thin layer chromatography of lipids izmir institute of technology molecular biology and genetics...

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