Biblid: 1821-4487 (2010) 14; 3; p.141-144
UDK: 582.661:547.458.88
Original Scientific Paper
Originalni naučni rad
INFLUENCE OF CU2+ AND AL3+ IONS ON ZETA POTENTIAL CHANGE OF
PECTIN AND PROTEIN PREPARATES EXTRACTED FROM SUGAR BEET
UTICAJ JONA CU2+ I AL3+ NA PROMENU ZETA POTENCIJALA PEKTINSKIH
I PROTEINSKIH PREPARATA IZOLOVANIH IZ ŠEĆERNE REPE
Tatjana KULJANIN*, Nevena MIŠLJENOVIĆ*, Gordana KOPRIVICA*, Ljubinko LEVIĆ*, Bojana FILIPČEV**
* Faculty of Technology, 21000 Novi Sad, Bulevar Cara Lazara 1, Serbia
** Institute for Food Technology, 21000 Novi Sad, Bulevar Cara Lazara 1, Serbia
e-mail: [email protected]
ABSTRACT
All models which are used to describe the behaviour of electrically charged colloidal particles are based on the presence of two
basic layers which surround a particle – stationary and diffused layer known as electric double layer. Electrokinetic phenomena are
generated by ions in the diffused layer and a potential across the diffused layer where hydrodynamic motion of liquid still exists is a
measurable parameter denoted as electrokinetic or Zeta potential. By adding positively charged ions Cu2+ and Al3+, Zeta potential
can be reduced to a zero point in which repulsive forces between the colloidal particles stop to exist enabling their aglomeration and
coagulation in aqueous solutions. It has been experimentally confirmed in all tested preparations that the addition of Cu2+ and Al3+
ions changed the sign of Zeta potential in such a way that a negative Zeta potential gradually decreased, proportionally to the increased concentrations of these ions. Electrokinetic measurements also showed that changes in the sign of Zeta potential occurred at
lower concentrations of coagulant CuSO4 as compared to coagulant Al2(SO4)3. The reason for this is a greater ability of surface
complexations of Cu2+ ions and coagulation enhancement effect of SO42- ions.
Key words: Zeta potential, pectins, proteins, sugar beet, CuSO4, Al2(SO4)3.
REZIME
Svi modeli koji opisuju ponašanje naelektrisanih koloidnih čestica baziraju se na postojanju dva osnovna sloja koja okružuju česticu - nepokretni i difuzioni sloj, koji zajedno nose naziv dvojni električni sloj. U elektrokinetičkim pojavama učestvuju isključivo joni
iz difuzionog sloja a potencijal u delu difuzionog sloja gde još postoji mogućnost hidrodinamičkog kretanja tečnosti, merljiva je
veličina koja se označava kao elektrokinetički ili Zeta potencijal. Dodavanjem pozitivno naelektrisanih jona Cu2+ i Al3+, može se Zeta
potencijal smanjiti do nule kada nestaju odbojne sile između koloidnih makromolekularnih čestica što omogućuje njihovu aglomeraciju i koagulaciju u vodenim rastvorima. U ovom radu, ispitivana su dva model-rastvora komercijalno raspoloživih pektina u koncentracijama koje odgovaraju koncentracijama u soku šećerne repe kao i jedan model-rastvor proteinskog preparata. Ispitivani sistemi su tretirani rastvorima CuSO4 i Al2(SO4)3. Eksperimentalno je potvrđeno da je kod svih ispitivanih preparata dodavanjem Cu2+ i
Al3+ jona, došlo do promene predznaka Zeta potencijala pri čemu se negativna vrednost Zeta potencijala smanjivala proporcionalno
povećanju koncentracije ovih jona. Elektrokinetičkim merenjima je utvrđeno da je do promene predznaka Zeta potencijala došlo pri
manjim količinama koagulanta CuSO4 u odnosu na koagulant Al2(SO4)3 što znači da CuSO4 pokazuje bolja koagulaciona svojstva.
