इंटरनेट मानक

Disclosure to Promote the Right To Information Whereas the Parliament of has set out to provide a practical regime of right to information for citizens to secure access to information under the control of public authorities, in order to promote transparency and accountability in the working of every public authority, and whereas the attached publication of the Bureau of Indian Standards is of particular interest to the public, particularly disadvantaged communities and those engaged in the pursuit of education and knowledge, the attached public safety standard is made available to promote the timely dissemination of this information in an accurate manner to the public.

“जान का अधकार, जी का अधकार” “परा को छोड न तरफ” Mazdoor Kisan Shakti Sangathan Jawaharlal Nehru “The Right to Information, The Right to Live” “Step Out From the Old to the New”

IS 3508 (1966): Method of sampling and test for [FAD 19: Products and Equipment]

“ान एक न भारत का नमण” Satyanarayan Gangaram Pitroda “Invent a New India Using Knowledge”

“ान एक ऐसा खजाना जो कभी चराया नह जा सकताह ै”ै Bhartṛhari—Nītiśatakam “Knowledge is such a treasure which cannot be stolen”

18 : 1501-1_ Indian Standard ~ METHODS OF SAMPLING AND TEST FOR ~ GHBB ()

Fourth Reprint AUGUST 1990 (Incorporating Amendments No. I and 2 ,

UDC 637-25: 543"05

o Copyright 1975 BURIAU or INDIAN STANDARDS WANAlt BBAYAN. , BAHADUR SHAH ZAFAR MARO NIW baHl 110002 Jlca, 1961 1113508 .1•• I ndian Standard METHODS OF SAM.PLING AND TEST FOR GHEE(BUTTERFAT)

Dairy Industry Sectional Committee, AFDC 12

ChairlrUln Representing DR K. C. SSN Indian Dairy Science Association, BangaJore

M~mbtrs

AaRlcuLTURAL ~IAaKETINo Directorate of Marketing & Inspection ( Ministry 0 ADVlaS8 TO THB GOVB8NIIBNT &. Agriculture), Nagpur 0 .. h'DIA S.al V. Cs A.~Da.MoULY ( Alternate) 5••1 B. R. BSDEICA.R Hindustan Milkfood Manufacturers Limited, Nabha SIQII I. C. E. DAUSON ( Alternate ) 8"1 C. V. CHANDRA SBItHAa T. T. ( Private) Limited, Bangalore S••I S. S. 1\(4:(1 (Allernat,) SOl H. ~I. DALAYA. " Kaira District Co-operative Producers' Union Ltd, Anand DaJ. D. COSTBACTOR (Alttrnalt) S.al C. D. DASTOOB Larsen & Toubro Ltd, Bombay SRal H. W. RAJICHANDASI ( Alumate ) na N. N. DAITUR National Dairy Research Institute, Karnal DB C. P. ANA.NTAKRI8H~ AS ( Alttrnill, ) S.B' V. A. DATAR Vulcan-Laval Ltd, Bombay SRBI A. DsvAIlIYA ( Altemat« ) DI.ECTOB, MILIT.BY FARMS Directorate of ~filitary Farm" Army Headquarten AaI8TAST DIRECTOR. !\!ILITABY FABII8 ( TECH) (AlttTtUllt) EXECUTIVE HEALTH OnlcBR Municipal Corporation, Bombay MUNICIPAL AN.ALY8T (Al"rnatt) CoL A. G. FBBN.NDu Food Inspection Organization, Quartennaster General's Branch, Army Headquarters Llf'·COL N. G. C. ISNo£B ( Allt,,,,,t, ) 5••1 G. S. GODBOL. Dairy Development Commissioner, Oovernmeet of Maharasbtra S••~Y. v. S"UIl:K•• ( Altt,.,..,,) Da K. K. !YA Minilltry of Food &. Agriculture Saar G. GoPIKATB (Alk",4l1) CoL P. C. KlUX.. Technical Standardization Committee, Foodstuff's (.~t:inistry or Food at Agriculture) Da S. S'. PRAll'oAK (Altemtll,) SIISI A. R. A. K.I••IIAlf Defence Production Orlaniaation [Ministry or Defence ( DGI ) ] SBaz K. P. Smo. ( ~11frMl' ) (C..tUtwi .. /NI" 2 )

BU_AU OF INDI'AN" STANDARDS YANAIt BHAVAN, 9 BAHADUIl SHAH ZAPAR. MARO NBW DBLHI 110002

Ilre_.I9&6

( dMI~tlf,.", /JIll' I) M""" &"..",., Da A. P. MAIUDPd HlDdUitan Lever Ltd, Bom"y Da K. K.. G. M ••o. (~IImNIII) MILK Oo1IJIII810... Milk Conuniaioner, Madra Smal P. VIRtA1fAftlA M..o. (AlImwJU) S.ar S. N. MJ'U4 Central Food Laboratory, Calcutta SOl B. K. Mua-rBY Indian Aluminium Co Ltd, Calcutta SBJd N. GoJt..u. KaJu.Alf '( AlImMI, ) SBaI B. E. NAJlQJILI Nesde'. Productl ( India) Ltd, "New Delhi SJUd .,: J. llYA1' (it/"""") 5...j. PADJUJrABBA1f The A. P. V. EDgineering Co Ltd, Calcutta SJIJU1. G. BBoWK (AI"",,) S•• S•.a.•.-WAIIY DirectOl'fte General of Technical Development' Da R. S. SUVAftAYA Central Committee (or Food StaDdariis ( MiDiatry 01 Health) S..I P. JAJlAJmAlfA' AIYA. ( AI,,,,..,, ) D. M. SWAIID'ATBAlf Ceutral Food TechDO'ogical R~lrch Institute (CSIR), Mysore SIIBI M. R. CBAlIDBdllKBA•• ( AI",,,.,,) SJIJU.R. H. V....IAV", Polson Limited, Bombay SJDU Be P. P.u.KBJWALLA (AlImNIII ) Da I. S. V..... nairyTechnology Division, National Dairy Research Institute,,·K.rnal 5••1M. R. SJIPQV"'A_ (AllnR1II6 ) SB&J JAil•• N. W...... 10 personal C8pathy (Allah_" A,rleul''''''' /1II'illll6, AUtl1uJbtul ) Da B ••I BBAQWAlf. Director General, lSI (~.f/itt. M,mHr) Deputy Director ( Agri It Food) $.,,111'.1 ~BSI v.s. ~fATB11. Assistant DJreotor (Agri It Food), lSI

Methods of Test for Dairy Products Subcommittee, AFDC 12.:4 c...",., DBN. N. Ouru. National Dairy Relearch Institute. Kamal M"""", D. S. C. CBAKBABAaftY Public Analyst, Go,·ernrJient of Wat Ben.al SBN V.CBANDRAMOVLY Directorate or Marketing &: I~ction ( Ministry or Food It Agriculture), Narur SJl1U !vI. R. CBAtm.AaKaA.~ Central Food Technologjca Research Jnititute (CSIR), MYlOre " .. SSRI H. lvl. D~L.y ... Kaira District Co-operative Milk Producers' Union Ltd, Anand 0. I. M. PAUL (~l"m'" ) (co,,~ .",." 62 ) 2 Da A-VII Ita.-S..Gun. Da R.. S. SalVAftAYA SIIaIJ.uDI N. W.....

62 11.,....\1- CONTBNTS PAoa O. FouwoaD ... .4. 1. SCOPB 5 2. SAMPLING ••• 5 3: Q,UALITY 0. RBAOBNn 10 PHYSICAL MlTHODI 4. DBURIIIKATION o. MoJlTUllB CoNTain' 10

5. DBTBIlMINATION OP COLOU& 11 6. DBnaloNATION oplUnAcnva IND.X 13 7. DaTBIUdNA.TlON OJ'TIT.. 15 8. DBTBIUIINATIOK OP MKLTlMo POIMT (Sup POINT) 19 9. SeoRB CARD Foa GBU :.. 20 CHIMICAL MITHODS JO. DauIUIINAT10N 0' INSOLUBLE IIIPVRmu 20 II. DBUlUIINATION OP ACIDITY 21

12. DaUlUIlNATION OP SOLU.U AND INIOLUBLB VOLAftLa ACIDI ( RBiCHERT-MEISIL, POLBNIO AND KIRSCHIfBR VALU') ... 22 13. DSTBIUIINATION 0' SAPONIPICATION VALUE 29 14. DBT...IIi_ATlON 01' IODUCB VALUE (WI)S' MBTBOD) 30 15. DBTBIUIINATION 01' UNIAPOMlPIABU MAna ••• ~34 16. naTBUlilfATlOK OP A 36 ,17. DSTBIUIDfATION OP TOCOPIIBIlOL ••• 59 18. DaT&cmON AJlQ DIITaaImIATION O. D.oLYBD Sa,w 41 19. Daa.....A11OJf 01' AImoxIDAK'n ••• 42 20. ».uan0ll o. V..TAIIU 'IN G••• BY THa Pa.... '!'UYL A.caTA.... Tur •.. 52 21 DaftalaNATIOM O. PaUBNca o. SBIAIm (1A1JDOUIIf TUT) •.• ••. •.. ••. ••• 5& 22. _ftOK o...... PaaoXIDa VALUB 56 23. DnaUIIICAftOM o. IJIOIC CoNT~1ft 59 3 11.3511·1111 I ndian Standard METHODS OF SAMPLING AND TEST FOR GHEE (BUTTERFAT)

o. FOR EWO RD 0.1 This Indian Standard was adopted by the Indian Standards Institution on 16 September 1966, after the draft finalized by the Dairy Industry Sectional Committee had been approved by the Agricultural and Food Products Division Council. 0.2 Ghee (milk-fat, butterfat, -oil), is the most important which enters inter-state trade. Due to variation in its composition from region to region and season to season, depending on the type of animal and the food given, the establishment of its purity often involves elaborate analysis, as well as tests for its keeping quality. The present standard describes the methods of sampling and quality control generally needed for such analysis and facilitates the interpretation of results. 0.3 In the formulation of this standard, considerable assistance has been derived from the following publications: FIL-IDF 6 Acidity of butterfat. International Dairy Federation. FIL-IDF 7 Refractive index. International Dairy Federation. FIL-IDF 8 . International Dairy Federation. Directorate of Marketing and' Inspection ( Ministry of Food and ARri- culture). Methods of sampling and testing of butterfat (gir,,) and butter under agmark. 1953. B.S. 627 : 1953 Sampling and fatty . British Standards Institu­ tion. B.S. 684: 1958 Methods ofanalysis ofoils and fats. British Standards Institution. Standard Methods of the Oils and Fats-Dh:ision of the International Union of Pure and Applied . 1964. Butterworths, London'. Official Methods of Analysis of the Association of Official Agricul. tural Chemists, 9th ed. WashingtoJ3. ~5t U.S.A. 0.3.1 Full use has also been made of the valuable information received from the National Dairy Research Institute, Kamal. '.4 In reporting the result of a test or analysis made in accordance with this standard, if the final value, observed or calculated. is to be rounded oft; it' shall be done in accordance with IS: 2.1960-. -Rula (orroUDdi"-.aumerleal n1ueI ( __). 4

1.lCOd 1.1 This standard prelCribes' the methods of "lDpJing, abalysis' anW __ generally used for evaluating the qualitVor ,hee. The specific methoc!l''to be used would depend on the object of the analysis. ·

2. SAMPLING

2.1 Sampling shall becarried out by an mtperieqeed ~n. It C&IIDOt be too Itron~ly empha,i~ed that correct sampling is a. Clifticult problean aad ODe ,vhich requires the mosl careful attention to details if the .-bsequent analysis is to be ofvalue. A. "1Dple which is representative of the bulk ~ asemial and is particalarly difficult to obtain from a consig~ent CODIiat­ in-, ora large number of packages. Ho,,·ever,·u. guide to the sdectioa of samples, useful information may be found in documents and certi&cata normally accompanying the consignments and usually include claai&cation markings. It is recommended that the method given should be adhered to wherever practicable. Particular circumstanees may render solDe m0di­ fications of the recommended method n~ry. 2.2 leale of S..pU., 2.2.1 lA, - All the containers irra single consignment belongm,-to the same batch ofmanufacture shall be grouped together to constitute a lot. If a consignment is declared to consist of different batches ofmanufacture, the batches shall be marked separately and the group ofcontainers in each batch shall constitute separate lots. 2.2.2 The number ofcontainers to be selected for sampling shall tlepald upon th~ lot size and shall be in accordance with Table I.

TABLE I NUMBER OF CONTAINERI TO BE S~LECTED FOa SAMPLINC

Sox•••o.eo• .,AIJfZ.. NUlDEa ow COYrAIX... IX '1'•• Lo'1' '1'0 •• SBLJtcrBD

II I I 2 to 40 2 4. " 110 S .111 tt ,300 ,5 101 It,' &00 101 andabove 10

5 11,'3501'.1. 2.2.3 These' containers shall be selected at random &om the lot. To ensure the randomness ofselection, a random ~umber table as agreed to between the purchaser and the supplier, shall be used. In case such' a table is·not available, the following procedure shall be adopted: I Starting f(om any container, count them u 1,2, 3,...up to,anCi so OD, in one order, where r'is equal to the integral part of N/", N being the total number of containers in the lot and'lI the number of containers to be selected (s" Table 1). Every rth container thus counted shall be withdrawn to give required number ofcontainen in the sample. 2._.~ I£th{t:e is a possibility ofwide variation among the different unita, for ~x~P'lfJ .~ the consignment of ghee from an individual producer, ever, unit shall ~ &IilRled. 2.3 Sa..ple CoDtala.... 2.3.1 Wide mouth jar and bottles and tin containers of 50, 100 and 200/250 ml capacities and of following approximate dimensions are con­ venient to use as sample containers: Nominal capacity 50 ml Height 60 X Width 48 X width ofthe mouth 50mm Nominal capacitJ 100 011 Height 70 X Width 60 X width ofthe mouth 44 mrn Nominal capacity 200/250 ml Height 97 X Width 70 X width of the mouth 60 mm 2.3.2 The jan shall .. be closed by means of a screw cap lined with butter paper, Bottles shall be gJ~-stoppered. Tin containers shall be closed with the press-en type of Iids, For chemical analysis, bottles may also be closed with rubber stoppers lined with butter paper iforganoleptic tests are not to be made. 2.4 Samplla, AppUaac.. 2.4.1 The sampling i'nstrument shall be such that it is possible to sample the contents throughout the whole depth ofthe containers by it. 2.4.2 Sampli", Cone",,", T"bls ( Fig. 1..4 )-A con~el1ient sampling instru­ ment consists oftwo concentric tubes closely fitted into each other through­ out their entire length, so that one tube may be rotated within the other. A longitudinal opening,of about one-third the circumference is cut in both tubc;s. In one. poIitjon the tube is open and ad~its the ghee; by tuming the Inner tube It becomes a sealed CODtainer. . The inner tube may be 19 to 38 mm in ~.ametcr and undivided Inill leJIIlh. The two tubes are provided with V-shaped porta at their lower 6 e CLOSEDo OPEN c3 SECTION XX

I 'ORTS~ ~ @ OPEN CLOSED BOTTOM VIEWS

Flo. lA SAMPUMO CONCBNTlUC TUBBS ends, 10 placed that ghee contained in the instrument ean be drained through them when the longitudinal openings are open. The instrument should be inserted closed; it is then opened to admit ghec and finally closed and withdrawn. 2.f.l SaaJlliJ14 Pia" rllk (Fil. IB) - The sampling tube may be used whea lhee it flUId and i. known to be quite uniform. It consists of• .-etal or ,thiCk-waJled gl.. tube which may_yar)' from 20 to 40 mm in diameter aucl ahould be 375 to 750 mm IODg_ The upper and lower end.arec:onicaI 7 Elo. 1B SAIIPUHO PLAIN' TUBE and narrowe4 down te about 6 to 12 mm. At the upper end there are two ringl to assist handling. To ta~e an individual sample the apparatus is tint closed at the top with the thumb, or stopper, and lowered until the desJred depth. reacbed; it is then opened for a shon time to admit the ghee and finally cloeecl .DeI withdrawn. 2.f.4 The samplingappliancessball be made preferablyor.taiaJaalteel. The lurface of the instruments shall be poli.hed. 2deS All aamplin~ equipment shall be perfectly cleaD and dry aDd ahall DOt impart any foreign odour or Savour. Samplinl iDitrumeDti may be c1eaDed with hot lOapywater or other detergent, care beiDI taken to Wash away the lut traces with scalding hot water. If. lOurce or Iteam it available the instruments may be giVeD a final cleaninl in ajet or..... 8 2.5 po., T...._ 2~.1 Sampliol shall be carried out in such a manner as to protect the sample, the lampling iDstrumentsand the containen in which the samples are placed from advenfitious contamination, such as rain and dust. 2.5.2 Material adhering to the outside of the sampling instruments shall be removed before the contents are discharged. 2.5.3 A sample shall be drawn Crom each container to be sampled with the sampliDg instrument which is inserted through. convenient opening in such a manner as to sample the entire depth of the contents. 2.5.4 All samples from _he same co~gnment .hall be put into a clean and dry receptacle, preferably of atainless steel. The contents of the receptacle shall be thoroughly mixed and the required sample drawn into a clean and dry sample container. 2.5.5 The sample container shall be closed, leaving sufficjent air space at the top for expansion. On the other hand this space shall not be too lup.... air e~ertl detrimental actton. 2.5._ All samples shall be protected from light and heat, and kept in • cool place. 2.1 P~d..oleo.poRte Sample - Taking equal amountofghee from each or the containen selected (2.2.2). collect at least 300 g of the material u described in 2.5 which shall be mixed and divided into three equal parts. Each part Ihan be transferred to a separate sample container. aae or these composite samples shall be for the purchaser, one for the veDCIor and the third for the referee. Store the containers at a cool and clark- plaee. 1.7 T_doa...Ito..... of Sampl•• - Samples should be sent u quickly as possible to the examiDing laboratory, and should be prGtect~d from light and contaminating odour. The sample shan be ktpt In a coo) and dark place. 2.1 Pnpua••o''''''e,... Score Canl ... A•••,.I. 2.1.1 s-,uftw &0" c.tl of GMI - Testing shaD be carried out soon after openiag the container. In cue oflarge containers. soOn after opening, thecontents shall be thoroughly mixed and about 200 , shall be transferred t~ ...... bottle with a well.fitting stopper. The sample shaJl not be heated bdbre the score card is prepared. 2.1.2 s.",fir "" D,lmIIi.,ion DfMoi,,,,, ad Gmnlll AMlysil 2.1.2.1 Mix the sample in the container in Which it is received until holDOFneous. Carry out thisoperatioDin a cool place, away from direct lumi,bt, and complete it in shortest possible time. In the event of any ICpUation takinl place in between, that is. mixilll and commencement

11.'.·1. or the analysis for moisture, remix the .mple. Use tbia (or the determination ofmoiature. 2.1.2.2 After the determination ofmoisture place the bottle in a water­ bath at a tem~rature not lUsher than 50·0 till completely melted. Filter through a dried, fluted open..texture IS em filter paper (for example, Whatman No.4) with the help of a hot water funnel, directly into the receiving bottle. Continue the filtration until it is complete, or not more than 3 or .. ml of ghee remainl. The filtered ghee lhould be bright and clear.

