Spinach Extracts and Chromatography

This is what happened during my Introductory Chemistry laboratory lesson today: Thin Layer Chromatography (TLC) of Spinach Extracts. I've taught this twice in the past years and today, I decided to document it.

How to extract pigments from spinach leaves:

Step 1 - Buy some Chinese spinach from the market (or steal a few leaves from your mum's kitchen). Tear the leaves into small pieces and place them in a mortar. Add a few millilitres of acetone and grind them up. Keep grinding and adding acetone until a dark green liquid extract is obtained.

Step 2 - Use a dropper to draw the dark green extract and collect them in a test tube. Add more acetone and grind to produce more extracts, until the test tube is about half filled. Add 2 ml of hexane (3rd picture from the left), cap the test tube and invert it a few times. Allow the mixture to stand and it will separate to two layers (rightmost picture). The water extracted from the leaves will settle below, leaving the dark green hexane extract on top. If a lot of fine pieces of solid leaves were also transferred into the test tube, they will suspend in the hexane layer, and only a very thin layer of dark green extract can be seen.

How to separate the pigments on thin layer chromatography:

Unfortunately, this experiment doesn't work on paper chromatography, it has to be done on silica gel (supported either on aluminium foil or glass). Draw a line, using a pencil, about 0.5 cm from the bottom of the silica gel plate. Use a capillary tube to spot the dark green extract in the middle of the pencil line. Place the plate into a beaker, which contains about 2 ml of 30% acetone / hexane solution. Cover the beaker with a watch-glass and allow the TLC to develop. Do not move the beaker. When the solvent is almost reaching the top of the plate, remove it from the beaker and immediately draw the solvent front with a pencil. The topmost yellow pigment that is really close to the solvent front is beta-carotene. The bright green one that is somewhere in the middle of the plate is chlorophyll a, while the dull green one that is slight below is chlorophyll b.

An extension of this experiment would be to perform a separation on a column chromatography. The following is the adapted mini version of a column chromatography. A glass Pasteur pipette is clamped on a retort stand, at a height such that a glass vial can be placed just below it. Place a small cotton ball into the pipette from the top and use a stick to push it in until it reaches the narrowing part. Fill the pipette with silica gel suspended in hexane. Condition the column using 30% acetone / hexane solution, allowing the solvent to drip from the bottom of the pipette. Do not let the silica gel run dry at any point. When the solvent level reach the top of the silica gel, add about 2 drops of the dark green hexane extract on the top of the silica gel. Quickly add 30% acetone / hexane and continue adding until all the pigments are separated.

The separated pigments are collected in small vials. From left to right: faint yellow being beta-carotene, green chlorophylls, yellow solution containing other pigments.

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