Measuring changes in ascorbic acid (vitamin C) concentration in ripening fruit and vegetables

We know a lot about ascorbic acid (vitamin C) in the human diet and are familiar with its role in controlling scurvy and as an antioxidant. But what is its role in the plant cell? Why do plants produce vitamin C? And how can we measure the amount of vitamin C in different fruits and juices?

Ascorbic acid may be found in all the compartments in the plant cell, where it plays diverse roles. It is known to be involved in cell division and cell wall synthesis and also acts as an inhibitor of dangerous compounds such as hydrogen peroxide and the dangerous radicals of oxygen generated as a by-product of respiratory and photosynthetic machinery of the cell.

We have recognised the presence of ascorbic acid in ripening fruits, particularly in citrus, but are less certain about its role. Other organic acids, notably citrate and malate, are often present in larger quantities and contribute to the sugar-acid balance. You probably know from experience how this changes (in favour of the sugars) as fruits ripen, but you can use this method to measure for yourself whether the vitamin C really does disappear during ripening and in storage in your own selection of fruits and vegetables.

The assay

The principle of this method is a titration with dichlorophenolindophenol (or phenol-indo-2:6-dichlorophenol, also known as DCPIP). Ascorbic acid reacts with DCPIP, changing the colour from blue to colourless. They react in a 1:1 fashion, so if a known quantity of DCPIP solution reacts with the plant tissue extract, the quantity of DCPIP used gives a direct measure of the quantity of ascorbic acid present.

• Grind up a measured mass of fruit or vegetable in a known volume of distilled water

Try a ratio of perhaps 1 g fruit to 5 cm³ water, though the precise ratio of fruit to water may need to be modified if, for instance, the plant material is particularly glutinous. The grinding can be done in a mortar and pestle, or using a blender for larger samples.

• Filter or centrifuge the plant macerate to provide a clear solution (of whatever colour!), then use this solution for the titration reaction.

• Transfer say 1 cm³ of the plant extract solution into a test tube. Place the test tube beneath a burette or vertically mounted 1 cm³ pipette (with pipette-pump attached) containing 0.1% DCPIP solution and add DCPIP dropwise to the extract in the test tube.

[When the first drop of DCPIP is added to the extract, the initial blue colour should quickly disappear – as a consequence of the reaction between any ascorbic acid present and the DCPIP. If this does not happen then the ascorbic acid concentration is very low (or even zero!) and a larger volume of extract may be needed in the test tube, if a measurement is needed].

• Add DCPIP until the blue colour does not disappear, and note the volume of DCPIP used. This volume is equivalent to the amount of ascorbic acid present in the extract, so the concentration of ascorbic acid in the original plant material can be calculated and expressed as units per gram of fresh mass

Calculating the concentration of ascorbic acid (vitamin C)

Here is an example of how you can measure the concentration of ascorbic acid (vitamin C) in your original sample of fruit.

Suppose you started with 10 g of tissue and made an extract in 50 cm³ of water and then in the titration a 1 cm³ sample of your extract required 1.5 cm³ of 0.1% DCPIP solution. As each cm3 of 0.1% DCPIP (MWt 290.08) solution is equivalent to 6.071×10-4 g ascorbic acid (MWt 176.12). Therefore 1 cm³ of extract must have contained 9.107×10-4 g of ascorbic acid. This means the original 50 cm³ extract contained 0.0455 g and this came from 10 g of fruit. So you can work out that each gram contained 0.00455 g ascorbic acid.

Note that some plant materials may be very acid, in which case the colour of the DCPIP becomes pink / magenta. In this case, look for the appearance of a permanent pink colour during the titration, rather than a blue colour.

(You can find further information in: Smirnoff N. Ascorbic Acid: metabolism and function of a multifaceted molecule. Current Opinion in Plant Biology 2000, 3:229-235.)