CHEMISTRY PROJECT

ESTIMATION OF ASCORBIC ACID

Vivek Chokhani

2014-15

ESTIMATION OF ASCORBIC ACID

2014-15

CONTENTS

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INTRODUCTION
Vitamins are accessory dietary factors required for healthy life in addition to the usual diet of fats.
carbohydrates, proteins and mineral salts. The word Vitamin' comes from the Latin word VITA'
meaning 'LIFE'. Subsequently the name 'VITAMIN' was coined by FUNK in 1911 as vitamine, who
believed that the anti-beriberi factor (thiamine) was an amine essential to the maintenance of life.
Although subsequent discoveries showed that not all vitamins are amines, the historical name has
been retained, however without the terminal `e'.
Vitamins are classified according to their solubility characteristic into two groups namely the Fat
Soluble vitamins [A, D, E &K] and the Water Soluble Vitamins [B-complex & C]. A normal individual
ingesting a well balanced diet gets adequate supply of these vitamins and needs no supplement.
However diseases caused by primary or dietary deficiency result from inadequate quantities of
vitamins in the diet. Disease such as 'scurvy' caused by deficiency in 'Ascorbic Acid' is alleviated
rapidly by improved diets or supplementary vitamin therapy. Ascorbic acid is the chemical name for
Vitamin C. The minimum daily requirement of vitamin C to prevent deficiency disease such as Scurvy
and disorders is 60 mg/ adult.
Vitamins in excess of the body's ability to utilize them, are generally excreted, but some untoward
effect of 'HYPER VITAMINOSIS' have been shown to occur. But these effects are primarily a function
of fat-soluble vitamin, which are more readily stored in the body.
Vitamin A causing skin Lesions and Irritability
Vitamin D causing calcification of soft tissues
Vitamin E causing increased clotting in patients of Anti-Coagulent therapy.

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Unlike the fat soluble vitamins, the water soluble vitamin like Ascorbic acid cannot be stored in the
body and should be regularly supplied. As a consequence their determination in food &
pharmaceutical preparations has assumed considerable importance.

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BIOLOGICAL ACTIVITIES
The biological role of vitamin C is extensive. It has been found to have effects upon germination and
root growth. Spraying with ascorbic acid has been found to be effective in the protection of plants
against the worst effects of ozone in the atmosphere, produced by photolytic action on polluted air,
particularly in big cities. Fish are unable to synthesize ascorbic acid and this results in the "Broken
Back Syndrome". Large amounts of ascorbic acid are used in Aquaculture.
When Ascorbic Acid is used in the food industry it increases its nutrient value, improves the taste of
fruit drinks and prevents oxidation as a preservative. The addition of ascorbic acid to meat, improves
its colour, flavor and odour, as well as lowering the amounts of nitrite which has to be used in curbing.
As a side effect it has been found that ascorbic alone in co-operation with Tocopherol when used in
meat curing, inhibits the formation of nitroso compounds which are believed to be carcinogenic, while
not interfering significantly with the inhibition by nitrite of a very dangerous micro organism
"Clostridium botulinum". L-ascorbic Acid is very widely used in bread baking, where it is present as a
"Flour Improver". L-Ascorbic Acid has also found a place in industrial processes like polymerization
reaction, in photographic developing and printing, in metal technology and even in intravaginal
contraceptives.

PROCTECTING ROLE OF ASCORBIC ACID

Ascorbic Acid plays a role in protecting against the following:

HEART DISEASE
Results of scientific studies on whether vitamin C is helpful for preventing heart attack or stroke are
mixed. Vitamin C doesn't lower cholesterol levels or reduce the overall risk of heart attack, but
evidence suggests that it may help protect arteries against damage.
Some studies -- though not all -- suggest that vitamin C, acting as an antioxidant, can slow down the
progression of atherosclerosis (hardening of the arteries). It helps prevent damage to LDL ("bad")
cholesterol, which then builds up as plaque in the arteries and can cause heart attack or stroke. Other
studies suggest that vitamin C may help keep arteries flexible.
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In addition, people who have low levels of vitamin C may be more likely to have a heart attack, stroke,
or peripheral artery disease, all potential results of having atherosclerosis. Peripheral artery disease
is the term used to describe atherosclerosis of the blood vessels to the legs. This can lead to pain
when walking, known as intermittent claudication. But there is no evidence that taking vitamin C
supplements will help.The best thing to do is get enough vitamin C through your diet. That way, you
also get the benefit of other antioxidants and nutrients contained in food. If you have low levels of
vitamin C and have trouble getting enough through the foods you eat, ask your doctor about taking a
supplement.

HIGH BLOOD PRESSURE

Population based studies (which involve observing large groups of people over time) suggest that
people who eat foods rich in antioxidants, including vitamin C, have a lower risk of high blood
pressure than people who have poorer diets. Eating foods rich in vitamin C is important for your
overall health, especially if you are at risk for high blood pressure. The diet physicians most frequently
recommend for treatment and prevention of high blood pressure, known as the DASH (Dietary
Approaches to Stop Hypertension) diet, includes lots of fruits and vegetables, which are loaded with
antioxidants.

COMMON COLD
Despite the popular belief that vitamin C can cure the common cold, the scientific evidence doesn't
support the notion. Taking vitamin C supplements regularly (not just at the beginning of a cold)
produces only a small reduction in the duration of a cold (about 1 day). The only other piece of
evidence supporting vitamin C for preventing colds comes from studies examining people exercising
in extreme environments (athletes such as skiers and marathon runners, and soldiers in the Arctic). In
these studies, vitamin C did seem to reduce the risk of getting a cold.

CANCER
Results of many population based studies (evaluating groups of people over time) suggest that eating
foods rich in vitamin C may be associated with lower rates of cancer, including skin cancer, cervical
dysplasia (changes to the cervix which may be cancerous or precancerous, picked up by pap smear),
and, possibly, breast cancer. But these foods also contain many beneficial nutrients and antioxidants,
not only vitamin C, so it's impossible to say for certain that vitamin C is protecting against cancer.
Taking vitamin C supplements, on the other hand, has not been shown to have any helpful effect.
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In addition, there is no evidence that taking large doses of vitamin C once diagnosed with cancer will
help your treatment. Moreover, some doctors are concerned that large doses of antioxidants from
supplements could interfere with chemotherapy medications. More research is needed. If you are
undergoing chemotherapy, talk to your doctor before taking vitamin C or any supplement.

OSTEOARTHRITIS
Vitamin C is essential for the body to make collagen, which is a part of normal cartilage. Cartilage is
destroyed in osteoarthritis (OA), putting pressure on bones and joints. In addition, some researchers
think free radicals -- molecules produced by the body that can damage cells and DNA -- may also be
involved in the destruction of cartilage. Antioxidants such as vitamin C appear to limit the damage
caused by free radicals. However, that said, no evidence suggests that taking vitamin C supplements
will help treat or prevent OA. What the evidence does show is that people who eat diets rich in
vitamin C are less likely to be diagnosed with arthritis.
Taking nonsteroidal anti-inflammatory drugs can lower your levels of vitamin C. If you take these
drugs regularly for OA, you might want to take a vitamin C supplement.

AGE-RELATED MACULAR DEGENERATION

Vitamin C (500 mg) appears to work with other antioxidants, including zinc (80 mg), beta-carotene (15
mg), and vitamin E (400 IU) to protect the eyes against developing macular degeneration (AMD), the
leading cause of legal blindness in people over 55 in the United States. The people who seem to
benefit are those with advanced AMD. It isn't known whether this combination of nutrients helps
prevent AMD or is beneficial for people with less advanced AMD. This combination includes a high
dose of zinc, which should be taken only under a doctors supervision.

PRE-ECLAMPSIA
Some studies suggest that taking vitamin C along with vitamin E may help prevent

pre-eclampsia

in women who are at high risk. Pre-eclampsia, characterized by high blood pressure and too much
protein in the urine, is a common cause of premature births. Not all studies agree, however.

ASTHMA
Studies are mixed when it comes to the effect of vitamin C on asthma. Some show that low levels of
vitamin C are more common in people with asthma, leading some researchers to think that low levels
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of vitamin C might increase the risk for this condition. Other studies seem to show that vitamin C may
help reduce symptoms of exercise-induced asthma.

OTHER
Although the information is limited, studies suggest that vitamin C may also be helpful for:

Boosting immune system function

Maintaining healthy gums

Improving vision for those with uveitis (an inflammation of the middle part of the eye)

Treating allergy-related conditions, such as asthma, eczema, and hay fever (called allergic rhinitis)

Reducing effects of sun exposure, such as sunburn or redness (called erythema)

Alleviating dry mouth, particularly from antidepressant medications (a common side effect from these
drugs)

Healing burns and wounds

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IMPORTANT DIETARY SOURCES OF VITAMIN C

