High Performance Liquid Chromatography(HPLC).ppt

High Performance Liquid Chromatography , Analysis, Principle, Types of HPLC, Instrumentation, Column Chemistry, Injector, Column, Mobile Phase, Guard column, Analytical Column, Pump, Degasser, Detector, Recorder, Application

1. Presented by …… Debabrata Dinda B.Pharm , 7th semester Roll No - 20801920056 Instrumental Methods of Analysis , PT 711 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

2. CONTENTS INTRODUCTION PRINCIPLE INSTRUMENTATION APPLICATION REFERENCES 1 2 3 4 5 6 TYPES OF HPLC

3. INTRODUCTION HPLC is a analytical technique used to separate, identify and quantify the mixture of component. Mobile Phase: liquid , Stationary Phase: Solid . It gives high performance due to smaller particle use as a stationary phase(3.5-10 ) . High pressure(1000-4000 psi) is applied for rapid separation of the component so it is also called as High Pressure Liquid Chromatography. The first commercially available HPLC system, ALC100 HPLC was developed by Waters Associates in 1967.  

4. TYPES OF HPLC Normal Phase HPLC Reverse Phase HPLC Stationary Phase: Polar Mobile Phase: Non Polar Non Polar or Hydrophobic component elute first. Polar or hydrophilic components elute late. Stationary Phase: Non Polar Mobile Phase: Polar Polar or Hydrophilic component elute first. Non Polar or Hydrophobic component elute late.

5. PRINCIPLE Adsorption It has solid stationary phase and liquid mobile phase. Each solute has its own equilibrium between adsorption on the surface of stationary phase. Solute has lower soluble or best adsorbed property travel slowly. Solute has higher soluble or poor adsorbed property travel fast. Affinity A specific ligand ,such as an antibody is bound to the inert stationary phase to achieve a highly selective separation.

6. Continue….. Size/Molecular Exclusion Ion Exchange In this technique ion exchange resin is used as stationary phase. Fixed ion (either cation or anion ) covalently bind with surface of column. Ion of the opposite charge reversible bind with fixed with electrostatic bond called as counter ions. Gel is used to separate the components of the mixture according to their molecular sizes. Gel matrix is made up of wide variety compound like- dextran, Agarose, polyacrylamide. This technique is use to separate of - protein, enzymes, polysaccharides.

7. INSTRUMENTATION Solvent Reservoir The function of solvent reservoir to hold the mobile phase. Binary system or Quaternary system reservoir are use. Solvents are flow through a tube made of PTFE, also known as Teflon . Internal diameter of tube is 1/8 inch. Solvents Commonly used solvents are water,acetonitrile,methanol (are HPLC grade solvents, highly pure pass through 0.45 filter).   Degasser Vacuum filtration Helium perging Ultrasonication HPLC Pump Constant pressure pump Constant flow rate pump

8. Continue….. Sample injector Three different modes of sample injection in HPLC. Septum Injectors Stop-flow Septumless Injectors Rheodyne Injector Rheodyne Injector Septum Injector

9. Continue….. Guard Column Internal diameter 4-5 mm. Length 2-10 cm. It contains same materials as the analytical column. Main function of guard column is protect analytical column from impurity. Analytical column External Material: Stainless-steel. External Diameter: 6.5 mm. Internal Diameter: 4-5 mm. Length: 10-30 cm. Stationary phase material packed under very high pressure (approximately 14000-15000 psi)

10. Normal phase Column chemistry Reverse phase Column Chemistry Continue….. Stationary Phase: Polar Silica gel (SiO2) is commonly used stationary phase & contains three Silanol group. Silanol group are responsible for polar nature. Stationary Phase: Non polar Silica gel is converting into non polar by react with organochlorosilane . R= C18 H37 then C-18 Column (ODS) R=C8 H17 then C-8 Column R=C4 H9 then C4 Column C Cl

11. Continue….. Detector UV Detector Refractive Index Detector Photodiode Array Detector Mass Spectral Detector Conductivity Detector Amperometric Detector Recoder & Integrators Recorder is used to record the responses obtained from detectors after amplification.It record the base line but not detect the area of individual peaks. Integrator can record the individual peaks with retention time,hight,width,peak area,percentage of area.

12. APPLICATION Assay of Active Pharmaceutical Ingredients of dosages form. To analyse the Content Uniformity of dosage form. Tablet dissolution study of Pharmaceutical dosages form. To analyse the R.S in dosages form. Self life determination of Pharmaceutical Products. Assay of Human Insulin in blood. Analysis of Antibiotics in blood plasma. Sugar analysis in fruit juices.

13. REFERENCES 1. G.r chatwal ,Sham k anand , Instrumental method of analysis 5 th edition, Himalayan Publishing House page 2.635-2.639. 2. Dr.S.Ravi Sankar, Instrumental method of analysis 5 th edition, Publications,Tirunelveli . Page 18.1-18.15 .