HPLC - High Performance Liquid Chromatography

HPLC 

High Performance Liquid Chromatography 
(Is an advance type of Liquid Chromatography)

INTRODUCTION

 It is a form of column chromatography used frequently in all field of Life science and Analytical Chemistry. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed and the chromatography column. We can analyzed the Vitamins, API, Enzymes, Photochemical, Toxins and Microbial toxin content with the help of HPLC. 

2.    Types of HPLC;-

2.1.    Normal phase HPLC

Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and separates 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 analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increase in analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength not only depends on the functional groups in the analyte molecule, but also on steric factors and structural isomers are often resolved from one another. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes while more hydrophobic solvents tend to increase retention times. Particularly polar solvents in a mixture tend to deactivate the column by occupying the stationary phase surface. This is somewhat particular to normal phase because it is most purely an adsorptive mechanism (the interactions are with a hard surface rather than a soft layer on a surface).

NP-HPLC had fallen out of favor in the 1970's with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HPLC bonded phases which utilize a partition mechanism which provides reproducibility.

2.2.    Reversed phase chromatography:-

Reversed phase HPLC (RP-HPLC or RPC) consists of a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe₂SiCl, where R is a straight chain alkyl group such as C₁₈H₃₇ or C₈H₁₇. The retention time is therefore longer for molecules which are more non-polar in nature, allowing polar molecules to elute more readily. Retention Time (RT) is increased by the addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent.

3.    PART OF HPLC

Generally all the HPLC have five modules.

3.1.    Solvent Tray (Solvent Reservoir):

Solvent reservoir is the upper part of HPLC that contains all the solvents which is used during analysis. For the buffer solution must be used amber colored glass bottle. All the solvent bottles filled with solvents connected to tubes and Outlet of tubes connected with degasser (some models of HPLC have same module for degasser and Pump). The inlet of tubes assembled with filtration kits and have a specific Marking (Like A, B, C and D).

Solvent Arrangement:

·         Always kept water in ‘’A’’ tube connected bottle because the Solvent parameter “A” is always default. If you don’t given the solvent % value in any solvent then automatically solvent tube “A” considered.......

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