BIOLOGY NOTE FOR CHROMATOGRAPHY

Chromatography

Chromatography is a technique used to separate mixtures (ofmacromolecules) into their components, such as, amino acids, proteinsand photosynthetic pigments (chlorophyll).

The mixture carried by a chromatography solvent (mobile phase e.g. ,ethanol) is made to flow through a porous chromatography medium(stationary phase e.g. paper, silica gel) which restrict the movementof macromolecules.

Component molecules are carried through the pores of the chromatographymedium depending on their size, solubility in solvent and differentialadsorptiof the component to the chromatography medium.

Some of the common types of chromatography are:
(a)
Column chromatography (NIS)
(b)
Planar chromatography
·
Paper chromatography
·
Thin layer chromatography

Paper Chromatography for the separatiof the chlorophyll

* A small spot of chlorophyll extract is placed on the base lineabout 1 cm from the end of the strip of the chromatography paper. Thespot is allowed to dry and the process is repeated until a smallconcentrated spot is obtained.
* The edge of the paper is then dipped in to a suitable solvent,taking care that the spot is above the su***ce of the solvent, andplaced in a sealed container.
* The solvent moves up the paper by capillary action, which occuras a result of the attractiof the solvent molecules to the paper. Withthis, the chlorophyll components are carried along at different rates.The rate of movement depends on the size of the component molecule, thedifferent solubilities in the solvent and the adsorptiof the solutes bythe paper.
* Before the solvent front reaches the opposite end of thechromatography paper, the process is stop and the chromatogram isdried. The solvent front is marked.
* Each compound in a mixture has a characteristic Rf value. Thisvalue is a constant for the same solvent at the same compound. Itherwords, by comparing the Rf value of the component of a mixture withthose of the known compounds, the component of the mixture can beidentified.


P/S: If the substance to be separated is non-colored andnon-fluorescent even under ultra-violet light (usually amino acids),then the chromatogram needs to be colored by spraying with certainchemicals. For amino acids, the coloring substance is ninhydrin.Ninhydrin reacts with amino acids to produce various purple coloredpatterns. The position and intensity of the color indicate the type andthe quantity of each constituent amino acid.




Two-dimensional paper chromatography

Two-dimensional paper chromatography is used to test complex mixturesof solutes that are indistinguishable by one-direction chromatography.(E.g. intermediate products of photosynthesis, various amino acids fromhydrolysis of protein)

Procedures:
1.
A square feet of filter paper is used, mixture is placed on thebaseline near one end of the paper and first separation is carried out.
2.
The chromatogram is takeut, dried, rotated 90̊ and second separation is carried out with different solvent/ same solvent.
3.
This separates out the various components with their different properties.
4.
The chromatogram is removed and the solute is identified by treatingwith suitable reagent. (spray with ninhydrin so that amino acid orprotein can present in purple color form)


Electrophoresis

Electrophoresis is a technique used to separate a mixture of chargedmolecules such as amino acids and protein in an electric field (E.g.paper chromatography and PAG gel [polyalamide])
All amino acids and proteins molecules have their own isoelectric. Theisoelectric point (pI) is the pH at which a particular molecule orsu***ce carries no net electrical charge. Amphoteric molecules calledzwitterions contain both positive and negative charges depending on thefunctional groups present in the molecule. They are affected by pH oftheir surrounding environment and can become more positively ornegatively charged due to the loss or gain of protons (H+).
The pI value can also affect the solubility of a molecule at a givenpH. Such molecules have minimum solubility in or salt solutions at thepH which corresponds to their pI and often precipitate out of solution.Biological amphoteric molecules such as proteins contain both acidicand basic functional groups. Amino acids which make up proteins may bepositive, negative, neutral or polar in nature, and together give aprotein its overall charge. At a pH below their pI, proteins carry anet positive charge; above their pI they carry a net negative charge.Proteins can thus be separated according to their isoelectric point(overall charge) on a polyalamide gel using a technique calledisoelectric focusing, which utilizes a pH gradient to separateproteins. Isoelectric focusing is also the first step in 2-D gelpolyalamide gel electrophoresis.
Used to separate and for identification of:
-Serum proteins -various amino acids in a protein hydrolysate – DNAfragment – DNA fingerprint – analysis of protein content in tissuefluid – genetic testing – paternity analysis – forensic – conservationbiology – animal behavior study –taxonomy – evolutionary biology