INTRODUCTIONGliadin proteins represent one of the gluten fractions.Most gliadin proteins are present as monomers. Theyaffect 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 lowpH, i.e. in acidic conditions of A-PAGE electrophoresismedium (acid polyacrylamide gel electrophoresis). Basedon research that was later conducted on amino acidsequences, α and β gliadins were classified in the samegroup (α/β) [3–5].Modern methods, such as two-dimensionalelectrophoresis and high-pressure liquid chromatographywith reversed phase, allow the separation of gliadinfractions into more than a hundred components. Basedon the analysis of amino acid sequences (completeand partial), amino acid composition and molecularweight, gliadins are divided into: ω5, ω1.2, α + β and γ[3, 6–8]. ω-gliadins are characterized by a high contentof glutamine, proline and phenylalanine. These aminoacids together make up about 80% of the total ω gliadincomposition. 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 disulfidebinding. These proteins consist of repetitive sequencesthat 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 andproline is much lower compared to ω-gliadin. Theydiffer in tyrosine content. Each of the two types hasan N- and a C-terminal region [3, 11]. The N-terminalregion (40–50% of total proteins) consists of repeatingamino acid sequences that are rich in glutamine, proline,phenylalanine, and tyrosine. The repeating sequencesof α + β gliadin are dodecapeptides. They are repeatedfive times. A typical unit of γ-gliadin is repeated upto 16 times. They are interspersed with additionalremains [12, 13]. Within the C-terminal region α + βand γ-gliadins are homologous. The sequences arenot repeting. They contain less glutamine and prolinethan the N-terminal region and have a more commoncomposition. α + β and γ gliadins contain six or eightcysteine residues. These residues are located in theC-terminal region. They form intramolecular disulfidebonds [14, 15]. Although the content of total gliadinproteins depends on the type of wheat and growthconditions (soil, climate, fertilization), α + β andγ-gliadins are the highest components. Ω-gliadins arepresent in lower amounts [16–18].To separate gliadin proteins, the followingtechniques are used: high performance liquid chromatographywith reversed phase RP-HPLC, exclusionchromatography SE-HPLC, high performance capillaryelectrophoresis HPCE, sodium dodecyl sulphatepolyacrylamide gel electrophoresis SDS-PAGE,and isoelectric focusing IEF [19]. One of the newertechniques for gliadin separation is the highperformanceSDS-GCE, which is based on thedifference in electrophoretic mobility of ions in solutionwithin the capillary. The molecule size affects themobility of ions [20].The number of people who are allergic to glutenproteins from wheat is increasing, which makes foodproducers give their consumers a guarantee thatproducts declared as “gluten free” really do not containgluten. The aim of this study was to investigate optimalconditions (solvent concentrations, capillary temperatureand voltage) for their separation by high performancecapillary gel electrophoresis.STUDY OBJECTS AND METHODSGliadin extraction. We analyzed gliadins inwheat flour samples (ash content: max 0.55%, moisturemax: 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 performedaccording to a modified Osborne method, as describedby Lookhart and Bean [21]. After the albumins andglobulins were removed (extraction was performed3 times with 8 mL of deionized water each, it wasobtained in laboratory conditions, on the apparatusSiemens water Technologies W3T199551, SiemensUltra Clear, at a conductivity of 0.055 mS/cm and at atemperature 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 