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High Performance Liquid Chromatography

High Performance Liquid Chromatography. The chromatogram is a record of detector output Vs time as the analyte passes through the chromatography system.

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High Performance Liquid Chromatography

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  1. High Performance Liquid Chromatography

  2. The chromatogram is a record of detector output Vs time as the analyte passes through the chromatography system. • It usually consists of a series of several peaks corresponding to the different times in which components of the analyte mixture emerge from the column. • The number of peaks corresponds to the minimum number of different substances (compounds or ions) contained in the analyte. • If the analyte is found to display only a single peak, it is an indication that it is composed of only a single component, i.e., it is pure.

  3. Advantages of LC • Wide range of possible properties of mobile phase • Wider choice of stationary phases and detectors • Suitable for non volatile components • Good resolution and selectivity • Useful for analytes that decompose at high temperatures

  4. Types Of Liquid Chromatography • Partition chromatography • Adsorption, or liquid-solid chromatography • Ion exchange chromatography • Size exclusion, or gel, chromatography

  5. Liquid –Solid Chromatography Adsorption Chromatography Stationary phase is solid and the separation occurs as a result of adsorption desorption steps Stationary phase is solid: either polar (silica gel) or nonpolar (polymer beads).

  6. Adsorption Chromatography • The separation mechanism in LSC is • based on the competition of the • components of the mixture sample for the • active sites on an absorbent such as Silica • Gel. • Both solute and solvent are attracted to • the polar sites on the stationary phase • If solutes have differing degrees of • atraction to the phase, a separtion is • possible

  7. Liquid-Liquid Chromatography Partition Chromatography • Separation of solutes is based on differences • in the relative solubility. • Mobile solvents will be retained to very little • extent compared to solutes • More highly retained species have greater • affinity (solubility) for the Stationary phase • compared to mobile phase

  8. Stationary phase Liquid-liquidChromatography Stationary phase Normal phase: Polar stationary phase and non-polar solvent (mobile phase) Reverse phase: Non-polar stationary phase and polar solvent (mobile phase) Inert support • The stationary solid surface is coated with a 2nd liquid (the • stationary Phase) which is immiscible in the solvent (Mobile) • phase. • Partitioning of the sample between 2 phases delays or retains • some components more than others to effect separation.

  9. Ion exchange chromatography Stationary phase has an ionically charged surface opposite to that of the eluent R+Y- + X-  R+X- + Y- (Anion exchanger) R-Y+ + X+ R-X+ + Y+ (Cation exchanger)

  10. Stationary phase is called ion-exchangers • The ion-exchangers are made of exchange groups bound to a support.

  11. (GPC) Separation is based on molecular size. Stationary phase is a material of Controlled pore size. Also called Gel permeation chromatography GPC

  12. Size exclusion chromatogrphy • Separation is based on molecular size. • Stationary phase is a material of Controlled pore size. • Also called Gel permeation chromatography GPC

  13. Gel-permeation Chromatography • Gel-Permeation Chromatography is a mechanical sorting of • molecules based on the size of the molecules in solution. • Small molecules are able to permeate more pores and are, therefore, • retained longer than large molecules. • Stationary phase is porous glass or silica or some polymers

  14. Columns can be made to separate specific size ranges • Larger species will elute first- they cannot pass through the pores thus their paths are short • This technique is useful for determining size and size range for polymers

  15. Choosing a Solvent (Mobile phase) in Liquid Chromatography • First the mode (normal or reversed phase) should be decided • If sample is water insoluble or non-polar: use normal phase; that is a nonpolar mobile phase (solvent) • If sample is water soluble or not soluble but polar: use reversed phase; that is a polar mobile phae (solvent)

  16. SOLVENTS Polar Solvents Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile Non-polar Solvents N-Decane > N-Hexane > N-Pentane > Cyclohexane

  17. Isocratic elution and Gradient elution • Isocratic elution The mobile phase is made of only one solvent or a mixture of solvents of known proportions • Gradient elution The mobile phase will have components of variable ratios.

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