层析法报告.pptVIP

V0 = retention volume of unretarded solute Vr1-3 = retention volume of substances in peaks 1-3 w1/2 = peak width at half peak height a = front distance at 0.1 x peak height b = tail distance at 0.1 x peak height Peak definitions Volume 1 2 3 A Volume Vr1 peak height, h half peak height w1/2 A a b V0 Vr1 Vr2 Vr3 2. 分离度 Rs 3.分离系数 α α代表了两个物质在相同的色谱条件下的分离选择性。 物质的化学性质或结构上的差异，反映在固定相和移动相之间的作用力也有差异，这就是层析分离的基础。 k2’ tr’2 α= —— = —— k1’ tr’1 α-1 k2’ Rs= ? N ( —— ) ( —— ) α 1+k2’ 提高分离度可以改变 α、k’、N三个参数来实现 3. 样品区带扩散的因素 涡流扩散 纵向扩散 传质速率、 柱内扩散 柱外扩散 样品、层析介质、装柱、层析条件、死体积 purity yield ( recoverable) specific activity Gradient slope Column: μRPC C2/C18 2.1/10 Sample: Lys-C digest of ADH, 50 pmol Gradient: 0.66% B / min 5 9, 11 20-21 31-34 2 2 5 9 11 20 21 34 31 Gradient: 0.23% B / min 20-21 31-34 9, 11 5 2 21 20 9 11 5 2 31 34 Gradient: 0.23% B / min Gradient: 0.66% B / min 5 0 1 0 0 1 5 0 0 0 . 0 0 0 . 0 2 0 . 0 4 0 . 0 6 0 . 0 8 2 0 . 0 4 0 . 0 6 0 . 0 0 . 0 0 . 0 0 0 . 0 2 0 . 0 4 0 . 0 6 0 . 0 8 5 0 1 0 0 1 5 0 0 0 . 0 0 0 . 0 2 0 . 0 4 0 . 0 6 0 . 0 8 5 0 1 0 0 1 5 0 0 0 . 0 0 0 . 0 2 0 . 0 4 0 . 0 6 0 . 0 8 min min min min AU (215 nm) AU (215 nm) AU (215 nm) AU (215 nm) * In ion exchange and other adsorptive techniques, flow rate has a small effect on the separation , especially when using modern, high resolution media, compared with gel filtration where flow rate is a critical parameter. However, there is an optimum flow rate for minimum band broadening. Optimal resolution for all peaks is seen here at 200 cm/h. Depending on the target peak and the level of purity required, flow rates up to 1800 cm/h could be used. Using those high flow rates, chromatographic separations within 2-5 minutes are possible. TIP: To optimise the separation for highest resolution/purity, reduce the flow rate to the maximum that provides sufficient resolution. TIP: To optimise