Chiral Development.ppt

1、Chiral Chromatographic Method Development,1.Chiral Chromatographic Analysis,1.1 Chiral Analytical Object,Starting materials Intermediates Final product,One chiral center Two or more chiral centers,1.2 Chromatographic Chiral Method,非色谱法,色谱法,液相色谱 气相色谱 超临界色谱,毛细管电泳,HPLC ：极性大，非挥发性，热稳定性差的样品 GC：有一定的挥发性，热稳定

2、性好的样品 SFC：分析速度快，时间短，消耗溶剂少，在进行柱筛选和制备分离方面有很大优势,Difference between the Chromatographic Method,1.3 Chromatographic Chiral Stationary Phase (CSP),纤维素,淀粉,Class of the CSPs,Derivation of the CSPs,3，5-二甲苯氨基甲酸酯,(S)-1-苯乙基氨基甲酸酯,(S)-1-萘乙基氨基甲酸酯,对甲苯基甲酸酯,（rigid & linear）,纤维素和淀粉类CSPs,- Cyclodextrin,环糊精类CSP,手性冠醚类CSP

3、,奎宁和奎纳定类CSP,Quinine衍生物 CHIRALPAKQN-AX：(8S, 9R) Quinidine衍生物 CHIRALPAK QD-AX：(8R, 9S) 反相条件下，相当于阴离子交换手性柱，分离酸性化合物 正相条件下，相当于刷型手性柱，可以分离碱性和中性化合物,1.4 Theory of the Chiral Selectivity of the CSPs,Mainly due to the difference of the interaction between the chiral stationary phase (CSP) in the column and the

4、two isomers. The enantioselectivity is the result of the corporation of the several interactions.,Cellulose,Amylose,Dipole-dipole,stacking,Hydrogen bond,-,O,O,O,O,O,R,R,R,R,R,R,N,H,H,H,H,H,N,N,Y,Hydrogen bond,Hydrogen bond, -, Dipole-Dipole interaction,Cyclodextrin,Derivatived Cellulose,Derivatived

5、Amylose,Chiral grooves,Spacial Affect,Hydrophobic Interaction,环糊精外缘亲水，内部形成疏水空腔,Hydrogen bond &Spacial affect,1.5 Commercial Chiral Chromatographic Column,Daicel 涂覆型手性固定相,Daicel 键合型手性固定相,流动相种类和溶解样品的溶剂有更多选择 溶剂耐受性好，寿命更长 柱效高 正、反相都可以使用 可以再生,ASTEC键合型手性固定相,耐压 (MPa)：010MPa 温度范围()：10 40 pH范围：2 8,HPLC手性柱的使用范围

6、,Agilent GC Chiral Column,hp-chiral 10 hp-chiral 10 -DEX 120 -DEX 110 CycloSil-B Rt-DEX,温度范围()：30 240 / 250 ,1.6 Mobile Phase,Mobile phase A: Hexane/Heptane Mobile phase B: Methanol/Ethanol/Isopropanol Acidic additive : TFA/Acetic acid 0.5% Basic additive: TEA/DEA 0.5% 涂覆型的手性色谱柱下列溶剂决不允许使用，包括样品溶剂：二甲基

7、甲酰胺、DMSO、二噁烷、甲苯、三氟醋酸、氯仿、氯化甲烷、丙酮和乙酸乙酯等,Normal Phase,Reverse Phase,Moblie phase for IA/IB/IC/QN-AX/QD-AX/ Chirobiotic Columns MTBE/Heptane/Hexane/Toluene/DCM/Chloroform/ Ethyl acetate/Ethanol/Isopropanol/THF/Acetone/1,4-dioxane,键合型手性色谱柱可以使用的流动相,特种手性色谱柱的流动相,Moblie phase for CR Column 0.1% HClO4 in wate

8、r/Methanol=100:085:15 storage: water Moblie phase for MA（+）/WH Columns 0.12.0mM CuSO4 in water/Methanol=100/085/15 storage:2mM CuSO4 in water,2. Chromatographic Chiral Method Development,2.1 Affective factors for the enatioselectivity,Chiral Columns,Chiral column is the heart of the chromatographic

9、chiral separation. Every type of CSP has the unique mechanism of enatioselectivity and is fit for the a kind of compounds having a sort of functional group or structure.,流动相 ：Hex./IPA/ AcOH = 90/10/0.1 流量：1.0ml/min. 温度 ：40 检测波长：UV254nm,Moblie Phase,The kind the Moblie Phase solvent Elution ability u

