These practical protocols are designed for students doing an Extended Project Qualification, Advanced Higher investigation, or IB investigation in biology.
They are linked to our Ripe First Time – Avoiding Food Waste project starter.
Areas for practical investigation
You can set up investigations to follow the changes in the concentrations of these pigments during ripening by trying to answer some of the questions below.
- How do the pigment levels change during the ripening process?How do the conditions of storage affect the colour changes?
- How do pigment levels change if a fruit / vegetable is allowed to become over-ripe?
- How do pigment levels change if the fruit / vegetable becomes infected by a fungal or bacterial pathogen?
- How do the pigment levels change during food preparation and cooking?
- How many anthocyanin pigments contribute to the colour of a particular fruit? In what sequence do they appear?
Measuring the relative amount of red versus green pigments (anthocyanins vs chlorophylls a & b) in plant tissues
With a colorimeter, it is possible to make comparative measurements of green and red pigments in plant tissues, using some of the broad band colour filters provided with the colorimeter.
A colorimeter filter passes a beam of light consisting of a whole range of wavelengths through a solution. Different coloured filters allow different sections of the visual spectrum (400 nm to 700 nm) to pass through, but the range of wavelengths might include as much as 20% of the spectrum. Green pigments get their colour from the fact that they absorb visual spectrum wavelengths other than green, and, as it happens, chlorophylls absorb red wavelengths particularly well. Red pigments get their colour from the fact that they absorb visual spectrum wavelengths other than red, and, as it happens, anthocyanins absorb blue wavelengths particularly well. Hence, by measuring the absorbance of a plant pigment extract, using a red and a blue filter, it should be possible to estimate the relative amounts of the “red-absorbing” and “blue-absorbing” pigments present.
Measuring chlorophyll content
- Use a small volume of absolute ethanol to extract chlorophylls from plant tissues, ground in a mortar with silver sand. Then centrifuge this solution to remove any solid debris.
- Keep the resulting clear supernatant and, using the blue filter and a green filter in the colorimeter, make two readings of the optical density of this solution. For each reading, first set the instrument to read zero absorbance with a tube containing ethanol alone (the blank). Then replace this tube with the tube carrying the coloured extract.
- If the absorbance reading is off the scale (greater than 1.0), dilute the extract with ethanol, by a known factor, until a reading can be made. Then use the dilution factor to calculate the original absorbance value.
- The figure for chlorophyll content is calculated by subtracting the value for absorbance with the green filter from that with the blue filter, as follows:
absorbance with blue filter – absorbance with green filter
Measureing anthocyanin concentration
- Use a small volume of acidified methanol (1 part concentrated HCl : 99 parts methanol) to extract anthocyanins from plant tissues, ground in a mortar with silver sand. Then centrifuge this solution to remove any solid debris.
- Keep the resulting clear supernatant and, using the blue filter, make two readings of the optical density of this solution. For each reading, first set the instrument to read zero absorbance with a tube containing acidified methanol alone (the blank). Then replace this tube with the tube carrying the coloured extract.
- Do the first reading with the original supernatant, which has a pH of much less than 3. At this pH, anthocyanins are coloured ions.
- Then add 1 M NaOH dropwise, until the pH of the extract is greater than 5 (which causes the anthocyanins to become colourless), and measure the absorbance again.
- Note the actual volume of NaOH added, and calculate its dilution effect. Once this is done, any remaining difference between the absorbance values at the two pH values is the result of anthocyanin concentration.
Measuring relative amount of chlorophyll a, chlorophyll b and carotene in plant tissues
If a spectrophotometer is available, measurements can be taken, at four key wavelengths, of the absorbance of light by a plant pigment extract. It is then possible to calculate the concentration of each pigment in mM.
- Grind up equal fresh masses of the fruit skin, in measured volumes of absolute ethanol (say 10 cm3).
- Place the ground material in centrifuge tubes (equal volumes in each tube) and spin at high speed for 5 minutes to remove debris. Decant the supernatants to clean small capped specimen tubes.
- Take absorbance measurements of the whole ethanolic extract, at the following crucial wavelengths, using absolute ethanol as the blank for zeroing the machine.
663 nm, 645 nm, 480 nm
Calculate concentrations of pigments as follows:
Chlorophyll a concentration in mM = 12.7 x A663 – 2.69 x A645
Chlorophyll b concentration in mM = 22.9 x A645 – 4.68 x A663
(where A663 and A645 are the values for absorbance at wavelengths 663 nm and 645 nm respectively)
Carotenoid concentration in mM = (A480 + (0.114 x A663) – (0.638 x A645))÷112.5
Once the value in mM is known, this information can then be used to estimate the total quantity of extracted pigment, by taking account of the mass of plant material used for extraction and the initial volume of extract that was made.
It is worth noting that the ratio of carotenoids to chlorophylls can be affected by environmental stresses experienced by the plant tissues. (Think, for example, about the changes that happen to colour of the grass on a lawn, if a paving stone is left in one place on it, say for a week.)
The basis for this protocol is taken from:-
pp148-152 in Methods in Comparative Plant Ecology – A Laboratory Manual. edited by GAF Hendry & JP Grime. Chapman & Hall 1993