Dosage Level

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MAINTENANCE OF HEALTH
The recommended daily dietary allowances [ RAD's ] over the past few decades have ranged from a
little as 30 mg per day to thousand times as much, depending on the changing individual, national
and international opinion.
The quantitative analysis of vitamin is particularly useful in the evaluation of human and animal
nutrition. The development of practical methods for the assay of vitamins eg. In blood and urine or in
the organs of experimental animals is of interest in medicine. Finally the increase use of vitamin in the
pharmaceutical and food stuff industries, has given a further stimuli to the perfection and wider
employment methods for their determination in the analytical laboratories of universities, Industries
and Government establishments.
There are a no of procedure available for the determination of Vitamins. These comprise physical,
chemical, bio-chemical, biological and quantitative determinations. The choice of the methods must
always be determined by the individual properties and composition of the material under examination.
So, there is a continuing interest in the development of newer analytical methods for the analysis of
vitamin.
One of the most fruitful developments in the biological field has been the study of methods for
measuring the potency of vitamins. Biological, bio-chemical and microbial assays procedures are
highly selective and sensitive and seldom require sample separation. The biological method which
has been used as a primary tool in the discovery of the vitamin still finds application for quantitative
vitamin assays and for the evaluation and standardization of other methods.
The primary requirements of a biological method is to feed a group of comparable animals a special
diet which is deficient only in the vitamin under study and is otherwise well balanced with respect to
other components. The biological methods for the estimation of vitamin C is specific for Antiascorbutic
activity and will measure the total amount of vitamin C present i.e. both in the reduced form (ascorbicacid) and the oxidized form (dehydro ascorbic acid). A method based upon the determination of
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minimum amounts of sample needed to prevent signs of Scurvy was originally devised by Sherman,
Lamer, Campbell and Smith.
Many modified procedures were subsequently suggested and of these the most frequently used are
the preventives dental histology method and the curative growth method. In all these cases guinea
pigs are used as experimental animals. A further quantitative bio assay has been devised, based on
the observation that the level of serum 'Alkaline Phosphate' i.e. the enzyme with an optimum activity
at Ph8.6 to 9.0, falls considerably in Scurvy and increases again when ascorbic acid is administered.
A special type of biological method for the determination of vitamin C is the Microbiological method in
which micro organisms are used as test objects. The amount of vitamin needed is considerably
smaller and this method is relatively short period of time. Biochemical methods for the determination
of vitamin are defined as procedure in which the vitamin is determined by the part it takes in certain
biochemical reactions. The above described biological, biochemical and microbial assay procedure
are highly selective and sensitive but are elaborate and time consuming. They are require special skill
in handling various reacting systems like animals, enzymes and micro organisms. In addition to the
above disadvantage, their limits of error are often large. So these methods are not suitable for routine
analysis of vitamins. However they are very much useful as Reference methods and for the
Standardization of various methods.
In the last 2 decades , chemical or physico-chemical methods have been increasingly replacing
biological methods for routine investigations i.e. in the pharmaceuticals industry. These methods
included gravimetric , volumetric electro analytical and optical methods . The chemical methods are
nearly always quicker and cheaper and their limits of error generally closer than in biological method.
However owing to their poor selectivity and sensitivity, chemical and physical methods must be used
only with certain precautions.
These are numerous titrimetric procedures available for the estimation of Ascorbic acid and are
based mainly on its reducing property. Earlier methods have been reviewed by Olliver. One group of
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methods is based on the colour change when the sample solution with or without preliminary
treatment is titrated against an indicator. Among the substances recommended for this purposes are
2,6 dichlorophenol indophenols , iodine, methylene blue , phosphotungsic acid , phosphomolybdic
acid , ferripyridyl thionine and chloranil.
Many other oxidising agents have also been used for the estimation of ascorbic acid. By direct
titration (using a suitable indicator) or by the lodometric method wherein the excess oxidizing agent
employed is estimated.

GENERAL PROPERTIES
The water soluble vitamin- ascorbic acid has been chosen for the present study and its important
general properties are given below in table.

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STRUCTURE OF ASCORBIC ACID C6H8O6

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When ascorbic acid is boiled with HC1, the crystals gave quantitative yield of furfural showing that
atleast five carbon atoms formed unbranched chain. Further tests showed, ascorbic acid to be a weak
monobasic acid and strong reducing agent. The product of such oxidation, involving the loss of two
hydrogen atoms produced C6H806 named as Dehydro Ascorbic Acid.
Although the crystals are quite stable, a water solution of ascorbic acid will undergo destruction when
exposed to air, heat, light or metals like Copper & Iron. It is unstable in alkali but relatively stable in
acidic medium.
The L isomer ( Dehydro Ascorbic Acid ) is physiologically another stable chemical form of the vitamin
erythrobic acid, ( D Arabo Ascorbic Acid ). This is sometimes used as an anti Oxidant in food
processing. The sum of the vitamin effect is from both Ascorbic Acid and Dehydro Ascorbic Acid.

