Methods of Estimation

PRACTICAL 7 METHODS OF ESTIMATION of Ascorbic Acid (Vitamin C)

OF ASCORBIC ACID
(VITAMIN C)
Structure
7.1 Introduction
7.2 Ascorbic Acid – Basic Concepts
7.3 Methods used for Estimation of Vitamin C
7.3.1 Titrimetric Method : 2, 6 Dichlorophenol Indophenol Method
7.3.2 Colorimetric Method: 2, 4 Dinitrophenylhydrazine Method

Experiment 1: Titrimetric Method: 2, 6 Dichlorophenol Indophenol Method for

Estimation of Vitamin C in a Given Solution
Experiment 2: Titrimetric Method: 2, 6 Dichlorophenol Indophenol Method for
Estimation of Vitamin C in Lemon Juice
Experiment 3: Titrimetric Method: 2, 6 Dichlorophenol Indophenol Method for
Estimation of Vitamin C in Chillies
Experiment 4: Colorimetric Method: 2, 4 Dinitrophenylhydrazine Method for
Estimation of Vitamin C in a Given Solution

7.1 INTRODUCTION
Ascorbic acid is also known as vitamin C which is a water soluble vitamin. As you
already know, vitamins are a group of small molecular compounds that are essential
nutrients in many multi-cellular organisms, and humans in particular. The name “vitamin”
is a contraction of “vital amine”, and came about because many of the first vitamins to
be discovered were members of this class of organic compounds. And although many
of the subsequently discovered vitamins were not amines, the name was retained. In
this practical we will learn about and practically carry out the methods of estimating
vitamin C, also known as ascorbic acid.

Objectives
After going through this practical and undertaking the experiments given herewith, you
will be able to:

describe the various methods of estimation of ascorbic acid, and

estimate and compare the ascorbic acid content of different kinds of related
vegetables or fruits.

7.2 ASCORBIC ACID – BASIC CONCEPTS

Ascorbic acid is a water-soluble vitamin, whose structure is shown in Figure 7.1. You
would have noticed that its structure resembles glucose.

O C

HO C
O
C

H C

H
C

CH2OH

Figure 7.1: Structure of vitamin C (ascorbic acid)

1750
Nutritional As the name suggests, ascorbic acid is acidic in nature and a pure solution of this white
Biochemistry crystalline substance has a pH of about 3. Ascorbic acid also have two possible
enantiomers – the L and the D form. The D form of ascorbic acid is generally inactive
as anti-scorbutic agents and naturally occurring ascorbic acid occurs in L form. Ascorbic
acid is stable in solid form and in acidic solutions but is easily destroyed in alkaline
solutions. Oxidative destruction of ascorbic acid is accelerated by increasing the pH.

Some mammals like rat can synthesize the vitamin from glucose by the uronic acid
pathway. However, primates like humans and some other animals like guinea pig, bats,
fish, birds cannot synthesize this vitamin. They lack the enzyme L gulonolactone oxidase
which prevents its synthesis from glucose. Since these species from the animal kingdom
cannot produce ascorbic acid, they have to obtain it entirely through the diet. Hence, its
importance.
The vitamin exists in the body largely in the reduced form with reversible equilibrium
with a relatively small amount of dehydro ascorbic acid. Both forms are physiologically
and metabolically active. We are already familiar with the functions of vitamin C. A
brief review of its functions is presented next.

Functions
Vitamin C is easily oxidized, and the majority of its functions in vivo rely on this property.
It plays a key role in the body’s synthesis of collagen and nor epinephrine by keeping the
enzymes responsible for these processes in their active reduced form.

