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.
– 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.
• 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.
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.
- Refractive index detector.
- UV/Visible detector.
- Fluorescence 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
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:
-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:
-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 THANKYOU : THANKYOU 20