资源描述
,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,2.Gel Chromatography,Separate Casein and Riboflavin by Sephadex G-75,Purpose,basic operation of,gel chromatography,(,凝胶层析,),principle of separating,riboflavin,(,核黄素,),and casein by Sephadex G-,75,welcome to use these PowerPoint templates,New Content design,10 years experience,principle,1.,Conception,Gel chromatography,inanalytical chemistry,technique for separating chemical substances by exploiting the differences in the rates at which they pass through a bed of,a porous,semisolid substance(gel),.The method is especially useful for separating,enzymes,proteins,peptides,and amino acids,from each other and from substances of lowmolecular weight.,Gel Chromatography,Gel Filtration,(Porath,Flodin,1959),Gel permeation chromatography(GPC),Size-exclusion chromatography(SEC),molecular sieve chromatography,(Hjertn,Mosbach,1962),exclusion chromatography,(Pedersen,1962),2.,固定相（凝胶）,三维空间网状结构,交联剂越多,交联度越大,网孔结构越紧密,交联剂越少,交联度越小,网孔结构越疏松,Sephadex,Beads are prepared by cross-linking,dextran,(,葡聚糖,),with,epichlorohydrin,(,环氧氯丙烷,),.Widely used in industrial processes.Also useful in desalting,(,脱盐,),and buffer exchange.,Sephadex,的分级范围,G10 700,G15 1,500,G25 1,000 5,000,G-50 1,50030,000,G-75 3,00070,000,G100 4,000 150,000,G150 5,000 400,000,G200 5,000 800,000,吸水性,*G,类的型号,每克干凝胶吸水量（,ml,）,x,10,*,如,G-75,每克干凝胶吸水量为,7.5 ml,样品,固,定,相,流,动,相,小分子,被,延滞,小分子,大分子,较,快流出,molecular sieve effect,(,分子筛效应,),V,e,-V,o,K,d,=,V,i,分配系数,K,d,：,特点：,（,1,）与被分离物质分子的大小和凝胶颗粒孔隙的大小有关,（,2,）与柱的长短粗细无关,每个溶质分子在流动相和固定相之间有一个特定的分配系数,(K,d,),应 用,脱 盐,测分子量,分离纯化,Ve,Albumin,NaCl,Ve,Ve,优点,缺点,凝胶层析的优缺点,凝胶是一种不带电荷的惰性载体，结构稳定,要求,样品和洗脱液的,粘度很低,操作条件较温和,凝胶颗粒网络,孔径大小有限,可被纯化物质的,分子量范围受限制,样品得率高,重复性好,可进行大量分离纯化,凝胶结构,对某些分子具有吸附作用,(1),棉花装填量及松紧度适中；,(2),凝胶不能有气泡和分层现象；,(3),装柱结束后保持凝胶表面平整。,1.,装柱,使用滴管，柱床表面以上洗脱液不得流干。,Notes,2.,洗脱,3.,加样,(1),洗脱液液面与凝胶柱床表面相切，关闭出口；,(2),加样时使用滴管，尽量不破坏凝胶表面；,(3),样品进入凝胶后再加缓冲液开始洗脱。,
展开阅读全文