In order to understand what HPLC, that is, High Performance Chromatography, it is necessary to understand the meaning of the term chromatography itself. Chromatography
Chromatography is defined as a procedure by which inert materials and drug principles encountered in pharmaceuticals preparations are separated by fractional extraction, adsorption, or ion exchange on a porous solid. Chromatography is particularly useful as a means of separating and purifying complex and closely related chemical substance which are difficult to distinguish. Chromatographic techniques are more applicable in those instances where small quantities are involved in the identification test. Objective
The principle objectives obtainable through the use of any type of chromatography, including HPLC are:
1.Resolution of mixtures into constituent parts.
2.Determination of homogeneity.
3.Comparison of substances suspected of being identical.
4.Purification of any substance or compounds.
5.Quantitative separation from complex mixtures.
6.Indication of molecular structure.
Recent developments in liquid chromatography have led to stationary phases that are chemically bonded to solid support .Due to the strength of the covalent bonds between the stationary phase and solid substance, these chemically bonded liquid chromatography columns are quite stable. They are used primarily in column liquid chromatography in which high pressures drive the mobile phase, a form of chromatography called high pressure liquid chromatography or high performance liquid chromatography. Therefore, HPLC or High Performance liquid chromatography may be defined as the advanced form of liquid chromatography which is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution. The mobile phase consists of the sample being separated or analyzed as well as the solvent that moves the sample through the column The stationary phase is the substance which is fixed in place for the chromatography procedure
Characteristics of HPLC:
HPLC as compared with the classical technique is characterized by:
small diameter (2-5 mm), reusable stainless steel columns;
column packing with very small (3, 5 and 10 mm) particles and the continual development of new substances to be used as stationary phases; relatively high inlet pressures and controlled flow of the mobile phase; precise sample introduction without the need for large samples; special continuous flow detectors capable of handling small flow rates and detecting very small amounts; automated standardized instruments;
Types of HPLC
According to the nature of the stationary phase and the separation process, HPLC can be classified into the following types:
a.Normal phase chromatography
b.Reversed phase chromatography
3.Size exclusion chromatography
4.Ion exchange chromatography
1) Adsorption chromatography
This the oldest type of chromatography and consists of a liquid mobile phase and a solid stationary phase. It is further classifies into the following types:
a) Normal phase chromatography
It is also known as Normal phase HPLC (NP-HPLC). It was the first kind of HPLC chemistry used, and separate analytes based on polarity.
This method uses a polar stationary phase and a non-polar mobile phase, and is used when the analyte of interest is fairly polar in nature. The polar analytes associates with and is retained by the polar stationary phase. b) Reversed phase chromatography
Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is silica which has been treated with RMe2 SiCl, where R is a straight chain alkyl group such as...