The employment of paper chromatography in the separation and identification of amino acids using spot comparison method

Experiment 2 de Leon Lomuljo

What are amino acids?

Amino acids are compounds that contain the amino group and the carboxylic group.

Chromatography

Technique capable of segregating the components of a mixture Relies on differences between the adsorption of a component on a mobile and stationary phase. The stationary phase may be a solid or a liquid on some inert backing material. The mobile phase may either be a liquid or a gas.

Compound is placed on stationary phase Mobile phase passes through the stationary phase. Mobile phase solubilizes the components. Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases.

Types of Chromatography

Thin-layer chromatography Liquid-solid column chromatography Gas-liquid chromatography High-performance liquid chromatography

Uses of Chromatography

For determining purity and authenticity of reagents For separating mixtures that are inseparable by distillation, recrystallization and sublimation For checking the final product of a reaction

Paper Chromatography

Form of Thin-Layer Chromatography Filter paper is the stationary phase Solvent is the mobile phase

Principles of Paper Chromatography

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 a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents.

Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

Paper Chromatography
Paper has a high affinity for water molecules. If the solute is hydrophobic, it will stay closer to the solvent front, or the line depicting the maximum advance of the solvent up the filer paper. If the solute is hydrophilic, it will not cover much distance.

Objectives
-to know the basic principles of and properly apply the techniques used in paper chromatography, -to compute and compare the Rf values of the given amino acids, to identify the unknown amino acids by comparison of Rf values -to determine the relationship between the chromatographic properties and the chemical structures of amino acids.

E x pe rime nta l

S olve nt Mix ture

12mL of butanol, 3mL glacial acetic acid and 5mL distilled water were mixed in a bottle
The bottle was then covered

A 19cm x 14cm filter paper was laid on a clean sheet of bond paper

Starting 2cm from the left edge, light pencil cross marks were on the line 3cm apart

A very light pencil line was drawn across the length of the chromatography paper about 1cm from the bottom

Clean capillary tubes were used to apply a spot of amino acid solution at the center of the cross marks
This was done 4 times

The spots (1-2mm in diameter) were dried with a hair dryer before applying more The paper was rolled and the edges were stapled (edges should be 1mm apart)

The bottle containing the solution was opened and the paper was placed inside The paper should not

After two hours, the paper was removed from the bottle and allowed to dry on a Petri dish cover

touch the sides of the bottle

The ninhydrin solution was brushed on the paper along the direction of the solvent flow

The staples were removed and the paper was placed front side on a clean sheet of bond paper

The paper was dried with a hair dryer

Using a pencil, the edges were traced of the resulting violet spots and the center with the darkest spot was marked

Procedure
The usage of an untouched filter paper was required in the experiment.
The amino acid was dried before applying the other one.

Rationale
This is because our hands contain amino acids that may transfer to the filter paper and might produce discrepancies. To minimize the diameter of the amino acid

Amino acids were applied four To ensure that adequate amount of amino acid is on the filter paper. times.
Diameter of the amino acids were limited to 1-2mm only.
Different capillary tubes were used for each amino acid solution.

To avoid the interference of adjacent amino acids To avoid contamination

To avoid a premature reaction that could cause a negative error
To make sure that the atmosphere in the container is saturated with solvent vapour

Spots on the filter paper didnt touch the solvent . The bottle was covered tightly.

Results

AMINO ACID STANDARDS

Distance Distance Travelled by the Travelled by the Amino Acid Solvent Mixture (cm) (cm)

Rf Values 0.32 0.27 0.75 0.54 0.58 0.75 0.27 0.42

GLYCINE LYSINE LEUCINE TYROSINE UNKNOWN A B C D

3.5 3.0 8.3 6.0 6.4 8.3 3.0 4.6

11 11 11 11 11 11 11 11

Ta ble 2.1: R f Va lue s of Amino Ac ids a nd U nk now n

Discussion

Paper chromatography is a method of separating and analyzing mixtures The filter paper, which contains a thin film of water trapped on it, is the stationary phase The solvent made up of 12:3:5 butanol, glacial acetic acid and water is the mobile phase

In the experiment, an ascending chromatography was performed since the solvent moves up against gravitational force and the only force that causes the rise of the solvent is capillary action. This is the reason why at least two hours was allowed for the solvent to rise. If the time was shorter, the component might not be sufficiently separated for easy identification.

When the filter paper was placed inside the bottle containing the mobile phase, it should be certain that the spots shouldnt touch the solvent since what were after is to see how far the amino acid would travel in the filter paper. If the solvent would come in contact with the spot, then a premature reaction will occur between the two, which can lead to a negative result when calculating for the retardation factor.

