seventh commiss direct march establish commun method analysi offici control feedingstuff eec commiss european commun regard treati establish european econom commun regard council direct juli introduct commun method sampl analysi offici control feedingstuff amend act access articl thereof direct requir offici control feedingstuff carri commun method sampl analysi purpos check complianc requir aris provis laid law regul administr action qualiti composit feedingstuff commiss direct eec june eec novemb eec april eec decemb eec march eec decemb establish number commun method analysi progress work make advis adopt seventh set method measur provid direct accord opinion stand committe feedingstuff adopt direct articl member state requir analys offici control feedingstuff content aflatoxin carri accord method annex direct gener provis set part introduct annex commiss direct eec june except part deal prepar sampl analyz applic method annex direct articl member state octob bring forc law regul administr provis compli direct forthwith notifi commiss thereof articl direct address member state brussel march commiss lardinoi member commiss oj oj oj oj oj oj oj oj annex determin aflatoxin dimension thin layer chromatograph method purpos scope method make determin level aflatoxin feedingstuff groundnut copra linse soya sesam babassu palm maizegerm oil cake cereal cereal product pea meal potato pulp starch lower limit determin mg kg ppb presenc interf substanc hinder determin start analysi method dimension thin layer chromatographi principl sampl subject extract chloroform extract filter aliquot portion purifi column chromatographi silica gel eluat evapor residu redissolv specif volum chloroform mixtur benzen acetonitril aliquot portion solut subject thin layer chromatographi tlc quantiti aflatoxin determin uv irradi chromatogram visual fluorodensitometri comparison quantiti standard aflatoxin ident aflatoxin extract feedingstuff confirm procedur reagent nb reagent quot analyt reagent quot qualiti state aceton chloroform stabil ethanol hexan methanol anhydr diethyl ether free peroxid mixtur benzen acetonitril mixtur chloroform methanol silica gel column chromatographi particl size mm absorb cotton wool previous defat chloroform glass wool sodium sulphat anhydr granular inert ga nitrogen hydrochlor acid sulphur acid kieselguhr hyflosupercel wash acid silica gel hr equival tlc standard solut aflatoxin ml chloroform benzen acetonitril mixtur prepar check section standard solut qualit test purpos aflatoxin ml chloroform benzen acetonitril mixtur concentr guid adjust obtain intens fluoresc aflatoxin develop solvent chloroform aceton unsatur tank diethyl ether methanol water unsatur tank diethyl ether methanol water satur tank chloroform methanol satur tank chloroform methanol satur tank apparatu grinder mixer shake apparatu magnet stirrer flute filter paper schleicher schüll equival diamet cm glass tube chromatographi intern diamet mm length mm ptfe cock ml reservoir rotari vacuum evapor ml round bottom flask ml conic flask ground glass stopper tlc apparatu glass plate tlc mm prepar quantiti suffici cover plate put silica gel hr conic flask add ml water stopper shake minut spread suspens plate obtain uniform layer mm thick leav dry air store desicc silica gel time activ plate keep oven ºc hour readi plate suitabl give result similar obtain plate prepar long wavelength nm uv lamp intens irradi make spot ng aflatoxin distinguish tlc plate distanc cm lamp ml graduat tube polyethylen stopper uv spectrophotomet fluorodensitomet option procedur prepar sampl quot observ quot part point grind sampl pass siev mm mesh accord recommend iso extract put ground homogen sampl ml conic flask add kieselguhr ml water ml chloroform stopper flask shake stir minut apparatu filter flute filter paper discard ml filtrat collect ml column clean insert lower end chromatographi tube cotton glass wool plug fill third tube chloroform add sodium sulphat check upper surfac sodium sulphat layer flat add silica gel small portion stir carefulli addit elimin air bubbl leav stand minut carefulli add sodium sulphat liquid fall upper surfac sodium sulphat layer mix ml extract collect ml hexan quantit transfer mixtur column liquid fall upper surfac sodium sulphat layer discard wash add ml diethyleth fall upper surfac sodium sulphat layer oper rate flow ml minut column run dry discard liquid elut ml chloroform methanol mixtur collect eluat evapor dryness temperatur exceed ºc stream inert ga rotari evapor quantit transfer residu chloroform benzen acetonitril mixtur ml graduat tube concentr solut stream inert ga adjust volum ml chloroform benzen acetonitril mixtur thin layer chromatographi spot tlc plate cm lower edg interv cm volum standard solut extract standard aflatoxin solut extract obtain superimpos point standard solut extract obtain develop chromatogram dark develop solvent choic solvent made deposit qualit standard solut plate check develop aflatoxin complet separ solvent evapor dark irradi uv light place plate cm lamp spot aflatoxin give blue fluoresc quantit determin determin visual fluorodensitometri visual measur determin quantiti aflatoxin extract match fluoresc intens extract spot standard solut spot interpol fluoresc obtain superimposit extract standard solut intens extract visibl spot fluoresc intens extract greater standard solut dilut extract time chloroform benzen acetonitril mixtur subject thin layer chromatographi measur fluorodensitometri measur fluoresc intens aflatoxin spot fluorodensitomet excit wavelength nm emiss wavelength nm determin quantiti aflatoxin extract spot comparison fluoresc intens standard aflatoxin spot confirm ident aflatoxin confirm ident aflatoxin extract process treatment sulphur acid sprai sulphur acid chromatogram obtain fluoresc aflatoxin spot turn blue yellow uv irradi dimension chromatographi involv format aflatoxin hemiacet aflatoxin nb oper carri carefulli diagram figur applic solut score straight line plate parallel contigu side cm side limit migrat solvent front spot solut plate capillari pipett microsyring