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QUANTITATIVE DETERMINATION
OF VITAMIN A AND VITAMIN E

VITAMINS
? Vitamins are naturally occurring organic compounds
that are essential to metabolic or other functions in the
body.
? Most vitamins cannot be synthesized by the body.
They must be supplied in the diet.
? Vitamins are usually classified as water soluble or fat
soluble

CLASSIFICATION OF VITAMINS
? Vitamins can be classified as either
? Water soluble
? Fat soluble.
? Water soluble vitamins are generally involved in
the cellular metabolism of energy supplying
nutrients.
? Fat soluble vitamins often have very specialized
functions

Water Soluble Vitamins
? Examples of water soluble vitamins
Vitamin C Vitamin B1 (Thiamine)

Fat Soluble Vitamins
? Common fat soluble vitamins include
? A,
? D, vitamin A
? E,
? K.

Vitamin A Sources
? Commonly found in cod liver oil, green vegetables,
and fruits.
? Carrots indirectly serve as a source of vitamin A
since they contain �� carotene which the body
readily converts to vitamin A

Vitamin A Functions
? Vitamin A is fat soluble.
? It is not readily broken down by cooking.
? Role in aiding in night vision.
? Vitamin A Deficiencies
? A deficiency in vitamin A results in night blindness.
? The most serious deficiency results in a condition
known as Xeropthalmia, a severe form of
conjunctivitis or blindness.

Vitamin E sources
? vitamin E can be found most abundantly in wheat
germ oil, sunflower, and safflower oils and also in nuts,
cereals, olive, tomato, spinach, blue crab.
olive nuts cereals

Vitamin E functions
? Vitamin E is used to refer to a group of fat-soluble
compounds that include both tocopherols and
tocotrienols.
? ��-Tocopherol is an important lipid-soluble
antioxidant.
? ��-Tocopherol has a regulatory effect on enzymatic
activities
? responsible for the repair of the wounds and
regeneration of the extracellular tissue, vitamin E also
plays a role in neurological functions, and inhibition of
platelet aggregation

Quantitative determination of
vitamin A
? Category: antixerophthalmic vitamin
? Structure:
? Standards: not less than 95% and not more than 110%
of standard number of units of vitamin A per g.

? Identification: exhibits a maximum at about 325-
327nm.
? QUANTITATIVE ESTIMATION METHOD:
? UV-Spectrophotometric method
? principle:
? it absorbs the UV-radiation ranges
from 200nm to 400nm.
? Valence electrons absorb the energy, there by molecule
undergo transition from ground state to excited state.
? By the absorption peaks the nature of electrons and
molecular structure can be elucidated.

? METHOD:
? Dissolve an accurately weighed quantity of the
substance in cyclohexane to give a solution containing
9 to 15 units of vit A per ml.
? Determine the wave length of maximum absorption.
? Measure the absorbance of the solution against cyclo
hexane at 328 nm.
? If the wave length of maximum absorption lies
between 326 and 329 calculate the vit A potency of the
sample from the expression
A328(A1%
1cm) �� 1900 = vit A potency in units per g.

? If the relative absorbances are not within the limits
then calculate a corrected absorbance at about 328nm
by applying the observed values to the equation
A328(corr.) = 3.52 (2A328 �CA316 �C A 340)
? If the wavelength of maximum absorbance lies out
side the range of 323 to 327 then the unsaponifiable
fraction of the sample must be further purified by
chromatography.

Quantitative determination of
vitamin E
? Category: anti oxidant
? Structure:
? Standards: it contains not less than 96% and not more
than 102% of C31H52O3.

QUANTITATIVE ESTIMATION METHOD:
Chromatographic method:
Principle:
? The principle of separation in GAS
CHROMATOGRAPHY is partition.
? They travel according to their partition coefficients
towards the stationary phase.
? More soluble travels slower
? Less soluble travels faster
? No two components have same partition coefficients.
? The components are separated according to their
partition coefficients.

? METHOD:
? Internal standard solution: dissolve an accurately
weighed quantity of hexa decyl hexa decanoate in n-
hexane to obtain a solution having a known
concentration of about 1mg per ml.
? Standard preparation: dissolve in internal standard
solution a suitable quantity of USP alpha tocopherol
RS to obtain a solution having known concentration of
1mg per ml.
? Assay preparation: transfer 50mg of vitamin E to a
50ml volumetric flask, dissolve in internal standard
solution, dilute with internal standard solution to
volume and mix.

Chromatographic system
? The GC instrument is
equipped with a flame
ionization detector.
? It contains 2m-4mm
borosilicate glass column
packed with 2% to 5%
liquid phase on 80 to 100
mesh support utilizing a
column injection.
? The column is
maintained at a temp.
between 245�� to 265��

? The injection port and detector block are maintained at
about 10�� higher than the column temperature.
? The flow rate of dry carrier gas is adjusted to obtain a peak
approximately 18 to 20 min after sample introduction.
? Procedure:
? Inject a suitable portion (2-5?l) of the assay preparation
into a suitable gas chromatograph.
? Record the chromatogram .
? Measure the areas under the 1st(alpha tocopherol) and 2nd
major(hexadecyl hexa decanoate)peaks, record the values
as aU and aD respectively.
? Calculate the quantity in mg, of vit E taken by the formula
(50CD/F)(aU/ aD)
Where CD is conc.in mg/ml
F is the relative response factor

REFERENCES
? INDIAN PHARMACOPOEIA
? BRITISH PHARMACOPOEIA
? UNITED STATES PHARMACOPOEIA
? www.googleimages.com
? www.wikipedia.com
? www.cyberlipid.org

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QUANTITATIVE DETERMINATION OF VITAMIN A AND VITAMIN E

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QUANTITATIVE DETERMINATION OF VITAMIN A AND VITAMIN E