HPLC (High Performance Liquid Chromatography)

LIQUID CHROMATOGRAPHY A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid. With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.

Schematic Diagram of Liquid Chromatography

High Performance Liquid Chromatography

FOUR BASIC LIQUID CHROMATOGRAPHY The 4 basic liquid chromatography modes are named according to the mechanism involved:  1. Liquid/Solid Chromatography (adsorption chromatography) A. Normal Phase LSC B. Reverse Phase LSC (uncommon)  2. Liquid/Liquid Chromatography (partition chromatography) A. Normal Phase LLC B. Reverse Phase LLC  3. Ion Exchange Chromatography  4. Gel Permeation Chromatography (exclusion chromatography)

HPLC columns (Stationary phase) Stationary phase: silica (SiO2ㆍxH2O) Active adsorption site: silanol (Si-O-H) Bare silica: Adsorption chromatography Boned stationary phase

Silica gel (SiO2) and alumina (Al2O3)

LIQUID SOLID CHROMATOGRAPHY Normal phase LS Reverse phase LS d- d+ Si - O - H 30 m Silica Gel 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.

The elution process In adsorption chromatography Elution can be described as a displacement of solute from the stationary phase by solvent. Elution strength

LIQUID-LIQUID CHROMATOGRAPHY ODPN (oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC NCCH 3 CH 2 OCH CN(Normal) (CH ) 16 (Reverse) 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.

Partition chromatography Columns for boned phase chromatography. Reverse and normal phase chromatography Reverse and normal chromatography are distinguishable based on the relative polarities of the mobile and stationary phase.

Elution strength

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

Effect of particle size on the HPLC analysis

Types of Chromatography LIQUID MOBILE PHASE Liquid-Solid Liquid-Liquid FORMAT Chromatography (Adsorption) Chromatography (Partition) Solid Liquid STATIONARY PHASE Normal Phase Reverse Phase Normal Phase Reverse Phase Mobile Phase - Nonpolar Mobile Phase - Polar Stationary phase - Polar Stationary phase - Nonpolar

HPLC Injector

Isocratic and gradient elution Isocratic elution Gradient elution.

ION-EXCHANGE CHROMATOGRAPHY SO 3 - Na + Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).

Ion exchange chromatography Packing materials Mobile phase Solute retention Application A pH is very important in Ion exchange chromatography.

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS + + SO 3 Na H N 3 COOH Ion-exchange Resin - + SO H N 3 3 - COO pH4.5 + Na

Chromatography of Amino Acids

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.

Detectors 1. Ultraviolet Detector 200-400nm 254 nm 2. Reflective Index Detector Universal Detector