separation of amino acids by paper chromatography

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Experiment 2 Separation of Amino Acids by Paper Chromatography Magbanua, Dianne B. Olabre, Ian Gabriel N.

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Page 1: separation of amino acids by paper chromatography

Experiment 2Separation of Amino Acids by

Paper Chromatography

Magbanua, Dianne B.Olabre, Ian Gabriel N.

Page 2: separation of amino acids by paper chromatography

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.

Page 3: separation of amino acids by paper chromatography

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.

Page 4: separation of amino acids by paper 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.

Chromatography

Page 5: separation of amino acids by paper 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)

Page 6: separation of amino acids by 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

* Two way paper chromatography - used to separate substances with very similar Rf values

Paper chromatography

Page 7: separation of amino acids by 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

Page 8: separation of amino acids by paper chromatography
Page 9: separation of amino acids by paper chromatography

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

Page 10: separation of amino acids by paper chromatography

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.

Page 11: separation of amino acids by paper chromatography

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

Page 12: separation of amino acids by paper chromatography

DISCUSSION Filter paper - made up of cellulose (non

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

Page 13: separation of amino acids by paper chromatography

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.

Page 14: separation of amino acids by paper chromatography

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

Page 15: separation of amino acids by paper chromatography

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.

Page 16: separation of amino acids by paper chromatography

DISCUSSION

Leucine: HO2CCH(NH2)CH2CH(CH3)2

2-Amino-4-methylpentanoic acid

Hydrophobic, aliphatic, neutral, non-polar

Page 17: separation of amino acids by paper chromatography

DISCUSSION

Glycine: NH2CH2COOH

Aminoethanoic acidAminoacetic acid

hydrophobic

Neutral, polar

Page 18: separation of amino acids by paper chromatography

DISCUSSION

Tyrosine: C9H11NO3

2-Amino-3-(4-hydroxyphenyl)propanoic acid

non-essential amino acid with a polar side group

Aromatic, hydrophilic, neutral

Page 19: separation of amino acids by paper chromatography

DISCUSSION

Lysine: HO2CCH(NH2)(CH2)4NH2

2,6-diaminohexanoic acid

basic essential amino acid

Polar, hydrophilic, charged (+)

Page 20: separation of amino acids by paper chromatography

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.

Page 21: separation of amino acids by paper chromatography

DISCUSSION

Page 22: separation of amino acids by paper chromatography

GUIDE QUESTIONS

Identify the stationary and mobile phases in paper chromatography.

stationary phase – water

mobile phase – mixture of butanol,

glacial acetic acid and

water

Page 23: separation of amino acids by paper chromatography

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

Page 24: separation of amino acids by paper chromatography

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

Page 25: separation of amino acids by paper chromatography

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

Page 26: separation of amino acids by paper chromatography

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

Page 27: separation of amino acids by paper chromatography

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

Page 28: separation of amino acids by paper chromatography

Amino Acid A TryptophanAmino Acid B HistidineAmino Acid C LysineAmino Acid D Phenylalanine

Page 29: separation of amino acids by paper chromatography

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

Page 30: separation of amino acids by paper chromatography

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

Page 31: separation of amino acids by paper chromatography

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

Page 32: separation of amino acids by paper chromatography

GUIDE QUESTIONS

üReversed Phase Chromatography

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

Page 33: separation of amino acids by paper chromatography

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.

Page 34: separation of amino acids by paper 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.

Page 35: separation of amino acids by paper chromatography

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.

Page 36: separation of amino acids by paper chromatography

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.

Page 37: separation of amino acids by paper chromatography

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.

Page 38: separation of amino acids by paper chromatography

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.html

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/Column%2520Chromatography.pps+basic+principle+of+column+chromatography&cd=3&hl=en&ct=clnk&gl=ph

http://www.separations.us.tosohbioscience.com/ServiceSupport/TechSupport/ResourceCenter/PrinciplesofChromatography/ReversedPhase/

http://orgchem.colorado.edu/hndbksupport/chrom.html

http://www.macalester.edu/~kuwata/Classes/2001-02/Chem%2011/Revised%20Amino%20Acids%20(9%201%2001).pdf

http://employees.oneonta.edu/knauerbr/chem226/226expts/226_expt11_pro.pdf

http://wiki.answers.com/Q/What_are_the_uses_of_paper_chromatography