Chromatography Experiment

Chromatography is a technique used to separate components in a mixture and can be used to partially identify the components.

In this experiment using paper chromatography, mixtures of food colours extracted from ‘Smarties’ will be separated and compared with a set of standard food dyes by observing the movement of the individual extracted colours up a sheet of paper using an aqueous salt solution.  There are two phases involved in chromatography; the ‘stationary phase’, in this case the paper, and the ‘mobile phase’, the salt solution.

Separation of colours using paper chromatography

REQUIREMENTS

  1. Large glass jam jar with cover (eg flat piece of plastic or glass)
  2. 6 sharpened wooden sticks (ie toothpicks)
  3. Hair dryer or warm oven
  4. Ruler
  5. Stapler
  6. Scissors
  7. Sellotape
  8. Paper clips
  9. Cotton thread
  10. Lollipop sticks
  11. Pencil
  12. Sheet of chromatography paper, or large filter paper
  13. 0.1%(w/v) aqueous sodium chloride solution (mobile phase)
  14. Distilled water
  15. Pack of assorted food colours (red, yellow, green and blue)
  16. Small test tubes
  17. Labels
  18. 2 tubes of ‘Smarties’ (mixed colours)​


METHODS

EXTRACTION OF THE COLOURS

Sort the ‘Smarties’ into piles of the same colour. Tip one of the piles into a small clean test tube and to the ‘Smarties’ add a small quantity of cold distilled water (approx 1cm3). Agitate the test tube and its contents for 2 minutes until the colour appears in the aqueous phase. Decant the coloured solution into a clean dry test tube.  Label the tube and its contents with name of the ‘Smarties’ colour.

Repeat the extraction process for each pile of ‘Smarties’.


PREPARATION OF CHROMATOGRAPHY PAPERS

Method 1: Using chromatography paper

Measure the height of the glass jar into which the chromatography will be carried out. From the large sheet of chromatography paper, cut a strip, which is 20mm less than the height of the jar.

Now form this strip into a loose cylindrical shape so that the diameter of the cylinder is approximately 20mm less than the internal diameter of the glass jar.  Do not staple the paper at this stage but flatten it out, on a clean dry surface, and rule a pencil line across the paper so that it is 10mm from the base of cylinder shape when formed.

This is the line onto which the colours are spotted.


Method 2:  Using filter paper

Obtain the largest filter paper possible and cut a strip whose width is at least 25 -50mm.  Cut the ends from this strip to maximise the length of paper to form a rectangle.

To one of the narrow ends of the paper strip stick a narrow strip of Sellotape. This will strengthen the top edge of the paper.

Flatten the strip onto a clean dry surface, and rule a pencil line across the paper so that it is 10mm from the base of strip at the opposite end to the Sellotape. This is the line onto which the colours are spotted.


SPOTTING THE COLOURS

Method 1: Using chromatography paper

Onto the pencil line and 10mm from the left edge of the paper, using a clean wooden stick, spot a small drop of the ‘Smartie’ extract. If the colour is not intense allow the spot to dry and repeat the spotting until colour is visible.

Then spot, using a clean wooden stick along the pencil line, 10mm apart, the following:

  1. red standard
  2. yellow standard
  3. ‘Smartie’ extract (same extract as spotted at the edge)
  4. green standard
  5. blue standard
  6. ‘Smartie’ extract (same extract as spotted at the edge)
  7. yellow standard
  8. red standard

Continue spotting until there is no space left along the pencil line.  Dry the spots thoroughly using a hair dryer or warm oven.


Method 2: Using filter paper

Using the same method as described above, spot a small drop of the ‘Smartie’ extract along the pencil line and 10mm in from the left edge of the strip of filter paper. Repeat this process with standard colours alternating with ‘Smartie’ extract until there is no space left on the pencil line. (eg if the width of the paper strip is 50mm it is possible to spot 2 standard colours and 2 ‘Smartie’ extracts. Dry the spots thoroughly using a hair dryer or warm oven.


