High Performance Liquid Chromatography

High PerformanceLiquid

Chromatography

• 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.

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

Types Of Liquid Chromatography

• Partition chromatography

• Adsorption, or liquid-solid chromatography

• Ion exchange chromatography

• Size exclusion, or gel, chromatography

Stationary phase is solid: either polar (silica gel)or nonpolar (polymer beads).

Liquid –Solid ChromatographyAdsorption Chromatography

Stationary phase is solid and the separation occurs as a result of adsorption desorption steps

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

• 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

Liquid-Liquid ChromatographyPartition Chromatography

Stationary phase Liquid-liquidChromatography

• 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.

Stationary phase

Inert support

Normal phaseNormal phase: Polar stationary phase and non-polar solvent (mobile phase)

Reverse phase:Reverse phase: Non-polar stationary phase and polar solvent (mobile phase)

R+Y- + X- R+X- + Y- (Anion exchanger)

R-Y+ + X+ R-X+ + Y+ (Cation exchanger)

Ion exchange chromatography

Stationary phase has an ionically charged surfaceopposite to that of the eluent

• Stationary phase is called ion-exchangers

• The ion-exchangers are made of exchange groups bound to a support.

)GPC(

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

Size exclusion chromatogrphy

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

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

• 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

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)

SOLVENTS

Polar SolventsPolar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

 Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane

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.

STATIONARY PHASES