Principles of chromatography
Principles of chromatography
|Mobile phase or carrier||solvent moving through the column|
|Stationary phase or adsorbent||substance that stays fixed inside the column|
|Eluent||fluid entering the column|
|Eluate||fluid exiting the column (that is collected in flasks)|
|Elution||the process of washing out a compound through a column using a suitable solvent|
|Analyte||mixture whose individual components have to be separated and analyzed|
- Take a few leaves and crush them in a mortar.
- Spot a drop of the leaf extract on a strip of chromatographic paper ~ 0.5 cm above the edge of the paper. Chromatographic paper is made of cellulose and is quite polar in nature.
- Place the strip of paper in a jar that contains a small volume of propanone (acetone). There should be just enough propanone that the edge of the paper dips in it comfortably. Place a lid on the jar to avoid any evaporation of the solvent.
- Let the solvent rise up the paper by capillary action. Remove the paper strip from the jar once the solvent has reached the ‘solvent front’ level. 5) What do you think you will notice?
- Higher the adsorption to the stationary phase, the slower the molecule will move through the column.
- Higher the solubility in the mobile phase, the faster the molecule will move through the column.
Different types of chromatography
|Technique||Stationary phase||Mobile phase||Basis of separation||Notes|
|*Paper chromatography||solid (cellulose)||liquid||polarity of molecules||compound spotted directly on a cellulose paper|
|*Thin layer chromatography (TLC)||solid (silica or alumina)||liquid||polarity of molecules||glass is coated with thin layer of silica on which is spotted the compound|
|*Liquid column chromatography||solid (silica or alumina)||liquid||polarity of molecules||glass column is packed with slurry of silica|
|Size exclusion chromatography||solid (microporous beads of silica)||liquid||size of molecules||small molecules get trapped in the pores of the stationary phase, while large molecules flow through the gaps between the beads and have very small retention times. So larger molecules come out first. In this type of chromatography there isn’t any interaction, physical or chemical, between the analyte and the stationary phase.|
|Ion-exchange chromatography||solid (cationic or anionic resin)||liquid||ionic charge of the molecules||molecules possessing the opposite charge as the resin will bind tightly to the resin, and molecules having the same charge as the resin will flow through the column and elute out first.|
|Affinity chromatography||solid (agarose or porous glass beads on to which are immobilized molecules like enzymes and antibodies)||liquid||binding affinity of the analyte molecule to the molecule immobilized on the stationary phase||if the molecule is a substrate for the enzyme, it will bind tightly to the enzyme and the unbound analytes will pass through in the mobile phase, and elute out of the column, leaving the substrate bound to the enzyme, which can then be detached from the stationary phase and eluted out of the column with an appropriate solvent.|
|Gas chromatography||liquid or solid support||gas (inert gas like argon or helium)||boiling point of the molecules||samples are volatilized and the molecule with lowest boiling point comes out of the column first. The molecule with the highest boiling point comes out of the column last.|
Thin layer chromatography (TLC): Retention factors (R)
- The component that travels the least distance on the TLC plate is the most polar, since it binds to the silica most tightly.
- The component that travels the maximum distance is the least polar; it binds to the silica least tightly and is most soluble in the non-polar solvent (mobile phase), and hence moves up the plate with the solvent.
|Component||Distance travelled by the component (cm)||Distance travelled by the solvent (cm)||Retention factor (R) of the component|
|C||1||5||R = 1/5 = 0.2|
|A||2||5||R = 2/5 = 0.4|
|B||3||5||R = 3/5 = 0.6|