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The employment of The employment of

paper chromatography in paper chromatography in

the separation and the separation and

identification of amino identification of amino

acids using spot acids using spot

comparison method comparison method

Experiment 2Experiment 2de Leonde LeonLomuljoLomuljo

What are amino acids?What are amino acids?

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

ChromatographyChromatography

• Technique capable of segregating the Technique capable of segregating the components of a mixturecomponents of a mixture

• Relies on differences between the adsorption of a Relies on differences between the adsorption of a component on a mobile and stationary phase.component on a mobile and stationary phase.

• The stationary phase may be a solid or a liquid The stationary phase may be a solid or a liquid on some inert backing material. on some inert backing material.

• The mobile phase may either be a liquid or a gas.The mobile phase may either be a liquid or a gas.

• Compound is placed on stationary phaseCompound is placed on stationary phase

• Mobile phase passes through the stationary Mobile phase passes through the stationary phase.phase.

• Mobile phase solubilizes the components.Mobile phase solubilizes the components.

• Mobile phase carries the individual Mobile phase carries the individual components a certain distance through the components a certain distance through the stationary phase, depending on their stationary phase, depending on their attraction to both of the phases.attraction to both of the phases.

Types of ChromatographyTypes of Chromatography

• Thin-layer chromatography Thin-layer chromatography • Liquid-solid column chromatographyLiquid-solid column chromatography• Gas-liquid chromatographyGas-liquid chromatography• High-performance liquid chromatographyHigh-performance liquid chromatography

Uses of ChromatographyUses of Chromatography

• For determining purity and authenticity of For determining purity and authenticity of reagentsreagents

• For separating mixtures that are inseparable by For separating mixtures that are inseparable by distillation, recrystallization and sublimationdistillation, recrystallization and sublimation

• For checking the final product of a reactionFor checking the final product of a reaction

Paper ChromatographyPaper Chromatography

• Form of Thin-Layer ChromatographyForm of Thin-Layer Chromatography• Filter paper is the stationary phaseFilter paper is the stationary phase• Solvent is the mobile phaseSolvent is the mobile phase

Principles of Paper Principles of Paper ChromatographyChromatographyCapillary ActionCapillary Action – – the movement of liquid within the spaces of a porous the movement of liquid within the spaces of a porous

material due to the forces of adhesion, cohesion, and surface tension. material due to the forces of adhesion, cohesion, and surface tension. The liquidThe liquid is able to move up the filter paper because its attraction to is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. itself is stronger than the force of gravity.

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

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

Paper ChromatographyPaper Chromatography

• Paper has a high affinity for water molecules.Paper has a high affinity for water molecules.• If the solute is hydrophobic, it will stay closer to If the solute is hydrophobic, it will stay closer to

the solvent front, or the line depicting the the solvent front, or the line depicting the maximum advance of the solvent up the filer maximum advance of the solvent up the filer paper. paper.

• If the solute is hydrophilic, it will not cover much If the solute is hydrophilic, it will not cover much distance. distance.

ObjectivesObjectives

-to know the basic principles of and properly apply -to know the basic principles of and properly apply the techniques used in paper chromatography, the techniques used in paper chromatography,

-to compute and compare the Rf values of the given -to compute and compare the Rf values of the given amino acids, to identify the unknown amino acids amino acids, to identify the unknown amino acids by comparison of Rf valuesby comparison of Rf values

-to determine the relationship between the -to determine the relationship between the chromatographic properties and the chemical chromatographic properties and the chemical structures of amino acids.structures of amino acids.

