Spectrophotometric Determination of Ascorbic Acid Concentration in an Unknown Solution

Topics: Ascorbic acid, Vitamin C, Vitamin Pages: 7 (1295 words) Published: April 16, 2012
Spectrophotometric determination of Ascorbic acid Concentration in an Unknown Solution


The naturally occurring organic compound with antioxidant properties is Ascorbic acid shortly referred as (AA). Normally the color of Ascorbic acid is white, but in some cases of impure samples it appears yellow in color. Ascorbic acid is solid in state. In water it dissolves well to give mildly acidic solutions. Ascorbic acid is one form ("vitamer") of vitamin C [1]. So, Ascorbic Acid (AA) in other terminology also called as Vitamin C. This ascorbic acid majorly helps in the maintenance of DNA, Lipids, Proteins and others in their original active, normal form.

The name of the anion Fe(CN)64− is mentioned as Ferrocyanide. This coordination complex is unreactive in aqueous solutions. Ferrocyanide is usually available as the salt potassium ferrocyanide, the formula is K4Fe(CN)6.[2]. [Fe(CN)6]4− is a diamagnetic species, compared to many salts of cyanide, these Ferro- and ferricyanides are less in toxicity, as they tend not to release free cyanide. Its reaction is its oxidation to ferricyanide:

[Fe (CN)6]4−   [Fe(CN)6]3− + e− Spectroscopically the conversion can be followed 

Now the main Aim of the procedure includes:

1. Determination of absorbance maximum ((max) of potassium ferricyanide in the range 350 nm to 550 nm, potassium ferricyanide has the chemical compound with the formula K3[Fe(CN)6] . The salt contains the octahedrally coordinated [Fe(CN)6]3− ion which is bright red in color and it is soluble in water and its solution shows some green-yellow fluorescence[3]

2. Production of calibration curve for ferricyanide at (max ,

3. Determination of stoichiometric number “n” in Eqn.(1) and

4. Determination of the concentration of Ascorbic acid (AA) in an unknown solution.

5. Study of a sample’s response to light is UV spectroscopy.

The Beer-Lambert law is the important principle involved in the absorption analysis.

Beer-Lambert’s law states that “for a given ideal solution, there is a linear relationship between concentration and absorbance provided that the path length is kept constant, the absorptivity (() is a constant for each molecule for each wavelength” [4]

”The ratio of the intensity of the light entering the sample (I0) to that exiting the sample (It) at a particular wavelength is defined as the transmittance (T)”.

T = (I0/It)

Absorbance may also be defined as “negative logarithm of transmittance”.

A = -log(T)

A = (cl ------- (1)


A = Absorbance

( = Absorptivity of the substance

c = Concentration and

l = Path length.


1. To the provided pre-weighed potassium ferricyanide Deionised water (50 ml) is added and is dissolved completely.

2. Deionised water (2 ml) is taken in a cuvette (B) and the baseline correction should be done at the wavelength range between 350 nm to 550 nm.

3. Take a Ferricyanide(400 µl) stock and made into 2 ml by water in a cuvette and is scanned at 600 nm/min speed, 100 nm/cm scale and with absorbance ranging from abs 0.00 to abs 1.50. The resulting wavelength ((max) is recorded.

4. By using the (max recorded, the baseline will be corrected .

5. For preparing the ferricyanide calibration curve, ferricyanide test solutions were prepared and read at specified absorbance as in part 1 in the Table: 1.

6. 50 ml of deionised water is added to the pre-weighed Vitamin C provided and dissolved completely to prepare Vitamin C stock solution (1 mM). For determining the stoichiometric number the test solution containing Vitamin C and deionised water is taken in a cuvette and the absorbance reading is recorded. The same step is repeated as in part 2 of the Table: 2. The solution containing the unknown Vitamin C and deionised water is also read at (max and was recorded. The same procedure was done...
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