Chromatogaphy Assignment Help
This technique is used for separating any mixtures of substance into individual components. These techniques are applicable in many processes such as for separation, for identification and for quantitative analysis of amino acid mixtures , peptides, proteins, nucleotides, lipids and carbohydrates . Here some types are explaining :
a) Column chromatography- This process is successfully used for differentiates proteins using their difference in protein charge , size, binding energy , affinity and other properties .
b) Ion-exchange chromatography- This technique utilize differences in the sign and magnitude of the net charge of proteins at a available pH. The column matrix is a synthetic polymer such as resin which containing bound anionic groups are called cation exchangers and those with bound cationic groups are called anion exchangers. By changing their pH , salt concentration separation will be maximized.
c) Size exclusion chromatography- This technique is also known as gel filteration which separates proteins according to size . In this method , large protein emerges from the column faster than the small ones. The solid phase having cross-linked polymer beads with engineered pores or cavities of a particular size . Large proteins cannot enter into the cavities and so they take a short path through the column , whereas small proteins enter the cavities and are slowed down by the more labyrinthine path through the column.
d) Affinity chromatography- This technique is based on binding affinity and energy . The beads which is present in the column have a easily attached to the chemical group which is ligands . When any protein mixture is added into the column , but here any protein have special affinity for the particular ligand in the column binds with beads , and its migration through the matrix is delayed.
e) High-performance liquid chromatography- This techniques is use always high-pressure pumps which increases the movement of the protein molecules down into the column , as well as higher quality of chromatographic materials which can withstand the compressing force of the pressurized flow .
The various techniques through which it is achieved are discussed in brief as follows:
- Spectroscopy
- Mass Spectrometry
- Chromatography
- Electrophoresis
- Isoelectric Focussing
- Microscopy
- Scatchard Analysis
- FRET
- Blotting
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