CARBOHYDRATE

LEARNING OUTCOMES
 After completing this topic, student should be able to:
 Explain briefly the definition and general classification
of carbohydrate.
 State the importance of carbohydrate determination.
 Explain the sample preparation of carbohydrate
analysis.
 Describe the carbohydrate determination whether by
chemical and physical methods.
INTRODUCTION
 Carbohydrates are the most abundant & widely distributed
food component in nature (55 – 60% of the caloric value
in human diet).
 Most forms of carbohydrate are composed of C, H and O;
in the ratio of 1 : 2 : 1; respectively.
 General formula : (CH2O)n; where n – represent numbers
of times the ratio is repeated
 Digestible CHO – provides an important source of energy
 Indigestible CHO – do not provide energy (known as
dietary fibre including lignin)
 Contributes to the sweetness, appearance & textural
characteristics of many foods
INTRODUCTION
 CHO may be present as isolated molecules or they may
physically associated or chemically bound to other
molecules
 Individual molecules – classified according to numbers of
monomers they contain e.g. monosaccharides,
disaccharides, oligosaccharides or polysaccharides
 Glycoprotein – CHO molecules are covalently attached
to proteins
 Glycolipids – CHO molecules are covalently attached to
lipids
CLASSIFICATION
Monosaccharides
 Also known as simple sugar
 Water-soluble crystalline compounds
 Aliphatic aldehydes or ketones which contain one carbonyl
group and one / more hydroxyl group

 Most naturally occurring monosaccharides have either 5

(pentoses) or 6 (hexoses) carbon atoms
 Reactive centers of monosaccharides : carbonyl & hydroxyl
groups
 E.g. monosaccharides : glucose, fructose, galactose
CLASSIFICATION
Monosaccharides

Galactose
CLASSIFICATION
Monosaccharides
 Reducing sugars
 Sugars posses free aldehyde or ketonic group in their
structure react with reducing agent (e.g. Fehling’s solutions
/ Benedict’s reagent)
 All monosaccharides are reducing sugar (free reactive
carbonyl group), and some disaccharides are also reducing
sugar (exposed carbonyl groups)
CLASSIFICATION
Oligosaccharides
 Relatively low MW polymers of monosaccharides ( <20 )
 Covalently bonded through glycosidic linkages

 Polymers that yield monosaccharides after hydrolysis

 Disaccharides – two monomers e.g. maltose, sucrose,

lactose)

Examples of disaccharides structure

CLASSIFICATION
Oligosaccharides
 Occur naturally in plants, animals and microorganisms

 Comprised of D-glucose, D-fructose and D-galactose

monomers
 Other important food oligosaccharides – raffinose,
stachyose, verbascose and fructo-oligosaccharides
CLASSIFICATION
Polysaccharides
 High MW polymers of monosaccharides (>20)
 Majority of CHO found in nature occur as polysaccharides
 Polymers that yield monosaccharides upon acid or specific
enzyme hydrolysis
 Homopolysaccharides – all contains the same monomer of
monosaccharides (e.g. starch, cellulose, dextrins and
glycogen)
 Heteropolysaccharides – contain more than one type of
monomer (e.g. pectin, hemicellulose and gums)
 Starch is the only polysaccharides that can be digested by
human and use as an energy source. Other polysaccharides
are indigestible.
CLASSIFICATION
Polysaccharides

Amylose

Starch

Amylopectin
CLASSIFICATION
Polysaccharides

Dextrins Pectin

 Polysaccharides play a crucial role in textural properties of

foods
IMPORTANCE OF CARBOHYDRATE
ANALYSIS
 Nutritional Labelling – to inform consumers of the
nutritional content of foods
 Standards of Identity – compositions conform to govt.
regulations
 Food Quality – physicochemical properties of foods e.g.
sweetness, appearance, stability and texture depend on
CHO type and concentration
 Food Processing – efficiency of food processing operations
depends on CHO type and concentration
CARBOHYDRATE CONTENT OF
FOODS
Food % Food %
Milk 4.78 Rice 30.00

Yogurt 5.60 Dates 48.5

Bread 52.20
Fruit juice 6-12
Honey 75.10
Apple 12.39
Flour 80-85
Grape 16.11
Brown sugar 86.60
Potato 19.80
Table sugar 99.40
Soy Bean 25.50
SAMPLE PREPARATION
 Important to get accurate and consistent results.
 Due to complex nature of food, sample must be purified to
remove possible interferences that may affect analysis.
 Preparation of sample for CHO analysis depends on the
nature of food being analyzed
 Aqueous solution - require very little preparation prior to
analysis
- e.g. fruit juices syrups and honey
 Solid sample - physically associated / chemically bound to
other components
- necessary to isolate CHO before it can be
analyzed
- e.g. nuts, cereals, breads and vegetables
SAMPLE PREPARATION
 Isolation technique :
 depends on CHO type, food matrix type and purpose of
analysis
 Foods are dried under vacuum (to prevent thermal
degradation), ground to fine powder (to enhance solvent
extraction), then defatted by solvent extraction. Extraction
of low MW CHO from foods is to boil defatted sample with
an 80% alcohol solution.
 Mono- and oligosaccharides – soluble in alcoholic solutions
Polysaccharides and dietary fibre – insoluble in alcoholic
solutions
SAMPLE PREPARATION
 Soluble components can be separated from insoluble
components by filtering the boiled solution and the filtrate is
collected.
 the two fractions are dried & weighed to determine their
concentrations
 Soluble components also contain other small molecules
that could interfere with subsequent analysis
 e.g. amino acids, organic acids, pigments, vitamins,
minerals etc.
 necessary to remove these components prior to CHO
analysis as the components may be coloured or produce
turbidity which will interfere with endpoint determinations
and spectorscopic analysis
SAMPLE PREPARATION
 The interfering chemical compounds can be removed by
treating the solution with :
1. Clarifying agents
2. Ion-exchange resins
 Clarifying agents
 Solutions are usually clarified prior to analysis
 Heavy metal salts (e.g. lead acetate) :
Forms insoluble complexes with interfering substances.
The complexes can be removed by filtration /
centrifugation.
Clarifying agent must not precipitate any CHO from the
solution (causing underestimation of CHO content)
SAMPLE PREPARATION
 Ion-exchange resins
 Many mono- and oligosaccharides are polar non-charged
molecules; therefore can be separated from charged
molecules by passing samples through ion-exchange
columns
 Non-polar molecules can be removed by passing a
solution through a column with non-polar stationary phase
 Proteins, amino acids, organic acids, mineral &
hydrophobic compounds can be separated prior to
analysis
 METHODS OF ANALYSIS
Chemical methods for Mono- and Oligosaccharides

 Based on mono- and oligosaccharides substances are

reducing agents
 react with other components to yield precipitates or
coloured complexes which can be quantified
 Titration : Lane-Eynon Method
 Gravimetric : Munson-Walker Method
 Colorimetric : Somogyi-Nelson Method
: Anthrone Method
: Phenol-Sulfuric Method
: DNS Method
METHODS OF ANALYSIS
Lane-Eynon Method
 Titration method of determining the concentration of
reducing sugar in a sample
 Non-reducing CHO are hydrolyzed to make them reducing;
then the concentration can be determined by using the
same method
METHODS OF ANALYSIS
Lane-Eynon Method
 Principle :
 Based on the reaction of reducing sugar with a solution of
copper sulfate followed by reaction with alkaline tartrate
(or by treatment with the Soxhlet solution, i.e. equal
volume mixture of copper sulfate solution and alkaline
tartrate solution)
 Mixture is boiled for a specific time, followed by addition of
methylene blue (as an indicator)
 Coloured solution is titrated until decolouration of the
indicator
METHODS OF ANALYSIS
Lane-Eynon Method
 Procedures :
 CHO solution in a burette is titrated in into a flask
containing known amount of boiling copper sulfate solution
(mixed Fehling’s solution) and methylene blue indicator.
Air excluded from reaction mixture by keeping liquid boiling
throughout titration process.
Reducing sugars in the solution will react with copper
sulfate, converted to insoluble cuprous oxide. Once all
copper sulfate in solution has reacted, indicator change
color from blue to colorless. Volume of sugar solution
required to reach end point recorded.
METHODS OF ANALYSIS
Lane-Eynon Method
 Applications :
 Determinations of reducing sugars in honey and other
high-reducing sugar syrups
 Disadvantages :
 The reaction is not stoichiometric – necessary to prepare a
calibration curve with a series of standard solutions of known CHO
concentration.
 Results depends on the precise reaction times, temp., & reagent
concentrations
 Cannot distinguish between different types of reducing sugar
 Cannot directly determine the concentration of non-reducing sugar
 Susceptible to interference from other types of molecules that act
as reducing agents
METHODS OF ANALYSIS
Munson-Walker Method
 Gravimetric method of determining the concentration of
reducing sugar in a sample
 can be applied to all reducing sugar

