Ascorbic acid is an enediol of a hexose sugar and the reduced or enediol form is readily oxidised to form dehyroascorbic acid. Both forms are biologically active, except after treatment with alkali.
In the method to be used here Ascorbic acid can be oxidised by the coloured dye 2,6 dichlorophenol indophenol (DCPIP) to dehydroascorbic acid. The dye is reduced to the colourless compound (leuco dye) so that the end point can be readily determined. DCPIP is available commercially in tablet form, where 1 tablet is equivalent to 1mg of ascorbic acid. In view of the fact that some coloured fruits such as strawberries will obscure the end point trichloromethane will be used to collect in the organic layer and enhance the colour. Further in the method there is the danger of interfering substances oxidising the L ascorbic acid, this can be minimised if the titration is carried out under acid conditions.
Aim and Hypothesis.
The aim of the experiment is to discover whether fruits kept for one week in a refrigerator show a significant degradation in vitamin C content compared with a control (a fresh sample) or not. The hypothesis will be that there is a significant degradation, this will be tested using the student’s ‘t’ test. Significant here implying that using school apparatus the difference will be detectable.
Plan and Method.
Apparatus. 10ml burettes, pipettes, beakers glass rod, Bunsen tripod and stand, pestle and mortar filter funnel, test tubes, measuring cylinder, volumetric flask (50 ml).
Materials tablets of DCPIP, fresh ascorbic acid solution, glacial ethanoic acid trichlomethane and samples of fresh fruit.
- Crush 1 tablet of DCPIP in about 40ml of warm water and dilute using distilled water to the 50ml mark I a volumetric flask. This should be sufficient for three replications of standard and fruit sample.
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A fresh ascorbic acid solution provided by the teacher on the day required. A standard solution of a 1% concentration i.e. 1mg per 100cm3
- The class will be divided into groups each group looking at a particular fruit from the list orange, strawberry, apple lime and lemon and blackcurrant. My individual results are for lemon.
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Samples of the fresh fruits will be crushed in a pestle and mortar. The mixture will be transferred to a centrifuge tube and centrifuged. A variable volume of distilled water will be added, to make up to the 30 cm3 mark and the mixture filtered for immediate use. Some texts recommend mineral wool and sand for this operation. Here these items were not used
- For those groups using a relatively non coloured fruit mixture e.g. lime or lemon pipette 5 ml of extracted juice into a boiling tube. Using a teat pipette add approximately 1ml of glacial ethanoic acid, Using a 10ml burette containing the DCPIP carefully titrate drop by drop until the dye solution becomes a permanent pink colour.
- Record the volume added in the normal manner by recording initial and final volumes on the burette. Call the titre T. if possible repeat these readings to reduce random errors.
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The titration should now be repeated using the standard ascorbic acid solution referred to as the standard. Record the titre as st. As DCPIP turns water a pink colour by itself an increase in accuracy can be obtained by counting the drops to make the same colour change as is observed in the original titration. This is known as a blank test. 5 cm3 of distilled water were placed in a boiling tube and DCPIP gradually added from the burette until a pink colouration was obtained The volume of DCPIP used call BL.
- For those groups using a coloured fruit such as strawberry or blackcurrant a modification is advised. Add 1ml of trichlormethane to the boiling tube before titration commences. The end point now is when a pink permanent colour is seen in the organic phase.
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Samples of fruit juice will be stored in the refrigerator labelled for subsequent use. A new titration has to be carried out in the subsequent analysis. A 30 cm3 diluted sample will be placed in a boiling tube, tightly bunged, labeled with the fruit and experimenter and left refrigerated for one week, around 5 degrees Celsius.
10. If time permits replicate all results at least three times.
The Vitamin C content is give by the formula
It C mg/100ml = (T-BL)*2/ (St-BL).* dilution factor. The dilution factor in this case is 3.33, because we only took 30cm3 samples of the fruit juice.
Class results are shown in table 1 on spreadsheet.
In my case Lemons were investigated, I did not dilute the juice as far as 30cm3 as my solution was considered already fairly clear I diluted by 5 cm3 of distilled water each sample of 5cm3 of unadulterated lemon juice I took for the runs.
