or High performance liquid chromatography is the technique by which separation of molecules in a mobile phase liquid by utilising the solid stationary phase. This could be used mainly for chemical analysis or for preparative applications.
The method is allowed work under high pressure of around 400 atmosphere to make the process work very fast instead of allowing the solvent work through capillary action. The high pressure atmosphere actually allows smaller particles for the column packing purpose and wide surface area for the stationary phase and mobile phase to interact.
The principle of High performance liquid chromatography
is the process of separation based mainly on distribution existing two distinct phases where the sample mixture components is actively acted upon by the mobile phase liquid which helps in spreading through the solid stationary phase. This helps in getting various liquids and their stationary phases utilised in chromatographic process.
Based on the manner in which the component mixture elute the liquid chromatography has been segregated into normal phase, reverse phase and flash chromatography.
1. Normal Phase Chromatography
In normal phase chromatography the component of the sample start eluting at varied rates depending mainly upon the comparative polarity. If the column which is getting used for the separation has more polarity compared to the mobile phase then it is considered as a normal phase chromatography.
In this type the stationary phase is found to be polar and hence more of the polar solutes which are separated adsorb to the stationary phase. Due to this the less polar molecules will now get eluted quicker than the ones which getting stuck to the polar stationary phase.
2. Reverse Phase Chromatography
In reverse phase chromatography the polarities are now reversed and the mobile phase solvent now will be considered for polar molecules and hence the gradient is carried in the order of most polar of the solvents first followed by the rest and the least polar going at the end.
The most common of the polar solvent mixtures are water and methanol while the non-polar stationary phase is silica gel coated with any non-polar liquid. The most polar of the molecules in the component mixture will get eluted first and followed by the least polar molecules.
3. Flash Chromatography
This is a modified technique of the same column chromatography process where due to the application of high pressure air or in vacuum, the mobile phase spreads quickly through the column. When a vacuum is created at one end the component mixtures try to move faster through faster in order to reach equilibrium state quickly. It is faster than the gravitational pull. The application of air pressure is manually controlled and hence difficult to control the flow and keep at constant rate.
Other forms of liquid chromatography are as follows.
4. Partition Chromatography
In case of partition chromatography both the phase of stationary as well as mobile are in liquid state. But both of these liquids taken for both phases are immiscible to each other.
5. Liquid-Solid Chromatography
This chromatography method is almost similar to partition but in place of liquid immiscible liquid for stationary phase, silica or alumina based components are taken into the column. The components in mobile phase having affinity for the stationary phase will get adsorbed and those which have les affinity will move faster with lesser retention factor.
6. Ion Exchange Chromatography
This type of chromatography is utilised for separating and determining the presence of ions on column with lower capacity in ion exchange. The working is based on the equilibria existing between ions of the solution and ions fixed to the stationary phase.
7. Size Exclusion Chromatography
The size exclusion method utilises the size of the particles and separates them based on their molecular size. The stationary phase is packed with smaller silica or polymer particles to turn this into a phase with uniform pores. Hence while the components move up the smaller of the particles get trapped while the bigger ones move along.
The entire retention factor is depended upon molecular size, larger the molecules lesser the retention time and smaller the size of particles the greater the retention time.
8. Affinity Chromatography
In affinity chromatography the reagent which binds to the analyte molecules with ligand would be retained in column while the free molecules will move along. The stationary phase is generally made up of porous glass beads which stops the molecules which are bonded.
The elution limits could be altered if the pH strength is changed and is often utilised for protein purification in the field of biochemistry.
9. Chiral Chromatography
The chiral chromatography helps in getting the racemic mixtures separate into their respective enantiomers and this is carried out by adding chiral additive to the mobile phase. Even a chiral stationary phase is opted which helps in identifying the chiral factor in the analyte.
For any liquid chromatography detector the following features are very much essential. The detector has to be sensitive to the components which move across the mobile phase, low noise level, lower limits of detection and bigger dynamic range.Based on these features the following detectors are utilised for liquid chromatography.
Due to the paucity of naturally occurring fluorescent compounds the post column derivation is carried out in fluorescence detectors.
- Photo Diode Array Detector
In this detector huge number of diodes are applied to serve as detector and helps in identifying many molecules at different wavelength. This helps in cost cutting as well as saving time.
- Mass Spectroscopic Detector
Mass spectroscopy helps in identifying molecules by fragmentation due to electric fields and separating these molecules on the basis of mass of the molecules to charge they carry.
In these type of detectors the sample ability to oxidise or reduce on respective electrodes is taken into consideration.
- Light Scattering Detectors
In the light scattering detectors, the ability of molecules to scatter light after passing over heated zone on being removed from the mobile phase is brought into action.