The other side of the filter paper was sprayed with ninhydrin such that we would be able to have a visual of how far the amino acids have traveled. Ninhydrin reacts with amino acids to form a blueviolet compound. The sprayed filter paper should show a number of different spots each corresponding to different amino acids. The further the spot from the starting line, the higher the affinity of the amino acid for the mobile phase and the faster is its migration.

The relative extent to which solute molecules move in a chromatography experiment is indicated by the retardation factor, or Rf. Its defined as the ratio of the distance moved by that particular component divided by the distance traveled by the solvent. Rf values range from 0-1 and can never be greater than 1.

Rf values range from 0-1 and can never be greater than 1. a substance which has a high Rf value means that it has a greater affinity for the mobile phase. On the contrary, a substance with a small Rf value has less affinity for the mobile phase. The affinity of the amino acid on the mobile phase is dependent on the functional group found in their structure.

Stationary Phase
Essential Structure:

made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose

These cellulose fibres attract and adsorbs water vapour from the atmosphere as well as any water that was present. Therefore, the stationary phase is considered to be polar.

The Rf values depend on the affinity of the amino acids to the mobile phase. Amino acids have the amino group, carboxyl group, another functional group R and a hydrogen atom bonded to a carbon atom. Amino acids have unique Rf values since they have varied functional groups attached to the carbon atom aside from the amino and carboxy group.

Amino acids with nonpolar hydrocarbon side chains: hydrophobic, lower water solubility Amino acids with polar but neutral R groups: hydrophilic, promote water solubility Amino acids with basic and acidic R groups: promote water solubility

Glycine

In glycine, the R is a hydrogen atom thus glycine is non-polar and from this one can predict that glycine would travel far in the filter paper.

Lysine

Lysine has a basic R group and both basic and acidic R groups promote watersolubility.

Leucine

Leucine is a hydrophobic amino acid and has an isobutyl group thus it also would travel far in the filter paper.

Tyrosine
Tyrosine on the other hand has an acidic R group thus its hydrophilic and would tend to travel less in the filter paper.

Ninhydrin
Indane - 1, 2, 3 - trione hydrate

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

Guide Questions

1. Identify the stationary and mobile phase in paper chromatography.

The stationary phase used is the filter paper. To be more precise, its the adsorped water in the filter paper, which is the stationary phase. The mobile phase is the 12:3:5 mixture of nbutanol, glacial acetic acid, and distilled water.

2. Explain briefly the differences in Rf values of the amino acid component of your mixture.

The difference in Rf values of the amino acid depends on the polarity or affinity of each substance to the mobile and stationary phases. If the amino acid is less polar, it will be attracted more to the less polar mobile phase and will travel at a longer distance across the filter paper; therefore, it will have a high Rf value. If the amino acid is more polar, it will be attracted more to the more polar stationary phase and will travel at a shorter distance; therefore, it will have a low Rf value.

3. What are the factors that could affect the Rf value of a solute?

-The polarity or affinity of the solute to the mobile and stationary phases: high polarity, low Rf value. -Molecular weight: High molecular weight, low Rf value. -Nature of stationary and mobile phase: increasing disparity in polarity between the stationary and mobile phase, increasing travel speed of solute along the solvent, increasing Rf value

5. A mixture of amino acids was separated into its components by two-dimensional chromatography using solvents S1 and S2. Draw clearly the twodimensional chromatogram and indicate the directions of solvent flow. Identify the amino acids A, B, C, and D.

Rf Values

6. Discuss briefly the principles of the following chromatographic techniques

Type
Thin Layer Chromatography

Mobile Phase
Liquid Liquid Gas Liquid Liquid

Stationary Characterist Phase ic

Solid Solid Liquid Solid Liquid
Principle of Capillary Action Principle of Separation is Adsorption Principle of Separation is Partition Eluent flows through a detector. Principle of Separation is Partition

Column Chromatography Gas Chromatography High Performance Chromatography Reversed Phase Chromatography

Conclusion and Recommendations

Based on the data gathered, the unknown amino acids were identified as A: tyrosine, B: leucine, C: lysine and D: glycine. These were based on the Rf values determined by measuring the distance travelled by the solvent in the filter paper. Differeces in Rf values could be explained by the molecular structures of each of the amino acids. Their R groups dictated their polarities. An increase in polarity corresponds to a decrease in the Rf value of amino acid.

To further improve this experiment, it is recommended that in the application of the amino acids to the paper, a smaller diameter should be aimed for. It was difficult to measure the spots since they were elongated. This could have led to inaccuracy. It is also recommended that the drying time be prolonged to reduce the chances of contamination and the overlapping of amino acid spots. Also, do not leave the containing chamber open for a long time since vapors would come out of it, making the travel time longer.