PP056 Thin layer chromatography of plant pigments PDF

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Helps students to yield proper knowledge on TLC...

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PP056 - Thin layer chromatography of plant pigments Why do this? Thin layer chromatography is a quick and effective method of separating pigments within leaves. The pigments run clearly on the chromatogram with little outward spread, allowing Rf values to be calculated and chlorophylls to be easily identified. This Practical Procedure draws on safety information from the following guidance. • Hazcards: 43A, 45B, and 85A

Suitability Ys 12 & 13

Method with control measures (including PPE) Wear eye protection Avoid inhaling vapour. Ensure the lab is well-ventilated. Minimise the escape of vapour from the bottle. Ensure no naked flames or other sources of ignition.

Procedure 1. Using sharp scissors cut a strip of TLC plate that will fit into a universal bottle (approx. 8cm x 1cm) and draw a line in pencil 5mm from the bottom edge of the plate. 2. Pour running solvent into the universal bottle so that is approximately 3mm high (it must be under the 5mm line of the TLC plate), and replace the lid for the solvent vapour to saturate the bottle. 3. Place a leaf, or leaves, (grass works well) on the TLC plate to cover the line. Place a ruler over the leaf so it lines up with the pencil line. 4. Using a penny coin, press down firmly and roll along the ruler edge to form a green line.

5. Allow the green line to dry. To extract more of the green mixture, repeat steps 3 & 4 several times with a new leaf until a narrow green band forms. 6. Using forceps, place the TLC plate into the universal bottle and replace the lid. Do not move the bottle while the chromatogram is running. 7. Once the ‘solvent front’ has moved 7/8ths of the way up the plate (around 10 minutes), use forceps to remove it from the bottle. Mark the position of the solvent front (before it evaporates) and leave the plate to dry on a tile. 8. Photograph the result as soon as the solvent has evaporated because the pigments are light-sensitive and will fade. To calculate Rf values, mark the location of each distinct pigment by drawing a dot in the centre of the line. This document is intended to support teachers when planning practical activities. It is not designed as a worksheet for classroom use. ® PP056 Oct 2018 ©CLEAPSS , The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ Page 1 Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk

Disposal Where possible, save the solvents for reuse in further chromatography activities. Up to 30ml total volume of leftover solvents can be rinsed down the sink with plenty of water. Plates can be stuck in lab books or disposed of via general waste.

Expected observations/results We ran TLC plates for grass which revealed yellow, green and grey pigments. Pheophytin (grey) was very faint; the yellow pigments faded after a few hours; the chlorophylls were very distinctive. From our tests using grass in the running solvent of 5:2:2 cyclohexane, ethyl ethanoate, and propanone, we calculated the Rf value ranges shown below.

Solvent front

Pigment

Colour

Rf value range

β-carotene

Yellow

0.96 - 0.98

Pheophytin

Grey

0.73 – 0.78

Chlorophyll a

Blue-Green

0.64 – 0.70

Chlorophyll b

Green

0.59 – 0.63

Lutein

Yellow

0.48- 0.53

Violaxanthin

Yellow

0.36 – 0.40

Neoxanthin

Yellow

0.22 – 0.24

Biology notes The pigments within the mixture moved at different rates depending on how well they dissolved in the solvent, therefore different plants and different running solvents will yield different Rf values. However you can identify the pigments from their order on the plate. Suggested apparatus and materials •

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Apparatus and materials notes

Plant leaves; grass, spinach or geraniums work well TLC plate with silica coating

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The chromatography works best with freshly made running solvent. It may keep for a few months but check that the solvent still works.

Universal bottle and lid Forceps

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If good separation of pigments is not achieved, adjust the ratio of the running solvent mix by one unit at a time. Cyclohexane will slow down some pigments, while propanone will move others quickly up the plate. The TLC plate has a silica coated side which can fracture when cut with scissors and flake off. Scoring the silica side with a scalpel beforehand then cutting with scissors can prevent this.

Penny coin (or equivalent) Scalpel, sharp scissors Ruler & pencil White tile Running solvent made up of a 5:2:2 mixture (by volume) of cyclohexane, ethyl ethanoate and propanone labelled with the following pictograms:

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Chromatography paper is not suitable because the pigments do not separate into distinct bands.

This document is intended to support teachers when planning practical activities. It is not designed as a worksheet for classroom use. PP056 Oct 2018 ©CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ Page 2 Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk...