OBJECTIVES
• To measure ascorbic acid level in food products
• Identify the sources of ascorbic acid
• Master of extraction and determination of
ascorbic acid (vitamin c) in food products
• Explain the clinical significance of Vitamin C
Outline of Today’s lecture
1. Introduction
2. Sources of Vitamin C
3. Functions of Vitamin C
4. Spectrophotometric determination
What is Ascorbic Acid?
• Ascorbic acid is a naturally occurring organic
compound with antioxidant properties. It is a
white solid, but impure samples can appear
yellowish. It dissolves well in water to give
mildly acidic solutions. Ascorbic acid is one form
of vitamin C.
Introduction
• Vitamin C is a water-soluble vitamin that is
necessary for normal growth and development.
• Water-soluble vitamins dissolve in water. The
body cannot store them. Leftover amounts of the
vitamin leave the body through the urine. That
means you need a continuous supply of such
vitamins in your diet.
Sources of Vitamin C
• It can be obtained from citrus fruits, tomatoes,
potatoes, and fresh vegetables, particularly from
red and green peppers. It is now made
synthetically. All animals except man, other
primates, and guinea pigs, are capable of
synthesizing their own ascorbic acid; man must
obtain it from food.
Functions of Vitamin C
• Vitamin C is required for the growth and repair
of tissues in all parts of your body.
• It is necessary to form collagen
• Vitamin C is one of many antioxidants in the
body
Symptoms of a Vitamin C Deficiency
Fatigue – Early on, someone with a vitamin C deficiency
will tend to get tired easily and experience reduced
energy.
Mood Changes – Individuals with a vitamin C deficiency
may become irritable or short tempered.
Weight Loss – As with other “wasting conditions”
someone with a vitamin C deficiency may experience
sudden weight loss.
Joint and Muscle Aches – Chronic pain in the limbs or
joints can be a symptom of a vitamin deficiency.
Continue….
Dental Conditions – Just as a healthy daily dose of
vitamin C contributes to healthy teeth and gums, a
deficiency can cause deterioration of the gums.
Dry Hair and Skin – A change in hair and skin
conditions can also signal that the body is not getting
enough of vitamin C and other essential vitamins and
minerals.
Infections – When an individual does not have enough
vitamin C over time, this can have a negative impact on
general healing of wounds and the fighting of infections.
Deficiency of Ascorbic acid
Deficiency of Ascorbic acid
Deficiency of Ascorbic acid
• “ Scurvy “
• Two of the chemical properties of ascorbic acid
are its acidity, due to the enolic hydrogen at C-3
(see Reaction 1) and its oxidation to
dehydroascorbic acid (see Reaction 2).
• The reducing power of ascorbic acid is the basis
for most of the quantitative analytical
procedures for estimation of this compound,
such
as
titration
of
the
dye,
2,6dichlorophenolindophenol
(DCPIP)
(see
Reaction 3).
Procedure
A- Preparation of samples:
1-Citrus fruit juice and bottled juices. The juice
from fresh fruit can be obtained with a lemon
squeezer followed by filtration through course
filter paper. These steps will remove fibrous
materials that might plug the tip of the pipettes.
Pipette 0.5 ml of fruit juice into a test tube and
dilute with 4.5 ml of 1% phosphate-citrus
solution. Cover the tube with parafilm and
invert several times to mix. Remember to
account for these dilution factors when
calculating your vitamin C concentration in the
sample.
2-Vegetables. Homogenize 1 gm (weight to
0.01gm) of vegetable with 5-10 ml of 1%
phosphate–citrate using a mortar and pestle.
Pour off the liquid into a filter funnel. Extract
the solids a second time with 10 ml of 1%
phosphate-citrate and filter. Combine the filtrate
into a volumetric flask. Add 1% phosphatecitrate to the filtrate until the volume is 25 ml.
Remember to account for these dilution factors
when calculating your vitamin C concentration
Preparation of Cuvettes
Blank
(only Reagent)
Standard
(Reagent+ Vit.C))
Sample
(Reagent+Sample
Reagents (ml)
Std.
S#1
S#2
Vitamin C Standard
0.05
-
-
Sample # 1
-
0.05
-
Sample # 2
-
-
0.05
0.45
0.45
0.45
1.0
1.0
1.0
Phosphate –Citrate
buffer
DCPIP
• Mix all the tubes; zero the spectrophotometer at
518 nm with the blank (only distilled water).
• Read all the standard and samples at 518 nm.
• Record your results for further calculations.
Concentration of vitamin C (mg/ml)=
Absorbance of Sample X Conc. of Standard X 10
Absorbance of Standard