INTRODUCTION
Gliadin proteins represent one of the gluten fractions.
Most gliadin proteins are present as monomers. They
affect the viscosity and extensibility of wheat flour [1, 2].
Gliadins are divided into four groups, namely α-, β-, γ-
and ω-gliadins. This division is based on mobility at low
pH, i.e. in acidic conditions of A-PAGE electrophoresis
medium (acid polyacrylamide gel electrophoresis). Based
on research that was later conducted on amino acid
sequences, α and β gliadins were classified in the same
group (α/β) [3–5].
Modern methods, such as two-dimensional
electrophoresis and high-pressure liquid chromatography
with reversed phase, allow the separation of gliadin
fractions into more than a hundred components. Based
on the analysis of amino acid sequences (complete
and partial), amino acid composition and molecular
weight, gliadins are divided into: ω5, ω1.2, α + β and γ
[3, 6–8]. ω-gliadins are characterized by a high content
of glutamine, proline and phenylalanine. These amino
acids together make up about 80% of the total ω gliadin
composition. ω5-gliadins have a higher molecular weight
(≈ 50 000 Da) than ω1.2 (≈ 40 000 Da). Most ω gliadins lack cysteine, so there is no possibility of disulfide
binding. These proteins consist of repetitive sequences
that are rich in glutamine and proline [3, 9, 10].
Molecular weights of α + β and γ-gliadins overlap
(≈ 28 000–35 000 Da). The content of glutamine and
proline is much lower compared to ω-gliadin. They
differ in tyrosine content. Each of the two types has
an N- and a C-terminal region [3, 11]. The N-terminal
region (40–50% of total proteins) consists of repeating
amino acid sequences that are rich in glutamine, proline,
phenylalanine, and tyrosine. The repeating sequences
of α + β gliadin are dodecapeptides. They are repeated
five times. A typical unit of γ-gliadin is repeated up
to 16 times. They are interspersed with additional
remains [12, 13]. Within the C-terminal region α + β
and γ-gliadins are homologous. The sequences are
not repeting. They contain less glutamine and proline
than the N-terminal region and have a more common
composition. α + β and γ gliadins contain six or eight
cysteine residues. These residues are located in the
C-terminal region. They form intramolecular disulfide
bonds [14, 15]. Although the content of total gliadin
proteins depends on the type of wheat and growth
conditions (soil, climate, fertilization), α + β and
γ-gliadins are the highest components. Ω-gliadins are
present in lower amounts [16–18].
To separate gliadin proteins, the following
techniques are used: high performance liquid chromatography
with reversed phase RP-HPLC, exclusion
chromatography SE-HPLC, high performance capillary
electrophoresis HPCE, sodium dodecyl sulphate
polyacrylamide gel electrophoresis SDS-PAGE,
and isoelectric focusing IEF [19]. One of the newer
techniques for gliadin separation is the highperformance
SDS-GCE, which is based on the
difference in electrophoretic mobility of ions in solution
within the capillary. The molecule size affects the
mobility of ions [20].
The number of people who are allergic to gluten
proteins from wheat is increasing, which makes food
producers give their consumers a guarantee that
products declared as “gluten free” really do not contain
gluten. The aim of this study was to investigate optimal
conditions (solvent concentrations, capillary temperature
and voltage) for their separation by high performance
capillary gel electrophoresis.
STUDY OBJECTS AND METHODS
Gliadin extraction. We analyzed gliadins in
wheat flour samples (ash content: max 0.55%, moisture
max: 15%, acidity: max 3, protein content 9.8 g/100 g)
purchased on the market of the Republic of Srpska,
Bosnia and Herzegovina by capillary gel electrophoresis.
Extraction of gliadin proteins was performed
according to a modified Osborne method, as described
by Lookhart and Bean [21]. After the albumins and
globulins were removed (extraction was performed
3 times with 8 mL of deionized water each, it was
obtained in laboratory conditions, on the apparatus
Siemens water Technologies W3T199551, Siemens
Ultra Clear, at a conductivity of 0.055 mS/cm and at a
temperature of 20°C and 3 times with 8 mL of 2%
solution of NaCl, NaCl, Lach-Ner, Czech Republic,
high purity, ≥ 99.00%) gliadin was extracted with 8 ml
of ethanol of different concentrations (50, 60 and 70%
v/v, refined REAHEM, 96% v/v ethyl alcohol, Srbobran,
quality corresponds to the quality property for ethyl
alcohol, contains a minimum of 96% v/v ethanol).
Samples were homogenized on a vortex (Advanced
Vortex Mixer ZX3, 3000 rpm) for 30 min. The samples
were then centrifuged in a centrifuge (Rotina 380 R,
Hettich Zentrifugen) for 5 min at 1,000 rpm. The
resulting supernatant was poured into a normal 25 mL
vessel, and after the third extraction the normal vessel
was made up to final volume with ethanol of various
concentrations (50, 60 and 70% v/v). The precipitate was
then washed with deionized water.
