Thin Layer Chromatography of

Lipids

Izmir Institute of TechnologyMolecular Biology and Genetics

DepartmentBiochemistry Lab.

Research Assistant Işıl ESMER

Thin Layer Chromatography

• TLC is one of the kinds of chromatography techniques

• Briefly Separation of two or more compounds or ions

• Solid – Liquid form of Chromatography

Why TLC ?

• Easy

• Inexpensive

• Quick in order to separate mixtures

• It is possible to identify unknown compound within a mixture

Specific Examples

• Determination of the components that plant contains

• Analyzing ceramides and fatty acids

• Detection of pesticides or insecticides in food and water etc.

Theory of TLC

• Mobile Phase (liquid)– Single solvent or combination of solvents

• Stationary Phase (Solid Absorbent)– Polar or non polar absorbent material

Competition between mobile and stationary phase

Slica Gel as a Stationary Phase

• Sheet of glass, plastic aluminum foil which is coated with Slica gel (SiO2)

• Absorbent such as slica gel is prepared by mixing small amount of inert binder like “ calcium sulfate “

– Small particules 6 to 13 µl– Smooth – Homogenous surface

Slica Gel as a Stationary Phase

Slica Gel as a Stationary Phase

• In case of slica gel Polar molecules will interact HIGHLY STRONGLY with polar Si – O bonds

• Polar molecules tend to stick to absorbent molecules (Stationary phase)

• Weakly polar molecules tend to move through absorbent more rapidly than polar species

Mobile Phases

Theory of TLC

• “ Different compounds travel at different rates due to the differneces in their attraction to the stationary and mobile phase “

• Commonly said strong solvents push the analyzed compounds up the plate while weak elutants barely move them

Retention Factor (Rf)

Retention Factor (Rf)

• Rf is constant if the chromatography conditions are constant ;

– Solvent system– Absorbent– Thickness of Adsorbent– Amount of material spotted– Temperature

2D - TLC

Equipment

Visualization

• If organic compounds are colored, it is easy to observe.

• If organic compound UV active You can visualize it with UV light

• If organic compound is not UV active you can stain with various dyes such as iodine or Naphtanol Blue Black

• Ethanolic phosphomolybdic acid reagent• α-naphthol- sulphuric acid reagent • Phosphate reagent of Dittmer and Lester• Ninhydrin reagent

Visualization

Terms

• Origin Line• Chromatogram• Developmnet chamber• Front • Capillary action• Stationary Phase• Solvent