Vitamin C Practical Experiment Hypothesis; Based on knowledge already at hand, My hypothesis will be that freshly squeezed juice will

contain more vitamin C compared to juices that are from concentrate and that juice from lemons will contain more vitamin C due to their acidity.

Vitamin C content in Juices

No of drops of juice need to decolourise the DCPIP solution 7 6 5 4 3 2 1 0 Orange Cherry Tropical Lychee Mango Vitamin C 1% Types of juices

Variables;

Variables such as temperature could affect the results that are collected, as much as can be done to control the temperature to make the results accurate will be done, holding samples with hands can increase the temperature slightly so they will be handled by the lip of the vial and placed into a rack. When testing samples multiple times I will ensure that the equipment cleaned and dried or if possible new as the pH of water might affect the reaction of DCPIP and ascorbic acid, water can also affect the results depending on whether it is hard water or soft water, hard water contains more mineral and more chlorine is present. Measuring will be as close to as possible accurate, this will include re-measuring samples.

I will be measuring the amount of juice sample solution it takes to decolourise 1cm of DCPIP, first I will use a controlled sample of 1% vitamin C to decolourise the DCPIP, and with this I can compare results from the other juice samples. The measurements will be taken with a

pipette and released into the DCPIP 1ml at a time, each time 1ml is released it will be shaken to allow a fair mix of solution, and this will be repeated on till decolourisation. Vitamin C (Ascorbic Acid) has a pH of under 4-5 when diluted with water DCPIP, 2,6-dichlorophenolindophenol, which is a dye used to monitor light reaction in photosynthesis

Apparatus & Chemicals; Pipette Fruit Juice Samples

DCPIP 1% Solution Test tubes Test tube rack Burette (Possibly) Glass Rod (Possibly)

I have decided to use test tubes because they provide a clear sight into the experiment at hand, they are also smaller then Pyrex beakers which would spread the solution over a large surface where as a test tube would make it easier to establish decolourisation. Pipettes are a fast way to measure the solution with a limited amount of time, it is a suitable and vital piece of apparatus however there might be a probably with used pipettes as they can have dents from previous experiments and would only be accurate to around 0.0ml-0.2ml when measuring solutions. A burette would allow an accurate measure of how much solution has been added into the DCPIP, probably a greater accuracy then using a pipette. A glass rod would allow a fair mixing of the solution but the effects on the results are unknown.

Safety;

Take care of glassware. Sanitise experimental area after. Wear eye protection at all times.. Care when handling juice samples or chemicals as they may stain. Do not drink juice samples as dangerous chemicals may be present after experimentation.

Pre-Experiment Method;

The method I will be conducting is simple. I will measure 1% DCPIP 1cm(3) from a beaker to a pipette, with another pipette I will add the 1% vitamin C solution which is the control of the experiment 1cm(3) at a time to a test tube, each time 1cm(3) is added, I will gently stir the test tube to make sure then the reaction takes place. I will repeat this on till the DCPIP solution is decolourised. To get a fair and accurate result I will repeat this 3 times and then average the results. Outliers will be excluded to keep the test accurate. The samples will be kept in a test tube rack to compare different colours.

The next juice two sample will be tested with the same amount of DCPIP, first placed into a test tube via pipette then another unused Pipette will extract the juice sample and will be added into the DCPIP 1ml at a time then stirred gently and this will continue on till the DCPIP is decolourised. The time allocated during this experiment is 1/2 hour. Accuracy could be improved but due to this some apparatus and time will be put aside in order to complete the experiment

There are some ethical issues with testing these chemicals and releasing them into the sewer as they are not helpful to the environment due to their pH, care will be taken so that any DCPIP and Vitamin C is not wasted and only what is needed is used.

Results;

T1+T2+T3 3

Once all of the data was collected, It was input into the table below, It shows the amount of juice required to decolourise the DCPIP, 3 tests were conducted and from that you can then work out the

average with a formula result from all 3 of the trials.

to get the average, the average can be used to conclude a fair

These results show that the juice sample that contains the most Vitamin C was 7.3ml 0.13g Vitamin C the Jiff, with an Solution average of 5.5ml 14ml 15ml 18ml 15.6ml 0.06g Pure Orange to turn the Juice DCPIP 5.5ml 5ml 6ml 5.5ml 0.18g Jiff decolourised compared to the 15.6ml of pure orange Juice. The Vitamin C. Solution took 7.3ml to decolourise the DCPIP which was 1.8ml more than Jiff, This suggests to me that Jiff contains less water than the Vitamin C. 1% Solution did. Vol. Of Juice Required to Decolourise 1cm(3) of 1% DCPIP Test 1 Test 2 Test 3 6ml 7ml 9ml Average Vitamin C. Content With the average, I then calculated the Content of Vitamin C in each of the samples by Dividing them by the 1ml of 1% DCPIP Solution which gave the sum then converted to grams. These results prove my hypothesis in that freshly squeezed fruit will contain more vitamin C, this is because it will not have been watered down by manufacturers for a higher quantity. The results also prove my hypothesis is right that lemons contain more Vitamin C. Due to their acidity which is the sign of ascorbic acid (Vitamin C) and their low pH levels.