Samples preparation for analysis at GCE. Prior
to analysis samples were diluted with sample buffer
(SDS-MW sample buffer, PA 800 plus, Beckman
Coulter, USA), so that the total volume was 95 μL
and the concentration was 1 mg/mL. Then 2 μL of
internal standard (10 kDa, PA 800 plus, Beckman
Coulter, United States) and 5 μL of 2-mercaptoethanol
(high purity, 99.00%, Sigma-Aldrich Chemie GmbH,
Germany) were added. The samples were then heated
on a thermo-shaker (Thermo-Shaker, TS-100, Biosan) at
100°C for 3 min. After cooling to room temperature for
5 min, the samples were ready for analysis by capillary
gel electrophoresis (Agilent, CE 7100).
Preparation SDS-MW standard for analysis
by capillary gel electrophoresis. Prior to the
preparation standard, based on the recommendation of
the kit manufacturer, the standard was taken to room
temperature for 15 minutes after removal from the
refrigerator. It was then carefully stirred on a vortex
(Advanced Vortex Mixer ZX3, 3000 rpm) for a few
seconds. After that, 10 μL of standard (SDS-MW
standard, PA 800 plus, Beckman Coulter, United
States) was pipetted into the vial. Then 85 μL of buffer
(SDS-MW sample buffer, PA 800 plus, Beckman
Coulter, USA) and 2 μL of internal standard (10 kDa,
PA 800 plus, Beckman Coulter, USA) were added. Then
5 μL of 2-mercaptoethanol (Sigma-Aldrich Chemie
GmbH, Germany, high purity, 99.00%) was added.
Then, it was heated on a thermo-shaker (Thermo-
Shaker, TS-100, Biosan), at a temperature of 100°C for
3 min. After heating, the standard vial was cooled to
room temperature over 5 min. Prepared in this way, the
standard is ready for analysis.
Gliadin proteins separation by capillary gel
electrophoresis. Separation of gliadin proteins by
capillary gel electrophoresis was performed on an
Agilent apparatus, CE 7100, with a capillary inner
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diameter of 50 μm, a total length of 33 cm, and an
effective length of 23.50 cm. The SDS-MW analysis kit,
PA 800 plus (2015 Beckman Coulter, USA) was used
for separation. SDS gel buffer (0.2% SDS, pH = 8) was
used to fill the capillary. The kit contains the following
chemicals: SDS-MW gel buffer (0.2% SDS, pH = 8),
SDS-MW sample buffer (100 mM Tris-HCl, pH = 9,
1% SDS), internal standard (10 kDa), external standard
(10 to 225 kDa), acid wash solution (0.1N HCl), base
wash solution (0.1N NaOH), as well as two capillaries
57 cm long, 50 μm ID. According to the manufacturer’s
instructions, the kit is stored at room temperature after
opening, except for the internal and external standards,
which are stored at a temperature of 2–6°C. Preparation
of the capillary electrophoresis (CE) instrument was
done according recommendations Agilent Technologies
[22–24].
Statistical data processing. Statistical data
processing was performed in IBM SPSS, Statistics 26.
Descriptive statistical analysis calculated the average
value, standard deviation and 95% confidence interval
of the average value. Variance analysis of different
groups was used to evaluate the effect of solvent
concentrations, capillary temperature and electrode
voltage on the number of detected proteins and the
relative concentration of each gliadin proteins.
RESULTS AND DISCUSSION
In order to determine molecular weights unknown
proteins, a calibration curve was obtained using 7
proteins in SDS-MW size standard.
Electrophoregram, the migration time, and the
calibration curve of MW standard proteins with known
molecular weight (10, 20, 35, 50, 100, 150 and 225 kDa)
are presented in Fig. 1, Table 1, and Fig. 2, respectively.
The proteins were separated by capillary gel
electrophoresis (CE, Agilent, CE 7100, internal capillary
diameter 50 μm, total capillary length 33 cm, effective
capillary length 23.50 cm, capillary temperature 25°C,
voltage –16.5 kV (reverse mode), duration of analysis
30 min, and absorbance measured at 220 nm).
The ratio of molecular weights (log MW) and
migration time (t) of proteins is represented by the
equation y = 0.08168x – 0.00098, where y represents
logMW and x represents the migration time of proteins
(t). R2 shows the correlation coefficient (0.9847).
A calibration curve was used to estimate the
molecular weight of unknown proteins. The coefficient
of correlation shows a high dependence of the logarithm
of the molecular weight of the protein and the migration
time of the protein.
The number of proteins in each gliadin fraction and
their relative concentration were obtained based on the
total number of identified proteins and the total relative
concentration.
Table 2 shows descriptive indicators of total proteins
and the number of gliadin proteins after extraction with
different concentrations of ethanol.
