SEMINAR ON ISOCRATIC AND GRADIENT ELUTION USED IN HPLCFacilitator:Dr. B.M.GurupadayyaProfessorMr. Chandan .R.SAsst. professorJ.S.S.C.P, Mysore
INTRODUCTION• High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture• The principle of separation in normal phase mode and reverse phase mode is adsorption. The component which has more affinity towards the adsorbent, travels slower. The component which has less affinity towards the stationary phase travels faster. Since no 2 components have the same affinity towards the stationary phase, the components are separated.
Mobile Phase Reservoirs• Inert container with inert lines leading to the pump are required.• Reservoir filters (2-10 mm) at reservoir end of solvent delivery lines• Degassed solvent - Vacuum filtration - Sparge with inert gas (N2 or He) - Ultrasonic under vacuum• Elevate above pumps
Isocratic elution: A separation that employs asingle solvent or solvent mixture of constantcomposition.Gradient elution: Here two or more solventsystems that differ significantly in polarity areemployed. After elution is begun; the ratio ofthe solvents is varied in a programmed way,sometimes continuously and sometimes in aseries of steps. Separation efficiency is greatlyenhanced by gradient elution.
HPLC Pump Criteria• Constructed of materials inert toward solvents to be used• Deliver high volumes (flow rates) of solvent (to 10 mL/min)• Deliver precise and accurate flow (<0.5% variation)• Deliver high pressure (to 6000 psi)• Deliver pulse free flow• Have low pump-head volume• Be reliable
Reciprocating Pumps• One, two, or three pump heads - more heads, less pulse• Small head volumes (50 to 250 mL)• Short piston stroke• Inert pistons (generally sapphire)• Continuous use (no refill time)• Pulse dampeners
Constant Pressure Pump• Constant pressure pump, not constant flow• Can deliver high pressures• Stable flow during delivery stroke• Stop flow on refill stroke• Low cost
Sample Introduction• Valve-type injectors - Six port fixed volume Rheodyne reproducible injection volumes variable loop size easy to use, reliable - Six port variable volume Waters variable injection volumes without loop change increased maintenance, operator skill required more expensive
Auto Injectors• Continuous injections operator free• Comparable precision and accuracy to manual• Much more expensive initially• Much more convenient Up 100 samples and standards with microprocessor control
Liquid-Chromatographic ColumnsLiquid-chromatographic columns areordinarily constructed from smooth-borestainless steel tubing, although heavy-walled glass tubing is occasionallyencountered. The latter is restricted topressures that are lower than about 600psi.
Analytical ColumnsLiquid-chromatographic columns range inlength from 10 to 30 cm. Normally, the columnsare straight, with added length, where needed,being gained by coupling two or more columnstogether. The inside diameter of liquid columnsis often 4 to 10 mm; the most common particlesize of packings is 5 or 10 µm. The mostcommon column currently in use is one that is25 cm in length, 4.6 mm inside diameter, andpacked with 5 µm particles. Columns of thistype contain 40,000 to 60,000 plates/meter.
Guard ColumnsA guard column is introduced before the analyticalcolumn to increase the life of the analytical column byremoving not only particulate matter and contaminantsfrom the solvents but also sample components that bindirreversibly to the stationary phase. The guard columnserves to saturate the mobile phase with the stationaryphase so that losses of this solvent from the analyticalcolumn are minimized. The composition of the guard-column packing is similar to that of the analyticalcolumn; the particle size is usually larger. When theguard column has become contaminated, it is repackedor discarded and replaced with a new one.
References• www.chromatographyonline.org• Willard Merritt, Dean Settle, Instrumwental method of analysis, 7th ed.• Kasture A V, Pharmaceutical Analysis vol-ii.• www.forumsci.co.il/hplc