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Research ArticleFour Derivative Spectrophotometric Methods forthe Simultaneous Determination of Carmoisine and Ponceau4R in Drinks and Comparison with High PerformanceLiquid Chromatography

Fatma Turak1 Mithat Dinccedil2 Oumlznur Duumllger1 and Mahmure Ustun Oumlzguumlr1

983089 Department o Chemistry Faculty o Science and Art Yıldız Technical University 983091983092983090983090983088 Istanbul Turkey 983090 Tekirdag City Food Control Laboratory 983093983097983088983088983088 Tekirdag Turkey

Correspondence should be addressed to Fatma urak furakyildizedutr

Received 983094 October 983090983088983089983091 Revised 983089983088 December 983090983088983089983091 Accepted 983089983088 December 983090983088983089983091 Published 983089983090 February 983090983088983089983092

Academic Editor Hian Kee Lee

Copyright copy 983090983088983089983092 Fatma urak et al Tis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Four simple rapid and accurate spectrophotometric methods were developed or the simultaneous determination o two oodcolorantsCarmoisine (E983089983090983090)and Ponceau983092R (E983089983090983092) in their binarymixtures andsof drinks Te1047297rst methodis basedon recordingthe 1047297rst derivative curves and determining each component using the zero-crossing technique Te second method uses the 1047297rst

derivative o ratio spectra Te ratio spectra are obtained by dividing the absorption spectra o the binary mixture by that o oneo the components Te third method derivative differential procedure is based on the measurement o difference absorptivitiesderivatized in 1047297rst order o solution o drink samples in 983088983089 N NaOH relative to that o an equimolar solution in 983088983089 N HCl atwavelengths o 983091983094983094 and 983092983093983089nm or Carmoisine and Ponceau 983092R respectively Te last method based on the compensation methodis presented or derivative spectrophotometricdetermination o E983089983090983090 and E983089983090983092mixtures with overlappingspectra By using ratios o the derivative maxima the exact compensation o either component in the mixture can be achieved ollowed by its determinationTese proposed methods have been successully applied to the binary mixtures and sof drinks and the results were statistically compared with the reerence HPLC method (NMKL 983089983091983088)

1 Introduction

Color is a vital constituent o ood and probably the 1047297rstcharacteristic perceived by the human senses Probablyor economic reasons brightly colored and stable syntheticcolorants have been widely used as ood additives Tesecolorants are used to supplement and enhance natural colorsdestroyed during processing or storage and substantially increase the appeal and acceptability o ood stuff Te termcolor additive can be applied to any dye pigment or othersubstances arti1047297cially made or obtained rom a vegetableanimal mineral or another natural source[983089] Some syntheticcolorants and their metabolic products are consumed in largeamounts maybe toxic and cause cancer deormation and soorth [983090]

Carmoisine (E983089983090983090) and Ponceau 983092R (E983089983090983092) are syntheticdyes that contain azo and aromatic ring structures and areofen used in dyeing ood drink medicine and cosmetics(Scheme 983089)

Te development o chemical and instrumental methodsor the separation identi1047297cation and quantitative analysiso synthetic ood colorants has become extremely importantor the ood and beverages industry and academic andgovernmental institutions to assess the quality and saety o ood products [983091] Although the amounts o synthetic colorsthat are added to ood and drinks are strictly controlledthey may exceed the authorized levels Tus monitoring o the levels o colorants in high consumption products such asbeveragesbecomes o great importance Te number o works

Hindawi Publishing CorporationInternational Journal of Analytical Chemistry Volume 2014 Article ID 650465 11 pageshttpdxdoiorg1011552014650465



983090 International Journal o Analytical Chemistry

N N

N N

OH

HO

NaO3S

NaO3S

NaO3S

NaO3S

SO3Na

Carmoisine (Azorubine E122) CI 14720 Ponceau 4R (Cochineal Red A E124) CI 16255

S983139983144983141983149983141 983089 Chemical structures common names European community numbers (E) and color index (CI) numbers o synthetic oodcolorants studied

published during the last years demonstrates the importanceo this problem andthe need ordevelopingast accurateand

selective techniques or synthetic colorants analysis [983092]Various techniques have been introduced or the deter-

mination o mixtures o colorants with three or more com-ponent combination in ood samples

Tese include capillary electrophoresis [983093] differentialpulse polarography [983092] high perormance liquid chromatog-raphy [983094ndash983097] electrospray mass spectrometry [983089983088] spec-trophotometry [983089983089ndash983089983091] partial least squares [983089983092] and spec-tro1047298uorimetry [983089983093] Chromatographic methods have beenused or colorant analysis in oods and they are recom-mended when the mixture contains many different colorants[983094 983095 983089983094 983089983095] However there are some potential drawbackssuch as usage o toxic solvents the need or complex samplepretreatments and the resulting waste product Capillary electrophoresis is also very suitable when the sample containsseveral colorants but oods beverages and pharmaceuticalscontain only one two or rarely three ood dyes and thesemethods need special equipment that may not be availablein certain quality laboratories All multivariate calibrationmethods including Partial Least Square (PLS) require thedata processing with powerul sofware as well as the manip-ulations o the abstract vector space and its application tothe regression analysis and various chemometric methodsare also ofen used or more complex mixtures Tus ana-lytical methods rom alternative technique are always useul

especially i the methods are simple cheap and compara-tively ast Spectrophotometry as alternative methodologyis suitable or routine laboratories especially or developingcountries and sometimes serious analytical problems canbe resolved by this common technique Among computer-controlled instruments derivative techniques (ratio deriva-tive difference derivative and compensation method) areplaying a very important role in the binary analysis o mixtures by UV-VIS molecular absorption spectrophotom-etry All approaches are useul in the resolution o bandoverlapping in quantitative analysis

In this present work selective sensitive simple andlow-cost procedures were developed or the simultaneous

determination o Carmoisine (C) and Ponceau 983092R (P) insynthetic mixtureand drink samples by using derivative spec-

trophotometry (DSM) ratio derivative spectrophotometry (RDM) derivative differential spectrophotometry (DDM)and compensation method (CM) Tese methods have beensuccessully applied to synthetic mixtures and drink samplesand the results obtained were compared with those obtainedby HPLC method according to NMKL 983089983091983088 [983089983096]

No extraction or evaporation step no complexationagent andno harmulchemicals are involvedin the suggestedmethods in thesuggested methods so canbe used orroutineanalysis o both colorants in quality control and routinelaboratories

2 Experimental983090983089 Apparatus All spectral measurements and treatment o data were carried out using a Shimadzu UV-983090983092983093983088 doublebeam UV-Vis spectrophotometer with two matched 983089-cmquartz cells connected to a Hewlett Packard compatiblepersonal computer and a HP 983089983089983088983090 Laser Jet Printer BundledUV-PC personal spectroscopy sofware o version 983090983090983089 wasused to process the absorption and the derivative spectraTe spectral band width was 983089 nm with wavelength scanningspeed o medium HPLC measurements were perormed withan Agilent 983089983089983088983088 series HPLC-DAD system with a vacuumdegasser quaternary pump auto sampler and injector with a983089983088983088 L loop volume Separation was achieved using a WatersSpherisorb C983089983096 column (983092983094 times 983090983093983088mm 983093 m) and variablewavelength UV-VIS DAD detector measuring at 983093983090983088 nm orCarmoisine and 983093983089983090 nm or Ponceau 983092R

983090983090 Chromatographic Conditions Separation was achievedusing a Waters Spherisorb C18 column (983092983094 times 983090983093983088mm983093 m)Te solvent system consisted o a mixture o an aqueoussolution containing 983088983088983088983093 M tetrabutylammonium hydro-gen sulphate (BA) and a phosphate buffer solution (pH983094983093) methanol (983093983088 983093983088 vv) It was pumped isocratically at

983088983095mLminminus1 or the separation Te eluents were monitoredat 983093983089983090 and 983093983090983088 nm or P and C respectively



International Journal o Analytical Chemistry 983091

983137983138983148983141 983089 Experimental parameters calculated or the simultaneous determination o Carmoisine and Ponceau 983092R in binary mixture by compensation method

Colorants Concentration range (g mLminus983089) Ratio Meana RSD ()

Carmoisine 983090ndash983089983088 983089D983092983095983090983089D983093983094983097 983088983094983088983088 plusmn 983088983088983088983090 983088983091983091983091

Ponceau 983092R 983090ndash983089983088 983089D983092983095983088983089D983093983093983092 983088983094983097983088 plusmn 983088983088983088983097 983089983091983088983092a

