ChromatographyChromatography

Tomáš MlčochTomáš Mlčoch

Pavel MoťkaPavel Moťka

ChromatographyChromatography

Described by Tswett in 1906Described by Tswett in 1906 He separated some pigments using a He separated some pigments using a

tube filled with CaCOtube filled with CaCO33

By washing the extract through the By washing the extract through the column with column with anan organic solvent organic solvent he he was able to produce several colored was able to produce several colored bandsbands

band = pásband = pás

column = sloupeccolumn = sloupec

ChromatographyChromatography

Analytical methodAnalytical method Separation of components of a mixtureSeparation of components of a mixture Stationary phaseStationary phase

– SolidSolid

– Liquid Liquid

Mobile phase (flow over stationary Mobile phase (flow over stationary phase)phase)– LiquidLiquid

– GasGas

Types of Types of ChromatographyChromatography

Column ChromatographyColumn Chromatography Thin Layer Chromatography (TLC)Thin Layer Chromatography (TLC) Paper ChromatographyPaper Chromatography Gas Chromatography (GC)Gas Chromatography (GC) High Performance Liquid High Performance Liquid

Chromatography (HPLC)Chromatography (HPLC)

high performance = vysoce účinnáhigh performance = vysoce účinná

Column Column ChromatographyChromatography

Vertical glass columnVertical glass column Mobile phase = liquidMobile phase = liquid Stationary phase = Stationary phase =

solidsolid

– Silica gel (SiOSilica gel (SiO22))

– AluminaAlumina (Al (Al22OO33))

Sample = Sample = ssolid or liquid olid or liquid

placed on columnplaced on column

Separatory funnel

Column Column ChromatographyChromatography Affinity = molecules adsorbed on solidAffinity = molecules adsorbed on solid

– Compound in liquid elute firstCompound in liquid elute first

– More polar molecules elute lastMore polar molecules elute last

Collect separated compounds Collect separated compounds

(fractions)(fractions)– Evaporate solventEvaporate solvent

– ID or further purifyID or further purify

evaporate = vypařitevaporate = vypařit

Thin Layer Thin Layer ChromatographyChromatography Similar to column Similar to column

chromatographychromatography Mobile phase = solventMobile phase = solvent Stationary phase = solid (silica Stationary phase = solid (silica

gel)gel)

Paper ChromatographyPaper Chromatography

Similar to TLCSimilar to TLC

Stationary phase = HStationary phase = H22O adsorbed O adsorbed

by celluloseby cellulose

Mobile phase = solventMobile phase = solvent

Frequently used to polar Frequently used to polar

compoundscompounds– Amino acids, carbohydrates, etc.Amino acids, carbohydrates, etc.

Gas ChromatographyGas Chromatography

Mobile phase = stream of inert gasMobile phase = stream of inert gas Stationary phase = high boiling liquid filmStationary phase = high boiling liquid film

– Polarity similar to compounds in mixturePolarity similar to compounds in mixture

– Supported on a solidSupported on a solid

– Packed in a columnPacked in a column hheated glass or metaleated glass or metal 2-3 m in length; 2-5 mm diameter2-3 m in length; 2-5 mm diameter

ddiameteriameter = průměr = průměr

Gas ChromatogramGas Chromatogram

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

Resources:Resources: http://www.labo.cz/sl/vy07.htmhttp://www.labo.cz/sl/vy07.htm www.rpi.edu/dept/chem-eng/ www.rpi.edu/dept/chem-eng/

Biotech-Environ/CHROMO/chromintBiotech-Environ/CHROMO/chromintro.htmlro.html

http://ull.chemistry.uakron.edu/http://ull.chemistry.uakron.edu/analytical/Chromatography/analytical/Chromatography/

Analytická chemie II, prof. Lumír Analytická chemie II, prof. Lumír SommerSommer

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