Descriptive analysis showed that the highest number
of proteins (23.50) was obtained after extraction with
70% ethanol, by the method of Lookhart and Bean. The
lowest number of proteins was obtained by extraction
with 50% ethanol (18.67). One-factor analysis of the
variance of different groups showed that there was
a statistically significant difference in the number of
Figure 1 Electrophoregram of MW standards of proteins of known molecular weight separated by capillary gel electrophoresis
Table 1 Migration time of proteins with known molecular
weight separated by capillary gel electrophoresis
Molecular weight (MW), kDa log MW t, min
10 1.00 13.36 ± 0.21
20 1.30 15.77 ± 0.18
35 1.54 18.13 ± 0.26
50 1.70 20.15 ± 0.29
100 2.00 24.25 ± 0.10
150 2.18 26.78 ± 0.36
225 2.35 29.41 ± 0.15
mAU
25
20
15
10
5
0
5 10 15 20 25 30 min
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Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Х
proteins, F (2.15) = 2 3.70, S ig. = 0 .000. The highest
number of within α + β gliadin fractions was obtained
after extraction with 60% ethanol (7.67). The lowest
number of those proteins was obtained after extraction
with 50% ethanol (6.00). A statistically significant
difference was found in the number of proteins,
F(2.15) = 8.58, Sig. = 0.003.
Extraction with 50 and 60% ethanol produced the
highest and the lowest number of proteins within the
γ-gliadins (5.33 and 4.67, respectively). There was
no statistically significant difference in the number
of p roteins, F (2.15) = 1 .15, S ig. = 0 .342. T he h ighest
amount of ω1.2-gliadins was obtained after extraction
with 60% ethanol (5.17), while the lowest after extraction
with 50 and 70% ethanol (4.33). One-factor variance
analysis showed no statistically significant difference,
F(2.15) = 2 .19, S ig. = 0 .146. T he h ighest n umber o f
ω5-gliadins was obtained after extraction with 70%
ethanol (6.67). The lowest amount was observed after
extraction with 50% ethanol (3.83). A statistically
significant difference in the number of proteins was
found, F(2.15) = 6.77, Sig. = 0.008.
According to Table 2, an increasing ethanol
concentration increased total proteins, increased and
then slightly decreased α + β gliadin fraction, decreased
and then increased γ-gliadins, increased and then
decreased ω1.2-gliadins, and increased ω5 gliadin
fractions.
Table 3 shows descriptive indicators of the total
relative concentration and the relative concentration
of gliadin proteins after extraction with different
concentrations of ethanol.
Descriptive analysis showed the highest relative
protein concentration of α + β gliadin fractions after
extraction with 50% ethanol (31.25%) and the lowest
concentration after extraction with 60% ethanol
(17.69%). One-factor variance analysis revealed a
statistically significant difference in the relative protein
concentration, F(2.15) = 174.13, Sig. = 0.000.
Extraction with 50 and with 60% ethanol produced
the highest and the lowest relative concentration of
γ-gliadins (27.72 and 18.55%). A statistically significant
Figure 2 Calibration curve obtained as the ratio of the logarithm
of molecular weight and migration times of known
proteins separated by capillary gel electrophoresis
Table 2 Descriptive indicators of total proteins and gliadin proteins separated by fractions (Agilent, CE 7100, capillary inside
diameter 50 μm, total capillary length 33 cm, effective capillary length 23.50 cm, capillary temperature 25°C, voltage –16.5 kV
(reverse mode), duration 30 min, absorbance measured at 220 nm)
Ethanol,
% (v/v)
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
Total number
of proteins
50 6 18.67 1.21 0.49 17.40 19.94 17 20
60 6 23.00 1.67 0.68 21.24 24.76 21 25
70 6 23.50 1.05 0.43 22.40 24.60 22 25
α + β gliadins 50 6 6.00 0.63 0.26 5.34 6.66 5 7
60 6 7.67 0.82 0.33 6.81 8.52 7 9
70 6 7.50 0.84 0.34 6.62 8.38 6 8
γ gliadins 50 6 5.33 0.82 0.33 4.48 6.19 4 6
60 6 4.67 0.82 0.33 3.81 5.52 4 6
70 6 5.00 0.63 0.26 4.34 5.66 4 6
ω1.2 gliadins 50 6 4.33 0.82 0.33 3.48 5.19 3 5
60 6 5.17 0.75 0.31 4.38 5.96 4 6
70 6 4.33 0.82 0.33 3.48 5.19 3 5
ω5 gliadins 50 6 3.83 0.98 0.40 2.80 4.87 3 5
60 6 5.50 1.22 0.50 4.21 6.79 3 6
70 6 6.67 0.52 0.21 5.12 7.22 5 7
ANOVA (TP) F(2.15) = 23.70, Sig. = 0.000, eta square = 84.78/111.61 = 0.76
ANOVA (α + β) F(2.15) = 8.58, Sig. = 0.003, eta square = 10.11/18.94 = 0.53
ANOVA (γ) F(2.15) = 1.15, Sig. = 0.342 > 0.05
ANOVA (ω1.2) F(2.15) = 2.19, Sig. = 0.146 > 0.05
ANOVA (ω5) F(2.15) = 6.77, Sig. = 0.008, eta square = 12.33/26.00 = 0.47
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difference in relative concentration was found,
F(2.15) = 111.01, Sig. = 0.000. The relative concentration
of ω1.2-gliadins was the highest after extraction with
70% ethanol (14.93%) and the lowest after extraction
with 60% ethanol (4.82%). The one-factor analysis of the
variance showed a statistically significant difference in
the relative concentration, F(2.15) = 472.47, Sig. = 0.000.
