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

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

Instrumentation of HPLC

Instrumentation of HPLC

Instrumentation of HPLC

Picture of HPLC instrument

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

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

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

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

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

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

HPLC column

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

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

U.V. & Visible light absorbing detector

U.V. & Visible light absorbing detector

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.

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.

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