IS 9967-3: Milk and milk products - Determinaion of nitrate and nitrite contents, Part 3: Method using cadmium reduction and flow injection analysis with in-line dialysis

2008·5.4 MB·English

by  Bureau of Indian Standards

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IS 9967-3 (2008): Milk and milk products - Determinaion of
nitrate and nitrite contents, Part 3: Method using cadmium
reduction and flow injection analysis with in-line dialysis
[FAD 19: Dairy Products and Equipment]
“!ान $ एक न’ भारत का +नम-ण”
Satyanarayan Gangaram Pitroda
““IInnvveenntt aa NNeeww IInnddiiaa UUssiinngg KKnnoowwlleeddggee””
“!ान एक ऐसा खजाना > जो कभी च0राया नहB जा सकता हहहहै””ै”
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“Knowledge is such a treasure which cannot be stolen”

IS 9967 (Part 3) : 2008
ISO 14173-3 : 2004

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Indian Standard
MILK AND MILK PRODUCTS
DETERMINATION OF NITRATE AND
NITRITE CONTENTS
PART 3 METHOD USING CADMIUM REDUCTION AND FLOW INJECTION
ANALYSIS WITH IN-LINE DIALYSIS (ROUTINE METHOD)
( Second Revision )
ICS 67.100.01
© 81S 2008
BUREAU OF INDIAN STANDARDS
MANAK SHAVAN. 9 SAHADUR SHAH ZAFAR MARG
NEW D ELHI 110002
July 2008 Price Group 5
Dairy Products and Equipment Sectional Committee, FAD 19
NATIONAL FOREWORD
This Indian Standard (Part 3) (Second Revision) which is identical with ISO 14673-3 :2004 'Milk and
milkproducts- Determinationofnitrateandnitritecontents- Part3:Methodusingcadmium reduction
andflowinjectionanalysiswithin-linedialysis(Routinemethod)'issuedbytheInternationalOrganization
forStandardization(ISO)wasadoptedbytheBureau ofIndianStandards ontherecommendation ofthe
Dairy Products andEquipment Sectional Committee andapproval ofthe Foodand Agriculture Division
Council.
This standard was first published in 1981 based on the earlier version of International Standard,
ISO4099:1978andsubsequently revisedin 1997toalignwith therevised ISO4099 :1984underdual
numbering and its scope covered determination of nitrate and nitrite contents in cheese using
cadmium reduction and spectrometry. Subsequently ISO 4099 was withdrawn and revised into three
parts of ISO 14673to include in its scope a variety of milk and milk products and also including two
more methods fordetermination of nitrateand nitrite contents.
Subsequently to align with ISO 14673 on the subject, IS 9967 is now being revised into three parts.
Otherparts are asfollows:
Part 1 Method usingcadmium reduction and spectrometry
Part2 Method usingsegmentedflowanalysis (routine method)
The text of ISO Standard has been approved as suitable for publication as an Indian Standard
without deviations.Certain conventions are, however,not identical to those used in Indian Standards.
Attention is particularly drawntothe following:
a) Whereverthe words'InternationalStandard' appear referring to this standard, they should
bereadas'IndianStandard'.
b) Comma (,) has been used as a decimal marker while in Indian Standards. the current
practice isto useapoint (.)asthedecimalmarker.
Inthisadoptedstandard.referenceappearstocertain InternationalStandardsforwhichIndianStandards
alsoexist,Thecorresponding IndianStandardswhicharetobesubstitutedintheirrespectiveplacesare
givenbelow alongwiththeirdegree ofequivalenceforthe editions indicated:
International Slandard Corresponding Indian Standard Degree of
Equivalence
ISO 1042 : 1998 Laboratory IS915:2006 One-mark volumetric flasks Technically
glassware One-mark - Specification (secondrevision) Equivalent
volumetric flasks
ISO14673-1:2004Milkandmilk IS 9967 (Part 1) : 2008 Milk and milk Identical
products - Determination of products - Determination of nitrate and
nitrate and nitrite contents nitrite contents: Part 1 Method using
Part 1 : Method using cadmium cadmium reduction and spectrometry :
reductionand spectrometry
In reporting the result of a test or analysis made in accordance with this standard, if the final value,
observed or calculated.is tobe roundedoff,it shall be done inaccordance with IS2 : 1960'Rules for
roundingoff numericalvalues(revised)'.
IS 9967 (Part3) : 2008
ISO 14673-3: 2004
Indian Standard
MILK AND MILK PRODUCTS
DETERMINATION OF NITRATE AND
NITRITE CONTENTS
PART 3 METHOD USING CADMIUM REDUCTION AND FLOW INJECTION
ANALYSIS WITH IN-LINE DIALYSIS (ROUTINE METHOD)
( Second Revision )
WARNING- The use ofthisInternationalStandard may Involvehazardous materials, opeqtions and
equipment This standarddoes notpurportto addressanthe safetyproblemsa.soclatedwithIts use.
It is the responsibility of the use, of this standard to estabilsh safety and health practices and
detenninethe applicabilityofregulatorylimitationspriortouse.
1 Scope
This part of ISO 14673110F 189 specifies a routine method for the determmauon of the nitrate and nttnte
contents of milk and milk products by cadmium reduction and flowinjectIOnanalysis (FIA) The method 1$
applicableto hard,semi-hardand soft cheesesofvarious ages.and processedcheese The cetecnonlimItsof
the methodare 0,5mg of nitrateionsperkilogram and 1,0mg ofnitriteionsperkilogram
Themethodis also applicabletowheypowder,milkpowderandmilk-basedInfantfood.
NOTE 1 The method closely resemblesthe FIAmethod deIGtibedinreference (2)forthedeterminatIonofnitrate aI'ld
nitrite inmilk andftuid dairy products.Adaptations_remedII toallow for the analySISof<:Melt! and toobla/Osuff>ot!nl
..nsilivityforthedeterminationoflowlevelsofnitnle /0 chee..andm,lk-based Infanl foods
NOTE2 For determinationofnitrillll and nitratefollowingcadmium reduction,use 'Imade ofltlelameCOlour~Kt1on
asdeIcriblIdinISO14873-1!IOF189-1.
2 Nonnativereferences
The following referenced documents are indispensable for the application of this document For dated
references, only the edition cited applies. For undated references. the latest edition of the referenced
document(inclUdinganyamendments)applies
ISO 1042, Laboratoryglassware- One-marl<volumetricflasks
ISO 14673-11IDF189-1, frfiIk and milk products - Determination of nitrate and nitrite contents - Part 1
Methodusingcadmiumreductionandspectrometry
3 renns and definitions
Forthe purposesofthis document,thefollowingtermsand definitionsapply
3.1
nitratecom.nt
massfraction ofnitratedetenninedbythe procedurespecifiedin thispartof ISO 14673!IOF 189
NOTE Thenitratecontentisexpressedasthemass tilmilhgramsofnttrateIOns(NO)-)perkIlogramofproduct

