This experiment was planned to study how different temperatures can affect the loss of vitamin C in orange juice stored for a fixed period of time. Equal volume of freshly squeezed orange juices with known (initial) vitamin C concentration were stored at different temperatures i.e. 10oC, 20 oC, 30 oC, 40 oC, 50 oC and 60 oC for a constant duration of 5 days. After the storage period, the vitamin C concentration for each temperature was measured by DCPIP titration and the difference relative to the initial concentration was calculated to calculate the amount of vitamin C reduced. The result of this experiment showed that the amount of vitamin C lost increases with the increase in temperature and 10oC was the best temperature that gave the least reduction in vitamin C concentration. An analysis using Pearson product-moment correlation coefficient has revealed a strong positive linear relationship between the two variables with the calculated r value exceeds the critical value at 5 % significant level thus, supporting the experimental hypothesis.
Keywords: vitamin C, ascorbic acid, DCPIP (dichlorophenolindophenol)
Research and Rationale:
Vitamin C is a remarkable compound derived from one of the ascorbic enantiomers, L-ascorbate. It is a water soluble vitamin that cannot be naturally synthesized in human body.1 Therefore, it is vital to have our diet balanced with adequate amount of it every day. There are two important roles played by vitamin C; antioxidant and collagen formation.2 These two features have placed vitamin C in a class of its own as a future potential in fostering better health.
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A previous research regarding relationship between vitamin C and human brain has shown that vitamin C could help drugs to pass the blood brain barrier.6 This would enable brain diseases such as Parkinson to be effectively treated as artificial dopamine can be delivered directly to the brain. At the same time, the biggest challenge in bone marrow treatment i.e. getting enough cells, could possibly be solved as a recent study by Dunagqing Pei13 on vitamin C, has found that it can boost the production and pluripotency of stem cells in human body.
However, Vitamin C is also known to be very vulnerable towards heat. The precursor of vitamin C, ascorbic acid, has molecular of C6H8O6. The proximity of highly electronegative oxygen atoms on the hydroxyl (OH) groups makes the hydrogen atoms to become easily detached from the structure. Meanwhile, the presence of heat causes the hydroxyl bond to break the ascorbic acid is said to undergo “destruction” or oxidation by losing hydrogen atoms, forming dehydroascorbic acid. Therefore, it is suggested that the rate of ascorbic acid destruction is significantly greater at higher temperature.*
Diagram 1. The structure of Ascorbic Acid.7
Diagram 2. Oxidation of Ascorbic Acid.8
Most of the studies on vitamin C degradation are principally based on the effect of storage condition and period, and very few were done on identifying the degradation trend within a specific range of temperature.
Previous research3, on the effect of storage methods and conditions on vitamin C retention in human milk revealed that freezing reduces the least amount of vitamin C as compared to other storage methods, followed by refrigeration which is better than cold water. Another study at Ankara University4, has shown an inverse relationship between temperature and retention rate of vitamin C in citrus fruit concentrates, while orange fruit was found to have higher retention ability than the others.
Therefore, this experiment was aimed to find a specific trend regarding the effect of temperature on the loss of vitamin C. Citrus fruits (orange) were used in this experiment due to appreciable amount of vitamin C that they have, thus, increasing the reliability of the results. The results from this study can be used to illustrate how certain temperatures can cause drastic change in vitamin C hence, increasing the awareness on appreciating the effort of preserving vitamin C content in food for optimum health benefit. For instance, in agricultural tropical regions that grow citrus fruits, it becomes very vital to preserve the fruits at low temperature if possible due to higher chance of vitamin C destruction than other regions.
The higher the temperature, the higher the degradation of vitamin C in freshly squeezed orange juice.
There is no significant relationship between the different temperature and the degradation of vitamin C in freshly squeezed orange juice.
