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Unit 2C: Chromatographic techniques to identify components in mixtures
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Part C of Unit 2, Learning and Using chromatographic techniques to identify components in mixtures
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Unit 2CChromatography of amino acids and plant extracts
I was required to find out the Rf values (Rf = Retention Factor) of 3 amino acids and a plant
extract. This can be used to identify unknown samples by comparing the Rf values and results
to published results of the samples. Chromatography is used in different industries for different
objectives. It is used to determine what antibodies are good at fighting various bacteria and
viruses. For example, it was used to find out which antibodies where best at neutralizing the
Ebola virus during the fight against the Ebola virus. Another example is it is used in competitive
sport because it is very effective and accurate at identifying substances in the bloodstream.
Because of this, it is used to test athletes for performance enhancing drugs. Chromatography is
a method where substances are separated for analysis. Substances are separated as they travel
in a mobile phase. The mobile phase is the solvent that transports the mixture up the material
that absorbs it. The solvent moves up the stationary phase, which is the absorbing material that
the mixture travels through. Different substances travel up the stationary phase (paper) at
different speeds, depending on the dyes or individual substances. Some move more than others
in a set time. In paper chromatography, the stationary phase is chromatography paper. The
mobile phase can be an aqueous liquid, or it can be a non-aqueous organic (carbon-based)
solvent. An example of an aqueous (water-based) liquid is water is toluene. An example of an
organic (carbon-based) solvent is acetone, which is used in nail polish removers. I was required
to use butanol-acetic acid-water which is an aqueous liquid. The separation of the mixture
depends on how strongly attracted the chemicals in each dye are to the stationary phase
(chromatography paper) and mobile phase (butanol-acetic acid-water). However, the results
could be affected due to the cellulose fibers, which are used when making the paper, the fibers
affect the results because they can absorb moisture from the room. Furthermore, water
molecules in the air could attach themselves to the paper giving it a thin layer of water
molecules. These both can alter the result, but it is only a minor change. To identify the
separate substances in the mixture the Retention factor (Rf) values would need to be
calculated. The Rf value is calculated by measuring the distance the solvent travelled and
measuring the distance the substance travelled, then dividing both the results when they have
separated.
The formula for this is: Rf = distance moved by the compound ÷ distance moved by the solvent
TLC or thin-layer chromatography is very similar to paper chromatography. It is also used to
analyse dyes from mixtures of substances. However, the stationary phase is a thin layer of an
unreactive substance on top of a flat inert surface/TLC plate. Examples of an unreactive
substance that could be used is silica or aluminum oxide. The stationary phase I used when
carrying out thin-layer chromatography had a thin layer of silica. Some substances in mixtures
, are carried further up the stationary phase than others due to the differences of each
substance in solubility and strength of absorption between them and the stationary phase. TLC
has advantages over paper chromatography, for example, in TLC the mobile phase moves
quicker and more evenly than in paper chromatography, because of this, TLC is more reliable at
making better chromatograms than paper chromatography (Chromatogram: paper or plate
produced showing the separation of a substance). TLC chromatograms show a clearer
separation of a mixture than the paper chromatograms do, this makes it easier to analyze. The
reliability of the results depend on how much solvent is use, the temperature of the
surrounding and the amount of substance put on the stationary phase are constant.
Paper chromatography for the separation of amino acids
Equipment:
1. Pencil
2. 10cm chromatography paper strip
3. Glass capillary tubes
4. Ruler
5. Beaker
6. Amino acids (aspartame, leucine, lysine)
7. Solvent (Butanol-Acetic Acid-Water)
8. Ninhydrin solution
9. Hairdryer
10. Wooden splint
Method
I had to draw a line with a pencil on one end of the chromatography paper. Pencil is used
because it is insoluble. I was also required to heat the capillary tube using a Bunsen burner I
avoided touching the paper to prevent sweat getting on the paper. Our sweat contains amino
acids that could interfere with the results, so to prevent this from happening I wore gloves and
used forceps to pick up the paper. The capillary tubes were heated with a Bunsen burner. I
made sure I did this safely by keeping a distance between me and the Bunsen burner and
keeping at a safe flame. The capillary tube was then used to add a small spot of amino acid onto
the chromatography paper. After the drop of amino acid was added I used a hairdryer to dry
the spot. I repeated this 2 times for the remaining amino acids, keeping about a centimeter gap
between each spot.
The chromatography paper strip was then placed into the beaker. 0.5cm3 of the solvent
(Butanol-Acetic Acid-Water) was already poured into the beaker before the paper strip was put
in. The chromatography paper was taken out the beaker from the solvent and after roughly an
hour, the solvent had moved up 75% of the paper. I marked the distance the solvent front
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