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書式設定,書式設定,第 2,第 3,第 4,第 5,*,書式設定,書式設定,第 2,第 3,第 4,第 5,*,1,/79,大赛璐（中国）手性事业部,手性分析中的注意事项,手性分析条件的建立和优化,2,/79,内容提纲,1.,手性,HPLC,分析条件的建立和优化,1,-1,正相色谱,2-2,反相色谱,2.,手性色谱柱的自动筛选,3.,手性分析中的注意事项,3,/79,Daicel,手性色谱柱一览表,正相,直链淀粉衍生物,CHIRALPAK,AD/AD-H,CHIRALPAK,AS/AS-H,纤维素,衍生物,CHIRALCEL,OD/OD-H,CHIRALCEL,OJ/,OJ-H,CHIRALCEL,OA,OB,OC,CHIRALCEL,OF,OG,OK,CHIRALCEL,CA-1,反相,CHIRAL,PAK,AD,-RH,CHIRAL,PAK,AS,-RH,CHIRALCEL,O,D-RH,CHIRAL,CEL,OJ,-RH,其它,CROWNPAK,CR(,),(,),CHIRALPAK,OT,(,),OP(,),CHIRALPAK,WH,MA(,),CHIRALPAK,QD-AX,CHIRALPAK,QN-AX,共价键合型新产品,CHIRAL,PAK,IA,CHIRAL,PAK,IB,4,/79,分析条件优化流程图,(1,),酸性,中性,碱性,1.,流动相中添加物的必要性,配制流动相,Hex/IPA/,TFA,=90/10/0.1,Hex/IPA/,DEA,=90/10/0.1,Hex/IPA,=90/10,样品,5,/79,碱性样品,添加物在分离中的作用,mV,Minutes,0.0,1.0,2.0,3.0,4.0,5.0,6.0,7.0,0,2,4,6,8,10,12,14,16,18,20,22,24,10.74,13.96,Hex/IPA/,DEA,=90/10/,0.1,流量,：,1.0mi/min.,温度,：,25,、,检测波长,：（,254,）,流动相,Hex/IPA=90/10,Hex/IPA/,AcOH,=90/10/,0.1,流动相,Hex/IPA=90/10,-0.10,-0.05,0.00,0.05,0.10,0.15,0.20,0.25,0.30,0.35,mV,0,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,Minutes,10.94,0.40,流量,：,1.0mi/min.,温度,：,40,、,检测波长,：（,254,）,0,5,10,15,20,25,30,35,mV,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,6.66,7.52,Minutes,0.0,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,mV,0,5,10,15,20,25,30,35,40,酸性样品,（没出峰,),（无法分离,),6,/79,添加物的种类和用量,样品酸碱性,添加物种类,添加量,中性,不需要,碱性,乙二胺,(DEA),(n-,丁胺,*,1,),(,乙醇胺,*,2,),0.5,通常,0.1,酸性,三氟乙酸,(TFA),醋酸,0.5,通常,0.1,*1,主要用于1级胺,*,2,主要用于1级氨基醇,7,/79,分析条件优化流程图,(2,),保留时间,?,长,适宜的分析时间,(10-30 min.),短,增加,异,丙醇,增加,正已烷,2.,配制好流动相,选择合适的手性柱,分析,8,/79,分析条件优化流程图,（3）,没有分离,部分分离,3.,选择合适的手性柱,尝试别的手性柱,OK!,基线分离,继续优化分析条件,分离？,9,/79,筛选结果,0,5,10,15,20,25,30,35,40,45,50,55,60,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,4.68,4.82,OB,15,mV,1,2,3,4,5,6,7,8,9,10,11,12,13,14,Minutes,6.71,6.87,OD,0,5,10,15,20,25,30,35,40,45,50,55,60,65,mV,0,-5,0,5,10,15,20,25,30,35,mV,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,6.66,7.52,AD,0,5,10,15,20,25,30,35,40,45,50,55,60,65,mV,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,OJ,mV,0,10,20,30,40,50,60,70,80,mV,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,4.70,90,AS,15,0,10,20,30,40,50,60,70,80,90,100,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,4.70,OC,Minutes,10,20,30,40,50,60,70,80,mV,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,4.36,15,OG,Minutes,0,0,10,20,30,40,50,60,70,80,mV,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,Minutes,4.36,OF,色谱条件,流动相,：,Hex./IPA/AcOH=,90/10/0.1,流量,：,1.0ml/min.,温度,：,40,检测波长,：,UV254nm,样品,：,先择效果最好的手性柱,然后继续优化分析条件,10,/79,分析条件优化流程图,（4-A）,部分分离,A.,降低异丙醇浓度,B.,调整温度,C.,更换醇类,4.,分析条件继续优化,11,/79,醇浓度的影响,0,5,10,15,20,25,30,35,mV,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,6.66,7.52,在多数情况下，降低醇的浓度可延长保持时间，改善分离度,Column,：,AD,Temp.,：,40,Flow