Vitamin C may also play a role in detoxifying byproducts of respiration. Occasionally

during respiration, O2 is incompletely reduced to superoxide ion (O2-) instead of being
reduced completely to its -2 oxidation state (as in H2O). Normally an enzyme called
superoxide dismutase converts O2- to H2O2 and O2, but in the presence of Fe2+, the
hydrogen peroxide may be converted into the highly-reactive hydroxyl radical (OH).
The hydroxyl radical can initiate unwanted and deleterious chemistry within a cell when
it removes a hydrogen atom (H) from an organic compound to form H2O and a new,
potentially more reactive free radical. Ascorbic acid can donate a hydrogen atom to a
free radical, and thus stop these reactions from occurring. The role of ascorbic acid as
an antioxidant, therefore, is of prime importance.

What would be the consequences of lack of vitamin C in the diet? Certainly we know of
the consequences. Let us recall together.

Deficiency diseases due to vitamin C

Vitamin C deficiency in human’s results in the disease called scurvy, whose symptoms
include hemorrhaging (especially in the gums), joint pain and exhaustion. In its final
stages a profound exhaustion, diarrhoea, and then pulmonary and kidney failure
characterize scurvy, which result in death.

So then what are the rich dietary sources of vitamin C? Let us recall and list all the
vitamin C-rich food sources.

Dietary Sources of Vitamin C

Fruits (particularly citrus fruits), vegetables are the best sources of ascorbic acid. Amla
is the richest source of ascorbic acid. The amount of ascorbic acid in plants varies
greatly, depending on such factors as the variety, weather and maturity.

Organ meats (e.g., liver and kidney) generally are also good sources of vitamin C.
However, muscle meats and most seeds do not contain significant amounts of ascorbic
acid.

176
Remember, the most significant determinant of vitamin C content in foods is how the Methods of Estimation
food is stored and prepared. of Ascorbic Acid (Vitamin C)
Next, how much vitamin C is required by our body to keep it fit and healthy and away
from the symptoms to scurvy? Let’s find out.

Requirements and Recommended Dietary Intake

A very small daily intake of vitamin C (10-15 mg/day for an adult) is required to avoid
deficiency and stave off scurvy. However, there has been, and continues to be, vigorous
debate on what the optimum daily intake of vitamin C. According to the Recommended
Dietary Intakes of Indians as recommended by the Indian Council of Medical Research
(ICMR), a daily intake of 40 mg for adult has been suggested.

With this basic understanding about the structure, properties, nature, food sources and
requirements of vitamin C, we now move on to the study of the methods which are
employed for the estimation of vitamin C in the laboratory.

7.3 METHODS USED FOR ESTIMATION OF VITAMIN C

The most commonly used methods to determine ascorbic acid in foods are:
1. Titrimetric methods - The 2, 6 dichlorophenol indophenol method is the common
titrimetric method used for determination of ascorbic acid.
2. Colorimetric methods - The 2, 4 dinitrophenyl hydrazine method is a common
colorimetric method used for estimation of ascorbic acid in solutions and foods.

Let us get to know about these methods in greater details.

7.3.1 Titrimetric Method : 2, 6 Dichlorophenol Indophenol Method

The titrimetric method is a redox titration method which depends on the reduction of the
blue dye 2,6 dichlorophenolindophenol to a colourless leuco compound by ascorbic acid
in solution or in extracts made out of foodstuff. Ascorbic acid, as you have already
studied, is a strong reducing agent because of which it reduces the dye 2,6
dichlorophenolindophenol and itself gets converted to dehydro ascorbic acid. After
equivalence point is reached the next drop of dye gives a pink colour to the solution
indicating the end point. The dye in this titration is coloured in the oxidized form and
colourless in the reduced form. Also the dye is pink in acidic solution and blue in alkaline
solution.

In acidic solution the pink dye is reduced to a colourless substance in presence of

ascorbic acid. The solution remains colourless as more dye is added until all the ascorbic
acid has reacted. As soon as the next drop of dye solution is added the solution becomes
light pink due to excess dye indicating that the end point of the titration has reached.

