I n t ern at i on al I N TE RN ATI ON AL ORG AN I ZATI ON To bacco an d h yd razi d e Tabac et Fi rst ed i t i on U DC FOR St an d ard S TAN D ARD I ZATI ON M E WH AP OAH Af l t obacco OPr AH 1 ALWl prod u ct s I - I O C TAH AAP TH AU WWORG AN l S ATl ON - Det ermi n at i on du - t abac - Dkt ermi n at i on d es rksi d u s d ’ h yd razi d e N ORM ALI S ATI ON mal ei c mal ki q u e - 4- Ref 66 : 643 : of DE resi d u es prod &s Descri pt ors I N TE RN ATI ON ALE t obacco, ch emi cal an al ysi s, d etermi n ati on of ten t, resi d u es, spect roph ot omet ri c an al ysi s, N o test I S0 76 - ( E) eq u i pmen t Pri ce based on pag es Foreword I S0 ( the I n t ern at i on al n at i on al s t an d ar d s n at i on al St an d ar d s bo d y i n t eres t ed ri g h t to an d be Org an i z at i on i n st i tu tes in is carri ed a s u bj ect r epr es en t ed on n on - g over n m en t al , Draf t I n t ern at i on al th e m em ber th e I S0 in f or ou t for t h at St an d ard i z at i on ) m e m ber th rou g h wh i ch wi t h I S0 by bef o r e t ech n i cal al s o the th ei r is Th e a wor l d wi d e wo r k t ake t ech n i cal h as f ed erat i on d evel opi n g E very be e n s et org an i z at i on s , part accept an ce of commi t t ees commi t t ee I n t ern at i on al I SO, ad o pt e d approval bod i es ) a t ech n i cal commi t t ee l i son S t an d ar d s bod i es for ( I S0 in the of I n t er- m em ber up h as the g o ver n m en t al wor k commi t t ees ar e as I n t ern at i on al ci rcu l at ed to S t an d ar d s by Cou n ci l I n t ern at i on al Tobacco St an d ar d an d t obacco I S0 76 was produ cts, an d d eve l o pe d was by Tech n i cal ci rcu l at ed to the Comm i t t ee m e m be r I SO/TC bod i es in 6, N o ve m be r 78 It h as been appr o ved the m e m be r bod i es the fol l owi n g cou n t ri es I tal y Brazi l Kor ea, Bu l g ari a M exi co Cz ech os l ovaki a N et h er l an d s U n i t ed Ro m an i a USSR Ar ab Rep of G r e ec e Sou th I ndi a Spai n m e m be r bo d y g rou n d s of the fol l owi n g Swi t z erl an d Rep Pri n t ed i n Th l an d of Tu rkey Afri ca, cou n t ry Rep ex pr es s ed Org an i zat i on Swi t zerl an d for F Stan dardi zati on , d i s approval R 980 Ki n g d o m Yu g os l avi a of : G er m an y, I ntern ati onal : S we d e n I rel an d Bel g i u m t ech n i cal of Au st ral i a Eg ypt , Th e by l of the d o cu m e n t on I S0 I N TE RN ATI ON ALSTAN DARD Tobacco an d h yd razi d e Sco pe Th i s an d of prod u ct s - Det ermi n at i on fi el d of Stan d ard mal ei c appl i cati on speci fi es h yd razi d e a resi d u es met h od in for t obacco th e an d deter- Pu ri fi cat i on Di ssol ve t obacco sol u te produ cts th e u sed i s appl i cabl e resi d u es on g f rom t obacco to t obacco mal ei c an d t obacco h yd razi d e prod u ct s su cker trol an d to ag en t s crops Dry Referen ces I S0 042, Laborat ory g l assware - On e- mark I S0 4793, Laborat ory pu rpose si n tered t h erm0met ers J ) cl assi fi cati on an d l fri ttedl 4674, of Tobacco an d fi l ters - t obacco prod u ct s - Gen eral if g re move to th e of vol ati l e porti on basi c redu ct i on su cci n i c by wi th sodi u m compou n d s n ascen t h yd razi d e l i berated i ts th e h yd roxi d e yel l ow co mpl ex is an d wi th Ad d i ti on h yd rog en wh i ch h yd razi n e sol u ti on n ecessary, carbon an aci d cl ean - u p of th en of of the g ran u l ated mal ei c h yd rol yzed spect roph ot omet ri c cryst al s over ml of yel l ow wi th 50 at cryst al s ml ph osph oru s( V) water an d of col d oxi d e “C on a water an d store of reag en t sol u ti on of th e pu ri fi ed sol u ti on ( 6) ; cryst al s fi l ter in 00 th rou g h ml of a si n t ered th e 0, g l ass M fi l ter i s stabl e for ot h erwi se up to fresh mon t h reag en