HPLC原理以及应用.ppt

* * * * * Not only is it important to use 缓冲液ed 流动相s, optimizing the 缓冲液 concentration can improve 色谱峰峰形. This is especially important at mid pH where the 缓冲液 can mask 硅羟基interactions between the analytes 和residual 硅羟基. The example here shows a group of basic compounds, antidepressants, at pH 7 with both 10 mM 和25 mM 磷酸盐缓冲液trength. The 拖尾因子s of these compounds are all lower at 25 mM 磷酸盐缓冲液than at 10 mM. The increased 缓冲液 strength 降低the effect of interactions with the charged 硅羟基. We typically recommend a 缓冲液 strength of 25 mM to start. This will give you more consistent control of the 色谱柱 硅羟基 和the 离子化 of your analytes resulting in a more reproducible method. * * * This slide shows just why we say that梯度 equilibration is not a limitation when using 短色谱柱. Most色谱柱 require “ ten时间s their色谱柱 volume” to obtain reproducible retention during the梯度 run. A 标准4.6 x 150 mm色谱柱 has an internal volume of 1.54 mL, which means that 15 mL of溶剂are requi红色 to equilibrate that色谱柱 –和at a 流速 of 1.0 mL/min. it takes 15 minutes to accomplish this task. For the 短 4.6 mm i.d.色谱柱 this is 短en considerably from 5 – 1.5 minutes.和for the 2.1 mm i.d.色谱柱 operated at 1.0 mL/min., equilibration时间 is 缩短d to a 60 to 18 seconds….it takes more时间 to ready the next injection. * Flush the column with stronger solvents than your mobile phase in order to clean it. If you are cleaning a reversed-phase column then some appropriate solvent choices are listed here. You should use at least 25 mL of each solvent for a standard 4.6 mm 内径 analytical column. Smaller 体积s can be used with smaller 内径 columns. ? The first step is to wash the column with the mobile phase without buffer salts. This will wash out the salts and eliminate any possibility of precipitation before going on to stronger solvents. Methanol and acetonitrile are the most common wash solvents and the first choice would be the one in your mobile phase. Then I like to use a mix of ACN and IPA. It will eliminate a lot of contamina