Transcript TOPIC 6: CARBOHYDRATE ANALYSIS

CARBOHYDRATE
ANALYSIS
SFA3023
FOOD ANALYSIS
INTRODUCTION
• Classification of carbohydrates (CHO)
– Monosaccarides
– Disaccharides
– Oligosaccharides
– Polysaccharides
• Digestible
• Non-digestible
Sample Preparation for mono-, di- and oligosaccharides
• Need to isolate the CHO first before the
measurement is made.
• Preliminary method commonly used to many
isolation techniques
1. Food are dried under vacuum to prevent
thermal degradation.
2. Ground to a fine powder to enhance solvent
extraction.
3. Defatted by solvent extraction.
• Most commonly used method for isolation
 Boil a defatted sample with an 80% alcohol
solution to extract low molecular weight CHO
from foods.
 Mono-, di- and oligosaccharides are soluble in
alcoholic solution while the other components
not.
 The soluble part are separated from the insoluble
part by filtration method.

Treating the filtrate with clarifying agents or by
passing it through one or more ion-exchange resins
– to remove minor components such as amino
acids;
1. Clarifying agents:
 Example: heavy metal salts
 Function: to form insoluble complexes with
interfering substances that can be removed
by filtration or centrifugation.
2. Ion-exchange:


Mono-, Di- and Oligosaccharides are polar.
Therefore possible to separate by using the
combination of a positively and negatively
charged columns.
METHODS OF ANALYSIS
• CHO content can be determined by
calculating the % remaining after all the other
components have been measured:
% CHO= 100 - % moisture - % protein - % of lipid
- % mineral
• Can lead to erroneous results due to
experimental errors in any of the other
methods.
MONOSACCHARIDES AND
OLIGOSACCHARIDES
Chromatographic Methods
1. HPLC
• Advantages: rapid, can tolerate a wide range of
sample conc., precise and accurate and requires
no prior derivatization
• Disadvantages: require micron-filter filtration
prior to injection
• Stationary phase used: Ion exhange
chromatography, reverse phase chromatography
and normal phase chromatography
2. GC
• Most be converted to volatile derivatives
• Involve two preparation steps
 Reduction of aldehyde groups to primary
alcohol groups
 Conversion of the reduced sugar into a volatile
peracetate ester or pertrimethylsilyl ether
derivative
• These steps must be complete 100% to ensure
the measurement is accurate and precise.
Chemical methods
• Based on the fact that many of mono-, di-, and
oligosaccharides are reducing agents that can react
with other components to yield precipitates or
colored complexes.
• Non-reducing CHO can be determined after
hydrolysis
• 3 categories of chemical method: titration,
gravimetric and colorimetric.
A. Titration Methods
• Example: Lane-Eynon method
• Use to determine the conc. of reducing sugar in a
sample
• Disadvantages:
1. The results depend on the precise reaction times, temp
and reagent conc. → must be carefully controlled
2. Cannot distinguishing bet different types of reducing
sugar
3. Cannot directly determine of non-reducing sugars
4. Susceptible to interference from other types of
molecules that act as reducing agents
Procedure
A known amount of boiling copper sulfate
solution and a methylene blue indicator
CHO solution
Color changes: blue to white
B. Gravimetric Methods
•CHO is heated in the presence of an excess of copper
oxide and alkaline tartrate (to keep Cu 2+ ion in
solution) under controlled conditions → leads to the
formation of a copper oxide precipitate:
Reducing sugar + Cu 2+ + base → oxidized sugar + CuO2
(precipitate)
•This method has similar disadvantages as Lane-Eynon
method
•However, it is more reproducible and accurate.
•
The concentration of precipitate present can be
determined
a. Gravimetrically – filtration, drying and weighing
b. Titrimetrically – redissolving the precipitate and
titrating with a suitable indicator
C. Colorimetric Methods
• Can be used to determine total sugar (reducing and
non-reducing sugars) due to the presence of
oxidizing agent (sulfuric acid)
• Example of methods: Anthrone method and Phenol
– Sulfuric Acid method
• The concentration of CHO is measured at specific
absorbance using spectrophotometer.
Anthrone Method Procedure
Sample + sulfuric acid +
anthrone reagent
Boiled until a blue-green
color is yielded
Measured the solution absorbance at 620 nm
Phenol-Sulfuric Acid Method
Procedure
CHO solution is added into a test tube
Phenol + sulfuric acid are added into the
CHO solution containing test tube
Yellow – orange color is
formed
Measured at 420 nm
Somogyi – Nelson Method
• Determine total reducing sugar
• Is based on the reduction of Cu2+ to Cu+ ions by
reducing sugars.
• Cu+ then reduced an arsenomolybdate complex
which produce blue color that is measured
spectrophotometrically.
D. Enzymatic Methods
• Relies on enzyme ability to catalyze specific reactions
• Rapid, highly specific and sensitive to low
concentrations
• Little sample preparation needed

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Liquid foods – directly tested
Solid foods – need to be dissolved in water first
• Two most common methods