t if stored sh ou l d in be th e d ark prepared in a d l y h yd razi d e, st an d ard sol u t i on correspon d i n g to to th e it n earest in 00 to 1 ml of mg , 0, l M mg of s od i u m pu re mal ei c h yd roxi d e h yd razi d e, sol u t i on di l u te 000 ml wi th ( 5) water zi n c h yd razi d e Di sti l l ati on of Gran u l at ed d en si t y d et ermi n at i on di sti l l ate of m ay th e be test porti on an d a of h as n ot been tan ce It ch ecked i n cl u d ed ti on s wi th th e 200 pal e sti r fu n n el zi n c, mo re of th an parti cl e , 70 si ze 500 pm, h avi n g a bu l k g /ems 4- d i met h yl ami n oben zal d eh yd e pre- d i g esti on th e was h or ab- to It If sti rri n g , wh i t e d esi ccat or Mal ei c an d an d th e Bu ch n er of an d pg /ml d i ssol ve test stan t a ml n ecessary reag en t Wei g h , th e t h rou g h 50 ch arcoal di - sampl i n g 2) of Fi l ter in act i vat ed Porosi t y Pri n ci pl e Boi l i n g powd ered d esi g n at i on 2’ wi th an d aci d refri g erator; I S0 mi n Preparat i on fu n nel , Th e g radi n g , of bottl e Di ssol ve g en eral g Fi l ter a vacu u m su l ph u ri c Sol i d - st em reag en t vol u met ri c fl asks 770, 5 an d fu n n el d ark I S0 mal ei c 4- d i met h yl ami n oben zal d eh yd e Ad d fi l trate in a of for sl owl y th e Bu ch n er in ti on s of mech an i cal l y met h od to 20 eth an ol add, Th e of ( E) resi d u es I ntern ati on al mi n at i on t obacco 4676- 960 of that is fou n d that th e reco mmen d e d by compari n g h yd razi n e from th e su l ph ate mal ei c g rad e of that th e col ou r an d h yd razi d e zi n c zi n c u sed to is be prod u ced by of pri me u sed i mpor- sh ou l d stan dard be sol u - 4- d i met h yl ami n oben zal d eh yd e after red u ct i on an d di sti l l ati on Reag en t s Du ri n g g rad e th e an d an al ysi s, di sti l l ed u se water on l y or reag en t s wat er of of eq u i val en t 4- d i met h yl ami n oben zal d eh yd e, 0, M su l ph u ri c II At presen t at the stag e of draft 2) At at th e stag e of draft presen t aci d recog n i zed 20 of So d i u m h yd roxi d e, So d i u m h ydroxi d e, Su l ph u ri c 2, M sol u ti on pu ri ty g /l sol u ti on I SO/R 0, l M sol u ti on in sol u ti on ( Revi si on 4 an al yti cal aci d, 0, M sol u ti on 770- 970) I S0 4676- 960 I ron Th e ( E) ch l ori de reag en t prod u ced sh ou l d wi t h tetrah yd rate be ch ecked to en su re that no 4- d i met h yl ami n oben zal d eh yd e An t i - f oam An t i - bu mpi n g ag en t ( paraffi n ( FeCl z 4H 20) wax col orati on Carry is in sol u ti on or refi n ed Sampl i n g veg etabl e Hi gh Tobacco, in poi n t oi l , for t h ermomet er kn own ch aracter to to that be free u n d er f rom test mal ei c ( see h yd razi d e th e n ote to an d Preparati on Gri n d 4) of the th e l aboratory apparat u s On e- mark an d cl ass 000 an d vol u met ri c ml , co mpl yi n g th e fol l owi n g fl asks, wi th th e of Test capaci ty25 ml , req u i remen t s of 00 I S0 ml proced u res speci fi ed in a the sampl e l aboratory g ri n di n g passes a mi l l mm so mesh that the screen wh ol e Mi x th e th orou g h l y the n earest 01 g, g of th e prepared sampl e Determi n ati on 042, A If Measu ri n g Beaker, Si n tered requ i red, al kal i n e cyl i n d er, of of capaci t y 250 capaci t y 00 ( see g l ass fi l ter ml fu n n el ( Bu ch n er fu n n el ) , P 00, compl yi n g wi th th e Al kal i n e req u i remen t s of I S0 ml di sti l l ati on apparat u s ( see fi g u re) , st eam compri si n g : g en erator; c) d en ser; d) vari abl e j oi nt fl at trol l ed h ot an d fl ask bott omed , a mo me t er pl ate sh al l th i ck- wal l ed t h ermomet er compl yi n g d esi g n ati on Start wel l wi th be fl ask st ru ct ed fi tted t n i n g th e a wi th req u i remen t s to from