 Principle
 Involving oxidation of the CHO in the presence of heat
and an excess of copper sulfate and alkaline tartrate,
under carefully controlled conditions – leads to the
formation of a copper oxide precipitate :
Reducing sugar + Cu2+ + base oxidized sugar + Cu2O (precipitate)

 Amount of precipitate formed is directly related to the

concentration of reducing sugar in the sample
METHODS OF ANALYSIS
Munson-Walker Method
 Concentration of precipitate present can be determined :
1. gravimetrically (by filtration, drying and weighing)

2. titrimetrically (be redissolving the precipitate and

titrating with a suitable indicator)
 Basic conditions (alkaline) are required to keep copper
solution as copper hydroxide (Cu+).
 The methods depends on the ability of reducing sugar to
react with copper solution
 Modification of Munson-Walker method : involves the use of
an excess alkaline copper citrate with sodium carbonate (base).
Following the reduction, excess copper citrate react with excess
potassium iodide. Liberation of iodine is titrated with sodium
thiosulfate.
METHODS OF ANALYSIS
Munson-Walker Method
 Advantages :
 More reproducible and accurate.
 Disadvantages :
 Same disadvantages as Lane-Eynon method.
METHODS OF ANALYSIS
Nelson-Somogyi Method

 Modification of Munson-Walker and Lane-Eynon Method to

measure reducing sugar
 Principle :
 The reducing sugar when heated with alkaline copper
tartrate reduce the copper, from cupric to cuprous state,
thus cuprous oxide is formed
Cuprous oxide is treated with arsenomolybdate reagent
(prepared by reacting ammonium molybdate
[(NH4)6Mo7O24)] and sodium arsenate (Na2HAsO7) in
sulfuric acid). Reduction of arsenomolybdate complex
produces an intense, stable blue-coloured solution.
METHODS OF ANALYSIS
Nelson-Somogyi Method
 Principle :
 The absorbance of the solution is determined at either 500
or 520nm against standard
 Require preparation of standard curve (using D-glucose at
levels of 50, 100 and 150 μg/mL)

 Applicable for samples containing low concentrations

of CHO (0.3 – 3 mg / tested aliquot)
METHODS OF ANALYSIS
Physical methods
 The methods are based on the physical properties of CHO
 Polarimetry
 Refractive Index
 Specific gravity / Density
 Infrared
 Limited for pure CHO syrups (e.g. honey, dextrose
syrups), requires clarified solutions and in some cases
require the use of conversion tables to obtain accurate
result.
METHODS OF ANALYSIS
Polarimetry
 Useful to measure chiral substances
 Most compounds that contain a chiral (asymmetric) carbon
atom will rotate the plane of polarization of polarized light
 The magnitude and direction of rotation of the plane of
polarized light by a chiral compound is a specific physical
property of the compound that can be used to
characterized it
 CHO contain chiral carbon atoms i.e. optically active
 Device to measure the angle that plane polarized light is
rotated on passing through a solution – Polarimeter
 Polarimetry makes use of the optical activity of CHO
METHODS OF ANALYSIS
Polarimetry
 Polarimeter consists of :
 Monochromatic light
 two prisms (polarizing prism and analyzer); polarizer used
to convert light waves in the beam of monochromatic light
into plane polarized light,
 cell or tube for sample solution
 scale (indicate no of degrees rotated)
METHODS OF ANALYSIS
Polarimetry
 Principle :
 Asymmetric carbon atoms have the ability to rotate plane
of polarization of polarized light
 Plane polarized light passed through solution exhibiting
optical activity, it rotated either to left (-) or right (+).
 Angle of polarization proportional to the concentration of
optically active molecules in solution.
 Prior to analysis, sample solution must be clarified.
 CHO able rotate plane polarized light through an angle of
rotation
METHODS OF ANALYSIS
Polarimetry
 Optical rotation depends on:
1. Light wavelength (λ)
2. Sample concentration (T)
3. Temperature
4. Amount of sample

 Wavelength (λ) often used : sodium D line (589 nm);

Cell / tube length : 1 decimeter (10 cm);
Operating temperature : 20oC
METHODS OF ANALYSIS
Polarimetry
 Formula that relates to the parameter :

(α)D20 = 100 α / lc
where : α = observed angular rotation;
l = length of tube (decimeter);
c = concentration of solution in g / 100 mL;
(α)D20 = specific rotation for the sugar at sodium D
line at 20oC
METHODS OF ANALYSIS
Polarimetry
 Concentration is determined from the specific optical
rotation value, when no other optically active compounds
are present and all other factors are held constant

 Concentration of an unknown sample is determined by

measuring the angle of rotation and comparing it with a
calibration curve
METHODS OF ANALYSIS
Polarimetry
METHODS OF ANALYSIS
Polarimetry
METHODS OF ANALYSIS
Polarimetry
 Precaution :
 Solution to be analyzed need to be clarified
 Allreducing CHO display mutarotation between α and ß
isomers. If CHO solution is freshly prepared / not
equilibrated, error may occur due to the phenomenon.
Therefore, CHO solution should be allowed to stand for
several hours to establish equilibrium; or add a few
drops of ammonia to establish equilibrium rapidly.