The solution of ascorbic acid or its extracts are prepared in metaphosphoric acid or
glacial acetic acid or a mixture of the two. The acid prevents the oxidation of ascorbic
acid due to oxygen in the atmosphere which is faster in presence of metal ions like Fe3+
and Cu2+. Metaphosphoric acid can also form complexes with metal ions which reduces
the catalytic oxidation of ascorbic acid. In foods, metaphosphoric acid releases the
ascorbic acid from proteins and inactivates ascorbic acid oxidase.
The reaction involved in this method is highlighted in Figure 7.2

1770
Nutritional OH
O C O C
Cl Cl
Biochemistry
HO C O C

O O
C + O C
N

H C H C

H
C H C

O CH2OH
CH2OH
L - Ascorbic acid 2,6 dichloro phenol L - dehydro ascorbic ac
indophenol
Figure 7.2: Conversion of ascorbic acid to dehydro ascorbic acid

A detailed step by step procedure for titrimetric method is given in experiment 1, 2 and
3 in this practical. We shall follow this method when we conduct these experiments in
the laboratory. Now let us get to know about the other method of estimating the vitamin
C content i.e. the colorimetric method.
7.3.2 Colorimetric Method: 2, 4 Dinitrophenylhydrazine Method
A detail step by step procedure is enumerated in experiment 4 later in this practical. We
shall use this method in estimating vitamin C in a given sample in the laboratory. Here,
let us look at the principle involved in the calorimetric method.
Reaction
O C O C COOH
O
HO C O C C
O C
O O C
C O C

H C Oxal
H C H C
H
H C
C H C

CH2OH
CH2OH CH2 OH

L - Ascorbic acid L - dehydro Diketogulonic acid

ascorbic acid

H H H C

O2 N N H C
N N H
NO2 NO2 O

O 2N N N C

NO2 H C
NO 2
Dinitrophenylhydrazine OH C H

CH2OH
Osazone
Figure 7.3: Osazone formation from ascorbic acid
Principle
Ascorbic acid is oxidized to dehydro ascorbic acid by copper(Cu++) ions. 2, 4 dinitrophenyl
hydrazine reacts with the ketone group of dehydro ascorbic acid under acidic conditions to
give osazone, as highlighted in figure 7.3. The depth of the yellowish orange coloured
osazone produced upon acidification with sulphuric acid is directly proportional to the
amount of ascorbic acid present and can be measured photometrically at 540 nm
wavelength.
As you carry out the procedure in the laboratory you will understand the method even
better. So then let’s get started and practically learn the two methods we have studied
178 above. There are four activities in this practical.
 Methods of Estimation
EXPERIMENT
of Ascorbic Acid (Vitamin C)
TITRIMETRIC METHOD: 2, 6 DICHLOROPHENOL
INDOPHENOL METHOD FOR ESTIMATION OF
VITAMIN C IN A GIVEN SOLUTION 1
Date: ..........................
Aim: To estimate the ascorbic acid content in the given solution by 2, 6 dichlorophenol
indophenol method

Principle
(Write the principle behind the titrimetric method in the space provided herewith along
with the reaction involved)

Reaction

Apparatus
Burette- 10 ml
Pipettes- 5 ml, 10 ml
Conical flask- 100 ml
Measuring cylinders
Beakers
Glass marker

Reagents
2,6 dichlorophenol indophenol solution
3% metaphosphoric acid
Stock solution of ascorbic acid
Ascorbic acid standard solution (1 mg/5 ml)

Procedure
The step-by-step procedure involved in titrimetric estimation of vitamin C is enumerated
herewith. Follow these instructions carefully while conducting the experiment.

Rinse all your apparatus including burettes and pipettes with 3% metaphosphoric acid
at the beginning of the experiment. Now begin with the standard titration.

Standard titration
Pipet 5 ml of standard ascorbic acid solution into a 100 ml conical flask. Fill the burette
with the dye solution. Titrate the solution against the dye solution till a light pink color
appears which persists for 30 seconds. Standardize the dye solution.

