Vitamin C Investigation

Concentration, in biochemistry terms, is “increasing the strength or proportion of (a substance or solution) by removing or reducing the water or any other diluting agent or by selective accumulation of atoms or molecules.” (Oxford English Dictionary). Therefore concentrated fruit juice has had its water removed (probably for ease of transportation) then had water re-added (reconstituted) before packaging.

Vitamin C is an important part of the human diet. It is found in fruits, green vegetables and potatoes, and is “necessary for the formation of blood and bone and for resistance to infection.” (Ramsden, 1995). It is an antioxidant which is capable of counteracting the damaging effects of oxidation. Vitamin C helps to prevent deficiency diseases, in particular scurvy, and is abundant in citrus fruits such as lemons, limes and oranges. Its chemical name, ascorbic acid, “is derived from a- and scorbuticus (Scurvy) as a shortage of this molecule may lead to scurvy.” (www.chemie.de).

The amount of vitamin C in a fruit juice can be estimated using a blue chemical die called DCPIP (common name 2,6-dichlorophenolindolephenol). It is reduced by an equal amount of vitamin C to a colourless, or sometimes pink, compound solution.

This experiment will aim to find out whether fresh orange juice really does contain more vitamin C than those made from concentrate. By first testing a known concentration of vitamin C solution it should be possible to calculate the vitamin C content in each fruit juice.

Method

Apparatus: Safety eye mask, lab coat, ascorbic acid solution 0.1%, DCPIP solution 0.1%, test tube holder, 21 test tubes (3 for standardisation test, 12 for juices from concentrate and 12 for fresh orange juices), pipette for measuring DCPIP solution, 2 x syringes (1 for measuring ascorbic acid and 1 to measure orange juice with), 3 brands of orange juice from concentrate, 3 brands of fresh orange juice, paper, pencil.

Take 3 of the test tubes, label them 1 to 3 and place them in the test tube holder. Use the pipette to put 1 ml DCPIP solution in each test tube. Using the syringe, drop 0.1ml ascorbic acid solution (vit C) into test tube 1, then gently shake the test tube. Repeat, adding 0.1ml vit C solution and gently shaking until the DCPIP solution is decolourised. Note the amount of vit C solution used to fully decolourise the DCPIP solution. Repeat the test for test tubes 2 and 3, noting how much vit C solution is required.

Repeat the experiment with each of the three brands of orange juice from concentrate and with each of the fresh orange juice brands. i.e. for each juice, label 3 test tubes 1 to 3 and place them in the test tube holder. Using the pipette, put 1 ml DCPIP solution in each test tube. Use the syringe to drop 0.1ml juice into test tube 1, then gently shake the test tube. Repeat, adding 0.1ml juice and gently shaking until the DCPIP solution is decolourised. Note the amount of juice used to fully decolourise the DCPIP solution. Repeat the test for test tubes 2 and 3, noting how much juice is required.

Risk assessment: As DCPIP solution is a die, a lab coat must be worn throughout the experiment. A safety eye mask will also be worn as an extra precaution.

Carrying out the practical

It became clear shortly after beginning the experiment that something was wrong. An equal concentration of vit C solution is supposed to reduce DCPIP solution to a colourless solution, but this was not happening. It actually took around 5 ml vit C solution before any visible reaction took place at all, and upwards of 30 ml for the DCPIP to lose its colour completely.

Whilst doing some additional research, it transpired that both DCPIP and vit C solutions need to be freshly prepared for the experiment; both solutions are readily oxidised with exposure to air, and therefore deteriorate. In addition, if DCPIP solution is stored for any length of time it should be kept in a dark-coloured container, which was not the case. Furthermore, vit C solution should be kept cool, as “the rate of oxidation increases rapidly with the temperature” (Ramsden, 1995). Once both solutions were freshly made the difference was obvious; the fresh DCPIP solution was blue rather than purple and the test worked much better.

Care was taken when measuring solutions into test tubes by keeping them in the test tube rack on a flat surface. Solutions were measured accurately by keeping the pipette and syringe vertical, and all measurements were taken at eye-level. The amount of ‘shake’ given to the test tubes after each drop of solution was added is difficult to quantify, but it was kept to a reasonable degree of consistency.

Due to time and buying constraints, it was only possible to purchase two brands of fresh orange juice. The decision was made to improvise, and use the juice of an orange, squeezed just prior to testing. This required additional equipment: pestle, mortar and a beaker. A few segments of the orange were placed in the mortar and crushed with the pestle until enough juice could be poured into the beaker, ready for use in the experiment. Whilst it would have been better to have used three brands of fresh orange juice, the freshly squeezed orange juice provided some interesting, unexpected results.