HPLC (High performance liquid chromatography) for Pharmaceutical Analysis.pptx

High performance liquid chromatography is basically a column chromatography in highly improved from.

1. Prepared by : Subhajit Dutta Research Scholar JSS College of Pharmacy, Ooty JSS Academy of Higher Education and Research, Mysuru

2. Overview Introduction Types Principle Instrumentation Advantages Disadvantages Applications

3. Introduction High performance liquid chromatography is basically a column chromatography in highly improved from. Instead of solvent being allowed to drip through a column under gravity. A force is applied on it under high pressure of up to 400 atm . That makes it much faster . Much better separation of the compound of the mixture.

4. Detection method can be used. It has ability to separate , identify and quantitate at the compound. HPLC can be employed to about any sample such a pharmaceuticals, food, nutraceutical, cosmetics, environmental, forensic sample industrial chemical and Ayurveda .

5. Types 1. Normal- Phase HPLC : Column filled by tiny silica particles + non polar solvent (ex : Hexane) Diameter - 4.6 mm Length - 150-250 mm 2. Reversed- Phase HPLC : Column size Same The silica particles are filled in the column. The particles are modified to make them non polar.

6. This is done by attracting long hydrocarbon chains ( 8-18 C atoms) to its surface. A polar solvent is utilized (ex : a mixture of water and an alcohol such as methanol ). Polar compounds in the mixture will pass more quickly .

7. Principle The principle of HPLC is based on the distribution of the analyte (sample) to between a mobile phase (eluent) and stationary phase (packing material of the column).

8. Depending on the chemical structure of the analyte, molecules are stopped while passing the stationary phase. The specific intermolecular interactions between the molecules of a sample and the packing material (on column). Hence, different constituents of the sample are eluted at the different time. A detection unit (ex: UV detector ) identifies the analytes after leaving the column.

9. Instruments

10. Instrumentation 1 . Solvent / Mobile Phase Reservoir : The solvents or buffers mixture of solvent & buffers in the form of homogeneous mixture are kept in reservoir and are allowed to enter the mixing chamber . This is made up of glass bottle with properly covered.

11. 2 . Pump : The HPLC pump runs the solvent and sample through the column. To minimize variation in the elution, the pump must maintain a constant , pulse free & flow rate . This is achieved in Multi piston Pump Two piston control the flow rate A syringe pump employed for greater control of flow rate.

12. 3 . Injector : Injector is placed next to the pump . Use a syringe and the sample is introduced to the flow of eluent. The auto injector system is widely used. It allows repeated injection in a set scheduled timing.

13. 4 . Column : The HPLC column is the foundation of this instrument. It contains the stationary phase media, two separate sample ingredients into their constituent parts. Inner diameter and length impact retention rates and elusion rates, as will the particle size and chemical composition of the stationary phase.

14. Number of separation supported by HPLC column. Stable column temperature is a critical factor. Column thermostats are used in order to maintain stable temperature.

15. 5. Column Heater : It helps to keep consistent temperature condition. The columns are generally put inside the column header.

16. 6. Detector : UV detector :

17. Absorption of UV light of various wavelength can be seen by many organic compounds . If you do have a beam of UV light shining through the stream of liquid coming out of the column and a UV detector on the opposite side of the stream you can obtain a direct reading of how much of the light is absorbed. The amount of light absorbed will be dependent upon the amount of a particular compound that is passing through the beam at the time.

18. Procedure Inject the liquid sample into column containing stationary phase. individual sample components are forced down the tube by high pressure from the pump. components are separated under the influence of various chemical or physical interaction with the particle in stationary phase.

19. The separated analytes are identified by detector. The detector measure the concentration of the components. Data from the detector is processed and a chromatogram is produced.

20. Advantages of HPLC Disadvantages of HPLC It is a rapid method Fast and precise quantitative analysis Highly reproducible It can be upgraded to mass spectroscopy Manage all areas of analysis to increase productivity. It is costly It shows complexity Its shows low sensitivity Co-elution is difficult to detect Irreversibly observed compounds cannot be detected

21. Applications 1 . Pharmaceutical : Stability testing dissolution study tablet quality control 2. Environmental : Detection of phenolic compound in drinking water . Biomonitoring of pollutants.

22. 3. Forensic : Quantitative analysis of biological sample . Identification of steroid in blood, urine etc. Textile dyes Determination of Cocaine and other drugs of abuse in blood, urine etc. 4. Clinical test : Urine Analysis, Antibiotic Analysis in blood. Analysis of Bilirubin and biliverdin.

23. 5. Food and flavour : Measurement of quality of soft drinks and water . Sugar analysis of Fruit juice . Analysis of Polycyclic compound in vegetables. Preservative analysis.