Experiment 2 Separation of Amino Amino Acids 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 hase •
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)
•Gas Chromatography (gas-liquid) •Column Chromatography (liquid-solid) •Paper Chromatography (liquid-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 Standards Travelled by Travelled by the Amino the Solvent Acid (cm) Mixture (cm)
Rf Values
Glycine Tyrosine
3.4
12. 5
0.272
6
12. 5
0.48
Leucine
11
12. 5
0.88
Lysine
3
12. 5
0.24
3.2 10.1
12. 5 12. 5
0.256 0.808
Unknown Glycine Leucine
Table I. The Rf Values of the Amino
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: ü
polarity, pH
ü
molecular weight
ü
structure
DISCUSSION Higher affinity to the stationary phase
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 •
•
Glycine: most polar Leucine: least polar Unknown: Glycine + Leu
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-1,3dione)
make
the amino acids visible
detects
ammonia, or primary and secondar 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? ü
polarity or affinity of the solute to the mobile and stationary phases
ü ü
molecular weight
nature of stationary and mobile 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
S1 (cm) 6.1
S2 (cm) 5.8
B
8.9
2.1
C
6.0
1.0
D
9.0
4.5
Amino Acid Ala Phe Lys Leu Glu His Trp
S1 (cm)
S2 (cm)
3.7 9.14 6.15 2.0 2.3 9.0 5.9
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. 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
ü 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
ü
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 ü High
Performance Chromatography
-the separation of compounds is carried out on the basis of their characteristic polarities
GUIDE QUESTIONS ü 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: ü
paper chromatography,
ü
thin-layer chromatography,
ü
column chromatography,
ü
gas chromatography,
ü
high 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
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.