Chromatography Techniques

Chromatography as Separation Technique

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High Performance Liquid Chromatography (HPLC) is a non-destructive procedure for resolving a complex mixture into its individual fractions or compounds. It is based on differential migration of solutes with the solvents. The solutes in a mobile phase go through a stationary phase. Those solutes with a high affinity for the mobile phase will spend more time in this phase than the solutes that prefer the stationary phase. As the solute rise up through the stationary phase they separate. The process is called chromatographic development. The fraction with greater affinity to stationary layer travels slower and shorter distance while that with less affinity travels faster and longer.

In normal-phase chromatography, the stationary phase is polar and the mobile phase is nonpolar. In reversed phase the stationary phase is nonpolar and the mobile phase is polar.

Flash Column Chromatography (FCC) or Flash Chromatography is a quickest and the easiest way to separate complex mixtures of compounds. It uses compressed air to push the solvent through the column. This provides better separation and reduces the amount of time required to run a column.

Ion exchange chromatography (IEC) uses an ion exchange mechanism to separate analytes based charge difference. It is performed in columns but can also be useful in planar mode. It uses a charged stationary phase to separate compounds including cations, anions, amino acids, proteins, lipids and peptides. Loading samples in buffers of low ionic strength makes ion exchange chromatography an excellent purification step after HIC.

Affinity chromatography is based on selective non-covalent interaction between an analyte and sample molecules. It is highly specific, but not robust method. It is used in biochemistry in the purification of proteins bound to tags. Fusion proteins are labelled with compounds such as antigens or biotin which bind to the stationary phase specifically. Later after purification, some of these tags are removed and the pure protein is obtained. Immobilized Metal Affinity Chromatography is used to separate molecules based on the relative affinity for the metal. Often these columns can be loaded with different metals to create a column with a targeted affinity.

Chiral chromatography includes the separation of stereoisomers. Enantiomers have no chemical or physical differences apart from being three-dimensional mirror images. For chiral separations to happen, either the mobile phase or the stationary phase must themselves be made chiral by inducing different affinities between the analytes.

Size-exclusion chromatography (SEC) also known as gel filtration chromatography, separates molecules according to the size or more accurately according to the hydrodynamic diameter or the hydrodynamic volume. Small molecules enter the pores of the media where they are trapped and removed from the flow of the mobile phase. Mean residence time in the pores depends upon the effective size of the analyte molecules. It is a low-resolution chromatography technique and thus it is used for determining the tertiary structure and quaternary structure of purified proteins.