HPLC Basics

high pressure liquid chromatography

1. HPLC HIGH PERFORMANCE LIQUID CHROMATOGRAPHY BY. Ayush sharma ( QA) HPLC 1

2. What is HPLC ? It stands for the “High performance liquid chromatography” & sometimes also referred as the High pressure liquid chromatography. HPLC is a chromatographic technique that can separate a mixture of compounds . It is also used in the biochemistry, separation of combination drugs and in analytical chemistry to identify , quantify and purify the individual components of a mixture. HPLC 2

3. HPLC Apparatus HPLC 3

4. Chromatography Physical method in which separation of components takes place between two phases i.e.. A stationary phase and a mobile phase. Stationary phase The substance on which adsorption of the analyte (the substance to be separated during chromatography ) takes place. It can be a solid, a gel, or a solid liquid combination. Mobile phase Solvent which carries the analyte (a liquid or a gas). HPLC 4

5. PRINCIPLE The main principle of separation is adsorption. The components which has a lesser affinity towards the stationary phase travels faster, where as the components which has the greater affinity towards the stationary , travels slower. No two components have the same affinity towards the stationary phase on the basis of that the components are separated . HPLC 5

6. HPLC is a separation technique that involves : The injection of the small volume of liquid sample into a tube packed with tiny particles (3 to 5 micron) in diameter called the stationary phase . where the individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump . HPLC 6

7. These separated components are detected at the exit of this tube (column) by a flow through device (detector) that measure their amount. The output from the detector is called a liquid chromatogram . LC & HPLC works in the same way except the speed ,efficiency , sensitivity and ease of operation of HPLC is vastly superior . HPLC 7

8. Working process of HPLC HPLC 8

9. HPLC 9

10. TYPES OF HPLC Based on mode of Separation Based on principle of Separation Based on Elution technique Based on scale of operation Based on type of analysis HPLC 10

11. Based on mode of separation 1- Normal based chromatography Stationary phase is polar (hydrophilic) & mobile phase if non-polar (hydrophobic). 2- Reverse phase chromatography Stationary phase is non-polar (hydrophobic) and mobile phase is polar (hydrophilic). Polar-Polar bonds and non polar-non polar bonds have more affinity that Polar –Non polar bonds. Reverse phase chromatography is used for the drugs which are usually hydrophilic . HPLC 11

12. Based on principle of Separation 1- Adsorption Chromatography 2- Ion exchange chromatography 3- Ion pair Chromatography 4- Gel permeation Chromatography 5- Affinity Chromatography 6- Chiral Chromatography HPLC 12

13. Adsorption Chromatography In the adsorption chromatography solute molecules bond directly to the surface of the stationary phase. The components which has the more affinity towards the mobile phase elutes first & the components which the less affinity towards the stationary phase elutes later. No two components have the same affinity towards mobile phase & stationary phase. HPLC 13

14. Based on Scale of Operation 1- Analytical HPLC No recovery of individual components of substance 2- Preparative HPLC Individual components of substance can be recovered HPLC 14

15. Based on type of Analysis 1- Qualitative analysis Analysis of a substance in order to ascertain the nature of its chemical constituents , here separation is possible but cannot assess the quantity in this analysis . 2- Quantitative analysis Determining the amounts and proportions of its chemical constituents . HPLC 15

16. Based on Elution Technique The process of the extracting one material from another by washing with a solvent, as in washing loaded ion exchange resins to remove captured ions. 1- Isocratic elution A separation in which the mobile phase composition remains constant throughout the procedure is termed as isocratic elution. 2- Gradient elution A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution. HPLC 16

17. APPLICATIONS 1- Pharma ceutical 2- Environmental 3- Forensics 4- Clinical 5- Food & Flavour HPLC 17

18. APPLICATIONS HPLC is one of the most widely applied analytical separation techniques . 1) Pharmaceutical Pharmaceutical quality control. Shelf life determination of pharmaceutical products. Tablet dissolution of pharmaceutical dosages. Identification of counterfeit drug products. HPLC 18

19. Environmental Phenols in Drinking water. Estrogens in coastal waters- The sewage source. Biomonitering of PAH (polycyclic aromatic hydrocarbons) pollution in high altitude lakes. HPLC 19

20. Forensics Identification of anabolic steroids in serum , urine , sweat and hair. Simultaneous quantification of psychotherapeutic drugs in human plasma. A mobile HPLC apparatus at dance parties on site identification and quantification of the drug Ecstasy. HPLC 20

21. Clinical Analysis of antibiotics. Quantification of DEET (N,N-diethyl-meta-toluamide) in human urine. Food & Flavor Ensuring soft drink consistency and quality Sugar analysis in fruit juices. Analysis of vicinal di-ketones in beer. Polycyclic aromatic hydrocarbons in Brazillien vegetables and fruits . HPLC 21

22. ADVATAGES OF HPLC Separation is fast & Efficient. Accurate quantitative measurement. Repetitive & reproducible analysis using the same column. It can be applied for the separation & analysis of the very complex mixtures. Both aqueous & non-aqueous samples can be analyzed. It provides a means for determining of multiple components in a single analysis. HPLC 22

23. Disadvantages Expensive Complex to operate Tedious to detect co-elution HPLC 23

24. Thank You Note .. Questions puchana mana ha . HPLC 24