Column chromatography (portable peristaltic pump)
In the past 30 years, various techniques and methods for the separation of biological macromolecules have been continuously developed. With the improvement and commercialization of various media, column chromatography based on fillers plays an important role in the purification of natural products after crude extraction.
1. Hydrophobic Interaction Chromatography (HIC)
HIC is based on protein hydrophobicity. Specifically, it is a column chromatography method that separates proteins by differences in surface hydrophobicity.
The formation of hydrophobic bonds has no specific requirements for non-polar groups, and the number, size, strength, distribution, and steric hindrance of hydrophobic zones on the surface and inside of different proteins are different, which is also the fundamental reason for HIC protein separation. Among the 20 amino acids, 8 are hydrophobic amino acids, and their strength order is Trp, Ile, Phe, Pro, ValLeu, Met, and Ala. When the medium and sample protein concentration is unchanged, more factors are affecting HIC, mainly salt, its role is to expose the non-polar base of the interface so that it is easy to form hydrophobic effect, followed by the increase of water polarity, improve the surface tension of the interface, the higher the salt concentration, promote hydrophobic effect is stronger. Generally, (NH4)2SO4 is commonly used to promote salt thinning. ② Miscible organic solvent, the function is to weaken the interfacial tension, reduce the hydrophobic effect. ③ Temperature, when the system temperature rises from 4℃ to 25℃, the hydrophobic force increases by 20% ~ 30%, which is mainly due to the increase of kinetic energy, improve the surface tension, generally when the temperature is reduced, can increase the salt concentration, so that the hydrophobic effect is not reduced. (4) pH, when pH≈pI can eliminate the repulsion of surface charge, and surfactants and denaturing agents also have an effect on it.