Determination of ascorbic acid by spectrophotometer

Spectrophotometer
 Spectrophotometer Principle. The spectrophotometer is an instrument which
measures the amount of light that a sample absorbs. The spectrophotometer works
by passing a light beam through a sample to measure the light intensity of a sample.

Device components
1. The lamp

2. Monochromator

3. Sample cuvette

4. The detector

5. The amplifier

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1.The lamp
Emits all colors of light (i.e., white light).

2.Monochromator
The selects one wavelength and that wavelength is sent through the sample.

3.Sample cuvette
We place the sample in it for analysis.

4.The detector
Detects the wavelength of light that has through the sample.

5.The amplifier
Increases the signal so that it is easier to read.

The Blank
Is a solution that contains the entire reagents except the substance to be measured.

It is used to adjust the device to zero.

Lambert Law
The amount of light decreases with increase in thickness of layer of solution through which
the light passes.

Or Transmittance of light is inversely proportional to the thickness of the cuvette at the

same concentration.

Beer's Law
The concentration of substance is directly proportional to the amount of light absorbed and
inversely to log of transmitted light.

The quantitative measurement of colorless materials is based on the principle that they will
be converted to colored substances in certain chemical or biological reactions.

It is based on Comparison the color in a solution containing the test substance with that in a
standard solution of known concentration.

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The two solutions are observed simultaneously in a colorimeter and quantities on the basis
of absorption of the light at a specific wave length.

The amount of light that is absorbed is proportional to the concentration of the substance.

The common law says: Concentration in the unknown =( Absorption intensity of the
unknown/ (Absorption intensity of the standard)) × Standard concentration

Action steps
1. 10 g blended sample was homogenized with about 50 mL of 5% metaphosphoric
acid-10% acetic acid solution.
2. Then it was quantitatively transferred into a 100 mL volumetric flask and was shaken
gently until a homogeneous dispersion was obtained.
3. Then it was diluted up to the mark by the 5% metaphosphoric acid-10% acetic acid
solution. Then the solution was filtered and the clear filtrate was collected for the
determination of vitamin C in that sample.
4. To the filtered sample solution few drops of bromine water were added until the
solution became colored (to confirm the completion of the oxidation of ascorbic acid
to dehydroascorbic acid).
5. Then few drops of thiourea (10%) was added to it to remove the excess
6. Then 2,4- dinitrophenyl hydrazine solution was added thoroughly with all standards
and also with the oxidized ascorbic acid.
7. Total vitamin C employing coupling reaction of 2,4-dinitrophenyl hydrazine dye with
vitamin C and followed by spectrophotometric determination.

8. Standard vitamin C (ascorbic acid) solution:

0.05 g standard crystalline ascorbic acid was dissolved in 100 mL of distilled water to
prepare 500 ppm standard stock solution.

9.Measurement is done at a wavelength 521nm

Reactions
Ascorbic acid is oxidized to dehydroascorbic acid by the action of bromine solution.

L-dehydroascorbic acid reacts with 2,4-dinitrophenylhydrazine and produces an osazone

which on treatment with 85% H2SO4 forms red colored solution.

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