1) Choosing the best citrus fruits:
4.2 Five types of citrus fruits were randomly chosen, namel: lemon, lime, grapefruit, Clementine and orange. The fresh juice of each fruit was obtained through cutting and squeezing. The vitamin C content for each juice was determined by titration against 1 ml of 1 % DCPIP. The titration was repeated two times to get an average volume of the juice needed to decolourise DCPIP. The result:
The least volume of orange was needed to decolourise DCPIP, indicating that it has the highest vitamin C content. Therefore, orange fruit was chosen as it would give significant response towards different temperatures.
3) Determining the best storage period:
Several orange fruits were squeezed to obtain fresh orange juice that would be enough for its vitamin C content to be measured on daily basis. Firstly, the initial vitamin C content of the juice was measured and 4.8 ml was needed to decolourise 1 ml of DCPIP solution. Equal volume of the remaining juice was divided into two beakers and each was stored in an incubator of different temperatures (10 oC and 60 oC). Two distinctive temperatures were chosen to allow easy comparison of the trend in each temperature. The result:
The result shows no more change in volume of juice required after 5 days for 60 oC. Therefore, storage period of five days were chosen for the main experiment.
Manipulated variable: Different temperatures (oC)
(6 incubators were set at different temperatures of
10 oC, 20 oC 0, 30 oC, 40 oC, 50 oC and 60 oC)
Responding variable: Amount of vitamin C lost
(By DCPIP titration, the difference between initial and final vitamin C concentration in each juice was
calculated to determine the concentration of vitamin C reduced)
Fixed variables : Storage period, volume and concentration of DCPIP, type of fruits
(The storage period was five days. 1ml of 0.1% DCPIP
Used for each titration)
Beakers, knife, test tubes, syringes, Parafilm, aluminium paper, incubators, mortar and pestle, measuring cylinder.
Orange fruits, 1% dichlorophenolinophenol (DCPIP) solution, distilled water, 500 mg vitamin C tablet.
Real Experimental Procedures:
Standardizing Vitamin C Concentration:
1) A tablet of 500 mg vitamin C tablet was crushed into fine powders using a mortar and a pestle.
2) The powdery form of vitamin C was then dissolved into 100 ml of distilled water in a beaker to form 5 mg/ml of ascorbic solution.
3) 1 ml of 1% DCPIP solution was measured and placed into a test tube by using a syringe.
4) 1 ml of 5 mg/ml of ascorbic acid solution was then taken using a syringe and added drop by drop into the measured DCPIP solution until decolourised.
5) The volume of ascorbic acid solution needed to decolourise the DCPIP solution was recorded.
6) The titration process was repeater three times to get an average volume.
The result of titration is as follows:
Volume of Juice Titrated / ml
Hence, 2.5 ml of 5 mg/ml of ascorbic acid solution was needed to decolourise 1 ml of 1% DCPIP solution
To find a formula to calculate vitamin C concentration in orange juice,
Conc. of orange juice (mg/ml) Vol. of orange juice (mg/ml)
5 mg/ml 2.5 ml
Since the volume needed to decolourise is proportional to vitamin C concentration. So.
Conc. of orange juice (ml) 2.5 ml
5 mg/ml Vol. of orange juice (mg/ml)
Concentration of orange juice (mg/ml) = X 5 mg/ml
Vol. of orange juice (ml)
Therefore, this calculation would be used to calculate the vitamin C concentration.
Determining vitamin C loss:
10 orange fruits were cut and squeezed to obtain fresh juice.
1 ml of 1% DCPIP solution was measured and placed into a test tube by using a syringe.
1 ml syringe was filled with the orange juice and added drop by drop into the DCPIP solution until it decolourised. The volume of juice added was recorded.
The titration was repeated five times to get an average volume and its vitamin C concentration was calculated using the derived formula.
The juice was then divided into 6 equal volumes and each placed into 100 ml beaker.
The top of each beaker was sealed with Parafilm and its surface was wrapped with aluminium paper and labelled with different temperatures.