This line graph shows the plot of the tests carried out. From this we can see that for pure orange juice and Vitamin C samples that there is a steady rise from test 1 to test 3, this could be due to a number of factors that I will include in my limitations, However the Jiff tests shows to be accurate and consistent from test 1 to test 3. There is a 4ml difference from test 1 to test 3 in the pure orange juice tests. There is a 2ml difference from test 1 to test 2 in the Vitamin C 1% Solution Test. This graph also shows standard deviation, from the bars we can see the average of the test, it measures the spread of data from the mean. The results conclude that the deviation is a Low standard deviation because the valves are not spread out so much. The equation for the standard deviation used is;

This pie chart shows the content of vitamin C. Using the average of the volume of solution required to turn DCPIP into. From the results we can see that the Jiff has the highest amount of Vitamin C. Present, It has 3 times more than the Pure Orange Juice Sample, it also contains 0.05g more than the 1% vitamin C solution. RECOMMENDED DIETARY ALLOWANCE (ADULT MALE) 9.0 G PER DAY RECOMMENDED DIETARY ALLOWANCE (ADULT FEMALE) 7.5 G PER DAY From this we can see that Jiff reaches two times the amount of RDA in males. The pure orange juice reaches 66.6% of the RDA. This RDA does include food intake, so these juice samples do quite well in the RDA perspective

The graph shows that on all samples tested,

Post-Experiment Method, Limitations and Variables; As planned the measurements of the juice solutions were accurately measured and did not obscure sight in the pipette, However the DCPIP was to a degree harder to measure due to the darkness of the

solution, it covered the pipette in a dark blue colour and was hard to tell where and how much solution was being measured. The experiment took slightly longer due to this as I had to wait for the solution to settle into the solution. As this was the first time carrying out an experiment like this, it was unknown that the 1% Vitamin C solution Control would turn into a brown colour when decolourised, Although this happened I still recorded the results and then continue to see if any difference would appear but after 5 minutes of no change they were placed into a test tube rack to be observed later on and compared with the other trials, the result was that they did turn brown and that was expected to be the end of the trial. The Pure Orange Juice also produced different than expected appearance, the solution did decolourise the solution but it turned to a yellow colour which was the original of the juice sample. One problem that I was faced with was the limited amount of apparatus, I ended up having to clean them with tap water, they were dried but to a certain extent, the pH of the water could affect the experiments outcome, however all the of solutions would have had some water in them so this shouldnt affect the results in a significant way.

Post-Experiment Method, Limitations and Variables-Continued; Other problem in the experiment that I later picked up on from observation was that the DCPIP and Juice solutions were left open meaning that oxidation could affect the results which is a loss of electrons and a gain in hydrogen, this will cause the DCPIP to turn blue again, as Vitamin C is a reducing agent it adds electrons therefore its used in this experiment because it will change colour. The limitation of this experiment is that it was only repeated three times due to the amount of time available, more results would have given better accuracy in results and given me the chance to spot outliers in the experiment. We also do not know if Vitamin C is lost during ageing or through heat treatment which often products go through to ensure that its free from harmful bacteria. One improvement that could be made to the experiment is the equipment, having a larger choice to use from, and enough so that other factors like water dont come into play. One modification that could used is that now we know that after a certain colour in the process of decolourisation we know that the trial is finished therefore saving us time for something else. Another Improvement is to be more careful with the juice samples and to keep them contained so that they are not affected by the elements.

In this investigation, I will analyse by titration, the vitamin C (ascorbic acid) content of 7 different types of fruit juices purchased from a supermarket. I will test for the presence of vitamin C using an organic indicator solution called DCPIP (dichlorophenol indophenol), which will be reduced to a colourless form from dark blue, at the presence of vitamin C. Reduction is where a molecule losses an oxygen molecule, gains a hydrogen atom or gains electrons. Oxidation is the opposite, i.e. gains an oxygen molecule, loses a hydrogen atom or loses electrons. The amount of juices used to turn the DCPIP colourless will be recorded and the vitamin C content will be determined by measuring the

