PRESENTED BY

HIBA.C.H

1ST YEAR M-PHARM

DEPT. OF PHARMACEUTICS

CHROMATOGRAPHY

• Chromatography Greek :chromatos -- color ,

"graphein" -- to write.

INTRODUCTION

INTRODUCTION cts……

DefinitionThe separation of a mixture by distribution of its

components between a mobile and stationary phase over time

ORThe separation of mxt of components into

individual components through equilibrium distribution b/w two phases

INTRODUCTION cts……

• Mobile phase - phase that moves through chromatograph

• Stationary phase - phase that is stationary in chromatograph

Purpose of Chromatography

• Analytical - determine chemical composition of a sample

• Preparative - purify and collect one or more components of a sample

Uses for ChromatographyReal-life examples of uses for chromatography:

• Pharmaceutical Company – determine amount of each chemical found in new product

• Hospital – detect blood or alcohol levels in a patient’s blood stream

• Law Enforcement – to compare a sample found at a crime scene to samples from suspects

• Environmental Agency – determine the level of pollutants in the water supply

• Manufacturing Plant – to purify a chemical needed to make a product

Classification of Methods

There are two classification schemes:

–Mobile phase

–Attractive forces

Mobile Phase

• Gas (GC)• Water (LC)• Organic solvent (LC)• Supercritical fluid (SCFC)

Classification based on Mobile Phase

Gas Chromatography

Gas - solid

Gas - liquid

Liquid chromatography (LC)

Liquid- solidColumn

(gravity

flow)

High performance(pressur

e flow)

Thin layer(adsorptio

n)

Liquid -liquid

Paper –partition

Column partition

Classification based on Attractive Forces

1. Adsorption2. Ion Exchange3. Partition4. Size Exclusion

ADSORPTION CHROMATOGRAPHY

Separation based on their adsorption onto the surface of solid (stationary phase).

the compound which has more affinity towards stationary phase travels slower.

Ex; Column chromatography (CC) , TLC, HPLC

Partition Chromatography

solute are separated based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support.

Component which is more soluble in mobile phase travels faster & more soluble in stationary phase travel slower. Ex : liquid chromatography, Paper Chromatography,

column partition chromatography

Ion Exchange Chromatography Use ionic stationary phase

– ions separated on the basis of their tendency to displace counter ions adsorbed on stationary phase (Depends on charge, hydration, “solubility”…)

Anionic stationary phases: used for cation separation Cationic stationary phases : for anion separation for ionic compounds - Ex : CC , HPLC

SIZE EXCLUSION CHROMATOGRAPHY

Separation is a result of “trapping” of molecules in the pores of the packing material

• Very large molecules can’t get into the pores – unretained

• Very small molecules get hung up in to pores for a long time - most retained – longest retention time

• stationary phase is a porous matrix

• Ex: CC, HPLC

Types of Chromatography• Liquid Chromatography – separates liquid

samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

• Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)

Types of Chromatography

• Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)

• Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

STATIONARY PHASEType of chromatography Material

Paper chromatography Filter paper, cellulose

Thin Layer Chromatography Silica gel, alumina, polyamide

Gas chromatography(GC)

Squalene, apezion, carbowax M

High Performance Liquid Chromatography

C-8, C-18, Licosorb, Silicone

MOBILE PHASEType of chromatography Solvent

Paper chromatography Air, alcohol

Thin Layer Chromatography Hexane, ether petroleum, alcohol.

Gas chromatography(GC)

He, Ar, N2

High Performance Liquid Chromatography

Cyclohexane, n-hexane, carbon tetrachloride, ethanol, methanol, air