3. Q,UALITY OF REAGENTS 3.1 Unless specified otherwise, pure chemicaJa and distiUed water (,,, IS: 1070-1960. ) shall be employed in tests. Non-' Pure chemieals • thanmcan cbemJcaltthat doDOt coataia impuritlea which afFect theexperimental results.

f. DETERMINATION OF MOISTURE CONTENT ~.1 Appa..." 4.1.1 Moil,." Dish- of aluminium 7 to 8 cm in diameter and 2 to 2·5 em deep; provided with tight-fitting slip-ever cover• ••1.2 DIM_- cbntaining an efficient desiCCAnt, such as phosphorul pentoxide. 4.1.3 A;"oOlll- preferably electrically heated, with temperature control device. 4.2 Pnceclve - Weigh accurately about 10 g of the sample into a moisture dish which has been dried previously, cooled in the desiccator and then ~hed. Place the dish in the air-ovcn for approximately one hour at lOS- ± I·C. Remove the dish from the , cool in the desiccator to room tenaperature and weigh. Repeat this procedure but keep the dish in the oven only for half an hour each time until the difrerence between the two luccessive weighings does not exceed 1 mg. Preserve the dried sample for the determination of insoluble impurities ( III 10 ). 4.3 Calaladea Moisture and volatile m~tter 100 ( HI; _ ~ ) content. percent by weaght - (WI .!. W) • where WI .. weight in g of the dish with Sbee before dryiDl, W. - weight in g ofthe duh with1_after dryinl. aacI W - weight in g or the empty dilh. _ for water, dWUed quality ( __). 10 J8.,..1IM 4.4 Accaraq otdI.Metlaod - The maximum deviation between dupU. cate determinatiOlllshall Dot exceed 0-1 (percent).

5. DBTBB.MINATION 0. COLOUR ,.I Two methods for measuring colour of ghee are prescribed. The fint method using a Tintometer is simple and suitable for routine work. Where more precise information is required the second method using a spectrophotometer shall be used. 5.1 Tlatometric Metlaocl 5.1.1 AHaratus 5.1.1.1 Tint,,,,,,,, - preferably with light attachment, 5.1.1.2 Tlurmomlltr - calibrated from oCt to 50·C. 5.I.l.S TintD""t" cells - O·S-em and I-em. 5.1.1.4 Wt,lItr-batA - maintained at 40° to 50°0_ 5.1.2 P,ocedllr, - Melt the sample as described in 2.1.2.2 and transfer it to the Tintometer . Keep the cell in a water-bath and Itir the contents with a thermometer, When the sample attains a temperature of we, match the colour against standard glasses in the Tintometer. Express the results as yellow units per r..rt\ at 40°0. ~.2 Spectrop.otometrlc Method 5.2.1 A/J/Jarflbu 5.2.1.1 S/Jeelro/JlaDlotlUlIr - A spectrophotometer capable ofadjustment to give the following readings on a standard nickel sulphate solution (SN 5.2.1.3 ) at 25° to SO·O, after setting the zero point and after adjusting the 100 percent transmittance point (0 absorbance) apinat tetrachloride in a cuvette having the outline specified in 5.2.1.2: Milli",iertnll T,IUIStIIiUlllte, 400 Less than 4-0 percent 460 '26·2 ± 2-0 percent 510 73-9 ± 1-0 percent S50 54-8 :J: 1-0 percent 620 5-2 ± 0·5 percent 670 I-I :J: 0-.5 percent 700 Leas than 2·0 percent 5.2.1.2 Mad. ,1Gs 9litulrietll ea.''''- Inaide. diameter approxi­ mately 21·a IDDlj outliClediameter approximately 24-5 mm. All cuvetta to II 11._.1_ ...wiala • pven iDltrument shouldbecheckecl with carboD tetndaIo­ ride ( ca.) and the nickellUlphate solution at 550 mp aad ~ witIIiD :j:G-6 ~Dt of the ..me transmittance. The cuvett. shoUld be kept deaD and Cree from ICratches. 5.2.1.3 S,.".tlidlw '" nIl~ 101,,,. - Dissolve 200 g of Dickel salphate (NiSO••6Ha0), analytical reagent grade, in' distilled water. AdcllO ml or coDcentrated hydrochloric acid. Dilute to exactly I 000 ml in • volumetric flask. The temperature of the solution should be between 25- and 30°0. ~ The nickel coetent of the IOlutioD lhall be between 4·40 aod ...·46 g oCnickel per 100 ml at 25° to 30°0. 5.2.1.4 Filln,.", - fine porosity, such al Whatmaa No ... 12. 5.2.2 RMI",." 5.2.2.1 C",6. ,.Ir.Alorid, - redistiUed If the transmittance diffen ftam distilled W.Ater by 0·5 percent at 400 mlA. 5.23 P,tJ&,d"" 5.2.3.1 Ctlli6,tJlioa oj I1tI s/Jetrophot,mttn - Tum on the spectrophoto­ meter and allow at least 20 minutes' warm-up period before standarclqing ormakingany measurements. After the initial warm-up period, rotate both the control knobs on top ofthe instrument counter-clockwise to their stop JM*tion. Adjust the galvanometer by means of the galvanometer adjtllt­ ment or by shding the scale 10 that an exact zero reading is obtained. Set tile wavelength dial to 460mf&- Re!eheck the zero reading of the instru­ IDeDt, insert a cuvette filled with carbon tetrachloride in the inatrumeDt · aDcIlet the 100 percent transmittance point exactly. Fill a cuvette with the ltandardizdlg nickel sulphate solution and read the transmittaDteor the 101ution. The reading should Call between 24-2 and 28·2. In a similar manner set the instrument at 550 mil ano read the traDIIDittance for the nickel sulphate solution. The readiDI lboald 1.:&11 between 53-8 and 55-S.

Ifthe reading at 460 ml& IS above 26-2t the reading at 550 mJl should be above 54·8; if the reading at 460 mil is below 26'2, the rflading at 550 IIlf& should be below 54·8; otherwise adjust the wavelength knob uodemeath the instrument until both readiDRs are in the same direction above or below the median values established, but within the specific limits. Set the instrument at 510 m,.., and read the transmittance for the nickel sulphate solution. The 510 mil reading shall be between 72·9 and 74-9 Read the other specified values. All should faU within the limita specified• 5.2.'.2 D,tmniuliM - The sample shall be rendered optically clear and tree from water and other suspended imput'itia. Adjult the tcm· peratue of the sample to S:i· to 40-0, fill the cuvette using a su8lcient 12 II.UII-IIII .mount of .mple to enaure a full column in the light beam. Place the &lied cuvette in the instrument and read the absorbance to the nearest 0·001 at 460, 550, 620 and 670 mIL 5.2.4 CIIbl.,i,1I Photometriccolour - 1·29 D_ + 69-7 D... + 41-2 D. - 56-4D.. where D II the ablorbance. 5.2.4.1 Special instrument scales for reading the four facton involved directly may be used.

I. DBTBRMlNATION OF REFRACTIVE. INDEX 6.1 Refractive index i. the ratio of the velocity of light in vacuum to the . velocity of light in the ample medium; more generally, it is expressed as the ratio between the sine of the angle ofincidence to the sine of the angle afftfraction when a ray of light of a definite known wavelength (usually aa9-3 mil the mean of the D-lines of sodium) passes from air into ghee, Refractive index ofghee i. measured at 40°0 to ensure that the' sample is completely melted. 1.1.1 Accurate resultl are obtained by using monochromatic light of a wavelength of 589·3 ~ ( the mean of the D-lines ofsodium). DiJFused white light may be uied provided the instrument used is fitted with a luitable compensator. Readings with white light are only accurate when a JJe!fectly colourleu and sharp line of demarcation is obtained between tbe dark and light ahades. 1.1.2 The refractive index should be read on an Abbe refractometer which gives the true refractive index or on a butyro-refractometer t which reacla on an arbitrary scale at constant temperature u near 40°0 as poaible.

1.2A...... tu 1.2.1 l'fieirio,. RI/r."""'" - fitted with an accurate thermometer ( reading from W to 50-a ). '.2.2 H" W." CitJlIltlli1l, DIlJie, - to maintain the temperature of the prism CODItQt at'W :t: 1-0. 1.2.1 S.... r...,- daylight can allO be used if the refractometer bu an achromatic compeasator. 1.3 _ .._ - ltandard 8uid for checking the accuracy of the instrument. '.4 ~.I.II .. 'A.! ~ IhaJI be rendered optically clear, and (ree from water aacI .. ilDpuritiea ( 1M 2.1.2.2 ). IS ...3508.1966 -6.4.2 The correctness of the instrum~Dt shall be tested before takin, reading by carrying out tests with fluid of known refractive index. At temperature of 40°0, or over, the prisms ofmost instruments never reach the temperature indicated by the registering thermometer, and at tempera­ tures greatly removed from the standard temperature for the instrument. there is a small error due to the change of the refractive index of the glass. At these high temperature, check the instruments experimentally with a liquid of known temperature coefficient, and apply the eorreenoa thus found to instrument readings given by the sample .. 6.4.3 It shall be borne in.mind that the presence of free fatty acids considerably lowers the refractive index.

6.4." Ghee shall completely 1111 the space between the two prisms, .nd .ball show no air bubbles. The reading shall be taken after ghee has been kept in the prism for 2 to 5 minutee and after it has been ensured that it has attained constant temperature by taking two or more readin...

'.1 ake care that the ghee has reached the temperature orthe instru. ment before lhe reading is taken. Before commencing to take readinp circulate throagh prisms a stream of water at constant temperature and measure accurately the constant temperature at which the readin.. are­ taken. 6.4.5 Us, oj Abb, RI/raetotMln - To charge the instrument. o~ double prism by means of screw head and place a few drops of the sample on prism, or if preferred, open prisms slightly by turning screw head aDd put a few drops of sample Into , funnel-shaped arrture between prisms. Close prisms firmly by tightening the screw head, Allow inatru­ ment to stand for few minutes before reading is taken, so that temperature nr sample and instrument will1'e same. 6.4.5.1 Method of measurement is based upon observation ofpoeitioD of border line of total reSection in relation to the facel ofa prism of fliat glan. Bring this border line into field of vision of telescope by rotating the double prism by means ofthe alidade in the following ~er: Hold sector firmly and mo~ alidade backward or forward until field of vision is divided into light and dark portiOb. Line divid~ these poraons i. the C border line" and, u a rule, will Dot be ••harp liDi but a band ofcolour. The colC'uts are eliminated by rotating acrew heacl • or compensator UDtil sharp, colour~eu line i. obtained. Adjust border JiDe 10 that it falls on point ofintenection or cro..~hain. Read refractive IDcIa of substance direcdy on scale or sector. Check COrrectness or iDstruJDeDt with water at 200C, the theoretical refractive index of water at we iI 1·S33 O. Anl COrrectiOD found f=rY should IDe made OD all reMlap. Maximum difFerence between d licate cleterminadolll .haD DOt .-..d 0·000 2 unit of the refractive 'ad 11.,..111&

'.4.' U" tU a,,'1"""'.""'" - Place 2 or S drops of the ....ple OIl .urtace or lower pnsm. Close the prism and acUUlt .. in ••~.s. '.4.7 For conversion ofrefractive index vallie. into butyro-rJractometer ~cIing, and ";', ""sll, usc Table 2.

TABLE 2 BVTYRo-aBFRAarOMETEll RBADINGI AND INDIca OF REFRACI'IOH

B.R. R.na6Oft.. B.R. aBnAC'1'IVB B.R...... CftnI R.~D"O JXD.% RJlADDrQ IlfDZ% R."DDG IJfDIIZ (I) \2) (I) (2) (I) (2) 35-0 1·4488 40·5 1·4527 ~,O 1·4565 35,5 1·4491 41·0 1·453J 46·5 1·4569 36·0 1-449.5 41·5 1·4534 4700 1·4572 36,5 1·4499 42·0 1·4558 47·5 1·4576 37·0 1·4502 42·5 1-4541 48·0 1·457, '7·S 1·4506 4S·0 1·4M-5 40·5 1·458' 38·0 1·4509 45·5 1·4548 49·0 1·f~6 SB·5 1·4513 44·0 1·4552 49·5 1·.590 59·0 1·451 7 44·5 1·4555 50·0 1·'59' 39·5 1·4520 45·0 1·4558 40·0 1·452.. 45·S 1·4562

'.4.8 The refractive index decreases with a rise, and increases with • fill in temperature. If the temperature i. Dot exactly at we, x is aclded to the observed reading (or each degree above or subtracted lor each degree below4000/IY '.11I, where X Cor butyro-refractometer·.. 0·55 X for... Abbe refi'actometer ..0-000 365 Normally the temperature of oblervation sball not deviate b¥ more than :1:2°0. 0.4.' A"",,,,,- oillu M"W.- The maximum difference between dupli­ cate determinations shall not exceed 0-000 2 unit Cor the refractive index and 0-1 for the butyro-refractometer reading.

7. DBTBRMINAnON 01' TITIlB

7.1 The titre of((heerepraeatl the higb.-t temperature reached when the liberated water-IDIOluble fat~ acid. are crystallized under arbitrarily controlled coaditiODl. TIae titre is ,.eraIIy taken to repraeDt thO 15 11.'•.1- solidification point of the .. although they actually IOliduy over • range oftemperature. 7.1.1 Gbee is aaponi&ed with .~talh solution. The resulting lOap is decompoeed with sUlphuric uid and the liberated water-insoluble ratty acids are separated, washed free from acid and dried. Titre it then determined on theae fatty acids. 7.2 Appan..- The assembly of the apparatus is shown in Fig. 2 and the detaila of the cOllltituent parts are u follows:

LOW-FORM BEAKER

CORK EQUALLY SPACED TO HOLD THE BOTTLt INTACT

ER·BATH

TITRE TEST-TUIE

WIOE-MOUTH BOTTLE

Flo. 2 TITD All8lDLy

II 7.2.1 n", T.bI - of gla!l, provided at the top with a rim or Sa.,and havinl the following dimensions: Extern:!l diameter 31-0 to 32-0 mm Length 90 to tOO mm Wan thickness 2·0 to S-O mm The tube carries a mark at 57 mm from the bottom to ihow the height to which the tube is lub~qumtly to be filled, and is inserted in a cor~ 10 that it may be held centrally in the wide-mouthed bottle. 7.2.2 Witle-MOII'1a Bo,tl, - ofglus, conforming to the following details: Diameter 60 to 100 mm Capacity 400 to 800 ml The bottle shall be loaded with just sufficient lead shots to make it sink and be reasonably stable when it is immersed in the water-bath• ..., .2~3 H'.,,,·B.th - any suitable bath complying with the following requirements: Water level 10 mm above the 5741D1 mark OD the titre tube Temperature or water 200 ± 1°0 surrounding the bottle 7.2.t ~tns 7.2.4.1 Tit" '''''mom'''' - range 00 to 6()OC in 0·1 degree ••, calibrated for 45 mm immersion for reading the titre. . 7.2.t.2 A general purpose thermometer ofsuitable range. 7.2.5 $t;":,- of g)alS, staiDlas steel, or suitable alloy rod of 2 to S mm eliameter. One end shall be beat ill the form ofa loop of 19 mm outside diameter at rrght angles to the ahaft oftbc stirrer. The stirrer i. used to aaitate the fatty acids immediately berore reading the titre. 7.2.' StI/JDfIifietllitlll Ytull - either a fluk or beakf'r of••table capacity. 7.3 ...... 7.1.1 Gl"rItol·P.,.. saliM-DiIsolw, with the aid of beat, IOlid potlllliam hydroxide in chemically pure glycerine ill the JII'OPO"ion I: 5 by weight.· To a~id ro.milll. do DOt heat above 145°0.