amount of drops of a known vitamin C concentration solution, used to turn the DCPIP colourless. Vitamin C Vitamin C plays a vital part in the correct functioning of the living body (most animals synthesise their own vitamin C. we don t). There are 4 ways in which vitamin C considerably benefits the body: 1. Antioxidant. Oxidation of haemoglobin in the blood is necessary to survive, but too much oxidation can be damaging to tissues and organs. Vitamin C prevents too much oxidation from occurring. 2. Homeostasis. When the body is exposed to stress, the adrenal grand uses vitamin C to produce adrenaline in response to the stress by which blood will be moved to the muscles from the digestive system, etc. This is for flight, fright or fight. 3. Collagen. This is the principal component of tendons, ligaments, skin, bone, teeth, and cartilage, in addition to being the ground substance between cells. Inadequate vitamin C levels will lead to weakening of these areas leading to skin lesions, blood vessel fragility, loosening of gums and teeth, characteristics of scurvy. 4. Immune system. Vitamin C is involved in the production of white blood cells, which are the main defence against disease. Without vitamin C, the body will be very susceptible to disease. Vitamin C has also been linked in the prevention of heart disease and cancer. List of equipment Volumetric flask to measure out the correct volume of DCPIP. Beaker, 1 to containing the juice you wish to test and 1 for the DCPIP solution. 2ml syringe, to measure out the DCPIP. 1ml syringe, to measure out the quantity of juice added to the DCPIP. 10 test tubes, into which the DCPIP and the juice is added. Test tube rack, to hold the test tubes. Electronic balance to weight the correct amount of DCPIP. Method The experiment was performed at room temperature.

The juices used were: Orange juice Grapefruit juice Lemon juice Pineapple juice Mixed fruit juice Cranberry and raspberry juice Apple juice The first task was to measure out 1% DCPIP solution in the volumetric flask and to make sure it is completely mixed (distilled water is used so not to effect the accuracy of the experiment and a squeeze bottle can be used to top up the measurement for further accuracy). The level was read from the bottom of the meniscus. The DCPIP can then be put in to a beaker to ease the filling of the syringes (DCPIP must not be left overnight to avoid oxidation which may affect the results). 2ml of DCPIP are then put into each test tube using the 2ml syringe. The 1ml syringe is then filled with the 0.05% vitamin C solution and is added drop by drop to each tube individually until the DCPIP has been reduced and the blue colour has disappeared. The amount of 0.05% vitamin C solution needed to reduce the DCPIP is recorded. This procedure was repeated 10 times to provide an adequate level of accuracy to the experiment. The test was then performed on the fruit juices of unknown vitamin C content. The relevance of testing the 0.05% vitamin C solution is to determine the vitamin C concentration in the fruit juices by dividing the amount of 0.05% vitamin C solution needed to reduce the DCPIP, by the amount of fruit juice needed to reduce the DCPIP. This is then multiplied by 0.05 (the known concentration of the first solution). By doing this, the vitamin C content of the fruit juices can be determined. Any difficulties or anomalies were also recorded. Calculation example: Volume of 0.05% vitamin C solution needed to reduce DCPIP = 0.39ml Volume of orange juice needed to reduce DCPIP = 1.55ml

Vitamin C content of orange juice = volume of 0.05% vit. C solution X 0.05% Volume of orange juice = 0.39 X 0.05 1.55 = 0.013% Questionnaire Put a tick in the box beside the juice that you think has the highest vitamin C content, and a cross in the box beside the juice with the lowest vitamin C content. Orange juice Apple juice Lemon juice Pineapple juice Grapefruit juice mixed fruit juice Cranberry and raspberry juice Questionnaire The questionnaire was given to a sample of 50 people to get an idea on general thoughts on fruit juices vitamin C content. Juice with the most vitamin C: 52% orange juice 34% lemon juice 10% mixed fruit juice 2% cranberry and raspberry juice 2% grapefruit juice (0% for the rest) Juices with the least vitamin C: 32% apple juice

28% cranberry and raspberry 24% pineapple juice 10% mixed fruit juice 6% lemon juice (0% for the rest) Generally, people believe the citrus fruits contain the most vitamin C. Graph showing questionnaire results Juices with the most vitamin C content Juice a = orange juice Juice b = lemon juice Juice c = mixed fruit juice Juice d = cranberry and raspberry juice Juice e = grapefruit juice Juices with the least vitamin C content Juice a = apple juice Juice b = cranberry and raspberry juice Juice c = pineapple juice Juice d = mixed fruit juice Juice e = lemon juice Students T-test 1 Calculating the significant difference between the means of orange juice and pineapple juice. To see if there is more or less than 95% confidence that the difference between the means has not occurred by chance alone.

