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Assam Agricultural
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College of Community Science

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 CONTENT
• Introduction
• Sample preparation
• Some commonly used methods
for carbohydrate analysis:
1) Chromatographic method
2) Electrophoretic method
3) Chemical methods
4) Enzymatic method
• References
 INTRODUCTION
 Carbohydrates are one of the most important
components of food. Carbohydrates may be
defined as polyhydroxy aldehydes or ketones or
compounds which produce them on hydrolysis.
They are often referred to as saccharides or
sugars.
 They are a major source of energy and dietary
fibre which influences physiological processes.
 Digestible carbohydrates, which are converted
into monosaccharides and absorbed by the body,
provide metabolic energy. Worldwide,
carbohydrates account for more than 70% of the
caloric value of the human diet
 Nondigestible polysaccharides (all those other
than starch) com- prises the major portion of
dietary fibre.
 On the basis of number of sugar units,
carbohydrates are divided into 4 categories:
monosaccharides, disaccharides, oligosaccharides
and polysaccharides.
 Carbohydrate analysis is of great importance in
the food sciences because of their significant role
of as macronutrients, as major constituents of
dietary fiber, and as food structure components
contributing to textural properties, and food
additives.
SAMPLE PREPARATION
Sample preparation for carbohydrate analysis
depends on the nature of the food being analysed.
Aqueous solutions, such as fruit juices, syrups and
honey, usually require very little preparation prior to
analysis.
In solid foods, carbohydrates are physically
associated or chemically bound to other components.
In these foods it is usually necessary to isolate the
carbohydrate from the rest of the food before it can
be analysed.
Preliminary method commonly used for isolation of
carbohydrates:
1. The first step is drying, which also can be used to
determine moisture content. Drying is done by
placing a weighed amount of material in a vacuum
oven and drying to constant weight.
2. Then, the material is ground to a fine powder to
enhance solvent extraction.
3. Lipids are extracted from the fine powder in a
Soxhlet extractor. Prior extraction of lipids makes
extraction of carbohydrates easier and more
complete.
4. The defatted sample is boiled with an 80% alcohol
solution. Monosaccharides and oligosaccharides are
soluble in alcoholic solutions, whereas proteins,
polysaccharides and dietary fiber are insoluble.
5. The soluble components can be separated from
the insoluble components by filtering the boiled
solution and collecting the filtrate and the retentante.
These two fractions can then be dried and weighed to
determine their concentrations.
6. In addition, to monosaccharides and
oligosaccharides various other small molecules may
also be present in the alcoholic extract that could
interfere with the subsequent analysis. It is usually
necessary to remove by treating the solution with
clarifying agents or by passing it through one or
more ion-exchange resins.
7. Prior to analysis, the alcohol can be removed from
the solutions by evaporation under vacuum so that
an aqueous solution of sugars remains.
SOME COMMONLY USED METHODS
FOR CARBOHYDRATE ANALYSIS:

1. CHROMATOGRAPHIC METHOD:
Chromatographic methods are the most powerful
analytical techniques for the analysis of the type and
concentration of monosaccharides and
oligosaccharides in foods. Types of chromatographic
methods for carbohydrate analysis are:
 TLC: TLC is a method for separating compounds
by their rate of movement through a thin layer of
silica gel coated on a glass plate. TLC is not very
suitable for carbohydrate analysis. Carbohydrates
are highly polar molecules and often require
derivatization to be analysed by TLC. This is
primarily because their polar nature makes it
difficult to separate these molecules on
commonly used polar supports, such as silica and
alumina.
 GC: GC provides both qualitative and quantitative
analysis of carbohydrates. For GC, sugars must be
converted into volatile derivatives. The most
serious problem with GC for carbohydrate
analysis is the reduction of aldehyde groups to
primary alcohol groups and conversion of the
reduced sugar into a volatile derivative.
  HPLC: HPLC is the method of choice for analysis
of mono- and oligosaccharides and can be used
for analysis of polysaccharides after hydrolysis
.HPLC gives both qualitative analysis. HPLC
analysis is rapid, can tolerate a wide range of
sample concentrations, and provides a high
degree of precision and accuracy. HPLC requires
no prior derivatization of carbohydrates.

2. ELECTROPHORETIC METHOD:
Carbohydrates can also be separated by
electrophoresis after they have been derivitized to
make them electrically charged, e.g., by reaction with
borates. A solution of the derivitized carbohydrates
is applied to a gel and then a voltage is applied
across it. The carbohydrates are then separated on
the basis of their size: the smaller the size of a
carbohydrate molecule, the faster it moves in an
electrical field.
3. CHEMICAL METHODS:
A number of chemical methods used to determine
monosaccharides and oligosaccharides are based on
the fact that many of these substances are reducing
agents that can react with other components to yield
precipitates or colored complexes which can be
quantified. Various chemical for carbohydrate
analysis are:
 Titration method: The Lane-Eynon method is a a
tritration method of determining the
concentration of reducing sugars in a sample. A
burette is used to add the carbohydrate solution
being analyzed to a flask containing a known
amount of boiling copper sulfate solution and a
methylene blue indicator. The reducing sugars in
the carbohydrate solution react with the copper
sulfate present in the flask. Once all the copper
sulfate in solution has reacted, any further
addition of reducing sugars causes the indicator
to change from blue to white. The volume of
sugar solution required to reach the end point is
recorded.
 Colorimetric method: The Anthrone method is
an example of a colorimetric method of
determining the concentration of the total sugars
in a sample. Sugars react with the anthrone
reagent under acidic conditions to yield a blue-
green color. The sample is mixed with sulfuric
acid and the anthrone reagent and then boiled
until the reaction is completed. The solution is
then allowed to cool and its absorbance is
measured at 620 nm. There is a linear
relationship between the absorbance and the
amount of sugar that was present in the original
sample. This method determines both reducing
 and non-reducing sugars because of the presence
of the strongly oxidizing sulfuric acid.
 Gavimetric method: The Munson and Walker
method is an example of a gravimetric method of
determining the concentration of reducing sugars
in a sample. Carbohydrates are oxidized in the
presence of heat and an excess of copper sulfate
and alkaline tartrate under carefully controlled
conditions which leads to the formation of a
copper oxide precipitate. The amount of
precipitate formed is directly related to the
concentration of reducing sugars in the initial
sample.
4. ENZYMATIC METHOD:
Analytical methods based on enzymes rely on their
ability to catalyze specific reactions. There are many
enzyme assay kits which can be purchased
commercially to carry out analysis for specific
carbohydrates. The two methods most commonly
used to determine carbohydrate concentration are:
(i) Allowing the reaction to go to completion and
measuring the concentration of the product,
which is proportional to the concentration of
the initial substrate;
(ii) Measuring the initial rate of the enzyme
catalyzed reaction because the rate is
proportional to the substrate concentration.
REFERENCE:
https://people.umass.edu/~mcclemen/581Carbo
hydrates.html
BeMiller JN (2007) Carbohydrate chemistry for
food. scientists, 2nd edn. AACC International, St.
Paul, MN