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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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