High performance (pressure) liquid chromatography is an improvement of the classical method of liquid or partition chromatography. In liquid chromatography, a solute (or solutes) is partitioned between two immiscible liquids.
In other words, both the mobile phase and the stationary phase are liquids.
The main components of HPLC are:
A reservoir system for the mobile phase: The solvent (mobile phase) reservoir System consists of one or More glass or stainless steel vessels which can contain 1 to 2 Litre of solvent. There are two types of elution techniques. If a single solvent is used, the separation is called isocratic elution. Separation efficiency can be increased by using two or more solvents. Such an elution is called gradient elution. This type of elution shortens the time of separation without seriously affecting the resolution of peaks.
Dissolved oxygen and nitrogen in the solvent form bubbles in the analytical column and the resolution of peaks are affected therefore dissolved gases are removed by degassers. If two or more solvents are used, the solvents from the reservoirs are mixed in the mixing vessel and pumped into a pre-column by means of a high pressure pump having an output of at least 100 psi.
The precolumn contains the same packing as is used in the analytical column. The functions of the precolumn are
(i) to remove impurities in the solvent and
(ii) to saturate the mobile phase with the liquid stationary phase.
This second step prevents removal of the stationary phase from the packing in the analytical column.
Then there is a flow splitter which splits the flow of mobile phase in two equal half.
The experimental sample is injected into the column by means of a self-sealing Teflon or neoprene septum. Sometimes, the sample can also be introduced, by stopping the flow of solvent for a moment, removing the cap on the column and injecting the sample on to the column.
The analytical column consists of a stainless tube about 15 to 150 cm in length. The selection of the column packing depends on the chemical nature of the sample components and the mobile phase to be used. The column is generally surrounded by a water jacket to maintain a constant temperature.
In general, three types of particles are used for column packing in HPLC.
1. Microporous particles consist of crosslinked networks with particle diameter about 510 µm. Only small molecules of the solute are accessible to the pores of these particles.
2. Macroporous particles have particle diameters of about 60 µm. These are accessible to small as well as big solute molecules.
3. Pellicular particles have an inert core surrounded by a film of the stationary phase. The particle diameter of these particles is 35 to 45 µm.
The high pressure and speeds, involved in HPLC, sometimes tend to mechanically remove the stationary liquid phase from the solid support. To overcome this difficulty, chemically bonded stationary phases have been developed. These consist of organic groups attached to silica gel.
E.g, a hydrocarbon surface can be formed by the reaction of trialkyl monochlorosilane with the -OH groups on the surface of silica gel. The organic parts stand on one end on the surface of the gel somewhat like the bristles of a brush. Such column packing are called brush type packing.
The sample gets separated on the column due to partition between the mobile phase and stationary phase and the components register on a detector. The signal from the detector goes to the recorder and the chromatogram of the sample is obtained. The most commonly used detector is a spectrophometer.