Sample titration
Take the given unknown solution provided to you in a 100 ml volumetric flask. Dilute the
sample ascorbic acid solution to the mark with 3% metaphosphoric acid. Mix well.
Pipette 10 ml of this dilute solution into a 100 ml conical flask and titrate against the dye
solution till a light pink colour appears which persists for 30 seconds. Calculate the
ascorbic acid content of the given solution based on the calculations described next. 1790
Nutritional Precautions
Biochemistry
1. Rinse all glassware with 3% metaphosphoric acid before you begin your practical
and subsequently each time you wash your flasks, cylinders, etc.
2. Make up all volumes with 3 % metaphosphoric acid.

Method of Calculation
Clearly note down the observations and the readings as indicated in the format below for
standard and sample titration.

1. Standard titration:
i) Strength of standard ascorbic acid solution = 1 mg/5 ml
ii) Volume of standard ascorbic acid solution = 5 ml
iii) Volume of dye solution required

Burette reading (ml)

S.No.
Initial Final Difference
Pilot
1
2
3

Titer value = A = ……..ml

iv) A) …….. ml of dye solution is reduced by 5 mL of standard ascorbic acid solution

But 5 ml of standard ascorbic acid solution contains 1 mg of ascorbic acid
... (A) …….ml of dye solution is reduced by 1 mg of ascorbic acid

2. Sample titration:
a) Flask No.......................
v) Given ascorbic acid solution is diluted to 100 ml
vi) Volume of dilute sample ascorbic acid solution = 10 ml
vii) Volume of dye solution required

Burette reading (ml)

S.No.
Initial Final Difference
Pilot
1
2
3

Titer value = B =……..ml

viii) (A) ……. ml of dye solution is reduced by 1 mg of ascorbic acid

... (B) ….…. ml of dye solution is reduced by:

1 × b = 1×…. = C = ……... mg ascorbic acid

a

ix) But (B) …. ml of dye solution is reduced by 10 ml of dilute sample ascorbic acid
solution
... 10 ml of dilute sample ascorbic acid solution contains (C) ..... mg ascorbic acid.
... 100 ml of dilute sample ascorbic acid solution contains:
180
 c × 100 = D = ……….. mg ascorbic acid Methods of Estimation
10 of Ascorbic Acid (Vitamin C)

Observed Value = D = …………..mg ascorbic acid

Expected Value = ………mg (Take from counselor)

% error = Expected value – Observed value × 100

Expected Value

Now calculate the % error using the formula given above.

Result
The given solution contains …………….. of ascorbic acid

Now, submit this experiment for evaluation.

..................................
Counsellor signature

1810
Nutritional
EXPERIMENT
Biochemistry EXPERIMENT
TITRIMETRIC METHOD : 2, 6 DICHLOROPHENOL
INDOPHENOL METHOD FOR ESTIMATION 2 OF
2 VITAMIN C IN LEMON JUICE
Date: ..........................

Aim: To estimate the ascorbic acid content in lemon juice by 2, 6 dichlorophenol

indophenol method.

Principle and Reaction

You have studied the principle and reaction in the previous experiment. Try writing the
principle and reaction on your own now in the space provided herewith .

Reaction

Apparatus
Burette – 50ml
Pipettes – 5ml, 10ml
Conical flask 100ml
Measuring cylinders
Beakers
Glass wool
Funnel
Wattman No1 filter paper
Glass marker

Reagents
2, 6 dichlorophenol indophenol
3% metaphosphoric acid
6% metaphosphoric acid
Stock solution of ascorbic acid
Ascorbic acid standard solution (1 mg/5 ml)
Lemon
182
Procedure Methods of Estimation
of Ascorbic Acid (Vitamin C)
Carry out the experiment following the steps enumerated herewith:
Rinse all your apparatus including burettes and pipettes with 3% metaphosphoric acid
at the beginning of the experiment.