a sph eri cal 360 of a of up OC I SO/R ( 61 , th e th er- n ect ed u sed to requ i red th e th rou g h pass an d a s t e am Th i s en abl es g en erator th e th ree to reacti on /di sti l l ati on way wast e a st ead y wh i l e 770, th e tap Th e t h rou g h boi l i n g a vessel th i rd ou tl et d en ser rate to reacti on /di sti l l ati on sh al l be sh al l wh en be be n ot mai n t n ed fl ask is in bei n g ch an g ed - Th e wh ol e apparatu s sh al l be su rrou n d ed by a screen Spect roph otomet er, cel l s 425, a ( 41 , u nti l aci d pre- fl ask a s mal l Ad d amou n t an t i - bu mpi n g t en ts to i n to poi n t th e few t h ermomet er boi l i n g th e abou t oi l the of g ran u l es t h ermomet er ( 0) an d occasi on al l y t h ermomet er su i tabl e 455 an d 485 nm for an d measu remen t s fi tted wi th th e mi n h eat th e Red u ce th e i n d i cates Al l ow to cool 65 for “C; mi n tai n the su re th e at 00 ml that en d an d of fi l ter th e wash i n g Ad d evaporate a th e ml of to th e an d on the th e fu n n el h ot pl ate di sti l l ate to m ay ( 7) the to to reac- th e th e 20 Ch i l l ( 4) to th e abou t abou t, be Mai n to mi n i n to the apfl ask di sti l l ati on di sti l l ati on wi th g ran u l es fl ask abou t di sti l l ate fu n n el bel ow d en ser th e in of Du ri n g th e is ch l ori de s t eam OC: to sol u ti on tu be th e d u ri n g an ti - bu mpi n g t en ts measu ri n g aci d ten ts admi t ti n g f ml i ron the g l ass cyl i n d er Al tern ati vel y, th e col l ected i nto si n t ered 00 d el i very wat er is a su l ph u ri c n ect 200 cool i n g di sti l l ate th e few to by t emperat u re a of i mmed i at el y d en ser th rou g h th e g ( 3) di sti l l ati on th i s of zi n c fl ow fl ask pl ace of d en ser Ad d ten t s rapi d ml th e aci d th e an d of fl ask start th e a en d th e 8) g en erator g ran u l ated h eati n g an n ex tai n i n g of an d ( see s t eam of mai n t n th an , of reacti on /di sti l l ati on sol u ti on an d OC hi gh fl ask ti on /di sti l l ati on rotary wavel en g t h s th e that g water safet y 360 swi rl i n g th e beaker, Warn i n g to th e t ake su rface parat u s g en erator ( 21 , so an d 91 75 s t eam th e ( 8) Di sti l l ati on an d Th e by fl ask; reacti on /di sti l l ati on ml a sh ou l d cyl i n d er Th e to h yd roxi d e ag en t carefu l l y, h eat porti on sodi u m t n i n g ten ts reacti on /di sti l l ati on test th e I n sert wel l th i s b) preced ed di g est i on an ti - foam fl ask a) th e of ( 9) St e am be 4793 th e 5 m ay A) porosi t y 50 g rad e di g est i on an n ex ml Tran sfer the porti on to di g est i on 500 of mat eri al mat eri al Wei g h , : s ampl e g rou n d Apparat u s U su al wi th wel l g rou n d accord an ce Proced u re si mi l ar in g ran u l es boi l i n g 1 sampl i n g 4874 oi l ) ou t I S0 di sti l l ate a 250 n ot cen t rated ml ml beaker bu t en - Ri n se of an d l ess in a evaporator at mm If req u i red, th er ( see th e an n ex di sti l l ate A) m ay be cl ean ed before proceed i n g fu r- Heat- resi stant To s t eam s u ppl y l ag g i n g - was te - To H ot pl ate Col d water ) r- I To was te ; II G radu ated I ’ I ’ I ’ recei ver II I L! * / I /Ah- Fi g u re 3 Reacti on wi th - Apparat u s for determ i n ati on of m al ei c M eas u re 4- d i m et h yl am i n oben z al d eh yde h ydraz i de th e th e cen t rated transfer ad d before mi x it ml qu anti tati vel y of th e m aki n g th e di sti l l ate a 25 ml as des cri bed vol u m etri c fl ask 4- di m et h yl am i n oben z al deh yde up ten ts to obtai n ed to wel l the m ark an d s tan d wi th it in water th e dark in for ( ) th e abou t 30 an d ( ) Cal cu l ate from Spect roph otom etri c a mm cel l wi th th e m eas u rem en t s test sol u ti on as des cri bed If ml of