E xperimenta lE xperimenta l

S olvent MixtureS olvent Mixture 12mL of butanol, 12mL of butanol,

3mL glacial acetic acid 3mL glacial acetic acid and 5mL distilled and 5mL distilled

water were mixed in water were mixed in a bottlea bottle

The bottle was The bottle was then coveredthen covered

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

paper paper

A very light pencil line was A very light pencil line was drawn across the length of drawn across the length of the chromatography paper the chromatography paper

about 1cm from the about 1cm from the bottombottom

Starting 2cm from Starting 2cm from the left edge, light the left edge, light pencil cross marks pencil cross marks

were on the line 3cm were on the line 3cm apartapart

Clean capillary tubes Clean capillary tubes were used to apply a were used to apply a

spot of amino acid spot of amino acid solution at the center solution at the center

of the cross marksof the cross marks

The spots (1-2mm The spots (1-2mm in diameter) were in diameter) were dried with a hair dried with a hair

dryer before dryer before applying moreapplying more

The paper was The paper was rolled and the edges rolled and the edges were stapled (edges were stapled (edges

should be 1mm should be 1mm apart)apart)

This was done 4 times

The bottle The bottle containing the containing the solution was solution was

opened and the opened and the paper was placed paper was placed

insideinside

After two hours, After two hours, the paper was the paper was

removed from the removed from the bottle and allowed bottle and allowed

to dry on a Petri to dry on a Petri dish coverdish cover

The staples were The staples were removed and the removed and the paper was placed paper was placed

front side on a front side on a clean sheet of bond clean sheet of bond

paperpaper

The ninhydrin The ninhydrin solution was solution was

brushed on the brushed on the paper along the paper along the direction of the direction of the

solvent flowsolvent flow

The paper should not touch the sides of the

bottle

The paper was The paper was dried with a dried with a

hair dryerhair dryer

Using a pencil, the Using a pencil, the edges were traced of edges were traced of the resulting violet the resulting violet

spots and the center spots and the center with the darkest spot with the darkest spot

was markedwas marked

ProcedureProcedure RationaleRationale

The usage of an untouched filter The usage of an untouched filter paper was required in the paper was required in the

experiment.experiment.

This is because our hands contain amino This is because our hands contain amino acids that may transfer to the filter acids that may transfer to the filter

paper and might produce discrepancies.paper and might produce discrepancies.

The amino acid was dried before The amino acid was dried before applying the other one.applying the other one.

To minimize the diameter of the amino To minimize the diameter of the amino acidacid

Amino acids were applied four Amino acids were applied four times.times.

To ensure that adequate amount To ensure that adequate amount of amino acid is on the filter paper.of amino acid is on the filter paper.

Diameter of the amino acids were Diameter of the amino acids were limited to 1-2mm only.limited to 1-2mm only.

To avoid the interference of To avoid the interference of adjacent amino acidsadjacent amino acids

Different capillary tubes were used for Different capillary tubes were used for each amino acid solution.each amino acid solution.

To avoid contaminationTo avoid contamination

Spots on the filter paper didn’t Spots on the filter paper didn’t touch the solvent .touch the solvent .

To avoid a premature reaction that To avoid a premature reaction that could cause a negative errorcould cause a negative error

The bottle was covered tightly.The bottle was covered tightly. To make sure that the atmosphere in the To make sure that the atmosphere in the container is saturated with solvent vapourcontainer is saturated with solvent vapour

ResultsResults

Table 2.1: Rf Va lues of Amino Ac ids and Unknow nTable 2.1: Rf Va lues of Amino Ac ids and Unknow n

AMINO ACID AMINO ACID STANDARDSSTANDARDS

Distance Distance Travelled by the Travelled by the

Amino Acid Amino Acid (cm)(cm)

Distance Distance Travelled by the Travelled by the Solvent Mixture Solvent Mixture

(cm)(cm)Rf ValuesRf Values

GLYCINEGLYCINE 3.53.5 1111 0.320.32LYSINELYSINE 3.03.0 1111 0.270.27LEUCINELEUCINE 8.38.3 1111 0.750.75TYROSINETYROSINE 6.06.0 1111 0.540.54UNKNOWN UNKNOWN AA

6.46.4 1111 0.580.58

BB 8.38.3 1111 0.750.75CC 3.03.0 1111 0.270.27DD 4.64.6 1111 0.420.42

DiscussionDiscussion

• Paper chromatography is a method of separating Paper chromatography is a method of separating and analyzing mixturesand analyzing mixtures

• The filter paper, which contains a thin film of The filter paper, which contains a thin film of water trapped on it, is the stationary phasewater trapped on it, is the stationary phase

• The solvent made up of 12:3:5 butanol, glacial The solvent made up of 12:3:5 butanol, glacial acetic acid and water is the mobile phaseacetic acid and water is the mobile phase

• In the experiment, an ascending chromatography In the experiment, an ascending chromatography was performed since the solvent moves up was performed since the solvent moves up against gravitational force and the only force that against gravitational force and the only force that causes the rise of the solvent is capillary action. causes the rise of the solvent is capillary action.

