3508 Ghee

Free Standard provided by BIS via BSB Edge Private Limited to RISHI FOOD TESTING LAB -
ERODE(rishifoodtestinglab@gmail.com) 49.37.211.194 [for non-commercial use only].

(Reaffirmed 2012)
(Reaffirmed 2018) 18 : 1501-1_
Indian Standard ~
METHODS OF SAMPLING AND TEST FOR ~
GHBB (BUTTERFAT)
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 Food &. 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 Milk Producers' Union
Ltd, Anand
Da J. 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. Dsv AIlIY A ( Altemat« )
DI.ECTOB, MILIT.BY FARMS Directorate of ~filitary Farm" Army Headquarten
AaI8TAST DIRECTOR. !\!ILITABY
F ABII8 ( 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~tl f,.", /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'f te 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
1
5•• M. R. SJIPQV
SB&J JAil•• N. W.......
"'A_ (AllnR1II6 )
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...",.,
DB N. N. Ouru. National Dairy Relearch Institute. Kamal
D. S. C.
M"""",
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 VITAMIN 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 FAT 'IN G••• BY THa Pa....
'!'UYL A.caTA.... Tur •.. 52
22. _ftOK
21 DaftalaNATIOM O. PaUBNca o. SBIAIm OIL (1A1JDOUIIf
TUT) •.• ••. •..
o...... PaaoXIDa V ALUB
23. DnaUIIICAftOM o. IJIOIC CoNT~1ft
••. ••• 5&
56
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, butter-oil), is the most important dairy
product 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 Iodine value. 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 fats and fatty oils. British Standards Institu-
tion.
B.S. 684: 1958 Methods of analysis of oils and fats. British Standards
Institution.
Standard Methods of the Oils and Fats -Dh:ision of the International
Union of Pure and Applied Chemistry. 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 wi th IS: 2.1960-.
-Rula (or roUDdi"-.aumerleal n1ueI ( _ _).
4
1.lCOd
1.1 This standard prelCribes' the methods of "lDpJing, abalysis' anW __
generally used for evaluating the qualitV or ,hee. The specific methoc!l' 'to
be used would depend on the object of the analysis. ·
2. SAMPLING
2.1 Sampling shall be carried 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 of value. 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 of manufacture shall be grouped together to constitute a lot. If
a consignment is declared to consist of different batches of manufacture,
the batches shall be marked separately and the group of containers in each
batch shall constitute separate lots.
2.2.2 The number of containers 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
101 It,' &00
,5
101 and above 10
5
11,'3501'.1.
2.2.3 These' containers shall be selected at random &om the lot. To
ensure the randomness of selection, 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 of containen in
the sample.
2._.~ I£th{t:e is a possibility of wide 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 if organoleptic 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 of the containers by it.
2.4.2 Sampli", Cone",,", T"bls ( Fig. 1..4 )-A con~el1ient sampling instru-
ment consists of two 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 In ill
leJIIlh. The two tubes are provided with V-shaped porta at their lower
6
e
CLOSED
o c3
OPEN
SECTION XX
'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. are c: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 preferably or .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 iDstruments and 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 _ d o a ... 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 of large 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 this operatioD in 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 of moiature.
2.1.2.2 After the determination of moisture 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 cbemJcalt that doDOt coataia impuritlea which
afFect the experimental 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 oven, 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 of the duh with 1 _ after dryinl. aacI
or
W - weight in g the empty dilh.
_ for water, dWUed quality ( _ _ ).
10
J8.,..1IM
4.4 Accaraq ot dI. 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 cell. 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 carbon
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 should be checkecl 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
or making any measurements. After the initial warm-up period, rotate both
the control knobs on top of the 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 transmittaDte or
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.
If the reading at 460 ml& IS above 26-2 t 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 imp ut'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
Photometric colour - 1·29 D_ + 69-7 D... + 41-2 D. - 56-4 D..
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 of incidence 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 of ghee 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 of sodium). 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."""'"-
( reading from W to 50-a ).
fitted with an accurate thermometer
'.2.2 H" W." CitJlIltlli1l, DIlJie, - to maintain the temperature of the
1.2.1 S....
prism CODItQt at'W :t: 1-0.
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 of most 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 read in.. 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 of poeitioD
