Fundamentals and Advances in Liquid Chromatographic Separation Techniques

Chromatography is a non-destructive procedure for resolving a complex mixture into its individual fractions or compounds. Chromatography 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 solutes rise up through the stationary phase they separate, the process 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 quick and easy way to separate complex mixtures of compounds. FCC uses compressed air to push the solvent through the column. This helps provide 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 on their respective charges. It is performed in columns but can also be useful in planar mode. IEC uses a charged stationary phase to separate charged compounds including cations, anions, amino acids, proteins and peptides. Conventional methods use stationary phase having an ion exchange resin that carries charged functional groups that interact with oppositely charged groups of the compound to retain. IEC is commonly used to purify proteins using fast protein liquid chromatography (FPLC).

Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size or more accurately according to their hydrodynamic diameter or 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. Molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. It is a low-resolution chromatography technique and thus it is often reserved for polishing step of purification and is also useful for determining the tertiary structure and quaternary structure of purified proteins as can be carried out under native solution conditions.

Affinity chromatography is based on selective non-covalent interaction between an analyte and sample molecules. It is highly specific, but not robust. It is used in biochemistry in the purification of proteins bound to tags. Fusion proteins are labelled with compounds such as His-tags, 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. Affinity chromatography utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Earlier affinity columns are used as a preparative step to flush out unwanted biomolecules. Some HPLC techniques utilize the properties of affinity chromatography. IMAC- Immobilized Metal Affinity Chromatography is used to separate aforementioned 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. Traditional chromatography or other separation processes are incapable of separating these. For chiral separations to happen, either the mobile phase or the stationary phase must themselves be made chiral by inducing differing affinities between the analytes.

Liquid Chromatography separation techniques are now mostly used due to their versatility, ease of handling and accurate chromatograms compared to solid-liquid techniques. These include HPLC, HPTLC and UHPLC being the major ones. Others include some of the Hyphenated HPLC methods.

  • Normal phase chromatography
  • Reverse phase chromatography
  • Flash chromatography
  • Ion exchange chromatography
  • Size exclusion chromatography
  • Affinity chromatography
  • Chiral chromatography
  • Capillary electrochromatography
  • Optical Force Chromatography

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