High performance liquid chromatography

HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution using a high pressure.

1. High Performance Liquid Chromatography By Mehwish Nawaz

2. Contents Introduction HPLC instrumentation Classification of HPLC HPLC Chromatogram Applications Advantages Disadvantages

3. HPLC It separates, identifies and quantifies the components dissolved in a liquid solvent by using a high analytical resolution. Sample is carried by a moving gas stream of helium and Nitrogen.

4. Introduction A type of column chromatography . HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. It is especially suitable for compounds which are: N ot easily volatilized T hermally unstable H igh molecular weights .

5. Important Points Mobile/Stationary phase Based on interactions of both phases Characteristics Based on separation of compounds Mechanism P olar & non-polar molecules

6. Principle Separation  of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the  separation.

7. HPLC instrumentation HPLC instruments consist of: A reservoir of mobile phase A pump An injector A separation column A detector

8. Components of HPLC Solvent Reservoir Isocratic elution - single solvent separation Gradient elution - 2 or more solvents, varied during separation Pumps C apable of pumping solvent up to a pressure of 4000 psi and at flows of up to 10 ml/min) Sample Injection System Sample valve, Syringe/injector Columns Straight , 15 to 150 cm in length; 2 to 3 mm packing - silica gel, alumina, Celite Detectors UV, fluorescence, Refractive index Data Processing Specific softwares connected to HPLC) Waste

9. HPLC

10. HPLC Column

11. Types of column Guard Column Guard Column Fast Column Fast Column Preparative column Capillary column Nano column Analytical column

12. HPLC Detectors UV/Vis Refractive index Fluorescence Evaporative light scattering (ELSD) MS Diode Array Detector (DAD) Electrochemical detectors.

13. Classification of HPLC Reverse phase HPLC Hydrophobic stationary phase Hydrophilic compounds will elute first Normal Phase HPLC Hydrophilic stationary phase Hydrophobic will elute first

14. HPLC chromatogram t o - elution time of unretained peak t R - retention time - determines sample identity

15. Factors affecting HPLC activity 1. Internal diameter of column the smaller in diameter, the higher in sensitivity 2. Pump pressure the higher in pressure, the higher in separation 3. Column dimensions 2.1–4.6 mm diameter, and 30–250 mm length 4. The polarity sample, solvent and column 5. Temperature the higher in temperature, the higher in separation

16. Applications Pharmaceuticals industry Analysis of natural contamination Forensic test Clinical test Food and essence manufacture

17. Advantages High speed High resolution High sensitivity Re-usable column No destruction of the components The instrumentation are automatic, computerized Sample is recovered completely Quantitative work is more easily and most sensitive

18. Disadvantages It can be costly, complex to operate and doesn't work for all samples. Need a skill to run the instruments Solvents consuming

19. H ow to resolve these problems????? The following table lists frequent problems in HPLC: Uncommon peak shapes Lack of sensitivity Poor sample recovery Pressure problems Baseline problems Leaks Changing retention times