of border line of total reSection in relation to the facel of a prism of fliat
glan. Bring this border line into field of vision of telescope by rotating
the double prism by means of the 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 of colour. The colC'uts are eliminated by rotating acrew heacl •
orcompensator UDtil sharp, colour~eu line i. obtained. Adjust border JiDe
10 that it falls on point of intenection or cro..~hain. Read refractive IDcIa
of substance direcdy on scale or sector. Check COrrectness or iDstruJDeDt
1·S33 O. Anl COrrectiOD found f=rY
with water at 200C, the theoretical refractive index of water at we
should IDe made OD all reMlap.
Maximum difFerence between d licate cleterminadolll .haD DOt .-..d
0·000 2 unit of the refractive 'ad
iI
11.,..111&
'.4.' orU"lower
.urtace
tU a,,'1"""'.""'"- Place 2 or S of the ....ple
drops
pnsm. Close the prism and acUUlt .. in •• ~.s.
OIl
'.4.7 For conversion of refractive 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 1·453 J 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 below 4000/IY '.11I, where
X Cor butyro-refractometer·.. 0·55
... Abbe refi'actometer .. 0-000 365
X for
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 ((hee repraeatl 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 fatty acid.. although they actually IOliduy over •
range oftemperature.
7.1.1 Gbee is aaponi&ed with glycerol.~talh solution. The resulting
lOap is decompoeed with sUlphuric uid and the liberated water-insoluble
ratty acids are separated, washed free from mineral 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, - of glus, 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 of suitable 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 of a 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 of distdled water.
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 soap is completely diasolved. Add carefully,
With stirring, SO ml of dilute sulphuric acid 101ution. Check for an ez~
of acid by the addition of a few drops of methyl orange 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 of hot 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.
Ti", -
7.f-2 D,lmftilUUioll 0/ 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 per minute, the stirrer mo~
through a vertical distance of about sa IDID- Continue ltirring in tbiI
manner until the temperature baa remaiaed cODstant for 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 glycerides. doe. not have a
lharp melting point but for control purposes it is possible to obtain a u.eful
eomparison of samples by determining the temperature at which a column
offat of fixed 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 1-2 to 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 of moisture. 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. about 11> 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 of the 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-
meter 10 that the lower end of the melting point tube is even with the bottom
of the bulb of the 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
below thq lurface of water. Heat the side tube o{the apparatul gently, so
that the temperature of the water rises slowly at the rate of 2°0 per min\lte
till the temperature reaches 25°0. and thereafter at the rate of 0-5·0 per
II
"".1_
or
minute. Note the temperature 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 of ghee 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 petroleum.
10.1 Appantu - Conical Suk of 25O-ml capacity" provided with
stopper.
10.2 . . . .eat - L i8ht 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 solvent 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. lime 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 Potassium 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 alcohol 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 of the flask. Bring them to boil and while it i, still hot,
titrate with O·J,..N sodium hydroxide, 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 of free 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:
--w
D egree 0 r aCIidi ty - 100 N
where
N ~ the quantity of alkali used, expressed as. ml of I N
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 of these 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 of ml of 0-1 N aCJueous alkali
solution r~uired to neutralize the water.iDsoluble steam "watile ratty 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 of 0·1 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 ,bee the
o
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'" - 50 mi.
12.3.3 The usembly of the appar~tus for the distillation is shown ill
Fi•• 3 and 4 and dctaUs of the 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 of Itill-head and vertical
A rubber stopper, fitted below the bulb of the longer arm of the still-
head, and used for connecting it to the flask shall have its lower surface
10 mm above the centre of the side hole of the still-bead. -
c) CoruiIIu" - The condenser shall be made of glass 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
water jacket
\\fan thickness of widened part 1-25 :f: 0-25 mm
Internal diameter oCwidened part 20 :I: 1 mm
External diameter orinner tube 12:1: &5 IBID
within water jacket
Wall thicbell or inDer tube 1-0 :l: 0-2 mm
or
. Wall thickoea outer jacket 1-25 :I: 0·25 IDDl
laternal cI1aaaeter of water jacket 30 ~2 IDID
II
11,3501·1_
no "'..
FlO. S Pouaua DanLLATIO~ AwAM",.
24
11.,..1.
G"
F~. 4 &ftlL-HaAD FOR POLBNIU DmnLLATION MPAaATUI