For Standard titration:

Pipette 5 ml of standard ascorbic acid solution into a 100 ml conical flask. Fill the
burette with the dye solution. Titrate the solution against the dye solution till a light pink
color appears which persists for 30 seconds. Standardize the dye solution.

For Sample titration:

Cut the lemon into two halves with a stainless steel knife. Remove the seeds. Squeeze
the juice from half a lemon, directly into a 25ml measuring cylinder containing exactly
10 ml of 6% metaphosphoric acid. Note down the exact volume of acid plus lemon
juice.Transfer the contents of the measuring cylinder into a 100 ml volumetric flask,
using 3% metaphosphoric acid for rinsing & transferring Mix. If the solution is not
clear, filter the solution through a dry filter paper into a dry flask. Pipette 10 ml of the
filtrate into a 100 ml conical flask. Titrate against the dye solution till a light pink color
appears which persists for 30 seconds. Calculate the content of ascorbic acid in the
lemon juice.

Precautions
1. Rinse all glassware with 3% metaphosphoric acid before you begin your practical
and subsequently each time you wash your flasks, cylinders, etc
2. Make up all volumes with 3% metaphosphoric acid.
3. Use a stainless steel knife for cutting the lemon.
4. After cutting the lemon, immediately squeeze the juice into 6% metaphosphoric acid.

Method of Calculation
Clearly note down the observations and the readings as indicated in the format below
for standard and sample titration and then do the calculations.

a) Standard titration
i) Strength of standard ascorbic acid solution = 1 mg/5 ml
ii) Volume of standard ascorbic acid solution = 5 ml
iii) Volume of dye solution required

Burette reading (ml)

S.No.
Initial Final Difference
Pilot
1
2
3

Titer value = A ml=…..ml

iv) A ….. ml of dye solution is reduced by 5 ml of standard ascorbic acid solution

But 5 ml of standard ascorbic acid solution contains 1 mg of ascorbic acid
... (A) ……….ml of dye solution is reduced by 1 mg of ascorbic acid

1830
Nutritional b) Sample titration
Biochemistry
v) Volume of 6% HPO3 = X ml = …….. ml
vi) Volume of 6% HPO3 + lemon juice = Y ml = ……….ml
vii) Volume of lemon juice = y - x = Z = ……….. ml
(Z) ……..ml of lemon juice diluted to 100 ml Volume of dilute lemon juice = 10 ml
viii) Volume of dye solution required
ix) (A) …….….ml of dye solution is reduced by 1 mg of ascorbic acid
... (E) ….….. ml of dye solution is reduced by:

1 × E = 1 ×… = F = …….. mg ascorbic acid

A ........
x) But (E)……. ml of dye solution is reduced by 10 ml of dilute lemon juice
... 10 ml of dilute lemon juice contains (F)……. mg ascorbic acid
... 100 ml of dilute lemon juice contains:
F ×100 = ….…×100 = G = …….. mg ascorbic acid
10 10
xi) But 100 ml of dilute lemon juice contains (Z)……..ml of lemon juice
(Z) ………ml of lemon juice contains (G) ……. mg ascorbic acid
... 100 ml of lemon juice contains:
G × 100 = …… × 100 = H = …….mg ascorbic acid
Z

Result

The given solution contains……………..of ascorbic acid.

Now, submit this experiment for evaluation.

..............................
Counsellor signature

184
 Methods of Estimation
EXPERIMENT
of Ascorbic Acid (Vitamin C)
TITRIMETRIC METHOD: 2, 6 DICHLOROPHENOL
INDOPHENOL METHOD FOR ESTIMATION OF
VITAMIN C IN CHILLIES 3
Aim: To estimate the ascorbic acid content in chillies by 2, 6 dichlorophenol indophenol Date: ..........................
method.

Principle and Reaction

Write the principle and reactions on your own.