As for ω5 gliadin fractions, they were found in the
highest concentration after extraction with 60% ethanol
(47.45%) and the lowest after extraction with 70%
ethanol (30.98%). There was a statistically significant
difference in the relative concentration, F(2.15) = 104.83,
Sig. = 0.000.
Based on the obtained results (Table 3), an increasing
ethanol concentration decreased and then increased the
relative concentration of α + β, γ- and ω1.2-gliadins and
increased and then decreased that of ω5-gliadins.
Table 4 shows descriptive indicators of the total
number of proteins and number of gliadin proteins
separated by fractions after extraction with 70% (v/v)
ethanol and separated at a capillary temperature of 20,
25, 30, 35 and 40°C.
Descriptive analysis revealed that the highest number
of proteins was obtained after extraction with 70%
ethanol and at a capillary temperature of 25°C (23.50),
while the lowest amount of proteins was observed at
35°C (18.83). The one-factor analysis of variance showed
a statistically significant difference in the number of
proteins, F(4.25) = 11.02, Sig. = 0.000.
The highest and the lowest numbers of α+β gliadin
fractions were obtained at 20°C (10.00) and 25°C
(7.50), respectively. There was a statistically significant
difference, F(4.25) = 6.24, Sig. = 0.001. The number of
γ-gliadins was the highest at 25°C (5.00) and the lowest
at 35°C (3.50). ANOVA test showed a statistically
significant difference in the number of proteins, F(4.25)
= 9.01, Sig. = 0.000. The highest amount of ω1.2-gliadins
was obtained at a capillary temperature of 25°C (4.33)
and the lowest at 20 and 35°C (2.67). A statistically
significant difference in the number of proteins was
found, F(4.25) = 9.08, Sig. = 0.000. ω5-gliadins
were identified in the highest number at a capillary
temperature of 25°C (6.67) and in the lowest number at
35°C (3.50). The one-factor analysis of variance revealed
a statistically significant difference, F(4.25) = 5.63,
Sig. = 0.002.
According to the results obtained, it can be seen that
with increasing capillary temperature, total proteins
increased, then decreased and increased slightly again.
α + β gliadin fractions decreased, then increased
and decreased slightly again. As for γ-, ω1.2- and
ω5-gliadins, their fractions increased, then decreased
and increased slightly again.
Table 5 shows descriptive indicators of the
total relative concentration of proteins and relative
concentration of gliadin proteins separated by fractions
after extraction with 70% (v/v) ethanol and separated at
different capillary temperatures.
According to the data, the highest relative
concentration of α + β gliadin fractions was obtained
after extraction with 70% ethanol and a capillary
temperature of 40°C (47.55%). The lowest concentration
was observed at 35°C (27.22%). One-factor variance
analysis revealed a statistically significant difference
Table 3 Descriptive indicators of the total relative concentration and relative concentration of gliadin fractions (Agilent, CE 7100,
capillary inside diameter 50 μm, total capillary length 33 cm, effective capillary length 23.50 cm, capillary temperature 25°C,
voltage –16.5 kV (reverse mode), duration 30 min, absorbance measured at 220 nm)
Ethanol,
% (v/v)
N RC, % SD Std. error 95% confidence interval of average Min Max
Lower bound Upper bound
Total relative
concentration
50 6 100.00 0.00 0.00 100.00 100.00 100 100
60 6 100.00 0.00 0.00 100.00 100.00 100 100
70 6 100.00 0.00 0.00 100.00 100.00 100 100
α + β gliadins 50 6 31.25 1.30 0.53 29.89 32.61 29.07 32.87
60 6 17.69 1.27 0.52 16.36 19.03 16.11 19.61
70 6 28.29 1.40 0.57 26.82 29.76 26.06 30.09
γ gliadins 50 6 27.72 1.15 0.47 26.51 28.92 26.50 29.57
60 6 18.55 0.97 0.40 17.53 19.57 17.21 19.99
70 6 26.66 1.35 0.55 25.25 28.08 24.75 28.13
ω1.2 gliadins 50 6 5.21 0.34 0.14 4.85 5.56 4.84 5.66
60 6 4.82 0.21 0.09 4.59 5.04 4.58 5.08
70 6 14.93 1.04 0.43 13.84 16.03 13.03 15.95
ω5 gliadins 50 6 36.16 0.70 0.29 35.42 36.90 34.97 37.01
60 6 47.45 1.37 0.56 46.01 48.89 45.81 49.39
70 6 30.98 3.13 1.28 27.70 34.27 29.55 37.36
ANOVA (α + β) F(2.15) = 174.13, Sig. = 0.000, eta square = 609.67/635.93 = 0.96
ANOVA (γ) F(2.15) = 111.01, Sig. = 0.000, eta square = 301.93/322.33 = 0.94
ANOVA (ω1.2) F(2.15) = 472.47, Sig. = 0.000, eta square = 394.02/400.27 = 0.98
ANOVA (ω5) F(2.15) = 104.83, Sig. = 0.000, eta square = 851.07/911.96 = 0.93
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in the relative concentration, F(4.25) = 193.61,
Sig. = 0.000. The relative concentration of γ-gliadins
was the highest at 20°C (43.88%) and the lowest at
30°C (24.48%). A statistically significant difference
in the relative concentration of different groups was
F(4.25) = 210.31, S ig. = 0 .000. A c apillary t emperature
of 35°C led to the highest relative concentration within
the ω1.2-group (27.21%), while 30°C provided the
lowest (14.03%). There was a statistically significant
difference in the relative concentration, F(4.25) = 165.39,
Sig. = 0 .000. T he h ighest r elative c oncentration o f ω 5-
gliadins was obtained after extraction with 70% ethanol
and at a capillary temperature of 25°C (30.98%) and
the lowest at 20°C (5.42%). The effect of capillary
temperature on relative protein concentration within ω5
gliadin fraction was examined by one-factor analysis
of variance. A statistically significant difference in the
relative concentration within the fraction was found,
F(4.25) = 195.85, Sig. = 0.000.