d) RIItiDIr - The receiver ih.lI be a 8ask with two graduation !DUb
on the neck, ~De at 100 ml and the other at 110 ml.
e) ~tl.·&",tl-- An ..bestos-board of 120 miD diameter and 6 mID
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 of the flask above
the Iiole. A new ubestos-board may conveniently be pre~
'br bevelling the ed,e of the bole, soaking in water, mouldin, the
edge with a flame, and drying. -
f) Gu B"",,, - The burner should be sufficiently 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 of accurately determined strength ( this solution is needed only
if ltinchner value is to be determined).
12.4.1 Sil"." Sul/JluJ" - powdered.
12.~.1' Fill" Ptl/Jtr- Whatman No.4 (or its tqui\'aJen t ') 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 parts ofthe 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 15 mlnutel. 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 thaD light yellow (iDdicatinC ewer-
heatiD, ), ~t the saponification with a Crah sample of lhee~
12.5.1.3 Adcl two g18ll beacII, followed by 50 ml 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 01 the 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. l l t ; 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" tbc lower (aqueoua) 'layer 1_", bebiacl iaIoIuble.... wIdcIa
.a..
have rileD to the lurface. AcIcl thae to the lIlain b;ik or tbe lDIoIuble
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 ,II Q.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 of the condenser. the cyliDder1Uad the liter.
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
III! 01
&1trate coatUDI...... the lOIable voIt:tUe acid~ into.ati.tieD ~ add 0-1
pbeDolpbtha1ein ·I~tor. aad titrate With the '-1um htdroxicle IOlu_
"'dee'''' be ~ Ior'dar: dtratioli If~e It

h"""
_ til the l~uicJ becoma plak. r1.... the 10G-ml1luk with the'aearlf aeutra-
."·liquid toWuda the Cad or the tltra~~N IGCIiUlil
..,.11 DOt required).
du-
~7
11.. '.·1_
12.5.2.1 If the KinchDer value is tp be obtained. the titratioa 1aIk'
.hall be dried before Ulej Dote the actual volume of barium 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 of phenol-
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 of the 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 of pumice 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
T1 . . volume in ml or 0'1 N barium ~r 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~ pressures, such
28
11.,.·1-
at may occur •• high altitudes. The rollo\ving (actors may he applied to
values determined at a barometric pressure to convert them to the values
determined at normal pressure.
· h t 1
Correc t R ere er va ue ..
(Observed value - .10 ) 101 760
Jog P + , 10
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~terminations shall not exceed 0·5 units.
13. DETBRMINATIOlO O. SAPONIFICATION VALUE

13.1 The saponification value denotes the weight of potassium hydroxide,
expressed as milligrams, required to saponify completely ore gram of ,hee.
Saponification value is useful in detecting the presence of mineral 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 of ghee
and from it can be calculated the saponification equivalent, which is the
amount of ghee saponified by one gram equivalent of potassium hydroxide,
and is equal to 56 100 divided by the saponification value.
13.2 A~... -Flat·bottom flask, 200 or 250 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 be colour_

or very pale yellow_Keep in • dark place.
NOT. - Alcoholic solution orpota.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 tor several 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 accurately standardized.
13.t Procecltare - Weigh accurately 2-0 ± 0·001 g of ghee into a 8ask.
Add 25 ml, accurately measured, of the alcoholic potassium hydroxide
solution. Add 1 or 2 glass beads and boil cont;nuously undor a re8ux
condenser for balf an 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,
T 1 == volume in ml of 0·5 N add required for the I • •e. and
W == weight in g of the sample taken.
U.5.2 Saponification Number - sapo au: ~c:

a I
nvaue
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'·.
ct..".
I~.s.a- 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 of water.
·u.,..- s,.,,, s.ltUiD,. - Triturate 5 I of ltarch 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 of sodium thiOluJphate crystals ( 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.~tiODof lOdium tbiosulpbate, pipetto 25 ml of
. tWs IOlutioD into • clean 1.,toP~red 250-m1 conical lask or bottle.
Add 5 ml of CODCeDtrated 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
, ~ volume in 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
.•,...""'
t...
1rw.......
a. . . .
bWaroaaate. techDicaI.cl ....IJllcai rapDt.
~ atdd (.".,.).
Sl
the .cet~c acid and 'tat it for reducing impurities as Collowa:

a) D,lmftin.t;oll oj""lt;", /IOi".' - Take a 15 em 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 until the .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,sl for r,duM, im/Jliri'ils (po'lUn,,'" /JtrrIUIIIlG""" IIs1 ) -Dilute
2 ml of the acetic acid 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 it the
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, if necessary, and determine the .~gth by titration. with
standard sodium thiosulphate 101ution. Set aside 50 to 100 ml
of the 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 of iodine 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) orthe lUIle lOClium thiOlwpbate
IOlution.
b) Dissolve 8 g of iodine trichloride in approximatel, 4SOml·of·aceti.c
acid. Dissolve separately 9 g of iodine .in 450 ml of acetic acid
using heat, if·necessary. Add gradual1~ iodine 101ution to the
iodine trichloride until the colour Iuu c 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 mJ of
standard thiOlulphate solution when the halogen content it esti-
mated by titration in the p~ce of an excess ofpotaaium iodide
and water•. Heat the solution to 10000 for 20 DlIDutea, 8Dd cool.
Prevent accea of water 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 of the 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 of a 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
of distilled 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 of tie titratiOD
and ·shake the conten"- vjgorously. Carry out a blank tat, usm,. the IUDe
quantities of the reaptl.
14.5_
12-69 ( B ~S)N
Iodine va1 ue- 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 of the ItaDdaftl lOClium thioIulphate IOllItioD,
aDd
,W - weiIfIt in I of ~e material takeD tbr the telL
14.1 A_err 01. 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
or
matter ghee are .~roJa, vitamin A. 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.mI cylindrical.
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 not darker 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 several hOun,
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.
eooo DOt exceecllDs &001 perceot.
fton-voIaI1le at
15.s.s ."'-raidue Don-_tiIe at eooo Dot aceediDc 0-01.
15.s.~
15.1.5 H~_ -
P."". . H ~ - approximately &5 N aqueous IOlutieD.
So-. 0·1 N 101ution in ethanol. DiIIOlve 5 to 6 I or
lOCIium hydroxide pellett in 1 ml of water aad mix. the .101ati~ with
10
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
ethanol.
P"""',,,,,...,.....-
1-0 percent lO_doD ia 95 perceIlt ( _/_ )
15.4 _ - Weigh accurately 5 s«

the ~ &0 wltbin ±O-Ol,
ia • 250-ml_lat-bot~m last, add -50 mI of alcobo1iC potuliam h ~
101udon and two p. beadI. Attach the Iuk 'to . . . . . . . . . . . . . .
Sf
JI.".~
~t GIl • bo~wa. .bath tor hour, swlrliD! at freqUeDt iDterVaIt·· to
ODe
e-.e complete .-poDiIication. .
15.4.1 Remove the contents 01 the 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. Draw off 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' -
~_ .leparating f~nnel c~taining 20 ml of water. Extract the &C\1IeOUi I
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 more with 20 ml of water. Continue wuhing with water until tile
wash water DO longer turns pink on addition of pheaolphtbaleiD 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 of a gentle current of air, the flaIk
being almOlt entirel)' immened, held obliquely aDd rotated in a boiling-
water-bath. Dry the flask aDd coatents to constant weilht at a tempera-
ture hot exceeding so-c. -.
15.4.5 Diaolye tho contents in 10 mJ 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 If there 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
of manipulative error reject the test and repeat the determinauOD from the
beginning.
15.sCal~
. UDlapon~able matter J percent by weight - I ~!!. .
where
WI - weight in g or the residue, and
W - weight in g of the sample taken.
35
1I._~UIt·
II. DB1'BUmfATION CJP VITAMIN A