Principle

Reaction

Apparatus
(In the last two experiments you have used the apparatus necessary for conducting this
method. Now you should be able to write the apparatus required on your own. Write
down the apparatus required in the space provided herewith)

Material and Reagents

(Write the material and reagents you would require in the space provided herewith)

Procedure
Carry out the experiment following the steps enumerated herewith:
Rinse all your apparatus including burettes and pipettes with 3% metaphosphoric acid
at the beginning of the experiment.

For Standard titration:

Pipette 5 ml of standard ascorbic acid solution into a 100 ml conical flask. Fill the
burette with the dye solution. Titrate the solution against the dye solution till a light pink
color appears which persists for 30 seconds. Standardize the dye solution.
1850
Nutritional For Sample titration:
Biochemistry
Take a clean and dry large chilli and weigh it. Note the weight of the chilli. Grind the
chilli to a fine paste in a pestle and mortar adding some sand to it for finer grinding. Add
a few drops of 6% metaphosphoric acid while grinding. Transfer the contents carefully
to a 50ml volumetric flask ensuring that not a drop spills. Rinse the pestle and mortar
with 3% metaphosphoric acid and transfer the rinsing to the volumetric flask. Repeat
this twice until all the paste has been transferred to the volumetric flask. Make the
volume to 50 ml mark with 3% metaphosphoric acid. Mix the contents and filter the
solution through glass wool and then through a dry filter paper into a dry flask or beaker.
Pipette 10 ml of this solution and titrate with 2,6 dichlorophenol indophenol solution till a
light pink colour is seen that persists for 30 seconds. Calculate the ascorbic acid content
in the chilli.

Precautions
1. Rinse all glassware with 3% metaphosphoric acid before you begin your practical
and subsequently each time you wash your flasks, cylinders, etc
2. Make up all volumes with 3 % metaphosphoric acid.
3. Use a stainless steel knife for cutting the chilli.
4. After weighing the chilli and cutting it for grinding, immediately add a few drops of
6% metaphosphoric acid.
Method of Calculation
Record your observations in the format given and do the calculations as indicated herewith.
A. Standard titration:
i) Strength of standard ascorbic acid solution = 1 mg/5 ml
ii) Volume of standard ascorbic acid solution = 5 ml
iii) Volume of dye solution required
Burette reading (ml)
S.No.
Initial Final Difference
Pilot
1
2
3

Titer value = A ml = ……… ml

iv) (A) ……. ml of dye solution is reduced by 5 ml of standard ascorbic acid solution.
But 5 ml of standard ascorbic acid solution contains 1 mg of ascorbic acid
... (A) ……… ml of dye solution is reduced by 1 mg of ascorbic acid
B. Sample Calculation:
v) Wt of chilli= X g
X g of chilli diluted to 50 ml
vi) Volume of the extracted solution of chilli taken for titration= 10 ml
vii) Volume of dye solution required

Burette reading (ml)

S.No.
Initial Final Difference
Pilot
1
2
3
186 Titer value = B ml = ……… ml
viii) (A) ………ml of dye solution is reduced by 1 mg of ascorbic acid Methods of Estimation
... (B) ……… ml of dye solution is reduced by: of Ascorbic Acid (Vitamin C)
1 × B = 1 × …. = C = …..…. mg ascorbic acid
A
ix) But (b) ..ml of dye solution is reduced by 10 ml of extracted solution of chilli
... 10 ml of extracted solution of chilli contains (C) ……. mg ascorbic acid
... 100 ml of extracted solution of chilli contains:
c ×100 = …×100 = D = …..….. mg ascorbic acid
10 10
x) But 100 ml of extracted solution of chilli contains (X) ….g of chilli
(X)….. g of chilli contains (D)…….. mg ascorbic acid
... 100g of chilli contains
d × 100 = …… × 100 = ……….mg ascorbic acid
X

Result
The given solution contains……..…….of ascorbic acid.

