Effects of Temperature, pH and Cu2+ on Abscorbic Acid Stability
NAME: Sophia Chai Lai Jin
 
Objectives:

To perform a selected redox titrimetric procedure.
To measure the stability of ascorbic acid under various conditions.

Introduction
L-ascorbic acid as known as Vitamin C is an essential nutrient for humans. L-ascorbic acid is one of the most potent compounds acting as an antioxidant in biological systems by scavenging active oxygen species and free radicals. L-ascorbic acid is a well-known water-soluble antioxidant that has a whitening effect and serves as a cofactor of prolinehydroxylase to promote synthesis of collagen. Low intakes cause a nutrient deficiency disease known as scurvy. Scurvy causes general weakness, anemia, gum disease, and skin hemorrhages. The effectiveness of ascorbic acid as a food additive depends on its oxidation to dehydroascorbic acid which effectively destroys ascorbic acid. Since it is so readily oxidized, it is frequently called the most unstable vitamin.
Ascorbic acid may degrade via a number of different mechanisms. Anaerobic and aerobic pathways have been identified; but when oxygen is present, oxidative degradation predominates. Factors that may influence the rate of ascorbic acid degradation include temperature, salt and sugar concentration, pH, oxygen concentration, metal catalysts, and enzymes.
Apparatus
Test tubes, beakers (20 and 250 mL), burets, pipets (1,5 and 10 mL), Erlenmeyer flasks (50 mL), graduated cylinders (10 and 50 mL), hot plate, boiling beads, pH meter, water bath.
Materials
Ascorbic acid solution (0.5 and 10 mg/mL), oxalic acid solution (0.25 M), 2,6-dichloroindophenol (dye) solution, cupric sulphate, (CuSO4.5H2O) solution (10 g/100 mL), glycine buffer (0.1 M, pH 2), carbonate buffer (0.1 M, pH 8), HCl (1 M).
Experimental Procedure

Abscorbic Acid Standard Curve

9 mL of oxalic acid solution and 1 mL of 1.0 M HCl was transferred to each of four Erlenmeyer flask.
0.5, 1.0, 1.5 and 2.0 mL of ascorbic acid solution (0.5 mg/mL) was added to each respectively.
Each flask rapidly with dye solution was titrate until a light but distinct rose pink colour persists for at least 5 s.
A volume o fdye versus miligrams of ascorbic acid was plotted.

Effects of Temperature, pH and Cu2+ on Ascorbic Acid Stability

10 mL (duplicate) was prepared of each of the following solution

Ascorbic acid in glycine buffer
Ascorbic acid in carbonate buffer
Ascorbic acid in glycine buffer + CuSO4
Ascorbic acid in carbonate buffer + CuSO4

1 mL of ascorbic acid (10 mg/mL), 0.5 mL of CuSO4 and sufficient buffer was added to bring the total volume to 10 mL and well mixed.

The pH was recorded for each solution.
They was cover and capped loosely and transfer to a boiling water bath. It was boiled for 15 minutes and cooled.
Titration was done.

Results:
Table 1 : Volume of dye vs miligrams of ascorbic acid

Ascorbic acid (0.5 mg/mL)

Ascorbic acid in mg

Volume of dichloroindophenol used (mL)

0.5

0.25

30.4

1.0

0.5

57.1

1.5

0.75

82.7

2.0

1.0

114.1

Table 2: pH value of each solution

Solution

pH value

1st set

2nd set

Ascorbic acid + glycine

2.06

2.09

Ascorbic acid + carbonate

7.24

7.27

Ascorbic acid + glycine + CuSO4

1.98

2.00

Ascorbic acid + carbonate + CuSO4

6.17

6.22

Table 3: Volume of dye vs different solutions

Solution

Volume of dichloroindophenol used (mL)

Ascorbic acid + glycine

5.8

Ascorbic acid + carbonate

3.3

Ascorbic acid + glycine + CuSO4

2.6

Ascorbic acid + carbonate + CuSO4

1.6

Calculation
Average amount of ascorbic acid to titrate with dichloroindophenol in 1 mL
Y = 113.21x
1 = 113.21x
X = 0.0088 mg
1 mL of dichloroindophenol is required to titrate with 0.0088 mg of ascorbic acid
Total volume of ascorbic acid solution = 10 mL

Ascorbic acid + glycine solution required 5.8 mL of dichloroindophenol:

0.0088 mg X 5.8 = 0.05104 mg
Concentration of ascorbic acid = 0.005104 mg/mL

Ascorbic acid + carbonate required 3.3 mL of dichloroindophenol:

