Experiment IX

Group VIB

VITAMIN ANALYSIS
 Learning Objectives
 At the end of this experiment, the
students should be able to:
○ Identify the corresponding vitamins
present in a variety of foodstuffs.
○ Gain understanding of how vitamins
from natural organic extracts
compared with commercially
prepared vitamins
Vitamin Analysis
Vitamin
Vitamin A
C

Vitamin
D

Niacin
Vitamin
E
 Vitamin A
 PRINCIPLE:
The sample containing Vitamin A,
when made to react with antimony
trichloride and pyrogallol, produces a
colored complex
 Vitamin A
 Vitamin A prepared  Vitamin A from
commercially natural organic
solutions
1 mg of Vitamin A
10 mL CCl4 Filtrate of peeled
and grated
medium-sized
carrot
 Vitamin A Analysis
 Test tube #1  Test tube #2
 5 mL of the  5 mL of extracted Vitamin
commercialized Vitamin A A solution
solution  20% solution of SbCl3 in
 20% solution of SbCl3 in CHCl3 drop by drop
CHCl3 drop by drop

UNAVAILABLE LIGHT ORANGE COLOR

WITH GLOBULE OF FAT
 Vitamin A
 What happened after the addition of
antimony trichloride?
 The 5 mL of extracted vitamin A added with 20%
solution of SbCl3 in CHCl3 produced a blue
colored complex.

 After addition of pyrogallol?

 The addition of pyrogallol to the extracted
vitamin A produced a rose-pink color reaction.
This test is also called Fearon’s test for vitamin
A.
 Vitamin A
 The use of direct flame is avoided
because antimony when heated ,
emits a very toxic fumes of chlorine
and Antimony oxide fumes

 In addition to this, Vitamin A in air

and light becomes unstable. So
retinol, when heated while exposed
to air and light
 will break down faster than retinol in
the dark, anaerobe environment. In
any situation, temperatures above 40
degress celsius will begin to slowly
degrade and break down retinol
Vitamin Analysis
Vitamin
Vitamin A
C

Vitamin
D

Niacin
Vitamin
E
 Vitamin D
 Vitamin prepared  Vitamin from natural
commercially organic substance
 1 mg of Vitamin D  1 mL cod liver oil
 10 mL CCl4  10 mL CCl4
  filtrate
 Vitamin D Analysis
 Test tube #1  Test tube #2
 2 mL pyrogallol in  Do steps 1-3 for the
benzene extracted vitamin D
 10 drops of commercial solution
Vitamin D solution
 Mix. Add 1 mL alcohol
solution of AlCl3
 Warm in water bath

Black (darker) Black

 Vitamin D is classified as a group of
fat soluble secosteroids responsible
for increasing intestinal absorption of
calcium, magnesium, phosphate and
multiple other boilogical effects. In
humans, the most important
compounds in this group are vitamin
D3 and D2. It can also function as a
hormone in the body
 Two forms of vitamin D
Cholecalciferol
○ Found in animl products and which the
body manufactures when exposed to the
sun
Ergocalciferol
○ Found in vegetables and
 Roles of this vitamin includes
Absorption of calcium in the intestines
and reduces loss in the urine
maintain our bones and teeth healthy
Regulates calcium and phosphorus
metabolism
Vitamin Analysis
Vitamin
Vitamin A
C

Vitamin
D

Niacin
Vitamin
E
 Vitamin E
 PRINCIPLE:
A sample containing Vitamin E has the
ability to react with nitric acid to produce
a colored chromogen.
 Vitamin E
 Vitamin prepared  Vitamin from natural
commercially organic substance
 1 mg of vitamin E  1 mL coconut oil
 10 mL of CCl4  10 mL CCl4
  residue is extracted
 Vitamin E Analysis
 test tube #1  test tube #2
 2 mL of nitric acid in  1 mL coconut oil
alcohol  10 mL CCl4
 5 drops of commercial Vit  Do steps 1 and 2 for
E extracted vitamin E
 Water bath

Red with formed fat Red with precipitate

globule (sediment) (particulates)
Best sources of Vitamin E
 Wheat germ oil
greatest source
Others
○ Almonds
○ Hazelnuts
○ Canola oil
○ Grapeseed oil
○ Olives
Vitamin Analysis
Vitamin
Vitamin A
C

Vitamin
D

Niacin
Vitamin
E
 Niacin
 PRINCIPLE
An aqueous solution containing this
vitamin produces a colored product if
made to react with cupric sulfate in an
alkaline medium
 Niacin
 Niacin prepared  Niacin prepared from
commercially natural organic
 1 mg of niacin substances
 10 mL H2O  1 g yeast
 10 mL H2O
 Niacin
 Test tube #1  Test tube #2
 5 mL aqueous solution of  Do steps 1-3 for extracted
niacin niacin sample
 0.1 M NaOH drop by drop
until litmus paper turns
neutral
 Mix 1 mL cupric sulfate
 Shake and stand for 5
mins.