Mean o 1047297ve separate determinations

983137983138983148983141 983090 Statistical parameters o the simultaneous determination o Carmoisine and Ponceau 983092R by DSM RDM and DDM

Methods AnalyteSelected

wavelength(nm)

Concentrationrange

(g mLminus983089)Regression equationsa

Correlationcoefficient

(1038389)

Detection limit(g mLminus983089)

Quanti1047297cationlimit

(g mLminus983089)

DSM C 983089D983091983091983089 983090ndash983089983088 156sdot10minus41103925+190sdot10minus5 983088983097983097983097983093 983088983088983096983094 983088983090983096983094

P 983089D983093983089983095 983090ndash983089983088 232sdot10minus41103925minus320sdot10minus5 983088983097983097983097983094 983088983088983097983089 983088983091983088983092

RDM C 983089D983091983090983091 983090ndash983089983088 413sdot10minus31103925minus323sdot10minus4 983088983097983097983097983092 983088983088983095983097 983088983090983094983091


DDM

C 983089D983091983094983094 983090ndash983089983088 325sdot10minus41103925+127sdot10minus4 983088983097983097983097983094 983088983088983095983089 983088983090983091983094

P 983089D983092983093983089 983090ndash983089983088 439sdot10minus41103925minus156sdot10minus4 983088983097983097983097983096 983088983088983096983089 983088983090983095983088aFive separate determinations were perormed and mean was calculatedlowast the concentration o the analyte (1038389g mLminus983089)

983090983091 Chemicals and Materials Distilled waterwas used to pre-pare the solutions and or mobile phase All chemicals usedwere o analytical reagent grade and were purchased romMerck (Darmstadt Germany) Ponceau 983092R (E983089983090983092 CochinealRed A molecular weight 983094983088983092983092983095 CI 983089983094983090983093983093 CAS number 983090983094983089983089-983096983090-983095) and Carmoisine (E983089983090983090 Azorubine molecular weight983093983088983090983092983092 CI 983089983092983095983090983088CAS number 983093983091983088983090983094-983094983097-983097) were rom Sigma-

Aldrich (Steinheim Germany) Sof drinks were purchasedrom commercial markets

983090983092 Standard Solutions and Calibration Graphs Stock solu-tions o Carmoisine and Ponceau 983092R (983089983088983088 g mLminus1) wereprepared by dissolving appropriate weights in distilled waterVarious aliquots o each stock solution within the concen-tration range stated in able 983090 were transerred into 1047297ve setso 983089983088 mL volumetric 1047298asks and the volume was completedwith distilled water Te stability o the working solutions o Carmoisine and Ponceau 983092R was studied by recording thetime-dependent absorptionspectra no changes in the spectra

were observed or at least one month when the solutions arestored at room temperature in the dark

For DDM method accurate volume aliquots o the stock solution were transerred into two sets o 983089983088 mL volumetric1047298asks and the volume was made up with 983088983089 N HCI and 983088983089 NNaOH to give a series o equimolar solutions containing 983090983088ndash

983089983088983088 g mLminus1 o both Carmoisine and Ponceau 983092R Calibra-tion solutions (907317 = 5) were used to construct the calibrationcurves in the standardization o the cited methods

For HPLC method stock solutions o (983090983093 mg983089983088983088 mL)Carmoisine and Ponceau 983092R were prepared in a 983089 983089 vv mixture o methanolmdashBA buffer solution (983088983088983088983093 M BA +983088983088983088983093 M phosphate buffer pH 983094983093) Te standard solutions

were prepared containing 983093ndash983090983088 g mLminus1 o Carmoisine andPonceau 983092R in the same solvent system All prepared solu-tions were 1047297ltered through 983088983092983093 m membrane 1047297lter beoreinjection

Commercial drink samples due to turbid are treatmentwith the Carrez I and II solutions

Carrez solution I it contains potassium hexacyano-errate (II) trihydrate (K4[Fe (CN)6] times 983091H2O) 983089983088983088 mL

concentration 983089983093 g times 983089983088983088mLminus1

Carrez solution II it contains zinc sulate heptahy-drate (ZnSO4 times 983095H2O) 983089983088983088 mL concentration 983091983088 g times

983089983088983088 mLminus1

983090983093 Spectrophotometric Measurements

(i) Final solutions or measurements were prepared overthe concentration ranges given in able 983090

(ii) Te derivative spectra were recorded over the wave-length range o 983091983088983088ndash983095983088983088 nm Te amplitude o the

derivative curve was measured at the selected wave-lengths or each method (able 983089)

(iii) For the compensation method the absorbance differ-ence spectra (sample versus reerence) were recordedover the wavelength range o 983091983088983088ndash983095983088983088 nm usingdifferent concentrations o the reerence solutionprepared rom the stock solution

(iv) For the DD method the Carmoisine and Ponceau 983092R solutions in 983088983089 N HCI difference derivative spectrawere recorded against corresponding 983088983089 N NaOHsolutions o the colorants as a blank Te amplitudeo the 1047297rst derivative spectra (able 983090) was measuredat the chosen wavelengths




983090983094 Procedures

983090983094983089 Derivative Spectrophotometric Method (DSM) Ali-quots o Carmoisine working solutions equivalent to 983090983088ndash

983089983088983088 g mLminus1 were placed into 983089983088 mL calibration 1047298asks andthe volume was made up with distilled water Te absorption

spectra were recorded versus distilled water Next the zero-order spectra were transormed into 1047297rst derivative Teamplitude rom the baseline to peak at 983091983091983089 nm (zero-crossingpoint or Ponceau 983092R) was related to the actual contents o Carmoisine

Appropriate aliquots o aqueous working solutions o Ponceau 983092R were placed into 983089983088 mL calibration 1047298asks andthe volume was made up with distilled water Te spectra o standard solutions in the concentrationrange 983090983088ndash983089983088g mLminus1

were recorded and subjected to the mathematical treatment

(1047297rst derivative) Te values o 1D517 measured at 983093983089983095 nm(zero-crossing point or Carmoisine) were used or construc-tion o calibration graph

983090983094983090 Ratio Derivative Spectrophotometric Method (RDM)Accuratelyaliquots o 983088983090 983088983092983088983094 983088983096 and983089983088 mL o colorantsstock solution were transerred into 983089983088 mL volumetric 1047298asksand the volume was made up with distilled water Te spectrao the prepared standard solutions were scanned rom 983091983088983088 to983095983088983088 nm and stored in the computer For the determination o Carmoisine the stored spectra o Carmoisine were dividedseparately by the spectrum o 983094 g mLminus1 Ponceau 983092R to obtainthe ratio spectra Te 1047297rst derivative corresponding to each

ratio spectrum (1DD) was recorded A calibration graph

relating the peak amplitudes (1DD323) to the corresponding

concentrations in g mLminus1

o Carmoisine was constructedIn order to determine Ponceau 983092R stored spectra o Pon-

ceau 983092R standard solutions were divided by a standard spec-

trum o Carmoisine o 983094 g mLminus1 Te 1047297rst ratio derivativespectra were calculated Te concentration o the Ponceau 983092R wasproportional to the amplitude o the maximum at 983091983092983092 nm(1D344)

983090983094983091 Derivative Differential Spectrophotometric Method (DDM) Te difference spectra between the acidic (983088983089 NHCI) solution and equimolar basic (983088983089 N NaOH) solutiono pure colorants and drink samples were recorded rom

983091983088983088983088 to 983095983088983088983088 nm by placing the acidic solution in thesample compartment and the basic solution in the reerencecompartment A 1047297rst derivative spectrum o each o thedifferential curves was subsequently recorded Te derivativeabsorbance values were measured at 983091983094983094 and 983092983093983089 nm orCarmoisine and Ponceau 983092R respectively

983090983094983092 Compensation Method (CM)

(983089) Determination o Standard Ratios Te 1047297rst derivativespectra or each set o standard solutions were recorded usingthe appropriate blank solution Te ratios o the 1047297rst deriva-

tive maxima and minima ( 1D11039251

1D11039252

) where appropriate at

the speci1047297ed wavelengths (1 and 2) as indicated in able 983089were calculated

(983090) Compensation Spectrophotometric Measurements Tesolution o the mixture (containing compounds Carmoisineand Ponceau 983092R) was placed in the sample cell A series o

solutions containing various concentrations (983090ndash983089983088 g mL

minus1

)o Carmoisine above and below that present in the mixturesolution were prepared and placed in succession in thereerence cell Te 1047297rst derivative spectra (1D) were recordedand the corresponding ratios (able 983089) in each instancewere calculated and ollowed the calculated ratio or purecolorant Ponceau 983092R Te exact balance point (the ratio o the mixture is equal to that o pure compound Ponceau 983092R)was determined at which the concentration o Carmoisine insample solution is equal to that in the reerence solution