7.1.2 ~ AM-approximately SO ~t (WIV)_ Add QDe volumeor aalphuric acid (Ip Kr .-84-) to four volumes ofdistdledwater. 7.1.1 All., Or."..,.-- H5 pen:eDt lIqaeG1ll lOla'" 17 III_.~­ 7.4 ...... {7.t.1 'p,,,.,,,,ioa ./FaU.7 ~ - Weighar.proXimatel,110 I of glycerol. potuh solution into the _ponification Yelle. Alitate, either by hand or mechanica'uy and heat to 150·0. Add about 50 mI of sample abd reheat to'lW to 15000. Continue agitation untilaaponificatioD is complete; this it iad~by the mixture becoming transparent and homogen~UI, and is accompanied by a foam which peniatl for a few minutes when the mixture is allowed to settle. 1.f.1.1 Cool sUghtly and add 200 to 300 mI of distilled wat~~ AJ!tate and heat until the is completely diasolved. Add carefully, With stirring, SO ml ofdilute sulphuric acid 101ution. Check for an ez~ ofacid by the addition of a few drops of methyl indicater solution; adel more acid if necessary. Heat the mixture until the fatty acids ~te in the form ora completely melted and clear layer; avoid boiling u the mote volatile fatty ac~ds may then be lost. 7.t.l.2 Siphon or draw off the aqueous layer as completely as poaible, wash the fatty acids with 500 ml ofhot distilled water, allow to settle and again .draw off the aqueous layer. Repeat the washings until the wash water is no longer acid to methyl orange. After removal of the last wuhing, al19w ~~.. fatty acids to settle for a few minutes and decant throup • dry filter"paperiioto .. small beaker, takin~ care to leave behind in t&e oripnal vessel any water remaining. If VIsible moisture is still present remove this water by again settling, decanting and filtering the fatty acidl; The fatty.acids must remain completely melted throughout the filtration. 7.4.1.3 Heat the fatty acids rapidly and momentarily to 130°0 to remove any traces of moisture meanwhile continue the stirring. The fatty .acids should neither be held at 130·0 nor heated to this temperature more than once. 7.f-2 D,lmftilUUioll 0/Ti", - Fill the titre tube to the 57-mm mark with the fatty acids; when these have cooled to about 15°0 above the expected titre. place the tube in the assembly with the flanged rim close to the top etC the cork. Insert the titre thermometer to the appropriate immenion mark, the thermometer being supported centrally by a cork, through which aIIo pUles the stirrer. 7.4.2.1 Before the temperature or the Catty acids dro~ to a value of 1000 above its expected titre, 'commence asitation in a vertical mannerat • rate of 100 complete up-and-down motioas perminute, the stirrer mo~ through a vertical distance of about sa IDID- Continue ltirring in tbiI manner until the temperature baa remaiaed cODstantfor SO seeonde, or hu begun to rise within 30 seconds of ceuiag to fall. Discontinue stiniDI iminediately and lift the stirrer out or the __pie. . 7.4.2.2 Observe the rise in temperature; the highest temperature reached after stirring hu ceued is the dtre. 11 1S.~.i.

~en makiog the titre reading, avoid all undue vibration\&1 this will cause the temperature to drop before reaching the maximum. \ I. DETERMINATION or MELTING POINT (SLIP POINT) \ 1.1 Ghee being a heterogeneous mixture of . doe. not have a lharp melting point but for control purposes it is possible to obtain a u.eful eomparison ofsamples by determining the temperatureat which a column offat offixed length rises in an open capillary tube. It is essential, how­ ever, that the comparison should be carried out under exactly the sarne conditions and the suitable conditions are described in the procedure. 8.2 Apparata8 8.2.1 M,lting-Poi", Tubes - thin-walled. uniformly bored capillary glass tubes open at both ends and with the following dimensions: ' Length 50 to 60 mm Inside diameter 0·8 to I-I mm Outside diameter 1-2to 1'5 mm 8.2.2 71ImntJm,'" - with 0·2°0 subdivisions and a suitable ,ange_ ·The thermometer should be checked against a standard thermometer which has been calibrated and certified by the National Physical Laboratory, New Delhi, or any other laboratory recognized for such work, , 8.2.3 Mllljn,·P"inI Apparatus - beaker with a side-tube heating arrange­ ment. Thiele melting point tube may also be used, 1.2.4 H,., Sour" - gas burner or a spirit lamp. 8.3 Proceclare - Melt the sample and filter it through a filter paper to remove any impurities and the last traces ofmoisture. Make sure that the sample is absolutely dry. Mix the sample thoroughly. Insert a clean melt­ ing point tube into the molten sample product so that a column of the material. about11> mm long, is forced into the tube. Chill. the sa·mple in the tube at once by placing the end of the tube containing the sample against a piece ofthe ice until the fat gets lOIidified. Place the melting point tube in a test,.tube and keep it for one hour either in .. refrigerator or In water maintained at 4° to l00C. Remove the melting point tube and I attach with a rubber band -or any othet suitable means to the thermo­ meter10 that the lower end of the melting point tube is even with the bottom ofthe bulb ofthe thermometer. Pour water at about 100e into the beaker or the Thiele tube, and suspend the thermometer in the centre of the apparatul. 10 that the lower end of the sample column is about 30 mm belowthqlurface ofwater. Heat the side tube o{the apparatul gently, so that the temperature of the water rises slowly at the rate of2°0 per min\lte till the temperature reaches 25°0. and thereafter at the rate of0-5·0 per II "".1_ minute. Note the temperature or the water when the sample column com­ IIleDces to rise in the melring point tube. Report the average of two such separate determinations as the meltiDg point, provided that the read&'.. do not dift'er by more than 0·5°0.

t. ICOU CARD POll GIIBB '.1 The score card system may be used Cor judling ghee for competitiODI ezhibitioDl, etc. A lugestecllCOre card for ghee is: Flavour 50 points Testura 20 " Colour 10 ,. Preedom fro. lUlJieacled impurities 15 'J Package , ,. Total 100 poiDts

1.1411 Gbee scoring 91 and above shall be graded as excellent, between­ 80 and 90 as very good, and between 70 and 79 as good. 8.2 Only pure product can be used for giving score card. The test shill be carried out by a small selected and trained panel of judges, A control IUDple-oftype ofghee examined shall be used for comparison.

II. D_ATION OF INSOLVIILB IMPURITIEI 11.0 IDIOluble impurities represent foreign matter exclusive of ~ which are not diIsolved by . 10.1 Appantu- Conical Suk of 25O-ml capacity" provided with stopper. 10.2 ....eat- Li8ht petroleum of boiling range 40° to 60°C. 10.3 P.....-...- Weip accurately 9bout 20 g of the moisture-free sample ( ,,, 4.2) into a conical ftaak. Add 200 ml or light petroleum. Stopper the &uk and lhake. Allow to stand atf'room temperature for 30 minutes. rllter the solution throuah a previousl)- dried and tared 12 em diuDeter&Iter paper. 11.3.1 Wash the filter paper and the tim with light petroleum till rat­ free. Remove the filter ~J all6w the to evaporate. and dry in aD oven at 98- to 100·0 (or I hour. Weiah the filter paper. R.~t dry" and -'~I_ uadl the 1011 or wei,ht between lucetllive weiahiDp doeIlIOt aceed 0-000 5 g. 20 II.SM·l- 10.4 Calc:alado. Insoluble impurities, percent by weight _ 100 ( W, - WI ) W where IW. -= weight in g of the filter paper and impurities, WI - weight in g of the dry filter paper, and w .. weight in g of the sample taken.

II. DETERMINATION OF ACIDITY 11.1 Apparata. 11.1.1 ConiealjltUks - 250 ml capacity. 11.1.2 }Jur,tt,- with soda. guard tube. 11.2 ••••ta 1112.1 Eth.,l AlcDhol or R,clf/itd Spirit - 95 percent (II/D), sp gr 0-8160, neutral to phenolphthalein. ·ll~2.2 Sodium H.,dro~id, or H.1tlrauu- 0'1 N aqueous solution aeeurately standardized against acid potaaium phthalate (AR) or oulic acid (AR)_ • 11.2.3 PlwtDlpAllaalli" I"die.or - 1-0 percent IC?lutlOD in 95 percent ('/') ethyl or rectified spirit.

II.S P...... - Weigh 10 f( df the sample in a 2SO·mI conical flail£. In • second 8uk brulg 50 ml of alcohol to the bolling point and while .tiIl above 700C neutralize it to phenolphthalein (using 0-5 ml) with 0'1 N sodium hydrox\de. Pour the neutralized alcohol on ghee in the &uk aDd mix the contents ofthe flask. Bring them to boil and while it i,still hot, titrate with O·J,..N , shaking vigorously during t~ titration. The end p'lint of the titration is reached when the addition or a lingle drop produces a slight but definite colour change persisting for at least 15 seconds. 11.-6 AeIcI V.... - The number of mg of KOH required to ne'Lltralize.the free fatty acid. present in I I of the sample,. •• 5-61 T ACid value - -,y- where T - volume oCtO'! N alkali required for titratioa in mi. and W - weilhl in I of ample taken.

21 11._-1. 11.5 Free • ..,.....- The acicUIJr or Pee i. frequently a~ .. the percentage offree fatty acida in the..pIe, calculated as elele acid. - 2-82 T Free fatty acids - ---w- 11.6 De..... 01 AeldIt7 - It is the total titratable acidity preaent in the .sample expressed as percentage: idi 100 N Degree 0 r aCI ty - --w where N ~ the quantity of alkali used, expressed as. ml of IN solution. 11.7 Ac....ay of til. Metlaocl- The maximum deviation between •duplicate' determination shall not exceed 0-2 degree of the acidity or equivalent. 12. DB~~ATION OF SOLUBLE AND INSOLUBLE VOLATILB ACIDS ( UICIIDT OR UICllBRT.MBmIL, POLBNSD AND KIRSCHNER VALUBS) ·12.1 The method does not determine the total quantities of volatile ratty acids, soluble and insoluble in water. present in combination in rat. 11Ie amount ofthese acids actually determined by the process are dependent on Itrict adherence to the dimensions or the apparatus and the details of the procedure, 12.2 DeSaldo•• 12.2.1 The Reichert-Meissl \'alue (R. M, ~alue) i. the number ofml or 0·1 N aqUeoUi alkali solution required. to neutralize the water-soluble.team volatile Catty acids distilled from 5 g or ghee under the precbe conclitioal lpeci&ed in the method. 12.2.2 The Polenske value is the number ofml of 0-1 N aCJueous alkali solution r~uired to neutralize the water.iDsoluble steam "watileratty aciell distilled from .5 I of Ibee under the precise conditioDi specified in the method. e 12.2.5 The Kirsc~er value is the Dumber of ml of0·1 N aqueous aIbIi solution required to neutralize the water-ldluble Iteam volatDe.r::r..,acicb whi~h form water-soluble silver salt. diltUled from 5 I of,beeo the precise conditiona lpecifiecl ill the method. 12.JA...... 12.s.1 Or-""q,IWIrl- 100 mI aDd 25 mI caPacities.

'21 11,'••1_ lU.2 PiJII'" - 50mi. 12.3.3 The usembly of the appar~tus for the distillation is shown ill Fi•• 3 and 4 and dctaUs ofthe constitucnt parts are given below: a) Flat-bo"om boilia,JItuk (Pol.,) - The flask shall be made of heat~resistanceglass and shall conform to the following detaiJs:· Volume contained to bottom or neck 310:1: 10 ml ~~~Dd ~±5~ Internal diameter of neck 21 ± 1 mm Overall height 160 ± 5 mm Diameter of base 4~:f: 5 mm b) S,ill-Mad - The still-head shall be made of glass tubing of wall thickness 1-25 ± 0-25 mm, and shall conform to the shape ShOWD in Fig- 4, and with the following dimtnsions: A 180 :f: 5 mm B 107-5 ± 2-5 mm C 80 ±5 mm D 70 ± 5 mm E 20 ± 2 DUD F 4 ±l !DID G (external diameter of bulb) 37-5 ± 2-5 mID Internal diameter of tubing S-O :I: 0-5 mm Acute angle between slopping 60 :I: 2° part ofItill-head and vertical A rubber stopper, fitted below the bulb of the longer arm ofthe still­ head, and used for connecting it to the flask shall have its lower surface 10 mm above the centre ofthe side hole ofthe still-bead. - c) CoruiIIu" - The condenser shall be made ofglass and conform to the following dimensions: Overall length 520 :I: 5 mm 'Length or waterjacket 300 :b 5 mm Length oCwideneci part above 70:J: 10 mm waterjacket \\fan thickness of widened part 1-25 :f: 0-25mm Internal diameter oCwidened part 20 :I: 1 mm External diameter orinner tube 12:1: &5 IBID within waterjacket Wall thicbell or inDer tube 1-0 :l: 0-2 mm . Wall thickoea or outer jacket 1-25 :I: 0·25IDDl laternal cI1aaaeter of waterjacket 30 ~2 IDID

II 11,3501·1_

no "'..

FlO. S Pouaua DanLLATIO~ AwAM",.

24 11.,..1.

c

A G"

F~. 4 &ftlL-HaAD FOR POLBNIU DmnLLATION MPAaATUI d) RIItiDIr - The receiver ih.lI bea 8ask with two graduation !DUb on the neck, ~De at 100 ml and the other at 110 ml. e) ~tl.·&",tl-- An ..bestos-boardof 120 miDdiameter and 6mID in tbickness, with.circular hDle of about 65 mm in diamet~r·shaU be ud to support the 8u) over th~ burLer. During eftstiJlation the Polenake flask shall fit sDugly into "the hole in the board to prevent tho lime from impinging on the lUI-raCe ofthe flaskabove the Iiole. A new ubestos-board may conveniently be pre~ 'br bevelling the ed,e ofthe bole, soaking in water, mouldin, the edge with a flame, and drying. - f) Gu B"",,, - The burner should besufficiently large to allow the distillation to be completed in the time specified in 12.5.1.3. 12.S.I.1 The .pparalUl shall be IUpported on a retort stand. 12.1.t GIaI FtIItM - of approximate diameter 6 em.

25, 11._.-. IU...... 11.4.1 0",,,.'- 98 percent (fIJ/_), confbrmiDa to AR. srade or 18: 1796-1961*. 12.4.2 Sod... H.,...- 50 ~t (tlJltIJ) solution. SodiUJD~ xide is dissolved in an equal weight .r water and the IOlatio}' is It in • bottle protected from carbon dioxide. The clear portion &ee from depoeit is used. 12.403 Diluk SaI/J/uIN Md- :groximately 25 mI of concentrated IUIpburic acid is diluled to I: J adJl»ted until 40 mJ neutralize 2 ml or the 50 percent sodium1aydroxide solutIon. 12.4.4 GlassB,tld, - approximately 1·5 to 2·0 mm in diameter or ground pumice powder, which passes throURh 250-micron IS Sieve (_IS: 460­ 1962t) and remains on 125-micron IS·Sieve ('11 IS: 46Q-1962t ). Q.U PMlIOljlhlJulllia 1.-.""...... 0-5 percent solution is 95 percent (pip) ethyl alcohol, or rectified spirit. 12.4.6 EIIt7l Aleolaol- 95 percent (11/11) neutralized to phenolphthalein­ immediately before use, or neutralized denatured spirit. 12.4.7 Sodium H,tlrou Sol"lUI" - approximately 0'1 N aqueous solution or aodium hydroxide of accurately determined strength. 12.4.1 BIJIi"", H.Jdrodd, Sol.'io" - approximately 0-1 N barium hydro­ xide solution ofaccurately determined strength ( this solution is needed only ifltinchner value is to be determined). 12.4.1 Sil"." Sul/JluJ" - powdered. 12.~.1' Fill" Ptl/Jtr- Whatman No.4 (or its tqui\'aJent ') of9 ern din­ JReter. 12.1 PNee••re 12.5.1 Weigh 5'00:1: 0·01 g of ghee into a Polenske flask. Add 20 g of glycerol and 2 ml of the 50 percent sodium hydroxide solution. Protect the burette containing the latter from carbon dioxide, and wipe its nozzle clean from carbonate deposit before withdrawing solution for the tests; reject the first few drops withdrawn from the burette. Heat the flask over a naked flame, with continuous mixing, until ghee, including any drops adhering to the upper partsofthe flask, is saponified. and the liquid becomes perfectly clear; avoid overlieating during this saponification. Cover the 8uk with a watch-glass. . 12.5.1.1 Make a blank test without ghee, but using the same quanti. ties of reagents and following the same procedure, again avoidirlg ·Specmcation for crude IlyceriDe and r.lned alycerine. tspeci&cation fOr tat sieve.("N,II) .