Razlog tome je velika sposobnost površinske kompleksacije koja je naročito izražena kod Cu2+ jona kao i uticaj SO42- jona na
poboljšanje koagulacije.
Ključne reči: Zeta potencijal, pektini, proteini, šećerna repa, CuSO4, Al2(SO4)3.
INTRODUCTION
Macromolecular compounds, above all pectins and proteins,
account for around 60% of the total non-sucrose compounds in
sugar beet juice. The most common purification practices using
milk of lime and carbon-dioxide can remove less than half of
non-sucrose compounds present in sugar beet juice. Since the
affinity of calcium to bind the undesirable macromolecules from
sugar beet juice is relatively low, this paper investigated the use
of compounds with di- or three-valent cations such as CuSO4 i
Al2(SO4)3 which chemically induce the charge neutralization of
macromolecules present in sugar beet juice causing their coagulation. Coagulation is more effective if, besides charge neutralization, reduced solvatation occurs, too.
The ability of cations to bind to macromolecules in sugar
beet juice depends on the presence of carboxyl groups nonesterified with methyl alcohol as well as the presence of carboxyl groups in proteins.
In the papers of Kohn (1987) and Dronnet et al. (1996), the
affinity of divalent metal ions to bind with pectins and derivatives of pectins isolated from citrus and sugar beet was investi-
Journal on Processing and Energy in Agriculture 14 (2010) 3
gated. In both studies, it was found that the affinity of metal ions
to form complexes followed the order:
Cu2+ ~ Pb2+ >> Zn2+ > Cd2+ ~ Ni2+ ≥ Ca2+
Interaction between cations and diverse protein macromolecules was investigated in the works of Dougherty (1996),
Gaucheron et al. (1997) and Philippe et al. (2005). They found
the following order regarding the binding ability of cations:
Cu2+ > Zn2+ > Ca2+ > Mg2+ > Fe2+
Such a diversity in the binding affinity of metal cations could
be explained by two types of bindings (Kohn, 1987): electrostatic binding of Ca2+, Sr2+, Zn2+ ions and formation of surface
complexes by Cu2+ and Pb2+ ions which explain the greater affinity of Cu2+ and Pb2+ ions toward pectins isolated from citrus and
sugar beet.
Cation-protein complexes can be formed by electrostatic
bonds (Ca2+, Mg2+, Zn2+) or much stronger coordinative bonds
with oxygen atoms (Cu2+).
On the basis of selectivity scales, it is evident that Cu2+ ions
are more efficient than Ca2+ ions because of marked surface
complexation ability (Wiedemer, et al., 2000; Kuljanin, Tatjana
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Kuljanin, Tatjana et al. / Influ. of Cu2+ and Al3+ Ions on Zeta Pot. Change of Pectin and Protein Prep. Extracted from Sugar Beet
et al., 2008; Garnier et al., 1994; Kuljanin, Tatjana, 2008a;
Lević et al., 2007).
In the above mentioned studies, Zeta potential of protein particles in the presence and absence of cations was measured. Zeta
potential reduction was observed in the presence of cations with
the exception of Zn2+ ions. Among other changes, it was found
that macromolecules hydration decreased in the presence of
cations which is an important prerequisite for protein coagulation besides charge neutralisation (Gaucheron et al., 1997).
In this paper, using a method of measuring Zeta potential, the
application of CuSO4 and Al2(SO4)3 salts as a source of Cu2+ and
Al3- ions in clarification of sugar beet juices instead of Ca2+ ions
was investigated. Optimal concentrations of these coagulants
needed for successful coagulation of macromolecules present in
sugar beet juice do not depend only on the magnitude of positive
charge on coagulant molecule but on numerous other factors to
be further investigated. Analyzing the experimental results,
based on tracking the changes in Zeta potential (Kuljanin, Tatjana et al., 2008; Kuljanin, Tatjana, 2008a), mechanism of
charge neutralisation of pectin and protein macromolecules by
Al and Cu can be elucidated.