Discussion.
For my lemon results I obtained a value of 28.3 mg/100cm 3 which is about 62.8% of the value quoted in the literature. After one week I obtained a 29.25% reduction . I had expected from my researches not to obtain a figure anywhere approaching 100% as DCPIP does not pick up the de-hydroascorbic acid content in the lemons. The class results indicated that similar reductions were obtained for pears, oranges apples and strawberries. Limes and blackcurrants appear to have withstood storage rather better, However one does not know if this is a real phenomenon or just due to group variation in precision and accuracy or even procedure. The students who conducted experiments on pears and blackcurrants obtained a similar percentage Vitamin C content to my determination on lemons. Apples and oranges came somewhat lower, with Limes and strawberries bringing up the rear.
Evaluation.
One of the problems with DCPIP is that it forms a pink colouration with water and therefore this is why it was necessary to carry out a blank test. Some of the groups may have disregarded this blank step, but for coloured juices this may have been even more important to gaining a more accurate result. The method appears to have given the right trend in terms of total vitamin C content of the various fruits as compared to the literature. Among those students having the harder task of a coloured solution, i.e. blackcurrants have nevertheless obtained a high Value for their Vitamin C content. Although as against that strawberry values came out low, as did limes. Limes have a fairly clear solution within which to work.
In all cases the value for the Vitamin C after 1 week was less than that for the original crushed juice. However there seems to be a mixed set of results when values obtained are compared with the literature. All groups underestimated the vitamin C content. There is good reason to suppose why an underestimate would have been more likely.
One of the errors is a systematic one. DCPIP oxidises L Ascorbic acid to dehydroascorbic acid, but dehydroascrobic acid is active and forms part of the total vitamin c content of the fruit. Therefore without some special procedure DSCPIP method cannot obtain all of the vitamin C content. Some texts in the literature recommending reducing the sample of Vitamin C with Hydrogen sulphide. Unfortunately we were not allowed to work with this gas. Hydrogen sulphide would reduce all of the dehydro- ascorbic acid to the L Ascorbic acid form, hence all of this could be oxidised by the DCPIP and all of the vitamin C content obtained , at least in theory. Secondly some groups had difficulty in crushing their fruits and may not have extracted all the vitamin C in them. Apple and pears might be subject to this difficulty. Also in my case and with oranges much of the Ascorbic acid is in the peel and I am not sure whether under the mashing and centrifuging procedure all of the acid would have been obtained.
Another problem was that although students obtained 30cm3 within experimental error of juice extract, the amount of fruit they started with could not possibly have been standardised. It appears then that like is not being compared with like. When compared to the literature some results express Vitamin C content as mg/100g of fruit. As the total volume obtained for the class results is now lost in the dilution it will not be possible to ascertain on that measurement the validity or otherwise of the method. However if the results in the literature quote concentrations in mg/100cm3 then our results are directly comparable.
An experimental difficulty arises with the DCPIP because the volumes being used are so low, then very careful experimentation is required. One drop is a significant item when working with such low overall volumes and therefore to underestimate or overestimate the end point by one or two drops is serious. Special care has to be taken to avoid cross contamination with the apparatus. Any conical flask that is used for the titration must be thoroughly washed with water, distilled water and dried. In between replicates some groups would not have achieved this ideal. In the burette when adding the DCPIP solution care must be taken that the level drains down to where it can be read.
Whilst working on the experiment time is allowing oxidation of the L Ascorbic acid by molecular oxygen. Thus inevitable underestimation will arise on that score.
It is unlikely that the vitamin C content of fruits degrades when ripe at similar rates across the plant kingdom. Some fruits such as apples for example degrade at a much slower rate than strawberries say. A hard cuticle prevents immediate aerial oxidation. When the fruit is crushed and filtered as in this experiment, the vitamin C should degrade at a more or less constant rate in all the specimens and to that extent this experiment is a well designed one. Another problem with some groups is that after one week they may not have re-standardised their vitamin C solution with fresh DCPIP. It is important to do this if in fact the tablets are manufactured with some degree of variation in them
