Laura's Vit C Project

THE UNIVERSITY OF THE WEST INDIES
ST. AUGUSTINE
DEPARTMENT OF CHEMICAL ENGINEERING
CHEMICAL ENGINEERING RESEARCH PROJECT
CHNG 3013
DETERMINATION OF THE VITAMIN C CONTENT

OF FOODSTUFF SOLD LOCALLY
SUPERVISOR: MR. J. SMITH
SUBMITTED BY:
LAURA CHARLES
ID#: 05714774
April 18, 2008
ABSTRACT
Vitamin C has been proven to be one of the most effective antioxidants and is considered to be
an essential part of the human diet. It is therefore important for individuals to ensure that their
Recommended Daily Allowance for the vitamin is obtained from the foods which they purchase.
This report was primarily carried out to determine the vitamin C content of foods produced
locally. Internationally manufactured foods were also tested and the two sets of findings were
compared. The companies being considered in this report are Fruta Kool Kidz, Orchard, KoolAid, Flavor Aid, Sunshine and Kelloggs. The three methods utilized for the analysis of cereals
and juices were: Iodine test, 2, 6-Dichloroindophenol Titration and a pH Potentiometry test. It
was observed that all products showed some level of deviation from their labeled values. It was
recommended that more accurate methodologies for the vitamins analysis such as the HPLC
techniques should be employed.
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TABLE OF CONTENTS
ABSTRACT......................................................................................................... II
LIST OF FIGURES............................................................................................... V
LIST OF TABLES...............................................................................................VII
CHAPTER 1........................................................................................... INTRODUCTION
9
1.1 OBJECTIVES................................................................................................................... 10
CHAPTER 2...................................................................................................... THEORY
11
2.1 INTRODUCTION............................................................................................................... 11
2.2 HISTORY....................................................................................................................... 12
2.2.1 Work of Pauling.................................................................................................. 13
2.3 PROPERTIES................................................................................................................... 13
2.3.1 General Properties............................................................................................. 13
2.3.2 Stability.............................................................................................................. 14
2.4 IMPORTANCE OF VITAMIN C IN NUTRITION............................................................................15
2.4.1 Biological Activities of Vitamin C........................................................................15
2.4.2 Recommended Intake of Vitamin C....................................................................17
2.4.3 Dietary Sources of Vitamin C.............................................................................17
2.5 METHODS OF ANALYSIS.................................................................................................... 18
2.5.1 Oxidation-Reduction Methods............................................................................18
2.5.2 Potentiometry using pH Analysis........................................................................19
2.5.3 High Performance liquid Chromatography.........................................................20
CHAPTER 3............................................................................. METHODS OF ANALYSIS
21
3.1 2, 6-DICHLOROINDOPHENOL TITRATION..............................................................................21
3.1.1 Reagents............................................................................................................ 21
3.1.2 Procedure........................................................................................................... 22
3.1.3 Preparation of Cereal Sample.............................................................................22
3.1.4 Assay of Extract................................................................................................. 22
3.2 IODINE TEST.................................................................................................................. 25
3.2.1 Reagents............................................................................................................ 25
3.2.2 Procedure........................................................................................................... 25
3.3 POTENTIOMETRY............................................................................................................. 27
3.3.1 Reagents............................................................................................................ 27
3.3.2 Procedure........................................................................................................... 27
CHAPTER 4.................................................................... RESULTS AND CALCULATIONS
29
4.1 2, 6-DICHLOROINDOPHENOL TITRATION..............................................................................29
4.1.1 Ascorbic Acid Standard......................................................................................29
4.1.2 Laboratory Results for Sunshine Cereals............................................................29
4.1.3 Results based on Calculations for Sunshine Cereals..........................................32
4.1.4 Laboratory Results for Kelloggs Cereal..............................................................35
4.1.5 Results based on Calculations for Kelloggs Cereal............................................37
4.2 IODINE TITRATION........................................................................................................... 40
4.2.1 Ascorbic Acid Standard......................................................................................40
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4.2.2 Laboratory results for Fruta Kool Kidz................................................................41
4.2.3 Results based on Calculations for Fruta Kool Kidz drinks....................................44
4.2.4 Laboratory results for Orchard...........................................................................48
4.2.5 Results based on Calculations for Orchard drinks..............................................49
4.2.6 Laboratory results obtained for powdered drinks...............................................52
4.2.7 Results based on Calculations for Powdered drinks............................................54
4.3 POTENTIOMETRY METHOD................................................................................................ 56
4.3.1 Laboratory results for Sunshine Cereals.............................................................56
4.3.2 Laboratory results for Juices...............................................................................59
CHAPTER 5........................................................................... DISCUSSION OF RESULTS
61
CHAPTER 6......................................................... CONCLUSION & RECOMMENDATIONS
65
6.1 CONCLUSION................................................................................................................. 65
6.2 RECOMMENDATIONS........................................................................................................ 66
REFERENCES.................................................................................................... 67
APPENDIX A..................................................................................................... 68
APPENDIX B..................................................................................................... 69
APPENDIX C..................................................................................................... 78
6.3 SAMPLE CALCULATIONS.................................................................................................... 78
6.3.1 Standardization of NaOH....................................................................................78
6.3.2 Batch 1 of Orange Juice by Fruta Kool Kidz.........................................................78
NOMENCLATURE...............................................................................................80
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LIST OF FIGURES
Figure 2- 1: Structure of L-ascorbic acid................................................................................12