1'.' The determiDaaon or vitaDiiD A ,·may .be carried oat ~ther by
1peCtrO~ Diethod o~ ~Price methocL· ID eMe of dlapute.
'*
the tpeetrophotometric method.' . 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 'A oC 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
1~1.2.s P.,.,..
not areater thaD 0-05 at 300.mf&. .
/gtIto_ _ liM - 50 percent ( _/.).
a- 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 acidi8ecl Copperlulphate IOludon for one miDu~
aacI.ublequeatly waahed with wa~.
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) red iDdicator 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

"'ce
,
ltaDd.ret eap.uJ.a O·S'to 1-0 I aad traasrer thea to tbe .~
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 100 m1
of. . . ~ ~ etker layer. without.ptatioD. When good ieparatioD
baa takea plaCe, After two miDutel, remove. tile aqueous layer. ShUt
~usly with ~ to 5-aaI ~ otwater, alI!"f to '~z:ate, remove &nd
~ the aqueous layer. If • ~hat reaaatant:emWsion fOnDI, dilute
with 100 mI of...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 100 qaI of water through the ether
layer and see that· the Inat water wuh is rlot alkaline to phenolphthalein.
B~rate the ether eztract on water-bath to about 50 mi. Add 5 to 10 lof
lOCIium .uJ~te, ~J and allow to lettIe. Decant into a 5O-ml graduated
IMk, riDie the ium .ulpbate with several additional portions of ether,
lOcI,'
- 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"""'''' " . . _Ow.. "'"- .
Evaporate a suitable
aliquot or the ether dation of the 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 chloroform to give absorbance valuei of
_. eo, 40, and 20 perceat or. the original" abJor~ce. Determine abaor-
baceI or the blue Colour formed when one-millilitre aliqUot ofeaclaottbese
lie IOlutioDa plUl ODe mIlIUitre of chlolOform 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 wlume oCtile 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 wiali e.da let of IalDpJe readJDp to eatabti.h the
I curve.. ID the
ca.e
latta- n eeablilh the calibntion" curve wbeDever variatioD lD the
..-pDt or otbel'Yariab~ ill 'proced~ occurs. , .
11.1.1.1 ~_- Weith ~ y • guantity 01 the 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~. ~ve the,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 and the fixed volume or the aotim.ony
I
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 100 g 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.
If cells 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 shall show
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 of chloroform. Add 5 g of calciulh 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 with 40 ml of ethyl 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 of ether 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 of water, allow to
separate, remove and discard die aqueous layer. If a 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 of vitamin 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 of ether 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 vitamin E CO.isb. of the. •~~ beta. gamma, aacI
delta-tocopherols. 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 cm l 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 112 g
of potassium 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:Joiling water-bath for one hour With
hydrochloric acid. Repeat with fresh· portions of acid at room temperature:
Wash with water until free of acid, 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 of methyl alcohol, &ltd 10 m1 of water, 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 of 0·2 percent solution of feme chloride in abso-
lute ethyl alcohol, prepared fush from ferric chloride hydrate, and mix. Add
I ml of a 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 tocopherol 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 of liquid ghee in a test-tube
and add an equal.quantity of hot 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 of added 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 0-816 0 ).
11.2.3 hoe,",- Prepare the test solution by adding ~5 ml
bromophenol blue indicator to each 100 ml of the aCJ,ueous acetone just
or the
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 of water, 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 yellow cOlour 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 ANTIOXIDANTS

