History of Chromatography n Early LC carried out in glass columns n diameters: 1-5 cm n lengths: cm n Size of solid stationary phase n diameters:  m n Due to this, the flow rates were low and the separation times long. n Decrease particle size of packing causes increase in column efficiency. n diameters 3-10  m n This technology required sophisticated instruments n new method called HPLC

Chromatography by M. Tswett ether CaCO 3 chlorophyll Chromato Chromatography colours

HPLC originally referred to: High Pressure Liquid Chromatography n high pressure to be able to use small particle size to allow proper separation at reasonable flow rates

HPLC Laterly referred to: High Performance Liquid Chromatography n high performance due to its reproducibility currently refers to: High Precision Liquid Chromatography

Introduction n Compounds are separated by injecting a sample mixture into the column. The different component in the mixture pass through the column at different rates due to differences in their partition behavior between the mobile phase and the stationary phase.

What is HPLC? n The most widely used analytical separations technique n Utilizes a liquid mobile phase to separate components of mixture n Uses high pressure to push solvent through the column n Popularity : n sensitivity n ready adaptability to accurate quantitative determination n suitability for separating nonvolatile species or thermally fragile ones

Principle of HPLC: The principle of HPLC are based on Van Deemter equation which relates the efficiency of the chromatographic column to the particle size of the column, molecular diffusion and thickness of stationary phase. The Van Deemter Equation is given as H or HETP = A + B + C υ H or HETP = A + B + C υ υ where, A represents eddy diffusion B represents molecular diffusion B represents molecular diffusion C represents rate of mass transfer C represents rate of mass transfer υ represents flow rate υ represents flow rate

Advantages to HPLC n Higher resolution and speed of analysis n HPLC columns can be reused without repacking or regeneration n Greater reproducibility due to close control of the parameters affecting the efficiency of separation n Easy automation of instrument operation and data analysis n Adaptability to large-scale, preparative procedures

Components Of A Liquid Chromatograph System n Mobile Phase / Solvent Reservoir n Degasser n Solvent Delivery System (Pump) n Injector n Precolumn n Column n Temperature Control n Detectors n Recorder (Data Collection)

HPLC Basic Instrumentation Mobile phase Pump Solvent Delivery Injector Sample Injection Column Separation Detector Data Processor 1. HPLC

HPLC system HPLC Solvent Reservoirs Solvent Delivery System Variable UV/Vis Detector Rheodyne Injector HPLC Column Computer Workstation

HPLC system n Solvent Reservoir n Degasser n Solvent Delivery System (Pump) n Injector n Column &oven n Detectors n Recorder (Data Collection)

Components Solvent Reservoir Usually one or more glass or stainless steel reservoirs each of which contains ml of solvent Usually one or more glass or stainless steel reservoirs each of which contains ml of solvent n Isocratic elution - single solvent separation technique Gradient elution - 2 or more solvents, varied during separation Gradient elution - 2 or more solvents, varied during separation

Degasser n Problems caused by dissolved air(O 2, N 2 )in mobile phase n Unstable delivery in pump n Bigger noise and large baseline-drift in detector cell  In order to avoid causing the problems, mobile phase should be degassed. mobile phase should be degassed. n vacuum pumping systems n distillation system n a system for heating and stirring the solvents n sparging system - bubbles an inert gas of low solubility through the solvent

Solvent Delivery System Three basic types of LC Pumps used are: n pneumatic pumps n motor driven syringe type pumps n reciprocating pumps

Injectors n For injecting the solvent through the column n Minimize possible flow disturbances n Limiting factor in precision of liquid chromatographic measurement n Volumes must be small n  L n Sampling loops n interchangeable loops (5-500  L at pressures up to 7000 psi)

Column n straight, 15 to 150 cm in length; 2 to 3 mm i.d. n packing - silica gel, alumina, Celite

Detector n Mostly optical n Equipped with a flow cell n Focus light beam at the center for maximum energy transmission n Cell ensures that the separated bands do not widen

Types of Detector n UV/Visible Fixed wavelength - Fixed wavelength - variable wavelength n Photo Diode Array n Refractive index n Fluorescence n Evaporative light scattering n Conductivity n Electrochemical