Figure 2- 2: Diagram of the oxidative and reduced structure of Vitamin C............................13
Figure 3- 1: L-ascorbic acid reactions important to vitamin C analysis. Fig. adapted from
Pearsons Composition and Analysis of Foods, pp 232..........................................................21
Figure 3- 2: Photo taken of the setup of apparatus and reagents used for the DCIP analysis
of vitamin C........................................................................................................................... 22
Figure 3- 3: Photo showing colour change of cereal. This is the colour indicating that reaction
is near its end point.............................................................................................................. 22
Figure 3- 4: Photo taken of setup of apparatus and reagents used in the Iodine test for
Vitamin C.............................................................................................................................. 24
Figure 3- 5: Photo showing the change to blue-black of sample indicating reactions end
point...................................................................................................................................... 24
Figure 3- 6: Photo taken of setup of apparatus and reagents used in the Potentiometry test
for Vitamin C......................................................................................................................... 26
Figure 4- 1: Graph demonstrating the deviation of the Vitamin C content in Sunshine Frosted
Flakes from the stated value (represented by the solid purple line) of 25%..........................32
Figure 4- 2: Graph demonstrating the deviation of the Vitamin C content in Sunshine Raisin
Bran from the stated value (represented by the solid purple line) of 30%............................33
Figure 4- 3: Graph demonstrating the deviation of the Vitamin C content in Sunshine Froot
Ooos from the stated value (represented by the solid purple line) of 20%...........................33
Figure 4- 4: Graph demonstrating the deviation of the Vitamin C content in Kelloggs Frosted
Flakes from the stated value (represented by the solid purple line) of 10%..........................37
Figure 4- 5: Graph demonstrating the deviation of the Vitamin C content in Kelloggs Froot
Loops from the stated value (represented by the solid purple line) of 20%...........................38
Figure 4- 6: Graph demonstrating the deviation of the Vitamin C content in Kelloggs Special
K from the stated value (represented by the solid purple line) of 25%..................................38
Figure 4- 7: Graph demonstrating the deviation of the Vitamin C content in Fruit Punch made
by Fruta Kool Kidz from the stated value (represented by the solid purple line) of 80%........45
Figure 4- 8: Graph demonstrating the deviation of the Vitamin C content in Orange Juice
made by Fruta Kool Kidz from the stated value (represented by the solid purple line) of
100%..................................................................................................................................... 45
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Figure 4- 9: Graph demonstrating the deviation of the Vitamin C content in Guava Pine made
by Fruta Kool Kidz from the stated value (represented by the solid purple line) of 80%........46
Figure 4- 10: Graph demonstrating the deviation of the Vitamin C content in Grape Juice
made by Fruta Kool Kidz from the stated value (represented by the solid purple line) of
100%..................................................................................................................................... 46
Figure 4- 11: Graph demonstrating the deviation of the Vitamin C content in Fruit Punch
made by Orchard from the stated value (represented by the solid purple line) of 52%.........50
Figure 4- 12: Graph demonstrating the deviation of the Vitamin C content in Black Currant
made by Orchard from the stated value (represented by the solid purple line) of 100%.......50
Figure 4- 13: Graph demonstrating the deviation of the Vitamin C content in Orange juice
made by Orchard from the stated value (represented by the solid purple line) of 70%.........51
Figure 4- 14: Graph demonstrating the deviation of the Vitamin C content in Pineapple made
by Flavor Aid from the stated value (represented by the solid purple line) of 10%...............54
Figure 4- 15: Graph demonstrating the deviation of the Vitamin C content in Lemonade made
by Kool-Aid from the stated value (represented by the solid purple line) of 10%..................54
Figure 4- 16: Graph demonstrating the standardization of NaOH using standard HCl...........59
Figure 4- 17: Graph showing pH curve obtained for Orange Juice by Fruta Kool Kidz.............59
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LIST OF TABLES
Table 2- 1:Dietary reference intake values for vitamin C in mg, by life stage and sex.............9
Table 2- 2: Physical Properties of L-Ascorbic Acid..................................................................12
Table 3- 1: Chemicals used in DCIP analysis of Vitamin C......................................................20
Table 4- 1: Titration results obtained for Ascorbic acid standard...........................................29
Table 4- 2: Titration results obtained for Frosted Flakes produced by Sunshine.....................29
Table 4- 3: Titration results obtained for Raisin Bran produced by Sunshine.........................30
Table 4- 4: Titration results obtained for Froot Ooos produced by Sunshine.........................30
Table 4- 5: Results obtained for the calculation of the Vitamin C content in three batches of
Frosted Flakes....................................................................................................................... 32
Raisin Bran............................................................................................................................ 32
Froot Ooos............................................................................................................................ 33
Table 4- 8: Titration results obtained for Kellogg's Frosted Flakes..........................................35
Table 4- 9: Titration results obtained for Kellogg's Froot Loops..............................................35
Table 4- 10: Titration results obtained for Kellogg's Special K................................................36
Frosted Flakes....................................................................................................................... 37
Froot Loops............................................................................................................................ 37
Special K............................................................................................................................... 38
Table 4- 14: Titration results for Ascorbic acid standard........................................................40
Table 4- 15: Titration results obtained for Fruit Punch produced by Fruta Kool Kidz...............41
Table 4- 16: Titration results obtained for Orange Juice produced by Fruta Kool Kidz............41
Table 4- 17: Titration results obtained for Guava Pine produced by Fruta Kool Kidz...............42
Table 4- 18: Titration results obtained for Grape Juice produced by Fruta Kool Kidz..............42
Fruit Punch............................................................................................................................ 44
Orange Juice.......................................................................................................................... 44
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Guave Pine............................................................................................................................ 45
Grape Juice............................................................................................................................ 45
Table 4- 23: Titration results obtained for Fruit Punch produced by Orchard.........................48
Table 4- 24: Titration results obtained for Black Currant produced by Orchard......................48
Table 4- 25: Titration results obtained for Orange Juice produced by Orchard.......................49
Fruit Punch............................................................................................................................ 49
Black Currant........................................................................................................................ 50
Orange juice.......................................................................................................................... 50
Table 4- 29: Titration results obtained for Pineapple produced by Flavor Aid........................52
Table 4- 30: Titration results obtained for Lemonade produced by Kool-Aid..........................53
Pineapple by Flavor Aid......................................................................................................... 54
Lemonade by Kool-Aid........................................................................................................... 54
Table 4- 33: pH results obtained for Batch 1 of Frosted Flakes produced by Sunshine...........56
Table 4- 34: pH results obtained for Batch 1 of Raisin Bran produced by Sunshine...............57
Table 4- 35: pH results obtained for Batch 1 of Frosted Flakes produced by Kelloggs...........58
Table 4- 36: pH results obtained for Orange juice produced by Fruta Kool Kidz.....................59
Table A- 1:Products tested (packaged mass/volume included)..............................................68
Table B 1: pH results obtained for Batch 2 of Frosted Flakes produced by Sunshine.............70
Table B 4: pH results obtained for Batch 2 of Raisin Bran produced by Sunshine..................73
Table B 7: pH results obtained for Apple juice produced by Fruta Kool Kidz...........................76
Table B 8: pH results obtained for Guava Pine produced by Fruta Kool Kidz..........................77
Table B 9: Standardization of NaOH using standard HCl........................................................78
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Laura Charles
ID#: 05714774
Chapter 1
INTRODUCTION
Vitamin C or L-ascorbic acid, as it is sometimes referred to, is an essential part of the human
diet. It is a powerful anti-oxidant and is thus necessary for increasing the bodys immunity to
diseases and maintaining a healthy lifestyle. In order for this to be achieved, the RDA
(Recommended Dietary Allowance) for adults for Vitamin C is:
75 mg per day for women,
90 mg for men.
In light of this, an important question which must be answered is: Does one truly receive
his/her RDA from manufactured products? Thus, one of the major aims of this report was to
determine the Vitamin C content in foods produced in Trinidad and Tobago. This was to ensure
the validity of the manufacturers labels.
Two types of foods from various local and international companies were tested. These
included cereals, fruit drinks and powdered drinks. The local companies investigated were
Sunshine Snacks, Fruta Kool Kidz and Flavor Aid. The international companies included
Kelloggs, Orchard and Kool-Aid.
This report also compared the consistency of the Vitamin C content in each product by
performing tests on various batches of the same flavor. Each local and international products
PDV (Percentage Daily Value) was compared to the RDA. Finally, each local products vitamin
content was compared to that of the international product in order to determine the better buy
with respect to Vitamin C content.
Three different methods which were used to obtain the vitamins content included:
Potentiometry via pH analysis
Indophenol method or DCIP
Iodine Test
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However, each of these methods was limited in their accuracy of the actual PDV
obtained. Such limitations are discussed in Chapter 2. The most accurate method which should
have been employed is known as the High Performance Liquid Chromatography test. This
method is used worldwide by pharmaceutical companies and for L-ascorbic analysis of
biological samples. Unfortunately, due to limitations within the laboratory this method could not
have been employed.
This report focuses not only on the analysis of Vitamin C content but also on its
importance in the diet. It highlights the importance of the PDV labeling on each product and
indicates how nutritious a product really is and if one is getting his or her moneys worth for the
purchase.
1.1 Objectives
In light of the recommended dosages for both children and adults, it was important to test for the
accuracy of the labeling for nutritional information placed on locally manufactured food and
beverages produced in Trinidad and Tobago. The following lists the objectives of this report.
1. To collect a variety of packaged foods and beverages produced locally in Trinidad and
Tobago which contains Vitamin C.
2. To perform laboratory tests for Vitamin C on such foods and beverages.
3. To determine the validity and consistency of a Companys nutritional labeling on each
product.
4. To compare the results of these tests in several ways such as:
a. Products from a specific company purchased in different batches from various
supermarkets throughout the country.
b. Products from local companies to that of international companies.
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Chapter 2
THEORY
2.1 Introduction
Ascorbic acid is present in all animals and higher plants. Humans and certain vertebrates such as
monkeys and certain fishes have special or specific requirements for the acid whereas other
species synthesize the compound. Large concentrations of ascorbic acid can be found in the
tissues of both plants and animals. A few dietary sources of the vitamin are: citrus fruits, peppers,
tomatoes, potatoes and leafy greens. Poor Sources include: dairy products, meats and cereal
grains.
The RDA (Recommended Dietary Allowance) can be defined as the dietary intake level
that is sufficient to meet nutrient requirements of 97-98% of healthy individuals in a particular
life stage and gender group. A breakdown of the RDA for various life stages and gender are
given in Table 2-1[1]. An additional amount of Vitamin C per day has to be added for smokers
due to an increase in their rate of oxidative stress and metabolic turnover of the vitamin.
Table 2- : 1Dietary reference intake values for vitamin C in mg, by life stage and gender
Adapted from Vitamin C: Functions and Biochemistry in animals and plants by H. Asard et al., 2004, pp. 299.
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2.2 History
The discovery of Vitamin C or Ascorbate (ASC) was first made by Charles King and Albert
Szent-Gyorgyi in 1932. It was later synthesized by Haworth and Hirst in 1933.
The importance of Vitamin C was observed many years before its actually discovery. In
fact, the benefits of citrus fruits were first reported by a British Naval physician, James Lind in
his Treatise on Scurvy in 1753. During the early days of the slave trade, the lack of the vitamin
led to a condition nicknamed calamity of sailors. Today this disease is termed Scurvy and is
characterized by weakness, small hemorrhages throughout the body which cause the bleeding of
gums and skin and the loosening of teeth.
The first trial of inducing the disease Scurvy on guinea pigs was made in 1907. An
animal analysis based on the pigs response was developed in 1917 in order to test the antiscorbic activity of foods. The acid was initially isolated from natural sources shown to be the
anti-scorbutic factor and then structurally characterized by Szent-Gyorgyi Haworth and King in
the 1930s.
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In 1956, the Free radical Theory of Aging was proposed. Free radicals from both nonenzymatic reactions in and outside of cells contribute to the aging process. Evidence attained in
the late 1990s implicates reactive oxygen and nitrogen species as contributing factors to both
chronic diseases and age associated functional declines.
ASC has been tested under many
different oxidizing conditions and has proven to be the most effective endogenous aqueous phase
antioxidant in human plasma.
In 2002, Ames et al discovered that the use of high concentrations of the vitamin
component of various co-enzymes 3 can remedy 50 genetic diseases involving defective enzymes.
This discovery was in support of Linus Paulings hypothesis of orthomolecular medicine. That
is, the preservation of good health and the treatment of disease by varying the concentrations in
the human body of substances that are normally present in the body [1]. This meant that some
types of illnesses such as mental illness may be effectively treated by providing the optimum
concentration of micro-nutrients such as Vitamin C and various types of Vitamin B in the brain.
According to Dr. Norton Nelson et al Vitamin C was shown to inhibit the formation of
nitrosamines [2]. These nitrosamines are the most powerful chemical carcinogens found in
laboratory animals and are formed by the reaction of nitrate and nitrite salts (used as food
preservatives) with chemicals in the body. It was also noted that one of the main sources of
dietary nitrates is vegetables and can help protect against the development of gastric cancer
possibly because of their Vitamin C content.
2.2.1 Work of Pauling
He focused his attention of Vitamin C in the mid-1960s due to the vitamins multiple
biochemical functions but lack of clinical literature in the previous years. By using not only
cross-species comparisons but also the Vitamin C content of diets based on raw plant foods, he
argued that a greater optimal intake of the vitamin should be ingested as opposed to the current
recommended dietary allowance4. He believed that adequate amounts of supplemented Vitamin
C would help prevent colds or shorten their duration [1].
Frei et al, 1989 and 1991

The non-protein portion of an enzyme required for the enzymes activities.
4
Pauling 1970
3
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2.3 Properties
2.3.1 General Properties
L-ascorbic acid is the active form of Vitamin C and is the trivial name for the vitamin which is
the accepted biochemical nomenclature. Its formula is C 6H8O6 and its structure is given in Fig.
(2-1). Vitamin C is the term given to compounds which exhibit full or partial biological activity
of L-ascorbic acid. This includes esters of ascorbic acid which exhibit 100% relative activity and
the oxidized form of L-ascorbic acid, dehydroascorbic acid with 80% relative activity. The
primary dietary sources of the vitamin are L-ascorbic acid and dehydroascorbic acid [3].
Figure 2- : Structure of L-ascorbic acid
Physical properties of L-ascorbic acid are summarized in Table 2-2. L-ascorbic acid is a
white to slightly yellow crystalline powder with high water solubility at room temperature. The
functional roles of this form of the acid include its uses as a nutritional food additive,
antioxidant, flavor and colour stabilizer, to name a few.
Table 2- : Physical Properties of L-Ascorbic Acid5