CHEMISTRY
The most important and prominent chemical property of ascorbic acid

(L-threo- 2, 3, 4

,5 ,6-pentahydroxy-2-hexanoic acid-4- lactone ) is its strong reducing property. This is due to the
liberation of hydrogen atom from the ene-diol-hydroxyl group, the ascorbic acid being oxidized to
dehydroascorbic acid (eg. By halogens in acidic or neutral solutions. This reaction is readily reversed
by reducing agents in vitro (eg. By (H2S)) and invivo( eg. By HS Compounds such as gluthathione). In
alkaline, neutral or acidic solutions ( above ph 5) dehydroascorbic acid undergoes hydrolysis, with the
splitting of the lactone ring to form diketoglutonoic acid which is biologically inactive. This reaction is
irreversible invivo but can be reversed invitro by HI. Diketoglutonoic acid undergoes further
irreversible oxidation to oxalic acid and

L-threonic acid. These reactions are represented in

the following figures.

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REACTIONS OF ASCORBIC ACID

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EFFECT OF DILUTION
The presence of chelating agents, reduces the oxidation of Ascorbic acid. Ascorbic acid reacts with
dioxygen in the presence of metals, particularly transition metals. Hence vitamin solution should not
be stored in copper containers. Addition of EDTA prevents oxidation of Ascorbic acid by reacting with
these metals.

ESTIMATION OF ASCORBIC ACID IN JUICES

Ascorbic acid is widely distributed in plant and animal tissue. The most important sources of this
vitamin are citrus fruits, tomatoes, ripe berries, potatoes and green leafy vegetables.
Its function in metabolic process is based on the fact that ascorbic acid and dehydro ascorbic, inter
convert readily in plant and animal tissues. Dehydroascorbic is a product of oxidation of ascorbic acid.
NaOH can bring about the hydrolysis of ascorbic acid as follows
C6H8O6 + NaOH

C6H7O6 + H2O

The above reaction has been used as basis for estimation of ascorbic acid.

EFFECT OF FOOD PRESERVATION

The particular manner in which a food is preserved, eg. By freezing, by canning or by drying (
dehydration ), has a distinct and significant effect on its ascorbic acid content. This fact can be well
illustrated by comparing the vitamin values of fresh, and frozen food stuff When frozen, the ascorbic
acid content decreases.

EFFECT OF STORAGE
The medium of storage and temperature of storage also affects the ascorbic acid content in food
stuff. For example, fruits such as cherries and Peaches when stored longer than 4 months at +10 F
have known to lose marked amount of ascorbic acid. However the same fruits when stored at 0 to -20
F did not suffer any appreciable loss of vitamin within the same period of time.
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Orange juice and other citrus fruit juices lose negligent amounts of ascorbic acid when stored in
refrigerators. This is due to the presence of citric acid in the juice which acts as a preservative.
Canned foods also lose appreciable amount of acid when stored at elevated temperature. Food stuff
exposed to light, lose more amount of ascorbic acid than those in closed containers.

MATERIALS REQUIRED
1. 500 ml measuring cylinder
2. 25 ml measuring cylinder
3. 100 ml coloured bottle
4. 100 ml colourless bottle
5. 100 ml standard flask
6. 500 ml standard flask
7. 500 ml beaker
8. 20 pipette
9. conical flask
10. burette
11. mortar and pestle.

CHEMICALS REQUIRED
I.

Fruit juice samples

II.

phenolphthalein

III.

Distilled water

IV.

NaOH(0.1N) solution.

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PREPARATION OF SOLUTIONS
1) SODIUM HYDROXIDE SOLUTION
Four grams of NaOH crystals were weighed into a standard flask. The solution was then made to one
liter by adding water. The resulting solution was that of 0.1 N NaOH.

2) FRUIT JUICE SOLUTIONS

The fruits were cut into slices and then squashed using mortar and pestle. Seeds were removed and
the solutions were diluted to 100cc using distilled water.

PROCEDURE
INDICATOR
Phenophthalein is used as the indicator for the titrations. End point is taken as the point of change of
clour from colourless to pink.

INITIAL PREPARATIONS
Take a burette fitted with a glass top and wash it with distilled water and then rinse it with Sodium
Hydroxide. Now clamp it to the burette stand. Using a funnel fill it with NaOH. The initial reading is
taken as the one against meniscus. The pipette is rinsed with distilled water and 20cc of juice is
pipetted out.

EXPERIMENT 1:
20 ml of the juice is taken in a conical flask and 0.1 N NaOH is taken in the burette and the titration
was carried out till the end point was reached.

