ASCORBIC ACID

DETERMINATION
IN FOOD
Prepared by: Arş.Gör. Duygu Özmen
Lecturer: Dr. Nur ÇEBİ
CONTENTS

INTRODUCTION

PRINCIPLE OF THE EXPERIMENT

EXPERIMENTAL PROCEDURE

CALCULATION OF THE RESULTS

 INTRODUCTION
• Vitamins are important organic substances
for normal growth and maintenance of life. If
any of the vitamins are not taken into the
body, there may be disruptions in growth
and functions of organism due to the • Helping grow
chemical reaction that the vitamin helps. Effects of
• Help create healthy
generations
• The effects of vitamins in functions of body vitamins on • Normal functioning of the
nervous and digestive
are related to the regulation of biochemical human health systems, proper use of
nutrients, and assistance to
reactions. The effects of vitamins on human body resistance
health can be divided into three groups;
1. Helping grow
2. Help create healthy generations
3. Normal functioning of the nervous and
digestive systems, proper use of
nutrients, and assistance to body
resistance.
 INTRODUCTION
• Vitamins are required in very low
amounts in all metabolic activities.
Today there are 15 compounds
identified as vitamins. These Vitamins
compounds:
• Active vitamin structure from the
body can be taken from food
Active
• Provitamins (vitamin precursors) Provitamin
after being taken into the body vitamin
undergoes a series of chemical
changes into one or more
compounds that show vitamin
activity.
 INTRODUCTION
Vitamins are present in different amounts in foods and show different
distributions. Although some foods are quite rich in one or several
vitamins, they contain some vitamins in trace levels.
• These compounds, which are involved in many functions such as
growth, development and repair in the body, should be taken
regularly in certain amounts daily. For this reason, one should create
a balanced diet from food.
• Inadequate intake of vitamins with food causes absorption disorders
or some metabolic disorders.
• Disease caused by vitamin deficiencies is defined as hypovitaminosis
(taking vitamins below the normal minimum). For example; without
fresh fruit and vegetables, sailors who had to manage dry foods for a
long time had scurvy due to vitamin C deficiency. Since vitamin pills
could not be made with the technology of the time, vitamin C had to
be obtained from foods other than fresh fruits and vegetables.
Vinegar, pickles, rum and lime raw lime began to be included in the
list of supplies on the ships for this purpose.
 INTRODUCTION
The amount of nutrients taken into the body differs from the
amount used in the body. This is related to the bioavailability
(bioavailability; absorption of any nutrient in the body and the
level of its use in the organism). The need for vitamins in very
low amounts increases the importance of bioavailability.
Bioavalibility of vitamins varies depending on;
• The composition of the individual's diet,
• Whether the vitamin is in active vitamin form or coenzyme
form,
• How the vitamin interacts with other nutrients.
In addition, the bioavailability of vitamins may be affected by
the way foods are processed and may also vary from individual
to individual.
 INTRODUCTION
Vitamins are generally classified according to
their physical properties as follows:
• Fat-soluble vitamins: A, D, E, K.
• Water soluble vitamins: B1 (Thiamine), B2
(Riboflavin), B6 ​(pyridoxine), B12 (cobalamin),
Niacin, Folic acid, Biotin, Pantothenic acid,
Vitamin C (Ascorbic acid).
Since vitamins are generally unstable substances,
they can be damaged during processing of foods.
In addition, care must be taken when preparing
the extract in vitamin analysis. The amount of
vitamins in foods can be expressed as gr /100gr,
USP unit or I.U.
 INTRODUCTION
Vitamin analysis in foods can be determined by
volumetric titration and instrumental analysis. Vitamin
analysis in most foodstuffs can be performed by
spectroscopic methods. For this purpose, vitamins in
foodstuffs are extracted with suitable solvents (mostly in
acidic medium) and taken into solution. The solution is
passed through a special chromatographic column to
separate the vitamins and quantify using fluometry or
other optical methods.
The most valid method for accuracy is HPLC. However, it
is not preferred because it is very expensive.
The most common vitamin analyzes in foods;
• Determination of vitamin A (beta carotene) in soda
and fruit and vegetable products
• Determination of vitamin A and D in vegetable
margarines
• Vitamin C determination in fruit and vegetable
products.
INTRODUCTION
For Vitamin C analysis there are usually three methods as follows:

