Chromatography Tomáš Mlčoch Pavel Moťka

Chromatography Described by Tswett in 1906 Described by Tswett in 1906 He separated some pigments using a tube filled with CaCO 3 He separated some pigments using a tube filled with CaCO 3 By washing the extract through the column with an organic solvent he was able to produce several colored bands By washing the extract through the column with an organic solvent he was able to produce several colored bands band = pás column = sloupec

Chromatography Analytical method Analytical method Separation of components of a mixture Separation of components of a mixture Stationary phase Stationary phase –Solid –Liquid Mobile phase (flow over stationary phase) Mobile phase (flow over stationary phase) –Liquid –Gas

Types of Chromatography Column Chromatography Column Chromatography Thin Layer Chromatography (TLC) Thin Layer Chromatography (TLC) Paper Chromatography Paper Chromatography Gas Chromatography (GC) Gas Chromatography (GC) High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) high performance = vysoce účinná

Column Chromatography Vertical glass column Vertical glass column Mobile phase = liquid Mobile phase = liquid Stationary phase = solid Stationary phase = solid –Silica gel (SiO 2 ) –Alumina (Al 2 O 3 ) Sample = solid or liquid placed on column Sample = solid or liquid placed on column Separatory funnel

Column Chromatography Affinity = molecules adsorbed on solid Affinity = molecules adsorbed on solid –Compound in liquid elute first –More polar molecules elute last Collect separated compounds (fractions) Collect separated compounds (fractions) –Evaporate solvent –ID or further purify evaporate = vypařit

Thin Layer Chromatography Similar to column chromatography Similar to column chromatography Mobile phase = solvent Mobile phase = solvent Stationary phase = solid (silica gel) Stationary phase = solid (silica gel)

Paper Chromatography Similar to TLC Similar to TLC Stationary phase = H 2 O adsorbed by cellulose Stationary phase = H 2 O adsorbed by cellulose Mobile phase = solvent Mobile phase = solvent Frequently used to polar compounds Frequently used to polar compounds –Amino acids, carbohydrates, etc.

Gas Chromatography Mobile phase = stream of inert gas Mobile phase = stream of inert gas Stationary phase = high boiling liquid film Stationary phase = high boiling liquid film –Polarity similar to compounds in mixture –Supported on a solid –Packed in a column heated glass or metal heated glass or metal 2-3 m in length; 2-5 mm diameter 2-3 m in length; 2-5 mm diameter diameter = průměr

Gas Chromatogram Retention time can be used to ID a component if compared to known sample under same conditions Retention time can be used to ID a component if compared to known sample under same conditions –Flow rate, column size, temperature, etc.

Resources: Biotech- Environ/CHROMO/chromintro.html Biotech- Environ/CHROMO/chromintro.html cal/Chromatography/ cal/Chromatography/ Analytická chemie II, prof. Lumír Sommer Analytická chemie II, prof. Lumír Sommer

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