Now, that you have carried out the titrimetric method for estimation of vitamin C, we
hope the principle and the concept of this method is clear to you. Let us test your
understanding on this topic once again. Answer the questions given in the review question
section next and submit the answers along with the experiment result calculations to the
counsellor for evaluation.

Review Questions
1. What acts as an indicator in ascorbic acid titration?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
2. What is the role of 3% metaphosphoric acid in this titration?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
3. How does the indicator functions in this titration?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

1870
Nutritional 4. Why can’t humans synthesize ascorbic acid?
Biochemistry
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

5. Why does the solution of ascorbic acid have an acid pH?

.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

6. Why is ascorbic a strong reducing agent?

.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

Submit for evaluation.

.................................
Counsellor signature

188
 Methods of Estimation
EXPERIMENT
of Ascorbic Acid (Vitamin C)
COLORIMETRIC METHOD:
2, 4 DINITROPHENYLHYDRAZINE METHOD FOR
ESTIMATION OFVITAMIN C INAGIVEN SOLUTION 4
Aim: To plot a standard curve for ascorbic acid and to estimate the amount of ascorbic
acid in the given solution
Date: ..........................

Principle
Write the principle in the space provided.

Apparatus
Test tubes Borosil glass (6×3/4 inch) & test tube stand.
Pipettes 1 ml, 5 ml, 10 ml
Volumetric flask,100 ml,50 ml
Conical flasks
Beakers
Measuring cylinder
Glass wool
Funnel
Watman No1 filter paper
Glass marker
Labels
cuvettes single pan balance butler paper foreighing
Spetrophotometer / colorimeter
tissue roll

Reagents
1. 2, 4 dinitrophenylhydrazine reagent _ Dissolve 2 g of dinitrophenyl hydrazine in 100
ml of 9N sulphuric acid and allow to stand overnight. Mix and use.
2. 5% thiourea solution _ Dissolve 25 g of thiourea in 500 ml of distilled water.
3. 9N sulphuric acid
4. 85% sulphuric acid
5. 0.6% copper sulphate
6. Colour reagent or Dinitrophenylhydrazine _ thiourea _ copper sulphate (DTC)
reagent _ Mix
7. 5% trichloroacetic acid (TCA)
8. 5% metaphosphoric acid in 10% acetic acid solution _ Mix 1000 ml of 5%
metaphosphoric acid and 500 ml of 10% acetic acid.
9. Ice for cooling.

Procedure
Carry out the experiment following the instructions given herewith:
1. Prepare a series of tubes for blank, standard and sample labeling them as you
have done in previous colorimetric experiments. 1890
2. Weigh 100 mg of ascorbic and transfer to a 100 ml dry volumetric flask. Make
Nutritional the volume to 100 ml mark immediately with the metaphosphoric acid (5%)-
Biochemistry acetic acid (10%) solution. This is stock standard solution.
3. Pipette 1 ml out of this stock standard solution into a 50 ml volumetric flask
and make up the volume with TCA solution to give a concentration of 20 µg
/ml ascorbic acid. This is the working standard solution.
4. Pipette different concentrations of the working standard solution of ascorbic
acid in different tubes (0.5, 1.0, 1.5, 2.0, 2.5 ml) -S1, S2, S3, S4 and S5 - labelled for
standard. Make the total volume to 3 ml with 5% TCA in each tube.
5. Make a blank (B) with 3 ml TCA. No ascorbic acid solution is added.
6. Add 1ml of DTC reagent to each tube (blank, standard) and stir well
7. Incubate all tubes for 1 hr at 60oCin a water bath.
8. After 1 hour remove the tubes and cool them immediately in ice for 15 minutes
9. Add 5 ml of 9 N sulphuric acid to each test tube with careful vigorous shaking.
10. Allow the tubes to stand at room temperature for 20 minutes after which they can
be read at 540 nm.
11. For unknown sample: Dilute the given sample to 100 ml with metaphosphoric
acid acetic acid solution. Take 1.5 ml of the unknown sample in duplicate and
proceed just like you did for the standard tubes ( adding TCA , colour reagent
and finally sulphuric acid).
12. Plot the graph of concentration Vs OD to obtain the standard curve. Calculate
the ascorbic acid content of the unknown solution from the standard curve as
well as from OD.

