1. High Performance Liquid Chromatography Instrumentation

2. solvent pump injector column detector HPLC Instrument

3. Schematic of HPLC Instrument

4. Mobile phase Quality  High purity  Reasonable cost (and disposal)  Boiling point 20-50 °C above column temperature  Low viscosity  Low reactivity  Immiscible with stationary phase  Compatible with detector  Safety – limited flammability and toxicity

5. Mobile phase selection k’ of 2-5 for two or three component mixture k’ of 0.5-20 for multicomponent mixture Match analyte polarity to stationary phase polarity Mobile phase of different polarity  Normal Phase: nonpolar solvent, polar stationary phase least polar component elutes first increasing mobile phase polarity decreases elution time  Reversed Phase: polar solvent (water, MeOH, ACN), nonpolar stationary phase most polar component elutes first increasing mobile phase polarity increases elution time most widely used

6. Solvent Treatment Systems For Dissolved gases (Irreproducible flow rates and band broadening)  Degassers i.Vacuum system and distillating system. ii.Sparging system (inert gas insoluble in mobile phase) For Dust and particulate matter (Damage the pumping or injection system and clog the column)  Filtering the mobile phase through millipore filters.

7. Solvent Reservoir and Elution  Elution : Isocratic : Elution with a single or mixed solvents of constant composition. Gradient Elution: Elution of mixed solvents, with different polarities with composition varied with time.  HPLC instruments equipped with proportioning valves to introduce solvents from different reservoirs.  The ratio is preprogrammed before elution.

8. Pumping Systems Why pressure? The typical particle sizes in HPLC is 3-10 μm. In order to achieve flow rates of 0.5 to 5 mL/min, for a 10-30 cm column, pressures of 1000 to 6000 psi are required. Requirements for HPLC pumping system  pressures to 6000 psi  pulse free output  control flow rate from 0.1 to 10 mL/min  resistance to corrosion by solvents

9. Different types of pumping Systems  Displacement syringe pump Pulse free Small capacity (250ml) Only for isocratic elution.  Reciprocating pumps Small internal volume High output pressure Adaptable for gradient elution Large capacity Constant flow rate Pump used in most commercial design.

10. Reciprocating pumps www.lcresources.com/resources/getstart/2b01.htm Disadvantage : Causes pressure pulses which leads to i.poor quatitative analysis ii.detection problems. Solution i.Dual pump system ii.Pulse dampers ( act as shock absorbers)

11. Injection ( Sampling Valves) Six-port sample injection system is used. It takes i.Small amount of sample (≤ 500µl of sample) ii.In a pressurized system

12. Injection ( Sampling Valves) http://www.restek.com/info_sixport.asp

13. Guard Column ( Pre-column)  Prevents the contamination of the expensive analytical columns with fine particles that can eventually clog the mobile phase flow.  Porous stainless steel column(0.5 -2µm)  Composition same as analytical column.  Particle size is large to minimize pressure drops.  When contaminated, discarded and replaced by new one.

14. Analytical Column Generally made of stainless steel or teflon components 10-30 cm long x 4-10 mm internal diameter Packing usually 5 or 10 µm diameter

15. Analytical Column Packaging Pellicular  Spherical, nonporous, glass or polymer beads  30-40- µm diameter  Thin porous layer of silica, alumina, or ion-exchange resin deposited on surface Porous  Most common  3-10- µm diameter  Silica (most common), alumina, or ion-exchange resin  Thin organic film bonded to surface solvent (mobile phase) and sample wax coated beads HPLC Column to detector

16. Normal vs Reverse phase nonpolar stationary phase 3  m microscopic view of bead Reverse phase(C 4, C 8 or C 18 ): Non-polar stationary phase Normal phase: Polar stationary phase

17. Detectors  Ideal Characteristic of a detector Adequate sensitivity Good stability and reproducibility Linear response to solutes Short response time Response to all solutes in a mixture Non-destructive Temperature stability

18. Types of Detectors  Bulk property Detectors (Mobile phase property) Refractive index Density Dielectric constant Electrochemical  Solute property Detectors UV-Visible Fluorescence Diode array Mass spectrometry Diode array 3D plot