Chromatography

Intended learning outcomes: • Uses & common types of chromatographic techniques • Principles of separation and key practice aspects • Analytical Applications in identification & assuring quality of pharmaceutical substances

what is Chromatography

A technique used to separate and analyze components of a mixture based on their affinity for a stationary phase and a mobile phase.

what are the basics of chromatography

• capture

• elute

• detect

explain process of capture

The process of capturing analytes from a liquid mixture in chromatography.

what is the Stationary phase

The phase in chromatography that remains fixed and interacts with the components of the mixture.

what is the Mobile phase

The phase in chromatography that moves and carries the components of the mixture.

list the 5 common type of chromatography?

• column

• thin layer

• gel filtration

• ion exchange

• high pressure liquid

Gel filtration chromatography

A type of chromatography used to separate large molecules from small molecules based on their size.

Ion exchange chromatography

A type of chromatography based on the charge properties of the components, commonly used for proteins and peptides.

RF values

The ratio of the distance traveled by a component to the distance traveled by the solvent front in thin layer chromatography.

Give a quick explanation of column chromatography

• column loaded with stationary and mobile phase together

• silica gel is stationary phase

• polar molecules bind to silica and elute more slowly, non- polar molecules elute more quickly

• collected in fractions and determine by methods of colour

what is Silica gel

A common stationary phase in column chromatography that binds polar molecules and elutes them slowly.

How is UV light method used for detection

A method used to detect colorless components in thin layer chromatography by illuminating the plate and observing fluorescence

where it doesn’t fluoresce presence of drugs which contain aromatics

How is Ninhydrin used for detection

In presence of amines will appears pink/purple .

how is Iodine used for detection

detect double bonds in thin layer chromatography, which shows up as brown spots.

how is Potassium permanganate used for detection

used to detect sugars and sugar-like molecules in thin layer chromatography.

How to find the mass of unknown substances (Gel filtration)

• Calibrate the column by having different known samples of molecular weight

• Make a calibration curve

• Run unknown sample - gather elution volume

• From elution volume can use curve to find out likely molecular weight of unknown sample

what are applications of ion exchange chromatography

• Bio pressing/ protein purification

• Separate neutral compounds from mixture of anionic and cationic resins

• Isolation of metabolites from biological fluids