Experiment 2

Separation of Amino Acids by Paper Chromatography

Magbanua, Dianne B. Olabre, Ian Gabriel N.

Objectives:
proper application of the techniques of paper

chromatography
the computation and the comparison of the

Rf values of the given amino acids

the identification of the unknown amino acid

by comparison of Rf values.

KEYWORDS:
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

Chromatography
a physical method of separation in

which the components to be separated are distributed between two phases:

stationary phase: solid or a liquid supported on a solid which is fixed in place mobile phase: liquid or gas which moves in a definite direction.

Types:
Thin-Layer Chromatography (liquid-solid) Reversed Phase Chromatography (liquidsolid)

High Performance Chromatography

solid)

(liquid-

Gas Chromatography (gas-liquid) Column Chromatography (liquid-solid) Paper Chromatography (liquid-liquid)

Paper chromatography
type of partition chromatography based on the rate of migration of the

compounds being analyzed while they are in simultaneous contact with the stationary phase.

stationary phase: water bound to the cellulose matrix of the filter paper mobile phase: solvent mixture

RESULTS

Amino Acid Distance Distance Rf Values Standards Travelled by Travelled by the Amino the Solvent Acid (cm) Mixture (cm) 3.4 12. 5 0.272 Glycine 12. 5 0.48 Tyrosine 6 Leucine Lysine Unknown Glycine Leucine
11 3 3.2 10.1 12. 5 12. 5 12. 5 12. 5 0.88 0.24 0.256 0.808

Table I. The Rf Values of the Amino Acids

THEORETICAL Rf VALUES OF AMINO ACIDS

Amino Acids A. Glycine B. Lysine C. Leucine D.Tyrosine Theoretical Rf Values 0.26 0.14 0.73 0.45

DISCUSSION
The separation and migration of the amino

acids are based on their affinities to the stationary and mobile phases.
Factors that affect the affinity of a

substance:
polarity,

pH weight

molecular

structure of the molecule

shape

DISCUSSION
Higher affinity to the mobile phase Stick to the paper Unimpeded by the paper Travel more slowly Travel with the solvent front Smaller Rf values Larger Rf values Polar compounds Nonpolar compounds Bond to the cellulose of the paper Remain dissolved in the mobile more quickly phase

Higher affinity to the stationary phase

Glycine: most polar Leucine: least polar Unknown: Glycine + Leuc

DISCUSSION
Filter paper - made up of cellulose (non

polar) fibers which attract and adsorb water vapor to its surface.

DISCUSSION
20 different amino acids - linked together

by peptide bonds to form proteins.

Different proteins vary in the order and

number of amino acids in their polypeptide chains.

DISCUSSION
Amino acids with nonpolar, neutral

hydrocarbon side chains: hydrophobic, lower water solubility

Amino acids with polar but neutral R groups:

hydrophilic, promote water solubility

Amino acids with polar and basic and acidic

R groups: promote water solubility

DISCUSSION
Solubility of all amino acids in water

depends on the pH of the solution.

Rate of migration of each amino acid also

depends on the pH of the mobile phase.

DISCUSSION
Leucine:
HO2CCH(NH2)CH2CH(CH3)2 2-Amino-4-methylpentanoic acid Hydrophobic, aliphatic, neutral, non-polar

DISCUSSION
Glycine:
NH2CH2COOH Aminoethanoic acid

Aminoacetic acid
hydrophobic Neutral, polar

DISCUSSION
Tyrosine:
C9H11NO3 2-Amino-3-(4-hydroxyphenyl)propanoic acid non-essential amino acid with a polar side

group
Aromatic, hydrophilic, neutral

DISCUSSION
Lysine:
HO2CCH(NH2)(CH2)4NH2 2,6-diaminohexanoic acid basic essential amino acid Polar, hydrophilic, charged (+)

DISCUSSION
Ninhydrin
(2,2-Dihydroxyindane-1,3-

dione)

make the amino acids visible detects ammonia, or primary

and secondary amines.

