Experiment 2:

Separation of Amino Acids by

Paper Chromatography
Chem 31.1 WAD 2

Fernandez, Mia Allyanna A. | Lingan, Josephine Estelle C.

Chromatography
A physical method of separation
in which the components to be
separated are distributed
between two phases:
Mobile Phase
Stationary Phase

Types:

Thin-layer Chromatography
Reversed-phase Chromatography
High Performance Chromatography
Gas Chromatography
Column Chromatography
Paper Chromatography

Type

Types

Stationary Phase

Mobile Phase

Remarks

Thin-Layer

solid (silica gel, alumina, or cellulose

on a flat, inert substrate)

liquid (solvent)

Column

solid (silica gel, alumina, or cellulose

on a flat, inert substrate)

liquid (solvent)

solid (adsorption) or
nonvolatile liquid (partition)

gas

solid (silica gel, alumina, or cellulose

on a flat, inert substrate)

liquid (solvent)

relatively high pressure to be

applied

solid (more polar)

liquid

similar to high performance

except polarity of SP is
reversed

Gas
High
Performance
Reversed
Phase

Chromatography
Chromatography can be used to:
Qualitatively analyze the components of a mixture
Qualitatively identify the components of a mixture using known
compounds
Quantitatively determine the amount of a component in a mixture using
standard samples
Purify individual components by separating them from the other
compounds in a mixture

Retention Factor, Rf
Ratio of the time spent by the analyzed compound in the
stationary phase relative to the time it spent in the mobile
phase
Rf value - affinity with the mobile phase
Rf value - affinity with the stationary phase

Retention Factor, Rf

http://www.macalester.edu/~kuwata/Classes/2001-02/Chem%2011/Revised%20Amino%20Acids%20%289%201%2001%29.pdf

Methodology
Preparation of Solvent Mixture

24 mL Butanol
6 mL Glacial Acetic Acid
10 mL Distilled Water

Preparation of Chromatography Paper

*Note: Use gloves in handling the chromatography paper to avoid contamination

Methodology
Paper Chromatography Setup

Application of Amino Acids

Bottle Cover

Bottle
Chromatography
Paper
x

*Note: Use a clean capillary tube for each amino acid.

Spots must be about 1-2 mm

Solvent

*Note: Do not move the setup while the solvent is rising.

Methodology
After Developing Time
Remove the paper from the bottle. Let it stand in a petri dish.

Mark the solvent front using a pencil.

Remove staples and place the paper front side down on top of a
bond paper.
Brush the paper with Ninhydrin solution. Apply it evenly and
along one direction only.

Trace the edges of the resulting violet spots using a pencil.

Results
4.5

Distance traveled by
solvent front (cm)

Amino Acid

Distance Traveled
(cm)

Rf value
Theoretical

Rf value
Experimental

Glycine

1.0

0.26

0.22

Lysine

0.6

0.14

0.13

Leucine

4.4

0.73

0.97

Tyrosine

0.5

0.45

0.11

Unknown

1.0

Identity of Unknown

0.22
Glycine; Tyrosine

Results
Distance traveled by
solvent front (cm)

12

Amino Acid

Distance Traveled
(cm)

Rf value

Glycine

2.9

0.242

Lysine

0.167

Leucine

0.750

Tyrosine

6.2

0.516

Unknown

0.333

Identity of Unknown

Glycine

Paper Chromatography
Principles:
Capillary Action the movement of liquid within the spaces of a porous
material due to the forces of adhesion, cohesion, and surface tension.
The liquid is able to move up the filter paper because its attraction to itself is
stronger than the force of gravity.
Solubility the degree to which solute dissolves into a solvent.

Separation of components depends on both their solubility in the

mobile phase and their differential affinity to the mobile phase and the
stationary phase.

Reagents and Materials Used

Butanol
- (Butan-1-ol) CH3CH2CH2CH2OH

Glacial Acetic Acid

- C2H4O2

BUTANOL + GLACIAL ACETIC ACID Polar Solvent; MOBILE phase

WATER + FILTER PAPER Polar; STATIONARY phase
Amino Acid
- comprised of a carboxyl group and an amino group attached to the same
carbon atom (the carbon)

Reagents and Materials Used

Ninhydrin Solution
2,2 - Dihydroxyindane - 1,8 - dione
Makes the amino acids visible
Reacts with the amines to form blue-violet
or brown color

Amino Acids
Amino Acid

Abbreviation

Property(ies)

Higher affinity with

which phase?

Glycine

Gly

non-polar

mobile/stationary

Lysine

Lys

basic

stationary

Leucine

Leu

non-polar

mobile

Tyrosine

Tyr

polar

mobile

Amino Acids
Each amino acid has at least one amine and one acid functional group
Different properties due to variations in the structure of the R groups
Non-polar side chains: pure hydrocarbon alkyl groups (alkane branches) or
aromatic (benzene rings)
Polar side chains: functional groups such as acids, amides, alcohols, and
amines
Polarity ranking of functional groups:
Amide > Acid > Alcohol > Ketone, Aldehyde > Amine > Ester > Ether > Alkane
*Note: The more alkyl groups present, the more non-polar the amino acid would be.