mlof 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 ethylalcohol, contains a minimum of 96% v/v ethanol).Samples were homogenized on a vortex (AdvancedVortex Mixer ZX3, 3000 rpm) for 30 min. The sampleswere then centrifuged in a centrifuge (Rotina 380 R,Hettich Zentrifugen) for 5 min at 1,000 rpm. Theresulting supernatant was poured into a normal 25 mLvessel, and after the third extraction the normal vesselwas made up to final volume with ethanol of variousconcentrations (50, 60 and 70% v/v). The precipitate wasthen washed with deionized water.Samples preparation for analysis at GCE. Priorto analysis samples were diluted with sample buffer(SDS-MW sample buffer, PA 800 plus, BeckmanCoulter, USA), so that the total volume was 95 μLand the concentration was 1 mg/mL. Then 2 μL ofinternal standard (10 kDa, PA 800 plus, BeckmanCoulter, United States) and 5 μL of 2-mercaptoethanol(high purity, 99.00%, Sigma-Aldrich Chemie GmbH,Germany) were added. The samples were then heatedon a thermo-shaker (Thermo-Shaker, TS-100, Biosan) at100°C for 3 min. After cooling to room temperature for5 min, the samples were ready for analysis by capillarygel electrophoresis (Agilent, CE 7100).Preparation SDS-MW standard for analysisby capillary gel electrophoresis. Prior to thepreparation standard, based on the recommendation ofthe kit manufacturer, the standard was taken to roomtemperature for 15 minutes after removal from therefrigerator. It was then carefully stirred on a vortex(Advanced Vortex Mixer ZX3, 3000 rpm) for a fewseconds. After that, 10 μL of standard (SDS-MWstandard, PA 800 plus, Beckman Coulter, UnitedStates) was pipetted into the vial. Then 85 μL of buffer(SDS-MW sample buffer, PA 800 plus, BeckmanCoulter, USA) and 2 μL of internal standard (10 kDa,PA 800 plus, Beckman Coulter, USA) were added. Then5 μL of 2-mercaptoethanol (Sigma-Aldrich ChemieGmbH, 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 for3 min. After heating, the standard vial was cooled toroom temperature over 5 min. Prepared in this way, thestandard is ready for analysis.Gliadin proteins separation by capillary gelelectrophoresis. Separation of gliadin proteins bycapillary gel electrophoresis was performed on anAgilent apparatus, CE 7100, with a capillary inner413Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Хdiameter of 50 μm, a total length of 33 cm, and aneffective length of 23.50 cm. The SDS-MW analysis kit,PA 800 plus (2015 Beckman Coulter, USA) was usedfor separation. SDS gel buffer (0.2% SDS, pH = 8) wasused to fill the capillary. The kit contains the followingchemicals: 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), basewash solution (0.1N NaOH), as well as two capillaries57 cm long, 50 μm ID. According to the manufacturer’sinstructions, the kit is stored at room temperature afteropening, except for the internal and external standards,which are stored at a temperature of 2–6°C. Preparationof the capillary electrophoresis (CE) instrument wasdone according recommendations Agilent Technologies[22–24].Statistical data processing. Statistical dataprocessing was performed in IBM SPSS, Statistics 26.Descriptive statistical analysis calculated the averagevalue, standard deviation and 95% confidence intervalof the average value. Variance analysis of differentgroups was used to evaluate the effect of solventconcentrations, capillary temperature and electrodevoltage on the number of detected proteins and therelative concentration of each gliadin proteins.RESULTS AND DISCUSSIONIn order to determine molecular weights unknownproteins, a calibration curve was obtained using 7proteins in SDS-MW size standard.Electrophoregram, the migration time, and thecalibration curve of MW standard proteins with