IJCS 2018; 6(4): 53-57 Development Of Spectroscopic Method .
International Journal of Chemical Studies 2018; 6(4): 53-57P-ISSN: 2349–8528E-ISSN: 2321–4902IJCS 2018; 6(4): 53-57 2018 IJCSReceived: 13-05-2018Accepted: 18-06-2018Desai Bhoomikaben SM.Tech. Scholars,Dairy Chemistry Department,SMC College of Dairy Science.Anand Agricultural University,Anand, (Gujarat), IndiaModi Zeel SM.Tech. Scholars, DairyChemistry Department, SMCCollege of Dairy Science. AnandAgricultural University, Anand,(Gujarat), IndiaAmit Kumar JainAssistant Professor, DairyChemistry Department,SMC College of Dairy Science,Anand Agricultural University,Anand, Gujarat, IndiaSC ParmarAssistant Professor, DairyChemistry Department, SMCCollege of Dairy Science. AnandAgricultural University, Anand,Gujarat, IndiaAI ShaikhAssociate Professor, DairyChemistry Department, SMCCollege of Dairy Science. AnandAgricultural University, Anand,Gujarat, IndiaKD AparnathiProfessor & Head, DairyChemistry Department, SMCCollege of Dairy Science. AnandAgricultural University, Anand,Gujarat, IndiaCorrespondenceDesai Bhoomikaben SM.Tech. Scholars, DairyChemistry Department, SMCCollege of Dairy Science. AnandAgricultural University, Anand,Gujarat, IndiaDevelopment of spectroscopic method forquantification of starch in milkDesai Bhoomikaben S, Modi Zeel S, Amit Kumar Jain, SC Parmar, AIShaikh and KD AparnathiAbstractA spectroscopic technique was utilized to develop method for quantification of starch in milk. Forapplication of spectroscopy, colour was developed by reaction between starch and iodine, absorption ofthe colour was measured at 549 nm and standard curve was constructed for concentration of starch versusabsorbance obtained. Recovery factor was developed from amount of starch spiked and amount of starchobtained from standard curve. After developing the method, it was validated for performance by true(known) sample approach. The concentration of starch obtained by application of the developed methodwas compared with the concentration of the starch actually added for preparation of the sample. Forstatistical analysis t-test: two sample assuming equal variances was applied. The validation of methoddeveloped for quantification of starch suggested that the difference between the estimated amount ofstarch and amount of starch actually added to milk was statistically non-significant.Keywords: Milk adulteration, starch, quantification, spectroscopyIntroductionAdulteration of milk is a serious problem to be tackled by the dairy industry. Among variousadulterants used in form of carbohydrates, starch is used mostly though it is not permittedlegally. Starch or cereal flours, may be added to makeup the density after water addition andmask its detection. Starch is seldom added in the pure form to adulterate the milk. Usuallypoor quality starch or starchy materials such as cereal flours, arrowroot, wheat flours, mashedpotato etc. are added. Starch is a common adulterant in milk products as it gets easily mixed upwith products like khoa, burfi because of the similarity in the colour. Starch adds to the weightof the products, and is, therefore, a cheap source of adulteration [1]. If test for starch is positive,quantitative determination of starch should be carried out for determination of SNF content inmilk sample.To detect adulteration of milk with starch, use of this iodine test is reported [2-6]. In this testfirst milk is acidified with acetic acid and filtrate is collected. The filtrate obtained is treatedwith a few drops of a dilute solution of iodine [7].For quantification of starch in milk a Lane and Eynon method is given [8]. A method for starchestimation in ice cream is described [9]. A polarimetric method is reported for quantitativeestimation of added starch in milk [10]. This method is also confirmed for its applicability in icecream, gulabjamun mix and gulabjamun [11]. It appears that no work is reported fordevelopment of spectroscopic method for quantification of starch in milk.There is very limited work reported in the area of quantification of starch in milk usingchemical methods in conjunction with spectroscopy. Available methods are tedious, lengthy,involve the use of highly sophisticated instruments and are not applicable for routine purpose.Moreover, dairy industry is also in need of simple and inexpensive methods for quantificationof starch in milk. Because of these reasons there is a long standing need to develop simplemethods for quantification of starch in milk which can be used for routine purpose withminimum requirement of time, labour and instrumentation.Materials and MethodsMilk samplesThe genuine cow milk was brought from Livestock Research Station and the genuine buffalomilk was brought from Reproductive Biology Research Unit, Anand Agricultural University, 53