Substance
Ascorbic acid
CAS No. 50-81-7
Molar
Mass
176.13
Solubility
Sol in water
30 g/100 mL
Melting
Point oC
190-192
Crystal
Form
Monoclinic
platelets and
needles
max nm
245
265
Solvent
Water, pH 2.0
Water, pH 6.4
Adapted from Vitamin Analysis for the Health and Food Sciences by R.R. Eitenmiller & W.O. Landen, Jr., pp. 227
Common or generic name; CAS No.- Chemical Abstract Service number
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White or
yellow
2.3.2 Stability
When water activity remains low, crystalline L-ascorbic acid is highly stable in the presence of
oxygen. However, in solution the strong reducing properties of the acid can lead to rapid and
excessive oxidation changes with conversion to dehydroascorbic acid [3]. Fig. 2-2 shows both
the reduced and oxidized form (dehydroascorbic acid) of the acid. It is one of the simplest
vitamins in structure and readily undergoes oxidation due its instability as a compound.
In foods, pH has a great influence on the compounds stability. Maximal stability usually
occurs between pH 4 and pH 6. Cooking losses depend upon degree of heating, surface area
exposed to water and oxygen, presence of transition metals and many other factors. Browning in
a food product can reduce vitamin C content significantly.
Figure 2- : Diagram of the oxidative and reduced structure of Vitamin C
2.4 Importance of Vitamin C in Nutrition

2.4.1 Biological Activities of Vitamin C
Vitamin C is a water soluble antioxidant and is very important for protecting the body.
Oxidizing species attack the body in a variety of ways, e.g. smog and smoke both contain high
levels of oxidizing molecules which can cause tissue damage. Thus in its defense, the body
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absorbs extra vitamin C in response to an infection which then aids in destroying the infecting
organisms.
Vitamin C is valued for its role as a reducing agent or electron donor. It is known to be a
specific electron donor for eight human enzymes [4]. The disease Scurvy is directly related to
the malfunctioning of three of those enzymes. The vitamin is necessary for the maximal activity
of two enzymes required for the biosynthesis of L-carnitine. L-carnitine transports activated
fatty acids across the inner mitochondrion membrane and as such plays critical roles in the
modulation of energy metabolism. Early signs of scurvy are fatigue and lethargy 6 and are likely
related to carnitine deficiency.
Other symptoms of the vitamins deficiency include
neuropsychiatric changes such as depression, mood swings and hypochondria7.

Vitamin C also plays a role in the metabolism of cholesterol to bile acids. Resent
findings indicate that sufficient vitamin C may contribute to the prevention of cardiovascular
diseases. Other studies suggest that the consumption of high intakes of vitamin C can help lower
the risks of cataracts, and that vitamin C therapy may promote mental function in people with
Alzheimers disease.
However, according to the Institute of Medicine Report, not enough
evidence is available to support the fact that vitamin C will help delay or prevent the onset of
such diseases. Many research programs are currently investigating these issues [A].
2.4.1.1
Antioxidant Activity
An antioxidant is defined as a substance that, when present at low concentrations compared

with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate
[4]. This oxidation process is desired to be prevented since it is responsible for the damage done
to cells in the body by oxygen.
As discussed previously, the oxidation of ascorbic acid produces dehydroascorbic acid.
However, an intermediate compound which is neither strongly oxidizing nor strongly reducing
exists between the two acidic compounds. This intermediate is known as the ascorbyl radical.
The reduction potentials of this radical and ascorbic acid enable them to react with and reduce
Extreme exhaustion
A delusional and obsessive condition in which one believes that he/she is ill when no objective signs of illness can
be observed.
7
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virtually all physiologically relevant reactive oxygen species (ROS) and reactive nitrogen species
(RNS) which includes ozone and nitrogen dioxide [4].
2.4.1.2
Iron Absorption
Iron deficiency is the most common nutrient deficiency in the world. Consistent findings show
that iron absorption is enhanced from a single meal in the presence of vitamin C. This may be
because insoluble complexes are less likely to be formed when ascorbate reduces iron. However,
further research is required to determine whether increasing the intake of vitamin C is an
effective strategy for improving iron nutritional status [4].
2.4.1.3
Excretion
The primary route of excretion for ascorbate and its metabolites in humans is urine due to its
solubility in water.
2.4.2 Recommended Intake of Vitamin C

As discussed in Section 2.1, Table 2-1 gives a breakdown of the RDA for various life stages and
gender. The previous RDA for both men and women was 60 mg/day. This was based in part on
the prevention of scurvy with a 4 week margin of safety. The current RDA is based on the
vitamin C intake required for 80% neutrophil saturation with little urinary loss in healthy men
[4]. The RDA for women as suggested by the Institute of Medicine is 75 mg. Recent results
indicate that the RDA for vitamin C for women be raised to 90 mg/day as well. However, all
products tested in this report based their PDV (Percentage Daily Value) on a 2000 calorie diet.
This diet includes an RDA of 75 mg for both men and women for most products. Thus in this
report it is assumed that all values quoted on the products labels are based on an RDA of 75 mg
and therefore all calculations will be based upon this value.
2.4.2.1
Upper Intake Level
The tolerable upper intake level of the vitamin is the highest level of daily intake that is likely to
pose no risk of adverse health effects to the general adult population, 18 years and over. For
vitamin C this is estimated as [A]:
2000 mg for adults
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400 mg for children ages 1 3
650 mg for children ages 9 13
1800 mg for ages 14 18
2.4.3 Dietary Sources of Vitamin C
The best way to increase ones daily supply of vitamin C is by eating lots of fruits and
vegetables. A daily vitamin C intake of 200 mg is easily obtained by consuming 5 servings of
fruits and vegetables. A cup of fresh orange juice can supply 120 mg or grapefruit juice can
supply 90 mg. Fresh fruits and vegetables such as: broccoli, cantaloupe, grapefruit, oranges,
tomatoes, papaya and mangoes are the richest sources of vitamin C.
Cooking or boiling
vegetables containing the vitamin should be avoided since it results in vitamin C losses from 50
to 80%. Prolonged storage and other cooking practices such as steaming and microwaving foods
can substantially deplete the vitamins content in foods.
2.5 Methods of Analysis

2.5.1 Oxidation-Reduction Methods
2.5.1.1
2, 6-Dichloroindophenol Titration
The official method of analysis for Vitamin C from juices is the 2, 6-Dichloroindophenol (DCIP)
titration method8. This method is used as a rapid, quality control test for a variety of food
products and was thus chosen as a means of analyzing the vitamin C content in cereals.
The DCIP method was introduced by Tillmans in 1930. Ascorbic acid reduces the dark
blue color of the indicator dye to a colorless solution. At the end point of titrating an ascorbic
acid-containing sample with dye, excess unreduced dye is a rose-pink color in the acid solution.
Extraction of the vitamin from dry materials can be achieved by the use of metaphosphoric acid
containing glacial acetic acid [3]. This was necessary for extraction of the vitamin from the
cereal products investigated in this report. Chapter 3 gives a step-by-step procedure of the
method.
Deficiencies of the method
AOAC International. 2000. Official Methods of Analysis. 17th ed. Method 967.21
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Several important deficiencies exist with this method. The titration is limited to the
quantitation of L-ascorbic acid (ASC). The L-ascorbic acid is oxidized to dehydroascorbic acid
and excess dye remains pink in the acid solution. However dehydroascorbic acid cannot be
measured unless it is reduced to ascorbic acid. Secondly, the titration is unable to distinguish
between L-ascorbic acid and isoascorbic acid (another form of the acid). Thirdly, the method can
be used for fresh juices, processed foods and multivitamins that do not contain copper, iron or
tin.
The most important factor which limits the use of this method is that highly colored
extracts from fruit and vegetables can mask the color change in the titrations end point. Though
the test can be used for analysis of fruit drinks, the end point of juices such as Guava pine or
Black currant is difficult to obtain. Thus this method was used to determine the vitamin C
content in cereals only. Also, the reduction of DCIP is not limited to L-ascorbic acid and as a
result any reducing substance present in the sample can reduce the dye. These interferences can
lead to erroneously high measurements, if not recognized [3].
2.5.1.2
Iodine Test
In this method the titrating solution is an iodine solution. When the ascorbic acid reacts with
iodine it is oxidized and the iodine is reduced. Reduced iodine is unable to react with starch.
Any added iodine will be able to react with the starch when all the ascorbic acid has reacted. A
purple colour then forms and remains when the end point is reached. The following reactions
demonstrate this:
I2 + ascorbic acid
oxidized ascorbic acid + 2I-
I- + I2 + starch
I3- + starch complex (purple to blue-black)
In this report, this method was used as a replacement for the analysis of fruit drinks by
DCIP method due to the colour change of the end-point. Unlike the DCIP method, the endpoints colour was not difficult to visualize even for dark coloured drinks such as black currant.
In order to improve on the accuracy of the observed endpoint, each juice sample was diluted by a
factor of 10. This value was then incorporated into the calculations for obtaining the vitamins
content.
Laura Charles
ID#: 05714774
xxi
Deficiencies of the method
The iodine solution tends to break down in the presence of sunlight and must be placed in
an amber bottle. However, during the actual experiment, this is impossible to do and so may be
deemed as a source of error.
2.5.2 Potentiometry using pH Analysis

The titrating solution used in this method is (NaOH) Sodium Hydroxide solution. As increments
of the base are added to the sample, the change in pH readings is measured using a pH meter
from which the end point is obtained. However, this method should only be used for products
such as Vitamin C tablets which contain only ascorbic acid plus an inert diluent that facilitates
pressing into tablets.
The method was attempted in this report.
However, due to the presence of other
compounds, the isolation of the vitamins pH was impossible. Thus the readings obtained by the
meter did not reflect that of the vitamin only. Hence, erroneously high values for the vitamins
content were obtained.
2.5.3 High Performance liquid Chromatography

HPLC methods were developed in order to simultaneously analyze L-ascorbic acid,
dehydroascorbic acid and isoascorbic acid from food. Thus excellent procedures have been
developed for accurate determination of the vitamins content in the presence of isoascorbic acid
due to the ability to accurately differentiate between L-ascorbic acid and isoascorbic acid.
One of the procedures which make use of this method is known as the Pre-Column
Derivatization using OPD derivatization. OPD or o-phenylenediamine condensation reaction
with dehydroascorbic acid represents one of the most useful derivatization reactions to quantitate
total vitamin C. The product of the reaction is a highly fluorescent compound. This along with
other more complex steps utilizes the accuracy of the HPLC method for the vitamins analysis.
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ID#: 05714774
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This method should have been employed in this report. However, due to the
unavailability of both chemicals and equipment, it was unable to be utilized.
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ID#: 05714774
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Chapter 3
METHODS OF ANALYSIS
Vitamin C is easily reduced or destroyed by exposure to heat and oxygen during processing,
packaging, and storage of food. The Vitamin C content is required by the U.S Food and Drug
Administration to be listed on the nutrition label of foods. The instability of the vitamin makes it
more difficult to ensure an accurate listing of its content on the nutrition labels. Thus the
Vitamin C content obtained from the various methodologies used within this analysis can only be
assumed to be a rough estimate of the actual value and may vary from that stated on the
products labels.
3.1 2, 6-Dichloroindophenol Titration