knownmolecular 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 gelelectrophoresis (CE, Agilent, CE 7100, internal capillarydiameter 50 μm, total capillary length 33 cm, effectivecapillary length 23.50 cm, capillary temperature 25°C,voltage –16.5 kV (reverse mode), duration of analysis30 min, and absorbance measured at 220 nm).The ratio of molecular weights (log MW) andmigration time (t) of proteins is represented by theequation y = 0.08168x – 0.00098, where y representslogMW and x represents the migration time of proteins(t). R2 shows the correlation coefficient (0.9847).A calibration curve was used to estimate themolecular weight of unknown proteins. The coefficientof correlation shows a high dependence of the logarithmof the molecular weight of the protein and the migrationtime of the protein.The number of proteins in each gliadin fraction andtheir relative concentration were obtained based on thetotal number of identified proteins and the total relativeconcentration.Table 2 shows descriptive indicators of total proteinsand the number of gliadin proteins after extraction withdifferent concentrations of ethanol.Descriptive analysis showed that the highest numberof proteins (23.50) was obtained after extraction with70% ethanol, by the method of Lookhart and Bean. Thelowest number of proteins was obtained by extractionwith 50% ethanol (18.67). One-factor analysis of thevariance of different groups showed that there wasa statistically significant difference in the number ofFigure 1 Electrophoregram of MW standards of proteins of known molecular weight separated by capillary gel electrophoresisTable 1 Migration time of proteins with known molecularweight separated by capillary gel electrophoresisMolecular weight (MW), kDa log MW t, min10 1.00 13.36 ± 0.2120 1.30 15.77 ± 0.1835 1.54 18.13 ± 0.2650 1.70 20.15 ± 0.29100 2.00 24.25 ± 0.10150 2.18 26.78 ± 0.36225 2.35 29.41 ± 0.15mAU25201510505 10 15 20 25 30 min414Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Хproteins, F (2.15) = 2 3.70, S ig. = 0 .000. The highestnumber of within α + β gliadin fractions was obtainedafter extraction with 60% ethanol (7.67). The lowestnumber of those proteins was obtained after extractionwith 50% ethanol (6.00). A statistically significantdifference was found in the number of proteins,F(2.15) = 8.58, Sig. = 0.003.Extraction with 50 and 60% ethanol produced thehighest and the lowest number of proteins within theγ-gliadins (5.33 and 4.67, respectively). There wasno statistically significant difference in the numberof p roteins, F (2.15) = 1 .15, S ig. = 0 .342. T he h ighestamount of ω1.2-gliadins was obtained after extractionwith 60% ethanol (5.17), while the lowest after extractionwith 50 and 70% ethanol (4.33). One-factor varianceanalysis showed no statistically significant difference,F(2.15) = 2 .19, S ig. = 0 .146. T he h ighest n umber o fω5-gliadins was obtained after extraction with 70%ethanol (6.67). The lowest amount was observed afterextraction with 50% ethanol (3.83). A statisticallysignificant difference in the number of proteins wasfound, F(2.15) = 6.77, Sig. = 0.008.According to Table 2, an increasing ethanolconcentration increased total proteins, increased andthen slightly decreased α + β gliadin fraction, decreasedand then increased γ-gliadins, increased and thendecreased ω1.2-gliadins, and increased ω5 gliadinfractions.Table 3 shows descriptive indicators of the totalrelative concentration and the relative concentrationof gliadin proteins after extraction with differentconcentrations of ethanol.Descriptive analysis showed the highest relativeprotein concentration of α + β gliadin fractions afterextraction with 50% ethanol (31.25%) and the lowestconcentration after extraction