By analogy the same steps were ollowed by using solu-tions o pure Ponceau 983092R in the reerence cell to determine itsconcentration in the mixture at the balance point

Te same principle was also applied or the analysis o drink samples

(983091) Graphical Method o avoid the excessive preparation o several standard solutions o either compound Carmoisine orin order to locate the exact balance point a graphical methodis recommended [983089983097 983090983088]

Te same steps as or the compensation method wereollowed andthe derivative ratio o the mixture (in the samplecell) was calculated at each time and plotted against the con-centration o pure compound Carmoisine (in the reerencecell) A line with slight curvature is usually obtained Teconcentration o Carmosine can easily be interpolated romthe graph by substituting the ratio o pure Ponceau 983092R At

the point at which the ratio o the mixture is equal to thato pure Ponceau 983092R the concentration o Carmoisine in thereerence cell is equal to that in the mixture in the sample cellBy analogy the concentration o compound Ponceau 983092R canbe obtained

983090983094983093 HPLC Method (Reerence Method NMKL 983089983091983088) Tisreerence method is described in detail in Nordic Committeeon Food Analysis [983089983096] About 983090983088 L o each sample wasinjected and the concentrations were calculated on the basiso the ratioso peak areaswith those o thestandard solutionsTe chromatograms o two compounds and drink samples

were plotted and stored in the computer Separation wascarried out in room temperature Te mobile phase wasprepared daily 1047297ltered through a 983088983092983093 m membrane 1047297lterAll chromatograms were run with the use o a (983093983088 983093983088 vv)mixture o methanol and BA buffer Other experimentalparameters were 1047298ow rate 983088983095 mL minminus1 retention times983093983093983092 min or Ponceau 983092R and 983090983089983094983090 min or Carmoisine

983090983094983094 Application o the Proposed Methods or the Deter-mination o Two Colorants in Drink Sample Commercialsamples were mixed with Carrez (I) and Carrez (II) solutionsOnce precipitated the samples were centriuged 983093983088 mL o the sample that is clari1047297ed by Carrez precipitation was




0175

0375

0575

0775

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 0

7 0 0

A b

s o r b a n c e

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

3 0 0

3 2 0

3 4 0

F i r s t

d e r i v a

t i v e

Wavelength (nm)

P

C

00002

00022

4 7 5

5 0 0

5 2 5

5 5 0

5 7 5

F i r s t

d e r i v a

t i v e

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)

F983145983143983157983154983141 983089 Zero-order spectra (a) o 983089983088 g mLminus1 Carmoisine 983089983088 g mLminus1 Ponceau 983092R and their binary mixture 1047297rst derivative spectra (b) o colorants (Carmoisine(mdash) 983090ndash983089983088 g mLminus1 Ponceau 983092R (- -) 983090ndash983089983088 g mLminus1)

transerred to 983089983088983088 mL volumetric 1047298ask and the volume wasmade up with distilled water A series o dilution wasprepared quantitatively with water rom this solution toobtain standardsolutions to reach the concentration ranges o calibration curves graphed or each o the proposed methods(DSM RDM and CM) For the DDM method the same

procedure was used but in this case the 1047297nal volume wasmade up with 983088983089 N HCI and 983088983089 N NaOH separately orthe determination o Carmoisine and Ponceau 983092R HPLCmethod the same sample preparation procedure describedabove was ollowed using a mixture 983089 983089 vv o methanolmdashBA buffer solution Afer dissolution process preparedsolutions were 1047297ltered using 983088983092983093 m disposable membrane1047297lters (Sartrious Minisart 983088983092983093 m) by using a syringe Te1047297nal solution was diluted to the working concentration range

983090983095 Method Validation Te validity and suitability o theproposed methods were assessed by accuracy (reported aspercentage recovered) precision (reported as RSD) lin-

earity (evaluated by regression equation) limit o detection(LOD) and limit o quanti1047297cation (LOQ) [983090983089]

LOD and LOQ were based on the standard deviation o response and the slope o the corresponding curve using theollowing equations

LOD = 3

LOQ = 10

(983089)

where the noise estimate is the standard deviation o thederivative amplitude o the blank and is the slope o therelated calibration graphs [983090983090]

Te linearity o the methods was established in the

concentration ranges o 983090ndash983089983088 g mLminus1 or Carmoisine and

Ponceau 983092R respectively In this study 1047297ve concentrationswere analyzed in 1047297ve replicates Te linearity was evaluatedby the least square regression method

In order to demonstrate the validity and suitability o the proposed methods intra- and interday variability studieswere perormed at three different concentrations (983090 983094 and

983089983088 g mLminus1) in the presence o the certain concentration (983090983094and 983089983088g mLminus1) o the other colorants three times in a day and also three different days Method efficiency was tested interms o RSDrsquos or both intraday and interday precisions

Te precision was ascertained by carrying out 1047297ve repli-cate determinations o synthetic mixtures at different con-centration ratios o Carmoisine and Ponceau 983092R Recovery test also con1047297rmed the accuracy and applicability o theproposed methods by analyzing several synthetic mixtureso Carmoisine and Ponceau 983092R which reproduced differentcomposition ratios within the linearity range Tese ratios arealso the ratios in drinks Te mean percentage recoveries andRSD values were calculated

Recovery o the colorants o interest rom a given matrixcan be used as a measure o the accuracy or the bias o themethod Te same ranges o concentrations as employed inthe linearity studies were used

In our study the applicability o the derivative spec-trophotometric methods was also validated by comparisonwith the HPLC technique

3 Result and Discussion

Figure 983089(a) shows the absorption spectra o CarmoisinePonceau 983092R andthe mixture o them in the wavelength rangeo 983091983088983088ndash983095983088983088 nm Zero-order absorption spectra o Carmoisine




983137983138983148983141 983091 Method validation or the simultaneous determination o Carmoisine and Ponceau 983092R in laboratory prepared mixtures by theproposed methods

Method Accuracy (meanlowastplusmn RSD ) Precision repeatability a

(mean plusmn RSD )Intermediate precisionb

(mean plusmn RSD )

DSM C (983089D983091983091983089) 1008 plusmn 087 1001 plusmn 096 9991 plusmn 102

P (983089D983093983089983095

) 991 plusmn 102 1005 plusmn 105 1001 plusmn 092

RDM C (983089D983091983090983091) 1010 plusmn 095 9998 plusmn 099 1006 plusmn 105

P (983089D983091983092983092 ) 9825 plusmn 093 9983 plusmn 098 1004 plusmn 096

DDM C (983089D983091983094983094) 1004 plusmn 107 9895 plusmn 105 9992 plusmn 098

P (983089D983092983093983089) 9955 plusmn 095 1005 plusmn 095 1007 plusmn 096

CM C (983089D983092983095983090983089D983093983094983097) 9975 plusmn 102 998 plusmn 101 1001 plusmn 097

P (983089D983092983095983088983089D983093983093983092) 985 plusmn 106 9975 plusmn 103 1003 plusmn 106aTe intraday (907317 = 3) average o three concentrations (983090 983094 and 983089983088 1038389g mLminus983089) or Carmoisine and Ponceau 983092R repeated three times within the daybTe interday (907317 = 3) average o three concentrations (983090 983094 and 983089983088 1038389g mLminus983089) or Carmoisine and Ponceau 983092R repeated three times in three successive dayslowastTe values o recovery are an average o 1047297ve replicates o each o 1047297ve synthetic mixtures at different concentration ratios o C and P (983090ndash983089983088 1038389g mLminus983089)RSD relative standard deviation

and Ponceau 983092R in distilled water gave rise to maximumpeaks at 983093983089983095 nm and 983093983088983096 nm respectively As can be seenthe spectra o the colorants were strongly overlapped in the983092983088983088ndash983094983088983088 nm wavelength region Hence the direct absorp-tion measurement or assaying binary mixture seems to beimpossible On the other hand the absorption spectra o thetwo components within a wavelength range o 983091983088983088ndash983091983096983088 nmwere less overlapped than the region 983092983088983088ndash983094983088983088 nm So in thiswork the region o 983091983088983088ndash983091983096983088 nm was chosen or the analysiso Carmoisine In this spectral range DSM RDM and DDMwere used or the determination o the studied colorants inlaboratory prepared mixtures and sof drink samples Tesemethods are also applied to analyze Ponceau 983092R in the region

