Thin Layer Chromatography of Lipids Izmir Institute of Technology Molecular Biology and Genetics Department Biochemistry 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 Competition between mobile and stationary phase 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

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

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