The beakers were placed in six incubators of different temperatures according to the label and left for five days.
After five days, the beakers were sealed off.
1 ml of 1% DCPIP solution was measured and transferred into a test tube by using a syringe.
The orange juice stored in 10 oC was taken by a syringe and added drop by drop into the DCPIP until it decolourised. The volume of juice added was recorded
The titration was titrated three times to get an average volume and its vitamin C concentration was calculated using the formula:
Concentration of orange juice (mg/ml) = X 5 mg/ml
Vol. of orange juice (ml)
Steps 8-10 were repeated but this time using the orange juices stored in oC, 30 oC, 40 oC, 50 oC and 60 oC.
The difference between the initial concentration and the final concentration of each juice was calculated to determine the amount of vitamin C lost.
The process of cutting the orange fruits required the use of knife, so it was done carefully to avoid any injury. The fruits were then squeezed very gently to minimise heat production. The juice was only prepared right before the experiment was about to be carried out. DCPIP is a strong dye which is hard to be removed so lab coat was worn. During the titration of juice against DCPIP, the test tube was not shaken vigorously to avoid oxygen from dissolving. The juice was discarded immediately after the experiment.
The initial vitamin C concentration:
Volume Needed to Decolourise DCPIP Solution (ml)
Therefore, 4.6 ml of the fresh orange juice needed to decolourise DCPIP. So,
Initial vitamin C concentration = X 5 mg/ml
= 2.7174 â‰ˆ 2.72 mg/ml
Hence, the vitamin C reduced:
= 2.72 – X
* x is Vitamin C concentration left.
Vitamin C concentration lost in different temperatures:
Based on the result from the table, it is known that there is an obvious trend and correlation between the temperature and vitamin C loss. Therefore, Pearson product-moment correlation coefficient (PMCC) was chosen to measure the strength of this relationship.
In this method, the value of correlation coefficient, r needs to be calculated which ranges from -1 to 1. The details of its values are as follow:
0 < r â‰¤ 1 = positive linear relationship
-1 â‰¤ r < 0 = negative linear relationship
âˆš (1750 X 3.68388)
* Critical values for PMCC in appendix 1
Therefore, the value of correlation coefficient, r, using Pearson product-moment correlation coefficient has shown a strong positive linear relationship between temperatures and loss of vitamin C. Hence, null hypothesis is rejected.
Table 4 shows the volume of the freshly-squeezed orange juice needed to decolourise 1ml of 1% DCPIP solution. The average volume was used to calculate the concentration of vitamin C present initially. Meanwhile, Table 5 shows the amount of vitamin C concentration reduced after being stored at different temperatures for five days. From the table, there is an inverse relationship between the vitamin C concentration left and the amount of vitamin C lost. There is also a huge difference in the amount of vitamin C left between the juice stored in 10 oC andt the one stored at 60 oC, which is 2.11 mg/ml. This represents 77.6% of the original concentration of vitamin C. The calculated statistical correlation coefficient, r of 0.9584 is absolutely a strong indicator to support this relationship.
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Graph 1 illustrates the trends and correlation between the two variables. From the graph, it can be concluded that generally, the higher the temperature, the higher the amount of vitamin C lost. 10 oC is the best temperature that gave the least reduction in vitamin C level with only 0.12 mg/ml (4.4%) decrease after five days. Meanwhile, 60 oC caused maximum drop in concentration after five days with 2.23 mg/ml (82%) of vitamin C had lost. The largest gap in vitamin C loss occurred between 40 oC and 50 oC with 1 mg/ml (37%) of increase recorded.
Based on the graph, the amount of degradation at 20 oC and 30 oC opposed the general trends when 0.6 mg/ml vitamin C had lost at 10 oC which is greater than 0.56 mg/ml at 30 oC. However, the difference is so small which suggests that this anomaly might be due to several reasons:
Higher rate of oxidation of ascorbic acid by oxygen in the atmosphere.