983091983090983088ndash983093983090983088 nm

983091983089 Derivative Spectrophotometric Method (DSM) Undercomputer-controlled instrumentation derivative spectro-photometry played a very important role in the resolutiono band overlapping in quantitative analysis [983090983091] Figure 983089(b)shows the 1047297rst derivative spectra o Carmoisine and Ponceau983092R As can be seen in Figure 983089(b) in the region o 983091983088983088ndash983093983090983088 nm the 1047297rst derivative spectra allowed the simultaneousdetermination o Carmoisine and Ponceau 983092R At this region

the 1D spectra (Figure 983089(b)) show a characteristic peak at983091983091983089 nm or Carmoisine while Ponceau 983092R shows no response

Tereore the absolute values o the 1D peak amplitudes at983091983091983089 nm can be used to quantiy Carmoisine in this mixtureOn the other hand Ponceau 983092R canbe assayed in thepresence

o Carmoisine by measuring its 1 D response at 983093983089983095 nm (zero-crossing point o Carmoisine)

Under the experimental conditions described the cal-ibration graphs obtained by plotting the derivative values

versus concentrations or Carmoisine and Ponceau 983092R inthe concentration range stated in able 983090 exhibited linearrelationships Statistical analysis o the data showed thatlinearity o the calibration graphs and compliance withBeerrsquos law were validated and the small values o the inter-cepts as illustrated by the excellent values o correlation

coefficients o the regression equations o prove the valid-ity and applicability o the proposed method laboratory-prepared mixtures were prepared at different concentrationratios o Carmoisine and Ponceau 983092R (able 983091) Te obtainedresults or the analysis o these mixtures in 1047297ve replicates by the described method showed high accuracy and precisiono the proposed method (able 983091)

983091983090 Ratio Derivative Method (RDM) While the main dis-advantages o the zero-crossing method in derivative spec-trophotometry or resolving a mixture o components withoverlapped spectra are the risk o small drifs o the working

wavelengths and the circumstance that the working wave-lengths generally do not all in correspondence o peaks o the derivative spectrum this may be particularly dangerouswhen the slope o the spectrum is very high with consequentloss o accuracy and precision and the working wavelengthis in proximity o the base o the spectrum which causespoor sensitivity Te main advantage o the RDM is thechance o doing easy measurements in correspondence o peaks so it permits the use o the wavelength o highest

value o analytical signals (a maximum or a minimum) andmoreover the presence o a lot o maxima and minima isanother advantage by the act that these wavelengths give anopportunity orthe determination o active compoundsin the

presence o other compounds and ingredients which possibly interere in the assay [983090983092ndash983090983095]

Tis method is based on the use o the 1047297rst derivativeo the ratio spectra Te ratio spectra were obtained by dividing the absorption spectrum o the mixture by thato one o the components As we depicted above directabsorption measurements are not possible due to spectraloverlap Tis spectral overlapping was sufficiently enough todemonstrate the resolving power o the proposed methodFigure 983090(a) shows the 1047297rst ratio derivative spectra o differ-ent Carmoisine standard solutions (spectra divided by the

spectrum o 983094 g mLminus1 Ponceau 983092R solution) Te ratio 1047297rst

derivative amplitudes at 983091983090983091 nm (1DD323) corresponding to




001

003

308 328 348 368

F i r s t

d e r i v a

t i v e

C a r m o

i s i n e

P o n c e a u

4 R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

F i r s t

d e r i v a

t i v e

P o n c e a u

4 R C a r m o i s

i n e

Wavelength (nm)

f

g

h

i

j

minus002678

minus001678

minus000678

(b)

F983145983143983157983154983141 983090 First derivative ratio spectra o Carmoisine (a) and Ponceau 983092R (b) or different concentrations (Carmoisine (983094 g mLminus1 Ponceau983092R was used as a divisor) a 983090 g mLminus1 b 983092 g mLminus1 c 983094 g mLminus1 d 983096 g mLminus1 e 983089983088 g mLminus1 Ponceau 983092R (983094 g mLminus1 Carmoisine was usedas a divisor) 983090 g mLminus1 g 983092 g mLminus1 h 983094 g mLminus1 i 983096 g mLminus1 j 983089983088 g mLminus1)

983137983138983148983141 983092 Assay results or the determination o Carmoisine and Ponceau 983092R in sof drinks using the proposed methods and the reerenceHPLC method (NMKL 983089983091983088)

Methods Analyte Selected wavelengths(nm)

Assay results mean plusmn SDlowast (calculated calculated)lowastlowast

Sof drink I (g983089983088983088mLminus983089) Sof drink II (g983089983088983088mLminus983089)

DSM C 983089

D983091983091983089 930 plusmn 022(068145) 628 plusmn 029(061144)P 983089D983093983089983095 220 plusmn 011(118118) 139 plusmn 013(040179)

RDM C 983089D983091983090983091 926 plusmn 023(037145) 636 plusmn 021(128120)

P 983089D983091983092983092 235 plusmn 011(044125) 143 plusmn 013(013169)

DDM C 983089D983091983094983094 931 plusmn 024(073169) 641 plusmn 024(153164)

P 983089D983092983093983089 249 plusmn 017(189302) 144 plusmn 016(023268)

CM C 983089D983092983095983090983089D983093983094983097 930 plusmn 026(063188) 636 plusmn 028(107211)

P 983089D983092983095983088983089D983093983093983092 230 plusmn 016(023256) 143 plusmn 019(010368)

HPLC C 983093983090983088 921 plusmn 018 620 plusmn 019

P 983093983089983090 232 plusmn 010 142 plusmn 010lowastResults obtained are the average o 1047297ve experiments or each method SD standard deviationlowastlowastTe corresponding theoretical value or and at 983088983088983093 (theoretical 983090983091983089 theoretical 983094983091983097)

a maximum wavelength are proportional to the Carmoisineconcentration (Figure 983090(a)) o determine the other com-ponent Ponceau 983092R an analogues procedure was ollowedFor determination o Ponceau 983092R the stored spectra o the mixtures are divided by a standard spectrum o 983094 mg

Lminus1 Carmoisine (Figure 983090(b)) Standard solutions o Ponceau983092R and the content o Ponceau 983092R were determined by

measuring the signals at 983091983092983092 nm (1DD344) Figure 983090(b)Under the described experimental conditions the cal-

ibration graphs obtained by plotting the derivative valueso each colorant versus concentration in the concentration

range stated in able 983090 showed linear relationships Con-ormity with Beerrsquos law was evident in the concentrationrange o the 1047297nal dilution cited in able 983090 For this methodthe characteristic parameters o regression equations andcorrelation coefficients are given in able 983090 Te correlationcoefficients were 983088983097983097983097983094 and 983088983097983097983097983092 indicating good linearityLOD and LOQ were calculated or each colorant and areshown in the same table Te detection limits were oundto be 983088983088983095983097 g mLminus1 or Carmoisine and 983088983088983096983090 g mLminus1 orPonceau 983092R while the quanti1047297cation limits were estimatedto be 983088983090983094983091 g mLminus1 or Carmoisine and 983088983090983095983091 g mLminus1 or




000988

008988

016988

024988

032988

300 400 500 600 700

A b

s o r b a n c e

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

D i ff e r e n c e

1047297 r s t

d e r i v

a t i v e

Wavelength (nm)

C

P

minus00068

minus00028

(b)

F983145983143983157983154983141 983091 Difference absorption spectra o 983089983088 g mLminus1 Carmoisine 983089983088 g mLminus1 Ponceau 983092R and their binary mixture (a) Difference 1047297rstderivative spectra (b) o colorants (Carmoisine 983090ndash983089983088 g mLminus1 Ponceau 983092R 983090ndash983089983088 g mLminus1)

Ponceau 983092R Te proposed method was also applied to thedrink samples (able 983092) Te accuracy o RDM was checkedby analyzing the laboratory prepared mixtures o Carmoisineand Ponceau 983092R at various concentration ratios ranging rom

983090 to 983089983088 g mLminus1 (able 983091) All data represent the average o 1047297ve determinations Low values o relative standard deviation

indicate very good precision Satisactory recoveries withsmall standard deviations were obtained which indicated thehigh repeatability and accuracy o the method

983091983091 Derivative Differential Method (DDM) Differential spec-trophotometry (ΔD1) based on pH changes has also beenreported to be useul in the determination o binary mix-tures Tis method that depends on utilization o differenceabsorption spectra corresponding to the same compoundobtained at two different pHrsquos has been investigated or theanalysis o binary mixtures Te procedure is comprised o the measurement o ΔD1 ood colorantsrsquo in acidic solutionsagainst their alkaline solutions as blanks Te ΔD1 has

been successully used to eliminate intererences rom drink samples Tere are ew reports on utilization o the abovetwo combined techniques (difference and derivative) or theestimation o individual drug substances and or combinedpreparations [983090983096ndash983091983088]