11.• ,..­ eNrlaead., d~ the .tID,' witb _I Nt ~hydl'Cllid~; suCh' overheatiDc ..aid be ii&cllcatea ~ clarkeDiq 01the IiOIUUOD. . 12.1.I~ Meuure 9S mI of boiJiDI distiIIecl water,. which bu been viprousl, bOiled for 15mlnutel. IDto • l08-ml..-duated cyliDdu. When the "P II lafBciendy cool to pennit addition 'Of the water without 1011, bat Wore the ..p hal m1icU8ecI, aclcl the water, drainiDl the cy1iDcler fbr 5 .eco.da, aad dillOlve the lOa". Ie the solution is Dot clear (iDdicatiDc ID(OIDpiete I&PO.p&catioD ), or II darker thaDlight yellow (iDdicatinC ewer­ heatiD, ), ~t the saponification with a Crah sample of lhee~ 12.5.1.3 Adcl two g18ll beacII, followed by 50ml Qfthe dilute aul~ acid, -and.connect the ftask at once with file distilling ap~tUl. Heat the &uk without boiling its. coatentl, until the insoluble acids are completely melted, then increase the lame and diltil 110 m1 in between 19 and 21 min·utea. Keep the water lowing in the condenser at a suflicient apeed to maintain tfte,temperature 01the issuing cliatillate between ISO and 21-0. 12.5.1.4 When the dittillate reacha the IIO-ml' mark, remove tbe lame and replace the IIO-ml 8uk by a cylinder of.bout 25 ml capacity, to catch draininp. Close IIG-mllask with its stopper, aDd without ~ the CODtents.'place it in water'at 15-0 for 10 minuteilO as to immene the IIO-ml mark. Remove the tluk from the water, dry the outaide, andlDvert the flask carefully. avoiding wetting the .to~ with insoluble acid.. Mix the distillate by four or five double inveruolil, without violaat lhUiaI. Filter through a dry 9.cm open-texture filter paper (WhatmaD No. f) which fita Inugly into the funnel. llt;the first runniap and coDect 100 ml itt • dry volumetric ftaak; cork the and retain the filtrate for ti~.

Non-11ae lit... IbouIcI be he from inIOlubie ratly ac:icII. When Iiq.-" .. I _uble fattyacicll ,.. throulll the liter, receiyetbe Iltrate iD...... _ ftaniiI, ...... don, draw"tbclower (aqueoua) 'layer 1_",bebiacl iaIoIuble ....wIdcIa have rileD to the lurface. AcIcl thae to the lIlain b;ik or tbe lDIoIuble.a.. 12.5.1~ Detach the still-head' and wuh the coadenaer with ·three IUCCellive '15-mI portions o~ cold diltUled w.le!~ ~I each ~ .eparatdy throup the cylinder. the ,IIQ.m! Balk, the filter aDCI the funnel, ~early 'IIJiDI the paper each time and·.draining each • __1 beCore filtering the, next. Discard the waahinp. DiIIolve the inIoIuhle acids by three limO. wuhiap ofthecondenser. the cyliDder1Uad theliter. with 15' iIIl of neutralised ethanol, collecting the lOIution 1ft the IIG-ml Iuk. and drainiDl _ ethaDol after each walhiar. Cork ~ 8uk, and.· retain the sq-..tion (or titradoa. u iD 12.5.3. . 12.5.2 1IMwf1.JMlul, .,s.wr. YiMli/, AM. V. - Pour 100 m1 of the &1trate coatUDI...... the lOIable voIt:tUe acid~ into.ati.tieD~add 0-1 III! 01 pbeDolpbtha1ein ·I~tor. aad titrate With the '-1um htdroxicle IOlu_ _ tilthe l~uicJ becoma plak. r1....the 10G-ml1luk with the'aearlf aeutra- ."·liquid toWudathe Cad or the tltra~~N IGCIiUlil h""" du- "'dee'''' be ~ Ior'dar: dtratioli If~e It ..,.11 DOt required). ~7 11.. '.·1_ 12.5.2.1 If the KinchDer value is tp be obtained. the titratioa 1aIk' .hallbe dried before Ulej Dote the actual volume ofbarium hydroxide IOlu­ tion used; drain the lOO-ml &uk into the titration flask. clOie with a cork aDd continue as in 12.5.4. 12.5.3 POUMU. " luol.bl, Yolalil, Acid, Ydl", - Titrate the alcoholic solution of the insoluble volatile acids after addition of 0'25 ml ofphenol­ phthalein indicator. with the 0-1 N barium or sodium hydroxide solution until the solution becomes pink. 12.5.4 Kirst],,,,, Yal"' ..... Add 0'5 g of finely powdered silver sulphate to the neutralized solution reserved in 12.5.2.1. Allow the flask to stand in the dark Cor one hour with occasional shaking and filter the contents in the dark through a dry filter. Transfer 100 ml ofthe filtrate to a dry Polenske Bask, add 35 ml of cold distilled water, recently boiled for 15 minutes, 10 ml of the dilute sulphuric acid and 0'1 g ofpumice powder or two gl.. beads. Connect the flask with the standard apparatus and repeat the procell as described above, that is, the distillation of 110 ml in 19 to 21 minutes, the mixing ( but without the cooling for 10 minutes), and the filtration and the tltrarion of 100 ml of the filtrate with the barium hydro­ xide solution, 12.1 CaIca1ado••

Reichert-Meissl value == 1'10 ( T 1 - T,) Polenske value == T. - T.

121 ( 100+ T1 ) ( T. - T. ) Kirschner value .. 10 000 where ~r T1 .. volume in ml or 0'1 N barium sodium hydroxide solution used for sample under 12.5.2, ' T. == volume in ml of 0'1 N barium or sodium hydroxide solution used for blank under 12.5.2; T. - volume in ml of 0-1 N barium or sodium hyd~ide solution used for sample under 12.5.3, T. == volume in ml of 0'1 N barium or sodium hydroxide solution used for blank UDder 12.1.3, . . T. - volume in ml of 0'1 N barium hydroxide solution used tor sample under 12.5.4, and T• .. volume in ml of 0-1 N barium hydroxide JOlution used for blank under 12.5.4. Polenlke values, and to a mu.ch slighter exteDt Reichert values, have beea foilnd to be low ,¥hen determined at low barometri~ , such

28 11.,.·1- at may occur ••high altitudes. The rollo\ving (actors may he applied to values determined at a barometric to convert them to the values determined at normal pressure. · h t 1 (Observed value - .10 ) 101 760 10 Correct Rere er va ue .. Jog P +,

760 - 45 Corrected Polenske value - Observed value X P _ 45

where P == barometric pressure in mm of mercury at the place aDd time of determination.

12.7 Accarae, 01 the Metlaocl 12.7.1 Rt;ela,r,·M';ul Val", - The maximum deviation between dupH. cate determinations shall not exceed 0·5 units. 12':7.2 Polnuk, V. - Tile ma~dmum deviations between duplicate determinations shall not exceed 0-3 units. ! 12.7.3 Kirs,h"" YIlI". - The maximum deviations between duplicate d~terminationsshall not exceed 0·5 units.

13. DETBRMINATIOlO O. 13.1 The saponification value denotes the weight ofpotassium hydroxide, expressed as milligrams, required to saponify completely ore gram of,hee. Saponification value is useful in detecting the presence ofmineral oils. such as liquid paraffin, in ghee as they are not acted upon by alkali and such a sample does not form a homogeneous solution on saponification. .. The saponification value is related to the molecular weight ofghee and from it can be calculated the saponification equivalent, which is the amount ofghee saponified by one gram equivalent ofpotassium hydroxide, and is equal to 56 100 divided by the saponification value. 13.2 A~...-Flat·bottom flask, 200 or250 ml capacity, resistant to alkali and fitted with a relux condenser. 13.3 R•••ean 1'.3.1 AleoltDlie PoIlU';"'" H7flt'- - approximately 0·5 N solution in 95 percent ( "Iv) ethyl alcohol. Dissolve 35 to iO g ofpowsium hydroxide pellets in alcohol or ifnecessuy, in a minimum quantitv o(,,·atcr (.~. mately 20 ml) aad dilute with ethyl alcnhol to one iitre. The streIII'h

29 11._.1_ abouId be approximate. but not 1eu thaD 0-5 N. It should becolour_ or very pale yellow_Keep in • dark place. NOT. - Alcoholic solution or pota.ium hydro.ide dcvelopa colour clue Cfr the preaence of a1d~hydea. Alcohol caD be made aldehyde-Cree by ODe of the tollowina method.: a) Saturate alcobol with sodiuJD hydroxide and atore torseveral clayl,shaW.., occuionally. Distil. b) Reflux about 1·2 litres of alcohol with 10 I oC potaniulD hydroxidt uad 6 I I!'anulated aluminium or aluminium foil, for an bour. Distil aDd collect one litre. diKardin. the firlt 50 mi. 13.3.2 Plwaolp"'hallin Indiedlor - 1·0 percent solution in 95 percent ( Ifl.) ethyl alcohol. 13.3.3 Hytlroehloric Acid- 0-5 N aqueous solution accuratelystandardized. 13.t Procecltare - Weigh accurately 2-0 ± 0·001 g of ghee into a 8ask. Add 25 ml, accurately measured, of the alcoholic solution. Add 1 or 2 glass beads and boil cont;nuously undor a re8ux condenser for balfan hour to ·one hour swirling file contents 01 the &uk at frequent intervals: Determine the excess of alkali while the solutlon is still hot by titration with 0-5 N hydrochloric acid. using 0·5 mlof phenol phthalein indicator. U.t.1 Make a blank determination upon the same quantity of the potassium hydroxide solution at the same time under the same conditions.. 13.5 CaI"doD 1S.5.1 Saponification value _ 28'05 (~I- 7i)

where T. == volume in ml "fO-5 N acid required for the bluk,

T1 == volume in ml of0·5 N add required for the I ••e. and W == weight in g of the sample taken. ~c: U.5.2 Saponification Number - sapoau:a nvaueI 14. DBTBRMINATION OF IODIMB VALUE (WlJS' MBTIIOD) It.1 The iodine value or ghee denotes the percent.by weilbt of balo­ po. calculated as iodine, abaorbed under the coDditioD ofthe tat. IU Appantll8 It.2.1 Con. Pltuk - 250 ml capacity, preferably with FOUDcl-1U ltopper. or with good new tilht fitting bark cOrks. R.2.2 ."",- 50 mI, sraduated to 0-1 mi.

10 11.,..111I 14.1_.... It.s.t AU...-.. abaIl ~ of aaalytical grade.. H.l.1 "".".. 1»• ..."- confbrming to IS : ~50-195'·. I~.s.a- ct..".1(1tlroe1llri .d&Ul- conforming to IS: 2~5-1962t. , 1f.J.s P,IiUI.- 1...s.,;,,.- prepare a freah SOiUtiOD by diaolviDg 10 I or potallium iodide free from potulium. iodate, in 90 ml ofwater. ·u.,..- s,.,,, s.ltUiD,. - Triturate 5 I ofltarch and. O'!OI B of mercuric iocIiele with· SO ...-.or cold water and slowly pour it with stirring into one litre orboWol water. BoU Cor three minutes. Allow to cool and decant • the .~taDt c;lear UquicL ' . 1f.J.5 Sta4.l ~ 71ionIl!Jlu* So"";OII - approximately 0·1 N. DiIIoIVo ~telJ 24·8 g ofsodium thiOluJphatecrystals ( N-.s.o.. Sil.O ) in water which baa been weD boiled to free it from carbOn dioxide aacI make up to 1 000 mi. Store the lOlution in • cool place in a dark­ coloured stock bottle with • guard-tube Siled with. soda lime. After .toriag the 101utioD for about two weeks•.filter, if necessary, and stand­ ardize u followa. 14.1.5.1 Weigh ~te1y about 5-0 g ·or finely ground potassium cHchroma~ which baa been previously dried to a constant weight at 105- :J: 2-0 into a cloan one-litre volumetric~. Dissolve in water, -.ke up to the mark; shake thoroughly and keep the IOlutioll in a cool dark place. For Itanclareu.~tiODoflOdium tbiosulpbate, pipetto 25 ml of . tWs IOlutioD into • clean 1.,toP~red 250-m1 conical lask or bottle. Add 5 ml ofCODCeDtrated hydrochlonc acid and 15 ml td a 10-percent ,otutium iodide solution. Allow 'to Itaud in the clark for 5 miDutei and titrate the·mixture with ·the solution of .9dium thiOlulpbate, using ltalch lOIution u an iDternal indicator towarda the end. The encl poitu: is taken when the blue colour chBDges to are6n. Calculate tho' nQftDality (X) of the lOdium thiOlUlphate solution ..follows: 25 W ~ 49-03 Y wbere ,W- weight in I of the potauium dichromate, and , ~ volumein ml of sodium thiosulphate soJution required . Cor the titratioD. It.3.1 ,.-er;"IMI -'re-lubUmed.· It.!.' A.e,';' ..- aJaciaI, 99 percent, having a me1tins point or IS-sec aDd free &om redudng impurities. Determine the meltiog point of .•,...""'1...... bWaroaaate. techDicaI.cl ....IJllcairapDt. t...a....rw ~ atdd (.".,.).

Sl the .cet~c acid and 'tat it for reducing impurities as Collowa: a) D,lmftin.t;oll oj""lt;",/IOi".' - Take a 15em IODg test-tube and &II it to,about two-thirds with the aeetie acid. Ins: rt into the acid • tbermometer latisfyinR the requirements I~ified uDder 14.4 through a cork stopper fittirg the test-tube. The amount 0( acidsbould be at least 'double the quantity required to cover th~ bulb of the thermometer when the bottom of the latter i. If'mm from the ,bottom of the test-tube, Susper:d this tube wilbin a J.r~r test-tube through a cork. Cool the acid by immersing the, assembly in ice water untilthe .temperature is 10"0, then with­ draw the assembly from the ice wat~r and stir the acid rather vigorously (or a few momenta, thus causing the luper-cooled liquid to crystallize parti.ny. Take thermometer readiDgs every 15 seconds .nd consider as the .true melting point that tempera­ ture at which the reading remai~ coDitant for at least 2 minuta. b)-T,slfor r,duM, im/Jliri'ils (po'lUn,,'" /JtrrIUIIIlG""" IIs1 ) -Dilute 2 ml ofthe witl:. 10 ml oftbe ~~ •..nd add 2 drops of 0·1 N 'potassium permanganete solution and maintain at 27° ± 2°0.. The test .ball be taken as having been satisfied itthe pink colour i. not discharged at the end.oftwO houn. If.S.a Chloria Gill - Dry. 14.3.' Jodi", T,iehloritl, Elei.) , -, 14.3.10 lodiM Mo"oMloritll- 98 percent, chemically pure. 14.3.11 W-vs' ItHl;ru MOIlO&Aloritl, Solu(ioll- Prepare this IOlutiQn by on~ of the following three methods, and store lna gl....toppered bottle iD ,_ cool place, proteeted from light: a) Dissolve 13 g of iodine in one litre Qf&cetic acid, using gentle heat, ifnecessary, and determine the .~gth by titration. with standard sodium thiosulphate 101ution. Set aside 50 to 100 ml ofthe solution and introduce chlorine I. iDto the remainder until the characteristic colour change ,occurs and the halogen­ content i. neatly doubl~ as ascertained apia by titratlon. If the halOgen content has been more than doubled, reduce' it by addiDI the requisite quantity of the iodine-acetic.ad ~lutioD. A alight excesa ofiodine cloa no harm. but avoid an excna or chlorine.

~I: Ir the titration of 20 ml or o~nal iodine-acetic acid sOlution !,"CIuira 22 ml of standard IocIIUID thioIuJpbate, 20 ml or the fiDished Wi)' IOluhon should require between 43 and 44.1 (aad ,

32 lIa'5II·1­ DOt more than 44 ml) or the lUIle lOClium thiOlwpbate IOlution. b) Dissolve 8 g ofiodine trichloride in approximatel,4SOml·of·aceti.c acid. Dissolve separately 9 g of iodine .in 450 ml ofacetic acid using heat, if·necessary. Add gradual1~ iodine 101ution to the iodine trichloride until the colour Iuuc ged to reddish brown. Add 50 ml more of iodine solution and dilute the mixture with acetic acid tiU 10 ml of the mixture is equivalent to 20 mJof standard thiOlulphate solution when the halogen content it esti­ mated by titration in the p~ce ofan excessofpotaaium iodide and water•. Heat the solution to 10000 for 20 DlIDutea, 8Dd cool. Prevent accea ofwater vapour in preparing the solution_ c) Dissolve 10mlofiodine monochloride in about I 800 m1 ofglacial acetic .acid (chemically pure) and shake vi~UI1y. IlPipette 5 ml or this, add 10 ml of potassium iodide solution and titrate with 0-1 N standard sodium thiosulphate solution. using starch solution .. iDdicator. Adjust the volume ofthe ioIution ti1I it i. apptoximately Q-2.N.

