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CHAPTER 8. VITAMIN FST

The document outlines a course on vitamin analysis, detailing learning outcomes, methods of extraction, and various physicochemical methods for analyzing vitamins A, C, B1, and B3. It emphasizes the importance of proper precautions during analysis due to the sensitivity of vitamins to environmental conditions. Specific analytical methods such as colorimetric, fluorometric, and HPLC are described for different vitamins, highlighting their sources and the principles behind each method.

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4 views21 pages

CHAPTER 8. VITAMIN FST

The document outlines a course on vitamin analysis, detailing learning outcomes, methods of extraction, and various physicochemical methods for analyzing vitamins A, C, B1, and B3. It emphasizes the importance of proper precautions during analysis due to the sensitivity of vitamins to environmental conditions. Specific analytical methods such as colorimetric, fluorometric, and HPLC are described for different vitamins, highlighting their sources and the principles behind each method.

Uploaded by

Izzat danial
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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VITAMIN

LEARNING OUTCOMES

At the end of this course students are expected


to be able:
• Apply the principles and procedure of chemical and
instrumental analysis of vitamin.
• Analyse vitamin components to provide scientific solutions.
• Display the analysis of vitamin components using chemical
and instrumental methods.
INTRODUCTION
• Definition :
Relatively low molecular weight compounds requires by
human body and any type of living organisms as a source
of nutrient for normal metabolisms.
• IMPORTANCE OF ANALYSIS
Assuring adequate supply from existing food regimen.
Assessment of vitamin bioavailability for its user.
METHOD

• Vitamin assays involving:


physicochemical assays which are spectrohphotometric, fluorometric,
chromatographic, enzymatic and others

• Precautions:
1) Since vitamins are sensitive to conditions like light, oxygen, pH and heat, proper
precautions need to be taken to prevent its deterioration throughout the analytical
process.

2) Proper sampling and sub-sampling and preparation of homogeneous sampling


are critical for vitamin analysis.
METHOD
• Extraction methods
Extraction - Involving extraction of vitamins from its biological matrix before
analysis
Extraction procedures are specific for each vitamin.
• Types of extraction methods are:
i) ascorbic acid- cold extraction with metaphosphoric acid/acetic acid.
ii)Vitamin B1 and B2-boiling or autoclaving in acid and enzyme treatment.
iii)Niacin-autoclaving in acid or alkali
iv)Vit. A, E or D - organic solvent extraction, saponification and re-extraction with
organic solvents
PHYSICOCHEMICAL METHODS
 Structure of Vitamin A
PHYSICOCHEMICAL METHODS

• Sources of vitamin A
Liver,the richest dietary source of vitamin A
Plants do not contain vitamin A, but many dark-green or
dark-yellow plants (e.g carrot, cabbage, corn etc.) contain
pro-vitamin A like carotenoids such as beta-carotene (pro-
vitamins converted to vitamin A are within the intestinal
mucosa to retinol during absorption)
PHYSICOCHEMICAL METHODS

VITAMIN A
Precautions:
Vitamin A sensitive to UV light, air, prooxidant, high
temperature and moisture. Therefore, need to use low
actinic glassware/cover glassware with aluminium foil,
nitrogen or vacuum, avoid excessively high temperature
and use antioxidant at the onset of procedure.
PHYSICOCHEMICAL METHODS

• Vitamin A
a) Colorimetric method
Principle:
• It measures the unstable colour at A620nm that results from reaction between
Vitamin A and antimony trichloride (SbCl 3)
• The intesity of blue colour proportional to the amount of retinol in food sample.
• The intensity of blue colour is measured against the set of known standards.
• The colour reaction does not differentiate between retinol isomers and retinol
esters.
PHYSICOCHEMICAL METHODS

• Vitamin A
b) HPLC method
This method involve chromatographic separation and quantitative
determination at 325 nm
PHYSICOCHEMICAL METHODS
 Vitamin C
Structure :