MATERIAL AND METHODS
Method of isolation of pectin and protein
preparation
Freshly sliced cossettes of sugar beet were washed out with
distilled water acidified with HCl to pH 5.5. The extraction of
pectin preparations was conducted discontinuously in an extractor (volume 2 dm3) using aqueous solution of HCl. The mass ratio of cossettess to solvent was 1:10. High molecular colloidal
fraction was isolated from extract by multistage precipitation
with 70 % ethanol. The precipitated colloids were left overnight
to deposit and the obtained sediment was washed out with 70 %
ethanol. Pectin preparations (denoted as P1 and P2), after precipitation and cleaning were dried in a vacuum drier for 12 hours
at 70 °C (Lević et al., 2007; Milić, Mirjana et al., 1992; Fares et
al., 2003).
The isolation of protein preparation (denoted as P3) was
conducted by sedimentation method at isoelectric point pI 3.5
and ambient temperature (AOAC Official Methods of Analysis,
2000). Fresh and washed beet root was chopped in a kitchen
mixer and juice was separated from pulp by pressing. Mechanical impurities were removed by centrifugation at 3000 r/min for
20 min. After precipitation with acetic acid at pH 3.5 and repeated centrifugation under identical conditions, the obtained
sediment was washed out with acidified water (pH 3.5). The obtained preparation was air dried and its protein content was determined according to Kjeldahl using a factor of 6.25 (Milić,
Mirjana et al., 1992).
Determination of molar mass, degree of polymerization and esterification
Mean molar mass of pectin and protein preparations was determined according to refractrometric and spectrophotometric
method as described in the paper of Kar and Arslan (1999). Degree of polymerization (DP) of pectin preparations was calculated by dividing the mean molar mass of the preparation with
the molar mass of dehydrated galactouronic residue (176
kg/kmol) whereas DP of protein preparation was derived by dividing the mean molar mass with average molar mass of amino
acids most frequently present in sugar beet proteins (136.3
kg/kmol) (Poel et al., 1998).
142
Degree of esterification was calculated using equivalents of
free (X) and esterified carboxy groups (Y) according to equation
(Kuljanin, Tatjana, 2008a):
Y
(1)
DE =
⋅100
X+Y
Preparation of materials for measuring Zeta
potential
In this experiment, 0.1% aqueous solutions of preparations
were used whereas coagulant solutions were prepared by dissolving 1 g of these salts in 200 cm3 of distilled water. A series
of coagulant concentrations in the range of 30, 60, 90, 120, 150,
180, and 210 mg/dm3 or ppm was prepared by adding the necessary volumes of the solution to 50 cm3 of 0.1% (w/w) pectin solution. Plan of the experiment and the volumes of coagulant solutions used are displayed in Table 1.
To accomplish the desired solution alkalinity (pH 7), Na2CO3
was added to Al2(SO4)3 (mass ratio of Na2CO3 to Al2(SO4)3 was
1 : 1.07, calculated on pure Al2(SO4)3).
Table 1. Plan of experiment
Name
Number of measurement
Volume of coagulant:
CuSO4 (cm3)
Number of measurement
Volume of coagulant:
Al2(SO4)3 (cm3)
50 cm3 Erlenmeyer flask with
0.1% (w/w) pectin (protein)
1
2
3
4
5
6
7
0.47 0.94 1.41 1.88 2.35 2.82 3.29
8
9
10 11 12 13 14
0.59 1.20 1.79 2.38 2.97 3.56 4.15
Zeta potential measurement
After the coagulant was added to the tested preparations, pH
was adjusted and the solution was stirred for 30 min on a highspeed magnetic stirrer (500 rpm). Then, the solution was stirred
for another 5 min at low speed and left to rest another 5 min to
prevent the disaggregation of the already formed floccules. An
aliquot taken from the supernatant was used to measure the zeta
potential. Measurements were performed at room temperature
for seven different solution concentrations.
