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Experiment 2Separation 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•Partition - the tendency for a compound to divide its time between two immiscible solvents.•Partition chromatography - paper chromatography which involves the use of a nonpolar solvent.
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.
Chromatography
Types:•Thin-Layer Chromatography (liquid-solid)
•Reversed Phase Chromatography (liquid-solid)
• High Performance Chromatography (liquid-solid)
•Gas Chromatography (gas-liquid)
•Column Chromatography (liquid-solid)
•Paper Chromatography (liquid-liquid)
• 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
* Two way paper chromatography - used to separate substances with very similar Rf values
Paper chromatography
RESULTSAmino Acid Standards
Distance Travelled by the Amino Acid (cm)
Distance Travelled by the Solvent
Mixture (cm)
Rf Values
Glycine 3.4 12. 5 0.272
Tyrosine 6 12. 5 0.48
Leucine 11 12. 5 0.88
Lysine 3 12. 5 0.24
UnknownGlycineLeucine
3.210.1
12. 5 12. 5
0.2560.808
Table I. The Rf Values of the Amino Acids
THEORETICAL Rf VALUES OF AMINO ACIDS
Amino Acids Theoretical Rf Values
A. Glycine 0.26
B. Lysine 0.14
C. Leucine 0.73
D.Tyrosine 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:
üpolarity, pH
ümolecular weight
ü structure
üshape of the molecule
ü temperature
ütype of paper to be used.
DISCUSSION•Higher affinity to the stationary phase•Stick to the paper•Travel more slowly•Smaller Rf values•Polar compounds•Bond to the cellulose of the paper more quickly
•Higher affinity to the mobile phase•Unimpeded by the paper•Travel with the solvent front•Larger Rf values•Nonpolar compounds•Remain dissolved in the mobile phase
Glycine: most polarLeucine: least polarUnknown: Glycine + Leucine
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 acidAminoacetic 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. reacts with the free amines to
form blue-violet or brown compounds
*CO2 is released. The carbon in the CO2 originates from the carboxyl group of the amino acid.
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 QUESTIONS Explain briefly the differences in Rf 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 less polar mobile phase
more polar amino acid more polar stationary phase
GUIDE QUESTIONS What are the factors that could affect the Rf value of a solute?
üpolarity or affinity of the solute to the mobile and stationary phases
ümolecular weight
ünature of stationary and mobile phase
GUIDE QUESTIONS Give reasons for the following procedure:
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 QUESTIONSb. The solvent mixture should be
allowed to saturate the chromatography chamber.
~allows for more effective development of the chromatograms
~prevents the evaporation of the solvent
d. The chromatography paper should not be touched with bare hands.
~avoid contamination of the chromatography paper
GUIDE QUESTIONS A mixture of amino acids was separated into its components by two-dimensional chromatography using solvents S-1 and S2.
Amino Acid
S1 (cm)
S2 (cm)
A 6.1 5.8
B 8.9 2.1
C 6.0 1.0
D 9.0 4.5
Amino Acid
S1 (cm) S2 (cm)
Ala 3.7 6.5Phe 9.14 4.9Lys 6.15 1.3Leu 2.0 9.6Glu 2.3 7.5His 9.0 2.2Trp 5.9 6.0
Amino Acid A TryptophanAmino Acid B HistidineAmino Acid C LysineAmino Acid D Phenylalanine
GUIDE QUESTIONS Discuss briefly the basic principles of
the following chromatographic techniques.
ü 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 solubility in the mobile phase
- cannot analyze volatile compounds
üColumn 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 QUESTIONSüGas Chromatography
-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
üHigh Performance Chromatography
-the separation of compounds is carried out on the basis of their characteristic polarities
GUIDE QUESTIONS
üReversed 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:
üpaper chromatography,
ü thin-layer chromatography,
ücolumn chromatography,
ügas chromatography,
ühigh performance chromatography,
ü reversed phase chromatography.
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 and form blue-violet compounds.
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 polarity, pH, molecular weight, structure, and shape of the molecules, temperature, and type of paper used.
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 glycine.
RECOMMENDATIONS For more effective development of the 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.
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