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IS 9967 (Part3): 2008
ISO 14673-3: 2004
3.2
nitritecontent
massfractionofnitntedeterminedbytheprocedurespecifiedinthispartofISO1467311DF 189
NOTE ThenIlnlecontentISexpressedasthe massInmilligramsofnitriteIons(N02-)perkilogramofproduct
4 Principle
4.1 A test portionis suspended in awarmextraction buffer solution Fat is separated by centrifuging and
rapidcooling.Analysesaremadeofsmallportionsofthede-fattedsolutionbyflowinjection analysis(FIA). In
hnedialYSIS IS used to remove protein and remaining fat. The nitrate ions are reduced to nitrite ions by
cadmium ThenitriteIOnsarereactedwithsl·'fanilamide andN-1-naphthyl ethylenediaminedihydroehloride to
give a red-coloured azo dye. The colour is measured in a flow cell at maximum absorption of the dye at
540nmwithreferencetotheabsorptionmeasuredat620nm.
4.2 The nitrite and nitrate contents of the test sample are calculated with reference to the measured
absorbancesforaseriesofstandardsolutionsofnitriteandnitrate,respectively. Ifthenitrite contentexceeds
0,5mgJ1<g. orexceeds10%ofthenitratecontent,correctionofthenitratecontent ismade bysubtracting the
nitntecontentfromtheobtainednitrateresults
5 Reagents
Useonlyreagentsofrecoqnized analyticalgradeunlessotherwisespecified.
5.1 Water. distilledordeiomzed,orwaterofequivalentpurity.freefromnitrateandnitriteions.
5.2 Cadmiumreductioncolumn, forexampleAquatec-Tecator"!
5.3 Extractionbuffersolutionorcarriersolution(C2)
DISsolve 26.69 of ammomum chloride (NH in 800ml water in a 1000ml conical flask By adding
4CI)
concentratedammoma.adjustthepHto8,5 Diluteto 1000mlwithwaterandmix.
5.4 Hydrochloricacid(HCI),(P20 ~ 1.19glml)
5.5 R.agentsolution(R1)
DISSOlve 5,09sulfanilamide (NH2CsH"S02NH2)In a mixture of 300ml water and 26ml of hydrochloric acid
(54)Ina500mlvolumetncflask(63) DilutetothemarkWithwaterandmix .
5.6 Reagentsolution(R2)
DISSolve 0.5g of N-1-naphthyl ethylenediaminedihydrochlonde (C10H7NHCH2CH2NH22HCI) in water in a
500mlvolumetncflask(63) Dilutetothemarl<WIthwaterandmix
Thesolutionmaybestoredforupto1weekInawell-stoppered brownbottleinarefrigerator.
5.7 Regenerationsolution.c(HCI)~0.1molll
Dilute80mlofhydrochloricsed (54)Withwaterto 1litreandmix.
1} Aquatec-Tecator.s an example of a SUitable product avatlable comrnercialty Ttus informahon is given for the
convenience ofusers ofltllSpartofISO14673i10F189anddoesnotconstitute anendorsementbyISOorIOFofthIS
product