As was discussed previously, the titrating solution is the dye made from 2, 6-Dichloroindophenol
sodium salt. The end-point of the reaction is determined by the appearance of a rose-pink colour
which persists for 15 seconds.
The titer of the dye can be determined using a standard ascorbic acid solution. Food
samples such as cereals in solution can then be titrated with the dye, and the volume for the
titration used to calculate the ascorbic acid content. The reagents and procedure used is given in
the following:
Table 3- : Chemicals used in DCIP analysis of Vitamin C
Chemicals
Acetic acid
Ascorbic Acid
2, 6-Dichloroindophenol sodium salt
Metaphosphoric acid
Sodium bicarbonate
Laura Charles
ID#: 05714774
CAS No.
Hazards
64-19-7
Corrosive
50-81-7
620-45-1
37267-86-0 Corrosive
144-55-8
xxiv
3.1.1 Reagents
1. 3% or 60g of Metaphosphoric acid (HPO3) was prepared by dissolving the pellets of
HPO3 in a beaker containing 800ml distilled water and 160ml acetic acid. The mixture
was then diluted to 2000ml with distilled water and then filtered into a bottle which was
then stoppered and refrigerated until it was ready to be used.
2. Ascorbic acid standard: This was prepared by weighing accurately 100mg of L-ascorbic
acid and was then made up to 100ml with the 3% HPO 3 from above. 10ml was then
extracted and further diluted to 100ml with 3% HPO3.
3. Dye solution: 500mg of the sodium salt of 2, 6-dichlorophenol-indophenol was dissolved
in approximately 500ml of hot distilled water containing 420mg of sodium bicarbonate.
The mixture was then cooled and diluted with distilled water to 2000ml. It was then
filtered into an amber bottle using fluted paper, stoppered and then refrigerated until it
was ready to be used.
3.1.2 Procedure
5ml of the prepared standard ascorbic acid solution was added to 5ml of HPO 3 and titrated with
the dye solution (using a burette) to a pink colour which persisted for 15 sec. The Dye factor
was then determined using the formula:
Dye factor =
0.5
titre
(1)
3.1.3 Preparation of Cereal Sample

5 g of sample was blended and then mixed with 3% HPO 3 and made up to either 50 or 100 ml
with HPO3. The mixture was then centrifuged.
3.1.4 Assay of Extract
An aliquot (5-10 ml) of the HPO 3 extract of the sample was titrated with the standard dye to a
pink end point which persisted for 15 sec.
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ID#: 05714774
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Figure 3- : L-ascorbic acid reactions important to vitamin C analysis. Fig. adapted from Pearsons Composition and Analysis
of Foods, pp 232.
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ID#: 05714774
xxvi
Indophenol Dye solution
Figure 3- : Photo taken of the setup of apparatus and reagents used for the DCIP analysis of vitamin C.
Colour change with

addition of dye
Figure 3- : Photo showing colour change of cereal. This is the colour indicating that reaction is near its end point.
Laura Charles
ID#: 05714774
xxvii
3.2 Iodine Test

A standard solution of vitamin C and a sample of fruit drink are both titrated and compared. This
comparison enables the calculating of the amount of Vitamin C present in the fruit juice. The
titration solution used is the iodine solution and the end point indicator is starch. The end-point
of the reaction is determined by the appearance of a purple to blue-black colour which persists
for 15 seconds.
3.2.1 Reagents
1. Vitamin C standard: 100 mg of ascorbic acid was dissolved in 75 ml of distilled water.
The solution was then diluted with distilled water to 100.0 ml. This resulted in a standard
solution with a vitamin C concentration of 1.0 mg/ml.
2. Iodine Solution: 0.6g of potassium iodide (KI) was dissolved in 500 ml of distilled water.
0.6g of iodine crystals (I2) was then dissolved in 50ml of ethyl alcohol. The two solutions
were then mixed and diluted to 1.0L with distilled water.
3. Hydrochloric acid (HCl): To prepare 1M solution of HCL, 8.3 mL of concentrated HCl
was added to 50 ml of distilled water. This mixture was then diluted to 100 ml with
distilled water. The 1M HCl solution was then placed in a marked dropper bottle.
3.2.2 Procedure
1. 10.0 ml of the vitamin C standard solution was added to 20 ml of distilled water in a
flask. The water does not interfere with the reaction.
2. 2 drops of 1M HCl followed by 15 drops of starch solution were added to the diluted
vitamin C standard solution.
3. The burette was filled with the iodine solution and the flask containing the Standard
sample was placed under the burette. Iodine was then added to the solution until a blueblack colour persisted for 15 sec. The volume of iodine used for the titration was then
recorded.
4. The procedure was the repeated for two additional trials.
5. The procedure was repeated for three trials of a 10 ml sample of fruit drink. All values
obtained were recorded.
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ID#: 05714774
xxviii
Iodine
Juice samples
Starch
HCl
Figure 3- : Photo taken of setup of apparatus and reagents used in the Iodine test for Vitamin C.
End point colour
Figure 3- : Photo showing the change to blue-black of sample indicating reactions end point.
Laura Charles
ID#: 05714774
xxix
3.3 pH Potentiometry Test

This procedure tested the change in pH readings when the vitamin C containing sample was
titrated against 0.1M (NaOH) Sodium Hydroxide solution.
3.3.1 Reagents
1.
2.
3.
4.
5.
0.1M NaOH solution

Standard HCl
Standard buffers of pH 4.00 and 7.00
0.4 - 0.5g of sample
Distilled water
3.3.2 Procedure
1. Individual cereal products were blended or crushed and placed in dry labeled bottles.
2. 0.4- 0.5 g of the ground sample was weighed accurately and transferred to a 250 ml
beaker.
3. The material was dissolved using 50 ml of water, filtered and then the filter paper was
washed with hot water to ensure that all the vitamin C was extracted.
4. The pH meter was set up and calibrated using standard buffers of pH 4.00 and 7.00.
5. A magnetic stirrer bar was placed into the beaker containing the weighed cereal sample.
6. With the beaker atop a magnetic stirrer, 0.1M NaOH was added in increments of 0.1 ml
via a burette, whilst being stirred. The pH value was recorded after each addition.
7. The procedure was repeated for a replicate sample for each food item tested.
8. NaOH was standardized using standard HCl via the same procedure.
9. Plots of pH versus Volume of NaOH were made. The end point is the point of greatest
slope and was thus determined for each plot. The average weight (mg) of ascorbic acid
was then calculated in each product.
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ID#: 05714774
xxx
electrode
NaOH sol
pH meter
sample
magnetic
stirrer
standard buffers
Figure 3- : Photo taken of setup of apparatus and reagents used in the pH test for Vitamin C.
Laura Charles
ID#: 05714774
xxxi
Chapter 4
Results and Calculations
4.1 2, 6-Dichloroindophenol Titration

4.1.1 Ascorbic Acid Standard
Table 4- : Titration results obtained for Ascorbic acid standard

Trial
Initial (ml)
Final (ml)
Actual (ml)
1
2
Average
0.4
18.0
17.5
35.0
17.1
17.0
17.05
4.1.2 Laboratory Results for Sunshine Cereals
Table 4- : Titration results obtained for Frosted Flakes produced by Sunshine

Sunshine
Frosted Flakes
Batch #
B1
Trial
1
2
Initial (ml)
1.3
3.4
Final (ml)
3.1
5.1
Actual (ml)
1.8
1.7
1.75
1
2
14.0
15.5
15.5
17.0
1.5
1.5
1.5
1
2
0.3
2.1
2.1
3.9
1.8
1.8
1.8
Average
B2
Average
B3
Average
Laura Charles
ID#: 05714774
xxxii
Table 4- : Titration results obtained for Raisin Bran produced by Sunshine
Sunshine
Raisin Bran
Batch #
B1
Trial
1
2
Initial (ml)
36.0
37.3
Final (ml)
37.3
38.6
Actual (ml)
1.3
1.3
1.3
1
2
30.5
33.2
33.2
36.0
2.7
2.8
2.75
1
2
26.0
28.2
28.2
30.5
2.2
2.3
2.25
Average
B2
Average
B3
Average
Table 4- : Titration results obtained for Froot Ooos produced by Sunshine

Sunshine
Froot Ooo's
Batch #
B1
Trial
1
2
Initial (ml)
9.4
11.3
Final (ml)
11.1
12.9
Actual (ml)
1.7
1.6
1.65
1
2
5.1
7.6
7.1
9.4
2.0
1.8
1.9
1
2
4.0
8.2
8.2
12.4
4.2
4.2
4.2
Average
B2
Average
B3
Average
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ID#: 05714774
xxxiii
4.1.2.1
Sample Calculations for Sunshine Cereals
In order to determine Vitamin C content using the DCIP method, the following calculations were
performed. Sample calculations are shown for Batch 1 of Frosted Flakes produced by Sunshine.
Dye factor =
(4.1)
(4.2)
Where,
Aliquot = 10 ml
Titre = 1.75 ml
Vol. made up = 100 ml
Wt/Volume of sample = 5 g = 5 ml
Ascorbic acid = ASC
From Eq. (4.1) we have:
`= 10.15 mg Ascorbic acid/ 100 g or ml
Mass of packaged cereal = 28 g
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ID#: 05714774
xxxiv
Amount of ASC in 28 g =
Vitamin C =
Stated Value on label is 25%

4.1.3 Results based on Calculations for Sunshine Cereals
Table 4- : Results obtained for the calculation of the Vitamin C content in three batches of
Frosted Flakes
Calculations for Frosted Flakes by Sunshine
Aliquot (ml)
B1
10
B2
10
B3
10
Titre
1.75
1.50
1.80
Vol. made up (ml)
100
100
100
5
10.15
5
8.70
5
10.44
Packaged Mass (g)
28
28
28
Required Vitamin C (mg)
75
75
75
Vitamin C in Packaged mass
2.84
2.44
2.92
% Vitamin C
Stated % Value
3.79
25
3.25
25
3.90
25
wt/vol of sample
mg ASC /100ml
Raisin Bran
Calculations for Raisin Bran by Sunshine
B1
5
1.30
Aliquot (ml)
Titre
Laura Charles
ID#: 05714774
B2
5
2.75
B3
5
2.25
xxxv
Vol. made up (ml)
100
100
100
wt/vol of sample
mg ASC /100ml
15.08
31.90
26.1
Packaged Mass (g)
35
35
35
60
60
60
5.28
7.04
11.17
14.89
9.14
12.18

