Assam Agricultural University, Jorhat

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- prise 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 analyzed. 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 analyzed.

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 removed 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 :

a. 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 analyzed 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.

b. 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.

c. 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 methods for carbohydrate
analysis are :

a. 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.

b. 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.

c. 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