BTEC Applied Science Level 3 Extended Diploma Unit 2: Practical Scientific Procedures and
Techniques
Learning Aim C: Using chromatographic techniques to identify components in mixtures
By Zohal Nadeem
Thin layer chromatography (TLC) of extracted plant pigments
INTRODUCTION:
I am a newly appointed technical assistant at Chemcalequip and as part of my induction period, I must
demonstrate skills that I have gained in a range of practical procedures and techniques. This means
that, I need to demonstrate the different chromatographic techniques to separate and identify
components in mixtures. In this case, I will be focusing on the TLC of extracted plant pigments.
In this report, I have correctly used chromatographic techniques to produce chromatograms, prepared
the equipment whilst using safe working practices and will be making conclusions based on the
analysis of my own results and measure the factors that affected the separation of mixtures. Using the
quality of results obtained in thin layer chromatography, I will suggest improvements to make sure the
outcomes is reliable enough to be compared to real investigated data in previous conducted
experiments.
Chromatography is a separation technique used to separate and identify substances within a solution.
Thin layer chromatography can be applied for real life uses such as analysing dyes in fibres, inks and
paints and used in the identification of pesticides or insecticides in food products which shows the
adaptiveness of this technique. There are several types of chromatographic methods that work on the
same principle with different mobile and stationary phases. In thin layer chromatography, the
stationary phase is a thin layer, usually silica gel (highly polar) or aluminium oxide coated onto an
adsorbent, inert surface such as glass, plastic or metal. The mobile phase (the running solvent,
Propanone) carries the components of the mixture whilst travelling up through the stationary phase by
capillary forces. Due to different components on the TLC plate moving at different rates, the
separation of different pigments is achieved. This suggests that adsorption relies behind thin layer
chromatography.
RISK ASSESSMENT:
Health and safety are crucial whilst handling the chemicals, therefore the student must conduct a risk
assessment to prevent harm. Personal protective equipment (PPE) such as lab-coats, goggles must be
worn and the hair must be tied up in the laboratory. Bags and coats must be kept in the cupboard away
during the experiment to prevent accidents. In addition, Propanone is highly flammable and is known
as an irritant to the eyes and skin. Acetone is volatile. There must be a ventilated area due to the
fumes which is why we place a lid on the beaker to prevent vapours being inhaled.
METHOD:
The method was extracted from Mr Barrett’s summary on TLC.
1. The leaf is crushed using a pestle and mortar. Whilst doing this, ensure 1 ml of Acetone is
added.
2. Using the thin layer chromatography (TLC) plate, a line approximately 1cm is drawn from the
bottom with a sharp pencil, exerted with minimum pressure across the paper.
, 3. Using a capillary tube, a small amount of supernatant is withdrawn from the watch glass. A
small dot of the extract is dotted onto the pencil line and then left to dry for 30 seconds. Then
a second spot of plant extract is added to the first.
4. This process is repeated 5 times.
5. A 0.5 cm^3 depth of solvent is poured into the 300ml beaker.
6. The TLC plate is lowered into the beaker until the bottom of the chromatogram is below the
surface of the solvent. Ensure the plant extract is above the surface of the solvent.
7. A wooden splint is used to keep the chromatogram vertical and support the slide so that the
pigments would travel vertically and not at an angle.
8. Keep a lid on the beaker to make sure the fumes are not escaping.
9. The solvent is allowed to run for 20 minutes, by this time the solvent has reached 2/3 of the
way up the strip.
10. The TLC plate was then removed from the solvent and the line of the solvent front was
marked with a pencil line at the furthest point of solvent travel.
11. The measurement of the distance travelled by the solvent was measured and recorded.
12. A picture of the results is taken and left to dry.
13. The retention factor (Rf) value is calculated.
Figure 1: Calculated the Rf values of the two separate leaf pigments
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