The terms vitamin C, ascorbic acid and ascorbate are


commonly used interchangeably.
PHYSICOCHEMICAL METHODS

• Sources of vitamin C
strawberries, tomatoes, broccoli, turnip greens and other leafy greens, sweet
and white potatoes

• Precautions:
The vitamin (L-ascorbic & -dehydroascorbic acid) is very susceptible to oxidative
deterioration, which enhanced by high pH and presence of ferric and cupric
acid. Therefore, the entire analytical procedure must be conducted at low pH
and with addition of chelating agent.
PHYSICOCHEMICAL METHODS
• Methods for vitamin C
i) 2,6-dichlorophenolindophenol titrimetric method.
Principle
 L-ascorbic acid is oxidizes to dehydroascorbic acid by indicator dye.
 It measures the decolourization of 2,6-dichorophenolindophenol dye by ascorbic acid.
 At the endpoint, excess of unreduced dye is rose pink in acid solution lasting at least 10 sec.
 This method is not suitable for highly coloured products (for example coloured fruit juices)
because of difficulty of determining the endpoint during titration.
 L- dehydroascorbic acid can be determined by first converting it to L-ascorbic acid with a
suitable reagent.
PHYSICOCHEMICAL METHODS
ii) Fluorometric method
Principle
This method measures both ascorbic acid and dehydroascorbic acid.
Ascorbic acid oxidised to dehydroascorbic acid upon reaction with o-
phenylenediamine.
Reaction between dehydroascorbic acid and o-phenylenediamine formed a
fluorescent quinoxaline compound.
The flourescent compound intensity proportional to the vitamin C content.
PHYSICOCHEMICAL METHODS
 Thiamine (Vitamin B1)
Structure:
PHYSICOCHEMICAL METHODS

• Thiamine (Vitamin B1)


Sources:
Green peas, Spinach, Liver, Beef, Nuts, Pinto beans, Bananas, Soybeans, Whole-
grain and Enriched Cereals, Breads, Yeast, aleurone layer of unpolished rice,
and Legumes.
PHYSICOCHEMICAL METHODS

• Methods for Thiamine (Vitamin B 1)


Thiochrome Fluorometric method

Principle
• In order to free the thiamine from the natural ester and protein bonds, the material
to be examined is digested with sulphuric acid and subsequently treated with a
phosphatase preparation.
• The thiochrome resulting from oxidation with potassium ferricyanide/hydrogen
peroxide in alkaline solution is extracted with isobutyl alcohol.
• The intensity of the blue fluorescence proportional to the thiamine concentration.

• The intensity of the blue fluorescence of the isobutyl alcohol extract is compared
with that of the standard solution. The intensity of fluorescence is measured.
PHYSICOCHEMICAL METHODS

• Methods for Thiamine (Vitamin B1)


Thiochrome Fluorometric method

Precaution :
• Thiochrome is light sensitive, therefore, analysis should be
performed under subdued light.
• Thiamine is sensitive to heat especially at alkaline pH. The
analytical steps starting from oxidation of thiamine until
flourescent measurement need to be carried out rapidly and
precisely according to the instructions.
PHYSICOCHEMICAL METHODS
 Niacin (Vitamin B3)

 Niacin is commonly refer to nicotinic acid &


niacinamide
 Sources : Liver, fish, peanuts, yeast, wheat germ,
whole grain, avocado, dates, prunes.
 Other sources : Milk & eggs
PHYSICOCHEMICAL METHODS

• Method for Niacin (Vitamin B3)


Colourimetric method

• Principle:
Involves a reaction between niacin (nicotinic acid) and cynogen
bromide, forms a coloured compound with an intensity proportional
to niacin concentration (under proper conditions)
• Critical : toxicity of cyanogen bromide, the analysis must be
carried out under fume hood.
• The result expressed as μg niacin / g sample
Other method of vitamin analysis

HPLC
• The extraction procedure are the same as outlined for the
vitamin determination.
• However, in this method, a common extract of the
vitamin is concentrated and separated by HPLC.

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