rate,：,1.0ml/min.,Hex./IPA/AcOH=,90/10,/0.1,Hex./IPA/AcOH=,100/1,/0.1,-0.5,0.0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,mV,0,5,10,15,20,25,30,35,40,45,50,55,60,Minutes,35.77,43.71,Sample,：,Flurbiprofen,12,/79,部分分离,B.,调整温度,C.,更换醇类,4.,分析条件继续优化,分析条件优化流程图,（4-B）,A.,降低异丙醇浓度,13,/79,温度的影响,Column,：,CHIRALPAK AD,Mobile phase,：,Hex./IPA/AcOH=90/10/0.1,Flow rate,：,1.0ml/min.,25,40,Temp.,：,5,降低柱温,会使峰形变宽，但可增大,值,升高柱温,会使峰形锋利，但,值却变小,-0.5,0.0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,8.72,12.13,:1.59,mV,Minutes,0,5,10,15,20,25,30,35,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,6.66,7.52,:1.23,mV,0,1,2,3,4,5,6,7,8,9,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,7.66,9.12,:1.32,14,/79,部分分离,C,.,更换醇类,B.,调整温度,4.,分析条件继续优化,A.,降低异丙醇浓度,分析条件优化流程图,（4-C）,15,/79,醇种类的影响,Hex./,IPA,/AcOH=90/10/0.1,IPA,和,EtOH,有时会使峰形和,值发生明显变化,Column,：,CHIRALPAK ADTemp.,：,40,Flow rate,：,1.0ml/min.,Sample,：,Flurbiprofen,0,5,10,15,20,25,30,35,mV,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,Minutes,6.66,7.52,a,=1.23,0,5,10,15,20,25,30,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,7.58,9.64,a,=1.45,mV,Minutes,Hex./,EtOH,/AcOH=95/5/0.1,16,/79,正相柱分析条件优化的总结,根据样品性质判断是否需要往流动相中加添加物,调整正己烷/醇的比例，从而选择适当的保留时间,其它优化方法,流动相的醇浓度,降低醇的浓度虽改善分离度，但会延长分析时间,色谱柱温度,升高柱温虽可使峰形锋利，但,值会降低,流动相中醇的种类,改换醇的种类,(IPAEtOH),，,有时可使分离发生一些变化,17,/79,1.,手性色谱柱分析条件的建立和优化,2-1,正相色谱,2-2 反相色谱,手性色谱柱的自动筛选,3.,手性分析中的有关注意事项,内容提纲,18,/79,RH,系列（反相用）的规格,流动相,耐压,(MPa),温度范围,(),pH,范围,OD-RH,水溶液/有机溶剂,*,1,=90/100/100,10,5,40,2,7,OJ-RH,AS-RH,5,40*,2,2,9*,2,3,AD-RH,*1,有机溶液,：,醇（甲醇，乙醇，异丙醇）或乙腈,*,2 pH,7,使用时，温度在,5,25,*3 pH,超过,8,时，使用硼酸缓冲液,19,/79,根据样品性质选择流动相,样品性质,OD-RH,、,OJ-RH,AD-RH,、,AS-RH,中性,水/乙腈,酸性,磷酸水溶液,(pH2)/,乙腈,0.1M,磷酸缓冲液,(pH2)/,乙腈,碱性,0.1M KPF,6,水溶液/乙腈,0.1M NaPF,6,水溶液/乙腈,20mM,硼酸缓冲液,(pH9)/,乙腈,20mM,磷酸缓冲液,(pH8)/,乙腈,20,/79,反相流动相的,pH,值对分离度的影响,(AS-RH),中性化合物,Benzoin,酸性化合物,N-CBZ-Norvaline,碱性化合物,Ofloxacin ethyl ester,水溶液,水溶液,/CH,3,CN=60/40,流速,：,1.0ml/min.,水溶液,/CH,3,CN=60/40,流速,：,1.0ml/min.,水溶液,/CH,3,CN=80/20,流速,：,0.5ml/min.,0,5,10,15,mV,0,2,4,6,8,10,12,14,Minutes,7.16,8.77,0,2,4,6,8,10,12,mV,0,1,2,3,4,5,6,7,8,9,10,Minutes,2.25,0.0,1.0,2.0,3.0,4.0,mV,0,5,10,15,20,25,30,35,40,45,50,55,60,Minutes,27.79,水,磷酸水溶液,(pH2),硼酸缓冲液,(pH9),0,5,10,15,mV,0,2,4,6,8,10,12,14,Minutes,7.11,8.70,0,5,10,15,20,mV,0.0,2.0,4.0,6.0,8.0,Minutes,4.17,4.69,0,20,40,60,80,mV,0,5,10,15,20,25,30,35,40,45,50,55,60,Minutes,4.00,4.48,4.77,0,5,10,15,mV,0,2,4,6,8,10,12,14,Minutes,6.97,8.53,0,10,20,30,40,50,60,70,mV,0,1,
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