Te difference absorption spectra o Carmoisine Pon-ceau 983092R and their binary mixture in 983088983089 N NaOH and in983088983089 N HCI are shown in Figure 983091(a) Figure 983091(b) shows the1047297rst derivative difference spectra o colorants in the concen-tration range 983090ndash983089983088 g mLminus1 Te spectra o both colorantsin Figure 983091(b) offer an advantage or their simultaneousdetermination by having zero-crossing points In particularthe 1047297rst derivative amplitudes at 983091983094983094 nm or Carmoisine and

983092983093983089 nm or Ponceau 983092R in the Carmoisine and Ponceau 983092R mixture were considered as the optimum working wave-lengths or their determination By measuring the values o the ΔD1 amplitudes at these wavelengths the concentrationo each colorant can be directly calculated since the di-erential 1047297rst derivative measurement cancels the irrelevant

absorbance due to the drinks matrix at these wavelengthsUnder the experimental conditions described the cali-

bration graphs obtained by plotting the derivative values o each colorant in the mixture versus concentration in therange concentration stated in able 983090 show linear relation-ships Te correlation coefficients were 983088983097983097983097983094 and 983088983097983097983097983096indicating good linearity Te LOD were 983088983088983095983089 g mLminus1 or

Carmoisine and 983088983088983096983089 g mLminus1 or Ponceau 983092R while the

LOQ were 983088983090983091983094 g mLminus1 or Carmoisine and 983088983090983095983088 g mLminus1

or Ponceau 983092R Te proposed procedure was success-ully applied or the determination o these colorants inlaboratory-prepared mixtures and drink samples Te resultsare presented in ables 983091 and 983092 Te values o relativestandard

deviation indicate very good reproducibility In additionapplication o the DDM was ound to be correct or the drink matrix intererence and to enhance the sensitivity

983091983092 Compensation Method (CM) Te compensation methodis a nonmathematical method or the detection and elimi-nation o unwanted absorption during spectrophotometricanalysis [983089983097 983090983088 983091983089 983091983090] Te accuracy o the method dependson the evaluation o the balance point Figure 983089(b) showsthe 1047297rst derivative spectra o Carmoisine and Ponceau 983092R in the concentration range 983090ndash983089983088 g mLminus1 Te 1047297rst derivativespectra were recorded or each reerence solution o thecomponents and the ratios o the 1D maximum and 1D




000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)

F983145983143983157983154983141 983092 (a) First derivative spectra between the mixture solution (983094 g mLminus1 Carmoisine + 983094 g mLminus1 Ponceau 983092R) in the sample cell anddifferent concentrations (983090ndash983089983088 g mLminus1) o Ponceau 983092R solutions in the reerence cell First derivative spectra between the mixture solution(983094 g mLminus1 Carmoisine + 983094 g mLminus1 Ponceau 983092R) in the sample cell and 983094 g mLminus1 Ponceau 983092R in the reerence cell at balance points andcomparison o these spectra with 983094 g mLminus1 concentration o Carmoisine (-- -) spectrum (b) First derivative spectra between the mixturesolution (983094 g mLminus1 Carmoisine + 983094 g mLminus1 Ponceau 983092R) in the sample cell and different concentrations (983090ndash983089983088 g mLminus1) o Carmoisinesolutions in the reerence cell Derivative spectra between the mixture solution (983094 g mLminus1 Carmoisine + 983094 g mLminus1 Ponceau 983092R) in thesample cell and 983094 g mLminus1 Carmoisine in the reerence cell at balance points and comparison o these spectra with 983094 g mLminus1 concentrationo Ponceau 983092R (- - -) spectrum

minimum were calculated able 983089 shows the mean valueso the ratios calculated or 1047297ve different determinations oreach solution Te ratios are constant andcharacteristic o thepure substance independent o concentration and presenceo another component For the determination o Carmoisineconcentrations in Carmoisine-Ponceau 983092R binary mixturesand drink samples the sample cell was 1047297lled with themixturesolution (983094 g mLminus1 Carmoisine + 983094 g mLminus1 Ponceau 983092R)and the reerence cell was 1047297lled in succession with aseries o reerence Ponceau 983092R with solutions in differentconcentrations Ponceau 983092R as shown in Figure 983092(a) Te

ratios o the mixture calculated rom the recorded 1D spectra

were compared with those o Carmoisine At the exactbalance point the ratio o the mixture corresponds to thato Carmoisine where the concentration o Carmoisine in themixtures in the sample cell is equal to that o the reerencesolution in the reerence cell For determining the othercomponent the same steps were ollowed using solutions o pure compound Carmoisine in the reerence cell to determineits concentration in the mixture (Figure 983092(b)) Conormity with Beerrsquos law was evident in the concentration range rom 983090

to983089983088 g mLminus1 o Carmoisine and Ponceau 983092R In this methodsynthetic mixtures were prepared at different concentrationratios o Carmoisine and Ponceau 983092R and the recoveries o the method were ound to be in the range o 9850 plusmn 105ndash

9975 plusmn 110 Te presented results in able 983091 are in goodagreement with the other proposed methods

983091983093 Application o the Developed Methods to the Sof DrinksTe developed and validated methods were successully applied to the determination o Carmoisine and Ponceau 983092R content in the commercial sof drinks Te obtained resultsare presented in able 983092 Good agreement was observed orthe assay results o the drinks by application o the ourmethods in this paper (able 983092) Te values o standarddeviation indicate very good reproducibility Te results o the proposed spectrophotometric methods were statistically compared with the results o the HPLC method given in

the literature [983089983096] at the 983097983093 con1047297dence level with studentrsquos -test and by the variance ratio -test (able 983092) Te calculated-and -values neverexceed the theoretical-and -values at983088983088983093 level o signi1047297cant difference Tere was no intererenceo ingredients in the products examined so no additiveextraction or separation procedures were required duringtheir assay Te results o all methods were very close toeach other Tis suggests that the our methods are equally applicable

983091983094 Validation o the Proposed Methods

983091983094983089 Linearity Te linearity o the proposed methods wasevaluated under different concentrations o Carmoisine andPonceau 983092R within the concentration range stated in able 983090Te good linearity o the calibration graphs and negligiblescatter o the experimental points is clearly evident by the

values o the correlation coefficients and variances around theslope (able 983090)

983091983094983090 Limit o Detection (LOD) and Limit o Quanti1047297cation(LOQ) LOD is expressed as the analyte concentration cor-responding to the sample blank value plus three standarddeviations and LOQ is the analyte concentration correspond-ing to the sample blank value ten standard deviations [983090983089]

All the proposed methods data was presented in able 983090Te LOD and LOQ values ound were between 983088983088983095983089 and983088983088983096983094 and 983088983090983091983094 and 983088983090983096983094 g mLminus1 or Carmoisine and 983088983088983096983089

and 983088983088983097983089 and 983088983090983095983088 and 983088983091983088983092 g mLminus1 or Ponceau 983092Rrespectively Te data shows that the methods are sensitiveor the determination o Carmoisine and Ponceau 983092R

983091983094983091 Precision (Repeatability) and Accuracy (Recovery )Te precision o the developed methods was expressedas a percentage o relative standard deviation (RSD) orrepeatability (intraday precision) and intermediate precision(interday precision) Te data obtained were less than 983089983088983094(able 983091) indicating reasonable repeatability o the proposed




methods Te intraday and interday accuracy ranges wererom 983097983096983097983093 to 983089983088983088983094 or Carmoisine and rom 983097983097983095983093 to983089983088983088983095 or Ponceau 983092R respectively Te results obtainedrom intermediate precision (interday) also indicated a goodmethod precision All the data were within the acceptancecriteria o con1047297rm the accuracy o the proposed methods

synthetic mixtures o different concentrationratios consistingo each ood colorant were prepared Te resulting mixtureswere assayed according to above proposed procedures in1047297ve replicates and the average results were calculated asthe percentage o analyte recovered Te percentages meanaccuracy or Carmoisine were 1008 plusmn 087 or method DSM1010 plusmn 095 or RDM 10040 plusmn 107 or DDM and 9975 plusmn102 or CM respectively And those o P were 9910 plusmn 102or method DSM 9825 plusmn 093 or RDM 9955 plusmn 095 orDDM and 9850 plusmn 106 or CM Te good recovery percent(RSD) assured the high accuracy o the proposed methods(able 983091) Te relative standard deviations were ound tobe less than 983089983088983095 and 983089983088983094 or Carmoisine and Ponceau983092R respectively indicating reasonable repeatability o theproposed methods