point volum purifi extract sampl obtain nm aflatoxin point standard solut develop develop chromatogram direct dark develop solvent cm layer unsatur tank solvent front reach solvent limit line remov plate tank dry dark ambient temperatur minut sprai hydrochlor acid band cm high cover point hatch area figur darken protect rest plate glass sheet react minut dark dry stream air ambient temperatur develop chromatogram direct ii dark develop solvent cm layer unsatur tank solvent front reach solvent limit line remov plate tank dry ambient temperatur interpret chromatogram examin chromatogram uv light check featur appear blue fluoresc spot aflatoxin origin standard solut appli migrat direct appear blue fluoresc spot unreact hydrochlor acid aflatoxin intens blue fluoresc spot aflatoxin hemiacet origin standard solut appli migrat direct ii appear spot match origin sampl extract appli posit spot defin migrat distanc aflatoxin point direct travel standard appli migrat distanc direct ii aflatoxin hemiacet travel standard appli fluoresc intens hemiacet spot origin extract standard appli match calcul result visual measur content microgram aflatoxin kg sampl ppb formula gt pic file quot quot gt volum microlitr standard solut aflatoxin extract similar intens fluoresc concentr microgram aflatoxin ml standard solut final volum extract microlitr allow dilut weight gramm sampl volum extract subject column clean fluorodensitometr measur content microgram aflatoxin kg sampl formula gt pic file quot quot gt volum microlitr extract spot plate quantiti nanogram aflatoxin extract spot proport deduc measur final volum extract microlitr allow dilut weight gramm sampl volum extract subject column clean prepar test standard solut determin concentr aflatoxin prepar standard solut aflatoxin chloroform benzen acetonitril mixtur concentr ml determin absorpt spectrum nm aid spectrophotomet measur optic densiti nm case chloroform solut nm case solut benzen acetonitril mixtur calcul concentr microgram aflatoxin ml solut formula gt pic file quot quot gt dilut dai light obtain work standard solut concentr aflatoxin ml refriger ºc solut stabl week test chromatograph puriti spot plate standard solut aflatoxin ml develop chromatogram uv light chromatogram show spot fluoresc percept origin deposit zone repeat differ result parallel determin carri sampl analyst exce relat highest result content aflatoxin kg absolut content greater kg relat highest content kg reproduc quot observ quot part point gt dimension thin layer chromatograph method purpos scope method make determin level aflatoxin feedingstuff fall scope method lower limit determin mg kg ppb method applic feedingstuff citru pulp principl sampl subject extract chloroform extract filter aliquot portion purifi column chromatographi silica gel eluat evapor residu redissolv specif volum chloroform mixtur benzen acetonitril aliquot portion solut subject dimension thin layer chromatographi tlc quantiti aflatoxin determin uv irradi chromatogram visual fluorodensitometri comparison quantiti standard aflatoxin ident aflatoxin extract feedingstuff confirm procedur reagent nb reagent quot analyt reagent quot qualiti state aceton chloroform stabil ethanol hexan methanol anhydr diethyl ether free peroxid mixtur benzen acetonitril mixtur chloroform methanol silica gel column chromatographi particl size mm absorb cotton wool previous defat chloroform glass wool sodium sulphat anhydr granular inert ga nitrogen hydrochlor acid kieselguhr hyflosupercel wash acid silica gel hr equival tlc develop solvent diethyl ether methanol water satur tank chloroform aceton unsatur tank standard solut aflatoxin ml chloroform benzen acetonitril mixtur prepar check point method apparatu point method gt pic file quot quot gt dimension tlc applic solut follow diagram figur score straight line plate parallel contigu side cm side limit migrat solvent front spot solut plate capillari pipett micro syring point purifi sampl extract obtain point standard solut point standard solut point standard solut point standard solut dry slow stream air inert ga spot obtain diamet mm develop follow diagram figur develop chromatogram direct dark develop solvent cm layer satur tank solvent front reach solvent limit line remov plate tank dry dark ambient temperatur minut develop chromatogram direct ii dark develop solvent cm layer unsatur tank solvent front reach solvent limit line remov plate tank dry dark ambient temperatur interpret chromatogram follow diagram figur irradi chromatogram uv light place plate cm locat posit blue fluoresc spot aflatoxin standard solut project imaginari line pass spot angl develop direct intersect line locat expect find aflatoxin spot origin sampl extract appli figur actual locat aflatoxin spot point intersect imaginari straight line form angl pass spot determin quantiti aflatoxin sampl extract supplementari chromatographi score straight line plate parallel contigu side diagram figur appli point figur purifi sampl extract obtain superimpos standard solut develop irradi chromatogram uv light check aflatoxin spot extract standard solut superimpos fluoresc spot intens aflatoxin spot develop plate quantit determin determin visual fluorodensitometri visual measur determin quantiti aflatoxin extract match fluoresc intens extract spot spot standard solut interpol fluoresc intens extract greater standard solut dilut extract time chloroform benzen acetonitril mixtur subject thin layer chromatographi measur fluorodensitometri measur fluoresc intens aflatoxin spot fluorodensitomet excit wavelength nm emiss wavelength nm determin quantiti aflatoxin extract spot comparison fluoresc intens spot standard solut confirm ident aflatoxin point method calcul result point method repeat point method reproduc quot observ quot part point observ method defat sampl fat defat light petroleum bp ºc prepar case analyt result express term weight defat sampl reproduc result reproduc result variat result obtain laboratori sampl estim valu aflatoxin kg kg valu greater kg valu kg appendix annex gt pic file quot quot gt gt pic file quot quot gt