Based on the obtained results (Table 5), it can be
seen that with increasing capillary temperature, the
relative concentration of α + β gliadins decreased, then
increased, decreased, and increased again. Within
γ-gliadins, the relative concentration decreased,
then increased, and decreased again. The relative
concentration of ω1.2-gliadins increased, then
decreased, increased again and finally decreased. Within
the ω5 gliadin fractions, the relative concentration
increased and then decreased.
Table 6 shows descriptive indicators of total
proteins and the number of gliadin proteins separated
by fractions after extraction with 70% (v/v) ethanol and
separated applying different electrode voltages (reverse
mode).
The highest number of proteins was obtained after
extraction with 70% ethanol, according to the method
by Lookhart and Bean and electrophoretic separation
at a voltage of –16.5 kV (23.50). The lowest number of
proteins was obtained at –14.5 kV (14.83). It was found
that there is a statistically significant difference in
the number of proteins, F(3.20) = 4 6.16, Sig. = 0.000.
The highest and the lowest amounts of proteins within
Table 4 Descriptive indicators of total proteins and the number of gliadin fractions (70% ethanol, Agilent, CE 7100, capillary
inside diameter 50 μm, total capillary length 33 cm, effective capillary length 23.50 cm, voltage –16.5 kV (reverse mode), duration
30 min, absorbance measured at 220 nm)
Column temperature,
°C
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower Bound Upper bound
Total number
of proteins
20 6 20.83 1.47 0.60 19.29 22.38 19 23
25 6 23.50 1.05 0.43 22.40 24.60 22 25
30 6 21.67 1.75 0.71 19.83 23.50 19 23
35 6 18.83 1.47 0.60 17.29 20.38 16 20
40 6 19.33 1.03 0.42 18.25 20.42 18 21
α + β gliadins 20 6 10.00 1.09 0.45 8.85 11.15 8 11
25 6 7.50 0.84 0.34 6.62 8.38 6 8
30 6 8.50 0.84 0.34 7.62 9.38 7 9
35 6 8.83 0.75 0.31 8.04 9.62 8 10
40 6 8.67 0.82 0.33 7.81 9.52 8 10
γ gliadins 20 6 4.00 0.00 0.00 4.00 4.00 4 4
25 6 5.00 0.63 0.26 4.34 5.66 4 6
30 6 4.50 0.55 0.22 3.93 5.07 4 5
35 6 3.50 0.55 0.22 2.93 4.07 3 4
40 6 3.67 0.52 0.21 3.12 4.21 3 4
ω1,2 gliadins 20 6 2.67 0.52 0.21 2.12 3.21 2 3
25 6 4.33 0.82 0.33 3.48 5.19 3 5
30 6 3.17 0.41 0.17 2.74 3.60 3 4
35 6 2.67 0.52 0.21 2.12 3.21 2 3
40 6 3.17 0.41 0.17 2.74 3.60 3 4
ω5 gliadins 20 6 4.50 0.84 0.34 3.62 5.38 4 6
25 6 6.67 0.52 0.21 5.12 7.22 5 7
30 6 4.83 1.17 0.48 3.61 6.06 3 6
35 6 3.50 0.55 0.22 2.93 4.07 3 4
40 6 4.33 0.82 0.33 3.48 5.19 3 5
ANOVA (TP) F(4.25) = 11.02, Sig. = 0.000, eta square = 84.33/132.17 = 0.64
ANOVA (α + β) F(4.25) = 6.24, Sig. = 0.001, eta square = 19.13/38.30 = 0.50
ANOVA (γ) F(4.25) = 9.01, Sig. = 0.000, eta square = 9.13/15.47 = 0.59
ANOVA (ω1.2) F(4.25) = 9.08, Sig. = 0.000, eta square = 11.13/18.80 = 0.59
ANOVA (ω5) F(4.25) = 5.63, Sig. = 0.002, eta square = 14.87/31.37 = 0.47
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Table 5 Descriptive indicators of the total relative concentration of proteins and relative concentration of gliadin fractions (solvent
70% ethanol, Agilent, CE 7100, capillary inside diameter 50 μm, total capillary length 33 cm, effective capillary length 23.50 cm,
voltage –16.5 kV (reverse mode), duration 30 min, absorbance measured at 220 nm)