Teal with 2 layers

Khaki green
Upper layer is lighter and clear while the
lower layer is turbid
 Stand the solution for 5 minutes to
facilitate the reaction of cupric sulfate
and sodium hydoxide yielding a
cupric hydroxide and sodium sulfate
as a product. These products did not
dissolve but instead form a solid
precipitates
Vitamin Analysis
Vitamin
Vitamin A
C

Vitamin
D

Niacin
Vitamin
E
 Vitamin C
 PRINCIPLE
This vitamin has the ability to react with
phosphomolybdic acid to produce a
colored product
 Vitamin C
 Vitamin prepared  Vitamin from natural
commercially organic substance
 1 mg vitamin C  Residue of peeled and
 10 mL of H2O mashed medium sized
potato
 10 mL H2O
 Vitamin C Analysis
 Test tube #1  Test tube #2
 5 mL phosphomolybdic  Do steps 1 and 2 for the
acid solution extracted vitamin C
 Add drop by drop , with sample
gentle shaking a 1 mL of
ascorbic acid solution

Clear with touch of aqua Hazy orange

blue
How are vitamins classified?
 Water soluble
 Cannot be stored in the
body and must be
replaced each day.
 Easily destroyed or
washed out during food
storage and preparation.
Ex. Vit. B- complex- found in
legumes, cereal grains, fresh
veggies, eggs, fruits, milk
Vit. C- found in citrus fruits
 Fat soluble
 Disolves in fat before
they are absorbed in the
boodstream to carry their
functions.
 Van be stored in the body
 liver  not needed
everyday in the diet.
 Easily absorbed in the
intestinal tract
Ex. Vit. A,D,E,K
 What are the effects of the
excessive intake of vitamins?
 Vitamins are organic compounds
needed in small quantities to sustain
life. We normally get the necessary
amount from our daily diet, but
sometimes, due to dietary restraints,
individuals will develop vitamin
deficiencies.
 What are the effects of the
excessive intake of vitamins?
However, taking too many
vitamins and supplements does have
negative consequences. Although
many water soluable vitamins are
excreted when too much is taken,
others remain in the body. These
vitamins can reach toxic levels and
cause adverse side effects.
 Vitamin A
 A high dose of vitamin A can cause
the following side effects: nausea,
vomiting, diarrhea, loss of appetite,
tiredness, headaches, dizziness,
blurred vision, poor muscle
coordination, itchiness and scaling of
the skin, bone pain, hair loss,
irregular menstruation in women,
 osteoporosis, and temporary or
permanent liver damage. High doses
of vitamin A are also known to
increase the risk of lung cancer in
smokers.
 Acute hypervitaminosis is a severe
condition resulting from consuming
too much vitamin A during a short
period of time.
 It was first documented in arctic
explorers who unknowingly
consumed vitamin A-rich livers of
many artic animals, such as seals,
husky dogs, and yes, even the polar
bear. The symptoms of the extremely
unpleasant condition are loss of skin
and hair, liver damage, hemorrhage,
coma, and even death.
 Vitamin D
 Vitamin D toxicity is usually caused
by megadoses of vitamin D
supplement not by diet or sun
exposure. That's because your
body regulates the amount of
vitamin D produced by sun
exposure, and even fortified foods
don't contain large amounts of
vitamin D.
 The main consequence of vitamin D
toxicity is a buildup of calcium in
your blood (hypercalcemia), which
can cause nausea and vomiting,
weakness, and frequent urination.
Symptoms might progress to bone
pain and kidney problems, such as
the formation of calcium stones
 Vitamin E
 Many adults take relatively large
amounts of vitamin E (alpha-
tocopherol 400 to 800 mg/day) for
months to years without any
apparent harm. Occasionally,
muscle weakness, fatigue, nausea,
and diarrhea occur. The most
significant risk is bleeding.
 However, bleeding is uncommon
unless the dose is > 1000 mg/day or
the patient takes oral coumarin
or warfarin. Thus, the upper limit
for adults aged ≥ 19 yr is 1000 mg for
any form of alpha-tocopherol.
 Analyses of previous studies report
that high vitamin E intakes may
increase the risk of hemorrhagic
stroke and premature death
 Vitamin C
For adults, the recommended daily amount for
vitamin C is 65 to 90 milligrams (mg) a day, and the
upper limit is 2,000 mg a day. Although too much
dietary vitamin C is unlikely to be harmful,
megadoses of vitamin C supplements might cause:
 Diarrhea
 Nausea
 Vomiting
 Heartburn
 Abdominal cramps
 Headache
 Insomnia
Ill effects of vitamin deficiency.
 Scurvy
 General malaise
 Lethargy
 Spotty skin
 Losing teeth
 Bleeding and swollen
gums
 Slow wound healing
 General weakness
 Vitamin C deficiency
Ill effects of vitamin deficiency.
 Rickets
 Softening of bones
in children resulting
in fractures and
deformities
 Vitamin D deficiency
Ill effects of vitamin deficiency.
 Beri beri
 Nerve, heart, brain
abnormalities
 Numbness and tingling
in hands and feet
 Memory loss
 Problems in breathing
and speaking
 Possible paralysis
 Confusion and
depression
 Severe Vitamin B1
deficiency
How may the chemistry of different
vitamins be described?
What are the physiological roles of
the different vitamis?
 Vitamin A
Maintenance of healthy epithelium
Bone development
Intermediary metabolism
Vision in dim light and cone vision
What are the physiological roles of
the different vitamins?

 Vitamin D
Induce proteins involved in active intestinal
calcium and phosphate absorption
Vitamin E
Antioxidant

Vitamin C
Protein, carnitine, collagen and
cathecolamines synthesis
Thank you!