Biochemistry

BIOCHEMISTRY PRACTICAL
Chromatography

Chromatography may be defined as "an analytical technique for the resolution of solutes, in which separation is made by differential migration in a porous medium and migration is caused by flow of solvent".

Put more simply, chromatography is a method for separating out two or more substances dissolved in a liquid, by the different ways these substances move when a liquid is passed through a medium like paper.

All substances are more or less soluble in water, alcohol, acetone, or a whole range of other liquids. A more soluble substance is more mobile, that is, it can be carried further and faster in the liquid. Another factor that determines a substances movement in a liquid is the size of a molecule of that substance. It seems reasonable that a small molecule is easier to carry than a large molecule, and will move faster.

For instance, if solutions of a red and blue dye are mixed, and a drop of the mixture is placed on filter paper (or ordinary blotting paper), the spreading patch will show separation of colours as one dye moves across the paper faster than the other.

Techniques like these are used extensively in the biological and chemical sciences to separate out such substances as sugars, organic acids, and amino acids.

In this prac we will be using a variety of coloured solutions and attempting to separate out the colours in them by running an alcohol/water mixture through them.

Part 1 - Food colourings, Inks etc.

Methods:

1. Cut a rectangle of filter paper approximately 12 cm long and 6 cm wide. Fold it lengthwise down the middle so that it can stand upright. Draw, in pencil, a line across the width of the paper about 1 cm from one end.

2. Using a fine pipette, place a drop of the food colouring mixture on the pencil line, avoiding the edges of the paper and the centre crease. A smaller drop will give a better separation than a large drop.

3. In the jar provided, place about 10 ml of the solvent (an alcohol/water mix).

4. Place the paper in the jar, being careful not to splash the sides of the jar, or the paper itself. The level of the solvent should always be below the pencil line drawn on the paper. Replace the lid of the jar carefully.

5. When the liquid has moved almost to the top of the paper, remove the paper and allow it to dry. Have the colours separated? Are the colours completely separated or are they smudged? Why might this be?

6. Provided no colouring has leaked into the solvent in the jar, it can be used again for other chromatography separations.

7. Repeat the steps above, but try using other coloured solutions. Biros and felt pens of various sorts may give good results. You can make overlapping spots on a piece of paper yourself, or try just one type of ink and see whether there is more than one colour in it. Experiment!!

8. What happens if you draw a line, write a word, compose a picture in coloured solutions and run it through a chromatography jar? Try it!!

Part 2 - Leaf Pigments

Methods

1. Place a silver beet or spinach leaf in a beaker of water and boil for about 1 minute. Remove the leaf with forceps and lightly dry with a paper towel.

2. Tear the leaf into small pieces and insert them into a test-tube about one third full with ethyl alcohol. Place the tube into the beaker of boiling water until the alcohol starts to turn green.

3. Remove the test-tube from the water bath. Crush the leaves with a glass stirring rod. Continue heating and crushing until the alcohol becomes a dark green colour.

4. Prepare a piece of filter paper in the same way as for the food colourings. Using a pipette, put a small drop of leaf pigment on the paper and let it dry. Repeat this several times, until you have a dark spot of pigment extract.

CAUTION: Before proceeding with the next part, make sure that ALL flames are extinguished.

5. Place a small amount of the solvent in a jam jar. Note that this solvent is a mixture of 1 part of diethyl ether and 3 parts of petroleum ether. It is FLAMMABLE and should not be in the same room as a naked flame! Allow the chromatogram to run for about 15 minutes, then remove it and allow it to dry.

6. Notice the faint bands of colour. From the top may be seen:
          carotenes (orange)
          xanthophylls (yellow)
          chlorophyll b (yellow-green)
          chlorophyll a (blue-green)
          anthocyanin (red)

7. If there is time, you may like to repeat the above exercise using leaves from a plant having an obvious red pigmentation.

To be handed in:

On a separate piece of paper, trace the shape of your filter paper strip.
Accurately sketch the bands of colour as obtained on your food colouring and leaf pigment chromatograms. Use appropriate colours or labels to indicate the colour of the pigments and identify each band.

Now answer the following questions.

(a) Why did separation of the bands occur?

(b) What pigments seemed to be present in the greatest amounts in the leaf?

(c) Why do leaves look green even though other colours like yellow are present?
(Hint: In the autumn the green chlorophyll molecule is broken down and removed by the plant).

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