Calculations and Standard Curve Plotting

Record your observations and do the calculations as suggested herewith:
I. Preparation of Standard Ascorbic Acid Solution
i) 100 mg of weighed ascorbic acid solution was dissolved in 5% metaphosphoric
acid, 10% acetic acid solution and the volume was made up to 100 ml.
ii) 1 ml of this solution was pipptted in 50 ml volumetric flask and volume made upto
50 ml using 5% trichloroacetic acid (TCA).
iii) ... Strength of prepared ascorbic acid solution = 20 µg/ml.
1 ml standard ascorbic acid solution contains = 20 µg ascorbic acid.
... 0.5 ml standard ascorbic acid solution contains =
... 1.5 ml standard ascorbic acid solution contains =
... 2.0 ml standard ascorbic acid solution contains =
... 2.5 ml standard ascorbic acid solution contains =

II. Colorimetric Reading for Standard Ascorbic Acid Solution

iv) Record the values as indicated in the format given herewith.

I II III IV V VI VII
Volume of Conc. of TCA DTC Sulphuric Optical Optical Density
Std. Sol. Ascorbic Acid (ml) (ml) Acid Density at for 30 µg
(mg) (ml) ascorbic acid
0.5

1.0

1.5

2.0

2.5

190 Mean OD for 30 µg ascorbic acid = A = …………………...

v) Now plot the standard curve for ascorbic acid (on a graph paper) with concentration Methods of Estimation
of ascorbic acid (figure included in item II above) on x- axis and the optical density of Ascorbic Acid (Vitamin C)
(figures in item VI) on y-axis. Stick the graph on page 192.
III. Colorimetric Reading for Unknown Solution
vi) Optical density of unknown solution, sample 1 =
sample 2 =
... Mean optical density of unknown solution = B =
IV. Determination of Ascorbic Acid Content From standard Curve
On the standard curve prepared earlier, plot the OD of the unknown solution on the
y-axis. Now check the corresponding concentration of ascorbic acid for this
OD on the x-axis. Say the value obtained is E.
So 1.5 ml of unknown solution contains E ml of ascorbic acid
... 100 ml of unknown solution will contain = E × 100 = F = ….…..mg.
1.5
Using this calculation now you calculate the ascorbic acid content in the unknown
sample(s) given to you. Write your calculations here in the space provided.
Observed Value = F = ………….
Expected value = D =…………(Take it from the counsellor)
Now calculate the % error in the space provided using the formula given
herewith:
.
. . % error (based on optical density) = D – F × 100
D

V. Determination of ascorbic Acid Content from OD

Mean O.D for 30 µg ascorbic acid = A ……… mg
... If OD of ascorbic acid solution is (A)…….., then concentration of ascorbic acid
is = 30 µg
if OD of the unknown solution is (B) ………, then concentration of unknown
Ascorbic acid solution = 30 × B = Z = …….mg
A

So Z …….. mg is present in 1.5 ml of unknown

... 100 ml of unknown solution contains = Z × 100 = …….. × 100 = C ……… mg.
1.5 1.5
Observed Value = C = ………….
Expected value = D =…………(Take it from the counselor)
Now calculate the % error in the space provided using the formula given herewith:
... % error (based on optical density) = D – C × 100
D

Result The given solution contains………………of ascorbic acid as determined from

OD and …………… as calculated from standard curve.
Submit for evaluation.

...................................
Counsellor signature 1910
Nutritional
Biochemistry

Standard curve between concentration of ascorbic acid in standard solution

192 and OD