with 60% ethanol(17.69%). One-factor variance analysis revealed astatistically significant difference in the relative proteinconcentration, F(2.15) = 174.13, Sig. = 0.000.Extraction with 50 and with 60% ethanol producedthe highest and the lowest relative concentration ofγ-gliadins (27.72 and 18.55%). A statistically significantFigure 2 Calibration curve obtained as the ratio of the logarithmof molecular weight and migration times of knownproteins separated by capillary gel electrophoresisTable 2 Descriptive indicators of total proteins and gliadin proteins separated by fractions (Agilent, CE 7100, capillary insidediameter 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.error95% confidence interval of average Min MaxLower bound Upper boundTotal numberof proteins50 6 18.67 1.21 0.49 17.40 19.94 17 2060 6 23.00 1.67 0.68 21.24 24.76 21 2570 6 23.50 1.05 0.43 22.40 24.60 22 25α + β gliadins 50 6 6.00 0.63 0.26 5.34 6.66 5 760 6 7.67 0.82 0.33 6.81 8.52 7 970 6 7.50 0.84 0.34 6.62 8.38 6 8γ gliadins 50 6 5.33 0.82 0.33 4.48 6.19 4 660 6 4.67 0.82 0.33 3.81 5.52 4 670 6 5.00 0.63 0.26 4.34 5.66 4 6ω1.2 gliadins 50 6 4.33 0.82 0.33 3.48 5.19 3 560 6 5.17 0.75 0.31 4.38 5.96 4 670 6 4.33 0.82 0.33 3.48 5.19 3 5ω5 gliadins 50 6 3.83 0.98 0.40 2.80 4.87 3 560 6 5.50 1.22 0.50 4.21 6.79 3 670 6 6.67 0.52 0.21 5.12 7.22 5 7ANOVA (TP) F(2.15) = 23.70, Sig. = 0.000, eta square = 84.78/111.61 = 0.76ANOVA (α + β) F(2.15) = 8.58, Sig. = 0.003, eta square = 10.11/18.94 = 0.53ANOVA (γ) F(2.15) = 1.15, Sig. = 0.342 > 0.05ANOVA (ω1.2) F(2.15) = 2.19, Sig. = 0.146 > 0.05ANOVA (ω5) F(2.15) = 6.77, Sig. = 0.008, eta square = 12.33/26.00 = 0.47415Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Хdifference in relative concentration was found,F(2.15) = 111.01, Sig. = 0.000. The relative concentrationof ω1.2-gliadins was the highest after extraction with70% ethanol (14.93%) and the lowest after extractionwith 60% ethanol (4.82%). The one-factor analysis of thevariance showed a statistically significant difference inthe relative concentration, F(2.15) = 472.47, Sig. = 0.000.As for ω5 gliadin fractions, they were found in thehighest concentration after extraction with 60% ethanol(47.45%) and the lowest after extraction with 70%ethanol (30.98%). There was a statistically significantdifference in the relative concentration, F(2.15) = 104.83,Sig. = 0.000.Based on the obtained results (Table 3), an increasingethanol concentration decreased and then increased therelative concentration of α + β, γ- and ω1.2-gliadins andincreased and then decreased that of ω5-gliadins.Table 4 shows descriptive indicators of the totalnumber of proteins and number of gliadin proteinsseparated 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 numberof 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 at35°C (18.83). The one-factor analysis of variance showeda statistically significant difference in the number ofproteins, F(4.25) = 11.02, Sig. = 0.000.The highest and the lowest numbers of α+β gliadinfractions were obtained at 20°C (10.00) and 25°C(7.50), respectively. There was a statistically significantdifference, F(4.25) = 6.24, Sig. = 0.001. The number ofγ-gliadins was the highest at 25°C (5.00) and the lowestat 35°C (3.50). ANOVA test showed a statisticallysignificant difference in the number of proteins, F(4.25)= 9.01, Sig. = 0.000. The highest amount of ω1.2-gliadinswas obtained at a capillary temperature of 25°C (4.33)and the lowest at 20 and 35°C (2.67). A statisticallysignificant difference in the number of proteins wasfound, F(4.25) = 9.08, Sig. = 0.000. ω5-gliadinswere identified in the highest number at a capillarytemperature of 25°C (6.67) and in the lowest number at35°C (3.50). The one-factor analysis of variance revealeda statistically significant difference, F(4.25) = 5.63,Sig. = 0.002.According to the results obtained, it can be seen thatwith increasing capillary temperature, total proteinsincreased, then decreased and increased slightly again.