Experimental Procedure

1. Dissolve a few grains of the powder in 1ml of water in a vial. The sample should be
concentrated.

2. Carry out column chromatography (N.B: Watch the top of the column always):

a) Ensure there is a small volume of mobile phase (deionized water) above the sand bed.

b) Attach syringe to outflow tube and clamp tubing.

c) Open the stopcock.

d) Pull the syringe gently, until the top of the mobile phase just enters the top of the sand
bed (i.e. it’s meniscus touches the sand).

e) Close the stopcock. Add sample using a pipet by touching the pipet to the inner rim of
the column, well above the sand bed.

f ) Gently squeeze pipet bulb while rotating the tip around the inner rim. This minimizes
disturbance to the sand bed.

g) Open the stopcock and pull the syringe gently, until the top of the sample just enters the
sand bed (i.e. it’s meniscus touches the sand). Close the stopcock then.

h) Add mobile phase solvent with a clean pipet. First, add it around the inner rim, then
slowly top up the rest of the mobile phase directly into the middle.

i) Open the stopcock and pull on the plunger so that the sample separates.

j) As first coloured layer approaches, close stopcock and empty out water from syringe.

k) Collect one layer, close the stopcock and transfer to a vial.

l) Wash syringe with mobile phase solvent and re-attach.

m) Continue collecting layers. Keep replenishing the mobile phase solvent if needed.

To clean the column, add and pull through 15ml of deionized water. Leave the column
with enough water to cover the upper sand bed.

3. Carry out colourimetry procedure to identify dyes and record the analytical wavelength:

a) Pick out standard dyes that look similar to the samples you extracted.

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b) With at least 10ml samples of the extracted dyes, carry out colorimetry. (Make sure your
extracted sample is not too dark; dilute it if so) (also wash the vials with deionized water
and allow to dry).

c) Record the absorption spectra data in the experimental form and then draw a graph.

Analytical Wavelength of Dye 1 =

Analytical Wavelength of Dye 2 =

4. Preparing of standard solutions:

a) Use a volumetric serological pipet to prepare standard solutions of concentrations:

- 100% 10ml solution

- 80% 8ml solution + 2ml water
- 60% 6ml solution + 4ml water
- 40% 4ml solution + 6ml water
- 20% 2ml solution + 8ml water
- 0% 10ml water

Concentration of 100% solution in mol dm-3:

Immerse the pipet below the surface of the liquid without the bulb.
Remove all the air from the bulb then fix it on.
Fill pipet to at least 2cm above the calibration mark.
Remove the bulb quickly and cover tip with your index finger.
Drain liquid by releasing pressure while rotating the pipet.
Reseal pipet with finger and transfer to new vessel.

5. Using Beer’s law:

a) Measure the absorbance at the analytical wavelength for each standard solution.

b) Plot an absorbance versus concentration (expressed as dilutions) graph for the blank and
the 5 diluted samples. Label the axes and title the graph; also draw a best-fit straight line
through the points.

6. Prepare sample for analysis:

a) Prepare a 50 mL sample of drink according to the package instructions. Use a top loading
balance to measure the mass of the powder that should be included.

b) Transfer about 10mL of this solution to a vial for the colorimetric measurement.

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c) Measure the absorbances at the analytical wavelengths for each dye in the juice sample.

Absorbance of Dye 1 =

Absorbance of Dye 2 =

d) Plot the absorbances of the dyes on the graphs and read the corresponding percentage
concentrations:

% Concentration of Dye 1 =

% Concentration of Dye 2 =

e) Calculate the molar concentrations of each dye by multiplying the % with the standard
concentration of the dye:

Molar Concentration of Dye 1 =

Molar Concentration of Dye 2 =

Calculations regarding ADI Values

Calculate molar concentration.

From volume of sports drink, calculate moles of dye.

Using molar mass of dye, calculate mass of dye.

Determine if dye exceeds limit.

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