10、nder normal phase: Hexane Heptane Methanol Ethanol Isopropanol Elution ability under reverse phase: ACNEthanol Methanol The enatioselectivity may be very different using different moblie phase. The concentration of the eluent Generally, the better resolution could be obtained when the concentration

11、of the eluent was decreased, but the peak would be more broad and tailed .,Column: OD-H 4.6*250 mm, 5 m Oven temperature: 20 Flow rate:1.0 mL/min,Hexane/Ethanol =99:1 (v/v),Hexane/Isopropanol =99:1 (v/v),水/乙腈 =50/50,水/乙醇 =35/65,水/甲醇 =25/75,2-Methyl-1-tetralone,2-Phenyl-2-butanol,手性柱：CHIRALPAK AD-RH

12、、流速：0.5ml/min、温度：25,Column：AD, Temp.：40, Flow rate：1.0ml/min.,Hex./IPA/ AcOH =90/10/0.1,Hex./IPA/ AcOH =100/1/0.1,Flurbiprofen,Additive of the moblie phase,The proper acidic or basic additive will improve the resolution and the peak shape.,碱性样品,Hex/IPA/DEA=90/10/0.1,流动相Hex/IPA=90/10,Hex/IPA/AcOH=90/

13、10/0.1,流动相Hex/IPA=90/10,酸性样品,（没出峰 ),（无法分离 ),水溶液,水溶液/CH3CN=60/40 流速：1.0ml/min.,水溶液/CH3CN=60/40 流速：1.0ml/min.,水溶液/CH3CN=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,水,磷酸水溶液 (pH2),硼酸缓冲液 (pH9),0,5,10,15,20,mV,0.0,2.0,4.0,6.0,8.0,Minutes,4.17,4.69,0.0,2.0,4.0,6.0,8.0,0,5,10,15,20

14、,25,30,35,40,45,50,55,60,Minutes,30.74,36.78,Column Oven,Generally, the better resolution could be obtained when the column oven was decreased, but the peaks would be more broad and tailed.,Column：CHIRALPAK AD Mobile phase： Hex./IPA/ AcOH =90/10/0.1, Flow rate：1.0ml/min.,25,40,Temp.： 5,Flurbiprofen,

15、升温程序：恒温/近似恒温的缓慢升温 分流比：分流要比大一些,Affective Factors for GC Chiral Method Development,色谱柱：Supleco Betadex 120（30m*0.25mm ID*0.25m） 检测器温度：FID 230 oC 柱流速：29 Psi，恒压模式 柱温：105 oC (30 min) 30 oC /min210 oC (6.5 min) 进样口温度：220 oC 进样分流比：100：1,2.2 Flowchart for Chromatographic Chiral Method Development,Choose one

16、 column and proper mobile phase,Add acidic or basic additiveChange the moblie phase,Change the column,Further modification,Further modification,Increase column oven Increase concentration of the eluent Mix two or several eluents togetherModify the percent of the additive,Choose the 25cm length of co

17、lumnDecrease column oven Decrease concentration of the eluentChange the moblie phase Mix two or several eluents together,Increase column oven Choose the 15cm length of column Increase concentration of the eluent Mix two or several eluents together Change the moblie phase,2.3 Strategy for Chromatogra

18、phic Chiral Method Development,一般首选正相，首选正己烷/乙醇 建议先采用高比例的洗脱剂将样品中所有组分冲出，然后再降低洗脱剂的浓度。例如：70%B50%B30%B10%B。尤其在样品组分复杂，纯度不高或化合物极性较大时。 当化合物出峰比较难洗脱，或在正相中难以得到理想的峰形时，改为反相进行尝试。 如果尝试了一根纤维素的柱子，分离效果比较差时，可以优先考虑尝试淀粉的柱子。 如果使用甲醇/乙醇或异丙醇的组合，必须保证甲醇乙醇/异丙醇（并且甲醇含量不宜超过8%）。 样品一般用烷烃/醇进行溶解，若很难溶解，可以使用键合型色谱柱，或尝试反相条件。,反相色谱中，建议优先尝试的水相与有机相的比例是50/50（v/v）。 反相色谱中，在已经实现分离的基础上，可以采用梯度以获得更好的峰形和合适的保留时间。 如果已有部分分离，也可以将两根相同的色谱柱进行串联；多个手性中心，不同的色谱柱表现出不同的选择性时，可以考虑进行串联。 在两个或多个手性中心的情况下经常碰到用酸用碱有不同的效果，可以将酸碱添加剂混在一起,2.4 Warning and No