α + β gliadin fractions decreased, then increasedand decreased slightly again. As for γ-, ω1.2- andω5-gliadins, their fractions increased, then decreasedand increased slightly again.Table 5 shows descriptive indicators of thetotal relative concentration of proteins and relativeconcentration of gliadin proteins separated by fractionsafter extraction with 70% (v/v) ethanol and separated atdifferent capillary temperatures.According to the data, the highest relativeconcentration of α + β gliadin fractions was obtainedafter extraction with 70% ethanol and a capillarytemperature of 40°C (47.55%). The lowest concentrationwas observed at 35°C (27.22%). One-factor varianceanalysis revealed a statistically significant differenceTable 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 MaxLower bound Upper boundTotal relativeconcentration50 6 100.00 0.00 0.00 100.00 100.00 100 10060 6 100.00 0.00 0.00 100.00 100.00 100 10070 6 100.00 0.00 0.00 100.00 100.00 100 100α + β gliadins 50 6 31.25 1.30 0.53 29.89 32.61 29.07 32.8760 6 17.69 1.27 0.52 16.36 19.03 16.11 19.6170 6 28.29 1.40 0.57 26.82 29.76 26.06 30.09γ gliadins 50 6 27.72 1.15 0.47 26.51 28.92 26.50 29.5760 6 18.55 0.97 0.40 17.53 19.57 17.21 19.9970 6 26.66 1.35 0.55 25.25 28.08 24.75 28.13ω1.2 gliadins 50 6 5.21 0.34 0.14 4.85 5.56 4.84 5.6660 6 4.82 0.21 0.09 4.59 5.04 4.58 5.0870 6 14.93 1.04 0.43 13.84 16.03 13.03 15.95ω5 gliadins 50 6 36.16 0.70 0.29 35.42 36.90 34.97 37.0160 6 47.45 1.37 0.56 46.01 48.89 45.81 49.3970 6 30.98 3.13 1.28 27.70 34.27 29.55 37.36ANOVA (α + β) F(2.15) = 174.13, Sig. = 0.000, eta square = 609.67/635.93 = 0.96ANOVA (γ) F(2.15) = 111.01, Sig. = 0.000, eta square = 301.93/322.33 = 0.94ANOVA (ω1.2) F(2.15) = 472.47, Sig. = 0.000, eta square = 394.02/400.27 = 0.98ANOVA (ω5) F(2.15) = 104.83, Sig. = 0.000, eta square = 851.07/911.96 = 0.93416Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Хin the relative concentration, F(4.25) = 193.61,Sig. = 0.000. The relative concentration of γ-gliadinswas the highest at 20°C (43.88%) and the lowest at30°C (24.48%). A statistically significant differencein the relative concentration of different groups wasF(4.25) = 210.31, S ig. = 0 .000. A c apillary t emperatureof 35°C led to the highest relative concentration withinthe ω1.2-group (27.21%), while 30°C provided thelowest (14.03%). There was a statistically significantdifference 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% ethanoland at a capillary temperature of 25°C (30.98%) andthe lowest at 20°C (5.42%). The effect of capillarytemperature on relative protein concentration within ω5gliadin fraction was examined by one-factor analysisof variance. A statistically significant difference in therelative concentration within the fraction was found,F(4.25) = 195.85, Sig. = 0.000.Based on the obtained results (Table 5), it can beseen that with increasing capillary temperature, therelative concentration of α + β gliadins decreased, thenincreased, decreased, and increased again. Withinγ-gliadins, the relative concentration decreased,then increased, and decreased again. The relativeconcentration of ω1.2-gliadins increased, thendecreased, increased again and finally decreased. Withinthe ω5 gliadin fractions, the relative concentrationincreased and then decreased.Table 6 shows descriptive indicators of totalproteins and the number of gliadin proteins separatedby fractions after extraction with 70% (v/v) ethanol andseparated applying different electrode voltages (reversemode).The highest number of proteins was obtained afterextraction with 70% ethanol, according to the methodby Lookhart and Bean and electrophoretic separationat a voltage of –16.5 kV (23.50). The lowest number ofproteins was obtained at –14.5 kV (14.83). It was foundthat there is a statistically significant difference inthe number of proteins, F(3.20) = 4 6.16, Sig. = 0.000.The highest and the lowest amounts of proteins withinTable 4 Descriptive indicators of total proteins and the number of gliadin fractions (70% ethanol, Agilent, CE 7100, capillaryinside diameter 50 μm, total capillary length 33 cm, effective capillary length 23.50 cm, voltage –16.5 kV (reverse mode), duration30 min, absorbance measured at 220 nm)Column temperature,°CN Xav SD Std.error95% confidence interval of average Min MaxLower Bound Upper boundTotal numberof proteins20 6 20.83 1.47 0.60 19.29 22.38 19 2325 6 23.50 1.05 0.43 22.40 24.60 22 2530 6 21.67 1.75 0.71 19.83 23.50 19 2335 6 18.83 1.47 0.60 17.29 20.38 16 2040 6 19.33 1.03 0.42 18.25 20.42 18 21α + β gliadins 20 6 10.00 1.09 0.45 8.85 11.15 8 1125 6 7.50 0.84 0.34 6.62 8.38 6 830 6 8.50 0.84 0.34 7.62 9.38 7 935 6 8.83 0.75 0.31 8.04 9.62 8 1040 6 8.67 0.82 0.33 7.81 9.52 8 10γ gliadins 20 6 4.00 0.00 0.00 4.00 4.00 4 425 6 5.00 0.63 0.26 4.34 5.66 4 630 6 4.50 0.55 0.22 3.93 5.07 4 535 6 3.50 0.55 0.22 2.93 4.07 3 440 6 3.67 0.52 0.21 3.12 4.21 3 4ω1,2 gliadins 20 6 2.67 0.52 0.21 2.12 3.21 2 325 6 4.33 0.82 0.33 3.48 5.19 3 530 6 3.17 0.41 0.17 2.74 3.60 3 435 6 2.67 0.52 0.21 2.12 3.21 2 340 6 3.17 0.41 0.17 2.74 3.60 3 4ω5 gliadins 20 6 4.50 0.84 0.34 3.62 5.38 4 625 6 6.67 0.52 0.21 5.12 7.22 5 730 6 4.83 1.17 0.48 3.61 6.06 3 635 6 3.50 0.55 0.22 2.93 4.07 3 440 6 4.33 0.82 0.33 3.48 5.19 3 5ANOVA (TP) F(4.25) = 11.02, Sig. = 0.000, eta square = 84.33/132.17 = 0.64ANOVA (α + β) F(4.25) = 6.24, Sig. = 0.001, eta square = 19.13/38.30 = 0.50ANOVA (γ) F(4.25) = 9.01, Sig. = 0.000, eta square = 9.13/15.47 = 0.59ANOVA (ω1.2) F(4.25) = 9.08, Sig. = 0.000, eta square = 11.13/18.80 = 0.59ANOVA (ω5) F(4.25) = 5.63, Sig. = 0.002, eta square = 14.87/31.37 = 0.47417Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–ХTable 5 Descriptive indicators of the total relative concentration of proteins and relative concentration of gliadin fractions (solvent70% 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,°CN RC, % SD Std.error95% confidence interval of average Min MaxLower bound Upper boundTotal relativeconcentration20 6 100.00 0.00 0.00 100.00 100.00 100 10025 6 100.00 0.00 0.00 100.00 100.00 100 10030 6 100.00 0.00 0.00 100.00 100.00 100 10035 6 100.00 0.00 0.00 100.00 100.00 100 10040 6 100.00 0.00 0.00 100.00 100.00 100 100α + β gliadins 20 6 36.38 1.11 0.45 35.21 37.55 34.90 37.8825 6 28.29 1.40 0.57 26.82 29.76 26.06 30.0930 6 33.86 1.29 0.52 32.51 35.21 32.27 35.3835 6 27.22 2.11 0.86 25.01 29.44 26.00 31.4940 6 47.55 0.99 0.41 46.50 48.60 46.01 48.99γ gliadins 20 6 43.88 1.14 0.47 42.68 45.08 42.39 45.3725 6 26.66 1.35 0.55 25.25 28.08 24.75 28.1330 6 24.48 1.59 0.65 22.81 26.15 22.24 26.7735 6 29.83 1.08 0.44 28.70 30.96 28.36 31.4440 6 27.18 1.34 0.55 25.77 28.59 25.07 28.89ω1.2 gliadins 20 6 14.75 0.89 0.36 13.81 15.69 13.28 15.8725 6 14.93 1.04 0.43 13.84 16.03 13.03 15.9530 6 14.03 1.34 0.55 12.62 15.44 12.07 15.7135 6 27.21 1.35 0.55 25.80 28.63 25.10 28.9540 6 14.46 0.58 0.24 13.85 15.07 13.71 15.13ω5 gliadins 20 6 5.42 0.34 0.14 5.06 5.77 5.02 5.9025 6 30.98 3.13 1.28 27.70 34.27 29.55 37.3630 6 27.28 2.04 0.83 25.14 29.42 24.49 29.6135 6 12.86 1.90 0.77 10.87 14.85 10.83 15.9540 6 10.67 1.07 0.44 9.55 11.80 9.20 12.22ANOVA (α + β) F(4.25) = 193.61, Sig. = 0.000, eta square = 1593.94/1645.39 = 0.97ANOVA (γ) F(4.25) = 