Instrumentation of HPLC, principle by kk sahu

INTRODUCTION
Instrumentation of HPLC
TYPES OF HPLC
PARAMETERS
APPLICATION
CONCLUSION
REFERENCE
High-performance liquid chromatography ( HPLC) is a specific form of column chromatography generally used in biochemistry and analysis to separate, identify, and quantify the active compounds.
HPLC mainly utilizes a column that holds packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules.

1. “ Instrumentation of HPLC ” By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. SYNOPSIS :- INTRODUCTION Instrumentation of HPLC TYPES OF HPLC PARAMETERS APPLICATION CONCLUSION REFERENCE

3. INTRODUCTION High-performance liquid chromatography ( HPLC) is a specific form of column chromatography generally used in biochemistry and analysis to separate, identify, and quantify the active compounds. HPLC mainly utilizes a column that holds packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase.

4. Picture of HPLC instrument

5. Instrumentation of HPLC HPLC instrumentation includes :- Solvent Reservoirs Pump Sample Injector Column(s) Detector Data System

6. 6 Pump Sample injection unit (injector) Column Column oven Detector Eluent (mobile phase) Drain Data processor Degasser Flow Channel Diagram for High Performance Liquid Chromatograph

7. Mobile Phase Reservoirs The type and composition of the mobile phase affects the separation of the components. Different solvents are used for different types of HPLC. For normal-phase HPLC, the solvent is usually nonpolar , and in reverse-phase HPLC, the solvent is normally a mixture of water and a polar organic solvent.

8. Pumps High-pressure pumps are needed to push the mobile phase through the packed stationary phase. Pumps are typically known to be robust, but adequate maintenance must be performed to maintain that characteristic. Inability to build pressure, high pressures or leakage could indicate that the pump is not functioning correctly. Proper maintenance of the pump system will minimize down time.

9. Pump

10. Sample Injector The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100mL of volume with high reproducibility and under high pressure (up to 4000 psi). For liquid chromatography, liquid samples can be directly injected and solid samples need only to be diluted in the appropriate solvent.

11. Column(s) The column or stationary phase is the core of any chromatographic system. Columns are commercially available in different lengths and packing materials. The use of the correct combination of length and packing material in correlation with the appropriate mobile phase can assist in the most effective separation of a sample compound.

12. Detector The detector is used to sense the presence of a compound passing through and to provide an electronic signal to a data-acquisition device. The main types of detectors used in HPLC are refractive index (RI), ultraviolet (UV-Vis) and fluorescence, but there are also diode array, electrochemical and conductivity detectors. Each detector has its assets, limitations and sample types for which it is most effective.

13. Data Display Systems Since the detector signal is electronic, use of modern data- acquisition techniques can aid in the signal analysis. The data- acquisition system of most HPLC systems is a computer. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret.

14. TYPES OF HPLC Normal phase chromatography : Also known Normal phase HPLC (NP-HPLC), this method separates analytes based on polarity . NP-HPLC uses a polar stationary phase and a non-polar mobile phase. Reversed phase chromatography : Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. RPC operates on the principle of hydrophobic interactions, which result from repulsive forces between a polar eluent , the relatively non-polar analyte , and the non-polar stationary phase.

15. Size exclusion chromatography : Size exclusion chromatography (SEC), also called as gel permeation chromatography or gel filtration chromatography mainly separates particles on the basis of size. It is also useful for determining the tertiary structure and quaternary structure of proteins and amino acids. Ion exchange chromatography : In Ion-exchange chromatography, retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded.

16. Bio-affinity chromatography: Separation based on specific reversible interaction of proteins with ligands . Ligands are covalently attached to solid support on a bio-affinity matrix, retains proteins with interaction to the column-bound ligands .

17. PARAMETERS For the accurate analysis of a compound, there are some parameters which are used as a standard for a particular compound. Internal diameter Particle size Pore size Pump pressure

18. APPLICATION Chemical Separations:- It is based on the fact that certain compounds have different migration rates given a particular column and mobile phase, the extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase.

19. Purification :- Purification is defined as the process of separating or extracting the target compound from a mixture of compounds or contaminants. Identification :- Generally assay of compounds are carried using HPLC. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph.

20. Other applications of HPLC Pharmaceutical applications Shelf-life determinations of pharmaceutical products . Identification of active ingredients of dosage forms. Pharmaceutical quality control Environmental applications Detection of phenolic compounds in Drinking Water Bio-monitoring of pollutant Forensics Quantification of the drug in biological samples. Identification of anabolic steroids in serum, urine, and hair Determination of cocaine and metabolites in blood

21. Clinical Quantification of ions in human urine Analysis of antibiotics in blood plasma. Estimation of bilirubin and bilivirdin in blood plasma in case of hepatic disorders. Food and Flavor Ensuring the quality of soft drink and drinking water. Analysis of beer. Sugar analysis in fruit juices. Analysis of polycyclic compounds in vegetables.

22. CONCLUSION It can be concluded that HPLC is a versatile, reproducible chromatographic technique for the estimation of drug products. It has wide applications in different fields in term of quantitative and qualitative estimation of active molecules.

23. REFERENCE High-performance liquid chromatography. Pdf file 208-528-1-pb.pdf file