Using chromatography to identify amino acids
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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.
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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
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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.
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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.
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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
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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.
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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.
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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
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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
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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
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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.
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Reference samplesWhat does this show about Unknown?
-Contains 2 substances (2 spots)
-They are E120 and E124 because the spots match the reference samples
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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.
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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
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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
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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
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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
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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
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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.
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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.
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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