% Vitamin C
Stated % Value
30
30
30
Froot Ooos
Calculations for Froot Ooo's by Sunshine
Aliquot (ml)
Titre
B1
10
1.65
B2
10
1.90
B3
10
4.20
Vol. made up (ml)
100
100
100
wt/vol of sample
mg ASC /100ml
9.57
11.02
24.36
23
23
23

75
2.20
75
2.53
75
5.60
% Vitamin C
Stated % Value
2.93
20
3.38
20
7.47
20
Packaged Mass (g)
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ID#: 05714774
xxxvi
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Sunshine Frosted Flakes from the stated value
(represented by the solid purple line) of 25%.
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ID#: 05714774
xxxvii
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Sunshine Raisin Bran from the stated value
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Sunshine Froot Ooos from the stated value
Laura Charles
ID#: 05714774
xxxviii
4.1.4 Laboratory Results for Kelloggs Cereal
Table 4- : Titration results obtained for Kellogg's Frosted Flakes

Kellogg's
Frosted Flakes
Batch #
B1
Trial
1
2
Initial (ml)
17.2
18.1
Final (ml)
18.1
19.1
Actual (ml)
0.9
1.0
0.95
1
2
19.1
22.8
22.7
26.4
3.6
3.6
3.6
1
2
17.0
21.5
21.5
26.0
4.5
4.5
4.5
Average
B2
Average
B3
Average
Table 4- : Titration results obtained for Kellogg's Froot Loops

Kellogg's
Froot Loops
Batch #
B1
Trial
1
2
Initial (ml)
12.50
13.50
Final (ml)
15.30
16.30
Actual (ml)
2.80
2.80
2.80
1
2
15.90
20.20
19.90
24.00
4.00
3.80
3.90
1
2
24.10
31.00
31.00
38.00
6.90
7.00
6.95
Average
B2
Average
B3
Average
Laura Charles
ID#: 05714774
xxxix
Table 4- : Titration results obtained for Kellogg's Special K

Kellogg's
Special K
Batch #
B1
Trial
1
2
Initial (ml)
26.40
27.40
Final (ml)
31.40
32.30
Actual (ml)
5.00
4.90
4.95
1
2
28.50
29.30
32.00
32.80
3.50
3.50
3.50
1
2
28.50
29.30
33.00
33.90
4.50
4.60
4.55
Average
B2
Average
B3
Average
4.1.4.1
Sample Calculations for Kelloggs Cereal
The following calculations are shown for Batch 1 of Kelloggs Frosted Flakes.
Aliquot = 5 ml
Titre = 0.95 ml
Vol. made up = 50 ml
Wt/Volume of sample = 5 g = 5 ml
Dye factor = 0.029
From Eq. (4.2) we have:
`= 5.51 mg Ascorbic acid/ 50 g or ml
Laura Charles
ID#: 05714774
xl
Mass of packaged cereal = 34 g
Amount of ASC in 34 g =
Vitamin C =
Stated Value on label is 10 %
4.1.5 Results based on Calculations for Kelloggs Cereal
Frosted Flakes
Calculations for Frosted Flakes by Kellogg's
B1
B2
B3
Aliquot (ml)
5
5
5
Titre
0.95
3.60
4.50
Vol. made up (ml)
50
50
50
wt/vol of sample
5
5
5
mg ASC /50ml
5.51
20.88
26.1
Packaged Mass (g)
34
34
34
75
75
75
3.75
14.20
17.75
% Vit. C
5.00
18.93
23.66
Stated % Value
10
10
10
Froot Loops
Calculations for Froot Loops by Kellogg's
Aliquot (ml)
Titre
Vol. made up (ml)
wt/vol of sample
B1
B2
B3
5
2.80
50
5
5
3.90
50
5
5
6.95
50
5
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mg ASC /50ml
16.24 22.62 40.31
Packaged Mass (g)
27
27
27
75
75
75
8.77
12.21 21.77
% Vitamin C
11.69 16.29 29.02
Stated % Value
20
20
20
Special K
Calculations for Special K by Kellogg's
B1
B2
B3
4.95
3.50
4.55
Vol. made up (ml)
50
50
50
wt/vol of sample
28.71
20.30
26.39
Packaged Mass (g)
23
23
23
75
75
75
13.21
9.34
12.14
% Vitamin C
17.61
12.45
16.19
25
25
25
Aliquot (ml)
Titre
mg AA / 50ml
Stated % Value
Laura Charles
ID#: 05714774
xlii
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Kelloggs Frosted Flakes from the stated value
Laura Charles
ID#: 05714774
xliii
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Kelloggs Froot Loops from the stated value
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Kelloggs Special K from the stated value
Laura Charles
ID#: 05714774
xliv
4.2 Iodine Titration

4.2.1 Ascorbic Acid Standard
Table 4- : Titration results for Ascorbic acid standard

Trial
Initial (ml)
Final (ml)
Actual (ml)
0.3
30.8
30.5
1.8
32.5
30.7
Average
4.2.1.1
30.6
Verification of Ascorbic acid concentration
Stated Concentration = 1.0 mg/ ml

Mass of ASC used = 0.101 g
X mg
average iodine used
for fruit juice mL 10.0 mg
average iodine used for std solution mL
Where,
X = mg of Vitamin C in sample
Using Eq. (4.3) we have:
30.6
10.0 10 mg / 10 ml ASC
30.6
Conc = 1.0 mg/ml

Therefore iodine solution can be used for testing of juice samples.
Laura Charles
ID#: 05714774
(4.3)
xlv
4.2.2 Laboratory results for Fruta Kool Kidz
Table 4- : Titration results obtained for Fruit Punch produced by Fruta Kool Kidz
Table 4- : Titration results obtained for Orange Juice produced by Fruta Kool Kidz
Laura Charles
ID#: 05714774
xlvi
Table 4- : Titration results obtained for Guava Pine produced by Fruta Kool Kidz
Table 4- : Titration results obtained for Grape Juice produced by Fruta Kool Kidz
Laura Charles
ID#: 05714774
xlvii
4.2.2.1
Sample Calculations for juices produced by Fruta Kool
Kidz
The following sample calculations are based on Batch 1 of Fruit Punch.

Avg. iodine used for std. sol. = 30.6 mL
Amt. of Vitamin C = X =
0.95 10
0.31 mg
30.6
Thus there is 0.31 mg of Vitamin C in 10 ml of dilute solution. However, amount of Vitamin C

in 10 ml of original sample is:
0.3110 3.1 mg
Therefore for 1 ml of juice, amount of Vitamin C =
3.1
0.31 mg / ml
10
Total vol. of drink = 200 ml

Hence total amount of Vitamin C in 200 ml is:
0.31 200 62.09 mg

% Vitamin C in sample =
62.09
100 82.79
75
Stated Value = 80%
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ID#: 05714774
xlviii
4.2.3 Results based on Calculations for Fruta Kool Kidz drinks
Fruit Punch
Orange Juice
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ID#: 05714774
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Laura Charles
ID#: 05714774
Guava Pine
Grape Juice
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ID#: 05714774
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Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Fruit Punch made by Fruta Kool Kidz from the
stated value (represented by the solid purple line) of 80%.
Laura Charles
ID#: 05714774
lii
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Orange Juice made by Fruta Kool Kidz from the
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Guava Pine made by Fruta Kool Kidz from the
Laura Charles
ID#: 05714774
liii
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Grape Juice made by Fruta Kool Kidz from the
4.2.4 Laboratory results for Orchard
Table 4- : Titration results obtained for Fruit Punch produced by Orchard
Table 4- : Titration results obtained for Black Currant produced by Orchard
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ID#: 05714774
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Table 4- : Titration results obtained for Orange Juice produced by Orchard
4.2.5 Results based on Calculations for Orchard drinks

Calculations made for drinks produced by Orchard were similar to those performed for Fruta
Kool Kidz.
Fruit Punch
Laura Charles
ID#: 05714774
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Laura Charles
ID#: 05714774
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Black Currant
Orange juice
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ID#: 05714774
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Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Fruit Punch made by Orchard from the stated
value (represented by the solid purple line) of 52%.
Laura Charles
ID#: 05714774
lviii
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Black Currant made by Orchard from the stated
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Orange juice made by Orchard from the stated
4.2.6 Laboratory results obtained for powdered drinks
Table 4- : Titration results obtained for Pineapple produced by Flavor Aid
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ID#: 05714774
lix
Table 4- : Titration results obtained for Lemonade produced by Kool-Aid
4.2.6.1
Sample Calculations for juices produced by Flavor Aid
The sample calculations given below are for Batch 1 of Flavor Aid.
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ID#: 05714774
lx
mg of Vitamin C in 1 ml of juice =
Amount of Vitamin C in 250 ml
% Vitamin C =
Stated value = 10%
4.2.7 Results based on Calculations for Powdered drinks
Pineapple by Flavor Aid
Laura Charles
ID#: 05714774
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Lemonade by Kool-Aid
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Pineapple made by Flavor Aid from the stated
Laura Charles
ID#: 05714774
lxii
Figure 4- : Graph demonstrating the deviation of the Vitamin C content in Lemonade made by Kool-Aid from the stated value
Laura Charles
ID#: 05714774
lxiii
4.3 Potentiometry Method

Only results obtained for the first batch of each cereal is recorded in this section. The results
obtained for Batches 2-4 can be found in Appendix B.
4.3.1 Laboratory results for Sunshine Cereals
Table 4- : pH results obtained for Batch 1 of Frosted Flakes produced by Sunshine

