Thin Layer Chromatography of Lipids

Presentation on theme: "Thin Layer Chromatography of Lipids"— Presentation transcript:

1 Thin Layer Chromatography of Lipids
Izmir Institute of Technology Molecular Biology and Genetics Department Biochemistry Lab. Research Assistant Işıl ESMER

2 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

3 Why TLC ? Easy Inexpensive Quick in order to separate mixtures
It is possible to identify unknown compound within a mixture

4 Specific Examples Determination of the components that plant contains
Analyzing ceramides and fatty acids Detection of pesticides or insecticides in food and water etc.

5 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

6 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

7 Slica Gel as a Stationary Phase

8 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

9 Mobile Phases

10 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

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15 Retention Factor (Rf)

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17 Retention Factor (Rf) Rf is constant if the chromatography conditions are constant ; Solvent system Absorbent Thickness of Adsorbent Amount of material spotted Temperature

18 2D - TLC

19 Equipment

20 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

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

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