the s u l ph u ri c aci d 4- d i m eth yl am i n oben z al deh yd e wi th thi s water sol u ti on in a vol u m etri c sol u ti on sol u ti on fl ask an d fi l l ( 6) ( 1 , th e to di l u te referen ce to cel l corrected - test of sol uti on 425, on 455 an d th e s pec- 485 nm abs orban ce, Acorr, of th e test sol u ti on ml 25 + 4s A425A455 an d test sol u ti on th e at abs orban ce sol u ti on the Aa 425, are 455 respecti vel y an d 485 th e abs orban ces of nm in 3 Ad d of wavel en g th s form u l a A42 45 wh ere obtai n ed the the mi n the Fi l l at fl ask, A ( 6) 2, sol u ti on Stopper t obacco abs orban ce t roph otom eter Cool in , of ml at fu rther 455 wi th 4- di m et h yl am i n oben z al d eh yde su l phu ri c aci d sol u ti on ( 61 , nm exceeds a 0, 8, m i xtu re sol u ti on di l u ted ( ) to 25 di l ute ml an d the test ml of IO wi th ml of of the water wi th Carry ou t t wo determ i nati on s on the s am e prepared s am pl e I S0 48 - ( E) Cal i brati on cu rve in th e sam pl e test sol u ti on as read fro m th e taken as cal i brati on cu rve ; Ad d al i q u ots h yd razi d e of 0, sol u ti on h yd razi d e, to d e scri bed in 1 , 2, ( 2) , g porti on s an d ml of correspon d i n g of an d absorban ce of th e to th e tobacco 3 above stan d ard to 80 ( 1 ) ug m al e i c of an d m, m al e i c treat i s th e m, Measu re th e prepared rel ati n g as th e i n d i cated in absorban ce mi cro g ram s, of th e th e stan d ard to Prepare th e stan d ard test mal e i c test a sol u ti on s cu rve ten t, in ti on - of I f no th e tobacco su i tabl e tobacco stan d ard A h yd razi d e for cal i brati on , sol u ti on in th en th e cal i bra- abse n ce as test por- averag e water ten t of th e sampl e sh ou l d be mad e for an y fu rth er d i l u ti on ( see th e resu l t, th e for m e an of repeatabi l i ty th e two ( 2) val u e s are provi d ed th at th e sati sfi ed Repeatabi l i ty of d i fferen ce carri ed resu l ts th e Meth o d of cal cu l ati on an d fro m presen t in th e cal i brati on th e sampl e test cu rve th e me an of m al e i c tobacco, is h yd razi d e, g i ven th e th e sam e sh al l val u e n ot is resu l ts ti m e or excee d g reater of d u pl i cate rapi d l y th an % on e of th ei r u s/g , d etermi n ati on s, after an oth er, me an or by val u e, pg l g th e wh en amou n t of mal e i c val u e is l e ss th an wh e n th e u g /g h yd razi d e sol u ti on expre sse d in m i cro g ram s Test by th e report per Th e of between at formu l a ten t ou t an al yst, m e an g ram th e of sam e Th e th e req u i remen ts Th e Read tobacco i s acceptabl e Expressi o n is correcti on 8 of sol u ti on s i s avai l abl e mal e i c g rams, 4) Take N OTE in th u s cal i brati on h yd razi d e m ass , ti on ; as test report sh al l sh o w th e meth od u sed an d th e resu l ts fo rmu l a obtai n ed 00 It speci fi ed in sh al l th i s al so men ti o n I n tern ati on al an y operati n g Stan d ard or d i ti on s reg ard ed as n ot opti on al -% 00 ma - ( for m, as e xampl e an y a speci al ci rcu m stan ce s proced u re th at may d escri be d h ave in an n ex i n fl u en ced th e A) , as we l l resu l t wh ere Th e m is th e m ass of m al e i c h yd razi d e, in mi cro g ram s, presen t test report i d en ti fi cati on of sh al l th e i n cl u d e sampl e al l d etai l s req u i red for co mpl e te I S0 An n ex Opti on al A extra Gen eral A l l presen t foami n g in d u ri n g t obacco the can al kal i n e s o me t i me s d i g esti on an d proced u res Proced u re Tran sfer th e test 50 the h yd roch l ori c ml Th e i n cl u si on of th e aci d pre- di g esti on stag e of A breaks d own th e protei n matt er an d to the aci d reacti on /di sti l l ati on ( A ) an d a fl ask smal l Ad d amou n t wax Heat th e fl ask an d