 Disadvantages :
 Polarimetry method unable to analyzed mixtures of CHO
(except sucrose in the presence of other CHO)
METHODS OF ANALYSIS
Polarimetry
METHODS OF ANALYSIS
Polarimeter
 Light can be thought as waves vibrating in all directions
perpendicular to the direction the light beam is traveling.

 A beam of light is like a wheel with infinite number of

wheels
 If the beam passed through a polarising filter, polarised
light will only vibrates in one direction.
 If another polarised light is set at 90o to the first, no light will
pass through the second filter
METHODS OF ANALYSIS
Polarimeter
METHODS OF ANALYSIS
Polarimeter
 If the sample tube is empty, light passes through the 1st
polarising filter, and the polarised light hits the analysing
filter (2nd polarising filter) at the same angle (plane) as it left
the 1st filter.
When an optically active solution is placed in the sample
tube, the polarised light is rotated by the solution and hits
the analysing filter at different plane whose angle relative to
the empty state that can be measured by rotating the
analysing filter.
 If analyzer rotates clockwise (+) dextrorotary
If analyzer rotates anticlockwise levorotatory
METHODS OF ANALYSIS
Polarimeter
METHODS OF ANALYSIS
Refractive Index (RI) Measurement
 Measures the content of dissolved solids in sugar solutions.
 Principle :
 RI (n) of a substance is the ratio of light velocity in a
vacuum to its velocity of a substance.
 When electromagnetic radiation passes from one medium
to another, it can change direction :
 it is either bent or refracted

 RI of a material can be determined by

measuring the ratio of angle of
incidence (i) to angle of refraction (r)
i.e. index of refraction (n)
METHODS OF ANALYSIS
Refractive Index (RI) Measurement
 Principle (cont’d) :
 RI of a substance depends on :
1. Concentration ([conc.] ; RI of CHO solution )
2. Temperature (T)
3. Wavelength of light
 RI standard measurement are made specific at T (20oC)
and wavelength (monochromatic sodium light at 589 nm).
 RI readings are normally expressed as % sugar wt./wt. or
alternatively oBrix (g sucrose / 100 g of sample).
 In practice, RI of CHO solution is usually measured at a
boundary with quartz.
METHODS OF ANALYSIS
Refractive Index (RI) Measurement
 Abbe refractometer – common type of refractometer.
 Precaution :
 Do not use ether or acetone to clean off samples from
prism because these solvents evaporate quickly and in that
process change the temperature.
 Applications :
 Method is quick & simple to carry out, gives direct reading
and require only one or two drops of sample.
 Performed with simple hand-held instrument.
 Analysis of food carbohydrates (total soluble solids) in variety
of products e.g. sugar syrups (honey, maple syrup, molsses),
tomato products & fruit products (juices, jams, jellies, etc.)
METHODS OF ANALYSIS
Refractive Index (RI) Measurement
 Hand-held refractometer
METHODS OF ANALYSIS
Refractive Index (RI) Measurement
 Abbe refractometer
METHODS OF ANALYSIS
Calculation of Carbohydrate by difference
 Amount of total CHO in food can be quantitated by
difference :
Total CHO = 100 – (% moisture + % protein + % fat/lipid + % ash)

 Disadvantages :
 Inaccurate result for CHO content due to experimental
error that may occur during determination of these major
food constituents
 Incomplete digestion / extraction of these major food
constituents – inaccurate result for CHO content
 Does not differentiate between available & non-available
CHO. Hence, specific analyses are necessary.
SUMMARY
 Many analytical techniques for CHO require sample
preparation that includes extraction & clarification
process
 Most of chemical methods for analysis of CHO are based
on the reaction of reducing sugars with chemical reagents :
 Yield precipitates of coloured complexes, which are
quantitated by solubilization then titration; or by specific
spectrophotometric determination.
 Some methods are non-stoichiometric, therefore require std
curves. Problems in accuracy when mixtures of sugars are
being determined.
 Physical methods (polarimetry, RI) are useful as rapid quality
control techniques for products such as syrups & juices.