Orange juice mean = 1.55 Pineapple juice mean = 6.43 Difference between them = 4.88 Standard deviation of orange juice = Sx =0.0750 Sx2 = 0.005625 Sx2 = 0.0005625 N Standard deviation of pineapple juice = Sy = 0.2744 Sy2 = 0.075295 Sy2 = 0.00753 N Sx2 + Sy2 = 0.0080925 NN Standard error = 0.08996 T = 54.247 There is 95% confidence that the difference has not occurred by chance alone. In fact, there is 99.9% confidence that the difference has not occurred by chance. Students T-test 2 Calculating the significant difference between the means of orange juice and grapefruit juice. To see if there is more or less than 95% confidence that the difference between the means has not occurred by chance alone. Orange juice mean = 1.55 Grapefruit juice mean = 0.93 Difference between them = 0.62

Standard deviation of orange juice = Sx =0.0750 Sx2 = 0.005625 Sx2 = 0.0005625 N Standard deviation of grapefruit juice = Sy = 0.0432 Sy2 = 0.0018662 Sy2 = 0.0001866 N Sx2 + Sy2 = 0.00749 NN Standard error = 0.2737 T = 22.653 There is 95% confidence that the difference has not occurred by chance alone. In fact, there is 99.9% confidence that the difference has not occurred by chance. Analysis During the experiment, there was a problem because one of the volumetric flasks had been filled with an incorrect amount of DCPIP. This greatly affected the results. Fortunately, the problem was swiftly rectified and the experiments were tested again with the correct DCPIP volume. This problem could have been avoided with more care and attention put into the measuring of the DCPIP. When the apple juice was tested, I found that after adding 15ml of juice to 2ml of 1% DCPIP, there was no change. 1ml was tested, but still there was no change. Eventually, 0.125ml of DCPIP was used in order for the complete reduction of the DCPIP. This alteration decreased the accuracy of the results because very small volumes of DCPIP had to be measured out. The standard deviation was calculated for the results obtained. This measures the spread of the individual measurements about the mean. The larger the spread, the larger the deviation. The results found had relatively small standard deviations except for the apple juice. This had a significantly higher standard deviation because the apparatus was not accurate enough to measure

such low levels of vitamin C. More repetitions could have been made with less amounts of DCPIP to reduce the spread and increase the accuracy. The levels of confidence found were 99.9% that difference between the two means were not by chance alone. The mixed fruit juice and cranberry and raspberry juice (the two highest results), had added vitamin C. The carton did not state how much vitamin C was added so it was not possible to allow for the addition. This means that the results did not show how much vitamin C is in those two juices, but how much is in the cartons. Vitamin C is added to some juice as an antioxidant to prevent the deterioration of the substance. This means that the cranberry and raspberry juice and the mixed fruit juice did not have sufficient vitamin C in it originally, so more had to be added. The lemon juice found to have low vitamin C content. However, it contained preservatives (e.g. E220 V E227) to prevent the growth of any bacteria and to be able to last a long time without decay. This preservation may have affected the results but damaging the vitamin C in the juice. Also, adding preservatives may prevent the decay of the juice, but it may not prevent the breakdown of the vitamin C. The modern diet may lack vitamin C because most produce is preserved. Ways in which to improve: Do more repetitions on different makes of juice in order to get a broader set of results. Use freshly squeezed juices so not to contain preservatives. Conclusion This experiment has shown the vitamin C content of a small variety of juices, but different brands contain different amounts of vitamin C so it has not proved a great deal. A more extensive investigation has to be carried out in order to get a better idea of the general trends in vitamin C content in fruit juices. I found from the data that I collected that my vitamin C reading were a great deal higher than the data shown in the table (Food Science, Nutrition and Health). This is probably from variations in procedure and the juices used. Their method in measuring the vitamin C content of juices could be very much different and so differences between the two experiments can occur. Blackcurrant juice was going to be tested because it is a well-known, non-citrus fruit drink, with a high vitamin C content. The reason why this test was not performed on this juice was because of its colour. It would have been extremely difficult to accurately measure when all the DCPIP had been reduced to its colourless form because of blackcurrants deep colour. The amount of vitamin C in different diets can vary tremendously. In MEDCs (more economically developed countries), the vitamin C content may slightly under the correct amount because of preservation techniques (explained earlier), but the ability to import fruits with high vitamin C content, and food supplements (vitamin tablets) make up for that lack. But, in LEDCs (less economically developed countries), where importation of fruit and food supplements are scarce,

there is a high deficiency of vitamin C. Generally, people do not understand the necessity of vitamin C (4 factors explained earlier) and the consequences of its deficiency (scurvy). The average person with a well balanced diet will be in the necessary requirement of vitamin C V between 75-100mg per day. However, a person with an unbalanced diet, may be under this level, e.g. 30mg of vitamin C per day. This will reduce the levels of vitamin C in the body eventually resulting in scurvy. Luckily, for a great deal of people, a lot of food contains vitamin C, but its deficiency could be reduced if people learned about its importance in the correct functioning of the human body. DCPIP (dichlorophenol-indophenol) is a blue dye which is decolourized by ascorbic acid on account of its reducing properties. DCPIP is a redox dye usually used as a monitor of the light reactions in photosynthesis as it is an electron acceptor that is blue when oxidized and colourless when reduced. The dye changes colour when it is reduced, due to its chemical structure.