14.3.12 C•• T"'*lIloritl, (Jr Chltwojorm - inert to Wi)- 101utloa. 14.4 _-Weigh accurately 0-40 toO-45g of the clear ghee in • clean dried conical flMk. Dissolve the fat in 15 ml carbon tetrachloride. and add by meina ofa burette exactly 25 ml of the Wij.'" reapnt. C101e the flask with it. stopper, mix carefully and leave it ltandiDg for ODe hour in the dark. Add 20 ID1 potaaium iodide solution and approximady 150 mI ofdistilled water, 8Dd mix. Titrate with 0·1 N lOdium tb~bate 1OIu­ tioD (use as indicator 2 ml of atarch solution), awirliDg_liquicl coDitaDdy. Add the starch iolutio~ shortly before the.ead oftie titratiOD and ·shake the conten"- vjgorously. Carry out a blank tat, usm,. theIUDe quantities of the reaptl. 14.5_ 12-69 ( B ~S)N Iodine va1ue- W where B ~ volume in mI of ltandard sodium thioIulphate .oludon , -required for the blank, S - voltame in m1 of.taDdard JIOdium .hiOlUlpbate IOI~ required Cor the -pie. K - normality ofthe ItaDdaftl lOClium thioIulphate IOllItioD, aDd ,W - weiIfIt in I of ~e material takeD tbr thetelL 14.1 A_err01.1IetIMMI- The renalu of dupUcate ~ .baIl DDt cliIer by IIIlON thaD 0-4.

II II.SIII·I- II. DBTBIlMINA'DON OP UNMPONIIIAaa MATID-. 15.1 The unsaponifiable matter colDpriseI of .uhltaacel IDIabIe ID pee which UttT aponification are inIoluble in water bat IOI~le ill the __t UIed (or the determiaatioa. The important CODItitueDti or UDIP....bIe matter or ghee are .~roJa, . caR)teDe, ancI toeopbelOla. 15.2 Appuaeu. 15.2.1 Fl4I-&IIoJII Fl_ - 250 ml capacity, fitted with • rdux OODden.er. 15.2.2 &1.-"lIDMl - 250.mIcylindrical. U.2.1 GlIuI"."",- 5 em cUameter. U.2.~ 0Na U.s _____ 1'.I.i MuW. P.~ H_OIMI- O·~, N IOlutiOll in 95 percent ( ~/' ) ethanol. The atreDgth shoufd be applOXImate but Dot Iell thaD &5· N. and ill ~lour notdarker than WlIe. yellow. DiIIolve 35 to 40 I ofpotulium h,droaide. peUeta in -20 m1 otWater and mix the dution with I 000 m1 or 95 ~t (,/-). ethanol. Allow the IOlutiOD to ltand for severalhOun, preferably overn~ then decant or filter off the dear'..upernatant Jicr4d. Keep the 81teree1lOluUon in a derk place. 15.1.2\~ B"",-Ip p- at 15-5C!/1~-5·o, &720 to '0-724, reIid. fton-voIaI1le at eooo DOt exceecllDs &001 perceot. 15.s.s ."'-raidueDon-_tiIe at eooo Dot aceediDc 0-01. 15.s.~ P.""..H~- approximately &5 N aqueousIOlutieD. 15.1.5 H~_ 0·1 N 101ution in ethanol. DiIIOlve 5 to 6 I or So-. - 10 lOCIium hydroxide pellett in 1 ml of water aad mix.the .101ati~ with 1 000 ml or 95 percent ('/') ethanol. Allow the lOlutioa to .tUMt-~ several hours. ~bly ~ then decant 01" 61ter 0« the clear IUpernatant I~uld. Keep the &It solution in • clark pJaee. 15.1.1 1I:1ft1tltJril.4ri4 - Ip If 1-18_ IS.lA E""-95 percent (".) Ip F 0·8160. 15.1.1 P"""',,,,,... ,.....- 1-0 percent lO_doD ia95 perceIlt ( _/_ ) ethanol. 15.4 _ - Weigh accurately 5 s«the ~ &0 wltbin ±O-Ol, ia • 250-ml_lat-bot~m last, add-50 mIof alcobo1iC potuliam h~ 101udon and two p. beadI. Attach the Iuk'to ...... Sf JI.".~

~t GIl • bo~wa..bath tor ODe hour, swlrliD!at freqUeDt iDterVaIt·· to e-.ecomplete .-poDiIication. . 15.4.1 Remove the contents 01the Jag to a 250-mJ separating Immel, ~ the Jlaak with, 50 ml of water. Rillle the ftaak with 50· ml.of diethyl ether and pour the ether cautioUsly into 1he funnel. St~per the ftiaDeI, shake vigorously, and allow the funnel to ltand until the tw() ~ c(J~uicl separate and clarify. Drawoff the aqueoui.. alcoholic layer ~~ the tiak ~ lor the "saponification. _5.4.2 Pour the ethereal layer from the top or the ftmne1 into a secoDd' ­ ~_ f~nnel c~taining .leparating 20 ml ofwater. Extract the I &C\1IeOUi ali:oboIlC soap SOlutiOD twiCe more. each time with SO ml of ether 1D the .me mannert and combine the three extracts in the second (unne1. l5.4-S Wash tl1e ethereal solution twice with 20 ml of Water, ahakiDa ftaaroualy OD each occaaion. Then auccasively wash'with 20 ml or 0·5 N aqueous potassium =xide solutioDt 20 ml or water, 20 m1 or &5 N ~1IeOUI potusi1UD h roxide solution, 20 mJ of water and apia with 20. m1 or 0-5 N aqueous ~tassium hydroxide IOlution, and at 1eMt twice morewith 20 ml of water. Continue wuhing with water until tile wash water DO longer turns pink on addition ofpheaolphtbaleiD i~dicator. 15.4.~ Transfer the ethereal solution to a weighed flask aDd diatil to ­ amaIl bulk. Add 2 to S ml f>f acetone and completely remove the IOIveat from the flask, torexample, by IQcaDI ofa gentle current of air, the flaIk being almOlt entirel)' immened, held obliquely aDd rotated in a boiling­ water-bath. Dry the flask aDdcoatents to constant weilht at a tempera­ ture hot exceeding so-c. -. 15.4.5 Diaolye tho contents in 10mJ or freshly boiJed and aeutraUsed 95 p:rcent etbaDOl aad titrate with the 0-1N alcoholic aodium hJdroxide solution, _Dg pheDOlpbthalein indicator. 15.f.6 The titration 10 obtained should not exceed 0-1 mi. It it doea, reject the test and-repeat the determination from the beginning. 15.4.7 Ifthere is any reason to sus~t the incomplete ~tioD of saponifilble matter, lubject the material, as weighed to re-aaponi&cation, re-estracUoo and Washing, under theeonditions specified in the method. It on this re-treatment, the amount'· of unsapdnifiable matter obtained is Dot the lame ..that weighed in the &nt determination, within the limits ofmanipulative error reject the test and repeat the determinauOD from the beginning. 15.sCal~

. UDlapon~able matterJ percent by weight - I~!!... where WI - weight in g or the residue, and W - weight in g ofthe sample taken. 35 1I._~UIt· II. DB1'BUmfATION CJP VITAMIN A 1'.' The determiDaaon or vitaDiiD A ,·may .be carried ~ther by 1peCtrO~ Diethod o~ ~Price methocL· ID eMe ofdlapute. the tpeetrophotometricmethod.' .'* UIed. 18.1 ~Pdee'~ __.1.1 ~.,., · 18.1.r.l Plio,."",. "tori,.",--- instrument with a clireet readiDg deflecdoD type galvanometer, IUitab1f. Cor m~ traDIIDittance or .bIorbeDce at 620 DIf&. 1_.1.2 ~ 18.1.2~O AU reapnta .han be or analytical pde. 18.1.2.1. Yi~.d rf/",.'""'4-A IOlub of cr,ttaWne vita- :miD 'AoC accurately mown Itrength. . 11.1.2.2' .,46,."", ..,tW ~ - or luch· apectraI padt, diat w~ measured in' one-centimetre quara ceJI apiDIt water. It ahaII ,how ablorbeace' DOt pater than 0-01 between 35O·.aad 120 '1IIf& and not areater thaD 0-05 at 300.mf&. . 1~1.2.s P.,.,.. /gtIto_ _ liM - 50 percent ( _/.). 11.1.2.4 BIIw - ~xide free, rediltilled or of auathetic ,racle (", IS: 336-196f·). Ether may be maintained tree from perOalcla by --wet ziDc roo. app~imately ~ emS per Htre, cut in Itripl IoDl to .reach at 1e8lt half way up the contaiDer, that h.. heeD·com­ pI y ~ in clilute acidi8eclCopperlulphate IOludon for one miDu~ a wa~. aacI.ublequeatly waahed with 11.1.2.5 Sotli.- -'J!!M* - anhydrous, granular. It shall D~ .bIorb vitamin A' under conclitioau of Ole, and 10 perceat lOIution .haII DOt be ~ to .,day) rediDdicator 101utiOD.. . 16.1.2.1 ~ IrWloritI, '"-prepared:''' cliIIolviDs 11S-4 I antimony trichloride iD 300\~ 400 m1 01chloroform. Add 5 I of calcium cbIoride &acI, &Iter while hot.· Dilute thefiltrate to 500 ml with cbI0r0t0rm. 11.1.2.7 aur./tJna-rediatilled. clilCareting the ~ aDd the last 10 perceat. '

11.1.1 "",., i , 11.1.1.1 ~ - Weigh. lICCUrateJy hili the vitamiD A "'celtaDd.ret eap.uJ.a O·S'to 1-0 I aad traasrer theatotbe.~ tioa IuL Rt6a fOr SO minuta with 40 m1 of~ alCohol ( IS paceD'. •/.) ~ 7 mt of ~UID hydrozicle' lO1ation ~'.u~ apparatus (n.b~ 1tOpperI' Uacl corb llaould DOt be used). ~ acid SO mI of -.podlc 1...... (.....,).

11._·-. water aacI eatrId·three ti_ with 5O-ml portMma of ether in • aparatiDr ' f1uuaeI. OombiDetbeedaetextractaiDaaotIaer~tingl\mDelJ ~dd 100m1 of...~~ etker layer.without.ptatioD. When good ieparatioD baa takea plaCe, After two miDutel, remove. tile aqueous layer. ShUt ~uslywith ~ to 5-aaI ~ otwater, alI!"f to '~z:ate, remove &nd ~ the aqueouslayer. If• ~hat reaaatant:emWsion fOnDI, dilute with 100mIof...ter to elimiaate or decreue tbiI euautifon before discard­ in, the aqueous portion. Wash with two addi~oba1 portion• .of S- to S-mI , 9( water. Again pour two portiODi or 100qaI of water through the ether layer and see that· the Inat water wuh isrlot alkaline to phenolphthalein. B~rate the ether eztract on water-bath to about 50 mi. Add 5 to 10lof lOCIium .uJ~te, ~J and allow to lettIe. Decant into a 5O-ml graduated IMk, riDiethe lOcI,'ium .ulpbate with several additional portions of ether, - pour into the·fIaK, dilaq to the mark with the final rinse. Tea, Coi ~ete estnction or fttamiD A &om sodium sulphate by adding a few ~ or the antimoDJ trIcb1~e IOlution to the resiaue. . 11.1.1.1"""'''' ".. Evaporate a suitable aliquot or the ether dation of the_Ow..unaponilable"'"-extract to about 5 mi. B~te off the nmaining ether at low heat under reduced pressure. T* up the raidue in .-fBdent chlorolbtm to give a concentratioD haviDg aD ablOrbenee or about 0·8 in the pho~lectric colorimeter. From thia dation make a aeriesoCdilutioaa in to give absorbancevaluei of _. eo, 40, and 20 perceat or. the original" abJor~ce. Determine abaor- baceI or the blue Colour formed when one-millilitrealiqUot ofeaclaottbese lieIOlutioDa plUl ODe mIlIUitre ofchlolOform iI treated with the volume of tile, ~timoDY trichloricte lOIution. that. is, suitable for tbe ~_ aDd hereinafter _erred ,to .. • the fixed volume'. The blaDk i_ adjusted to 100 ~t trAnlmittanee ~ • tube CODtainin~ 2 ml of chlorcd>rm and tile bed wlumeoCtile an~y trichloride IOlutioa. . U-ins. 'rectangular co-orcliDate ~per, plot the five ~bIorbanca obtaiaed .,uDat knoWD quantities or vitamin A aDd draw up the 'bat IIIOOda ,curve from the origin thrOUlh thae points. Do not attempt to clraw atraIPt Une un_·the curve'iI in filet ,••trai,ht line with tile origin at 8en». t. thOle iQltnuneDti that provide other than straight.liae cune, cMck thiI curve at ~UeDt intetvala. 'For thOle iastrumentl that do ~ ~t·IiDe calibration curft. make ODe reading ,of the. ref'ereDce loI.doD I wiali e.da let of IalDpJe readJDp to eatabti.h the curve.. ID the latta- ca.e n eeablilh the calibntion"curve wbeDever variatioD lD the ..-pDt or otbel'Yariab~ ill 'proced~ occurs. , . 11.1.1.1 ~_-Weith ~y• guantity 01the material COIl~",tD 451.V. Ot. n A enOt more than 5 g ofthematmaJ.)., tIleD .. Ia 11.1.1.1 ud obtain the raidue after ~tiDI the·etlter .....1IIOCIerAte beat ..reduced~. ~vethe,raidue hi·. dllnltI wi.....c:hlOr*m 10 that 2 inI of the chloroform IoIutIoa ...... at. -dmoDY trichloridcl dudoa ..... __.aD 17 11. __.1_

ablorbance of about 0-5 to ~2. Set the -instrument at 100 PfteDt ·trans­

mitlance with 2 ml of chloroform I and the fixed volume or the aotim.ony trichloride as blank. Place the tube containing 2 m1 of the chlorofOrm solution of the residue and add rapidly the fixed volume of the antimony trichloride sohstion. Record the maximum colorimetric reading·. Deter­ mine vitamin A from the standard curve and calculate units of"vitamin A per 100g of the sample.' 16.2 Specuophotometrlc Metlaod 16.2.1 AP/laralus 16.2.1.1 S/JIelroplaolf!1l'lUr - any reliable spectrophotometer with any suitable source of ultra-violet light (incandescent lamp is Dot a suitable source under 320 mu }. Direct reading spectrophotometer equipped with continuous spectrum source and reading 220 m.... is recommended. 16.2.1.2. Cells for mI(isurin, absorb""" in rdlra-lIiolll Iii'" - matched quartz cells with one-centimetre internal light path are preferable, but cella of other materials may be used, provided they are sufficiently transparent. Ifcells are not matched, suitable corrections shall be made. 16.2.2 Rlag,nls 16.2.2.0 All reagents stall be of analytical grade. 16.2.2.1 AblOlu" aleollol IJr iso/Wo/Jfl1UJI- of such spectral purity that when meesured in one-centimetre quartz cell against water, it shallshow absorbanee not greater than 0·01 between 350 and 320 mil and Dot greater than 0-05 at 300 m&&. 16.2.2.2 PDtlUSium "Jdroxid, sol"'itm ..... 50 percent (wi"). 16.2.2.3 EtMr - peroxide-free, redistilled or of anaesthe~(: ..... (". IS: 336-1964"). Ether may be maintained free from peroxide by the method given in 16.1.2.4. ..' 16.2.2~4 Sodi.",ndfhall - anhydrous, granular. It shall DOt ablord vitamin A under conditions of use, aDd 10 percent solution shall ... be acid to methyl red indicator solution. .' 16.2~.5 Cltlorojorm - redistilled, discarding the firlt and l..t 10 per" cent, 11.2.2.6 Alllimo1l.1 'rielaloiith IOlutio" - Prepare by dissolving I lief •• 300 to 400 ml ofchloroform. Add 5 g ofcalciulh chJo(ide and &Iter wbile, hot. DiJute filtrate to 500 ml with the chloroform. 16.2.3 Pr_M" 1&.2.3.1 Weigh accurately a quantity of·the material CODtainiDI20 to 45 I.U. of vitamin A ('not more tJian 5 r of the material) and traIiater it to the aponification fIUk. Reflux for 30 ~Duta with40 ml ofethyl aJcaJ .pla.c'doa (oretber. C....) IS.,.·IHI (95 percent til") and 7 m1 oCpotassium hydroxide solution using a1J~ apparatus (rubber stoppers and corb should Dot be used). (bo), add 30 ml of water and extract three times with :JO-ml portions ofether in a separating Cu·nnel. Combine the ether extracts' in another separating funnel, add 100 ml of water through the ether layer without agitation. When good separation has taken place, after two minutes, remove the aqueous layer. Shake vigorously with 3 to 5 ml portion ofwater, allow to separate, remove and discard die aqueous layer. Ifa somewhat resistant emulsion forms dilute with 100 ml water to eliminate or decrease this emulsion before discarding the aqueous portion. Wash with" two additional portions of 3- to 5-ml of water. Again pour two portions of lOO.. ml water through the ether layer and see that .he final water wash is not alkaline to phenolphthalein. Evaporate the ether extract on water-bath to about 50 ml, Add 5 to 10 g of anhydrous sodium sulphate, stir and allow to settle. Decant into a 50·ml volumetric ftask; rinse the sodium sulphate with several additional portions of ether and pour into the flask, diluting to the mark with the final rinse. Test for complete extraction ofvitamin A from sodium sulphate by adding a few drops of the antimony trichloride solution to the residue. 16.2.3.2 Evaporate a lO-nil aliquot ofether solution of unsaponi6able estract to about 2 mi. Evaporate the ether using moderate heat and reduced pressure. Take up the residue in sufficient isopropanol or absolute aicohol to give the concentration ~xpccted to yield absorbance reading of 0·4 to 0-8 at 325 1J1l.L. Determine absorbance of ~hil solution at 310, 325 and 334 mp. 16.2.4 CaleultltiD. Vitamin A content in I.U. per 100 g == .A ( corr;wted )'·7 X 333 where A ( corrected) == 7 A.. - 2-625 X A... - 4·375 X ~. (A ••, All. and A... represent ablorbanees at ~. 325, 310 and 334 m,& Feipectively ). L == length of absorption cell in centimetres, and W -= decimal fraction of unit of sample in one millilitre '1OIution whose absorbance is deter-' ~n~ . 17. DBTBDCINATION 01' TOCOPIIBROL 17.1 The I"~Up of CO.isb. of the.•~~ beta. gamma, aacI delta-. The tocopherols are potent aDtioxadanu. 17.2 Appa.... 17.2.1. Till.TuN - -15 X 2·5 em wid) ~ux co~. 17.2.2 0Iiu r.H-12 X 30!DID. It 11,3501.1_ n .2.'. PAtto"",,, ",;,,,Fil", 17.2." Waler-Ba'" 17.3 Re.,..t. 17.3.1 M,'h.!' AlcoW 17.3.2 Ethyl E'Mr- peroxide-free (ether may be maintained free from peroxide by adding wtt· zinc foil, approximately 80 cml per litre, cut in strips long enough to reach at least half w.ay up the container, that has been completely immersed in dilute acidified copper sulphate solution for one minute and subsequently washed with water ).