International Journal of Chemical StudiesAnand. The ratio of cow milk to buffalo milk was taken as40:60 to prepare mixed milk sample. Milk Samples wereprepared by spiking the raw milk with starch at the suitablelevel.Results and DiscussionFor quantitative determination of added starch in milk,qualitative test based on reaction with iodine is used. In thistest, iodine reacts with the amylose component of starch andgives blue coloured complex. The acidic condition in thereagent mixture accentuates the blue colour [12]. Attempt wasmade to use this reaction for developing a spectrophotometricmethod to estimate amount of starch mixed in the milk.This study was divided into 4 parts: to develop colour andfind out the maxima of the coloured complex formed,construction of standard curve, estimation of the recovery inmethod and validation of the method.Chemicals and ReagentsIodine (Loba Chemie Pvt. Ltd.), Potassium ferrocyanide (AR,S.D. Fine Chemicals Ltd., India), Starch (Qualigens Finechemicals), Trichloroacetic acid (Loba Chemie Pvt. Ltd.),Zinc acetate (ExcelaR, Qualigens Fine Chemicals, GlaxoIndia Ltd.)InstrumentSpectrophotometer (Micro ControllerSpectrophotometer CL-1320, Chemiline)BasedUV-VisSpectral CharacteristicsIn order to get spectra for starch mixed in milk, starch wasadded to milk to make adulterated sample. 20 ml portion ofmilk was heated for 10 min to dissolve the starch properly inmilk. 28 ml of hot distilled water was added in milk and wascoagulated by addition of 6 ml zinc acetate and 6 mlpotassium ferrocynide followed by filtration throughWhatman filter paper grade 1. To 5 ml of filtrate, 1 ml of 15per cent TCA was added to reduce the pH (as starch-iodinecomplex is more stable at low pH). 50 μl of 1 per cent iodinesolution was added to develop the colour and the content wasscanned for absorption maxima in visible region (400-800nm) in spectrophotometer. The spectrum obtained from thescan is presented in Fig. 1.Test procedure developed for determination of starch inmilkA) Preparation of standard curve1. In a beaker, 100 ml milk was taken and 0.03 g of starchwas added to it and mixed well.2. It was heated in boiling water bath for 10 min to dissolvestarch properly and cooled to room temperature.3. An aliquot of 20 ml milk was taken from the abovemixture.4. 28 ml of hot distilled water, 6 ml zinc acetate and 6 mlpotassium ferrocynide were added to it and mixedthoroughly.5. The content was filtered through Whatman filter paperNo. 1.6. Steps 1 to 5 were performed for pure milk (Control)simultaneously.7. Filtrates of sample (with starch) and control (withoutstarch) were collected in clean and dry conical flasks.8. For preparation of standard curve, different volumes offiltrate (1, 2, 3, 4, 5 ml) containing starch were taken indifferent test tubes so as to provide a concentration of0.1, 0.2, 0.3, 0.4 and 0.5 mg starch per test tube. Totalvolume was made up to 5 ml in each test tube withcontrol filtrate. Test tube containing 5 ml control filtrate(obtained from pure milk) served as blank.9. In each test tube 1 ml of 15 per cent TCA was added andthe content was mixed.10. 50μl of iodine solution (1 %) was added to each test tubeusing micropipette and mixed well to develop the bluecolour.11. The absorbance was measured at 549 nm against blank ina spectrophotometer.12. A standard curve was plotted using absorbance (along yaxis) and starch concentration (in mg) (along x-axis).Fig 1: Absorption maxima (λmax) of starch iodine complexDifferent peaks were obtained during scanning, e.g. 536, 540,549, 557 nm, among which the most prominent and highestpeak was obtained at 549 nm, therefore this maxima (549 nm)was selected for further study.Construction of standard curveAfter finding the maxima, in the second part, standard curvewas prepared. For preparation of standard curve, 0.03 g starchwas added in 100 ml milk and filtrate was prepared accordingto the procedure described in Section A. Different aliquots (0,1, 2, 3, 4, 5 ml of filtrate from sample mixed with starchcontaining 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mg starch) were takenand total volume was made to 5 ml with the filtrate of controlmilk sample, wherein, no starch was added. To these testtubes, colour was developed by following the procedure asdescribed in Section A. The intensity of the colour obtainedwith varying amount of starch is presented in Fig. 2.B) Estimation of starch in milk samples1. Adulterated milk samples added with starch wereprepared by mixing different amounts of starch (in therange of 0.0025 to 0.027 g per 100 ml of milk).2. To obtain the filtrate, steps 1 to 5 of the procedure used inSection A were followed.3. Aliquots of 5 ml filtrate were collected in test tubes andsteps 9 to 10 of Section A were followed.4. The absorbance was measured at 549 nm against blank ina spectrophotometer.5. The standard curve prepared as above was used fordetermination of added starch in milk. 54