• This is the reason why at least two hours was This is the reason why at least two hours was allowed for the solvent to rise. allowed for the solvent to rise.

• If the time was shorter, the component might not If the time was shorter, the component might not be sufficiently separated for easy identification.be sufficiently separated for easy identification.

• When the filter paper was placed inside the When the filter paper was placed inside the bottle containing the mobile phase, it should be bottle containing the mobile phase, it should be certain that the spots shouldn’t touch the solvent certain that the spots shouldn’t touch the solvent since what we’re after is to see how far the since what we’re after is to see how far the amino acid would travel in the filter paper. amino acid would travel in the filter paper.

• If the solvent would come in contact with the If the solvent would come in contact with the spot, then a premature reaction will occur spot, then a premature reaction will occur between the two, which can lead to a negative between the two, which can lead to a negative result when calculating for the retardation factor. result when calculating for the retardation factor.

• The other side of the filter paper was sprayed The other side of the filter paper was sprayed with ninhydrin such that we would be able to with ninhydrin such that we would be able to have a visual of how far the amino acids have have a visual of how far the amino acids have traveled. traveled.

• Ninhydrin reacts with amino acids to form a blue-Ninhydrin reacts with amino acids to form a blue-violet compound. violet compound.

• The sprayed filter paper should show a number The sprayed filter paper should show a number of different spots each corresponding to different of different spots each corresponding to different amino acids. amino acids.

• The further the spot from the starting line, the The further the spot from the starting line, the higher the affinity of the amino acid for the higher the affinity of the amino acid for the mobile phase and the faster is its migration. mobile phase and the faster is its migration.

• The relative extent to which solute molecules The relative extent to which solute molecules move in a chromatography experiment is move in a chromatography experiment is indicated by the retardation factor, or Rindicated by the retardation factor, or Rff..

• It’s defined as the ratio of the distance moved by It’s defined as the ratio of the distance moved by that particular component divided by the that particular component divided by the distance traveled by the solvent. distance traveled by the solvent.

• RRff values range from 0-1 and can never be values range from 0-1 and can never be greater than 1. greater than 1.

• • Rf values range from 0-1 and can never be Rf values range from 0-1 and can never be

greater than 1. a substance which has a high Rf greater than 1. a substance which has a high Rf value means that it has a greater affinity for the value means that it has a greater affinity for the mobile phase. mobile phase.

• On the contrary, a substance with a small Rf On the contrary, a substance with a small Rf value has less affinity for the mobile phase. value has less affinity for the mobile phase.

• The affinity of the amino acid on the mobile The affinity of the amino acid on the mobile phase is dependent on the functional group phase is dependent on the functional group found in their structure.found in their structure.

Stationary PhaseStationary PhaseEssential Essential Structure:Structure:

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

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

• The Rf values depend on the affinity of the amino The Rf values depend on the affinity of the amino acids to the mobile phase. acids to the mobile phase.

• Amino acids have the amino group, carboxyl Amino acids have the amino group, carboxyl group, another functional group R and a group, another functional group R and a hydrogen atom bonded to a carbon atom. hydrogen atom bonded to a carbon atom.

• Amino acids have unique Rf values since they Amino acids have unique Rf values since they have varied functional groups attached to the have varied functional groups attached to the carbon atom aside from the amino and carboxy carbon atom aside from the amino and carboxy group.group.

• Amino acids with nonpolar hydrocarbon side chains: Amino acids with nonpolar hydrocarbon side chains: hydrophobic, lower water solubilityhydrophobic, lower water solubility

• Amino acids with polar but neutral R groups: Amino acids with polar but neutral R groups: hydrophilic, promote water solubilityhydrophilic, promote water solubility

• Amino acids with basic and acidic R groups: Amino acids with basic and acidic R groups: promote promote water solubilitywater solubility

GlycineGlycineIn glycine, the R is a hydrogen atom thus In glycine, the R is a hydrogen atom thus

glycine is non-polar and from this one glycine is non-polar and from this one can predict that glycine would travel far can predict that glycine would travel far in the filter paper.in the filter paper.

