Basics of High Performance Liquid Chromatography

 Basics of High Performance Liquid Chromatography

Introduction:

                    Liquid chromatography is a very important separation method used in practically all chemistry fields. For many decades, it has played a key role in academic and industrial laboratories where it is used to analyze or purify components from complex mixtures. For example, it is used to separate proteins/drugs from impurities and to analyze drugs and endogenous components in biological materials. Most breakthroughs in biochemical and pharmaceutical sciences would probably not have been possible without chromatography.

"PRINCIPLE OF HPLC: The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase."

Scope of HPLC:

Flow Diagram of HPLC

Important Terms:

• Ø  Chromatogram: A plot of detector signal output versus time or elution volume. 
• Ø  Mobile phase: The liquid that moves the solute through the column.
• Ø  Stationary phase: The packing material of the column, which is the immobile phase involved in the chromatographic process.
• Ø  Peak: The visual representation on the chromatogram based on the 8 detector's electrical response due to the presence of a sample component inside the flow cell.
• Ø  Retention time: The time taken by the analyte peak to reach the detector after sample injection.
• Ø  Qualitation: An analysis process which is designed to identify the components of a substance or mixture.
• Ø  Quantitation: An analysis process which is designed to determine the amounts or proportion of the components of a substance.

What is Chromatogram?

Separation Modes:

                            1. Normal phase chromatography 

                            2. Reversed phase chromatography 

                            3. Ion chromatography 

                            4. Size exclusion chromatography 

                            5. Affinity chromatography

 

Conclusion:

                    Above mentioned are the very basic instrumentation and separation principle of High Performance Liquid Chromatography(HPLC). Different type of stationary phase and  mobile phase optimized during the development of the analytical method. HPLC having the large variety of   application in all the industries including pharma. compare to other chemical industries, pharmaceutical industries have an large usage of HPLCs. Also the results obtained from these methods are very accurate and precise.