Hplc

1. … HPLC Presented by; Salman Shehzada

2. CONTENTS Introduction History Instrumentation Operation Types Advantages Disadvantages

3. Introduction Abbreviated for; High-performance liquid chromatography  OR High-pressure liquid chromatography Defined as; “ A chromatographic technique used to separate components of mixture for the purpose to identify, quantify or purify the individual components of the mixture “. Widely used in field of biochemistry and analytical chemistry .

4. History Invented by Martin and Synge as extension of gas chromatography. The main idea was that; “ Small sorbent particles and pressure could produce fast liquid chromatography techniques”. The method was used practically in late 1960s for separation amino acids .

5. Instrumentation HPLC involves following functional instruments. Mobile phase reservoir Pump Injector Stationary phase ( Column ) Detector Computer

6. Mobile phase reservoir : Commonly glass bottles with caps are used . Teflon tubings and filters are connected to purge gas ( helium ) for degassing . Vaccum for 5-10 min is also used for degassing . Pump : It forces the mobile phase to pass through column . Flow rate is 1-2 ml/ min. Trypical pressure is 6000 – 9000psi. Injector : Can be manually (syringe) or automated . Sample volume 5-20µl. Ideal to stand pressure of mobile phase. Autosampler is used for analysis of many samples automatically .

7. Stationary phase ( Column ): Heart of HPLC. Separate sample components on basis of physical and chemical parameters . Lenght 10-30cm. Diameter 4-10nm. Packing material 5-10nm thick . Detector: Detection of elutes from column . Quantitative analysis of sample components. Output transferred to recorder/ computer. Computer: Data system that controls modules of HPLC. Signals from detector are interpreted to determine elution time, quantitative and qualitative analysis of sample .

8. Figure displaying instrumentation of HPLC

9. Figure displaying complex instrumentation of HPLC

10. Operation Operation strategies are explained as follows ; Sample : The sample to be analyzed is introduced into stream of mobile phase in minute quantity , mostly in microliters . The components of sample moves through different velocities reaching the column where it physically interacts with the sorbent . Velocity of sample components depends on; Chemical nature of sample Nature of column Composition of mobile phase

11. Column : Also known as stationary phase. Composed of sorbent material varying in particle size, and surface chemistry. Size of particles can be smaller which improves chromatographic resolution. In terms of surface chemistry, the column can be hydrophobic and polar in nature.

12. A B Plant extracts separated by HPLC by passing through stationary phase ( column ) Columns used in HPLC device

13. Eluent : Also known as mobile phase. Type of solvent used : Commonly it is composed of water miscible with organic solvents . For example methanol . Some HPLC utilizes water free mobile phase and uses acids such as formic acid or phosphoric acid . Many mobile phase works well by using salts such as sodium phosphate.

14. Composition of eluent : It depends on; Intensity of interactions between analytes and stationary phase. For example; Hydrophobic interactions in reversed-phase HPLC Polar interactions in normal phase HPLC Ionic interaction in ion exchange HPLC Often a series of trial runs are performed with the sample in order to find the HPLC method which gives adequate separation.

15. Figure showing mobile phase reserviors

16. Figure showing operation of HPLC

17. Figure showing operation of HPLC and analysis of analyte present in the sample

18. Types Partition chromatography Normal phase chromatography Displacement chromatography Reversed phase chromatography Size-exclusion chromatography Ion-exchange chromatography Bioaffinity chromatography Aqueous normal-phase chromatography

19. Advantages Advantages of HPLC is ; Seapartion of voltile and non- voltile components. Thermally unstable compounds isolated . Quick analysis High resolution Less cumbersome More reproducibility

20. Disadvantages Disadvantages of HPLC are; Tedious to detect co-elution . High cost . Complex to operate .

21. Thank you …