Chromatography

By- Bhavya, Harsh, Harshvardhan, Namrata, Ronit and Vidhatri

Contents

• Definition of chromatography• Different kinds of chromatography• Uses and applications of each

What is Chromatography?

Chromatography is the science which studies the separation of molecules based on differences in their structure and/or

composition.

The word was first derived from the Greek word chroma meaning “colour”.

Glossary…• Chromatograph: Instrument employed for a chromatography.

• Eluent: Fluid entering a column.

• Eluate: Fluid exiting the column.

• Elution: The process of passing the mobile phase through the column.

• Flow rate: How much mobile phase passed / minute (ml/min).

• Linear velocity: Distance passed by mobile phase per 1 min in the column (cm/min).

Different types

• Thin layer chromatography• Gas Chromatography• HPLC• Electrophoresis • Paper Chromatography

Thin Layer Chromatography

• TLC is using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic.

• The silica gel (or the alumina) is the stationary phase.

• The mobile phase is a suitable liquid solvent or mixture of solvents.

Uses

• Determination of the components a plant contains

• Monitoring organic reactions.• Detection of pesticides or insecticides in food and

water• Analyzing the dye composition of fibers in

forensics, or• Identifying compounds present in a given

substance

Gas Liquid Chromatography

• Involves a sample being vaporized and injected onto the head of the chromatographic column.

• The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

In the animation below the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.

Gas Chromatography

Uses

• Determination of volatile organic compounds (gases & liquids)

• Determination of partition coefficients and absorption isotherms

• Isolating pure components from complex mixtures

HPLC• High Performance Liquid Chromatography.

• Done at room temperature.

• Advantageous for chemicals that are heat sensitive or volatile.

• During this process, high pressure was used to generate the flow required for liquid chromatography in packed columns.

Uses

• It has the ability to separate, identify, and quantitate the compounds that are present in any sample that can be dissolved in a liquid.

• HPLC can be, and has been, applied to just about any sample, such as pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices, forensic samples, and industrial chemicals.

Electrophoresis

• Similar to TLC, but the substance is separated through a gel by electric current.

• It is reasonably easy and inexpensive.

• Electrophoresis may be the main technique for molecular separation in today's cell biology laboratory.

Uses

• Used in cell biological laboratory for molecular separation.

• A method of separating substances, especially proteins.

• It is used for analyzing molecular structure based on the rate of movement of each component in a colloidal suspension while under the influence of an electric field.

Paper Chromatography• Paper chromatography is an analytical chemistry

technique for separating and identifying mixtures that are or can be colored, especially pigments.

• In paper chromatography, the stationary phase is a very uniform absorbent paper.

• The mobile phase is a suitable liquid solvent or

mixture of solvents.

Uses

• They are used in many scientific studies to identify unknown organic and inorganic compounds.

• They are used in crime scene investigation.

• They are used for DNA and RNA sequencing as well.

Rf Factor

• Retention Factor

• It is a number that represents how far a compound travels in a particular solvent.

• It is measured by measuring the ratio of the distance traveled by the compound and the distance traveled by the solvent.

Thank you…