疏水相互作用色谱讲述

■ Sample preparation • Sample composition • Sample volume • Sample viscosity ■ Sample application ■ Batch Separation
Advantages of HIC

Large volume of sample can be loaded
Principle
•
Source of protein
•
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.
OH O C H 2 C H CH 2 O ( C H 2) 3 CH 3 Butyl
OH Oຫໍສະໝຸດ BaiduCH 2 C H CH 2 O
( C H 2) 7
CH 3
Octyl
OH O C H 2 C H CH 2 O
Phenyl
Effects of ligand density
Degree of substitution • ‰ Binding capacity of protein to HIC increases with increased alkyl chain length (A) and increased degree of substitution of immobilized ligand (B) • ‰ Caution: protein can bind via multipoint attachment, thus difficult to elute
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
Factors affecting HIC
Type
and concentration of ligand Type of base matrix. Type and concentration of salt pH Temperature Additives
HIC Ligands
Contents
→ Purpose → Principle of HIC → Advantages of using HIC → What are the factors affecting HIC → Conclusion
Source of protein
Extraction
Separation
Hydrophobic interaction chromatography
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
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.
Extraction
Separation
• How is this achieved?
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
Purity & characterization
■ Purpose
• Downstream purification • Separation of biomolecuoles • Exploits differences in hydrophobicity. → Number of hydrophobic aminoacids. → Distribution of these aminoacids.


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 chromatography step