Column temperature,
°C
N RC, % SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
Total relative
concentration
20 6 100.00 0.00 0.00 100.00 100.00 100 100
25 6 100.00 0.00 0.00 100.00 100.00 100 100
30 6 100.00 0.00 0.00 100.00 100.00 100 100
35 6 100.00 0.00 0.00 100.00 100.00 100 100
40 6 100.00 0.00 0.00 100.00 100.00 100 100
α + β gliadins 20 6 36.38 1.11 0.45 35.21 37.55 34.90 37.88
25 6 28.29 1.40 0.57 26.82 29.76 26.06 30.09
30 6 33.86 1.29 0.52 32.51 35.21 32.27 35.38
35 6 27.22 2.11 0.86 25.01 29.44 26.00 31.49
40 6 47.55 0.99 0.41 46.50 48.60 46.01 48.99
γ gliadins 20 6 43.88 1.14 0.47 42.68 45.08 42.39 45.37
25 6 26.66 1.35 0.55 25.25 28.08 24.75 28.13
30 6 24.48 1.59 0.65 22.81 26.15 22.24 26.77
35 6 29.83 1.08 0.44 28.70 30.96 28.36 31.44
40 6 27.18 1.34 0.55 25.77 28.59 25.07 28.89
ω1.2 gliadins 20 6 14.75 0.89 0.36 13.81 15.69 13.28 15.87
25 6 14.93 1.04 0.43 13.84 16.03 13.03 15.95
30 6 14.03 1.34 0.55 12.62 15.44 12.07 15.71
35 6 27.21 1.35 0.55 25.80 28.63 25.10 28.95
40 6 14.46 0.58 0.24 13.85 15.07 13.71 15.13
ω5 gliadins 20 6 5.42 0.34 0.14 5.06 5.77 5.02 5.90
25 6 30.98 3.13 1.28 27.70 34.27 29.55 37.36
30 6 27.28 2.04 0.83 25.14 29.42 24.49 29.61
35 6 12.86 1.90 0.77 10.87 14.85 10.83 15.95
40 6 10.67 1.07 0.44 9.55 11.80 9.20 12.22
ANOVA (α + β) F(4.25) = 193.61, Sig. = 0.000, eta square = 1593.94/1645.39 = 0.97
ANOVA (γ) F(4.25) = 210.31, Sig. = 0.000, eta square = 1448.88/1491.94 = 0.97
ANOVA (ω1.2) F(4.25) = 165.39, Sig. = 0.000, eta square = 773.27/802.49 = 0.96
ANOVA (ω5) F(4.25) = 195.85, Sig. = 0.000, eta square = 2949.03/3043.13 = 0.97
the α+β gliadin fractions were obtained at a voltage of
–17.5 Kv and –14.5 kV (8.17 and 5.17, respectively).
A statistically significant difference in the number of
proteins was revealed, F(3.20) = 12.50, Sig. = 0.000.
Within γ-gliadins, the highest amount of proteins was
obtained at a voltage of –16.5 kV (5.00) and the lowest
at –14.5 kV (2.50). ANOVA test showed a statistically
significant difference in the number of proteins,
F(3.20) = 2 6.82, S ig. = 0 .000. T he h ighest n umber o f
proteins within the ω1.2 gliadin fractions was obtained
at a voltage of –16.5 kV (4.33). The lowest amount
of ω1.2 gliadins was obtained at –17.5 kV (2.50). A
statistically significant difference in the number of
proteins w as f ound, F (3.20) = 1 0.85, S ig. = 0 .000.
Descriptive analysis showed that the highest number
of proteins within the ω5 gliadin fractions was at
–16.5 kV (6.67) and the lowest at –17.5 kV (3.00). There
was a statistically significant difference in the number of
proteins, F(3.20) = 12.83, Sig. = 0.000.
We can seen that with increasing voltage, total
proteins increased, then decreased and increased slightly
again. Within the α + β and γ gliadin fractions, the
number of proteins increased and then decreased. Within
the fraction of ω1.2- and ω5-gliadins, the amount of
proteins increased, then decreased and increased slightly
again.
Table 7 shows descriptive indicators of the
total relative concentration of proteins and relative
concentration of gliadin proteins separated by fractions
after extraction with 70% (v/v) ethanol and separated by
applying different electrode voltages (reverse mode).