Amino Acids
-

NH2CH2COOH
Aminoethanoic acid
Neutral, non-polar

HO2CCH(NH2)CH2CH(CH3)
2

2-Amino-4methylpentanoic acid
Neutral, non-polar

Amino Acids
-

HO2CCH(NH2)(CH2)4NH

2,6-Diaminohexanoic
acid
Polar

HO2CCH(NH2)(CH2)4NH
2

2-Amino-3-(4hydroxyphenyl)propanoi
c acid
Polar

Movement of Amino Acid

Depends on the affinity of the amino acid with the stationary and
mobile phases.
AFFINITY to the MOBILE PHASE
- Amino acid travels with the solvent
front
- Unimpeded by the paper
- Higher Rf value
- Non-polar compounds

AFFINITY to the STATIONARY

PHASE
Amino acid travels more slowly
Bonds to the cellulose of the paper
easily
Lower Rf value
Polar compounds

Factors Affecting Rf Values

a) Molecular weight Lower molecular weight results to greater velocity of
migration which in turn, results to a higher Rf value.
b) Polarity or affinity to mobile and stationary phase Higher polarity
results to lower Rf value.
c) Nature of stationary and mobile phase Greater difference in polarity
results to greater velocity of migration which would result to a higher Rf value.
d) Temperature Higher temperature results to a higher Rf value.

Guide Question #4
Give reasons for the following procedures:
a)
The diameter of the amino acid spots should be about 1 mm only.
To prevent the spots from overlapping with one another. If the
spots overlap, it would be hard to differentiate among the amino
acids because ninhydrin causes the same color change for all the
amino acids used.
b)
The solvent mixture should be allowed to saturate the chromatography
chamber.
For effective capillary action
To prevent evaporation of volatile solvents

c)
The chromatography paper should not be touched with bare
hands.
Hands contain large amounts of amino acids which could
contaminate the chromatogram thereby interfering with the
results and so it should not touch the chromatography
paper.

Guide Question #5
A mixture of amino acids was separated into its components by two-dimensional chromatography using solvents S1 and S2.
The data obtained are given below:
Distance traveled by the Amino Acid standard in S1 and S2:
Distance traveled by solvent fronts:
Amino Acid
S1 (cm)
S2 (cm)
S1 (Butanol, acetic acid, water) = 11.5 cm
S2 (Phenol, Water) = 12.0 cm
Ala
3.7
6.5
Distance traveled by the amino acid in S1 and S2:

Phe

9.14

4.9

Amino Acid

S1 (cm)

S2 (cm)

Lys

6.15

1.3

6.1

5.8

Leu

2.0

9.6

8.9

2.1

Glu

2.3

7.5

6.0

1.0

His

9.0

2.2

9.0

4.5

Trp

5.9

6.0

Draw clearly the two-dimensional chromatogram

and indicate the directions of solvent flow. Identify
the amino acids A, B, C and D.
Rf = distance traveled by the amino acid
distance traveled by the solvent

Amino Acid

Rf1

Rf2

0.53

0.48

0.77

0.18

0.52

0.08

0.78

0.38

Ala

0.32

0.54

Phe

0.79

0.41

Lys

0.53

0.11

Leu

0.17

0.80

Glu

0.20

0.63

His

0.78

0.18

Trp

0.51

0.50

A Tryptophan
B Histidine
C Lysine
D Phenylalanine

His
B

Lys
C

Phe

A
Trp
Ala
Glu

Leu

Conclusion
Separation of amino acids depend on their affinities with the
stationary and mobile phases
Polar amino acids: higher affinity with stationary phase:
lower Rf value
Non-polar amino acids: higher affinity with mobile phase:
higher Rf value

Recommendations
Dont touch the filter paper with bare hands to avoid contamination of
the chromatogram.
Whatman No.1 filter paper is advisable to use for more accurate and
observable results.
Apply the ninhydrin solution evenly on the back of the paper.
Be careful in putting the amino acids. Size matters.

References
Amino Acids. Retrieved 24 March 2015 from
http://www.elmhurst.edu/~chm/vchembook/213organicfcgp.htm
Functional Groups. Retrieved 24 March 2015 from
http://www.elmhurst.edu/~chm/vchembook/213organicfcgp.htm
Paper Chromatography. Retrieved 18 March 2015 from
http://faculty.pepperdine.edu/jfritsch/Fritsch/120%20web/Paper%20Chromatography%20S13%20
upload.pdf
Clark, Jim. Paper Chromatography. Retrieved from
http://www.chemguide.co.uk/analysis/chromatography/paper.html
Royal Society of Chemistry. Chromatography. Retrieved from
http://media.rsc.org/Modern%20chemical%20techniques/MCT5%20Chromatography.pdf