Zeta potential was determined by electrophoretic method using a commercial apparatus ZETA-METER ZM 77 (Riddick,
1975). The instrument consists of an electrophoretic cell with
platinum electrodes connected to direct current and a stereoscopic microscope equipped with a special ocular micrometer.
After adjusting the voltage, an electric recording device was
used to measure time needed for a colloidal particle to pass a distance of a standard micrometer division. An average value of
100 readings was used to derive the Zeta potential of colloidal
particles in the tested solutions using a diagram based on the
Helmoltz-Smoluchowski equation for electrophoretic mobility of
colloidal particles.
For each tested solution, measurements were repeated 3
times. Experiments were conducted at 6-fold magnitude on a
stereoscopic microscope and voltage adjusted at 200 V. Immediately before Zeta potential measurements, solution temperatures
were measured. Zeta potential was read from the diagram and
multiplied by a correction factor for a given temperature.
RESULTS AND DISCUSSION
Results related to the composition of pectin preparations (P1
and P2) are given in Table 2. Data on mean molar mass and degree of polymerization are also included in the table. The content
Journal on Processing and Energy in Agriculture 14 (2010) 3
Kuljanin, Tatjana et al. / Influ. of Cu2+ and Al3+ Ions on Zeta Pot. Change of Pectin and Protein Prep. Extracted from Sugar Beet
Table 2. Basic physico-chemical composition of pectin
preparation
Solid
Pre- content,
par.
SC
(g/100g)
Degree
Equivalent Equival. of
Degree Mean
Content of
of
of free esterified
of
molar
galactupolymCOOH
COOH
esterifi- mass,
ronic
erizagroups,
groups
cation,
MWsr
acid (%)
tion,
5
5
X · 10
Y · 10
DE (kg/kmol)
DP
P1
82.25
10.60
27.55
66.31
72.21
119048
676
P2
80.35
24.58
16.05
72.24
39.50
87720
498
Solid content,
SC (g/100g)
P3
86.25
Protein Total nitrogen
content,
content,
(g/100g)
N (g/100g)
56.10
8.86
Mean molar Degree of
mass,
polymerization,
MWsr
DP
(kg/kmol)
82645
606
Changes of mean values of Zeta potential (mV) of the tested
preparations after adding various quantities of coagulants CuSO4
and Al2(SO4)3 in the form of pure salts (mg/dm3 ili ppm) are
shown in Figures 1 and 2.
30
Zeta potential (mV)
20
10
0
-10
0
30
60
90
120
150
180
210
30
20
10
0
0
30
60
90
120
150
180
210
240
-10
-20
-30
-40
-50
Concentration of coagulant A l2(SO4) (mg/dm3)
Fig. 2. Dependence of the electrokinetic potential of pectins and
protein solution on the Al2(SO4)3 concentration:
♦ - preparation P1; ■ - preparation P2; ▲– preparation P3
Table 3. Basic physico-chemical composition of protein
preparation
Preparate
tion, marked specific adsorption of Cu2+ and Al3+ ions occurred
by surface complexation with COO- groups of pectin and protein
macromolecules.
Zeta potential (mV)
of galacturonic acid (degree of purity) in the tested preparations
is in agreement with the mean content of pectin found in raw
sugar beet juices from diffuser reported in literature. In accordance with the described extraction conditions, the obtained
preparations differed in the degree of esterification; the pectin
preparation P1 was classified as high esterified pectin (DE > 50)
whereas the preparation P2 was low esterified pectin (DE < 50).
The content of protein preparation P3 isolated by precipitation at
pI 3.5, mean molar mass and degree of polymerization are displayed in Table 3. Mean molar mass determined refractometrically and spectrophotometrically by measuring 5 different concentrations of pectin and protein preprarations (P1, P2 and P3):
0.0025; 0.005; 0.010; 0.015 and 0.020 g/cm3.
