液相色谱方法开发策略

Xterra RP18 At pH 3
0-80% gradient ACNH20, 10mM buffer, pH 3 0-80% gradient MeOHH20, 10mM buffer, pH 3
成功分离?
否
是
方法再优化
成功分离?
是
方法再优化
©2005 Waters Corporation
SunFire C18柱上的成功故事: Lansoprazole (等度分离)
pH 5.6
9.00
10.00
分析方法对pH敏感度的测试- 糟糕的结果
1. p-Toluamide 2. Lidocaine 3. Ibuprofen
0.40
Column: XTerra® RP18 4.6 x 100 mm, 5 µm Mobile Phase A: 20mM Ammonium Acetate (pH 5.0 to 5.8) or 20mM Ammonium Bicarbonate (pH 6.8 to 7.0) 0.30 Mobile Phase B: ACN Flow Rate: 0.5 mL/min Isocratic Mobile Phase Composition: 40% A; 60% B Injection volume: 10µL Temperature: 30oC Detection: UV @ 230 nm Instrument: AllianceTM 2690, 996 PDA 0.40 0.30 0.20 0.10 0.00 1.00 0.40 0.30 0.20 0.10 0.00 1.00
©2005 Waters Corporation
转向高 pH分离!
如何实施低pH下的三柱分析方案?
换柱前谨记探索流动相溶剂所带来的分离选择性
Sunfire C18 At pH 3 0-80% gradient ACNH20, 10mM buffer, pH 3 0-80% gradient MeOHH20, 10mM buffer, pH 3 成功分离?
©2005 Waters Corporation
1 2
1.00 2.00 3.00
pH 5.0
3
0.10 0.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
0.40 0.30 0.20 0.10 0.00 1.001Βιβλιοθήκη pH 5.23 2
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
0.080
Lansoprazole
0.070
pH 3.0 73%A: 27%B PA = 0.090 PT = 0.293
0.060
0.050
0.040 AU
0.030
0.020
Chromatographic Conditions : Columns: SunFireTM C18 4.6 x 150 mm, 5.0 µm Mobile Phase A: 20 mM NH4COOH, pH 3.0 Mobile Phase B: Acetonitrile Flow Rate: 1.4 mL/min Isocratic: as indicated Injection Volume: 2.0 µL Sample Diluent: 57:43 H2O:ACN Sample Concentration: 1.42 mg/mL Temperature: 30 oC Detection: UV @ 254 nm Sampling rate: 10 pts/sec Time Constant: 0.1 Instrument: Waters Alliance® HT 2795, with 2996 PDA Degradation Conditions: Temperature: ambient 50 mg of Lansoprazole + 5 mL of 0.4N HCl stirred for ~ 30 seconds Lansoprazole degraded ~ 43%
1
pH 5.4
3 2
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
1. p-Toluamide 2. Lidocaine 3. Ibuprofen
1 3 2
2.00 3.00 4.00 5.00 6.00 7.00 8.00 Grumbach, Diehl
峰拖尾: 28%
Peak Waters Tailing Retention Reproducibility
Peak Tailing
分离不佳: 24% Retention
Reproducibility
保留时间不重现: 25%
©2005 Waters Corporation
影响分离度的若干因素
柱效
N ↑ 2, R ↑ 40%
Xterra RP18 At pH 3
仍不成功?
SymmetryShield RP18 At pH 3
• For 2 liters of 10mM buffer, weight out 1.26g of
NH4COOH
• Add 2 liters of water and stir until dissolved • Adjust to pH 3 with formic acid
保留因子
K最佳=5
k2 N (α − 1) Rs = ´ ´ ÷ α (k 2 + 1) 4
分离选择性
(方法开发归根结底是如何使得α ≠ 1)
©2005 Waters Corporation
如何在实践中调控 ‘α’?
这是方法开发的根本!
流动相溶剂选择
流动相pH
α
分离柱
©2005 Waters Corporation
是否属亲水 性化合物?
柱寿命长 pH3为稳定保留区， 为稳定保留区，方法稳 定可靠
Atlantis dC18 At pH 2-5
不成功?
不肯定?
无充 分保 留!
Sunfire C18 At pH 3
不成功?
Atlantis HILIC At pH 2-5
Preparation of 10mM NH4COOH buffer at pH 3:
pH 7.0
2
3
2.00
3.00
4.00
5.00
6.00
7.00 8.00 9.00 Grumbach, Diehl
10.00
分析方法对pH敏感度的测试- 优异的结果
©2005 Waters Corporation
杂化颗粒柱对创建稳定可靠的 HPLC分析方法的独特价值
稳定保留区 稳定保留区
100
0.050
0.040
pH 3.0
0.030
AU
73%A: 27%B tR = 9.3 min N = 12,100 T = 1.08 k’ = 8.3
0.020
Chromatographic Conditions : Columns: SunFireTM C18 4.6 x 150 mm, 5.0 µm Mobile Phase A: 20 mM NH4COOH, pH 3.0 Mobile Phase B: Acetonitrile Flow Rate: 1.4 mL/min Isocratic: as indicated Injection Volume: 2.0 µL Sample Diluent: 75:25 H2O:ACN Sample Concentration: 350 µg/mL Temperature: 30 oC Detection: UV @ 254 nm Sampling rate: 10 pts/sec Time Constant: 0.1 Instrument: Waters Alliance® HT 2795, with 2996 PDA
(未离子化)
(未离子化)
碱1
中性化合物
碱 1+2
(完全离子化)
0 2 4 6 8
酸
(完全离子化)
pH
10
12
14
B1 B2
N
N
pH 5.5
A, B1
B2
A
pH6.0
©2005 Waters Corporation
分析方法对pH敏感度的测试- 糟糕的结果
0.40 0.30 0.20 Column: XTerra® RP18 4.6 x 100 mm, 5 µm Mobile Phase A: 20mM Ammonium Acetate (pH 5.0 to 5.8) or 20mM Ammonium Bicarbonate (pH 6.8 to 7.0) Mobile Phase B: ACN Flow Rate: 0.5 mL/min Isocratic Mobile Phase Composition: 40% A; 60% B Injection volume: 10µL Temperature: 30oC Detection: UV @ 230 nm Instrument: AllianceTM 2690, 996 PDA 0.40 0.30 0.20 0.10 0.00 1.00 0.40 0.30 0.20 0.10 0.00 1.00
©2005 Waters Corporation
0.20 0.10 0.00 1.00
1 3 2
2.00 3.00 4.00 5.00 6.00 7.00 8.00
pH 5.8
9.00
10.00
1 3 2