International Journal of Chemical StudiesFig 2: Intensity of the colour obtained with varying amounts ofstarchFig 3: Standard curve for starchIt is evident from the Fig. 2 that intensity of the blue colourformed gradually increased with the increase in concentrationof the starch mixed in the milk.The resulted intensity of the colour was determined bymeasuring the absorbance of the each test tube at 549 nm.Three replications were conducted and average values of thisexercise are given in the Table 1 below. The graph (standardcurve) of absorbance versus concentration of starch wasplotted, the standard curve so obtained is presented in Fig. 3.It was evident from data given in Table 1 and Fig. 3 thatabsorbance values of the reaction mixture were linearlyincreasing with the increase in concentration of the starchmixed in the milk and coinciding very well with increasingintensity of the colour. The slope (R2 0.999) indicated thatthe regression line was very well fitted in the data.Estimation of RecoveryAfter developing the standard curve, experiments wereconducted to estimate amount of starch obtained fromstandard curve after mixing known amount of starch in themilk. The starch was mixed in milk in the range of 0.0025 to0.027 g per 100 ml of milk. Different amounts of starch wereselected to cover the entire range of standard curve. Theamount of starch in each case was estimated from the standardcurve. The per cent recovery of starch obtained in theexperiment was derived from amount of starch expected inthe 5 ml of filtrate and the amount of starch estimated in 5 mlof filtrate from standard curve. Three replications wereconducted. The data obtained for the recovery of starch arepresented in Table 2.Table 1: Observations taken for preparation of standard curve(starch)Sr. No. Starch (mg/5 ml filtrate) Absorbance (549 .50.551The values are average of three replicationsTable 2: Estimation for recovery of starch in the experimentReplication Amount of starch expected (mg/5 ml unt of starch estimated (mg/5 ml .3260.4120.0470.1670.2150.3160.415It appeared from the results that the recovery of starch in theexperiments varied from 88.82 to 100 per cent, with anaverage of 95.58 per cent. Therefore, to estimate the actualamount of starch mixed in the milk, results obtained from theexperiment need to be multiplied by the recovery factor. Thisrecovery factor was obtained through dividing 100 by averageper cent recovery observed in the experiment. Hence, therecovery factor for the starch in the present study was 1.046(i.e. 100/95.58).Recovery .5594.00100.099.0799.6899.5095.58The amount of starch recovered in the filtrate was lower thanthe amount of starch mixed in the milk. This might beattributed to some losses of starch during preparation of thefiltrate. The losses may occur due to retention of starch in thecoagulum. The starch may retain in the coagulum due to twopossible reasons. One is interaction of the starch with casein.Another reason may be retention of starch in the serumportion remained in the coagulum as moisture. The finding ofthe present study is in the line with views expressed by theauthor [13]. These authors stated that both protein and starch 55
International Journal of Chemical Studiesare typical polyelectrolytes and that the interaction in acidiccondition must be caused by the attraction of opposite chargeswhich are caused by the dissociation of some ionizable groupsof the components.ml milk) of starch derived from standard curve was multipliedby recovery factor to get amount of starch mixed in 100 ml ofmilk by this experiment. The data obtained for amount ofstarch actually mixed in the milk and corresponding amountof starch practically estimated in the experiment, along withtheir statistical analysis are presented in Table 3.Validation of the MethodAfter developing the recovery factor, the amount (g per 100Table 3: Amount of starch mixed and estimated in 100 ml of milkS. No Starch from std. curve (A) (g/100 ml milk) Starch estimated (A x 1.046*) (g/100 ml milk) Starch mixed (g/100 ml age0.01440.01510.0151t Stat0.0043P(T t) two-tail0.9965TestNS* Recovery factorNS Non-significantThe starch was mixed in the milk in the range of 0.0025 to0.027 g per 100 ml of milk, with an average of 0.0151 g per100 ml of milk. The amount of starch estimated in the milk bythe method developed in this study ranged from 0.0025 to0.0273 g per 100 ml of milk, with an average of 0.0151 g per100 ml of milk. The comparison between actual amount ofstarch mixed per 100 ml milk and amount of starch estimatedper 100 ml milk by the method of the study revealed that thedifference between the two was statistically non-significant.The results suggested