17.3.3 Potassium HydrD%id, Solutio", ~tJUI()"S - 2 percent. 17.3.4 Potassium H,dro,id, Solution in Mlilayl Alcohol- 2 N. Dissolve 112g ofpotassium hydroxide pellets in methyl alcohol and dilute to one litre_· 17.3.5 Sodium Sulplallll, AM.1drolU - analytical reagent grade.

173.6 ex fl.' Dipyrit/.JI- 0·5 percent solution in absolute alcohol. 17.3.7 IIJdroehlorie Acid-Ip gr 1-16. 17..3.8 B~,,,, - analytical reagent grade. 17.3.9 Floridin XS Column - Fill a 12 X 30 mm tube with the purified absorbent. To purify, 'digest on a l:Joilingwater-bath for one hour With hydrochloric acid. Repeat with fresh·portions of acid at room temperature: Wash with water until free ofacid, then with ethyl alcohol, and with ben­ zene. Dry at room temperature. 17.3.10 Litla' P,t,.""", - boiling range 40° to 60°0. 17.3.11 E'''yl·AleohDl- absolute, aldehyde-tree (IN note UDder 1'.1.1 ). 17.3.12 F,,,ie clalorUh (F, CI•.6H.O )- analytical reagent grade. 17.f Procecllll'e-Saponify-J g of the sample in a test-tube attached to a reflux condenser with 2 ml of 2 N methyl alcohol solution of potassium hydroxide for 10 minutes at 72° to 74°e in an atmosphere of nitrosen. Dilute with 8 ml ofmethyl alcohol, <d 10 m1 ofwater, and extract 3 times with 50 ml o(peroxide free ether. Wah the combined ether extracts with water, with two percent &CJueoUi po~auium hydroxide solution, and ..ain with water until the alkali II removed. Dry the e~tract which consisa of the unsaponifiable matter over anhydrous sodium lulphate and eyaporate under vacuum in a~ atmosphere of·carbon dioxide. 17.-t.l C",otnll RnMIHIl - Diuolve the reaiclue in 5 ml of benzene aDd pa. the IOlution through the Floriclin XI column previously wetted with beasene, Wash with benzene uatO the elute volume iI 25 mi. TIle' 40 absorbent earth is coloured a -greenish blue by 'carotenoidl and dark blue by vitamin A. · 17....2 To 1 ml or a greater volume of the earotene-free UIlIaponifiable matter solutioD, add 1 1111 of0·2 percent solution offeme chloride in abso­ lute ethyl alcohol, prepared fush from ferric chloride hydrate, andmix. Add I ml ofa 0-5 percent solution of (lex' dipyridyl in absolute ethyl alcohol, adx, and make up to the volume of 25 mi. Prepare a blank in a similar JDaDDcr. Allowt~ stand for 10 to 15 minutes and compare the colours in. photometer with a standard solution prepared from pure treated with the same amount of reagents. Correct the known and unknown for the blank determination.

11. DETECTION AND ~ETBRMINATION OF DISSOLVED SOAPr 11.1 Petecd- ., Ioap - Take about 5 mI ofliquid ghee in a test-tube and add an equal.quantity ofhot water. Stopper the test-tube and shake the ceatents well. Separate the aqueous layer and add a few drops of phenolphthalein solution. A pink colouration indicates the presence of soap. la~ DeteraWaad..01 m...lv" Ioap 11.2..1 A,/HIN11lS 11.2.1.1 Tlst-tuN - approximately 150 X 40 mm of heat-resistant alass, fitted with ground-glass stopper. 11.2.1.2 Min,."t, - 5 mI capacity. 11.2.2 &tlgmts 11.2.2.1 Dis'ill••,IDIII - containing 2 percent ofadded water. 11.2.2.2 Hydroeltloric tl&id - 0-01 N, accurately standardiZed. • 11.2.2.3 B,o_p/IIttol blru ituli&alor - one percent solution in 95 pereeat ( vIp) e1J1anol ( sp gr 0-8160 ). 11.2.3 hoe,",- Prepare the test solution by adding ~5 ml or the bromophenol blue indicator to each 100 ml of the aCJ,ueous acetone just before use and titrating with 0·,01 N acid or alkali until it is just yellow in colour. Weigh 40 g of the sample into the test-tube, which shall have been previously well rinsed with the test solution. Add I ml ofwater, warm on the steam-bath and shake vigorously. Add 50 mJof the neutralized aqueous acetone and after warming on the steam-bath, ~hake the veael well and allow the contents to stand until they separate into two layen.-. IflCNlp is present, the upper Iayer will be coloured p-een or blue. Then add 0-01 N acid, preferably from a microburette, until the yeUow coIour'is restored. ContiDue the process ofwarming and shaking until the yellowcOlour of the upper.layer remains permanent.

41 11.3511.1_ 11.2.1.1 It i. convenient, but not essential, to run, at the aame time a blank on a soap-free sample. Any difference in colour between the upper I.yen can then readily be perceived. 11.2.4 CtJIeultJIitm 0·304 T Dissolved s6ap, as sodium oleate. percent by weight - W

where T .. volume in mJ orO·Ol N acid required, and W .. weight in g of the sample taken. NO'1'II - The above method it suitable for the determination or soap in gbee up to 0·05 percent. At bieher concentratioDl it is better to take 4ssbee and use 0-01 N acid. 19. DETERMINATION OF 19.1 Q,aaUtadve Te.t. lor B.tyktecl Hyclros,aal.ole ( BRA ), Batylatecl B,clrosytolae.e (BRT ), Gallate. aM Nordilly..... paiaredc acid (NOGA ) 19.1.1 R,ag,nls 19.1.1.1 EJuolieh "agtnt- A 0·5 percent solution of sodium nitrite (NaNO.) in glass distilled water, and a 0-5 percent solutio" of sulphanilic . acid in glass distilled water containing 50 percent concentrated hydro­ chloric acid, are kept refrigerated. The nitrite solution should be freshly prepared every 3 weeks. The 2 solutions are mixed each working day in a ratio of I : 100 of nitrite to suJphanilic acid to perform the reagent ( diazobenzenesulphonic acid). , 19.1.1.2 EIIa.11 aleoMI-72 percent ( ). 19.1.1.3 Sodium IIydro%uu - I N. 19.1.2 P'oetdu" 19.1.2.1 One ml of melted rat.is shaken in a test-tube with 2 ml of 72 percent ethyl alcohol. The emulsion formed is shaken with 1 mJ of Ehrlich reagent, then immediately with I ml ofnormal sodium hydroxide solution: . a) BH~ - The development of a red-purple colour indicates the presence: ofBHA and shows an absorption maximum at 535 mil_ b) BHT- Presence o! BHT giva a distinct -pink colour with an absorption maximum near 505 mf&e 'The colour produced with BHT develops very slowly under the CODditions mentioned for BIL\. c) ProI!Il ,''"- Under the conditio. of the telt, propyl pilate is fOunCI to pve a yellowcolour. which facla rapidly Ul~ IIVCI DO

42 measurable maximum in the visible range. Other gallates behave p~ly. ' d) NDGA - NDGA yields a red colour, which rapidly changes to brown and brown yellow. , 1• .2 B.tlmadOD or AadosJclaat8 19.2.1 Methods are prescribed for determining the antioxidants butylated hydroxytoluene (BHT ). butylated hydroxyanisole ( BHA ), propyl gallate (PG ), nordihydroguaiaretic acid (NDGA), and all combinations except those containing both propyl gallate and nordihydroguaiaretic acid. Butylated hydroxyanisolc and butylated hydroxytoluene are separated from the fat and the other antioxidants by distillation with superheated Iteam. The distillate is analyzed for the sum of butylated hydroxyanisole and butylated hydroxytoluene with ferric chloride-Z, 2'-bipyridinc and for butylated hydroxyanisole with 2, 6-dichlorequinonechloroimide, there­ by permitting butylated hydroxytoluene to be determined by difference. Nordihydroguaiaretic acid and propyl gallate are extracted from a carbon tetrachloride solution of the fat using 50 percent ethyl alcohol and are determined with ferrous sulphate buffered to an appropriate pH. Butylated Ia~roxyanisoleand butylated hydroxytoluene, although partially extracted with 50 percent ethyl alcohol, do. not react with ferrous sulphate. 19.2.1.1 In a separate outline propyl, octyl and dodecyl gallate are determined absorptiometrically in a sodium acetate buffer solution with ferrous tartrate, which il specific for the gallates. After solution'~ the fat in lightpetroleum, propyl gallate is extracted with water and the hl~her gallate. with absolute methanol.: With the extraction methods described 95 to 97 percent recovery ofantioxidant was possible. 19.2.2 AHa,Glru .. 19.2.2.1 Dis,illaliM GJI/Hl,alflS for BRA and BHT- The distillation apparatus consists ofa Iteam generator consisting ofa 1 OOO-ml Erlenmeyer flask containing water and several glass .beads. The rubber stopper in the ftask has an outlet tube ending in a 12/5 socket joint. Water is boiled on an electric heater. The superheater consists of a glass coil placed in a 1 000-m1 beaker half full of (mp60°C, 27°0, flash point 36°0), and providtd with a thermometer. The distilling flask is made from a 50/50 ltahdard-taper joint, a 12/5 and a 28/15 ball joint. The distilling flask is heated in a 1 OOO-mi beaker containing approximately 800 m1 ofbath wax. During a distillation the inlet and outlet tubes of the IUperheater and distilling Sask are.wrapped in glass wool; The condenser ;S 61 cenihnetre long with a 28/15 .socket joint. The distillate is filtered / and celleeted in ~ 250-mI glass-s~oppered graduate. ' 11.2.2.a '"",-'-bV • .,."..- with 515, 530, 550 and 620 IDf& ilcen,' aacl celli. ' 43 II.'501· 1_ 1'.2.2.3 Sl/HJrating!unnllJ - 150 ml and 250 ml capacities. 19.2.3 Rlag,nit 19.2.3.0 All reagents should be of analytical grade. 19.2.3.1 Carbon t,'raehloride 19.2.3.2 Eth]l alcohol - Add approximately 0-1 percent, potauium hydroxide and .pctassium permanganate to commercial absolute alcohoJ and distil in all-glass apparatus. The distillate is 100 percent alcohol and is diluted volume for volume to obtain the 50 and 25 percent alcohol.. 19.2.3.3 Barium h7droxitl, - Prepare one percent barium hydroxide [Ba(OH)•• 8.0] in boiled distilled water. Thll reagent shall be kept ~ a tightly stoppered bottle. 19.2.3.4 Ammonium Aydroxidl - concentrated. 19.2~.5 ·Fmous IlIlp"",,-O-04 percent ferrous sulphate (reSo•• 7H.0) in distilled water, freshly prepared. 1'.2.3.& $H_ ear6'''I.IIHietlrlHnuztl 6".1" - Prepare .5-3 ~ent anhydrous sodium carbonate and 4-2 percent sodium bicarbonate in dU­ tilled water. 1'.2.3.7 Ammonium fl&,'(l1I - Prepare a solution containing 2 percent or ammonium acetate in distilled water. 19.2.3.1 Ctlltium ,IIlori. - 20 mesh. 19.2.3.9 F"ric cltloritk - Prepare fresh 0·2 percent of ferric chloride (FeCla• 6H.O) in distilled water. . 19.2.3.10 2,2'·BiJ!1ridi",- Dissolve 200 mg of2,2'-bipyridinein 0·5 ml or 100 percent ethyl alcohol and dilute to 100 ml with dIStilled water. If the 2,2'·bipyridine is brownish, sticky, or possesses a strong odour, it should be purified as follows: . Dissolve 10 g of2~2'.bipyridine in 10 ml ofwarm, purified ethyl alcohol and add approximately 250 ml ofcold distilled water. Allow to stand in a refrigerator overnight, Filter the flake like bipyridine crystals and wash with cold distilled water, The purified 2,2 .bjp~. dine should be white flakes with a faint sweet odour, Retain the supernatant in a refrigerator where a second crop of. erys". is obtained upon evaporation of the water. Purify ~;thae crystall 81 above. . .1~.23.11 Bor" buff" - Prepare 2 percent borax (MatB.o,. IOHp) In distilled water. . 1'.23.12 "·B,,I,1l_W 1'.2.1.13 M,tlumol- Boil one litre of absolute methaDol UDder rdlux (or I hour with 8 g of solid potassium hydroxide and 5 I of aluminium powder and then distil. .

44 1I.,..1NI 11.2.3.14 F",., ,."." ItlJdiM - Dissolve 100 mg of ferrous sulphate and 500 mg of aaalytical ~t grade ~tassium sodium tartrate' ( Rochelle salt) in 100 ml ofdistilled water: This reagent' shouldbe freshly prepared (or each series ofdeterminations. 11.2.3.15 Sotliaa flU"'" sol.,i.,. - One pereeat, Dissolve 109 of sodium acetate ( CH. COONa.3H.O) in one titre or distilled water. 1'.2.3.16 1s...)1 .oW 19.2.'.17 Li""1"In""', Hili., '''''I' W 10 we- Shake one litre of the light petroleam with small amounts of concentrated sulphuric acid until colourless alid .then, wash it several times with one percent sodium hydroxide solution and finally with distilled water until it is Cree'from acid. Dry the solvent with anhydrous sodium sulphate, filter and distil. 11.2.4 Proe" 1'.2.4.1 AMl.1sil of/In/J.1I,tIllal, tuUl IItWtliJt.1tlro".w,••Ul- Weight .20 g ofthe ghee into a 250 ml separatory funnel and diaolve in 40 ml of carbon tetrachloride. Gentle warming may be necessary to complete the solution. Extract thia solution with 70 ml of 50 percent ethyl alcohol by repe.tedly inverting the funnel for 5 minutes, at the rate of approximately 120 inversions per minute. Run the entire contents of the separating I\IDnel into a 250 ml centrifuge bottle and ~ntrifuge for 10 minutes at ,I '500 rev/min. Pour the upper alcoholic layer into a beaker. A portion 'of this 50 percent alcoholic extract is used for the qualitative analysis and the remainder is then diluted to 25 percent alcohol for the quantitative analysis: ' a) o._i,.,;", tI1UJ!1ns I) Nortlihydroptliar," MUl- Pipette 10 ml of the 50 percent alcoholic extract into. test-tube ( 15 X 1·5 cm). ~dd 1 ml of barium hydroxide reagent, shake immediately, and look down the length of the tube against a white background, If there is more than 0·001 percent of. nordihydroguaiaretic acid in the Cat, a blue colour will form and fade rapidly. If there is more than 0'002 percent of propyl gallate in the fat, a transitory green colour forml. If both propyl gall,ate and nordihydropaiaretic acid are present in the same sample, a green colour' forms fint and quickly fadel; then the blue nordihydroguaiaretic acid colour forms and fades. To obtain • positive test in the presence or propyl gallate, at leat 0·003 ~ent o( Dordihydroguaiaretic acid should be present in thetat. 2) PrtIbl,.u.tl- Pipette another 10 ml ofthe 50 percent alcohol atract into • teat-tube aDd add 1 mI of concentrated .m....ium hydroxide. Iro·ooo 1 percent otpropyl_"pilate is pnMDt iD the fiat .' pink to red cOlour forma. This colour ia