210.31, Sig. = 0.000, eta square = 1448.88/1491.94 = 0.97ANOVA (ω1.2) F(4.25) = 165.39, Sig. = 0.000, eta square = 773.27/802.49 = 0.96ANOVA (ω5) F(4.25) = 195.85, Sig. = 0.000, eta square = 2949.03/3043.13 = 0.97the 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 ofproteins was revealed, F(3.20) = 12.50, Sig. = 0.000.Within γ-gliadins, the highest amount of proteins wasobtained at a voltage of –16.5 kV (5.00) and the lowestat –14.5 kV (2.50). ANOVA test showed a statisticallysignificant difference in the number of proteins,F(3.20) = 2 6.82, S ig. = 0 .000. T he h ighest n umber o fproteins within the ω1.2 gliadin fractions was obtainedat a voltage of –16.5 kV (4.33). The lowest amountof ω1.2 gliadins was obtained at –17.5 kV (2.50). Astatistically significant difference in the number ofproteins w as f ound, F (3.20) = 1 0.85, S ig. = 0 .000.Descriptive analysis showed that the highest numberof proteins within the ω5 gliadin fractions was at–16.5 kV (6.67) and the lowest at –17.5 kV (3.00). Therewas a statistically significant difference in the number ofproteins, F(3.20) = 12.83, Sig. = 0.000.We can seen that with increasing voltage, totalproteins increased, then decreased and increased slightlyagain. Within the α + β and γ gliadin fractions, thenumber of proteins increased and then decreased. Withinthe fraction of ω1.2- and ω5-gliadins, the amount ofproteins increased, then decreased and increased slightlyagain.Table 7 shows descriptive indicators of thetotal relative concentration of proteins and relativeconcentration of gliadin proteins separated by fractionsafter extraction with 70% (v/v) ethanol and separated byapplying different electrode voltages (reverse mode).Descriptive analysis showed that the highest relativeconcentration of α + β gliadins was obtained at a voltageof –17.5 kV (65.13%). The lowest concentration withinthis fraction was at –16.5 kV (28.29%). A statisticallysignificant difference in relative concentration wasF(3.20) = 851.47, Sig. = 0.000. The h ighest a nd thelowest relative concentrations of γ-gliadins wereobtained at –14.5 kV and at –18.5 kV (27.37 and 21.87%,respectively). A statistically significant differencein the relative protein concentration was found,F(3.20) = 2 0.47, S ig. = 0 .000. A v oltage o f – 14.5 k V418Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–ХTable 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,kVN Xav SD Std.error95% confidence interval of average Min MaxLower bound Upper boundTotal numberof 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 19α + 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 8γ 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 3ω1.2 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 4ω5 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 6ANOVA (TP) F(3.20) = 46.16, Sig. = 0.000, eta square = 242.33/277.33 = 0.87ANOVA (α + β) F(3.20) = 12.50, Sig. = 0.000, eta square = 30.00/46.00 = 0.65ANOVA (γ) F(3.20) = 26.82, Sig. = 0.000, eta square = 22.12/27.62 = 0.80ANOVA (ω1.2) F(3.20) = 10.85, Sig. = 0.000, eta square = 11.12/17.96 = 0.62ANOVA (ω5) F(3.20) = 12.83, Sig. = 0.000, eta square = 22.12/33.62 = 0.66caused the highest (26.73%) and –18.5 the lowest (3.91%)relative concentration of ω1.2-gliadins. The one-factoranalysis of variance showed a statistically significantdifference, F(3.20) = 1316.91, Sig. = 0.000. Relativeconcentration of ω5-gliadins obtained at a voltage of–18.5 kV was the highest (40.30%) and at –17.5 kV thelowest (4.91%). There was a statistically significantdifference in the relative concentration, F(3.20) = 549.81,Sig. = 0.000.According to the results obtained, the increasingvoltage decreased then increased and decreased againthe relative concentration of α + β gliadin proteins.Within the fraction of γ- and ω1.2- gliadins theconcentration decreased, and ω5-gliadins increased thendecreased and increased again.Lookhart and Bean performed separation andcharacterization of wheat proteins by high-pressurecapillary electrophoresis (HPCE) [21]. Gliadinswere extracted with 70% (v/v) ethanol. Separation ofproteins was performed at a voltage of 22 kV and at atemperature of 45°C. The detection wavelength was 200nm. Based on the obtained results, the retention timeof gliadin proteins was: α gliadins 3-4 min (molecularweight 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 fromwheat by capillary electrophoresis [25]. Gliadinswere extracted with ethanol and methanol of differentconcentrations (30, 40, 50, 60 and 70% v/v), withand without the reducing agent dithioerythritol. Thetemperature of the capillary ranged from 30 to 50°C, andthe voltage from 8 to 12 kV. The detection wavelengthwas 200 nm. Capillary temperature of 40°C and voltage10 kV showed optimal conditions. Ethanol proved to be abetter 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 foundthat the optimal conditions for separation of gliadinproteins were 70% ethanol concentration, a capillarytemperature of 25°C, and a voltage of –16.5 kV (reversemode) (Fig. 3).Our results are in agreement with Lookhart andBean and Bietz and Schmalzried [21, 25]. Although thementioned authors separated gliadin proteins by usingdifferent techniques of capillary electrophoresis, 70%ethanol proved to be the optimal solvent, which linesup with our results [21]. The gliadin proteins in thiswork were separated in less than 10 min, which is inagreement with Lookhart and Bean [21].419Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–ХCONCLUSIONBased on the results obtained, the optimal conditionsfor gladin separation were 70% ethanol concentration, acapillary temperature of 25°C, and a voltage of –16.5 kV(reverse mode). Under these conditions, total proteinswere 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 (solvent70% 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,kVN RC, % SD Std.error95% confidence interval of average Min MaxLower bound Upper boundTotal relativeconcentration–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 100α + 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 34.95γ 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 24.38ω1.2gliadins–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 4.52ω5gliadins–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 41.25ANOVA (α + β) F(3.20) = 851.47, Sig. = 0.000, eta square = 4935.49/4974.13 = 0.99ANOVA (γ) F(3.20) = 20.47, Sig. = 0.000, eta square = 107.68/142.75 = 0.75ANOVA (ω1.2) F(3.20) = 1316.91, Sig. = 0.000, eta square = 2000.54/2010.67 = 0.99ANOVA (ω5) F(3.20) = 549.81, Sig. = 0.000, eta square = 5014.18/5074.98 = 0.99RC = 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 greatlycontribute to the prevention of the incorrect declarationof the “gluten free” products, reduction of health risksfor people who are sensitive to gluten proteins, as well asthe 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 gelelectrophoresis at a capillary temperature of 25°C and at a voltage of –16.5 kV420Grujić R. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–ХIn addition, the results of the research are offundamental importance in the study of glutenproteins and the influence of technological procedureson their change and the possibility of reducing theallergic effect of individuals gluten proteins, duringprocessing.CONTRIBUTIONAuthors are equally related to the writing of themanuscript and are equally responsible for plagiarism.CONFLICT OF INTERESTThe authors declare no potential conflict of interest.