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FAR 142/3
BASIC PHARMACOLOGY AND BIOCHEMISTRY
EXPERIMENT 3:
DETERMINATION OF ASCORBIC ACID IN VITAMIN C TABLETS USING
2,6-DICHOLOROPHENOLINDOPHENOL
NAMES
: BEATRICE GILBERT
(123864)
(123877)
(123889)
(123901 )
LECTURERS NAME
OBJECTIVES
1. To determine the ascorbic acid content in vitamin C tablets using titration with the
redox indicator DCPIP.
2. To determine the effect of ascorbate oxidase on the sample of vitamin C tablets.
3. To investigate the effect of standing at room temperature, boiling and hydrogen
peroxide on the stability of ascorbic acid.
OVERVIEW
Pure ascorbic acid is a white, crystalline solid with a molecular formula
C6H8O6. It is water-soluble. Its main chemical property is that it oxidizes extremely
easily in solution, thus reducing anything that can accept electrons. The oxidation
process is impeded in acidic environment and at low temperatures. Ascorbic acid is a
form of vitamin C which is easily reduced or destroyed by exposure to light, heat,
oxygen, in basic solutions and in presence of metal ions such as Cu2+ or Fe3+.
The ascorbic acid content can be determined by 2,6-dicholorophenolindophenol
(DCPIP) titrimetric method for food production.DCPIP is used as the titrant because
the titrant should only oxidized ascorbic acid and not other substances that may be
present in the samples. It is also because DCPIP act as self-indicator where it only
must be in one color in the presence of excess ascorbic acid and another color when
all ascorbic acid has reacted. Ascorbic acid is determined by oxidizing it in acidic
medium with DCPIP as indicator to dehydroascorbic acid.
Ascorbic acid reduces the DCPIP,indicator dye from oxidized form(red in acid) to a
reduced form, colorless solution. At the end point of titration excess unreduced dye is
a rose-pink color in the acid solution. The titer of the dye can be determined by using
a standard ascorbic acid solution. Then the ascorbic acid in the food sample can be
determined by the calculation using the volume of the titration.
Before DCPIP is used to determine the ascorbic acid, we must first know the
concentration of ascorbic acid solution. In order to know that, we can use the reaction
of DCPIP solution with a known concentration with a solution of ascorbic acid which
is called as standardization. Then, the stability of ascorbic acid is studied by treating
with air, boiling for 15 minutes and hydrogen peroxide. From this study, we can know
the amount of ascorbic acid present before and after the treatment. Thus, it will shows
the factors that influence the ascorbic acid reduction and how it affects the ascorbic
acid.
In part C where vitamin C is analysed, ascorbate oxidase solution is added to the
second aliquot to be titrated with DCPIP to minimize the interference of other
compounds that may be present in the samples which allows the determination of
other reducing substances other than ascorbic acid. The first aliquot is titrated directly
and total content of all reducing substances present is determined.
RESULTS
Experiment
a. Preparation and standardization of ascorbic
acid standard solution
b. Study on the stability of ascorbic acid
standard solution to the following treatments:
i. Air
ii. Boiling for about 15 minutes
iii. Hydrogen peroxide
c. Analysis of vitamin C or multivitamin tablets
i)
Incubation of an hour with ascorbate
ii)
14.20
13.90
14.43
13.10
13.40
13.25
13.10
12.40
12.75
15.20
14.90
15.05
3.50
3.70
2.90
3.37
24.90
24.80
25.20
24.97
CALCULATIONS
A) Preparation and standardization of ascorbic acid standard solution
Weight of ascorbic acid = 0.02 g
0.02 g
176 g /mol
= 1.14x 104
mol
n
1.14 x 104
x 1000 =
x 1000
V
100
= 1.14x 104 mol dm3
Air
Volume of DCPIP used
( 13.10+13.40 ) mL
=
2
= 13.25 mL
Ascorbic acid content:
From part (a),
14.43 mL DCPIP is reduced by 2.00 mg ascorbic acid.
13.25 mL DCPIP is reduced by (13.25 mL 14.43 mL) 2.00 mg
= 1.84 mg ascorbic acid
Reduction of ascorbic acid
= 2.00 mg 1.84 mg
= 0.16 mg
Percentage reduction of ascorbic acid
= (0.16 mg 2.00 mg)100%
= 8.00%
ii)
Hydrogen Peroxide
Volume of DCPIP used
( 15.20+14.90 ) mL
=
2
= 15.05 mL
806.40 mg
ii)
805.50 mg
iii)
804.40 mg
21.4 0
14.43
x 2 = 2.97 mg
Second trial =
21.1 0
14.43
x 2 = 2.92 mg
Third trial
22.3 0
14.43
x 2 = 3.09 mg
First trial
= 29.70 mg
2nd trial
= 29.20 mg
3rd trial
= 30.90 mg
29 .7 0
100
x 805.43 = 239.21 mg
Second trial =
29.20
100
x 805.43 = 235.19 mg
Third trial
30.90
100
x 805.43 = 248.88 mg
First trial
239.21+235.19+248.88
3
=241.09mg
The standard deviation of ascorbic acid content in the tablet
=
(239.21241.09)2 +( 235.19241.09)2+(248.88241.09)2
3
99.03
3
= 5.75
DISCUSSIONS
A) Preparation and standardization of ascorbic acid standard solution
In part A, a standard ascorbic acid solution is prepared and standardized before
determining the ascorbic acid in order to know its concentration. The concentration of
ascorbic acid can be determined by titrating the 2,6-dichlorophenolindophenol or
DCPIP (0.025%). Titration of the sample must give the same pink color as the pink
color of titration product of ascorbic acid standard solution. Oxalic acid is used in this
experiment to dissolve ascorbic acid and also as a stabilizer which helps to acidify
ascorbic acid. This is because ascorbic acid is not stable in alkaline solution. Oxalic
acid is also used to prevent further oxidation of dehydroascorbic acid which is the
product of oxidation of ascorbic acid. From part A, we can see that the concentration
of ascorbic acid is 1.14x 104 mol dm3 and the volume of DCPIP reduced by
0.02 g ascorbic acid is 14.43mL.
pink in colour if there is no ascorbic acid content in it. As the titration continues, we
can find that the pink colour of the titrated solution will remain which mean that there
is no more content of ascorbic acid in the solution.
PRECAUTION:
1
The conical flask should be swirled continuously and gently during the titration
process.
The intensity of light pink colour, indicating the end point of titration should be
consistent in every experiment done to ensure accuracy of results obtained. Thus,
it is best if only one person doing all the titrations.
REFERENCES
1. http://chemlab.truman.edu/CHEM130Labs/VitaminC.asp
2. Voet, D., Voet J.D. and Pratt, C.W.: Fundamentals of Biochemistry(2 nd ed),
John Wiley, 2006.
3. Practical Manual of FAR 142/3 Biochemistry and basic principles of
molecular biology (pg 14-18)
4. David L.Nelson, Michael M.Cox. Lehninger-Principles of Biochemistry,4 th
edition.
5. http://www.rxlist.com/ascorbic_acid-drug.htm