False end-point titration
Apart from that, the results obtained have also shown that there is only a little change in vitamin C concentration from 10 oC to 30 oC. However, drastic change in concentration started to occur after 40 oC. This suggests that orange fruits should be kept below 30 oC with better vitamin C retention at lower temperature.
From the results of the experiment, the huge difference in vitamin C lost between 40 oC and 50oC could be explained by the presence of enzyme ascorbate oxidase in citrus fruits. The function of this enzyme is still not fully understood, but one best suggestion is that it might involve in controlling the oxidation process of ascorbic acid.12 Just like any other enzymes, when the optimum temperature is exceeded, the bonds holding the ascorbate oxidase together start to break and it is said to be denatured. Therefore, the “destruction” of ascorbic acid takes place without any control.
Measurement of vitamin C in this experiment was done by titrating the juice against dichlorophenolindophenol (DCPIP). It is a strong oxidizing agent with distinctive blue colour and decolourised when being reduced by vitamin C.15 Therefore, the destruction of vitamin C by heat means that more is needed to decolourise DCPIP solution.
Oxidation of DCPIP by oxygen in the atmosphere is one of the limitations of this experiment. Hence, the test tubes were not shaken vigorously during titration in order to minimise this limitation as vigorous moves can increase the rate of oxygen dissolving in a solution.
The other limitation could be the side decomposition of vitamin C due to the presence of light and air. Just like temperature, ultraviolet ray from incidence light causes the hydroxyl bond in ascorbic acid to break, thus become oxidised. To reduce this limitation, the beakers containing orange juice to be stored at different temperatures were neatly wrapped with aluminium paper which is a good reflector of light and heat. The surfaced of the beakers were also sealed with Parafilm. This would prevent the entry of air and significantly reduce unnecessary oxidation of vitamin C in the juice.
Several modifications can be made in the future to improve the accuracy and reliability of the results of this experiment. Iodine titration could be used as an alternative for DCPIP titration. Although this technique may require the use of more reagents, the result from the trial experiment has shown that the end point of titration is much easier to be identified. Besides, orange juice could be obtained by blending the peeled orange fruits instead of squeezing them. This ensures maximum amount of juice obtained from each fruit as well as preventing destruction of vitamin C by heat. Finally, percentage of vitamin C degraded can be used as the response variable instead of concentration. This would allow clearer illustration of the amount of vitamin C reduced for the readers and also make the comparison process easier.
Based on the result of this experiment, it can be concluded that the higher the temperature, the greater the degradation of vitamin C in freshly squeezed orange juice. The statistically calculated r value using Pearson product-moment correlation coefficient, 0.9584, is significantly higher than the critical value at 5% significance level thus, providing a strong evidence to support the hypothesis.
The table of significance values for PMCC
Several recourses were used in providing me important information in completing this assignment. Sources 1 and 2 are books that are specially made about fruit management and also detailed information about vitamin C as well as compelling research on it. Both books were published after year 2005, so the information available is mostly up to date.
Besides, I have also accessed journals available online. Sources 3 and 4 come from two well known websites for food and nutrition based journals. The American Journal of Clinical Nutrition is a trustable website with over 3100 members, publishing up to dates research related to nutrition and human.
Sources 7, 8 and 9 are chemistry based websites, serving enormous information on the structures and reaction of biological compounds. Chemwiki is a virtual online based chemistry textbook, accessed by millions of people searching for chemistry knowledge. Sources 10 and 11 are websites exclusively made for vitamin C. The Vitamin C Foundation, for instance, is recognised by Internal Revenue Service, IRS in protecting vast information about vitamin C for public reference.
Source 12, Scientific American magazine is a popular scientific magazine established for nearly two centuries since 1845 with more than 3.5 million readers all over the world. Therefore, there would be no doubt in reliability of the content in this magazine.
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