AKTA--实验室层析柱装柱技术介绍.pptVIP

实验室层析柱装柱技术介绍 Column packing 好的柱效是层析成功的关键 不同种类的凝胶、不同的层析柱有不同的装柱方法 柱效测定的方法 层析柱的维护 Column packing Column packing Column packing Column packing 检测装柱效果一般用0.5% (30um介质)或1% (90um介质) 柱体积的1%丙酮测柱效和峰形 The linear flow rate should be 30 cm/h for 34 μm and 50 μm media and 20 cm/h for 90 μm media 线性流速 对体积不同的柱子来说，体积流速不能直接用作比较， 一般都换算成线性流速，再作比较： 体积流速 (ml/min) x 60 线性流速 (cm/hr) = 揪揪揪揪揪揪 柱子横切面积 (cm2) Column packing It is important that the column is properly equilibrated before the packing is evaluated (~ 2 column volumes). It is preferable to run three test runs to see whether the test values are stable. If a poor result improves during this test, the reason can be that the column was not properly equilibrated. To check that the bed is stable, run the column at 70% packing pressure for 20 hours and test it again (preferably three test runs). Note that pressure spikes may cause poor packing (cracking). If this happens, an air trap and a pressure relief valve are needed between the pump and the column. The pressure relief valve should be placed between the air trap and column. Column packing 装柱注意点 选择一根设计良好的柱子 仔细混合均匀凝胶浆 在生产时使用柱子的温度下装柱 用平缓的动作将凝胶浆一次性加入柱子中 用恒压或恒流装柱 稳定和平衡凝胶柱床 检查柱效，必要时重装 用空柱子须留意. 使用调节器(flow adaptor) - 保护柱床的同时更可提高分辨率，节省凝胶 柱子物料须生物兼容，并有优良的化学抗性 加上装柱器(packing reservoir)，可一次把凝胶装满柱子，大大提高柱效，尤其是凝胶过滤柱。 柱子的过滤网孔径应该是凝胶颗粒的1/3, 一般10mm孔 径就够用，如用SOURCE15介质装C柱和XK柱，须换上5mm的网 Column packing Sepharose FF based gel: Sepharose Q, DEAE, SP, Phenyl 1. the 75 % slurry just before packing. 2. Ideally, Fast Flow media are packed at a con stant pressure not exceeding 3 bar (0.3 MPa) for XK columns. 3. If the packing equipment does not include a pressure gauge, use a packing flow rate of at least 400 cm/h (15 cm bed height, 25℃). If recommended pressure or flow rate can not be obrained, use the maximum flow the pump can deliver, this should also give a easonably well-packed gel. Do not exceed 75 % of the packing flow rate in subsequent chromatographic procedures