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ReagentsAshingbuffer:10gMg(NO3)2100mlEtOH1.5NHClstock:119.7mlconcentratedHCl(11.6M@36%byweight)880.3mlH2O1NSulfuricacidstock:28.9mlconcentratedsulfuricacid(18M/36N@96%byweight)971.1mlH2OAmmoniummolybdatestock:4.2mlAmmoniummolybdate1000ml1NH2SO4
Procedure1)Preparestandardsinduplicate.-->standards:0,3,5,7,10,12,and15µlofa1mMNaH2PO4stocksolution2)Aliquotlipidunknowns.3)Add100µlashingbuffertoeachsample.4)DryEtOHandchloroformfromsamplesbyheating@80°Cfor~20minutes.5)Flamesamplesusingastrongflameonthebunsenburner.6)Removesamplesfromflamewhenthebrowngasceasestobereleased.-->Thereshouldbeawhitepercipitateremainingatthebaseofthetubes.Iftheashingprocessisallowedtoproceedfortoolong,thepercipitatewillappearbrown/charredandthiswillskewthespectrophotometerreadings.7)Add0.3mlof0.5NHCltoeachsampleandboilfor15minutes.8)Toeachtube,add:0.6mlammoniummolybdate0.1mlascorbicacid(10%w/v,madefreshEACHtime)VORTEX!!9)Incubatefor30minutes@45°Corfor60minutes@37°C.10)ReadOD820.