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Seminar onHPLC Instrumentation and Solvents Used in HPLC : 

Seminar onHPLC Instrumentation and Solvents Used in HPLC Guided by: Presented by: Mr. Sumit Bansal Lakhvir Kaur 1

Contents : 

Contents • Instrumentation. → Solvent Reservoir and Degassing system. → Pumps and Sample injection system. → Column, Detector and Recorder. • Solvent Selection. • Solvent Quality. • References. 2

Instrumentation : 

Instrumentation Main components of HPLC are: 1. Solvent system and degassing system. 2. Pressure, flow and temperature. 3. Pumps and Sample injection system. 4. Columns and Detectors. 5. Recorder. 3

Instrumentation : 

Instrumentation 4

Solvent Reservoir and Degassing System : 

Solvent Reservoir and Degassing System Solvent Reservoir – Convey mobile phase to the degassers. • Degassers – Remove air bubbles or atmospheric gases. 5

Pressure, Flow and Temperature : 

Pressure, Flow and Temperature Pressure developed in column depends upon: – length of column. – particle size of stationary phase. – viscosity of mobile phase. – flow-rate of mobile phase. • Flow • Temperature 6

Pumps and Sample Injection System : 

Pumps and Sample Injection System Pumps: Features of HPLC pumps: 1.Solvent flow must be non-pulsing. 2.Interior of pump must not be corroded by any solvent. 3. Easy to dismantle and repair. Types: Constant pressure pump Constant flow pump 7

Pneumatic Pump : 

Pneumatic Pump 8

Single Piston Reciprocating Pump : 

Single Piston Reciprocating Pump 9

Dual Piston Reciprocating Pump : 

Dual Piston Reciprocating Pump 10

Injection System : 

Injection System Involves the transfer of sample by means of syringe from atm.pressure to a sample chamber. Sample loop valves: -Internal sample loop valve -External sample loop valve - Autosamplers 11

Internal & External Sample Loop Valve : 

Internal & External Sample Loop Valve 12

Column,Detector and Recorder : 

Column,Detector and Recorder Columns: - Solvent conditioning column. - Guard column. - Analytical column. • Detector - Refractive index detector. - UV/Visible detector. - Fluorescence detector. -Conductivity detector. • Recorder 13

Solvents : 

Solvents Solvent selection: Strain(1942) arranged solvents in order of eluting power. It increases with increasing polarity. Solvents: Petroleum ether Carbon tetrachloride Increasing Cyclohexane eluting power Acetone Benzene Alcohols Water 14

Slide 15: 

Contd…. •In normal phase partition chromatography,stationary phase is more polar than mobile phase. •In reverse phase HPLC,mobile phase is more polar. • Relative polarity depends on the method used to measure it. Greater the adsorption energy,higher the polarity. • Solvent compatibility with detector should be considered. 15

contd….. : 

contd….. Ranking of solvents by Synder is based on combination of parameters: -Dipole moment -Proton acceptor or donor properties -Dispersion forces 16

Solvent Quality : 

Solvent Quality Selected solvent should be reasonably constant in physical and chemical properties. Solvents are optimized to give good spectra and have uniform chromatographic properties. Problem with solvents is - Stability - Shelf life 17

contd….. : 

contd….. Non-polar and intermediate polarity solvents have troublesome properties or preservatives present. Solvents in this category: -Hydrocarbons -Chlorinated solvents -Ethers 18

References : 

References 1. A.H. Beckett, Stenlake J.B, “Practical Pharmaceutical Chemistry”, 4th Edition, Page no. 90,163-164. 2. Kar A., “Pharmaceutical Drug Analysis”, Page no. 162-165. 3. Munson J.B., ”Modern Pharmaceutical Analysis”, Part-2, Vol-2, Page no.108-110. 4. Chatwal G., “Instrumental Method of Analysis”, 2nd Edition, Page no. 2.625-2.630. 19



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