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IS 99&7(Part 3) :2008
ISO 14673-3: 2004
5.1 Sodiumnlnlltstocksolution,c(N0 -).. 1000 mgJI
3
Beforeuse, dry sodium nitrate (NaNOy to constantmass at 110 ·C to 12O·C DISSolve137.1mg ofthe dry
NaN03inwaterina 100mlvolumetricflask(63).DIlutetothe mll/1(with waterand mix.
5.9 SodiumnlnlltC11l1mtionsolutions.
Preparethe sodium nitrate calibration solutions onthe day ofuse.Pipette amounts of 25~, SOIJO. 100~.
1SO~g and 250 IJ9 of sodium nitratestocksolution (5.8)into separate SOmlvoIumetnc flash (63) DtIuteto
the mark with extraction buffer solution (5.3) to obtain sodium nitrate calibration solutions corresponding to
O,SOmgJI,1,00mgJI,2,00mgJI,3,00 mglland5,00 mgllnitrateions respectiYely.
5.10 Sodiumnitritestocksolution,c(N02-)=1000 mgll.
Beforeuse, drythe sodium nitrite (NaN02)to constant mass at 110·C to 120·C.Prepare the sodium nltrrte
stock solutioh on the day of use.Dissolve 1SO,Omg of the dry NaN02in water in a 100ml volumetric ftask
(6.3).Dilutetothe markwith waterandmix.
5.11 Sodiumnltrilltworkingsolution,c(N02-)=SO,Omgll.
Pipette 5,00ml ofthe sodium nitritestock solution(5.10)intoa100mlllOlumetncftask Dilute tothe ma"WIth
water and mix.
5.12 SodiumnitriteC11l1brationsolutions.
.ao
Shortly before use, pipette amounts of 25~I, SO~I, 100~l, 200 ~I and ~I of the sodium nltnte W()(1(lng
solution (5.11) into separate 50ml lIOIumetric flasks (6.3). Dilute to the ma,. with extraction buffersolutIOn
(5.3) to obtain sodium nitrite calibration solutions c:orresponding to 0,025 mgll, O.OSOmgll, 0.100mgll,
0.200 mgJIand0,400 mgJInitriteionsrespectively.Mixwell.
=
5.13 SodiumnitriterefeNncasolution,dN02-) 1,<48mgJI.
Pipette 1480 ~I of the sodium nitriteW()(1(lng solution (5.11) into a SOml volumetric fIuk (63).Dilute to the
mark with extraction buffersolution (5.3) and mix.Preparethe sodium nitrite reference solutIOnshortlybefore
use.
6 Apparatus
Clean allglasswarethoroughlyand rinse withdistilledwaterto ensurethat Itisfreefromnitrateandnitnte tOnS.
Usuallaboratoryequipmentand, in particular,the following.
6.1 AnalytiCliIbalance,capableofweighingtothe nearest 1mg,with areadabilityof0,1mg
6.2 Samplecontainer,providedwith anairtight lid.
'.3 Volurnatrtc finks,of nominalcapacity SOml, 100ml and 500 ml,complying withthe requirements of
ISO 1042, dassB.
6.4 Pipettes, capable of delivering 25 ~I, SO~. 100~I. 1SO~I, 200 IJI.2SO~1. .aoIJI and 1480IJ/ (semI
automaticpipettes).
'.5 Grinding device, appropriateforgrinding the test sample,if necessary To avoid loss ofmOIsture.the
deviceshouldnotproduceundueheat. AhammermiRshall not be used

