ANALYTICAL TECHNIQUES FOR

VITAMINS.
PRERNA JAIN

MSc. SEMESTER III

PS.CHE. 303 (Unit I)

WILSON COLLEGE
 Vitamins Vitamins
Definition: Vitamins are organic
compounds which are found in
natural food and are essential for the
nutrition, growth and development
Water Fat
of human body.
soluble soluble

Vitamin B Vitamin A
And Vitamin D
Vitamin C Vitamin E
Vitamin K
Fat Soluble Vitamins
•They are soluble in fat
•Bile salts are and fats are essential for their absorption.
•They are generally stored in liver.
•Normally they are not excreted in urine.
Vitamin A
NUTRITIONAL SIGNIFICANCE &
PHYSIOLOGICAL SIGNIFICANCE DEFICIENCY.

• Important for growth and tissue repair.
• Vision.
• Helps to reduce cancer.
• Its role in electron transport is
suggested.
• If liver stores of Vitamin A are
depleted, it causes defective night
vision.
• Keratinization of epithelial tissue.
• Xerophthalmia.
Vitamin D
PHYSIOLOGICAL SIGNIFICANCE
•Plays an important role in
mineralisation of bones as it
promotes synthesis of calcium
binding parts.
•Promotes renal absorption of
calcium and phosphates.
NUTRITIONAL SIGNIFICANCE
& DEFICIENCY.
•Defective bone formation which
results in bowlegs and pigeon
beast.
•Causes Muscle weakness.
Vitamin E
NUTRITIONAL SIGNIFICANCE &
PHYSIOLOGICAL SIGNIFICANCE DEFICIENCY.

• Antioxidant Property •Vitamin E deficiency result in an

• It helps in normal function and
anaemia to new born baby.
development of male and female •Tocopherol deficiency.
reproductive organs.
•Vitamin E deficiency can result
• It also protects enzymes in muscles, in neurological symptoms.
nerve or gonads from destructions.
Vitamin K
PHYSIOLOGICAL SIGNIFICANCE
• Plays an important role in blood
clotting mechanism.
• Vitamin K helps in deposition of
calcium in bones, kidney.
• Vitamin K is also associated with
the electron transport chain and
oxidative phosphorylation.
NUTRITIONAL SIGNIFICANCE &
DEFICIENCY
• Deficiency of this vitamin result in
hypoprothrombinaemia and result in
uncontrolled haemorrhages.
• Vitamin K deficiency may result in
internal bleeding (in the skull),
malformed fingers, and
under-developed facial features in
fetus.
Water Soluble Vitamins
• They are soluble in water.
• They are easily absorbed.
• They are not stored in body
• They have a threshold for urinary excretion.
Vitamin B₁(THIAMINE)
PHYSIOLOGICAL SIGNIFICANCE
• Thiamine plays a key role in
carbohydrate metabolism for the
generation of energy.
• The important function of thiamine is
its role as a coenzyme.
NUTRITIONAL SIGNIFICANCE & DEFICIENCY
• Thiamine deficiency results in Beri
Beri disease.
• Deficiency of this vitamin may lead
to loss of appetite, weakness,
paralysis, nervous irritability,
insomnia, loss of weight mental
depression and constipation
Vitamin C
PHYSIOLOGICAL SIGNIFICANCE
• Vitamin C is involved in oxidation
reduction reactions of the cell.
• Vitamin C is also a highly effective
water-soluble antioxidant.
NUTRITIONAL SIGNIFICANCE &
DEFICIENCY
• Certain cancers, Atherosclerosis are also
some causes due to vitamin C deficiency.
• Bleeding inside the brain and around the
heart can cause death in some people
with untreated vitamin C deficiency.
• Dry and splitting hair, Dry red spots on
the skin, Rough, dry, scaly skin,
Nosebleeds, Weakened immune
system,etc.
Estimation of β-carotene in carrots
1. Freshly ground carrot is allowed to stand overnight in a mixture of
Petroleum ether:Acetone (1:1), which is then filtered and again
extracted twice till all yellow colour is extracted. This is then
washed, dehydrated and concentrated.
2. Prepare a column using 6g calcium hydroxide and wet the column
using Petroleum ether. Load the concentrated extract to facilitate
the flow of slovent. Develop the column.
3. Collect 10mL of each fraction and dilute with developing solvent.
Take graded volume of 0.2-2.0mL of standard β- carotene solution
and dilute with Petroleum Ether:Acetone (1:1) mixture.
4. Record the absorbance of standard and sample at 450nm. Determine
the quantity of β-carotene in each of the collected fraction of
sample from the curve.
CALCULATIONS:
The true value of vitamin A in sample is expressed in terms of IU.
1 IU of vitamin A= 0.6μg of β- carotene.
Determination of thiamine in foods and
cereals.
1. Grind 2g of sample with 20mL 0.1N H2 SO4. Centrifuge the
suspension for 20mins. Decant the supernatant fractions into 50mL
flask and dilute to mark with 0.1N H2 SO4.
2. Add 3mL potassium ferricyanide and 3mL of 15% NaOH to suitable
portion of thiamine extract. Shake well.
3. Add 10mL iso-butanol and shake the mixture. Allow two phases to
separate completely.
4. Collect the upper organic layer. Using this, prepare graded volume
of standard solution containing 0 to 5μg thiamine hydrochloride and
proceed with steps 2 and 3.
5. Using fluorometer, measure the fluorescence intensity of standards
and sample using primary filter at 345nm and secondary filter at
435nm.
6. From the reference curve, determine the amount of thiamine in
terms of mg of thiamine per 100g of sample.
Estimation of ascorbic acid in lemon juice.
1. Squeeze fresh lemons and strain the lemon juice. Dilute it 10 folds
with 6% metaphosphoric acid.
2. Transfer 20mL standard vitamin C solution in flask and titrate
against 2,6-dichlorophenol indophenol solution till the appearance
of light pink colour. Note down the end point.
3. Similarly, titrate 20mL diluted lemon juice and note the end point.
CALCULATIONS:
Conc. Of vitamin C in standard soln = 10μg/mL
Conc of vitamin C in 20mL = 200μg
‘V’mL of dye is required for oxidation of vitamin C in 20mL standard
solution then 1mL of the dye causes oxidation of 200 μg of
vitaminC
20mL of diluted lemon juice is oxidized by ‘Y’ mL of dye.
Amount of vitamin C in 20mL diluted juice= 200* Y μg
V
 References
1. Food Chemistry by Alex. V. Ramani
2. Fundamentals of Biochemistry by AC Deb
3. Analytical Biochemistry by D.J Homes and H. Peck, Longman
4. https://patient.infro/in/health/vitamin-deficiency-leaflet
Thank You