L13.

VITAMINS DETERMINATION

Introduction
Vitamins are bioorganic compounds needed in small quantities for the maintaining of
the normal function of the organism. The vitamins obtained predominantly from food are A,
B6, B12, C, E, thiamine, riboflavin, folic acid, pantothenic acid; the ones obtained by
endogenous synthesis, vitamin D and niacin. Under the action of the intestinal flora, humans
obtain predominantly vitamin K AND biotin.
A great part of the vitamins fulfill the function of enzymatic cofactors, having an
essential role in the vital metabolic pathways. From the point of view of their solubility, there
are two big classes of vitamins:
- hydrosoluble vitamins: the vitamins of complex B (thiamine, riboflavin, niacin, B 6,
pantothenic acid, B12, biotin and folic acid) and vitamin C.
- liposoluble vitamins, derivatives of isopren: A, D, E and K.

The daily need of vitamins is very low. If the vitamin intake is too low, metabolic
disturbances appear; in extreme cases they can produce death. The maintaining of the vitamin
level at normal values is done by a corresponding food intake or by vitamin supplements as
pharmaceutical formulations. The vitamin excess must be avoided; some toxic phenomena
can appear, especially in the case of liposoluble vitamins.
Both deficit and excess are detected by laboratory analyses of vitamins in blood. Due
to their structural complexity, the chemical methods of determination are difficult and take
long time; in most cases, they are replaced by high performance physico-chemical methods.

Practical part: Vitamin C determination in fruit juices by iodimetry

In order to determine the vitamin C content of various fruit juices or soft drinks, their
titration with iodine solution can be used. Iodine reacts with ascorbic acid producing iodide
ions (I-) and dehydroascorbic acid, according to the following reaction:

The ascorbic acid is easily oxidized by atmospheric oxygen in neutral or alkaline

environment. In order to prevent this process, an acid medium is maintained by the addition
of acetic acid. Starch is added to the titrated solution so that excess iodine, which is not
consumed in the reaction with ascorbic acid, gives a color reaction. The formation of the blue
color is generally used as an indicator for starch, but in this experiment, it will indicate the
excess of iodine remaining at the end of the titration.
The purpose of the practical work is to compare the vitamin C content of fresh orange
juice and of commercially available orange juice.

Reagents

1. Standard ascorbic acid solution, 1 mg/ml.

2. Fresh juice obtained from an orange.
3. Commercially available orange juice.
4. Iodine solution 0,005 M.

1
5. Acetic acid solution 6M.
6. Starch solution 1%.

Procedure:

1. Determination of the volume of iodine solution required for the titration of a

standard ascorbic acid solution
Add the following solutions in two Erlenmeyer flasks:
- 10 ml distilled water
- 1 ml acetic acid solution 6M
- 5 ml standard ascorbic acid solution
- 1 ml starch solution 1%.

Titrate slowly with the 0.005M iodine solution, stirring the solution in the Erlenmeyer
flask, observing the appearance of a blue color. When the blue color persists for 30 seconds,
the titration is stopped and the final titration volume is written down.

2. Determination of the volume of iodine solution required for the titration of an

ascorbic acid sample
a. Fresh juice obtained from an orange is considered the first sample - solution A of
ascorbic acid (unknown concentration)
b. Commercially available orange juice is considered the second sample - solution B
of ascorbic acid (unknown concentration)

Add the following solutions in two Erlenmeyer flasks:

- 10 ml distilled water
- 1 ml acetic acid solution 6M
- 5 ml ascorbic acid solution of unknown concentration (A, respectively B)
- 1 ml starch solution 1%.

The titration procedure is similar with the one for the standard ascorbic acid solution.
The obtained volumes are noted: VA and VB.

Calculation
Vitamin C concentration from the two samples is calculated using the following
formulas:
CA = (VA/ Vm stand.) x 100 (mg/ml)
CB = (VB/ Vm stand.) x 100 (mg/ml)