GE公司培训—凝胶过滤层析技术资料.pdfVIP

1 / GE / Gel filtration 凝胶过滤层析技术 Principles of gel filtration 凝胶过滤原理 Optimize : How to get the expected results 优化 Products and applications 产品和应用 Practical considerations 实际需考虑的因素 Summary 总结 Content 内容 Principles of gel filtration 凝胶过滤原理 Optimize : How to get the expected results 优化 Products and applications 产品和应用 Practical considerations 实际需考虑的因素 Summary 总结 Content 内容 What is gel filtration凝胶过滤定义? Gel filtration (GF) is a simple and reliable chromatographic method for separating molecules according to size and shape. 凝胶过滤层析是根据分子大小和形状的差异进 行分离的一种简单可靠的层析技术。 AGAROSE 琼脂糖凝胶 A good gel for gel filtration contains about 95% water 凝胶过滤填料的 95%为水 Gel structure 填料结构 - pores filled with eluent 孔内充满洗脱液 - carefully controlled size range 孔径精密控制 - chemically /physically inert (lack of adsorptive properties) 惰性 1. Spherical particles packed into a column. 2. Sample applied. 3. Buffer (mobile phase) and sample move through column. Molecules diffuse in and out of matrix 4. Large molecules leave the column first followed by smaller molecules in order of size. Molecules larger than the matrix pores pass straight through. Diffusion Buffer Diffusion into the pores Diffusion out of the pores Buffer 凝胶过滤分离原理: 空间排阻 2 / GE / Steric exclusion leads to early elution 体积排阻程度决定洗脱位置 Molecules elute in order of size. 不同分子按照大小顺序洗出 The largest molecules elute first; the smallest ones elute last. 大分子先洗出, 小分子后洗出 Separation volumes 分离体积 void volume Vo volume of the gel matrix Vs pore volume Vi Vo = void volume 外水体积 VR = retention volume or elution volume 保留体积/洗脱体积 Vt = total liquid volume of the bed 总液体体积 Vi = inner pore volume = Vc - Vs – Vo 内孔体积/内水体积 Vc = total geometric volume of the column 柱体积 Vs = volume of gel matrix 固相凝胶的体积 Vo Vt Vc VR1 VR2 VR3 VR4 Vo VR volume co nc en tr at io n 非常大的分子在外水体积洗出, Vo The void volume Vo 外水体积 volume co nc en tr at io n Vt Vc Vo VR 非常小的分子的洗脱体积为 内水+外水 Vt = V0+Vi 柱床的几何体积, Vc Vt and Vc 总液体体积和柱体积 Distribution coefficient, Kd 分配系数 -VR : 外