Trial 1
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
pH
6.40
8.60
9.88
10.2
4
10.3
9
10.5
0
10.5
5
10.6
3
10.6
7
10.7
6
10.8
6
10.9
1
10.9
3
11.0
0
11.0
2
11.0
8
11.1
Trial 2
Actual
NaOH
(ml)
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.0
4.1
4.2
4.4
4.5
4.7
4.8
pH
6.36
7.48
9.81
10.2
3
10.3
2
10.4
9
10.6
0
10.6
8
10.7
6
10.8
2
10.4
6
10.9
3
10.9
9
11.0
1
11.0
4
11.0
8
11.1
Laura Charles
ID#: 05714774
NaOH
(ml)
Averag
e pH
0.0
0.1
0.2
6.38
8.04
9.85
0.3
10.24
0.4
10.36
0.5
10.50
0.6
10.58
0.7
10.66
0.8
10.72
0.9
10.79
1.0
10.66
1.1
10.92
1.2
10.96
1.3
11.01
1.4
11.03
1.5
1.6
11.08
11.12
lxiv
17
18
19
20
21
22
23
2
11.1
5
11.1
9
11.2
2
11.2
5
11.2
9
11.3
3
11.3
6
1
11.1
9
11.2
0
11.2
4
11.2
8
11.3
2
11.3
6
11.3
9
4.9
5.1
5.2
5.4
5.5
5.7
6.0
1.9
11.17
2.0
11.20
2.2
11.23
2.4
11.27
2.6
11.31
2.8
11.35
3.0
11.38
Table 4- : pH results obtained for Batch 1 of Raisin Bran produced by Sunshine

Trial 1
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
pH
4.94
5.14
5.68
6.28
6.85
7.33
7.87
8.48
9.07
9.49
9.77
9.87
10.0
3
10.1
6
10.2
6
10.3
5
Trial 2
NaOH
(ml)
0
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
pH
4.86
5.04
5.53
6.14
6.71
7.22
7.72
8.29
8.61
9.35
9.67
9.89
10.0
5
10.1
7
10.2
7
10.3
5
Laura Charles
ID#: 05714774
NaOH
(ml)
Average
pH
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
4.90
5.09
5.61
6.21
6.78
7.28
7.80
8.39
8.84
9.42
9.72
9.88
1.2
10.04
1.3
10.17
1.4
10.27
1.5
10.35
lxv
16
17
18
19
20
21
22
23
24
25
10.4
2
10.4
9
10.5
4
10.5
9
10.6
2
10.6
6
10.7
0
10.7
4
10.7
7
10.8
0
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
10.3
9
10.4
6
10.5
2
10.5
7
10.6
4
10.6
6
10.7
0
10.7
3
10.7
5
10.8
0
Laura Charles
ID#: 05714774
1.6
10.41
1.7
10.48
1.8
10.53
1.9
10.58
2.0
10.63
2.1
10.66
2.2
10.70
2.3
10.74
2.4
10.76
2.5
10.80
lxvi
Table 4- : pH results obtained for Batch 1 of Frosted Flakes produced by Kelloggs

B1 Sample: Frosted Flakes by Kellogg's
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Trial 1
NaOH
pH
(ml)
Trial 2
NaOH
pH
(ml)
5.3
2
8.9
6
9.7
0
10.
00
10.
21
10.
35
10.
45
10.
53
10.
61
10.
68
10.
73
10.
78
10.
82
10.
86
10.
91
10.
93
10.
96
10.
99
11.
02
5.1
4
9.0
5
9.9
5
10.
18
10.
33
10.
39
10.
50
10.
58
10.
64
10.
71
10.
76
10.
81
10.
85
10.
88
10.
92
10.
95
10.
99
11.
00
11.
03
0
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
Laura Charles
ID#: 05714774
Average
pH
0.0
5.23
0.1
9.00
0.2
9.83
0.3
10.09
0.4
10.27
0.5
10.37
0.6
10.48
0.7
10.56
0.8
10.63
0.9
10.70
1.0
10.75
1.1
10.80
1.2
10.84
1.3
10.87
1.4
10.92
1.5
10.94
1.6
10.98
1.7
11.00
1.8
11.03
lxvii
Laura Charles
ID#: 05714774
lxviii
4.3.2 Laboratory results for Juices
Table 4- : pH results obtained for Orange juice produced by Fruta Kool Kidz
Trial 1
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
pH
4.05
4.23
4.41
4.85
5.40
6.12
6.87
9.19
10.0
0
10.3
2
10.5
0
10.6
4
10.7
4
10.8
2
10.8
5
10.9
4
10.9
7
11.0
3
11.0
4
11.0
8
11.1
0
Trial 2
NaOH
(ml)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
pH
4.08
4.51
4.94
5.51
6.18
6.62
9.48
9.85
10.3
5
10.5
1
10.6
4
10.7
4
10.7
8
10.8
8
10.9
4
10.9
8
11.0
3
11.0
7
11.1
0
11.1
6
11.1
8
Laura Charles
ID#: 05714774
NaOH
(ml)
Average
pH
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
4.07
4.37
4.68
5.18
5.79
6.37
8.18
9.52
0.8
10.18
0.9
10.42
1.0
10.57
1.1
10.69
1.2
10.76
1.3
10.85
1.4
10.90
1.5
10.96
1.6
11.00
1.7
11.05
1.8
11.07
1.9
11.12
2.0
11.14
lxix
Figure 4- : Graph demonstrating the standardization of NaOH using standard HCl
Laura Charles
ID#: 05714774
lxx
Figure 4- : Graph showing pH curve obtained for Orange Juice by Fruta Kool Kidz
Laura Charles
ID#: 05714774
lxxi
Chapter 5
DISCUSSION OF RESULTS
The various juices and cereals tested in this project were obtained from different
supermarkets nationwide in order to obtain a greater sense of variety for each product. In turn
this would help to reduce the biasness of results obtained from experiments. Thus if a product is
found to contain less vitamin C than what is stated by its nutrition label, the reasons for this
occurrence would not include the social or economic status of the grocery store from which it
was purchased but instead would be based on other factors.
In order for a comparison between findings to be valid, at least 3 batches of the same
substance should be considered for testing. The higher the number of batches, the better is the
statistical analysis. However, due to time and economic constraints, the minimum amount was
used.
Chapter 4 gives a detailed record of the findings for all the experiments performed for
vitamin C analysis. Fig. (4-1) to (4-15) are column graphs which aid in demonstrating the
deviation of the vitamin C content as stated on the products nutrition label to that obtained in the
various experiments. Each column represents 1 batch of a product tested and the value of the
vitamin C content determined for such a product. A horizontal purple line was inserted on each
figure which provides a visual indication of the PDV of vitamin C based on a 2000 calorie diet as
stated by the label. These graphs were utilized for both the DCIP titration and the iodine test.
However, Fig. (5-16) and (5-17) are pH curves from which the vitamin C content was obtained
for the pH Potentiometry test.
pH Test
Among the three methods used in the analysis of vitamin C, the pH test was discovered to
be the least reliable. Appendix C, Section 5.1.2 shows a sample calculation based on results
obtained from this test for Fruta Kool Kidzs Orange juice. The results show that the drink
contained 1936 mg of the vitamin. However, the iodine test showed that the value of the vitamin
C content was 124.1 mg, a vast difference from the pH test.
Laura Charles
ID#: 05714774
lxxii
Erroneous values such as were obtained for the orange juice were also obtained for the
other products.
One of the main reasons for this occurrence is the interference of other
substances. The presence of other acidic groups such as citric acid or folic acid was also
incorporated into the pH readings. These groups were not removed or isolated prior to testing;
since the test is based on the reaction of an acid with a base, it can be assumed that not only did
the ASC react with NaOH but the other groups as well. Therefore, the test is best used on pure
substances or substances free of other acid groups such as vitamin C tablets. In light of this, the
test was considered invalid for the vitamins analysis.
Iodine Test
The iodine test was considered valid since the reaction involved focused only on the
oxidation of ASC by iodine. Thus it is assumed that the presence of other substances did not
interfere with the experimental values obtained.
The local company, Fruta Kool Kidz was first considered for analysis. From Fig. (4-7),
only batch 1 for Fruit Punch was approximately equivalent in vitamin C content to the labeled
value of 80%. The other 2 batches contained equal amounts of vitamin C which were slightly
lower than the stated value. In general, 3 of the juices tested, namely: Fruit Punch, Orange Juice
and Guava Pine had 2 batches containing nearly the same amount of vitamin C as stated by their
labels. However, Grape Juice contained nearly double the amount stated, yet all three batches
showed evidence of consistency in their vitamin C content. Though this flavor contained a
higher value of the vitamin than what was stated, its vitamin content is well below the critical
value for any age range and can thus be considered a good source of the vitamin.
The international company considered was Orchard.
Fig. (4-11) shows a similar
resemblance to Fig. (4-7). As with Fruta Kool Kidz, 1 batch contained a higher vitamin C
content whereas the other 2 batches contained nearly the equivalent amount of the vitamin to that
stated on the label. Black Currant was similar to Fruta Kool Kidz Grape Juice in that all 3
batches tested contained a much higher value than what was stated. Thus Orchards Black
Currant is also a very good source of vitamin C. However, the 2 companies differed in their
content of the vitamin for Orange Juice. Unlike Fruta Kool Kidz, none of the batches of Orange
juice tested for Orchard came near the amount stated. In fact their values were nearly half of
Laura Charles
ID#: 05714774
lxxiii
what was labeled. Orange juice by Fruta Kool Kidz is thus the better buy when considering
vitamin C content.
Reasons for Deviation
Several factors may have contributed to the deviation in the vitamins content. One main
reason for obtaining higher values than what was labeled is that companies such as Fruta Kool
Kidz admit to adding an excess of vitamin C to their products during manufacturing. This is done
to ensure that after a shelf life of approximately 8 months, the vitamin C content of the product
will contain the labeled value, since vitamin C oxidizes and thus depletes over time.
Another cause may be due to testing of the product after being opened for at least 1 day.
During the performance of the experiments it was not always possible to complete all tests of the
juices on the same day. Thus products which were unable to be tested were refrigerated over
night. Since vitamin C so easily oxidizes, this resulted in a loss of the vitamin and thus a lower
content at the time of testing.
Slight errors may have occurred due to human error when obtaining the endpoints. Also,
iodine decomposes in the presence of sunlight and thus must be kept in an amber bottle at all
times. However this was not possible at the time of testing.
Similar reasons could be considered for the deviation of the vitamins content in both
Flavor Aid and Kool-Aid. Both companies showed similar deviations (higher vitamin C content
but not a significant deviation) from the labeled values. Thus both their labels can be considered
reliable. However, their content of the vitamin is very low in comparison to the juices produced
by both Orchard and Fruta Kool Kidz and as such they (powdered drinks) are not considered a
good source of the vitamin.
DCIP Test
The DCIP Titration tests were used for testing cereals.
The first company to be
considered was Sunshine. None of the 3 cereals tested contained the amount of vitamin C as
stated by their labels. In fact their vitamin C content was much less than what was stated. With
the exception of Frosted flakes, there was a fluctuation in the vitamins content, revealing a
considerable lack of consistency (especially for Raisin Bran).
Laura Charles
ID#: 05714774
lxxiv
Kelloggs Cereals however, revealed a greater level of consistency in the vitamins
content. Frosted Flakes showed the greatest level of deviation from the stated value. That is, 2
batches tested contained nearly double the vitamin C content as stated. Though none of the
batches tested for Special K were equivalent to the labeled value, it showed the greatest level of
consistency. In general, Kelloggs cereals can be seen as the better and more reliable cereal
between the two companies tested, when considering vitamin C content.
Reasons for Deviation
Vitamin C can easily be lost during grinding. Thus some of each of the cereals content of
the vitamin may have been compromised during the process.
. This technique was more difficult than the iodine test in identifying the endpoint point.
Thus human error would be present when obtaining the correct end point colour.
In cases such as for Sunshine where the values were much lower than what was labeled,
it can be assumed that the manufacturers claims of the vitamins content were either untrue or
that during manufacturing, the product may have been exposed to oxidizing conditions thus
reducing the vitamins content.
Laura Charles
ID#: 05714774
lxxv
Chapter 6
CONCLUSION & RECOMMENDATIONS
6.1 Conclusion
1. The iodine test and DCIP titration were reliable methods for the determination of vitamin
C content in juices and cereals respectively.
2. The pH test was found to be the least reliable method for vitamin C analysis.
3. It was found that in the determination of the vitamin C content for juices produced by the
local company Fruta Kool Kidz, three flavors namely Fruit Punch, Orange juice and
Guava Pine were consistent with their stated PDV of vitamin C. Thus the companys
labels can be considered reliable.
4. Juices produced by Fruta Kool Kidz are a good source of vitamin C, especially Grape
Juice.
5. It was found that out of the three flavors tested from Orchard, only Fruit Punch showed a
level of consistency with its labeling of the vitamins content.
6. Orchard Black Currant is a good source of Vitamin C.
7. In comparison with values obtained for both companies, Fruta Kool Kidz was determined
to be the more reliable source for the labeling of its vitamin C content and as such is the
better buy.
8. Both Flavor Aid and Kool-Aid contained a higher percentage of vitamin C than what was
stated on the nutrition label. However the deviation was not significant and thus both
labels can be considered reliable.
9. Kool-Aid and Flavor Aid are not good sources of vitamin C.
10. It was found that the labels for Sunshine cereals are very unreliable. None of the cereals
tested revealed a vitamin C content near the stated value. All three cereals had very low
vitamin C content and are considered a very poor source of vitamin C.
11. Kelloggs Cereals revealed a great level of consistency in the vitamins content with its
labeled value and can be considered reliable.
12. Cereals are a poor source of vitamin C.
6.2 Recommendations
1. In order to improve on the accuracy of the analysis of the vitamin C content in products,
more accurate methodologies should be employed. One such method involves the HPLC
technique.
Laura Charles
ID#: 05714774
lxxvi
2. The pH test should be avoided. However, if methods can be obtained whereby all other
acidic groups from products being tested can be removed, then the method could be
utilized.
3. Test liquids immediately upon opening of their package to avoid any oxidation of the
vitamin.
Laura Charles
ID#: 05714774
lxxvii
REFERENCES
[1] Asard, Han, J.M. May and N. Smirnoff. Vitamin C: Functions and biochemistry in
animals and plants. Department of Biochemistry, University of Nebraska-Lincoln, BIOS
Scientific Publishers Ltd, Oxford, 2004.
[2] Nelson, Norton, Dr., and Sidney S. Lee. Assessment of Technologies for Determining
Cancer Risks from the Environment. Department of Environmental Medicine, New York
University Medical School, New York, 1981, pp.81.
[3] Eitenmiller, R.R., W.O. Landen, Jr. Vitamin Analysis for the Health and Food Sciences.
Department of Food Science and Technology, University of Georgia, CRC Press, New York,
1999.
[4] Parker, L., M.G. Traber, K. Kraemer and B. Frei. The Antioxidant Vitamins C and E.
AOCS Press, Illinois, 2002.
Websites
[A] http://healthlink.mcw.edu/article/959704173.html
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ID#: 05714774
lxxviii
APPENDIX A
Table A- : Products tested (packaged mass/volume included)