boi l the t en ts at of a mod erat e d escri bed rate in porti on cau s e di sti l l ati on paraffi n stag es ( E) A A 2 Prot ei n s exces si ve 4676- 980 red u ces the u n ti l th e vol u me h as been red u ced to bet ween 20 an d foam25 ml Was h d own th e wal l s of th e vessel wi th abou t 25 ml of i ng wat er an d bet ween A So m e porti on wi th of tobacco, bases, th e especi al l y tai n s col ou r reacti on an d 4- d i met h yl ami n oben zal d eh yd e act i vated carbon as that n atu ral t n i n g su bst an ces g i ve a Treat men t d escri bed in A a th at red of the hi gh 20 N OTE - Th e wi th stag e, di sti l l ate wi th compl et ed th i s bu t Ad d , Nei th er reg ard s of it t h ese as proced u res n ecessary for sh ou l d th e be u sed parti cu l ar ml parti al l y on ce th e Al l ow d i g est ed s od i u m as soon sl owl y sol u ti on A 25 u n ti l vol u me to h as been red u ced to cool as t obacco h yd roxi d e m ay h as been be l eft ad d ed overn i g h t th e met h od at th i s mu s t be possi bl e i nter- feren ce an al yst an d boi l pro- i nterfere col ou r el i mi n at es ag n u n l ess sampl e s an d wi th ( A 2) an d swi rl i n g , 56 proceed wi t h ml of the th e s od i u m al kal i n e h yd roxi d e di g esti on as in g of the bei n g tested A A Aci d Carbon A A Ad d i ti on al reag en t s Add i ti on al H yd roch l o ri c aci d , approxi mat el y M to 270 000 ml ml of wi th h yd roch l ori c wat er an d Act i vat ed aci d, Q approxi mat el y 1 Proced u re mi x Sh ake ) So d i u m Su i t abl e Cen tral mat eri al Secretari at carbo n 1 g /ml , the act i vat ed A req u i red sol u ti on A Di l u te reag en t req u i red A A cl ean - u p pre- d i g esti on h yd ro xi d e, i s avai l abl e 700 g /l sol u ti on commerci al l y Detai l s trati n g m ay be obtai n ed f rom the di sti l l ate carbon to ml Secretari at as of obtai n ed ( A ) di rect ed I SO/TC as for in d escri bed mi n an d in fi l ter wi th before cen - 26 ( DI N, German y, F R ) or f rom the I S0 I S0 46 - ( E) An n e x N o te s B l A safe t y scr e e n sh al l be pl ace d in on B d i sti l l ati on po si ti o n d u ri n g d i sti l l ati on proced u re B h ave Th e so d i u m B- After paratu s d i sti l l ati on , u si n g Re m o ve th e h eat re mo ve resi stan t th e rmomete r th e g l o ve s an d h ot an d seal fl ask weari n g th e we l l fro m th e safe ty th e ste am i n l et tu be free of so d i u m wi th a sm al l h yd roxi d e ten ts of th e zi n c d i sti l l ati on g ran u l es fl ask an d i n to ri n se th e an fl ask i ron si e ve th ree in twi ce th e si n k wi th th e wi th % en cru sted ( V/v) h yd roch l ori c cau sti c an d zi n c aci d g ran u l es sol u ti on Fi l l reg u l arl y acti on i n spected of th e as th ey cen trated sol u ti on Hi g h vacu u m j oi n ts of si l i e th e g rease apparatu s sh ou l d Wh e n be u sed on th e i ron ad d i n g al l to an d zi n c g ran u l es to th e ten ts of th e reac- water th e fl ask, care sh ou l d be taken th at no parti cl es to ad h ere re mo ve be th e th e ti on /d i sti l l ati on an d to cork Pou r ti mes sh al l o wi n g ap- ch l ori d e th e fl asks l i fe h yd roxi d e sph eri cal trap l i mi te d g l asse s B Ri n se d i sti l l ati on a ve ry to th e g rease an d th u s preven t proper cl osi n g of th e fl ask j oi n t wi th th e h yd roch l ori c Before re- u se ti al re mo ve to d e co mposi ti o n th e n ext ri n se al l of aci d th e fl ask resi d u al m al e i c d etermi n ati on sol u ti on th ree zi n c u n ti l ti me s as h yd razi d e it in n ext wi th wo u l d th e u sed water cau se pre- d i g est It is esse n - prematu re stag e of B parti cl es Care sh ou l d becau se be of exerci se d th e d an g er in of th e fi re d i spo sal of resi d u al zi n c