1. BIO 306.01/02 EXPERIMENT 5 Separation of Amino Acids by Paper Chromatography
A. NAZLI BAŞAK

2. The methodology of chromatography includes now several procedures.
Separation of Amino Acids by Paper Chromotography
Chromatography was first developed in the 19. century as a means of studying and separating plant pigments (hence the name “chroma”).
The methodology of chromatography includes now several procedures.
All separate on the same general principle; uninterrupted flow of a moving phase thru a region of stationary phase (substance) which contains the sample to be analyzed.

3. Separation of Amino Acids by Paper Chromotography
Compounds in the sample to be analyzed interact to different degrees with the stationary and moving phases, thus affecting the separation.
The type of interaction between the stationary phase and the compounds in the sample is what distinguishes the different procedures.
The interaction is based on one of the following principle: ion exchange; solubility; adsorption; affinity or sieving. Some procedures utilize a combination of factors.

4. Paper chromatography:
Separation of Amino Acids by Paper Chromotography
Paper chromatography:
One of the early designs in the development of chromatography techniques was the use of a sheet of filter paper as an inert stationary support for a liquid.
The paper absorbs water vapor, and the stationary phase is a polar liquid (the absorbed water) and not the paper itself.
Another solvent with more non-polar constituents, such as n-butanol / water, is allowed to migrate up or down the paper by capillary action.

5. Separation of Amino Acids by Paper Chromotography
When this so-called developing solvent reaches the location, where the sample was applied onto the paper(origin), the sample will partition to different extents between the moving, non-polar phase and the stationary polar phase.
This partioning will continue as long as the developing solvent moves along the paper.

6. Separation of Amino Acids by Paper Chromotography
Origin of application

7. The extent of migration is expressed by the Rf value;
Separation of Amino Acids by Paper Chromotography
Substances that are more hydrophilic will move less than those that are more hydrophobic.
The extent of migration is expressed by the Rf value;
Both distances measured from the origin of sample application

8. Experiment Procedure:
Separation of Amino Acids by Paper Chromotography
Experiment Procedure:
In paper chromatography substances are distinguished by their relative position in the paper after solvent has moved a given distance.
The sample is placed at a marked spot on the paper and allowed to dry; this is the origin.

9. The paper is then placed into the chromatography tank (closed chamber)
Separation of Amino Acids by Paper Chromotography
The paper is then placed into the chromatography tank (closed chamber)
One end is immersed in the mobile phase (n-butanol / water also called developing solution)
Capillary draws the solvent thru paper, dissolves the sample as it passes the origin, and moves the components in the direction of flow.

10. Separation of Amino Acids by Paper Chromotography
After the “solvent front” has reached a point near the top end of the paper, the sheet is removed and dried. The ratio of distances traveled by the sample and by the solvent is the Rf value.
Value of Rf depends on the substance, the paper and the solvent. The Rf value is always < 1.

11. Detection of samples by Ninhydrin reactions
Separation of Amino Acids by Paper Chromotography
Detection of samples by Ninhydrin reactions
A very widely used reaction of the α-amino group is the Ninhydrin reactions, which is utilized to estimate amino acids quantitatively in very small amounts.
An α-amino acid reacts with two molecules of Ninhydrin on heating to yield an intensively colored product. Amino acids and peptides with a free α-NH2 group give a purple color, whereas Proline with its NH group (imino) yields a yellow color.

12. Separation of Amino Acids by Paper Chromotography
Oxidative decarboxylation

13. Separation of Amino Acids by Paper Chromotography

14. Separation of Amino Acids by Paper Chromotography
Ninhydrin brings about the oxidative decarboxylation of the amino acid.
The ammonia and Hydrindantin so formed react with a second molecule of Ninhydrin to yield a purple pigment of which only the nitrogen atom arises from the amino acid.
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