45 11.,... 116&

stable for 3 to 5 minutes depending upon the propyl galJate concentration. b) (lptl1lliltlli", tUUI!1sis- Pipette 25 ml of the 50 percent alcoholic extract into a 50 ml centrifuge tube. Add 25 ml ofwater, mix and 'centrifuge at 2000 rev/min for 15 minutes, or until a clear solution is obtained. Pour off the clear 25 percent alcoholic solu­ tion into a beaker and use this solution for the quantitative analysis. I) Nordila.1dropaiar,'ie acid - Pipette three different aliquot! ofthe diluted alcoholic extract (25 percent ethyl alcohol) into 18-mm colorimeter tubes and make up to 12 ml with 25 percent ethyl alcohol. Add 1 ml offerrous sulphate reagent-and 1 ml ofsodium carbonate-bicafbonate buffer. Measure the absorb­ ency after 10 minutes with a photo-electric colorimeter using a 515 mIL filter. All absorbencies should be measured relative to a reagent blank. Prepare a reference curve over a range of50 to .500 pg of nordihydroguaiaretic acid by replacing the alcoholic extract in the above procedure with aliquots of a standard nordihydroguaiaretic acid solution in 25 percent ethyl alcohol. Under these conditions, the observed absorbency' divided by a k-value of 0·001 41 gives the concentration of nordihydroguaiaretic acid in micrograms per aliquot used. 2) Prop,lgalltlt, - Pipette' three different aliquots ofthe diluted alcoholic extract (25 percent ethyl alcohol) into 18-mm colorimeter tubes and make up to 12 ml with 25 perccnt ethyl alcohol. Add 1 ml of ferrous sulphate reagent and I mJ of ammonium acetate buffer, and mix. Measure the absorbency after 10 minutes with a photo-electric colori­ meter using a 515 ml£ filter. All absorbencies should be measured relative to a water blank. Prepare a reference curve over the range of 30 to 300 fA.g of propyl gallate per aliquot. Using this procedure, the observed absorbency divided by a k-value of 0'002 05 gives the concentration of propyl, gallate in micrograms per aliquot used. 19.2.4.2 ~lIal.1lir ofIJulylautl "ydroxytmisol, andIJu'.1'atttlla,1dfV.1lo''''''' a) Dis'iUolion- Place 16 grams of anhydrous calcium chloride (reagent grade) and 10 ml of distilled water in the distilling ftask; cool to approximately room temperature and weigh 5 grams ofthe ghee sample in the flask. Liibtly grease the ground. glassjoint and place' the top on the distilling flask. Before startio! the distillatioD, heat the J-th for the di.tiJlin. Sask to 160° ± 10 a, and the superheater bath ~:J: ~C and 46 11,3508·111I adjust the.lteam generator to distil approximately 4 ml of water per minute. Maintllin these cOnditions throuRbout the entire distillation. . As seon ~ these conditions are fulfilled, connect the super­ heater and the condenser to the disullio, ..ftuk. Start the dis­ tillation by connecting the Iteam generator. to" the superheater and immediately place the' bath around the distilling flask. Collqct the distillate in a 25().ml glass-stoppered graduate, filter­ ing the distillate' through a 9-cm Whatman filter paper No. 54. or- equivalent al it collects. The rate ofdistillation should be such that 125 ml ofdistillate collects in 30 ± 5 minutes. When 125 ml ofdistillate has been collected. st~p the distilla­ tion by disconnecting the distilling flask from the superheater and . removing the bath around the distilling flask. When the mouth ·of the condenser has cooled, disconnect it from the distilling flask and drain the water from the water jacket. Wash the condenser and filter paper thoroughly, Using six 100mi portionl' of hot ( &a- ± 5°0) 100 percent ethyl alcohol, allowing the alcoholic wa.hings to filter into the distillate. Continue washing the filter with hot alcohol 'uptil the combined volume of distillate aDd wubings is 250 ml when cooled to room temperature. b) AMI.1sir qf'dblilltlll 1) B"'.1liJt,d /f1dr'9tmis.oll (2, 6-di&!alo'tHJUiuJUeAlorimitl, tIII"")­ Pipette thre~ ~ifFerent aliquots .of the distillate (50 per­ cent alcohol) Into 18-mm colorimeter tubes and make up to 12 ml with 50 percent ethyl alcohol. Add 2 ml of the 2, 6-dichloroquinoDechlorimlde reagent and 2 ml of borax buffer and mix. After l~ minutes, add 5 ml of n-butyl alcohol to each tube, mix, and measure the absorbency with a rhoto-electric colorimeter using a 620 mil filter. Measure .1 absorbencies relatlve to a reagent blank. ,~. . Prepare a reference curve over a J..nge of 10 to 50 I« orbutylated hydroxyanilOle. The concentration of butylated hydroxfuisole, in' mic~am per aliquot used, i. obtained by dividing the observed absorbency by a i-value of 0·0102. 2) . B_f1l*I ~oqdllisol, II", ht,lalM ")'1''';'''01,"", (1m ,1Ilori8-2, 2 .6i/J:1ridi", ""Mod) - AJlsolutlons Ihall be cooled to room temperature belore starting thi. analysis. Pipette duplicate aliquots of the alcoholic distillate ( 50 percent ethyl alcOhol) into "5G-ml.I.....toppered Erlenmeyer flasks render­ ed 1m~ou. to light with black tape, and make up to 8.~ WIth 50 percent ethyl alcohol. Add 2 ml of ferric chIaneSe ~nt and 2 mJ of 2, 2'-bipyridine reagent to each ftaak anil mix. Thirty minutes after the addition of the 47 11.,.·1_ faTic chloride reapDt acidS mI,ofJl-butylalcohol aDd mix. Thirty-&ve minutes after the additiaD or the remc chloride re&pt, t»oUf the conteDts oftbe flak iD~ all ~8-mm colori­ meter tube and, after a further 2 DUDuttI, me8lure. the abeorbency in a photo-elec:tric colorimeter UIiDg.. 51S·mil filter. All measurements are made relative to a re8pDt blank. The absorbency is a meas~ ofthe sum ofbutylated. hydrosyaniaole and butylatecl hyclrozytoluene. Prepare a reference curve for butylated hyclrosytol~eDeover a raage or 10 to 50 N with each set oranalyaia. This is 'D~ aiDce the i-value for butylated hydroxytolueDe van. with tem­ perature; i-valua were (ouad to rancrfl! from O~OlO 8 at 22-0 to 0·0142 at SOOO. . Prepare a reference curve for butyl.ted hydroayaailole overa range of 10 to 50 1&1. Under the above conditioal. k-value or 0·0114 wu obtained. Divide the ablorbea~ obtained with the 2, 6edichloroquinonechlorimide reageat ~ the aliquot volume and by the 2, &.dichloroquinonechJorimicie k-vatue for butylated hydroxyariilale to obtain the concentra­ tion of butylated hydrosyanisole in mic-:ogrUDI per milUlitre of distillate, Multiply this value by' the ferric chloride-2. 2'-bipyridine i-value for butylated hydroxyanisole and by the number of miUDitres of diltillate used in the ferric chloride-z, 2'-j)ipyridine reaction. This, &g'lI~e repraena the absorbency aue to, butylated hyd~xyanisole in the ferric chloride-2, 2'-bipyridine, reaction. Subtract this latter &auae from the measured absorbency in the ferric chloricfe-2. 2'-bipyridine reaction to find the absorbency due to butylat;{ hydroxytoluene. Calculate the amount of butylated hydroxy­ toluene in' the distillate.

Examl": Weight offat sample == 5·0 I Total volume, distillate +washinp -= 250 ml 2, 6-Dichloroquinonechlorimide == 0-102 reaction KBHA Aliquot volume :- 12 mI Absorbency :.- 0·260 Ferric chloride-2, 2'-bipyridine -= &011 f aliquot volume reaction KBHA ,-8m1 OHT - 0-0122 aJ.orbeDcy - 0-311

11.3511.1_

c.I"""':O»DceDtratioa or BRA per ml or the . 00-260 ~te· - 0-0102 X 12 .. 2-27 III A~ per aliquot due to BRA in - 2-21 X 0-114 X 8 ~ 0-207 PeCl.-2, 2'-bipyridine reaction . .

Absorbency ~ aliquot due to BHTin - 0-398 - 0-207 &ell 0-191 FeCla-2.2 -bipyridine reaction Microsrama or BHT in the distillate ()e191 X 250 489 . 0·0122 8 == Concentration of BHT in the ghee 489 sample - 5x 101 X 100 == O·~ 8 percent Concentration or BBA in the ghee 2-27 X 250 100 _pie -= 5 X 10' X . == 0·0113 percent NO'nI-Tbe praeace olallA, BIlT, tocopherol in Ibee causes no error in the "'JIia 01propyl pnate ad NDGA, or • MM. 1'.2.4.3 D"..;"'Ii." of"'.1.11) oel7l ad tl0U9l g"U.lIs 'a) &IrtIt"- Dissolve 50 glofghee in light petroleum. Extract the solution 5 times successively with 2O-mJ portions ofdistilled water at 30°0. taking 2 minutes over each extraction. separate the phases and filter the' aqueous layer into a 11O-ml calibrated lull.: then wash the filter with water and add the washing to the filtrate in the 8ask until the mark is reached. Shake the fat-1isht petroleum layer, from which the wate hu, as far u poss!ble, been separated and removed, fint with 55-ml portion and then with four 15-ml portions of methanol, taking 2 minutes over each extraction. With some samples partial crystallization ofthe glycerides may occur owing to cooling. Ifso, the extraction should be carried out at 25°0, for example, by gentl.heating of the separating funnel. It is essential to wait at least 5 minutes after each extraction in order to obtain good separation of the layers, which may be assisted by swirling the separatiog funnel. At least 30 minutes shall be allowed after the last extraction before running off the lower layer. Transfer each extract as completely as possible to a 150-ml separating funnel. Add 3 ml of distilled water to the combined extracts and shake wdl. After 30 minutes have elapsed add the final residue of methanol that has separated from the fat-light petroleum layer and shake the separating funnel again. Run off the clear lower layer into a 110-ml calibrated flask, add sufficient methanol to make up to the mark, When necessary, a 125-m1 calibrated flask may be used iostead.

~9 11'·..._- b) A".!?su of,III ""ael I) Est;"..';,,. ofpr'b'ltlll." - Pipette 10 ml or the water ex­ tract in a ~o ml calibrated fluk. Add 1 ml offerroUi tartrate solutlen, fill up to the mark with sodium acetate IOlution and

mix well. After 10 minutes measure the 0rtical density of I the solution. at 530 ml£ relative to that 0 water, using an absorptiometer and a 2-cm cell. To det~ine the optical density of the blank, dilute 10 ml ofwater extract, without ferrous tartrate solution, to 50 ml with sodium acetate solu­ tion and measure the optical density after 10 minutes. ~ter. mine the reference value for propyl gallate by treating 10 ml portions ofa solution contalning 5 mg of propyl gallate per 100 ml of methanol in a similar manner. The percentage of propyl gallate present i. given by:

.~I - ~:.l. X _1_ X ~ E. - E. 2p 100 where

E1 == optical density of the water extract with ferrou- tartrate·, solution, E. == optical density of the w'ater extract without ferrous tartrate solution, E. == optical density of standard propyl gallate IQlutiOD ( 500 1£8 ) with ferrous tartrate solution, E. -= optical density ofstandard propyJ gaUate solution wit~. out ferroUi tartrate solution, and /J c: weight in g oC ghee taken. 2) Estimation ofDe~l ad tl-:Jl ,allal'- Pipette 20 mI of the water extract in a 150 ml separating fUDDel, Add I ml of ferrous tartrate solution and 40 ml ofsodium acetate solution. and mix well. After 10 minutes add 20 ml of a mixture or equal parts oC' isoamyl alcohol and light petroleum, aDd shake vigorously for 2 minutes. Extract the lower layer, after separation, again for 2 minutes with another 2(J.m1 portion of the mixture ofequal parts ofisoamyl alcohol and light petroleum. Run off the remaining water rrom the combined extract. as completely as possible, and transfer to a 5O-mJ 8ask. Add 3 ml of methanol to obtain a clear . IOluticy' dilute to volume with is,amyl alcohol aDd mix wtD. Measure the op~i~al density of the solution at 550 DlIi· relative to that ofueamyl alcohol, \IIi.., an ablorptiometer and 2-em cell. Determine the optical dcDIity of the fat '50 estract alone by 20 m1 of the methanol extract in the aame way. omittiDI the ferrous tartrate solution. Determine the refereDce value f9r either' octyl or dodecyl gallate by tratin' 10 m1 po~~iODS of a solution containing 5 mg ofthe gallic acid in-100 ml ofmethanol in the same way. If it is not known which g,-iate is present, octyl and dodecyl gallate may be distinguished fr~m each other by adding 1·5 ml of ferrous tartrate reagent solution to 5 m1 of the methanol extract. After 5 minutes add I m1 ofa mixture of' equal p.rts of isoamyl alcohol and light petroleum. Sh8.ke the mature carefully (shaking too violently considerably delays the separation of the phases). If the upper Ja~ becomes violet-blue in colour dodecyl gallate is indicated, since DO colour i. formed with octyl gallate.: Note that, ifthe optical density of the methanol extract is more than one and a half times' that of the reference solution, the procedure should be repeated with 2 ml instead of 1 ml of ferrous tar­ trate lolution. The percentage ofgallate present in the ghee i. given by: Et-E. I VI 10 81- E. X 2" X 100 X V. where 8. = optical density of the methanol extract )Yith ferrous tartrate solution, E. - optical density of the methanol extract without ferrous tartrate solution, E, == optical density of standard gallate solution (500 1&1) with ferrous tartrate solution, E. == optical density of standard gallate solution without ferrous tartrate solution. p. == weight in g ofghee taken, J.l~Yl == final volume of the methanol extract ( 100 or 125 ml ), and VI == volume of the methanol extract taken for the determlna­ tion, Nor. - Ifother antioxidantl, luch as butylated bydroxyaDilOle or butylated hydroxytolueneo, were also praent in .hee, they pall into tbe methanol phale together with the pllata OD beiDl abUm with . lIIethaool. Both autioxidanta, however, at tM Ulual concentradoa ~(H)2 ~centJ had no adverse efFect oa determinabODI of the pllie ackIaten. TIle ialu~ of the IJDeIPti dtric acid, ucarbic acid aDd p~ acid 011 the ~ 01 tM 1aiP- pllata WI

51 g·I'.·lK6

completely ~tical with their dFecta Oft thedetenniDation or DfOD9I gallate and will only be apparent If Ihee • atractecl directly wlala methanol. Normally tbe.e materiall will be removed la the water extract with the propyl plIa,tc.

20. DETECTION OP VBGftABLE PAT IN GIlD BY TID PHYTOSTBRYL ACETATE TEST

20.0 The method is suitable for the detection of the pretence of the' most' commonly used rats in ghee. The sensitivity depends upon the character of the vegetable rat 'used for admixture. The content is determined gravimetrically after I&ponificat~onof the fat and precipitation of the by adding an aleoholic digitonine solution to the soap solution. The melting point of the sterol acetate is determined after acetylatiDg the sterol digitonide by acetic anhydride. The crystal form of the sterols is microscopically examined after converting the sterol acetates into the steroids by saponification with an alcoholic potassium hydroxide solution.

28.1 ...... t. 20.1.1 Pot.,S;."" H.,druidl (Aul.1ae., Grath) Solation - dissolve 400 g of potassium hydroxide in 600 ,g ofdistilled water. 20.1.2 Digiloll;1II ( AMl,lietll arlld, ) Sol"tiDn - dissolve 10 g of digitonine in one litre of96 percent ethanol ( "III ). 20.1.3 Elhanol- redistilled, 95-96 percent ( v'/v) and 80 percent (IJ/v). 21.1.4 Di"".1 ElAn ' 20.1.5 k,," AnA.Jdritll

20.2 Apparata. 20.2.1 Conical Flask - of.250 m1 capacity wIth air-cooled conde~r. 20.2.2 Mie,ojil",ing D,llie, 20.2.3 MIlling.Point Apparduu 20.2.4 Melling-Poin' Tubes-·intemal diameter 0·8 to 1-0 mm, length 50mm. 20.2.5 Microscop, Slidts dnd Cow,Sli/1J 20.2.6 Mi,ros&oPe -linear magnification 200 X. '20.2.7 rll"momt'" - reading up to 150·0 with 0-,1 deg graduations. 52 20.3 Proced....· 20.3.1 D,,,,mi,,.liD,, of,,,, To"" $"101 Co"",,' - Weigh accurately to the nearest 10 mg about 15 g ofthe ghee in a conical flask of250 ml capaoity. Add 10 ml ofpotassium hydroxide solution and 20 ml of 95 to 96 percent ethanol ( vlv). Add 2 glass beads. Place the air-cooled condenser on the flask, heat on a boiling water-bath until the solution has become clear•. and continue boiling for half.an hour. Add 60 ml of water and then 180 ml of 96 percent ethanol ( vi" ), and raise the temperature to about 40°0. 20.3.1.1 Add 30 ml of the alcoholic digitonine solution ( one percent), shake and allow to cool. Place the Bask in a refrigerator at about 5°C Cor about 12 hours. Collect the precipitate of sterol digitonide by filtering through a filter paper ( Whatman No.1 or equivalent) in a Buchner funnel (diameter 8 cm }. Wash out the precipitate with water at about 5°C until the filtrate stops foaming, then once with 25 to 50 ml of 96 percent ethanol ( vIp) and at last once with 25 to 50 ml ofdiethyl ether. Dry the filter paper with precipitate on a watch-glass in a drying oven at 1020 ± 2°0 for about 10 to 15 minutes. Fold the filter into two, allowin~ the precipitate to come off as a pellicle, ftansfer the precipitate into a weIghing bottle and .eigh. 20.3.2 Preparation of 1M Sterol Acetates and Determination of the M,lling P"iIII- Transfer 100 ± 5 mg of the sterol digitonide to a test-tube, add I ml of acetic anhydride, and heat the tube in a glycerol-bath at 145°0 until the precipitate has dissolved. Do not use direct heat, since spattering may occur. Continue heating for 2 minutes and allow to cQOI at about 800e. Add 4 ml of 96 percent ethanol (vlv), mix, heat slightly to dissolve any steryl acetate which may tend to crystallize out. Filter the still warm solution through asmall medium speed filter paper impregnated with ethanol, and collect the filtrate in another test-tube. Heat the filtrate in the latter test-tube and carefully bring to gentle boiling. While still boiling add carefully, drop by drop from a pipette I to 1"5 mI oC water until the steryI acetate is just about to precipitate but still remains in solution. Avoid superheating. Add a few drops of 95 to 96 percent ethanol ( vIv) to dissolve' again any precipitated sterol acetate. Allow to cool in air for 2 hour~t and finally in ice-water for half an hour. Filter the crystallized stery) acetates on a small disc of fast speed filter paper by suction in a glass .micro6Jtering device and rinse the crystals with 1 ml of 80 percent ethanol (,,/,,). Redissolve the crystal cake by heating on a microburner in 1 ml ofethanol (96 percent) in a short heat-resistant glass test-tube (diameter 12 mm,-Iength 35 mm }, Allow to cool first in air for 15 minutes and then in ice-water for 5 minutes. Filter the crystallised sterol acetates as described above. Repeat redissolving. crystallization and filtration to obtain the third, occasionally the fourth or fifth recrystalliza­ tion. Dry the crystal cake on the paper first at about 30°0 in a drying oven and then at 102°:!: 2°0 in drying oven for 10 to 15 minu~.