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IS 9967(Par1 3) : 2008
150·14673-3: 2004
6.6 Laboratorymixerorhomogenizer(e.g an UItra-turrax2) .wIthglasscontainers of capacity250 ml or
400mi.suitablefor suspendingtest pornonsofcheese.
6.7 Tablecentrifuge. with centrifugetubes ofcapacity50ml, capableofcentrifuging at 1500f!
6.8 Flow injection analyser (FIA) (e g a Tecatcr-").with a Fiastaranalyser,controller. dialysis module,
sampler. provrdeo with sample cups of 4ml, with specific instrument control and evaluation software,
rlItote/nltratemamfold (see Figure 1).asampleloopvolumeof 200 ~1. two mixingcoils of length60em and of
dtameter0,7mm.connectingtubingsand pump tubingsprovidingthe flow ratesgiveninTable 1.
Other flow Injection analysers may use other procedures. The instructions of the manufacturer. therefore.
should be strictly followed
Table 1- Flowrates
ValuesInmilitlitresperminute
Solution Flowratefornitrate Flowratefor nitrite
Sampie.wash 2 2
CarnerC1I,waten(5 1) 2 1.2
CarnerC2(53) 2 1,2
ReagentRl (551 0.6 0.4
ReagentR2(56) 0.6 0,4
6.9 Matrixspectrophotometer(eg SKAlAR21).equippedwith interference filter 540nm and interference
filler 620nrn.aflaw cell ofpath length 5emandof volume40 ~I
6.10 FIA programparametersfornitrite
6.10.1 Programparameters
Evaluation peak height
Cahbranon equation linear
Numberofstandards 5
FIA·patternconecnon lime/cycletime 120s
6.10,2 Timingparameters
INlndowforpeak maximum 30 sto50s
Signal fIltenng maximum 2s
Wmdow forbaseline detection oto 10s
Inlectlonpenod oto60 s
Sampleloop wash 60 sto80 s
Sample loop fllhng 80 sto 120s
2) These Dro<lucts are e.amples or a SUitable products available commercially Tt-IIS mforrnatmn IS given for the
convenoenceofusers ortruspartorISO14673'IDF 189 anddoesnotconstitute anendorsementbyISOorIOFofthese
prooucts

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IS 9967 (Pert 3): 2008
ISO 14673-3: 2004
6.10.3 Calibrationcheck
Tolerance ±5%
Repeatafter(samples) 10samples
Standard used 0.100mgll00 -
2
6.11 FIAprogramparametersfornitrate
6.11.1 Programparameters
Evaluation peakheight
Calibrationequation linear
Number ofstandards 5
FIA-pallemcollectiontime/cycle time 60s
6.11.2 Timingparameters
Windowforpeakmaximum 25stooWs
Signalfiltering time 1s
Windowforbaseline detection oto15s
Injection period oto20s
Sampleloopwash 20 sto35s
Sampleloopfilling 35sto60s
6.11.3 Calibrationcheck
Tolerance ±5%
Repeatafter(samples) 10samples
Standardused 2.00mgll N03-
7 Sampling
Sampling is not part of the method specified in this part of ISO 1467311DF 189 A recommended sampling
methodisgiveninISO 707.
Itisimportantthatthe laboratoryreceiveasamplewhich IStrulyrepresentatrveandhasnotbeendamaged or
changed duringtransportorstorage.
Storethetestsample insuchawaythatdeteriorationandchange incomposmonareprell'entH
8 Preparation oftestsample
8.1 Cheese
8.1.1 Prior to analysis. remove the rind or mouldy surface layer of the test sample 'n such a way as to
obtainasamplerepresentativeofthe cheeseasitisusuallyconsumed
8.1.2 Grind the test sample by means ')fanappropriate grinding device (65) Milethe groundmassquiclcly
and.ifpossible. grind asecond time andagainmixthoroughly.Cleantheoevceaftergnndlngofeachsample
Ifthetest sample cannot be ground.mix itthoroughlybyintensive stirringandkneading

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