4 Conclusion

Four derivative spectrophotometric methods or the simul-taneous analysis o two common ood colorants have beensuccessully researched developed applied and compared by reerence HPLC method Proposed methods provide simpleaccurate and reproducible quantitative determination o Carmosine and Ponceau 983092R in synthetic mixtures and sofdrinks without any intererence rom ingredients presentTe developed methods are simple (as there is no need

or pretreatment) rapid (as they require measurements o D983089 and D983089 values at a single wavelength) and direct (asthey estimate each colorant independently o the other) UVmethods offer a cost effective and time saving alternative toother methods or example colorimetric complexometricand chromatographic analyses Te methods used in thisstudy are more versatile and easy to apply than the HPLCpolarographic and voltammetric methods Also the methodsdid not require any sophisticated instrumentation such asHPLC which requires organic solvents and time or advancedmethodologies (like chemometric methods)

We conclude that the sensitivities o the proposed meth-odsare almostcomparable andcan be used orthe determina-tion o the two colorants in commercial drinks in the absenceo official method

Conflict of Interests

Te authors declare that there is no con1047298ict o interestsregarding the publication o this paper

Acknowledgment

Tis research has been supported by Scienti1047297c ResearchProject Coordination Center o Yildiz echnical University (Project no 983090983088983089983090-983088983089-983088983090-KAP983088983095)

References

[983089] A Branen P Davidson and S Salminen Food AdditivesAcademic New York NY USA 983089983097983096983097

[983090] J Vries Food Saety and Toxicity CRC London UK 983089983097983097983094

[983091] M S Garcıa-Falcon and J Simal-Gandara ldquoDetermination o ood dyes in sof drinks containing natural pigments by liquid

chromatography with minimal clean-uprdquo Food Control vol 983089983094no 983091 pp 983090983097983091ndash983090983097983095 983090983088983088983093

[983092] S Chanlon L Joly-Pottuz M Chatelut O Vittori and J LCretier ldquoDetermination o carmoisine allura red and Ponceau983092R in sweets and sof drinks by differential pulse polarographyrdquo Journal o Food Composition and Analysis vol 983089983096 no 983094pp983093983088983091ndash983093983089983093 983090983088983088983093

[983093] N Dossi R oniolo A Pizzariello S Susmel F Perennes andG Bontempelli ldquoA capillary electrophoresis microsystem orthe rapid in-channel amperometric detection o synthetic dyesin oodrdquo Journal o Electroanalytical Chemistry vol 983094983088983089 no 983089-983090pp 983089ndash983095 983090983088983088983095

[983094] S Dixit S K Khanna and M Das ldquoSimultaneous deter-mination o eight synthetic permitted and 1047297ve commonly encountered nonpermitted ood colors in various ood matrixesby high-perormance liquid chromatographyrdquo Journal o AOAC International vol 983097983091 no 983093 pp 983089983093983088983091ndash983089983093983089983092 983090983088983089983088

[983095] S Dixit S K Khanna and M Das ldquoA simple method orsimultaneous determination o basic dyes encountered in oodpreparations by reversed-phase HPLCrdquo Journal o AOAC Inter-national vol 983097983092 no 983094 pp 983089983096983095983092ndash983089983096983096983089 983090983088983089983089

[983096] N Yoshioka and K Ichihashi ldquoDetermination o 983092983088 syntheticood colors in drinks and candies by high-perormance liquidchromatography using a short column with photodiode array detectionrdquo Talanta vol 983095983092 no 983093 pp 983089983092983088983096ndash983089983092983089983091 983090983088983088983096

[983097] S P Alves D M Brum E C Branco de Andrade and AD Pereira Netto ldquoDetermination o synthetic dyes in selected

oodstuffs by high perormance liquid chromatography withUV-DAD detectionrdquo Food Chemistry vol 983089983088983095 no 983089 pp 983092983096983097ndash983092983097983094 983090983088983088983096

[983089983088] M Ma X Luo B Chen S Su and S Yao ldquoSimultaneous deter-mination o water-soluble and at-soluble synthetic colorantsin oodstuff by high-perormance liquid chromatography-diodearray detection-electrospray mass spectrometryrdquo Journal o Chromatography A vol 983089983089983088983091 no 983089 pp 983089983095983088ndash983089983095983094 983090983088983088983094

[983089983089] E C Vidotti J C Cancino C C Oliveira and M D C ERollemberg ldquoSimultaneous determination o ood dyes by 1047297rstderivative spectrophotometry with sorption onto polyurethaneoamrdquo Analytical Sciences vol 983090983089 no 983090 pp 983089983092983097ndash983089983093983091 983090983088983088983093

[983089983090] M Hajimahmoodi M R Oveisi N Sadeghi B Jannat andE Nilroush ldquoSimultaneous determination o carmoisine and

Ponceau 983092Rrdquo Food Analytical Methods vol 983089 no 983091 pp 983090983089983092ndash983090983089983097983090983088983088983096

[983089983091] N Pourreza and M Ghomi ldquoSimultaneous cloud point extrac-tion and spectrophotometric determination o carmoisine andbrilliant blue FCF in ood samplesrdquo Talanta vol 983096983092 no 983089 pp983090983092983088ndash983090983092983091 983090983088983089983089

[983089983092] G Keser Karaoglan G Gumrukcu M Ustun Ozgur ABozdogan and BAsci ldquoAbilities o partial least-squares(PLS-983090)multivariate calibration in the analysis o quaternary mixture o ood colors (E-983089983089983088 E-983089983090983090 E-983089983090983092 E-983089983091983089)rdquo Analytical Letters vol983092983088 no 983089983088 pp 983089983096983097983091ndash983089983097983088983091 983090983088983088983095

[983089983093] C C Wang A N Masi and L Fernandez ldquoOn-line micellar-enhanced spectro1047298uorimetric determination o rhodamine dyein cosmeticsrdquo Talanta vol 983095983093 no 983089 pp 983089983091983093ndash983089983092983088 983090983088983088983096




[983089983094] N Dossi R oniolo S Susmel A Pizzariello and G Bontem-pelli ldquoSimultaneous RP-LC determination o additives in sofdrinksrdquo Chromatographia vol 983094983091 no 983089983089-983089983090 pp 983093983093983095ndash983093983094983090 983090983088983088983094

[983089983095] K S Minioti C F Sakellariou and N S Tomaidis ldquoDeter-mination o 983089983091 synthetic ood colorants in water-soluble oodsby reversed-phase high-perormance liquid chromatography coupled with diode-array detectorrdquo AnalyticaChimicaActavol983093983096983091 no 983089 pp 983089983088983091ndash983089983089983088 983090983088983088983095

[983089983096] Nordic Commitee on Food Analysis (NMKL) ldquoColours syn-thetic water soluble liquid chromatographic determination inoodsrdquo UDC 983094983094983095983090983096983093983092983091983093983092983092 983089983091983088 983089983097983096983097

[983089983097] A M Wahbi O Abdel-Razak A A Gazy H Mahgoub and MS Moneeb ldquoSpectrophotometric determination o omeprazolelansoprazole and pantoprazole in pharmaceutical ormula-tionsrdquo Journal o Pharmaceutical and Biomedical Analysis vol983091983088 no 983092 pp 983089983089983091983091ndash983089983089983092983090 983090983088983088983090

[983090983088] F A El Yazbi M E Mahrous H H Hammud G M Sonji andN M Sonji ldquoComparative spectrophotometric spectro1047298uoro-metric and high-perormance liquid chromatographic study or the quantitative determination o the binary mixtureelodipine and ramipril in pharmaceutical ormulationsrdquo Ana-lytical Letters vol 983092983089 no 983093 pp 983096983093983091ndash983096983095983088 983090983088983088983096

[983090983089] ICH Harmonized ripartite Guideline ldquoValidation o analyticalprocedures text and methodology Q983090 (R983089)rdquo in Proceedingso the International Conerence on Harmonization o Technical Requırements or Registration o Pharmacetıcals or Human Usepp 983089ndash983089983091 983090983088983088983093

[983090983090] D arinc and A Golcu ldquoDevelopment and validation o spectrophotometric methods or determination o somecephalosporin group antibiotic drugsrdquo Journal o Analytical Chemistry vol 983094983095 no 983090 pp 983089983092983092ndash983089983093983088 983090983088983089983090