Titration with Tillman Indicator (2,6-

dichlorophenolindophenol dye)

Titration with iodine

Spectrophotometric method
 INTRODUCTION
Vitamin C: Vitamin C, composed of colorless crystals with a
melting point of 192oC and a molecular weight of 176, is an
antiscorbut factor. It contains a dienol group which has both
reducing power and acidic properties.
• Easily soluble in water, methanol and ethanol.
• Insoluble in benzene, ether, petroleum ether, chloroform and
oil.
• Vitamin C (Ascorbic acid) is the most vulnerable of vitamins.
• Very sensitive to alkalis and oxidation, especially when
catalysts such as Cu and Fe are present.
• More resistant as dry crystals.
• Very resistant to acid solutions (below pH 4).
• Ascorbic acid is also oxidized by the oxygen of the air. As a
result of this oxidation, the molecule loses its vitamin activity.
• Available in nature as reduced (C6H8O6) and oxidized (C6H6O6).
 INTRODUCTION
Deficiency:
• Capillary vessels can have a weak structure,
• Teeth gums can bleed easily, teeth can be lost
• It causes joint diseases.
• It is also necessary for the normal formation of collagen and protein, an
important element of the skin and connective tissues.
• Vitamin C increases the body's resistance to many diseases. Daily vitamin C
requirement is 30–40 mg in adults.
• Vitamin C is the main vitamin contained in foods of plant origin.
Richest resources:
• Fresh fruits, especially citrus fruits such as oranges, tangerines, rose hips,
strawberries
• Fresh vegetables are dark green leafy vegetables, tomatoes, green and red
peppers, and potatoes.
 INTRODUCTION
• Vitamin C is very easily broken down by oxidation and also
thermally, especially at high temperatures. Vitamin C is the
most lost during processing, storing and cooking foods. As it is
so sensitive to various processes, the amount of loss in
ascorbic acid is used as a criterion of determining the negative
effect of many processes applied to foods. In the products
made from vegetables and fruits, vitamin C is determined to
investigate whether vitamin C is present, how much is the
amount, and whether it will contribute to nutrition.
• Some products may contain vitamin C or ascorbic acid to
facilitate processing or to prevent oxidation. In this case, it is
investigated whether the quantity in the product is as much as
the allowable amount. The findings are compared with the
standards (TS 6397).
 PRINCIPLE OF THE EXPERIMENT
Dye
• It is a pink color produced by dehydro-ascorbic solution
acid formed by titration with 2,6-
dichlorophenolindophenol dye solution of
ascorbic acid which is a strong reducing agent.
2,6-dichlorophenolindophenol dye is blue in
alkali enviroment and pink in acidic enviroment

