thin layer chromatography of lipids izmir institute of technology molecular biology and genetics...
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Thin Layer Chromatography of
Lipids
Izmir Institute of TechnologyMolecular Biology and Genetics
DepartmentBiochemistry Lab.
Research Assistant Işıl ESMER
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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
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Why TLC ?
• Easy
• Inexpensive
• Quick in order to separate mixtures
• It is possible to identify unknown compound within a mixture
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Specific Examples
• Determination of the components that plant contains
• Analyzing ceramides and fatty acids
• Detection of pesticides or insecticides in food and water etc.
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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
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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
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Slica Gel as a Stationary Phase
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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
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Mobile Phases
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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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Retention Factor (Rf)
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Retention Factor (Rf)
• Rf is constant if the chromatography conditions are constant ;
– Solvent system– Absorbent– Thickness of Adsorbent– Amount of material spotted– Temperature
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2D - TLC
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Equipment
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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
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• Ethanolic phosphomolybdic acid reagent• α-naphthol- sulphuric acid reagent • Phosphate reagent of Dittmer and Lester• Ninhydrin reagent
Visualization
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Terms
• Origin Line• Chromatogram• Developmnet chamber• Front • Capillary action• Stationary Phase• Solvent