Descriptive analysis showed that the highest relative
concentration of α + β gliadins was obtained at a voltage
of –17.5 kV (65.13%). The lowest concentration within
this fraction was at –16.5 kV (28.29%). A statistically
significant difference in relative concentration was
F(3.20) = 851.47, Sig. = 0.000. The h ighest a nd the
lowest relative concentrations of γ-gliadins were
obtained at –14.5 kV and at –18.5 kV (27.37 and 21.87%,
respectively). A statistically significant difference
in the relative protein concentration was found,
F(3.20) = 2 0.47, S ig. = 0 .000. A v oltage o f – 14.5 k V
418
Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Х
Table 6 Descriptive indicators of total proteins and the number of gliadin fractions (solvent 70% ethanol, Agilent, CE 7100,
capillary inside diameter 50 μm, total capillary length 33 cm, effective capillary length 23.50 cm, capillary temperature 25°C,
duration 30 min, absorbance measured at 220 nm)
Voltage,
kV
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
Total number
of proteins
–14.5 6 14.83 1.17 0.48 13.61 16.06 13 16
–16.5 6 23.50 1.05 0.43 22.40 24.60 22 25
–17.5 6 17.33 1.21 0.49 16.06 18.60 15 18
–18.5 6 17.67 1.75 0.71 15.83 19.50 15 19
α + β gliadins –14.5 6 5.17 0.75 0.31 4.38 5.96 4 6
–16.5 6 7.50 0.84 0.34 6.62 8.38 6 8
–17.5 6 8.17 1.17 0.48 6.94 9.39 6 9
–18.5 6 7.17 0.75 0.31 6.38 7.96 6 8
γ gliadins –14.5 6 2.50 0.55 0.22 1.93 3.07 2 3
–16.5 6 5.00 0.63 0.26 4.34 5.66 4 6
–17.5 6 3.00 0.63 0.26 2.34 3.66 2 4
–18.5 6 3.00 0.00 0.00 3.00 3.00 3 3
ω1.2 gliadins –14.5 6 3.00 0.00 0.00 3.00 3.00 3 3
–16.5 6 4.33 0.82 0.33 3.48 5.19 3 5
–17.5 6 2.50 0.55 0.22 1.93 3.07 2 3
–18.5 6 3.00 0.63 0.26 2.34 3.66 2 4
ω5 gliadins –14.5 6 4.17 0.75 0.31 3.38 4.96 3 5
–16.5 6 6.67 0.52 0.21 5.12 7.22 5 7
–17.5 6 3.00 0.63 0.26 2.34 3.66 2 4
–18.5 6 4.67 1.03 0.42 3.58 5.75 3 6
ANOVA (TP) F(3.20) = 46.16, Sig. = 0.000, eta square = 242.33/277.33 = 0.87
ANOVA (α + β) F(3.20) = 12.50, Sig. = 0.000, eta square = 30.00/46.00 = 0.65
ANOVA (γ) F(3.20) = 26.82, Sig. = 0.000, eta square = 22.12/27.62 = 0.80
ANOVA (ω1.2) F(3.20) = 10.85, Sig. = 0.000, eta square = 11.12/17.96 = 0.62
ANOVA (ω5) F(3.20) = 12.83, Sig. = 0.000, eta square = 22.12/33.62 = 0.66
caused the highest (26.73%) and –18.5 the lowest (3.91%)
relative concentration of ω1.2-gliadins. The one-factor
analysis of variance showed a statistically significant
difference, F(3.20) = 1316.91, Sig. = 0.000. Relative
concentration of ω5-gliadins obtained at a voltage of
–18.5 kV was the highest (40.30%) and at –17.5 kV the
lowest (4.91%). There was a statistically significant
difference in the relative concentration, F(3.20) = 549.81,
Sig. = 0.000.
According to the results obtained, the increasing
voltage decreased then increased and decreased again
the relative concentration of α + β gliadin proteins.
Within the fraction of γ- and ω1.2- gliadins the
concentration decreased, and ω5-gliadins increased then
decreased and increased again.
Lookhart and Bean performed separation and
characterization of wheat proteins by high-pressure
capillary electrophoresis (HPCE) [21]. Gliadins
were extracted with 70% (v/v) ethanol. Separation of
proteins was performed at a voltage of 22 kV and at a
temperature of 45°C. The detection wavelength was 200
nm. Based on the obtained results, the retention time
of gliadin proteins was: α gliadins 3-4 min (molecular
weight according to SDS-PAGE 35–38 kDa), β 4–6 min
(37–43 kDa), γ 5–6 min (43–47 kDa), and ω 6.8–10 min
(48–63 kDa).
Bietz and Schmalzried analyzed gliadins from
wheat by capillary electrophoresis [25]. Gliadins
were extracted with ethanol and methanol of different
concentrations (30, 40, 50, 60 and 70% v/v), with
and without the reducing agent dithioerythritol. The
temperature of the capillary ranged from 30 to 50°C, and
the voltage from 8 to 12 kV. The detection wavelength
was 200 nm. Capillary temperature of 40°C and voltage
10 kV showed optimal conditions. Ethanol proved to be a
better solvent than methanol.
