细胞生物化学实验教材Exp 2-Chromatography- Jiao Li.pptx

Chromatography——1. Gel chromatography （ separating hemoglobin and protamine ） 2. Ion exchange chromatography ( separating amino acid );Overview; Objective To understand the principle of chromatography and basic operation of column chromatography. ;Principle;3.Principle: The separation is based on physicochemical features of components in analyte mixture. ;B. Techniques by chromatographic bed shape: ① Paper chromatography ② Thin layer chromatography ③ Column chromatography;5. How can we evaluate Chromatography ?;②. Peak width ( W ) : an index that indicates the efficiency of separation. ;③. Partition coefficient: The ratio of concentrations of a compound in mixture between the two phases at equilibrium.;④. Resolution (Rs)： Degree of separation between components of an analyte on a chromatography system.;Which separation is good?; ; ;In this experiment: (2) Analyte: protein mixture of hemoglobin(mw:64500, red ) and protamine (mw:2000-12000, yellow- dinitrofluorobenzene treated) (3) Mobile phase: distilled water;3. Procedure ① Filling the column ② Loading the samples ③ Collecting the samples ; ;Experiment 2 (2) Ion exchange chromatography;In this experiment: (1) Stationary phase: Dowex 50 (cation exchange resin) (2) Analyte: amino acid mixture of Asp (pI:2.97) and lys (pI:10) (3) Mobile phase: citric acid solution (pH:4.2), NaOH ; ; 3. Procedure : ①. Filling the column ②. Loading the samples ③. Collecting the samples(6 tubes for each eluent) ;Results and analysis;Notes;Thought questions