When molecules in a mixture are very similar, direct quantification becomes difficult. This is a separating technology used to separate very similar
molecules from a mixture and then quantify them accurately. It is a member of a group of technologies known as "CHROMATOGRAPHY".
In chromatography, the components are separated on the basis of their chemical composition and structure. Any molecule present in the mixture will
have its own unique composition and structure and therefore will have different interaction with its surrounding. These interactions make them all move
uniquely on the support provided to it. This support is known as "stationary phase" and the energy to move is provided by a liquid or a gas
On the basis of the material being used as either phase, the chromatographic technique is titled accordingly.
- Use of paper as the stationary phase: paper chromatography
- Use of immobilized silica gel layer on a glass plate: Thin Layer Chromatography
- Use of volatile gases: Gas Chromatography
HPLC (High Performance Liquid Chromatography) is a mode of chromatography wherein the stationary phase is used in form of fine particles packed in a
cylindrical column made up of inert material like stainless steel. Stationary phase adsorbs the analytes and holds them reversibly for particular time.
All the molecules in the mixture will spend different time with different stationary phase and therefore come out at the end one by one. Adsorption
being the base of separation, usage of fine particles offers more surface area for the molecules to interact with. The time that a molecule takes to
travel from one end to other end of the stationary phase is known as the RETENTION TIME. Hence all molecules will display their own unique retention
time for a particular stationary-mobile phase combination.
Selection of stationary phase and mobile phase is done on the basis of the molecule of interest to get optimum retention time. As a result, HPLC
acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical
Silicagel is the most frequently used adsorbent. The adsorbent is packed in a column. The density of the packing directly affects the resolution. Hence
the HPLC columns are densely packed with the adsorbent to achieve significant degree of resolution. But very high density of packing demands for the
mobile phase to be introduced at very high pressure to generate effective flow. So the technique is also sometimes named as HIGH PRESSURE LIQUID
At the end of the column, a detector is kept. As the molecules come out from the end, the detector detects and gives signal. Till the molecules keeps
traveling through the detector's path it keeps detecting it and gives a peak on CHROMATOGRAM. The peak area calculation quantifies the molecule of
interest on the basis of standard values.
Remarkable interference can be observed by minor changes in the stationary and mobile phase composition, purity and/or density. Therefore it is must to
get standard grade reagents for HPLC. Such quality control requirements makes HPLC a little expensive technology as compared to other similar