Experiment 6

IDENTIFYING AMINO ACIDS THROUGH PAPER CHROMATOGRAPHY

The qualitaAve analysis of the composiAon of protein hydrolyzates can be carried out through
paper chromatography. In this method, a liquid sample flows down a verAcal strip of adsorbent paper,
on which the components are deposited in specific locaAons. The components of the sample are
distributed in the chromatographic paper according to the differences in their solubility in the staAonary
phase and the mobile phase. Substances that are soluble in water will move slower than those that are
soluble in the moving organic phase.

The extent of migraAon of the compound is expressed in terms of the Rf value.

Distance traveled by the solute

Rf = ----------------------------------------------------------
Distance traveled by the developing solvent

In this acAvity, known samples of amino acids will be used as standards to which an unknown
amino acid will be compared.

OBJECTIVE

To idenAfy amino acids through paper chromatography.

MATERIALS

chromatographic paper or filter paper hair blower

1000 mL beaker pencil
10 mL graduated cylinder ruler
capillary tube stapler
watch glass bond paper
test tube brush rubber band

Reagents:
ninhydrin spray reagent 2 standard amino acids
ethanol (C2H5OH) (glycine and alanine)
ammonia (NH3) unknown amino acid (to be
disAlled water provided by the instructor)

PROCEDURES

1. Get a chromatographic or a filter paper.

2. Using a pencil, draw, from the edge, a 3-cm margin on one side of the paper. Draw 1-cm
margins on the remaining 3 sides.
Note: Refrain from touching the surface of the paper while drawing the margins.
3. Using a capillary tube, make 10 to 15 applicaAons of the unknown amino acid in the
designated spot on the 3-cm margin. Make sure that the first applicaAon has dried before
applying the next.

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4. Do the same with the 2 standard (known) amino acids. Observe even spacing between
spots.

IllustraAon:

1 cm

1cm 1cm

UNKNOWN

● ● ●
STANDARD STANDARD

3 cm

5. Prepare 30 mL of the developing solvent in the beaker by mixing together 24 mL of C2H5OH,

3 mL of NH3 and 3 mL of H2O.
6. Staple both ends of the paper to make a paper cylinder.
7. Slowly insert the paper cylinder into the beaker by holding the 1-cm top margin with your
fingers taking care not to crumple the paper nor touch the surface of the inner side of the
beaker.
8. Cover the beaker with a piece of bond paper. Secure with a rubber band.
9. Allow the solvent to rise slowly in the paper for an hour.
10. Remove the paper cylinder from the beaker.
11. With a pencil, mark the distance traveled by the solvent.
12. Dry the paper using a hair blower.
13. Spray the paper with the ninhydrin soluAon. Develop the colors by allowing the paper to dry
once more using of the hair blower.
14. Encircle the spots with a pencil. Measure the height of the column from the encircled spot
up to the Ap of the colored column. Measure also the distance traveled by the solvent from
the spot to the pencil mark made in no. 11.
15. Calculate the Rf values of the amino acids. IdenAfy the unknown amino acid by comparing
its Rf value with those of the standards.

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 REPORT SHEET

Group No.: _______________________________ RaAng: _______________

Name: __________________________________ Date Performed: ________
C/Y/S: __________________________________ Date Submiied: ________

Experiment 6
IDENTIFYING AMINO ACIDS THROUGH PAPER CHROMATOGRAPHY

DATA and RESULTS

Parameters Glycine Alanine Unknown

Distance traveled by
the solvent
Distance traveled by
the amino acid
Computed
Rf Value

IdenAty of the unknown amino acid: ___________________________________

POST LABORATORY DISCUSSION

1. Is there a difference between the distances traveled by the solvent and the amino acids?
Why?
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2. What is the importance of the precauAons observed while preparing the paper chromatograph?
_________________________________________________________________________
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3. Give one importance of chromatography in Biochemistry.

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4. Can chromatography be used in idenAfying organic compounds other than amino acids?
Explain briefly.
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_________________________________________________________________________
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5. Illustrate the appearance of the paper chromatogram produced in the acAvity.

CONCLUSION

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CLINICAL APPLICATION

Discuss briefly how chromatographic techniques can be used as basis for the diagnosis of diseases.
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