HPLC-Assay

AssayYieldFeiZhang2007-03-08WithHelpFrom:Dr.ZhibinLuoandZhanpingWangNSSHHOHONH2FKSOEtSDMF,90-100oCA1A2A34-Amino-3-fluorophenolA1:4.42gKSCSOEt:12.3gDMF:25mL,23.75gTotalweightW=40.47gEstimatedproductamountof100%yield=6.368gZhibinLuoNSSHHOHONH2FKSOEtSDMF,90-100oCA1A2A31)PreparationofstandardsampleofA3withhighpurity.Preparestandardsolution:c1=71.6mg/250mL=0.2864;c2=164.7mg/250mL=0.6588.2)Undermethod070205B,determinethecorrespondingHPLCareaofsolution1and2:Area1=6046.2;Area2=13345.3)CalculatetheResponseFactor:RF1=Area1/c1=6046.2/0.2864=21111;RF2=Area2/c2=13345/0.6588=20257.RF=(RF1+RF2)/2=20684ZhibinLuoNSSHHOHONH2FKSOEtSDMF,90-100oCA1A2A3Afterreactionof0.5h.1)Sampling:Reactionaliquot66.8mg，dilutewithCH3CNin25mLvolumetricflask,HPLCarea=872.2)ConcentrationofA3involumetricflask=area/RF=872/20684=0.042mg/mL;TotalmassofA3inthe66.8mgaliquot:0.042mg/mL*25mL=1.05mg;TotalmassofA3inthereactionmixture(Totalweightofreactionsolution=40.47g):40.47gx(1.05mg/66.8mg)=0.636g3)Assayyield=0.636/6.368*100%=10%•Bufferelutingsystemdoesn’tdifferentiatesaltandfree-baseform……………………..Evaporatethesolutiontodrynessat40ºC185.0gofIPACwasaddedintothemixture.Extractedwith221.2gofH2O.SeparatedtheaqueouslayerBack-extractedwithanother60.9gofIPAC.WashedtheIPACsolutionwithanother152.6gofwater.Separateandevaporate…………….RecrystalizationwithIPACandheptane……………………….Afterrecrystailzation,filtratetoget8.00gofmotherliquid.Washthesolidwithanother10mlofheptane.Filtratedtoget4.30gofwashingsolution.Evaporatedtoget8.29gofyellowsolid.NClOClNClONH.HClPLE000430PLE000431M=176.0,36.45M=170.60ZhanpingWangTheoreticalyield:12.04gPreparationofstandard1.Defineastandard(workingstandardvsARDstandard)。2.QuantitativelyprepareA25mLsolutioninvolumetricflaskSampleAmount=21.4mgArea=5393Vol=25.00mlConcentration=21.4mg/25.00ml=0.856(mg/ml)ResponseFactor：K=area/C=5393/0.856=6300.2NClONH……………………..Evaporatethesolutiontodrynessat40ºC185.0gofIPACwasaddedintothemixture.Extractedwith221.2gofH2O.SeparatedtheaqueouslayerBack-extractedwithanother60.9gofIPAC..SeparatetheaqlayerWashedtheIPACsolutionwithanother152.6gofwater.Separatethisaqlayer…………….…RecrystalizationwithIPACandheptane……………………….Afterrecrystailzation,filtratetoget8.00gofmotherliquid.Washthesolidwithanother10mlofheptane.Filtratedtoget4.30gofwashingsolution.Evaporatedtoget8.29gofyellowsolid.Washingliquorassay1:Amount:227.09gWeightwosamples:A:835mgB:590mgWashingliquorassay2:Amount:222.85gWeightwosamples:C:1038mgD:895.5mgWashingliquorassay3:Amount:157.46gWeightwosamples:E:1324.6mgF:1757.3mgNClOClNClONH.HClPLE000430PLE000431M=176.0,36.45M=170.60Idealamount=12.04gAqueousextractionassay1).Aqueousextractionassay1SampleA:Dilute835mgaliquotto25.00mlArea=155.1)Concentrationofsolutioninvolumetricflask=155.5/6300=0.0246(mg/ml)2)Totalmassofsubstrateinvolumetricflask=0.0246(mg/ml)*25.00(ml)=0.616(mg)3)Concentrationoftheoriginalaqueousaliquot=0.616(mg)/835(mg)=0.000738mg/mgsolution4)Aqueouslayercontainssubstrate=0.000738x227.09=0.168gAssay%=0.168/12.04=1.39%Aqueousextractionassay1).Aqueouslayerassay1(ExtractionwithIPAc)Assay:1.36%2)aqueouslayerassay2(ExtractionwithIPAc)Assay%=0.25%3)Aqueouslayerassay3(IPAclayercombinedandwashedwithwater)SampleEArea=703.9Assay%=0.332/12.04=2.76%Conclusion:WashingtheIPAcextractionwithwaterLostmaterialtotheaqueouslayer……………………..Evaporatethesolutiontodrynessat40ºC185.0gofIPACwasaddedintothemixture.Extractedwith221.2gofH2O.SeparatedtheaqueouslayerBack-extractedwithanother60.9gofIPAC.WashedtheIPACsolutionwithanother152.6gofwater.Separateandevaporate…………….RecrystalizationwithIPACandheptane……………………….Afterrecrystailzation,filtratetoget8.00gofmotherliquid.Washthesolidwithanother10mlofheptane.Filtratedtoget4.30gofwashingsolution.Evaporatedtoget8.29gofyellowsolid.motherliquidassay:Amount:8.00gWeightwosamples:a:83.8mgb:131.7mgwashingliquidassay:Amount:4.30gWeightwosamples:c:231.9mgd:506.5mgNClOClNClONH.HClPLE000430PLE000431M=176.0,36.45M=170.60Idealamount=12.04gMotherliquorassay1)motherliquorassay:thesamemethodasaqueousextractionassay.Samplea:Fromthemotherliquor(8.0g),83.8mgaliquotwastakentoprepare25.00mlsolution。HPLCArea=1864Concentrationofthesolutioninvolumetricflask=1864/6300=0.296(mg/ml)Volumetricflaskcontainssubstrate=0.296(mg/ml)*25.00ml=7.397mgConcentrationoforiginalaliquot=7.397(mg)/83.8(mg)=0.088mg/mgsolutionTotalmassofsubstrateinthemotherliquor=0.088*8.0g=0.706gAssay=0.706/12.04=5.86%TheoreticalyieldWashingassay2)Washingliquorassay2Assay=0.72%Conclusion:1%ofmaterialwaslosttothewashing……………………..Evaporatethesolutiontodrynessat40ºC185.0gofIPACwasaddedintothemixture.Extractedwith221.2gofH2O.SeparatedtheaqueouslayerBack-extractedwithanother60.9gofIPAC.WashedtheIPACsolutionwithanother152.6gofwater.Separateandevaporate…………….RecrystalizationwithIPACandheptane……………………….Afterrecrystailzation,filtratetoget8.00gofmotherliquid.Washthesolidwithanother10mlofheptane.Filtratedtoget4.30gofwashingsolution.Evaporatedtoget8.29gofyellowsolid.Weightpercent:Amount:8.29gWeightwosamples:c:13.7mgd:19.8mgNClOClNClONH.HClPLE000430PLE000431M=176