1. High Performance Liquid Chromatography (H. P. L. C. ) dr. Sham b
High Performance Liquid Chromatography (H.P.L.C.) dr. Sham b. misal S.M. Joshi College, Hadapsar, Pune

2. Liquid chromatography is modified for faster separation
G.C. limitations
Compd. must be in vapor state
St. phase is imp, carrier gas as a transport medium
Diffusion of liquid mobile phase is serious problem thus takes more time for the separation
Liquid chromatography is modified for faster separation
Use of low viscous liquid, reduction in particle size of stationary phase, application of high pressure

3. Instrumentation of HPLC

4. Instrumentation of HPLC

5. Instrumentation of HPLC

6. Picture of HPLC instrument

7. Components of HPLC instrument
Solvent reservoir & degasifying system
Glass or stainless steel reservoir of cap. 1 – 2 Lt.
Equipped with degasifying system to remove dissolved gases from solvent
Types of ellution
Isocratic elution – same composition
Gradient elution – 2 or more solvents, ratio changed in programmed manner

8. Components of HPLC instrument
Pump
Pressure range – 1000 – 6000 psi
Flow rate – 3 ml/min
Screw driven syringe type pump
Provides pulse free delivery, Suffer from limitation of capacity, Unsuitable for gradient elution
Reciprocating pump
Provide pulsed delivery, Infinite solvent capacity
Pneumatic pump
Simple, inexpensive, provides pulse free delivery, Suffers from limited capacity & can’t use for gradient elution

9. Components of HPLC instrument
Precolumn (Guard column)
Contains same packing material as in main column but of larger particle size
Used to remove impurities from the solvent and to saturate the mobile phase with stationary phase

10. Components of HPLC instrument
Sample injection system
Rotary valve or slider valve, part of sample injection system
Capacity – 2 to 100 μL
Self sealing silicon, teflon or neoprene septum are used for the delivery of the sample

11. Components of HPLC instrument
Column
Stainless steel or glass column
Length – 10 to 150 cm
Diameter – 2 to 5 mm
Water jacketed column available commercially
Longer columns used for preparative separation of larger components
Solid adsorbent is used as packing material
Polyamide (nylon – 6 ) used for separation of non polar low molecular wt. solute
Polar bonded phase material
Pellicular material (thin layer or coating on surface) coating of 1 μm on glass beads of diameter of 40 μm

12. Components of HPLC instrument
Column
Polar bonded phase material
Pellicular material (thin layer or coating on surface) coating of 1 μm on glass beads of diameter of 40 μm
High molecular wt. compounds – Stationary liquid phase chemically bonded to the inert support
Ion exchange resins chemically bonded to inert solid suport
Efficiency of column increases with decreases in the particle size of packing material.
Special devices are used for packing of HPLC columns

13. HPLC column

14. Detectors Choice of detector depends on nature of sample component
Detectors based on absorption of U.V. & Visible light are most commonly used
250 nm line from mercury source are absorbed by most of the organic compounds

15. Types of Detectors
Absorbance (UV with Filters, UV with Monochromators)
IR Absorbance
Fluorescence
Refractive-Index
Evaporative Light Scattering Detector (ELSD)
Electrochemical
Mass-Spectrometric
Photo-Diode Array

16. U.V. & Visible light absorbing detector

17. U.V. & Visible light absorbing detector

18. Refractive index detector
Sense the difference in refractive index between column effluent and a reference mobile phase. Cell contains sample and reference chamber separated by diagonal sheet of glass. The collimating beam of light passes through both cells to mirror. The mirror reflects the beam back through the reference and sample cell to a lense which focuses on a photocell. The location of beam is determined by the angular deflection of the beam resulting from the refractive index difference between the contents of the 2 cells. As the beam changes its position of focus on photocell, the out put changes and the difference signal is electrically modified to give a signal proportional to the concentration of solute in the sample cell.

19. Application of HPLC
This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.

20. Applications of H.P.L.C. Pharmaceutical and Pesticides industries
Separation of polar substances is possible
Morphine, Heroin etc. can be separated on special type of resin with H.P.L.C.
Water soluble vitamins can be separated