Stated PDV for
Manufacturer
Product
Vitamin C based
on a 2000 cal.
Diet
Sunshine
Raisin Bran (35 g)
30
Frosted Flakes of Corn (28 g)
25
Froot Ooos (23 g)
20
Frosted Flakes (34 g)
10
Froot Loops (27 g)
20
Special K (23 g)
25
Fruit Punch Juice Drink
80
Guava Pine Juice Drink
80
Orange Juice drink
100
Grape Juice Drink
100
Black Currant Drink
100
Fruit Punch Drink
52
Orange Drink
70
Flavor Aid
Pineapple
10
Kool-Aid
Lemonade
Kelloggs
Fruta Kool Kidz

(200 ml)
Orchard
Laura Charles
ID#: 05714774
10
lxxix
APPENDIX B
Table B 1: pH results obtained for Batch 2 of Frosted Flakes produced by Sunshine
B2 Sample: Frosted Flakes of Corn by Sunshine
Trial 1
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
pH
5.60
9.06
9.91
10.0
5
10.2
6
10.4
2
10.5
2
10.6
0
10.6
8
10.7
1
10.7
9
10.8
4
10.8
9
10.9
3
10.9
6
10.9
8
11.0
2
11.0
5
Trial 2
Actual
NaOH
(ml)
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.0
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
pH
5.57
9.02
9.80
10.0
6
10.2
7
10.4
0
10.5
2
10.5
9
10.6
6
10.7
3
10.7
8
10.8
4
10.8
8
10.9
2
10.9
6
10.9
9
11.0
2
11.0
6
Laura Charles
ID#: 05714774
NaOH
(ml)
Average
pH
0.0
0.1
0.2
5.59
9.04
9.86
0.3
10.06
0.4
10.27
0.5
10.41
0.6
10.52
0.7
10.60
0.8
10.67
0.9
10.72
1.0
10.79
1.1
10.84
1.2
10.89
1.3
10.93
1.4
10.96
1.5
10.99
1.6
11.02
1.7
11.06
lxxx
18
19
20
11.0
9
11.1
0
11.1
3
5.9
6.0
6.1
11.0
9
11.1
2
11.1
4
Laura Charles
ID#: 05714774
1.8
11.09
1.9
11.11
2.0
11.14
lxxxi

Trial 1
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
pH
5.70
8.89
9.76
10.0
4
10.2
5
10.4
0
10.4
9
10.5
8
10.6
6
10.7
3
10.7
8
10.8
3
10.8
8
10.9
2
10.9
6
10.9
9
11.0
2
11.0
5
11.0
8
11.1
1
Trial 2
Actual
NaOH
(ml)
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
pH
5.59
9.17
9.87
10.1
6
10.2
7
10.4
2
10.5
3
10.6
6
10.7
3
10.7
9
10.8
2
10.8
7
10.9
4
10.9
8
11.0
1
11.0
5
11.0
8
11.0
9
11.1
3
11.1
6
Laura Charles
ID#: 05714774
NaOH
(ml)
Average
pH
0.0
0.1
0.2
5.65
9.03
9.82
0.3
10.10
0.4
10.26
0.5
10.41
0.6
10.51
0.7
10.62
0.8
10.70
0.9
10.76
1.0
10.80
1.1
10.85
1.2
10.91
1.3
10.95
1.4
10.99
1.5
11.02
1.6
11.05
1.7
11.07
1.8
11.11
1.9
11.14
lxxxii
Laura Charles
ID#: 05714774
lxxxiii

Trial 1
Readin
g
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
pH
5.63
8.84
9.43
9.90
10.2
3
10.3
3
10.4
5
10.5
4
10.6
2
10.6
8
10.7
1
10.7
7
10.8
0
10.8
5
10.8
8
10.9
2
10.9
6
10.9
9
11.0
1
11.0
4
Trial 2
Actual
NaOH
(ml)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
pH
5.46
9.01
9.71
9.97
10.2
0
10.2
4
10.3
1
10.4
2
10.5
0
10.5
7
10.6
3
10.6
9
10.7
3
10.7
8
10.8
2
10.8
5
10.9
0
10.9
1
10.9
4
10.9
7
Laura Charles
ID#: 05714774
NaOH
(ml)
Average
pH
0.0
0.1
0.2
0.3
5.55
8.93
9.57
9.94
0.4
10.22
0.5
10.29
0.6
10.38
0.7
10.48
0.8
10.56
0.9
10.63
1.0
10.67
1.1
10.73
1.2
10.77
1.3
10.82
1.4
10.85
1.5
10.89
1.6
10.93
1.7
10.95
1.8
10.98
1.9
11.01
lxxxiv
Table B 4: pH results obtained for Batch 2 of Raisin Bran produced by Sunshine

B2 Sample: Rainsin Bran by Sunshine
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Trial 1
NaOH
pH
(ml)
Trial 2
NaOH
pH
(ml)
4.88
5.19
5.95
6.29
6.89
7.44
8.07
8.80
9.35
9.68
9.90
10.1
0
10.2
2
10.3
1
10.3
9
10.4
6
10.5
2
4.68
5.12
5.41
6.16
7.03
7.30
7.91
8.86
9.36
9.69
9.93
10.1
0
10.2
4
10.3
4
10.4
2
10.5
1
10.5
4
0
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
Laura Charles
ID#: 05714774
Average
pH
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
4.78
5.16
5.68
6.23
6.96
7.37
7.99
8.83
9.36
9.69
9.92
1.1
10.10
1.2
10.23
1.3
10.33
1.4
10.41
1.5
10.49
1.6
10.53
lxxxv
17
18
19
20
21
22
23
24
10.5
9
10.6
6
10.7
0
10.7
3
10.7
6
10.8
0
10.8
3
10.8
6
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
10.6
0
10.6
5
10.7
0
10.7
4
10.7
7
10.8
1
10.8
3
10.8
7
Laura Charles
ID#: 05714774
1.7
10.60
1.8
10.66
1.9
10.70
2.0
10.74
2.1
10.77
2.2
10.81
2.3
10.83
2.4
10.87
lxxxvi