DISCUSSION

GUIDE QUESTIONS
Identify the stationary and mobile phases

in paper chromatography. stationary phase water mobile phase mixture of butanol, glacial acetic acid and water

GUIDE Explain briefly the differences in Rf QUESTIONS values of the amino acid component of
your mixture. ~depends on the polarity or affinity of each substance to the mobile and stationary phases.
less polar amino acid mobile phase less polar

GUIDE What are the factors that could affect the QUESTIONS Rf value of a solute?
polarity

or affinity of the solute to the mobile and stationary phases weight of stationary and mobile

molecular nature

phase

GUIDE Give reasons for the following procedure: QUESTIONS

a. The diameter of the amino acid spots should

be about 1mm only. ~spots will usually be larger than the original spots after drying

GUIDE b. The solvent mixture should be QUESTIONS

allowed to saturate the chromatography chamber.
~allows for more effective development of the chromatograms ~prevents the evaporation of the solvent

d. The chromatography paper

GUIDE A mixture of amino acids was separated QUESTIONS into its components by two-dimensional
chromatography using solvents S-1 and S2.
Amino Acid A B C D S1 (cm) 6.1 8.9 6.0 9.0 S2 (cm) 5.8 2.1 1.0 4.5 Amino Acid Ala Phe Lys Leu Glu His Trp S1 (cm) 3.7 9.14 6.15 2.0 2.3 9.0 5.9 S2 (cm) 6.5 4.9 1.3 9.6 7.5 2.2 6.0

Amino Acid A Amino Acid B Amino Acid C Amino Acid D

Tryptophan Histidine Lysine Phenylalanine

GUIDE Discuss briefly the basic principles of QUESTIONS the following chromatographic
techniques.
Thin-Layer Chromatography

- solvent moves up the plate due to capillary action - components of the sample mixture get separated based on their attraction to the stationary phase and the difference in

 Column Chromatography

-liquid-solid chromatography - form of adsorption chromatography -different substances will adsorb or adhere onto the surface of fine particles of a solid adsorbent

GUIDE Gas Chromatography QUESTIONS -stationary phase is a high-boiling liquid

-mobile phase is an inert gas -boiling point of the compound (but not always) that determines how fast a component travels through the column

High Performance Chromatography

-the separation of compounds is carried out on the basis of their characteristic polarities

GUIDE QUESTIONS
Reversed Phase Chromatography

-results from the adsorption of hydrophobic molecules onto a hydrophobic solid support in a polar mobile phase

CONCLUSION
Chromatography is a set of laboratory

techniques used for the separation of mixtures:

paper

chromatography, chromatography, chromatography,

thin-layer column gas high

chromatography, performance chromatography, phase chromatography.

reversed

CONCLUSION
Paper chromatography involves a solvent

moving along a filter paper.

The interaction of the analyzed

compounds, the filter paper, and the solvent mixture determines the rate of separation and the distances travelled by the substances.
Since the amino acids are colorless,

ninhydrin is necessarily applied on the filter paper to react with the amino acids

CONCLUSION
The difference in affinities of the amino acids

leads to their separation on the filter paper and to the formation of a column of spots on the direction of the movement of the mobile phase.
The retention factor (Rf) values of the amino

acids is calculated by dividing the distance travelled by the amino acid by the distance travelled by the solvent mixture.
The Rf values may be affected by the

CONCLUSION
Leucine (Rf=0.7990) is the least polar and

has the highest affinity to the mobile phase.

Glycine (Rf=0.2626) is the most polar and

has the highest affinity to the stationary phase.

The higher the RF value the less polar is the

amino acid.
The unknown is a mixture of lysine and

RECOMMENDATION For more effective development of the S chromatogram, the solvent mixture must be
covered and allowed to stand overnight.
The students must wear gloves and be very

careful in handling and preparing the filter paper to be used.

The drying time should be prolonged to

further reduce the chances of contamination and the overlapping of amino acid spots.

REFERENCES
http://www.doggedresearch.com/chromo/chromatography.htm http://people.clarkson.edu/~jimbo/2004/controlled.htm http://www.wpi.edu/Academics/Depts/Chemistry/Courses/General/tlc.ht

ml
http://www.buzzle.com/articles/thin-layer-chromatography.html

Buzzle.com
http://74.125.153.132/search?

q=cache:A6d46j_WiWQJ:gcopamravati.ac.in/STUDYMATERIAL/Colum n %2520Chromatography.pps+basic+principle+of+column+chromatograp hy&cd=3&hl=en&ct=clnk&gl=ph

http://www.separations.us.tosohbioscience.com/ServiceSupport/TechSu

pport/ResourceCenter/PrinciplesofChromatography/ReversedPhase/
http://orgchem.colorado.edu/hndbksupport/chrom.html