Organic Chemistry

Section 23.5 Separation of Amino Acids 969

Chromatography

Leu

Ala

Glu

most polar amino acid

least polar amino acid

origin

>Figure 23.2

Separation of glutamate, alanine,

and leucine by paper

chromatography.

Movie:

Column chromatography

Paper Chromatography and Thin-Layer Chromatography

Paper chromatographyonce played an important role in biochemical analysis be-

cause it provided a method for separating amino acids using very simple equipment.

Although more modern techniques are now more commonly used, we will describe

the principles behind paper chromatography because many of the same principles are

employed in modern separation techniques.

The technique of paper chromatography separates amino acids on the basis of po-

larity. A few drops of a solution of an amino acid mixture are applied to the bottom of

a strip of filter paper. The edge of the paper is then placed in a solvent (typically a mix-

ture of water, acetic acid, and butanol). The solvent moves up the paper by capillary

action, carrying the amino acids with it. Depending on their polarities, the amino acids

have different affinities for the mobile (solvent) and stationary (paper) phases and

therefore travel up the paper at different rates. The more polar the amino acid, the

more strongly it is adsorbed onto the relatively polar paper. The less polar amino acids

travel up the paper more rapidly, since they have a greater affinity for the mobile

phase. Therefore, when the paper is developed with ninhydrin, the colored spot closest

to the origin is the most polar amino acid and the spot farthest away from the origin is

the least polar amino acid (Figure 23.2).

O

O

O

O

O

O

O

N

O

O

O

+ H 2 O

O−

N

purple-colored product

+ H 2 O

H

HO−

The most polar amino acids are those with charged side chains, the next most polar

are those with side chains that can form hydrogen bonds, and the least polar are those

with hydrocarbon side chains. For amino acids with hydrocarbon side chains, the larg-

er the alkyl group, the less polar the amino acid. In other words, leucine is less polar

than valine.

Paper chromatography has largely been replaced by thin-layer chromatography

(TLC). Similar to paper chromatography, TLC differs from it in that TLC uses a plate

with a coating of solid material instead of filter paper. The physical property on which

the separation is based depends on the solid material and the solvent chosen for the

mobile phase.

PROBLEM 12

A mixture of seven amino acids (glycine, glutamate, leucine, lysine, alanine, isoleucine,

and aspartate) is separated by TLC. Explain why only six spots show up when the chro-

matographic plate is sprayed with ninhydrin and heated.

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