疏水相互作用色谱.ppt

 Umair Saleem Methods in protein chemistry ■ Alternatives ? Gel filtration chromatography ? Ion exchange chromatography ? Reverse phase chromatography Why HIC? ? Different basis of separation ? Weaker interactions → Less structural damage → Maintain high activity Contents → Purpose → Principle of HIC → Advantages of using HIC → What are the factors affecting HIC → Conclusion Source of protein Extraction Separation Purity characterization ■ Purpose ? Downstream purification ? Separation of biomolecuoles ? Exploits differences in hydrophobicity. → Number of hydrophobic aminoacids. → Distribution of these aminoacids. Principle ? Separation of substances is based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix ? Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix. ? How is this achieved? Source of protein Extraction Separation Purity characterization General Concept Hydrophobic Interaction Chromatography Experimental Technique ■ Choice of column → XK colums for HIC. ? Column dimensions → Short bed height (5-15 cm) suitable for HIC ■ Packing of Column: a modern, highly crosslinked agarose-based gel such as Sepharose Fast Flow is however easier than packing a gel filtration column since the bed height required is much smaller. ■ Sample preparation ? Sample composition ? Sample volume ? Sample viscosity ■ Sample application ■ Batch Separation Advantages of HIC Large volume of sample can be loaded Samples with high ionic strength can be used Well suited to use before gel filtration, ion-exchange and affinity chromatography Sample eluted with low salt Purification steps that generate large sample volume can be coupled with this method Good for samples after ammonium sulfate fractionation. These techniques may require pretreatment of samples (e.g. reducing ionic strength) Sample can be used in ion exchange c