH is added to the solution until pH 3.5, a
protonation of the dissociated functional groups may
occur, thereby leading to formation of intermolecular Hbonds and increase of molecular sizes. Conversely, addition of CH3COOH and CH3CH2COOH provided decreasing Mw values (Figure 2). As previously observed [12, 13],
the loosely-bound supramolecular nature of HA1 and its
pH-extracted fractions may account for the decrease of Mw
values after addition of acetic and propionic acids. In fact,
both protons due to low pH and alkyl chains present in the
added organic acids may act together in disaggregating
amphipilic humic matter and lowering its Mw. Conversely,
the polyphenolic HA5 did not change its molecular size
when HCl, formic, acetic or propionic acids were used
(Figure 2). This behavior can be again explained by considering that Mw is a mathematical elaboration that summarizes the full size-exclusion chromatogram without accounting
for relative changes of molecular size profiles [15, 23]. In
fact, Figure 3 shows that notwithstanding the similar Mw
values calculated for HA5 for different acid additions, the
chromatograms are significantly different. After addition of
HCl and HCOOH, the chromatographic profile appeared
broader and of lower intensity than for control solution.
Conversely, addition of acetic and propionic acids leads to
chromatographic profiles attributable to more uniform and
lighter material.
Differences in Mw values can be attributed to the different chemical composition of the humic fractions. The
bulk HA1 contains all the molecular characteristics of
HA2, HA3, HA4, and HA5, and its molecular size can be
used as a reference to explain the conformational behavior
of the humic fractions solubilized from HA1 at different
pHs. Moreover, it is obvious that the Mw values of the
fractions derived from HA1 cannot be higher than the bulk
material itself as the calculations of Mw seem to indicate.
The explanation of such a contradiction should be attributed to the hydration of each material in solution that, in
turn, depends on its molecular composition. Due to the pH
of their extraction, the HA2, HA3, and HA4 fractions had a
higher acidic nature than HA5 that was solubilized only at
pH 10 and implies a contribution of hydration water to the
molecular size. It should be assumed that the hydration
sphere of the materials isolated at pH 7.0, 8.0 and 9.0 fractions is larger than that of HA5. In fact, the higher the
amount of acidic functional groups, the larger is the hydration sphere of a water soluble organic system [20]. On the
other hand, the polyphenolic character of HA5 confers a
larger degree of hydrophobicity to this fraction, thereby
preventing the formation of a bulky hydration sphere.
The Mw values of Table 2 appear to confirm this
view, since HA2, HA3, and HA4 revealed higher Mw
values than HA5. Conversely, the bulk HA1 had a molec-
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© by PSP Volume 12 – No 7. 2003
Fresenius Environmental Bulletin
Mw
Control
20000
HCl
15000
Formic
10000
Acetic
Propionic
5000
0
HA1
HA2
HA3
HA4
HA5
Sample
FIGURE 2
Changes in weight-averaged molecular weights (Mw) of humic samples with addition of acids.
FIGURE 3 - UV-detected HPSEC chromatograms of HA5.
A. HA5 dissolved at pH 7.0.
B. As in A but added with formic acid to pH 3.5;
C. As in A but added with acetic acid to pH 3.5;
D. As in A but added with propionic acid to pH 3.5;
E. As in A but added with HCl to pH 3.5.
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© by PSP Volume 12 – No 7. 2003
Fresenius Environmental Bulletin
[9]
CONCLUSION
Experimental data indicated that sequential pHfractionation of a humic acid from lignite produced fractions of different chemical composition. While the use of
phosphates buffers of increasing pH provided samples
with decreasing aromaticity and polyacidity, pyrophosphate preferably extracted less acidic and polyphenol-like
humic molecules. The molecular size distributions followed by HPSEC pointed out the importance of water
hydration in the determination of weight-average molecular weight of humic fractions.
[10] Conte, P., Piccolo, A., 1999a. High pressure size exclusion
chromatography (HPSEC) of humic substances: molecular
sizes, analytical parameters, and column performance.
Chemosphere 38 (3), 517-528.
[11] Conte, P., Piccolo, A., 1999b. Conformational arrangement
of dissolved humic substances. Influence of solution composition on association of humic molecules. Environmental Science and Technology 33, 1682- 1690.
The significant changes in chromatographic profiles
and Mw values induced by addition of small amounts of
either organic or mineral acids suggested that also these
humic samples from lignite and its pH-separated fractions
behaved as weakly-bound supramolecular associations in
solution. These findings indicated that sequential pHseparations of humic matter do not influence the molecular size of the extracted fractions but rather their chemical
composition.
[12] Piccolo, A., 2001. The supramolecular structure of humic
substances. Soil Sci. 166, 810-832.
[13] Piccolo, A., 2002. The Supramolecular structure of humic
substances. A novel understanding of humus chemistry and
implications in soil Science. Adv. Agron., 75, 57-134.
[14] Piccolo, A., Nardi, S., Concheri, G., 1996. Macromolecular
changes of humic substances induced by interaction with organic acids. European Journal of Soil Science 47, 319-328.
[15] Piccolo, A., Conte, P., Cozzolino, A., 1999. Conformational
association of dissolved humic substances as affected by interactions with mineral and monocarboxylic acids. European
Journal of Soil Science 50, 687-694.
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Fresenius Environmental Bulletin
[22] Stevenson, F.J., Butler, J.H.A., 1969. Chemistry of humic
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(Eglinton G. and Murphy M.T.J., eds.) Springer–Verlag,
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Received for publication: December 27, 2002
Accepted for publication: January 13, 2003
CORRESPONDING AUTHOR
A. Piccolo
Dipartimento di Scienze del Suolo della Pianta
e dell’Ambiente
Università di Napoli Federico II
via Università 100
80055 Portici (Na) - ITALY
Phone: 0039 081 2539160
Fax: 0039 081 2539186
e-mail: alpiccol@unina.it
FEB/ Vol 12/ No 7/ 2003 – pages
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