Sample

2,6-dichlorophenolindophenol dye is preperad

Begining of During with sodium bicarbonate (alkali envoriment),
End of
the titration
titration and at the end of the analyse it gives pink cause
titration (colorless))
of metaphosphoric acid in the enviroment
 EXPERIMENTAL PROCEDURE
1. Preparation of Sample (Extraction):
Acids Used For
If the amount of ascorbic acid in the foodstuff to be determined is solid: Extraction&
•Approximately 200 - 300 g of sample is weighed and put into a blender,
Stabilization Of The
•Add 6% metaphosphoric acid solution up to the amount of sample
weighed, Ascorbic Acid
•Blender is turned into a homogenous crush by operating. (It is enough
to run the blender for 2 minutes) Metaphosphoric acid is used as
stabilizer to prevent enzymatic oxidation of ascorbic acid during this
process. Metafosforic Acid (%6)
• AD(+):
•After weighing 10 - 40 grams of the mixture in the blender, add 3% • Better protection from metallic
metaphosphoric acid solution to a 100 ml measuring flask and fill the
line with the same solution and filter through filter paper. ions
• Better protection in general
If the sample to be examined is a homogeneous liquid such as fruit pulp, • DISAD(-):
juice or tomato paste:
• Expensive
• Take 20 g or 20 ml directly and transfer to a 100 ml flask. Make up to • Hard to dissolve
volume with metaphosphoric acid solution. • Low Stability
• The sample is thoroughly mixed and filtered. In this way, the sample
can be stored without oxidation until titration.
 EXPERIMENTAL PROCEDURE
• 2. Standardization of dye solution:
• The aim of the standardization: In order to determine the factor of dye solution which is the
amount of dye solution (ml) used to to reduce 1 mg ascorbic acid the following steps should
be taken:
• Preperation of standard ascorbic acid solution: Take 10 mg ascorbic acid and solve it in 50 ml
MFA solution. According to this 5 ml standard ascorbic acid solution contains 1 mg ascorbic
acid.
• 5 ml of MFA solution and 5 ml standard ascorbic acid solution is put into a flask.
• Titrate to pink color with dye solution in the burette.
• The amount spent is saved. (V0) =… .m
• 3. Titration of the sample:
• Take 20 ml of the test sample and place 20 ml of MFA solution on top.
• Mix well and filter.
• 5 - 10 ml is taken from the filtrate and titrated to the light pink color with the
dichlorophenolindophenol solution in the burette. (The resulting color should be permanent
for at least 15 seconds and the titration should be completed as soon as possible.)
• The amount spent is saved. (V1) =… .ml
 CALCULATION OF THE RESULTS
M2: Amount of original sample in titrated filtrate (g)
M1: Amount of sample taken (g)
Vt: Amount of filtrate taken for titration
V: Completed flask volume (ml)

V0 :Spent dye solution amount for standard vitamin C

solution (ml)
F: Factor
1: Amount of ascorbic acid in the flask (1 mg)
 CALCULATION OF THE RESULTS
ANOTHER WAY :
V0 :Spent dye solution amount for
If V0 volume of dye solution oxidise 1 mg of ascorbic acid standard vitamin C solution (ml)
M: Amount of original sample in
titrated filtrate (g)
V1: Amount of dye solution spent
V1 volume of dye solution oxidise ??? mg of ascorbic acid for the sample

??? =(V1*1)/ V0

This is for the amount of sample in the flask! If you made further dilution you
should take those into consideration.

If M g of sample has ??? Amount of ascorbic acid

100 g of sample has X amount of ascorbic acid

X=(100*???)/M
 FOR REPORT
Spent dye solution amount for standard vitamin C solution (ml)=10
PARALLEL 1
Amount of original sample in titrated filtrate (g)=5,1
Amount of dye solution spent for the sample=20,3 ml
PARALLEL 2
Amount of original sample in titrated filtrate (g)=5,3
Amount of dye solution spent for the sample=20,6 ml
PARALLEL 3
Amount of original sample in titrated filtrate (g)=5,0
Amount of dye solution spent for the sample=20,1 ml

Calculate the amount of ascorbic acid in the sample (mg/100g) ?

Discuss the results with the proof of academic journals, take sample as «lemon juice»
USE AT LEAST 2 SOURCE FROM ACADEMIC JOURNALS!
 VIDEO SOURCES
• https://www.youtube.com/watch?v=ghvF0eYi6rA
• https://www.youtube.com/watch?v=oI33svVr82U
• https://www.youtube.com/watch?v=4y-zqxOHJKQ
• Iodine Method : https://
www.youtube.com/watch?v=m-oVOTTmbdE
• Merck Device Quick New Method: https://
www.youtube.com/watch?v=D6rsfw_tICo
THANK YOU FOR YOUR
ATTENTION
Dr. Nur ÇEBİ