Changing ethanol concentration (50, 60, and 70%
v/v), capillary temperature (20, 25, 30, 35, and 40°C),
and voltage (–14.5; –16.5, –17.5, and –18.5 kV), we found
that the optimal conditions for separation of gliadin
proteins were 70% ethanol concentration, a capillary
temperature of 25°C, and a voltage of –16.5 kV (reverse
mode) (Fig. 3).
Our results are in agreement with Lookhart and
Bean and Bietz and Schmalzried [21, 25]. Although the
mentioned authors separated gliadin proteins by using
different techniques of capillary electrophoresis, 70%
ethanol proved to be the optimal solvent, which lines
up with our results [21]. The gliadin proteins in this
work were separated in less than 10 min, which is in
agreement with Lookhart and Bean [21].
419
Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Х
CONCLUSION
Based on the results obtained, the optimal conditions
for gladin separation were 70% ethanol concentration, a
capillary temperature of 25°C, and a voltage of –16.5 kV
(reverse mode). Under these conditions, total proteins
were 23.5, including α + β gliadin proteins (Xav = 7.50,
relative concentration 28.29%), γ-fractions (Xav = 5.00,
Table 7 Descriptive indicators of the relative concentration of proteins and relative concentration of gliadin fractions (solvent
70% ethanol, Agilent, CE 7100, capillary inside diameter 50 μm, total capillary length 33 cm, effective capillary length 23.50 cm,
capillary temperature 25°C, duration 30 min, absorbance measured at 220 nm)
Voltage,
kV
N RC, % SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
Total relative
concentration
–14.5 6 100.00 0.00 0,00 100.00 100.00 100 100
–16.5 6 100.00 0.00 0,00 100.00 100.00 100 100
–17.5 6 100.00 0.00 0,00 100.00 100.00 100 100
–18.5 6 100.00 0.00 0,00 100.00 100.00 100 100
α + β gliadins –14.5 6 35.42 1.27 0,52 34.09 36.75 33.06 36.81
–16.5 6 28.29 1.40 0,57 26.82 29.76 26.06 30.09
–17.5 6 65.13 1.90 0,78 63.13 67.13 61.63 67.05
–18.5 6 34.08 0.73 0,29 33.31 34.85 33.09 34.95
γ gliadins –14.5 6 27.37 1.03 0,42 26.29 28.45 26.10 29.22
–16.5 6 26.66 1.35 0,55 25.25 28.08 24.75 28.13
–17.5 6 25.51 1.24 0,51 24.20 26.81 23.81 26.96
–18.5 6 21.87 1.61 0,66 20.18 23.56 20.02 24.38
ω1.2
gliadins
–14.5 6 26.73 0.74 0,30 25.95 27.51 25.98 28.10
–16.5 6 14.93 1.04 0,43 13.84 16.03 13.03 15.95
–17.5 6 5.34 0.37 0,15 4.95 5.73 4.99 5.89
–18.5 6 3.91 0.50 0,20 3.39 4.43 3.21 4.52
ω5
gliadins
–14.5 6 10.66 0.75 0,31 9.87 11.45 9.92 11.98
–16.5 6 30.98 3.13 1,28 27.70 34.27 29.55 37.36
–17.5 6 4.91 1.01 0,41 3.85 5.97 3.99 6.73
–18.5 6 40.30 0.88 0,36 39.37 41.22 38.88 41.25
ANOVA (α + β) F(3.20) = 851.47, Sig. = 0.000, eta square = 4935.49/4974.13 = 0.99
ANOVA (γ) F(3.20) = 20.47, Sig. = 0.000, eta square = 107.68/142.75 = 0.75
ANOVA (ω1.2) F(3.20) = 1316.91, Sig. = 0.000, eta square = 2000.54/2010.67 = 0.99
ANOVA (ω5) F(3.20) = 549.81, Sig. = 0.000, eta square = 5014.18/5074.98 = 0.99
RC = 26.66%), ω1.2-gliadins (Xav = 4.33, RC = 14.93%),
and ω5-gliadins (Xav = 6.67, RC = 30.98%).
The results obtained in this paper can greatly
contribute to the prevention of the incorrect declaration
of the “gluten free” products, reduction of health risks
for people who are sensitive to gluten proteins, as well as
the cost of treating the ones with celiac disease.
Figure 3 Electrophoregram of gliadin proteins extracted from wheat flour using 70% (v/v) ethanol and separated by capillary gel
electrophoresis at a capillary temperature of 25°C and at a voltage of –16.5 kV
420
Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Х
In addition, the results of the research are of
fundamental importance in the study of gluten
proteins and the influence of technological procedures
on their change and the possibility of reducing the
allergic effect of individuals gluten proteins, during
processing.
CONTRIBUTION
Authors are equally related to the writing of the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.

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