that method worked out in the studywas suitable for practical purpose.From review of literature it appears that method (based onLane and Eynon method) is reported for quantification ofstarch in milk [8]. In this method, milk is coagulated byethanol; while in the present study milk was coagulated bycombination of zinc acetate and potassium ferrocyanide. Inthis reported method, analysis is conducted on precipitatesafter washing with ethanol to make them free fromlactose/sugar; while in the present study filtrate is used foranalysis. In this method, starch is hydrolyzed to glucose byhydrochloric acid with heating for 2.5 hours and the reducingsugars before and after hydrolysis are measured by Lane andEynon method; while in the present study the filtrate wasacidified with TCA and colour was developed by addition of1 per cent iodine solution, which was measuredspectrophotometrically at 549 nm.developing the colour with starch at the rate of 0, 0.1, 0.2, 0.3,0.4 and 0.5 mg per 5 ml milk serum. After constructingstandard curve work was carried out to know actual recoveryobtained from the standard curve. From the average per centrecovery obtained in the experiment (95.58 %), the recoveryfactor was derived (1.046). The amount of starch obtainedfrom standard curve was converted into amount of starch in100 ml of milk by back calculation. The amount of starch thusobtained per 100 ml of milk was multiplied by the recoveryfactor to compensate the losses of starch occurring during theprocedure followed in the experiment. Finally, performanceof the method was tested by comparison of amount of starchobtained in milk by the developed method with actual amountof starch mixed in the milk. The difference between the twowas statistically non-significant. The results suggested thatmethod worked out in the study was simple, relatively fasterand suitable for practical purpose.References1. ants in common food products,Indian Standards: 15642 (Parts 1 and 2), Bureau ofIndian standard, Manak Bhavan, 9 Bahadur Shah ZafarMarg, New Delhi, 2006.2. BIS. Methods of test for dairy industry, Part- I: Rapidexamination of milk, Bureau of Indian standard, ManakBhavan, 9 Bahadur Shah Zafar Marg, New Delhi, 19603. BIS. Handbook of Food Analysis - Part XI: DairyProducts, Bureau of Indian standard, Manak Bhavan, 9Bahadur Shah Zafar Marg, New Delhi, 19814. Vishweshwar SK, Krishnaiah N. Quality Control ofMilk and Processing (for the course of dairying). Stateinstitute of vocational education director of intermediateConclusionFor quantification of starch, spectroscopic method based oniodine test used to detect starch in milk was used. Afterdeveloping the blue colour by reaction between starch andiodine reagent in milk serum, the maxima of colourabsorption was obtained in visible region and it was found tobe 549 nm. Then standard curve was constructed by 56
International Journal of Chemical Studies5.6.7.8.9.10.11.12.13.education govt. of Andhra Pradesh sindoor graphics,India, 2005.Draaiyer J, Dugdill B, Bennett A, Mounsey J. MilkTesting and Payment Systems Resource Book - Apractical guide to assist milk producer groups. FAO,2009, 38-43.Singh A, Chandra G, Aggarwal A, Kumar P.Adulteration Detection in Milk. Research News for U2012; 5:52-55.Sommerfeld P. Methods for the examination of milk:For chemists, physicians and hygienists, FB & C Ltd.Dalton house, 60 Windser Avenue, London SW19 3R;1901.Food Safety and Standards Authority of India. Manualof methods of analysis of foods (milk and milk products)- A Laboratory Manual. Food safety and standardsauthority of India, Ministry of health and family welfare,Government of India, New Delhi, 2015.Unnikrishnan V, Narayanan KM. A method forestimation of starch in ice cream. Indian Journal ofDairy Science. 1987; 40:440-444.Charles AS, Sharma R, Rajput YS, Singh RR. Apolarimetric method for quantitative estimation of addedstarch in milk. Indian Journal of Dairy Science. 2006;59(5):287-290.Charles AS, Sharma R, Rajput YS, Singh RR.Estimation of starch in ice cream and gulabjamun bypolarimetric method. Journal of Food Science andTechnology (Mysore). 2009; 46(3):207-211.Pimstone NR. A Study of the Starch-Iodine Complex: AModified Colorimetric Micro Determination of Amylasein Biologic Fluids. Clinical Chemistry. 1964;10(10):891-906.Takeuchi I, Shimada K, Nakamura S. The formation ofnitrogenous compounds in moto mash (starter) of sake.VII. Interaction between protein and starch ofrice. Journal of the Agricultural Chemistry Society ofJapan. 1968; 42:294-299. 57
with products like khoa, burfi because of the similarity in the colour. Starch adds to the weight of the products, and is, therefore, a cheap source of adulteration [1]. If test for starch is positive, . (as starch-iodine complex is more stabl
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