53 11.--·1- Grind the cryIta1 caire in • IIDaI1 &pte mortar .6IieIL.~v,:w ~wder.~ &11. melting-point tube••,height mahoutSmm.to'" . iDe the melting point in the melting-point appara~ raiaiDg ·the temperature very slowly in th~ last phue of the melting procas at a rate of0-5 degree per minute. Take the ~on the tbermometeratlhe'momenttbat.tbe lut crystal srain hujust disappeared, ., the ~tmspoint. 20.1.3 Minti. &_iutitnt oj1M S,.,,, - Dissolve .~ut 10~ o(the sterol acetate in a'small test-tuDe in 1 mI oC'96 pe.rcent ethanol ("/D) an4 add , or 2 dropl of pataaium 'hydroxide iolution. Heat on a boiling water-bath until boiling begins and the ~teryl acetate hu dissolved. Add 10 ml of distilled·water. transler tl;le .olution to a 125-mI separating funnel and .hake with 25 ml ofdietbyl ether, After 1e~.tiODt dr.in and discard the ~ueoUi layer. Wah, the ether layer with three 5-ml portioDI of dis­ tilled water. Transfer the ether- _ayer to a 5O-ml beaker and evaporate to dryness~ DislQlve the 'residue in 10 ml of 80 percent ethanol ("/"). Place a drop of the clear IOlution on a microscope cover slip, wait until crystallization ltartl on the periphery of the drop, then invert the cover dip and lay it Oft a microeCo~ slide. Examine the crystals. under the microscope at' ~ X linear ~tatiOD. A diap-am of the crystal abaJ* of sterol.is given in Fig. 5. . lI.t ...... "'..... 20.t.1 eM"'''. ' Total sterol content, pereent _ 0-25 ,.X 6 X 100 where 4 • weipt in g of1_ sample, and II -" weipt in I of the sterol digitonide. ~int 20.4.2 If the melting of the steryl acetate it found to' be 115°°1 the ghee sample is cOlilidered to be free from ~ble rat. If the melting point of the sterol acetate i. found to be hieher than 117·0, the fat sample II considered to contain vesetable fat. If the melting point ofthe sterol acetate is found to be lower than 117°0 and higher than 115°C, the rat sample is oDly considered. to contain vegetable fat it the melting point is increased after replicate 'recrystallization. 20.4.3 Ifunder the mttroscope the sterol crystalaoaly show the form of a pa~lIelOFam with an obtuse angle of 100·, which is c~racteri.tic for . the fat sample is.considered to be free &om..table fat. 'If under ,the microscope the sterol cryatalJ allo ahow the el-fated hexagonal form with an apIcal angle of IOS·,.which is _tIC fOt ., .!lr if ~me.of the Ita:ol. crystal~. ·have • re-entry aagle (lwaHow'. tad).· which II charaetenatic for IDistUIeI fA daoIeIteaI aDd phyto.teroll, the Cat sample iI cODiidered to. ~ta1a veptabIelat. ~ CHOLESTEROL 108-=>

PHYTOSTE ROl

CHOLESTEROL-PHYTOSTEROL MIXTURES Flo. 5 CaYITAL SB.u.. or SnaoL .55 III,'501. 1966 20.4.4 T,sI lUpo,' 20.4.4.1 The telt report shall state the total .terol content expreuecl , as a percent by weight, the melting point of the steryl acetate and the number ofrecrystalliaasions, and a description of the microscopic ilDAle of the sterol crystals. 21. DETERMINATION OF PRESENCE 0. OIL (BAUDOUIN TEST) 21.0 The development of a permanent pink colour with furfurallQlution in the presence of hydrochloric acid indicates the presence ofsesame oil. 21.1 Realeat. 21.1.1 H.1droehloric A,id - fuming, sp gr 1'19. 21.1.2 Furfural Solution - 2 percent solution of fttrfural, distilled Dot earlier than 24 hours prior to the test, in rectified spirit ( conforming to IS: 323·1959* ). 21.2 Proceclare - Take 5 ml of the melted ghee in a 25-ml'measuring cylinder (or test-tube) provided with a glass stopper, and add 5 ml of Ilydrochloric acid and 0·4 ml of furfural solution. Insert the glusltopper and shake vigorously for two minutes. Allow the mixture to separate. The. dev~lopmentof a pink or red colour in the acid layer indicates presence of .same oil. Confirm by adding 5 ml of water and shaking again. IC the colour in acid layer persists. is present. Ifthe colour disappean it is absent. 22. DETERMINATION or THE PEROXIDE VALUB 22.0 The peroxide value is a measure of the oxidative rancidity in ,hee and is expressed as millilitres of 0·002 N sodium thiosulphate per gram ofsample, or as milllequivalents ofperoxide per kilogram of~ple. Two methods are recommended. 22.1 lociometric.Metla~ 22.1.1 4P/HWtJ'''s 22.1.1.1 T,s,·",6" -150'x 2·5 mID. Before use, wash theae th~h1y with soap solution, rinse with hot water and allow to stand in chromic acid mixture for a few hours. Then rinse thoroughly (tile.Jut tilDe: with distilled water) and dry in an oven before use. 22.1.1.2 Rabb" 611111- To fit the test ...tube with a bole in the caltre through which is inserted a amall glass rod (or S to 4 DUD diameter) flattened at one end and rounded offat the other• •Specificationror rccti&ecltplrit ("fib.). 1S.'508·1_ ~.1.1.s W.",-..IA 22.1.1.4 Ctmietlljluk-250 ml capacity. 22.1.2 lUag"," 22.1.2.0 All reagentlshaJl be ofanalytical grade. 22.1.2.1 Sol""" mix"", - a mixture of 2 volumes ofglacial acetic acid and 1 volume ofchloroform. 22.1.2.2 Sodium thiosul/JIuJII ~ 0·002 N solution, freshly prepared dilution ~rom an accurately standardized 0·1 N solution. 22.1.2.3 P,tassium iodide - freshly powdered. '22.1.2.4 PoltUsium ioditl, - 5 percent solution, freshly prepared. 22.1.2.5 S"',h indicator - as in 14.3.4. 22.1.2.6 CtJrlJon dioxitJ, 22.1.3 Proc,dur,- The test should preferably be carried out in artificial lilht free from ultra-violet radiation. Weigh quickly but accurately a suitable quantity of t~e sample ( the weight ofthe sample taken for the test should be such that the titration does not exceed 10 ml ) into the test-tube and while still liquid add 1 g of powdered potassium iodide and 20 ml of the solvent mixture. Gently bubble carbon dioxide through the mixture of the ghee' and solvent (for routine tests, this is unnecessary ). Heat the contents of the tube to boiling within 30 seconds, preferably in a steam­ bath, and allow them to boil vigorously for not more than 30 seconds. As the solvent vapours begin to escape from the hole in' the bung, close tho opening with the glus rod. Cool immediately under a tap and transfer into conical Bask containing 20 ml of5.percent aqueous solution of potas­ sium iodide and wash out the test-tube twice with 25 to 30 ml ofdistilled water, Titrate the ablution with the sodium thiosulphate solution using starch indicator. Do not add the starch until the end point is almost reached. Perfornl.a blank test. This titration should not be more than 0·1 ml, 22.1.4 Ctdeultlli,1I ~.1.4.1 Peroxide value _ 8 000 A: where .If == volume of sodium thiosulphate solution required for the sample, l N _ exact normality ofthe solution, and M -= mua in g of the sample taken. 22.1.4.2 Results may be expreued in. milUmolecules of oxygen per q of fat. To obtain this, divide the peroxide value by 16. 22.1.U The results ex~din milli~valenti ofoxygen per kg of fat "ball be obtained '" dividins the peroxiae value by 8. 57 lS, 3501. 1966

22.2 O.Yle. Ab.orptlOD Metlaod 22.2.1 Appllrtltus 22.2.1.1 Oxygln a6sorp"io" tl/J/HI,alfU - as shown in Fig. 6. 22.2.1.2 Oxygm las 22.2.1.3 Oil-btlth - maintained at 790 ± 1°C• •15em CAPILLARY

GRADUATED ~OXVGEN 0 PLASTIC INLET 10 SCALE 18 20 " em _ SPHER BULB 30 40 50 MERCURV FILLEO OPEN TUBE eo MANOMETER 70 80 50 ml CONICAL FLASK 90' FOR HOL'DINO EXPeSE"D \00 GHEE

All dimensioDi in centimetres. FlO. 6 A~LAII Oxyour AaIollPl'loN AnAaATUI 22.2.2 Proud"" - Clean aQ ,I.. parts' with chromic acid. ItiDIe in diStilled water and dry. Weigh accurately 5 g ofghee into conical &uk and attach the manometer assembly. Open the inlet Jbr oxypn and c10ae connection to the manometer. Leave the outlet fbr oaygen open. CoD- .neer the inlet fbr the oxygen to the cylinder aDd replate theftowofoxyp t(),bubble slowly through the melted ghee, continue 8wda, with oxy~

58 lSI S5OI-l_ 5 minutes. Close the inlet and disconnect the oxygen cylinder. Set the Bask in the oil-bath at 7go ± 1°C. Open the flask to the manometer and periodically release the pressure inside the flask by opening the outlet for oxy«en. Continue releasing the arms pressure inside the flask until equilibrium is reached between two arms of the manometer and the flak has attained the temperature of th~ oil-bath 15 to 20 minutes from the time of introduction of the flask in the oil-bath. . 22.2.3 Note time when equilibrium is reached. Record reading of the manometer at the intervals of 2 hours initially in the fresh samples and one hour in case of old samples as well as the samples from stored butter and . 22.2.f Note down the time when the manometer level in limbs con­ nected to the flask starts its progressive rise, continue recording reading or level in manometer for another one hour. The number of hours elapsed .ner the equilibrium in the manometer was reached and thetime when mercury level in manometer started progressive increase (up to 10 mm ) corresponds to the induction period of the sample. 22.2.5 Value for induction period of 20 hours and over appears to cor­ respond to a marketable life of 6 months. Samples having an induction p~riod below 6 hours are found to be unmarketable. 22.3 IDterpretatloD ofRe••lts - Whilst either the iodometric peroxide value or the induction period as determined by the oxygen absorption method could be used to measure the keeping quality (shelf life) of ghee • C'ombination ofboth gives the most reliable results: P"ox;th Valru Int"Jmlaiion I"due/ion Period Peroxide Jl'alu« ml olO-OOiN of OJUIli'.1 at 79°C in Hours oj EX/JOs;d Sampl, StHlium at tlll·End of ThioJulph,,'e Inductio,. Period Solutionlg Below 1-5 Very good Above 20 Below 18-0 1-6 to 2·0 Good 16·20 Below 21·0 f:-' 2·1 to 2-5 Fair 11·15 Below 24·0 2·6 to 3-5 Poor 6·10 Below 27-0 3·6 to 4-0 Not acceptable Below 6 M>ove 30-0 23. DETERMINATION OF IRON CONTENT 23.0 Geaeral-This method is considered satisfactory for the determi­ nation of iron occurring normally in ghee. A~'id extraction suffices 'for this case and has been found preferable to the ashing technique. Where, however, the presence of iron from extraneous sources in less soluble inorganic forms, for example, ferrosilicate, is suspected, total iron should be determined by ashing and alkali fusion. 59 ts.3508.1966

23.1 Prladple 01 Method 23.1.1 Colorimetry of the pink colour formed by reaction of Fe+++ with thioglycollic acid. 23.2 Realeat. . 23.2.0 The reagents used shall be of analytical reagent quality and free from iron. Distilled water, re-distilled ~om all-glass. apparatus, shall be used throughout. 23.2.1 H.1tlrochlorie Acid- Sp gr 1·18. 23.2.2 Ammonia Solution - Sp gr 0·88. 23.2.3 rhiogl.!eollic Acid

23.2.4 Bromine WtJler- saturated. 23.2.5 Standard Iron Solution - Dissolve 8-635 g of ammonium ferric sulphate, NH.Fe(SO.)2_12H,O in distilled water containing 5 0ml of dilute ( 1: 3) sulphuric acid and dilute to I 000 ml, Before use, dilute this stock solution one hundred times by successive dilutions. One millilitre of the resulting solution is equivalent to 10 microgrammes Fe. 23.3 Apparatu. 23.3.1 Flask- of 250 ml capacity, with interchangeable conical ground glass joints. 23.3.2 Sp,ctrophotometer or Photoelectric Absorptiomet". - With a blue-green filter having a maximum transmission at approximately 480 nm; all­ glass cells should be used and should be, of such size (I-cm cells are usually satisfactory) that the optical density of-the solution under test lies lt~tween 0-1 and 0-8. or 23.3.3 Nusl" CJlitUlns - of SG-ml capacity. 23.4 Proceclare 23:4.1 Weigh 25 g of the sample into the flask. Add 15 mJ of water and 20 ml of hydrochloric acid. Use a few glass beads to regulate the .boillng. Reflux for one hour. Transfer to a separating funnel, allow to settle and run off the aqueous layer through a double, acid-washed, 0 medium texture filter paper into a' 9()().ml beaker (a Whatman No. 40 paper or equivalent, washed with hydrochloric acid of the lame concentration as that used for the extraction immediately beCore use; is suitable). Wash the sample again in the sep~ting funnel with two 5O-ml portions of hot water, using these to rinse the flask and pouring them through the filter after washiDg the sample. Evaporate the combined aqueous and acid extracts to about 5 ml and add 5 ml or bromine water. au Bon pad)' ,~~~l·the excess broDiine is driven o~. '. ~ .~I. traDlr~. ~ ~ Neuler c:ynD~er~ .. ~dd .2, to 3 drops' o~ ,thiOiry~lIi~ add, maa' Just albline'to litmus by the ClropwlM .add~tlon 'of: .tnonia IOlution and dilute to 50 mJ with water. R.ead the colour ip the .bIorptiomf:ter or in the .spectrophotometer. 25.5' cialCllladoa ...... of a....a 13.5.1 Determine the iron content of the' solution from a ltandarcl curve prepared as follows: ~easure into five separate 5O-ml graduated flasks by me4&.lIa of a burette, nil, 5, 10, 15 and 25 ml of the standard iron splution. Add 5 ml of water, 3 ml of the hydrochloric acid, 2 Or S drops or tbioglycollic acid and ammonia solution dropwise until the solutions are just alkaline to litmt1? paper. Cool, dilute to the mark with distilled water' and mix well. Determine the coloun of the solutions in the approptiate instrument, using in the reference cell the -control solution containing no iron. Plot a curve correlating transmission or optical density against micrograms of iron. 23.5.2 Alternatively, the iron content may be determined by matching with standards in Nessler cylinders, as follows: Place in a So-ml Nessler cylinder, 35 ml of water, S ml of hydrochloric acid and 2 to 3 drops of thioglycollic acid and add ammonia solution until the solution is just alkaline to litmus paper. Cool, dilute to 50 ml with water and add, from a burette, with constant stirring, standard iron solution until the colour of the solution matches that of the test solution. Note the volume of standard iron solution used. Repeat the preparation of the matching solution, but place the volume of standard iron solution previously determined in the Nessler tube before adding the reagents. If the standard colour is now different from tbat of the test lolutiuo, prepare-other standards containing appropriately more Dr less iron until a perfect match is obtained, The best visual matches are obtained when the solution in the Nessler ~ylinder contains 1 to 2 m1 of standard iron solution. .' 23.5.3 Expl'~sS the result. as mg iron/kg of the sample.

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