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 ( DIll).
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 of normal sodium hydroxide
solution: .
a) BH~ - The development of a red-purple colour indicates the
presence: of BHA and shows an absorption maximum at 535 mil_
b) BHT- Presence o! BHT giva a distinct salmon-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 yellow colour. which facla rapidly Ul~ IIVCI DO
42
measurable maximum in the visible range. Other gallates behave

p~ly. '
d) NDGA - NDGA yields a wine 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 hydroxy toluene, 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 light petroleum, propyl gallate is extracted with water and the hl~her
gallate. with absolute methanol.: With the extraction methods described
95 to 97 percent recovery of antioxidant was possible.
19.2.2 AHa,Glru
. . 19.2.2.1 Dis,illaliM GJI/Hl,alflS for BRA and BHT- The distillation
apparatus consists of a Iteam generator consisting of a 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 wax (mp60°C, smoke point 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 of bath 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
(FeCl a• 6H.O) in distilled water. .
19.2.3.10 2,2'·BiJ!1ridi",- Dissolve 200 mg of2,2'-bipyridine in 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 of warm, purified ethyl
alcohol and add approximately 250 ml of cold 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~.
supernatant in a refrigerator where a second crop of. erys".

dine should be white flakes with a faint sweet odour, Retain the
obtained upon evaporation of the water. Purify ~;thae crystall 81
is
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 of distilled water: This reagent' should be freshly
prepared (or each series of determinations.
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.
11.2.4Proe"
1'.2.4.1 AMl.1sil of/In/J.1I,tIllal, tuUl IItWtliJt.1tlro".w,• •Ul- Weight
.20 g of the 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 the tat.
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 of water, 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! of the
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 of ferrous 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 of 50 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,l galltlt, - Pipette' three different aliquots of the 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 of IJulylautl "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 of the ghee sample in the flask. Liibtly grease the ground.
glass joint 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 of distillation should be
such that 125 ml of distillate collects in 30 ± 5 minutes.
When 125 ml of distillate 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«
or butylated 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 remcchloride
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~ of the sum of butylated .
hydrosyaniaole and butylatecl hyclrozytoluene. Prepare a
reference curve for butylated hyclrosytol~eDeover a raage or
10 to 50 N with each set or analyaia. 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
over a range of 10 to 50 1&1. Under the above conditioal.
k-value or 0·011 4 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;{
of
hydroxy toluene. Calculate the amount butylated hydroxy-
toluene in' the distillate.
Examl":
Weight of fat 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 orBRA 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
0·0122
X 250 489
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 01 propyl pnate ad NDGA, or • MM.
1'.2.4.3 D"..;"'Ii." of"'.1.11) oel7l ad tl0U9l g"U.lIs
'a) &IrtIt" - Dissolve 50 gl ofghee in light petroleum. Extract the
solution 5 times successively with 2O-mJ portions of distilled 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 of the glycerides may occur owing to cooling. If so,
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;"..';,,. of pr'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 of water 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 of a 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 of standard 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 of sodium 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 of equal parts of isoamyl 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 of ueamyl 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 of the gallic
acid ester in- 100 ml of methanol 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 of a 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, if the
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 of gallate 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 of ghee 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. - If other 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 vegetable rats in ghee. The sensitivity depends upon the
character of the vegetable rat 'used for admixture. The sterol content is
determined gravimetrically after I&ponificat~on of the fat and precipitation
of the sterols 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 of distilled 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"" $"101Co"",,' - Weigh accurately to the
nearest 10 mg about 15 g of the ghee in a conical flask of 250 ml capaoity.
Add 10 ml of potassium 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 of diethyl 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
800 e. 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 of ethanol (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

~wder.~ &11. melting-point tube• • ,height mahoutSmm.
to'" .6IieIL.~v,:w
. 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 of 0-5 degree
per minute. Take the ~ on the tbermometeratlhe'momenttbat.tbe
lut crystal srain hu just disappeared, ., the ~tms point.
20.1.3 Minti. &_iutitnt oj 1M 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 of dietbyl 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"'''.sterol
'
Total ,.
content, pereent _ 0-25 X 6 X 100
where
4 • weipt in g of 1_ sample, and
II -" weipt in I of the sterol digitonide.
20.4.2 If the melting ~int 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 of the 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 If under the mttroscope the sterol crystala oaly show the form of
a pa~lIelOFam with an obtuse angle of 100·, which is c~racteri.tic for
cholesterol. 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 _ t I C fOt
phytosterol., .!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 veptabIe lat.
~
CHOLESTEROL
108-
=>
PHYTOSTEROl
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 of recrystalliaasions, and a description of the microscopic ilDAle of
the sterol crystals.
21. DETERMINATION OF PRESENCE 0. SESAME OIL