[983090983091] K K Srinivasan J Alex A A Shirwaikar S Jacob M RSunil Kumar and S Prabu ldquoSimultaneous derivative spec-trophotometric estimation o acecloenac and tramadol with

paracetamol in combination soliddosage ormsrdquo Indian Journal o Pharmaceutical Sciences vol 983094983097 no 983092 pp 983093983092983088ndash983093983092983093 983090983088983088983095

[983090983092] F Salinas JJ BNevadoand A E Mansilla ldquoA newspectropho-tometric method or quantitative multicomponent analysisresolution o mixtures o salicylic and salicyluric acidsrdquo Talanta vol 983091983095 no 983091 pp 983091983092983095ndash983091983093983089 983089983097983097983088

[983090983093] R Corbella ena M A Rodrıguez Delgado M J Sanchez andF Garcia Montelongo ldquoComparative study o the zero-crossingratio spectra derivative and partial least-squares methodsapplied to the simultaneous determination o atrazine andits degradation product desethylatrazin-983090-hydroxy in groundwatersrdquo Talanta vol 983092983092 no 983092 pp 983094983095983091ndash983094983096983091 983089983097983097983095

[983090983094] A El-Gindy A Ashour L Abdel-Fattah and M M ShabanaldquoSpectrophotometric determination o benazepril hydrochlo-

ride and hydrochlorothiazide in binary mixture using secondderivative second derivative o the ratio spectra and chemo-metric methodsrdquo Journal o Pharmaceutical and Biomedical Analysis vol 983090983093 no 983090 pp 983090983097983097ndash983091983088983095 983090983088983088983089

[983090983095] R R Ambadas and P D Bari ldquoRatio spectra derivative andzero-crossing difference spectrophotometric determination o olmesartan medoxomil and hydrochlorothiazide in combinedpharmaceutical dosage ormrdquo An Official Journal o the Amer-ican Association o Pharmaceutical Scientists vol 983089983088 no 983092 pp983089983090983088983088ndash983089983090983088983093 983090983088983088983097

[983090983096] C V N Prasad A Gautam V Bharadwaj and P ParimooldquoDifferential derivative spectrophotometric determination o phenobarbitone and phenytoin sodium in combined tabletpreparationsrdquo Talanta vol 983092983092 no 983093 pp 983097983089983095ndash983097983090983090 983089983097983097983095

[983090983097] F Salinas A Espinosa-Mansilla A Zamoro and A Munoz DeLa Pena ldquopH-induced difference spectrophotometry in theanalysis o binary mixturesrdquo Analytical Letters vol 983090983097 no 983089983092pp 983090983093983090983093ndash983090983093983092983088 983089983097983097983094

[983091983088] N Erk ldquoDerivative-differential UV spectrophotometry andcompensation technique or the simultaneous determination o zidovudine and lamivudine in human serumrdquo Pharmazie vol983093983097 no 983090 pp 983089983088983094ndash983089983089983089 983090983088983088983092

[983091983089] N Erk ldquoSpectrophotometric analysis o valsartan and hydro-chlorothiaziderdquo Analytical Letters vol 983091983093 no 983090 pp 983090983096983091ndash983091983088983090983090983088983088983090

[983091983090] N Erk ldquoAnalysis o binary mixtures o losartan potassium andhydrochlorothiazide by using high perormance liquid chro-matography ratio derivative spectrophotometric and compen-sation techniquerdquo Journal o Pharmaceutical and Biomedical Analysis vol 983090983092 no 983092 pp 983094983088983091ndash983094983089983089 983090983088983088983089



Submit your manuscripts at

httpwwwhindawicom




N N

N N

OH

HO

NaO3S

NaO3S

NaO3S

NaO3S

SO3Na

Carmoisine (Azorubine E122) CI 14720 Ponceau 4R (Cochineal Red A E124) CI 16255

S983139983144983141983149983141 983089 Chemical structures common names European community numbers (E) and color index (CI) numbers o synthetic oodcolorants studied

published during the last years demonstrates the importanceo this problem andthe need ordevelopingast accurateand

selective techniques or synthetic colorants analysis [983092]Various techniques have been introduced or the deter-

mination o mixtures o colorants with three or more com-ponent combination in ood samples

Tese include capillary electrophoresis [983093] differentialpulse polarography [983092] high perormance liquid chromatog-raphy [983094ndash983097] electrospray mass spectrometry [983089983088] spec-trophotometry [983089983089ndash983089983091] partial least squares [983089983092] and spec-tro1047298uorimetry [983089983093] Chromatographic methods have beenused or colorant analysis in oods and they are recom-mended when the mixture contains many different colorants[983094 983095 983089983094 983089983095] However there are some potential drawbackssuch as usage o toxic solvents the need or complex samplepretreatments and the resulting waste product Capillary electrophoresis is also very suitable when the sample containsseveral colorants but oods beverages and pharmaceuticalscontain only one two or rarely three ood dyes and thesemethods need special equipment that may not be availablein certain quality laboratories All multivariate calibrationmethods including Partial Least Square (PLS) require thedata processing with powerul sofware as well as the manip-ulations o the abstract vector space and its application tothe regression analysis and various chemometric methodsare also ofen used or more complex mixtures Tus ana-lytical methods rom alternative technique are always useul

especially i the methods are simple cheap and compara-tively ast Spectrophotometry as alternative methodologyis suitable or routine laboratories especially or developingcountries and sometimes serious analytical problems canbe resolved by this common technique Among computer-controlled instruments derivative techniques (ratio deriva-tive difference derivative and compensation method) areplaying a very important role in the binary analysis o mixtures by UV-VIS molecular absorption spectrophotom-etry All approaches are useul in the resolution o bandoverlapping in quantitative analysis

In this present work selective sensitive simple andlow-cost procedures were developed or the simultaneous

determination o Carmoisine (C) and Ponceau 983092R (P) insynthetic mixtureand drink samples by using derivative spec-

trophotometry (DSM) ratio derivative spectrophotometry (RDM) derivative differential spectrophotometry (DDM)and compensation method (CM) Tese methods have beensuccessully applied to synthetic mixtures and drink samplesand the results obtained were compared with those obtainedby HPLC method according to NMKL 983089983091983088 [983089983096]

No extraction or evaporation step no complexationagent andno harmulchemicals are involvedin the suggestedmethods in thesuggested methods so canbe used orroutineanalysis o both colorants in quality control and routinelaboratories

2 Experimental983090983089 Apparatus All spectral measurements and treatment o data were carried out using a Shimadzu UV-983090983092983093983088 doublebeam UV-Vis spectrophotometer with two matched 983089-cmquartz cells connected to a Hewlett Packard compatiblepersonal computer and a HP 983089983089983088983090 Laser Jet Printer BundledUV-PC personal spectroscopy sofware o version 983090983090983089 wasused to process the absorption and the derivative spectraTe spectral band width was 983089 nm with wavelength scanningspeed o medium HPLC measurements were perormed withan Agilent 983089983089983088983088 series HPLC-DAD system with a vacuumdegasser quaternary pump auto sampler and injector with a983089983088983088 L loop volume Separation was achieved using a WatersSpherisorb C983089983096 column (983092983094 times 983090983093983088mm 983093 m) and variablewavelength UV-VIS DAD detector measuring at 983093983090983088 nm orCarmoisine and 983093983089983090 nm or Ponceau 983092R

983090983090 Chromatographic Conditions Separation was achievedusing a Waters Spherisorb C18 column (983092983094 times 983090983093983088mm983093 m)Te solvent system consisted o a mixture o an aqueoussolution containing 983088983088983088983093 M tetrabutylammonium hydro-gen sulphate (BA) and a phosphate buffer solution (pH983094983093) methanol (983093983088 983093983088 vv) It was pumped isocratically at

983088983095mLminminus1 or the separation Te eluents were monitoredat 983093983089983090 and 983093983090983088 nm or P and C respectively











wavelength(nm)

Concentrationrange



(1038389)



(g mLminus983089)





DDM















983089983088983088 mLminus1





























1D11039252





minus1













0175

0375

0575

0775

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 0

7 0 0

A b

s o r b a n c e

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

3 0 0

3 2 0

3 4 0

F i r s t

d e r i v a

t i v e

Wavelength (nm)

P

C

00002

00022

4 7 5

5 0 0

5 2 5

5 5 0

5 7 5

F i r s t

d e r i v a

t i v e

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)







LOD = 3

LOQ = 10

(983089)


















(mean plusmn RSD )