0.0088 mg X 3.3 = 0.02904 mg
Concentration of ascorbic acid= 0.0029 mg/mL

Ascorbic acid + glycine + CuSO4 required 2.6 mL of dichloroindophenol:

0.0088 mg X 2.6= 0.02288 mg
Concentration of ascorbic acid = 0.002288 mg/mL

Ascorbic acid + carbonate + CuSO4 required 1.6 mL of dichloroindophenol:

0.0088 mg X 1.6= 0.01408 mg/mL
Concentration of ascorbic acid = 0.001408 mg/mL
Discussion
In this experiment, redox iodometric titration assay was used to determine the amount of concentration of ascorbic acid in each buffer solution. CuSO4 act as the oxidizing reagent in the reaction. Firstly, the dichloroindophenol act as the blue dye will oxidized the ascorbic acid when titration begin. The ascorbic acid will be oxidized into another form which is called dehydroascorbic acid. The following figure shows the equation of redox reaction of ascorbic acid and converted to dehydroascorbic acid in the titration.

In acid condition, the blue dye will turn the solution into pinkish colour, if the ascorbic acid is present, it will reduce the solution back into colourless compound. The higher the concentration of ascorbic acid, the more dichloroindophenol is needed to oxidize it. Until the titration end point is reached, a persist pink colour solution will be obtained. This indicates that the availability of ascorbic acid has used up for reduction and being oxidized quantitavely by dichloroindophenol. Therefore, from the result in part A standard curve calibration, a straight linear line was achieved. The 2.0 mL of ascorbic acid solution (0.5 mg/mL) required a huge amount of dichloroindophenol to neutralize it.

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From the result obtained, the ascorbic acid-glycine solution tend to have higher concentration of ascorbic acid. Meanwhile in ascorbic acid-carbonate solution has a lower concentration of ascorbic acid. This might due to in alkaline condition it will be more easily to oxidized into dehydroascorbic acid. In alkaline condition, the O2 will act as the oxidant to breakdown the ascorbic acid and destabilize it. Therefore, a slightly acidic pH, the antioxidant properties of ascorbic acid can be maintained.
Moreover, in the presence of CuSO4 will degrade the ascorbic acid easily as CuSO4 is a strong oxidizing agent. Cu2+ will catalaze the oxidation and heating also will increase the rate of oxidation. However, at a slight acidic pH such as in citrus juice, the vitamin is more stable.
Besides, thermal treatment to the ascorbic acid can degrade and destruct the antioxidant properties of the structure. In boiling water bath, most of the ascorbic acid are believed to be degraded. Therefore, the amount of the concentration of ascorbic acid are found to be lower than the beginning of the experiment.
Conclusion
The higher the concentration of ascorbic acid in a solution, the more amount of dichloroindophenol are required to neutralized it. In a very acidic condition, it will degrade the ascorbic acid faster. However in a neutral or alkaline condition in the presence of air, the ascorbic acid will oxidized fast due to the O2 which is an oxidant. High temperature can degrade the antioxidant properties of the vitamin C.
References
http://www.nlm.nih.gov/medlineplus/ency/article/000355.htm
http://chemistry.about.com/od/demonstrationsexperiments/ss/vitctitration_4.htm
http://www.sussexvt.k12.de.us/science/Textbook/LIM_LowRes_Unsecured/NCInv13D.pdf
Questions

Explain the difference in the results between the ascorbic acid-glycine and the ascorbic acid-carbonate buffers, and the effect of pH on ascorbic acid.

The ascorbic acid-glycine solution tend to have higher concentration of ascorbic acid. Meanwhile in ascorbic acid-carbonate solution has a lower concentration of ascorbic acid. This might due to in alkaline condition it will be more easily to oxidized into dehydroascorbic acid. In alkaline condition, the O2 will act as the oxidant to breakdown the ascorbic acid and destabilize it. Therefore, a slightly acidic pH, the antioxidant properties of ascorbic acid can be maintained.

Explain the difference in the results between ascorbic acid-glycine buffer and the ascorbic acid-glycine buffer with CuSO4; between ascorbic acid-carbonate buffer and the ascorbic acid-carbonate buffer with CuSO4 model systems.

The presence of metal ions such as copper and iron increase the rate of oxidation of ascorbic acid. CuSO4 is strong oxidizing agent. Ascorbic acid form complex with metal ions and reduce metal ion into metal. Therefore, the addition of CuSO4 has lowered the amount of dichloroindophenol which required to titrate with the ascorbic acid solution.