(BAUDOUIN TEST)
21.0 The development of a permanent pink colour with furfurallQlution
in the presence of hydrochloric acid indicates the presence of sesame 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~lopment of 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. sesame oil is present. If the 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
of sample, or as milllequivalents of peroxide oxygen 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 off at the other•
• Specification ror 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 of analytical grade.
22.1.2.1 Sol""" mix"", - a mixture of 2 volumes of glacial acetic acid
and 1 volume of chloroform.
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 of the 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 of 5. percent aqueous solution of potas-
sium iodide and wash out the test-tube twice with 25 to 30 ml of distilled
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
where
Peroxide value _ 8 000 A:
.If == volume of sodium
thiosulphate solution required for the
sample,
l
N exact normality of the 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~d in milli~valenti of oxygen 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
eo OPEN TUBE
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 of ghee 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
cream.
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
orlevel 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 of Re••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 of both 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 occurr ing 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 of
0ml
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.
61
BUREAU OF INDIAN STANDARDS

Headquarters,·
Manak Shaven, 9 8ahadur Shah Zatar Marg, NEW DELHI 110002
Telephones: 331 01 31, 331 13 75 Telegrams: Manaksansthe
( Common to all offices)
Reg/onal Offices: ' Telephones
Central : Manak Shavan, 9 Behadur Shah Zafar Marg, 331 01 31
[ 3311376
NEW ,DELHI·11 0002
*e8stern : 1/14 C.I.T. Scheme VII M, V. I. P. Road, . ~6 2499
Manlktola, CALCUTTA 700054 '
Northern : SCQ 445-446, Sector 35-C, 2 18 43
[ 31641
CHANDIGARH 160036
41 24 42
Southern: C. I. T. Campus, MADRAS 600113 { 41 2519
41
2916
fWestern : Manakelaya, E9 MIDC, Marol, Andheri (East), 6329295
BOMBAY 400093
Branch Offices:
'Pushpak' Nurmohemed Shaikh Marg, Khanpur, 2 63 48
AHMEDABAD 380001 . [ 26349
*Peenya Industrial Area, 1st Stage, Bangators Tumkur Road 38 49 65
BANGALORE 660058 [ 384966
Gangotri Complex, 5th Floor, Bhadbhada Road, T. T. Nagar, 66716
BHOPAL 462003
Plot No. 82/83, Lewis Road, BHUBANESH'WAR 751002 63627
63/5, Ward No. 29, R. G. Berua Boed. . 6th Byelane, . 331 77
GUWAHATI781003 .' '.,
6-8-56C L. N. Gupta Marg (Nampally Station Road ), 231083
HYDERABAD 500001 , .
R14 Yudhister Marg, C Scheme, JAI PU~ J()2005 6 3471
[ 69832
• lI', .
21 68 7~
117/418 B Sarvodaya Nagar, KANP(jR'208005 [ 21 8292
Patliputra Industrial Estate, PAT.NA 800013 62305
T.C. No. 14/1421, University P.'O:, .vam 6 21 04
TRIVANDRUM 695035
[ 62117
Inspection Office (With Sale Point) :
Pushpanjali, 1 st Floor, 205·A West High Court Road, 261 71
Shankar Nagar Square, NAGPUR 440010
Institution of Engineers ( India) Building, 1332 Shivaji Nagar, 62436
PUNE 411005 .
------,-- .
·Sa188 Office In Calcutta is at 5 Cnowrlnghee Approaeh~' P~O. Prtncep
."
278809
Street, Calcutta 700012
tSales Office In Bombay Is at Novelty Chambers, Grant' Road, sa 85 28
Bombay 400007 ~, .
:tSalea Office In Bangalore I. at Unity -Building, NaraslmharaJa Square 22 3e 71
Sangalore 560002 . .
~Pr~ln-t.-:-d-.t-=8~1m-c-o"'='Pr-:-ln~tl-nCl...l.::P~r.-••- .~O-.lh-:--J~"l-l\d~l.