240
-20
-30
-40
-50
Concentration of coagulant CuSO4 (mg/dm3)
Fig. 1. Dependence of the electrokinetic potential of pectins and
protein solution on the CuSO4 concentration:
♦ - preparation P1; ■ - preparation P2; ▲– preparation P3
In all tested preparations, charge inversion of Zeta potential
from negative to positive was observed within the whole series
of tested coagulants concentrations. Total net charge of Cu2+ and
Al3+ ions (including H+ ions in the solution) increased in magnitude in comparison to the magnitude of negative charge on the
surface of macromolecule. This proves that, besides ionic exchange and lowering of surface potential by charge neutraliza-
Journal on Processing and Energy in Agriculture 14 (2010) 3
According to the Schulze-Hardy rule, ions with high valence
like Al+3 should be able to decrease Zeta potential to zero point
at much lower concentration. However, from the results presented, it is obvious that in the preparations tested, less amount
of Cu+2 ions compared to Al+3 ions was required for lowering
Zeta potential to zero point. This can be explained by higher
binding ability of these ions considering that Cu+2 ions are firstranged in the previously given selectivity order which describes
the binding ability of divalent cations to pectins and proteins of
botanical origin. But, it is possible that other mechanisms also
take part:
- Cu2+ ions have greater electrophoretic mobility. Since they
have higher density than Al3+ ions, their kinetic energy is also
higher enabling their easier location in the stationary part of
double electric layer (Stern layer), neutralising the surface
charge of pectin and protein macromolecules.
- Cu2+ ions are less hydrated due to larger ionic diameter in
comparison to Al3+ ions which affects the velocity of their distribution into the diffuse layer of the double electric layer and consequently, into Stern layer on the macromolecule surface. However, the volume of layer occupied by adsorbed Cu2+ and Al3+
ions depends not only on the hydrodynamic radius of the ions
and their dehydration ability but on the ability of hydration coverings to overlap in the stationary Stern layer.
The lowest concentration of CuSO4 (82.0 mg/dm3) to neutralize Zeta potential was determined in the preparation P2. In the
case of other preparations, higher concentration was required in
P1 (100 mg/dm3) and in P3 (114 mg/dm3) (Fig 1). P2 preparation
showed the best coagulation properties because of lower degree
of esterification (39.50) and longer polygalacturonic chains
compared to the preparation P1 (esterification degree, 72.21).
Protein preparation P3 was high above its isoelectric point (pI
3.5) and showed the highest stability presumably due to lower
proportion of free COO- groups and unfavourable conformation
of macromolecules.
It is known from published data that charge inversion is affected by difference in size of cations and anions (in our case
SO42-; Cu2+ i Al3+). Since Cu2+ ions have smaller hydrodynamic
143
Kuljanin, Tatjana et al. / Influ. of Cu2+ and Al3+ Ions on Zeta Pot. Change of Pectin and Protein Prep. Extracted from Sugar Beet
diameter than Al3+ ions, coagulant CuSO4 will have higher destabilizing effect.
The increased slope of Zeta potential curve observed in the
CuSO4 coagulant concentration range from 60 mg/dm3 to 120
mg/dm3 for pectin preparations (P1 and P2) and from 90 mg/dm3
to 150 mg/dm3 for protein preparation (P3) can be explained by
formation of hydrolysis products (monomeric and polymeric)
which are carriers of large positive net charges causing coagulant complexation on the surface of pectin and protein macromolecules (Fig. 1). In this concentration range of coagulant, besides mechanism of surface complexation, mechanism of surface
precipitation is also possible.
CONCLUSION
Studying the charge inversion mechanisms on molecular
level, Cu+2 ions have larger influence on surface charge of macromolecules and consequently higher destabilizing effect in
comparison to Al+3 ions.
Ca+2 ions are most frequently used in classical separation
procedures of macromolecule compounds in sugar beet juice,
but, in binding these ions, weak, pure electrostatic reactions have
a determinate role. The concentration of these ions needed to
neutralize pectin and protein macromolecules is much higher as
compared to that of Cu+2 and Al+3.
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Received:30.06.2010.
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Accepted:20.08.2010.
Journal on Processing and Energy in Agriculture 14 (2010) 3