Mobile phase is constantly fed into the column inlet at a constant rate by a pump. The sample is injected from a sample injector, located near the column inlet. The injected sample enters the column with the mobile phase and the components in the sample migrate through it, passing between the stationary and the mobile phases. Compounds move in the column only when they are in the mobile phase. Compounds that tend to be distributed in the mobile phase therefore migrate faster through the column while compounds that tend to be distributed in the stationary phase migrate slower. In this way, each component is separated on the column and sequentially elutes from the outlet. Each compound eluting from the column is detected by a detector connected to the outlet of the column.
PRINCIPLE OF RP-HPLC
In RP-HPLC compounds are separated based on their hydrophobic character.retension is the result of interaction of then nonpolar components of the solutes and the non-polar stationay phase. Figure 2 High Performance Liquid Chromatography (Schematic) When the recorder, starting at the time the sample is injected monitors the separation process, a graph is obtained. This graph is called a chromatogram. The time required for a compound to elute (called retention time) and the relationship between compound concentration (amount) and peak area depend on the characteristics of the compound. Retention time is therefore used as an index for qualitative determination and peak surface area (or height) as an index for quantitative determination. There are two modes of elution processes: Isocratic elution and Gradient elution. 1.4.1 Isocratic elution: In an isocratic elution, a sample is injected onto a given column and the mobile phase remains unchanged through the time required for the sample components to elute from the column. Isocratic elution in HPLC is one in which the solvent composition remains constant. 1.4.2 Gradient elution: It is one in which the composition of solvent is changed continuously or in a series of steps. In this, two (and sometimes more) solvent system that differs significantly in polarity is used. The ratio of the two solvents is varied in a pre-programmed way during the separation, sometimes continuously and sometimes in a series of steps. 1.5 Instrumentation The main components of HPLC are: • A solvent reservoirs and solvent systems • Pumping systems • Sample injection system • Columns • Detectors 1.5.1 A solvent reservoirs and solvent systems A modern HPLC apparatus is equipped with one or more glass reservoirs, each of which contains solvent more than 500 ml. It is advisable to use deaerated mobile phase solvent mixture using a vacuum pump. Degassers may consists of vacuum pumping system, a distillation system, a device for heating and stirring, or, e system for sparging, in which the dissolved gases are swept out of solution by fine bubbles of inert gas that is not soluble in the mobile phase. 1.5.2 Pumps Pumps are most important component of HPLC and their performance directly affects the retention time, detector reproducibility and detector’s sensitivity. Most of work in HPLC is done using pressures between about 400-1500 psi. The requirement for chromatographic pumps includes (1) ability to generate pressures upto 6000 psi, (2) Pulse-free output, (3)flow rates ranging