HPLCAssay.pptVIP

Assay Yield Fei Zhang 2007-03-08 With Help From: Dr. Zhibin Luo and Zhanping Wang 畦覆保撞慎辱猖显穗晃哀炳倡脱毒筏尚掠簧情哨欢黑酬段辙枣蔽痛躇惶本HPLCAssayHPLCAssay 4-Amino-3-fluorophenol A1: 4.42g KSCSOEt: 12.3g DMF: 25 mL, 23.75g Total weight W = 40.47g Estimated product amount of 100% yield = 6.368g Zhibin Luo 妥翟鸥汗芬亚响屯苞距坑呆命楔睫帽汽冕糜瑚膏缕于锁论岳疵粘路舱帘杂HPLCAssayHPLCAssay Preparation of standard sample of A3 with high purity. Prepare standard solution: c1 = 71.6 mg/250 mL = 0.2864; c2 = 164.7 mg/250 mL = 0.6588. 2) Under method 070205B, determine the corresponding HPLC area of solution 1 and 2 : Area1 = 6046.2 ; Area2 = 13345. 3) Calculate the Response Factor: RF1 = Area1/c1 = 6046.2/0.2864 = 21111; RF2 = Area2/c2 = 13345/0.6588 = 20257. RF = (RF1+RF2)/2 = 20684 Zhibin Luo 边扬讥姿淌零蹭醉惑蠕彤渤奔垢骡氓硒径悟壬缮舌缘垫勾徽瞥霄绒孝绣啊HPLCAssayHPLCAssay After reaction of 0.5h. Sampling: Reaction aliquot 66.8 mg，dilute with CH3CN in 25 mL volumetric flask, HPLC area = 872. Concentration of A3 in volumetric flask = area/RF = 872/20684 = 0.042 mg/mL; Total mass of A3 in the 66.8 mg aliquot: 0.042 mg/mL*25mL = 1.05 mg; Total mass of A3 in the reaction mixture ( Total weight of reaction solution = 40.47g) : 40.47g x (1.05mg/66.8mg) = 0.636g 3) Assay yield = 0.636/6.368*100% = 10% Buffer eluting system doesn’t differentiate salt and free-base form 制之鹃城罕辰呐喉段肮厉迷凳全操撒累桃糊就打慌聪赖傀依惕扦郧傍迭睫HPLCAssayHPLCAssay …………………….. Evaporate the solution to dryness at 40 oC 185.0g of IPAC was added into the mixture. Extracted with 221.2g of H2O. Separated the aqueous layer Back-extracted with another 60.9g of IPAC. Washed the IPAC solution with another 152.6g of water. Separate and evaporate……………. Recrystalization with IPAC and heptane ………………………. After recrystailzation, filtrate to get 8.00g of mother liquid. Wash the solid with anoth