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Free Standard provided by BIS via BSB Edge Private Limited to RISHI FOOD TESTING LAB -

ERODE(rishifoodtestinglab@gmail.com) 49.37.211.194 [for non-commercial use only].

Fourth Reprint AUGUST 1990

UDC 637-25: 543"05

BURIAU or INDIAN STANDARDS

BU_AU OF INDI'AN" STANDARDS

Methods of Test for Dairy Products Subcommittee, AFDC 12.: 4

Da A-VII Ita.- S. . Gun.

16. naTBUlilfATlOK OP VITAMIN A 36

TABLE I NUMBER OF CONTAINERI TO BE S~LECTED FOa

101 and above 10

Starting f(om any container, count them u 1,2, 3, ...up to,anCi

ends, 10 placed that ghee contained in the instrument ean be drained

Elo. 1B SAIIPUHO PLAIN' TUBE

f. DETERMINATION OF MOISTURE CONTENT

I. DBTBRMlNATION OF REFRACTIVE. INDEX

'.2.2 H" W." CitJlIltlli1l, DIlJie, - to maintain the temperature of the

'.4.7 For conversion of refractive index vallie. into butyro-rJractometer

TABLE 2 BVTYRo-aBFRAarOMETEll RBADINGI AND INDIca OF

7. DBTBRMINAnON 01' TITIlB

CORK EQUALLY SPACED

TITRE TEST- TUIE

Flo. 2 TITD All8lDLy

7.2.2 Witle-MOII'1a Bo,tl, - of glus, conforming to the following details:

I. DETERMINATION or MELTING POINT (SLIP POINT) \

t. ICOU CARD POll GIIBB

1.1411 Gbee scoring 91 and above shall be graded as excellent, between-

II. D_ATION OF INSOLVIILB IMPURITIEI

II. DETERMINATION OF ACIDITY

11.5 Free • . . , ..... - The acicUIJr or Pee i. frequently a~ ..

12. DB~~ATION OF SOLUBLE AND INSOLUBLE

precise conditiona lpecifiecl ill the method.

b) S,ill-Mad - The still-head shall be made of glass tubing of wall

FlO. S Pouaua DanLLATIO~ AwAM",.

F~. 4 &ftlL-HaAD FOR POLBNIU DmnLLATION MPAaATUI

_uble fattyacicll , . . throulll the liter, receiyetbe Iltrate iD......._ ftaniiI, . .

"'dee'''' be ~ Ior'dar: dtratioli If~e It

12.7 Accarae, 01 the Metlaocl

12.7.3 Kirs,h"" YIlI". - The maximum deviations between duplicate

13. DETBRMINATIOlO O. SAPONIFICATION VALUE

abouId be approximate. but not 1eu thaD 0-5 N. It should be colour_

U.5.2 Saponification Number - sapo au: ~c:

the .cet~c acid and 'tat it for reducing impurities as Collowa:

14.3.10 lodiM Mo"oMloritll- 98 percent, chemically pure.

1 000 ml or 95 percent ('/') ethanol. Allow the lOlutioa to .tUMt-~

15.4 _ - Weigh accurately 5 s«

II. DB1'BUmfATION CJP VITAMIN A

· 18.1.r.l Plio,."",. "tori,.", --- instrument with a clireet readiDg

a- 11.1.2.4 BIIw - ~xide free, rediltilled or of auathetic ,racle

11.1.1.1 ~ - Weigh. lICCUrateJy hili the vitamiD A

trichloride as blank. Place the tube containing 2 m1 of the chlorofOrm

absorbent earth is coloured a -greenish blue by 'carotenoidl and dark

11. DETECTION AND ~ETBRMINATION OF DISSOLVED SOAPr

19. DETERMINATION OF ANTIOXIDANTS

measurable maximum in the visible range. Other gallates behave

supernatant in a refrigerator where a second crop of. erys".

the solution. at 530 ml£ relative to that 0 water, using an

estract alone by 20 m1 of the methanol extract in the aame