Trial 1
NaOH
pH
(ml)
Trial 2
NaOH
pH
(ml)
0
1
2
3
4
5
6
7
8
9
4.77
4.97
5.49
6.22
6.78
7.31
7.87
8.48
9.00
9.49
0
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
10
9.91
10.0
6
10.1
8
10.2
9
10.3
8
10.4
5
10.5
2
10.5
8
10.6
3
10.6
9
10.7
2
10.7
5
10.7
9
10.8
2
1.00
5.07
5.81
6.43
7.16
7.44
8.08
8.77
9.32
9.71
9.94
10.1
2
10.2
5
10.3
5
10.3
9
10.4
7
10.5
7
10.6
0
10.6
7
10.6
9
10.7
3
10.7
7
10.8
1
10.8
4
10.8
5
Readi
ng
11
12
13
14
15
16
17
18
19
20
21
22
23
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
Laura Charles
ID#: 05714774
Average
pH
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
4.92
5.39
5.96
6.69
7.11
7.70
8.32
8.90
9.36
9.72
1.0
10.02
1.1
10.16
1.2
10.27
1.3
10.34
1.4
10.43
1.5
10.51
1.6
10.56
1.7
10.63
1.8
10.66
1.9
10.71
2.0
10.75
2.1
10.78
2.2
10.82
2.3
10.84
lxxxvii
24
25
10.8
4
10.8
8
2.40
2.50
10.8
8
10.9
1
Laura Charles
ID#: 05714774
2.4
10.86
2.5
10.90
lxxxviii

Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Trial 1
NaOH
pH
(ml)
Trial 2
NaOH
pH
(ml)
4.74
5.11
5.81
6.56
7.14
7.43
8.08
8.83
9.34
9.67
9.88
10.0
4
10.1
6
10.2
5
10.3
3
10.4
0
10.4
6
10.5
2
10.5
7
10.6
2
10.6
6
10.7
0
10.7
3
10.7
5
10.7
4.64
4.94
5.43
5.99
6.69
7.24
7.82
8.49
9.15
9.58
9.88
10.0
5
10.1
9
10.3
1
10.4
2
10.4
7
10.5
3
10.5
8
10.6
4
10.6
8
10.7
3
10.7
6
10.8
0
10.8
4
10.8
0
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
Laura Charles
ID#: 05714774
Average
pH
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
4.69
5.03
5.62
6.28
6.92
7.34
7.95
8.66
9.25
9.63
9.88
1.1
10.05
1.2
10.18
1.3
10.28
1.4
10.38
1.5
10.44
1.6
10.50
1.7
10.55
1.8
10.61
1.9
10.65
2.0
10.70
2.1
10.73
2.2
10.77
2.3
2.4
10.80
10.83
lxxxix
25
9
10.8
0
2.50
7
10.9
0
Laura Charles
ID#: 05714774
2.5
10.85
xc
Table B 7: pH results obtained for Apple juice produced by Fruta Kool Kidz
Trial 1
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
pH
3.68
4.16
4.57
5.11
5.75
9.28
10.0
1
10.2
6
10.4
2
10.5
4
10.6
6
10.7
6
10.8
5
10.8
9
10.9
6
10.9
8
11.0
2
11.0
6
11.0
9
11.1
2
11.1
5
11.1
8
11.2
1
Trial 2
NaOH
(ml)
pH
NaOH (ml)
0.0
0.1
0.2
0.3
0.4
0.5
3.73
4.00
4.19
4.82
5.46
6.70
0.0
0.1
0.2
0.3
0.4
0.5
0.6
9.62
0.6
0.7
10.15
0.7
0.8
10.42
0.8
0.9
10.58
0.9
1.0
10.72
1.0
1.1
10.77
1.1
1.2
10.85
1.2
1.3
10.92
1.3
1.4
10.98
1.4
1.5
11.03
1.5
1.6
11.07
1.6
1.7
11.10
1.7
1.8
11.14
1.8
1.9
11.17
1.9
2.0
11.20
2.0
2.1
11.23
2.1
2.2
11.23
2.2
Laura Charles
ID#: 05714774
xci
23
11.2
3
2.3
11.24
Laura Charles
ID#: 05714774
2.3
xcii
Table B 8: pH results obtained for Guava Pine produced by Fruta Kool Kidz
Readi
ng
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Trial 1
NaOH
pH
(ml)
3.94
4.37
4.56
5.08
5.70
6.44
7.85
9.95
10.3
0
10.4
9
10.6
3
10.7
3
10.7
6
10.8
8
10.9
1
10.9
6
11.0
1
11.0
3
11.0
9
11.1
0
11.1
3
11.1
6
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
2.1
Trial 2
pH
NaOH (ml)
3.80
3.95
4.55
5.00
5.24
5.88
6.61
9.04
10.0
5
10.3
3
10.5
2
10.6
4
10.7
4
10.8
2
10.8
9
10.9
5
11.0
0
11.0
5
11.0
9
11.1
2
11.1
5
11.1
9
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Laura Charles
ID#: 05714774
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
2.1
xciii
Table B 9: Standardization of NaOH using standard HCl

Readi
ng
pH
NaOH
(ml)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1.13
1.14
1.16
1.19
1.21
1.24
1.27
1.30
1.34
1.37
1.40
1.44
1.48
1.53
1.57
1.63
1.68
1.75
1.82
1.90
2.00
2.11
2.26
2.44
2.79
8.72
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
21.0
22.0
23.0
24.0
25.0
Laura Charles
ID#: 05714774
xciv
11.1
1
11.1
6
11.2
3
11.3
0
11.3
5
11.4
0
11.4
3
11.4
6
11.4
9
11.5
2
11.5
4
11.5
7
26
27
28
29
30
31
32
33
34
35
36
37
26.0
26.2
26.4
26.6
26.8
27.0
27.2
27.4
27.6
27.8
28.0
28.2
APPENDIX C
6.3 Sample Calculations
6.3.1 Standardization of NaOH
Reaction:
NaOH(aq) + HCl(aq)
NaCl(aq) + H2O(l)
From Fig. (4-16) vol. of NaOH at endpoint = 24.7 ml

Ratio of NaOH(aq) to HCl(aq) is 1:1
Conc. of HCl = 0.1M
Vol. of HCl used = 25 ml
Laura Charles
ID#: 05714774
xcv
25 ml of HCl contains
mols
24.7 ml of NaOH contains
mols
Therefore # of moles in 1000 ml =
mol
Conc. of NaOH = 0.101M
6.3.2 Batch 1 of Orange Juice by Fruta Kool Kidz
Vol. of juice tested = 1 ml in 50 ml water

Vol. of NaOH as obtained from Fig. (4-17) = 0.55 ml
# moles of ASC = # moles of NaOH in 0.55 ml
=
moles
Molar mass of ASC = 176

Mass of ASC =
Thus mass of ASC in 200 ml of juice =
g = 9.68 mg/1 ml juice
mg = 1936 mg
Stated value = 100% Vitamin C = 75 mg
Laura Charles
ID#: 05714774
xcvi
NOMENCLATURE
ASC -
Vitamin C or Ascorbate
DCIP -
2, 6-Dichloroindophenol (titration method)
HCl -
hydrochloric acid
HPLC -
High Performance Liquid Chromatography
I-
iodine
NaOH -
sodium hydroxide
PDV -
Percentage Daily Value
RDA -
Recommended Daily Allowance
(aq) -
aqueous
(l) -
liquid
Laura Charles
ID#: 05714774

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THE UNIVERSITY OF THE WEST INDIES

DETERMINATION OF THE VITAMIN C CONTENT

SUPERVISOR: MR. J. SMITH

April 18, 2008

Figure 2- 1: Structure of L-ascorbic acid................................................................................12

75 mg per day for women,

ASC has been tested under many

Frei et al, 1989 and 1991

Figure 2- : Structure of L-ascorbic acid

Table 2- : Physical Properties of L-Ascorbic Acid5

Figure 2- : Diagram of the oxidative and reduced structure of Vitamin C

2.4 Importance of Vitamin C in Nutrition

Other symptoms of the vitamins deficiency include

neuropsychiatric changes such as depression, mood swings and hypochondria7.

However, according to the Institute of Medicine Report, not enough

An antioxidant is defined as a substance that, when present at low concentrations compared

2.4.2 Recommended Intake of Vitamin C

Upper Intake Level

2.5 Methods of Analysis

oxidized ascorbic acid + 2I-

I3- + starch complex (purple to blue-black)

2.5.2 Potentiometry using pH Analysis

However, due to the presence of other

2.5.3 High Performance liquid Chromatography

3.1 2, 6-Dichloroindophenol Titration

3.1.3 Preparation of Cereal Sample

Indophenol Dye solution

Colour change with

3.2 Iodine Test

End point colour

3.3 pH Potentiometry Test

0.1M NaOH solution

Results and Calculations

4.1 2, 6-Dichloroindophenol Titration

Table 4- : Titration results obtained for Ascorbic acid standard

4.1.2 Laboratory Results for Sunshine Cereals

Table 4- : Titration results obtained for Frosted Flakes produced by Sunshine

Table 4- : Titration results obtained for Froot Ooos produced by Sunshine

Sample Calculations for Sunshine Cereals

From Eq. (4.1) we have:

`= 10.15 mg Ascorbic acid/ 100 g or ml

Mass of packaged cereal = 28 g

Stated Value on label is 25%

Vol. made up (ml)

Packaged Mass (g)

Required Vitamin C (mg)

Vitamin C in Packaged mass

Packaged Mass (g)

Required Vitamin C (mg)

Vitamin C in Packaged mass

Vol. made up (ml)

Required Vitamin C (mg)

Packaged Mass (g)

4.1.4 Laboratory Results for Kelloggs Cereal

Table 4- : Titration results obtained for Kellogg's Frosted Flakes

Table 4- : Titration results obtained for Kellogg's Froot Loops

Table 4- : Titration results obtained for Kellogg's Special K

Sample Calculations for Kelloggs Cereal

From Eq. (4.2) we have:

`= 5.51 mg Ascorbic acid/ 50 g or ml

Stated Value on label is 10 %

4.1.5 Results based on Calculations for Kelloggs Cereal

Vol. made up (ml)

Packaged Mass (g)

Laura's Vit C Project

Laura's Vit C Project