Using chromatography to identify amino acids

Key words:SOLVENT – a liquid in which chemicals dissolve to make a solution (e.g. water, ethanol)

SOLUTE – a substance that is dissolved in a solvent.

SOLUTION – a solute dissolved in a solvent

AQUEOUS SOLUTIONS – solutions where a solute is dissolved in water

NON-AQUEOUS SOLUTIONS – solutions where the solute is dissolved in a solvent that isn’t water (e.g ethanol)

May 6, 2023

https://youtu.be/J8r8hN05xXk

Learning Outcome•explain the principle of chromatography• identify amino acids in a mixture• interpret chromatograms and calculate Rf values from chromatograms.

Chromatography

• A technique used to separate and identify chemicals in a mixture.

• Relies on the movement of a gas or liquid through a medium.

• The liquid or gas that moves is the mobile phase.

• The medium that does not move is the stationary phase

Paper Chromatography

• The stationary phase is water trapped between the fibres in the paper.

• The mobile phase is a solvent• The choice of solvent depends on what is

being separated.

Thin Layer Chromatography

• The stationary phase is a thin coating of absorbent solid on a sheet of plastic or glass.

• The mobile phase is a solvent.• TLC is quicker than paper chromatography

and can be used for more substances.• Dyes in food products and clothing, drugs,

medicines etc.

Substances to be identified are “spotted”

near one end of the filter paper

As the solvent moves up the paper, different molecules move at different rates with the smallest molecules moving the fastest

The technique is used for small molecules such as amino acids, small peptides and sugars

Paper chromatography isa technique used for the

separation & identificationof relatively small chemicalsubstances by a moving

solvent on sheets or stripsof filter paper

Filter papercylinder

clip

Solvent

Concentratedspot of

chemicals tobe separatedand identified

Direction ofsolvent

movement

Paper Chromatography

Developing the Chromatogram• The chromatogram is the paper or thin layer

plate.• The solvent front is marked with a pencil line.• Coloured spots are marked in case the colour

fades.• Invisible spots can be viewed using a UV lamp

and then marked.• Some chemicals need to be developed –

sprayed with a chemical that causes a colour change.

Stationary phase

Test sample

SIDE VIEW

FRONT VIEW

When doing the paper chromatography with food colours, the stationary phase was the water in the paper.

When doing the thin layer chromatography with painkillers, the stationary phase was solid coating on the card.

Sample moves from stationary phase to mobile phase.Mobile

phase (e.g water, ethanol)

Solvent front – how far the solvent has moved

SIDE VIEW

FRONT VIEW

This substance has moved further because it is more attracted to the mobile phaseSo,

chromatography separates substances in a mixture because they are more or less attracted to the mobile phase.

SIDE VIEW

FRONT VIEW

origin

Solventfront

solvent

Amino acidspots

Mark the solventfront & allowpaper to dry

Spray the drypaper with locatingagent (ninhydrin)to make the spots

visible

Dry the paperwith gentle heatto developthe amino acidspots

Purple spotsdevelop located atdifferent distancesfrom the origin line

Identify Amino Acids In A Mixture

Reference materials

• Testing a sample alongside a set of reference samples can be used to identify the chemicals present.

• The spots in the sample can be compared with the spots from the known reference samples.

Reference samplesWhat does this show about Unknown?

-Contains 2 substances (2 spots)

-They are E120 and E124 because the spots match the reference samples

Retardation Factors (Rf)

• A chemical can also be identified by its retardation factor (Rf)

• The formula is:

Rf = distance moved by sample distance moved by solvent

The answer is never greater than 1.

Calculate Rf for the three spots.

Substance Distance moved by sample (cm)

Distance moved by solvent front (cm)

Rf

E120 7.2 10 0.72E133 4.1 10 0.41E124 9.5 10 0.95

origin

Solventfront

The Rf Value

X1

X5

X4

X3

X2

Y

frontsolvent origin to from distance(spot) solute origin to from distanceR f

YXR f

The Rf value isalways a value less

than one as thesolvent front alwaysmoves further thanthe solute molecules

Rf values

• Any substance has a particular Rf value when used with a particular mobile and stationary phase.

• This can be used to identify substances if you know the Rf values of the reference samples

Rf• E.g.

A sample moves 3cm, whilst the solvent front moves 12cm. Which chemical is present?

Chemical 1 Rf = 0.44Chemical 2 Rf = 0.12Chemical 3 Rf = 0.25Chemical 4 Rf = 0.60

origin

Solventfront

arg leu alamet

Mixture ofunknown amino

acids

Identifying Unknown

Amino Acids

The mixture ofunknown aminoacids is seen to

contain four different amino acids

Of these fouramino acids,

two can be positivelyidentified

The mixture containsfour amino acids; two

unknown together with arginine & leucine

Qualitative Analysis

• Paper and TLC are both examples of qualitative analysis.

• They don’t show how much chemical is present – instead they just show what is or isn’t present.

• TLC gives better results and can be used for a wider range of chemicals.

• TLC is quicker, more sensitive and produces a clearer separation.

Quantitative Analysis

• Paper and TLC can give a rough idea of how much based on how intense the coloured spot is.

• Sometimes the spots can be removed and processed to find an amount – giving quantitative data.

Mixture ofamino acids onorigin line

Paper dried and rotated

clockwise through

90o

Solventfront

First solventSecond solvent

TWO-WAY PAPER CHROMATOGRAPHY

Two-way chromatography provides better separation ofsubstances that behave in a similar fashion in the first solvent.

A second run in a different solvent resolves two very close spotsmore clearly

Solventfront