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EXPERIMENT 2:
EFFECT OF REFRIGERATION
20 ml of the juice which was kept in a refrigerator is taken in a conical flask and 0.1 N NaOH is taken
in the burette and the titration was carried out till the end point was reached.

EXPERIMENT 3:
EFFECT OF STORAGE
20 ml of the juice which was kept in the open is taken in a conical flask and 0.1 N NaOH is taken in
the burette and the titration was carried out till the end point was reached.

EXPERIMENT 4(ORANGE-A):
15 ml of the juice was diluted with water and 20 ml was taken in a conical flask and

0.1 N NaOH is

taken in the burette and the titration was carried out till the end point was reached.

EXPERIMENT 5(ORANGE-B):
20 ml of the juice solution from orange B is taken in a conical flask and 0.1 N NaOH is taken in the
burette and the titration was carried out till the end point was reached.

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SAMPLES OBTAINED BY TITRATING DIFERRENT JUICES

AMLA JUICE

AMLA JUICE(2)

LIME JUICE(COLD)

LIME JUICE(STORAGE)

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ORANGE JUICE(COLOUR BOTTLE)

LIME JUICE(COLD)(2)

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ORANGE JUICE(COLD)

ORANGE JUICE(COLD)(2)

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EXPERIMENTAL RESULTS
TITRATION OF 20ml OF AMLA JUICE
SHORT PROCEDURE
PIPETTE SOLUTION: Amla juice
BURETTE SOLUTION: Sodium hydroxide
REACTION TEMPERATURE: Room Temperature
REACTION MEDIUM: Neutral
INDICATOR: Phenolphthalein
END POINT: Colourless to Pink

V1N1=V2N2
V1= 14.5 ml
N1= 0.1 N
V2= 20 ml
N2= 0.0725 N
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TITRATION OF 20ml OF LIME JUICE AFTER COLD STORAGE

SHORT PROCEDURE
PIPETTE SOLUTION: Lime Juice
BURETTE SOLUTION: Sodium hydroxide
REACTION TEMPERATURE: 100C to 150C
REACTION MEDIUM: Neutral
INDICATOR: Phenolphthalein
END POINT: Colourless to Pink

V1N1=V2N2
V1=16 ml
N1=0.1 N
V2=20 ml
N2=0.08 N

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TITRATION OF 20ml OF LIME JUICE KEPT EXPOSED TO AIR

SHORT PROCEDURE
PIPETTE SOLUTION: Lime Juice
BURETTE SOLUTION: Sodium hydroxide
REACTION TEMPERATURE: Room Temperature
REACTION MEDIUM: Neutral
INDICATOR: Phenolphthalein
END POINT: Colourless to Pink

V1N1=V2N2
V1=10 ml
N1=0.1 N
V2=20 ml
N2=0.05 N

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TITRATION OF 20ml OF ORANGE JUICE AFTER COLD STORAGE

SHORT PROCEDURE
PIPETTE SOLUTION: Orange Juice
BURETTE SOLUTION: Sodium hydroxide
REACTION TEMPERATURE: 100C to 150C
REACTION MEDIUM: Neutral
INDICATOR: Phenolphthalein
END POINT: Colourless to Pink

V1N1=V2N2
V1=8 ml
N1=0.1 N
V2=20 ml
N2=0.04 N

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TITRATION OF 20ml OF ORANGE JUICE KEPT IN COLOURED BOTTLE

SHORT PROCEDURE
PIPETTE SOLUTION: Orange Juice
BURETTE SOLUTION: Sodium hydroxide
REACTION TEMPERATURE: Room Temperature
REACTION MEDIUM: Neutral
INDICATOR: Phenolphthalein
END POINT: Colourless to Pink

V1N1=V2N2
V1=6 ml
N1=0.1 N
V2=20 ml
N2=0.03 N

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CONCLUSION
It was observed that the concentration of Ascorbic Acid decreased with dilution.
The concentration also decreased with the increase in temperature
Juices stored in coloured bottle lost some amount of Ascorbic Acid

Thus, we have estimated the effect of temperature, dilution, light and freezing on the content of
Ascorbic Acid in citrus fruits

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BIBLIOGRAPHY

BOOKS
1. THE DETECTION AND MEASUREMENT OF VITAMIN C by J.R CROOKE and R.E.D
MOXON
2. VITAMIN 'C' - ITS BIOCHEMISTRY by ROBERT.C
3. HANDBOOK OF VITAMINS AND HORMONES by KUTSY.R.J
4. ANALYTICAL BIOCHEMISTRY by RICHARD.C.R AND DAVID.C.N

WEBSITES
1. www.google.com
2. www.wikipedia.com

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