Experiment 2:Separation of Amino Acids by Paper Chromatography

Chem 31.1 WAD 2Fernandez, 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

TypesType 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)

Gas solid (adsorption) ornonvolatile liquid (partition)

gas

High Performance

solid (silica gel, alumina, or cellulose on a flat, inert substrate)

liquid (solvent) relatively high pressure to be applied

Reversed Phase

solid (more polar) liquid similar to high performance except polarity of SP is reversed

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

MethodologyPreparation of Solvent Mixture

24 mL Butanol6 mL Glacial Acetic Acid

10 mL Distilled Water

Preparation of Chromatography Paper

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

x x x x x x x x x

MethodologyApplication of Amino Acids

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

Spots must be about 1-2 mm

Paper Chromatography Setup

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

x x x x x x x x x

Bottle Cover

Bottle

ChromatographyPaper

Solvent

MethodologyAfter Developing Time

x x x x x x x x x

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.

ResultsDistance traveled by solvent front (cm)

4.5

Amino Acid Distance Traveled(cm)

Rf valueTheoretical

Rf valueExperimental

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 0.22

Identity of Unknown Glycine; Tyrosine

ResultsDistance traveled by solvent front (cm)

12

Amino Acid Distance Traveled(cm)

Rf value

Glycine 2.9 0.242

Lysine 2 0.167

Leucine 9 0.750

Tyrosine 6.2 0.516

Unknown 4 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 AcidsAmino 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 aminesPolarity 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-4-methylpentanoic acid

- Neutral, non-polar

Amino Acids

- HO2CCH(NH2)(CH2)4NH2

- 2,6-Diaminohexanoic acid

- Polar

- HO2CCH(NH2)(CH2)4NH2

- 2-Amino-3-(4-hydroxyphenyl)propanoic 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 Valuesa) Molecular weight Lower molecular weight results to greater velocity ofmigration which in turn, results to a higher Rf value.b) Polarity or affinity to mobile and stationary phase Higher polarityresults to lower Rf value.c) Nature of stationary and mobile phase Greater difference in polarityresults 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 #4Give 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 aminoacids because ninhydrin causes the same color change for all theamino acids used.

b) The solvent mixture should be allowed to saturate the chromatographychamber. For effective capillary action To prevent evaporation of volatile solvents

c) The chromatography paper should not be touched with barehands.

Hands contain large amounts of amino acids which couldcontaminate the chromatogram thereby interfering with theresults and so it should not touch the chromatographypaper.

Guide Question #5A 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 solvent fronts:

S1 (Butanol, acetic acid, water) = 11.5 cmS2 (Phenol, Water) = 12.0 cm

Distance traveled by the amino acid in S1 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

Distance traveled by the Amino Acid standard in S1 and S2:Amino Acid S1 (cm) S2 (cm)

Ala 3.7 6.5

Phe 9.14 4.9

Lys 6.15 1.3

Leu 2.0 9.6

Glu 2.3 7.5

His 9.0 2.2

Trp 5.9 6.0

Amino Acid Rf1 Rf2

A 0.53 0.48

B 0.77 0.18

C 0.52 0.08

D 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

Draw clearly the two-dimensional chromatogramand indicate the directions of solvent flow. Identifythe amino acids A, B, C and D.

Rf = distance traveled by the amino aciddistance traveled by the solvent

A Tryptophan B Histidine C Lysine D Phenylalanine

Trp

ALysC

B DPhe

His

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.

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