P (983089D983093983089983095









983091983090983088ndash983093983090983088 nm


















001

003

308 328 348 368

F i r s t

d e r i v a

t i v e

C a r m o

i s i n e

P o n c e a u

4 R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

F i r s t

d e r i v a

t i v e

P o n c e a u

4 R C a r m o i s

i n e

Wavelength (nm)

f

g

h

i

j

minus002678

minus001678

minus000678

(b)






DSM C 983089



P 983089D983091983092983092 235 plusmn 011(044125) 143 plusmn 013(013169)


P 983089D983092983093983089 249 plusmn 017(189302) 144 plusmn 016(023268)













000988

008988

016988

024988

032988

300 400 500 600 700

A b

s o r b a n c e

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

D i ff e r e n c e

1047297 r s t

d e r i v

a t i v e

Wavelength (nm)

C

P

minus00068

minus00028

(b)



















000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)





















4 Conclusion






Acknowledgment


References












































httpwwwhindawicom











wavelength(nm)

Concentrationrange



(1038389)



(g mLminus983089)





DDM















983089983088983088 mLminus1





























1D11039252





minus1













0175

0375

0575

0775

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 0

7 0 0

A b

s o r b a n c e

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

3 0 0

3 2 0

3 4 0

F i r s t

d e r i v a

t i v e

Wavelength (nm)

P

C

00002

00022

4 7 5

5 0 0

5 2 5

5 5 0

5 7 5

F i r s t

d e r i v a

t i v e

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)







LOD = 3

LOQ = 10

(983089)


















(mean plusmn RSD )


P (983089D983093983089983095









983091983090983088ndash983093983090983088 nm


















001

003

308 328 348 368

F i r s t

d e r i v a

t i v e

C a r m o

i s i n e

P o n c e a u

4 R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

F i r s t

d e r i v a

t i v e

P o n c e a u

4 R C a r m o i s

i n e

Wavelength (nm)

f

g

h

i

j

minus002678

minus001678

minus000678

(b)






DSM C 983089



P 983089D983091983092983092 235 plusmn 011(044125) 143 plusmn 013(013169)


P 983089D983092983093983089 249 plusmn 017(189302) 144 plusmn 016(023268)













000988

008988

016988

024988

032988

300 400 500 600 700

A b

s o r b a n c e

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

D i ff e r e n c e

1047297 r s t

d e r i v

a t i v e

Wavelength (nm)

C

P

minus00068

minus00028

(b)



















000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)





















4 Conclusion






Acknowledgment


References












































httpwwwhindawicom























1D11039252





minus1













0175

0375

0575

0775

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 0

7 0 0

A b

s o r b a n c e

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

3 0 0

3 2 0

3 4 0

F i r s t

d e r i v a

t i v e

Wavelength (nm)

P

C

00002

00022

4 7 5

5 0 0

5 2 5

5 5 0

5 7 5

F i r s t

d e r i v a

t i v e

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)







LOD = 3

LOQ = 10

(983089)


















(mean plusmn RSD )


P (983089D983093983089983095









983091983090983088ndash983093983090983088 nm


















001

003

308 328 348 368

F i r s t

d e r i v a

t i v e

C a r m o

i s i n e

P o n c e a u

4 R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

F i r s t

d e r i v a

t i v e

P o n c e a u

4 R C a r m o i s

i n e

Wavelength (nm)

f

g

h

i

j

minus002678

minus001678

minus000678

(b)






DSM C 983089



P 983089D983091983092983092 235 plusmn 011(044125) 143 plusmn 013(013169)


P 983089D983092983093983089 249 plusmn 017(189302) 144 plusmn 016(023268)













000988

008988

016988

024988

032988

300 400 500 600 700

A b

s o r b a n c e

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

D i ff e r e n c e

1047297 r s t

d e r i v

a t i v e

Wavelength (nm)

C

P

minus00068

minus00028

(b)



















000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)





















4 Conclusion






Acknowledgment


References












































httpwwwhindawicom




0175

0375

0575

0775

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 0

7 0 0

A b

s o r b a n c e

Wavelength (nm)

C

Mix

P

minus0025

(a)

00002

00022

3 0 0

3 2 0

3 4 0

F i r s t

d e r i v a

t i v e

Wavelength (nm)

P

C

00002

00022

4 7 5

5 0 0

5 2 5

5 5 0

5 7 5

F i r s t

d e r i v a

t i v e

Wavelength (nm)

C

P

minus00098

minus00078

minus00058

minus00038

minus00018

minus00098

minus00078

minus00058

minus00038

minus00018

(b)







LOD = 3

LOQ = 10

(983089)


















(mean plusmn RSD )


P (983089D983093983089983095









983091983090983088ndash983093983090983088 nm


















001

003

308 328 348 368

F i r s t

d e r i v a

t i v e

C a r m o

i s i n e

P o n c e a u

4 R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

F i r s t

d e r i v a

t i v e

P o n c e a u

4 R C a r m o i s

i n e

Wavelength (nm)

f

g

h

i

j

minus002678

minus001678

minus000678

(b)






DSM C 983089



P 983089D983091983092983092 235 plusmn 011(044125) 143 plusmn 013(013169)


P 983089D983092983093983089 249 plusmn 017(189302) 144 plusmn 016(023268)













000988

008988

016988

024988

032988

300 400 500 600 700

A b

s o r b a n c e

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

D i ff e r e n c e

1047297 r s t

d e r i v

a t i v e

Wavelength (nm)

C

P

minus00068

minus00028

(b)



















000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)





















4 Conclusion






Acknowledgment


References












































httpwwwhindawicom







(mean plusmn RSD )


P (983089D983093983089983095









983091983090983088ndash983093983090983088 nm


















001

003

308 328 348 368

F i r s t

d e r i v a

t i v e

C a r m o

i s i n e

P o n c e a u

4 R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

F i r s t

d e r i v a

t i v e

P o n c e a u

4 R C a r m o i s

i n e

Wavelength (nm)

f

g

h

i

j

minus002678

minus001678

minus000678

(b)






DSM C 983089



P 983089D983091983092983092 235 plusmn 011(044125) 143 plusmn 013(013169)


P 983089D983092983093983089 249 plusmn 017(189302) 144 plusmn 016(023268)













000988

008988

016988

024988

032988

300 400 500 600 700

A b

s o r b a n c e

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

D i ff e r e n c e

1047297 r s t

d e r i v

a t i v e

Wavelength (nm)

C

P

minus00068

minus00028

(b)



















000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)





















4 Conclusion






Acknowledgment


References












































httpwwwhindawicom




001

003

308 328 348 368

F i r s t

d e r i v a

t i v e

C a r m o

i s i n e

P o n c e a u

4 R

Wavelength (nm)

a

b

c

d

e

minus005

minus003

minus001

(a)

000322

001322

002322

300 325 350 375

F i r s t

d e r i v a

t i v e

P o n c e a u

4 R C a r m o i s

i n e

Wavelength (nm)

f

g

h

i

j

minus002678

minus001678

minus000678

(b)






DSM C 983089



P 983089D983091983092983092 235 plusmn 011(044125) 143 plusmn 013(013169)


P 983089D983092983093983089 249 plusmn 017(189302) 144 plusmn 016(023268)













000988

008988

016988

024988

032988

300 400 500 600 700

A b

s o r b a n c e

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

D i ff e r e n c e

1047297 r s t

d e r i v

a t i v e

Wavelength (nm)

C

P

minus00068

minus00028

(b)



















000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)





















4 Conclusion






Acknowledgment


References












































httpwwwhindawicom




000988

008988

016988

024988

032988

300 400 500 600 700

A b

s o r b a n c e

Wavelength (nm)

P

C

Mix

minus015012

minus007012

(a)

-00028

00012

00052

335 365 395 425 455 485

D i ff e r e n c e

1047297 r s t

d e r i v

a t i v e

Wavelength (nm)

C

P

minus00068

minus00028

(b)



















000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)





















4 Conclusion






Acknowledgment


References












































httpwwwhindawicom




000011000211000411000611

401 451 501 551 601

Wavelength (nm)

6C6P 2P6C6P 4P

6C

6C6P 8P6C6P 10P

minus000789

minus000589

minus000389

minus000189

6C6P 6P = 6C

F i r s t

d e r i v a

t i v e

(a)

00008

00028

00048

400 440 480 520 560 600

F i r s t

d e r i v a

t i v e

Wavelength (nm)

6C6P 2C6C6P 4C

6P

6C6P 8C6C6P 10C

minus00072

minus00052

minus00032

minus00012

6C6P 6C = 6P

(b)





















4 Conclusion






Acknowledgment


References












































httpwwwhindawicom






4 Conclusion






Acknowledgment


References












































httpwwwhindawicom


























httpwwwhindawicom

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