ASSEMBLY OF COMPONENTS AND START OF THE CHROMATOGRAPHY

Method 1:  Using chromatography paper

When the spots are dry, form the cylinder and staple the top and bottom of the vertical edges of the paper to form a cylinder with the ‘Smartie’ extracts and standard colours at the bottom.  Do not allow the sides of the paper to overlap but leave a small (say 2 mm) gap up the side of the cylinder.

Pour a small volume of the aqueous sodium chloride solution (mobile phase) into the glass jar to a depth of 5mm and carefully lower the cylinder into the liquid so that the pencil line and spots are above the level on the liquid.

Quickly slide on the lid and allow the liquid to move up the paper cylinder.  Do not move the jar and its contents during the chromatography.


Method 2:  Using filter paper

Once the spots are dry, fix two paper clips to the Sellotape layer at the top of the paper strip. Tie a piece of cotton thread to each of the paper clips (these will act as supports for the paper during chromatography).

Tie the other ends of the cotton thread to a lollipop stick, which is large enough to lie flat across the neck of the glass jar. The length of the cotton threads must be such that the bottom of the paper strip is approximately 2mm from the bottom surface of the glass jar.

Remove the paper and its attachments from the jar and pour a small volume of the aqueous sodium chloride solution (mobile phase) into the glass jar to a depth of 5mm.

Carefully lower the paper strip into the solution rest the lollipop stick across the neck of the jar and cover with the lid. Do not move the jar and its contents during the chromatography.

Finishing the chromatography (chromatography paper and filter paper)

When the mobile phase has moved an appreciable distance above the pencil line (this will depend on several factors including temperature and type of paper used), remove the paper from the jar and quickly dry the paper using either a hair dryer or warm oven.  The separated colours and standards should be seen clearly on the paper.


MEASUREMENTS

It may be easy to see, by eye, the similarity of movement up the paper of ‘Smartie’ components when compared to standards.  However, sometimes the mobile phase does not migrate evenly up the paper and measurements will need to be made, to obtain a greater degree of certainty of matching.

So, measure the average distance (mm), which the mobile phase rises above the pencil line at the bottom of the paper (let this be ‘A’)

For each component of the colour from the ‘Smarties’, measure the distance (mm), which the component moves above the pencil baseline (let these be ‘C1’, ‘C2’, ‘C3’ etc)

For each of the standard colours measure the distance (mm) moved by the colour above the pencil baseline (let these be ‘S1’, ‘S2’, ‘S3’ etc)


CALCULATIONS

For each ‘Smartie’ colour extracted calculate the ratio of ‘C/A’ (ie ‘C1/A’, ‘C2/A’, ‘C3/A’) for the individual components.

For each of the standard colours calculate the ratio of ‘S/A’ (ie ‘S1/A’, ‘S2/A’, ‘S3/A’) of the individual components. It should be noted that some standards will consist of a single colour, others maybe two or more colours.  Calculate the ratio of ‘S/A’ for each standard component.

For each of these ‘Smartie’ colours and standards component(s) record these values in a table.


ATTEMPTED IDENTIFICATION OF ‘SMARTIE’ COLOURS

Compare the values obtained for the ‘Smartie’ colours (ie ‘C/A’) with those obtained for the standards (ie ‘S/A’) and attempt to identify the components in the ‘Smartie’ colours by matching the values.

There may be a slight variation in these values, but if the experiment is repeated several times, (eg by several groups in the class), the average of each ratio can be determined as a class exercise. You will then find that the average value obtained, for a given component in the ‘Smartie’ extract, should be more consistent with the value of one of the standard component(s).

Notes

  • Some standard colours may contain more than one component.
  • If components are not visible after chromatography, the experiment will have to be repeated using a greater loading of standard colour or ‘Smartie’ extract along the pencil line.  The original aqueous extract may have to be concentrated by evaporation of water by heating over a flame.
  • If the coloured spots move slightly sideways across the filter paper, there is probably a paper overlap in the cylinder or touching of the paper against the walls of the glass jar.
  • If the mobile phase does not rise high up the paper, a greater volume may be required in the bottom of the jar. In this case stop the experiment when the aqueous phase stops moving up the paper.

More information

Explanatory YouTube videos from Free Science Lessons and Smart Learning For All

More detailed explanation from Chemguide: helping you understand chemistry