LysineLysineLysine has a basic R group and both basic Lysine has a basic R group and both basic

and acidic R groups promote water-and acidic R groups promote water-solubility. solubility.

LeucineLeucineLeucine is a hydrophobic amino acid and Leucine is a hydrophobic amino acid and

has an isobutyl group thus it also would has an isobutyl group thus it also would travel far in the filter paper. travel far in the filter paper.

TyrosineTyrosineTyrosine on the other hand has an acidic Tyrosine on the other hand has an acidic

R group thus it’s hydrophilic and would R group thus it’s hydrophilic and would tend to travel less in the filter paper. tend to travel less in the filter paper.

NinhydrinNinhydrinIndane - 1, 2, 3 - trione hydrateIndane - 1, 2, 3 - trione hydrate

•Higher affinity to the Higher affinity to the stationary phasestationary phase•Stick to the paperStick to the paper•Travel more slowlyTravel more slowly•Smaller Rf valuesSmaller Rf values•Polar compoundsPolar compounds•Bond to the cellulose of the Bond to the cellulose of the paper more quicklypaper more quickly

• Higher affinity to the Higher affinity to the mobile phasemobile phase•Unimpeded by the paperUnimpeded by the paper•Travel with the solvent Travel with the solvent frontfront•Larger Rf valuesLarger Rf values•Nonpolar compoundsNonpolar compounds•Remain dissolved in the Remain dissolved in the mobile phasemobile phase

Guide QuestionsGuide Questions

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

• The stationary phase used is the filter paper. To The stationary phase used is the filter paper. To be more precise, it’s the adsorped water in the be more precise, it’s the adsorped water in the filter paper, which is the stationary phase. filter paper, which is the stationary phase.

• The mobile phase is the 12:3:5 mixture of n-The mobile phase is the 12:3:5 mixture of n-butanol, glacial acetic acid, and distilled water.butanol, glacial acetic acid, and distilled water.

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

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

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

-The polarity or affinity of the solute to the mobile -The polarity or affinity of the solute to the mobile and stationary phases: high polarity, low Rf and stationary phases: high polarity, low Rf value. value.

-Molecular weight: High molecular weight, low Rf -Molecular weight: High molecular weight, low Rf value.value.

-Nature of stationary and mobile phase: increasing -Nature of stationary and mobile phase: increasing disparity in polarity between the stationary and disparity in polarity between the stationary and mobile phase, increasing travel speed of solute mobile phase, increasing travel speed of solute along the solvent, increasing Rf valuealong the solvent, increasing Rf value

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

RRff Values Values

6. Discuss briefly the basic principles of the 6. Discuss briefly the basic principles of the following chromatographic techniques.following chromatographic techniques.

a. Thin Layer Chromatographya. Thin Layer Chromatography

6. Discuss briefly the principles of the following 6. Discuss briefly the principles of the following chromatographic techniqueschromatographic techniques

TypeType Mobile Mobile PhasePhase

Stationary Phase

CharacteristCharacteristicic

Thin Layer Thin Layer ChromatographyChromatography

LiquidLiquid SolidSolid Principle of Principle of Capillary ActionCapillary Action

Column Column ChromatographyChromatography

LiquidLiquid SolidSolid Principle of Principle of Separation is Separation is AdsorptionAdsorption

Gas Gas ChromatographyChromatography

GasGas LiquidLiquid Principle of Principle of Separation is Separation is

PartitionPartitionHigh High

Performance Performance ChromatographyChromatography

LiquidLiquid SolidSolid Eluent flows Eluent flows through a through a detector.detector.

Reversed Phase Reversed Phase ChromatographyChromatography

LiquidLiquid LiquidLiquid Principle of Principle of Separation is Separation is

PartitionPartition

Conclusion and Conclusion and RecommendationsRecommendations

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

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