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Allowing complete reaction and measure the product
conc.
Measuring the initial rate of enzyme catalyzed reaction
i.
D-glucose/D-Fructose
• Glucose is converted to glucose-6-phosphate
(G6P) by enzyme hexakinase and ATP
• G6P is oxidized by NADP+ in the presence of
G6P-dehydrogenase (G6P-DH)
G6P + NADP+ → gluconate-6-phosphate + NADPH + H+
• The amount of NADPH formed is proportional to
the G6P conc. and the absorbance can be
measured at 340 nm
• Fructose needs to be converted to glucose first
before the analysis.
ii. Maltose/Sucrose
• Maltose and sucrose are broken down into their
constituent monosaccharides by α-glucosidase
enzyme
• Conc. of glucose and fructose are determined using
the previous methods
• Problem: oligosaccharides are also converted to
monosaccharides by α-glucosidase enzyme
E. Physical Methods
i. Polarimetry
• A device that measures the angle that plane
polarized light is rotated on passing through a
solution
• The conc. of CHO in an unknown sample is
determined by measuring its angle of rotation
and comparing it with the calibration curve.
ii. Refractive Index (RI)
• Is velocity of light in a vacuum divided by the
velocity of light in the material
• RI of CHO solution increases with increasing conc.
• Temp (20°C) and w/length (589.3 nm) dependent
• Used routinely in industry to determine sugar
conc. of syrups, honey, molasses, tomato
products and jams
iii.Density
• Density of an aqueous solutions increases as CHO
conc. increases
• Routinely used in industry for determination of
CHO conc. of juices and beverages.
iv.Infra Red
• A material absorbs infrared due to vibration or
rotation of molecular groups.
• Measurements are normally carried out by
measuring intensity of an infra red wave reflected
from the surface of a sample.
• Advantages: non-destructive and rapid.
F. Immunoassays
• Low molecular weight CHO are developed by
attaching the CHO of interest to a protein and then
injecting it into an animal
• Antibodies specific to CHO molecule is developed
then and can be extracted for determining the
specific CHO concentration.
• Advantages: extremely sensitive, specific, easy to use
and rapid
POLYSACCHARIDES
• Digestible
– Important source of energy. E.g. starch
• Non-digestible
– Cellulose, hemicellulose and pectins
ANALYSIS OF STARCH
• Starch properties
– Insoluble in water
– High density
• It is therefore possible to separate from other soluble
and less dense materials.
• Methods of starch separation for processed foods;
– Is similar to isolation of mono- and oligosaccharides using
80% hot ethanol solution
– Take the sediment as starch components due to
insolubility of starch in ethanol
• For semi-crystalline starch, the sample can be
dispersed in water and heated to a temp where the
starch gelatinizes.
• Addition of perchloric acid or calcium chloride to the
water prior to heating facilitates to solubilization of
starch.
•
Methods of starch determination
1. Specific enzyme is added to the starch solution to
breakdown the starch to glucose. The glucose
concentration is then analyzed using the methods
described previously.
2. Iodine can be added to the starch to form an insoluble
starch-iodine complex that can be determined
gravimetrically by collecting, drying and weighing the
precipitate formed or titrimetrically by determining the
amount of iodine required to precipitate the starch
Analysis of Fibers
• Fiber is also known as resistant starch
• The basis of many fiber analysis techniques is
therefore to develop a procedure that mimics the
processes that occur in the human digestive system.
• Major components of dietary fiber
– Cell wall polysaccharides
– Non cell wall polysaccharides
Sample Preparation and Analysis
Lipid removal
Fiber analysis
Protein removal
Starch removal
Selective precipitation of
fibers
Gravimetric Methods
Crude Fiber Method
• Gives an estimate of indigestible fiber in foods
• Determine by sequential extraction of a defatted sample with
1.25% H2SO4 and 1.25% NaOH
• The insoluble residue is collected by filtration, dried, weighed
and ashed to correct the mineral contamination of the fiber
residue
• Crude fiber measures celllulose and lignin in the sample but
does not determine hemicelluose, pectins and
hydrocolloidsbecause they are digested by the alkali and acid
Total, insoluble and soluble fiber method
• The basic principle: to isolate the fraction of interest
by selective precipitation and then to determine its
mass by weighing
• A gelatinized sample of dry, defatted food is
enzymatically digested with α-amylase,
amyloglucosidase and protease to break down the
starch and protein components.
Footnote: During gelatinization, starch granules are swell, loss their
crystallinity and birefringence and become much more susceptible to enzymecatalyzed hydrolysis.
• Total fiber content of the sample is determined by
adding 95% ethanol to the solution to precipitate all
the fiber.
– The solution is then filtered and the fiber is collected,
dried and weighed.
• Water - soluble and water - insoluble fibers can be
determined by filtering the enzymatically digested
sample.
• Soluble fiber in the filtrate solution and the insoluble
fiber trapped in the filter
• The soluble component is precipitated from solution
by adding 95% alcohol to the filtrate and is then
collected by filtration, dried and weighed.
• Official method for determine fiber content and is
widely used in food industry
• Disadvantages: tends to overestimate the fiber
content of foods containing high concentrations of
simple sugars such as dried fruit, possibly because
they get trapped in the precipitates formed when
the ethanol is added.
Chemical Methods
Englyst-Cummings Procedure
• A defatted food sample is heated in water to
gelatinize the starch
• Enzymes are then added to digest the starch and
proteins
• Pure ethanol is then added to the solution to
precipitate the fiber which is separated from the
digest by centrifugation and is then washed and
dried
• The fiber is then hydrolyzed using a concentrated
sulfuric acid solution to break it down into its
constituent monosaccharides
• The concentration is then determined using the
previous methods mentioned.
• Total mass of fiber in the original sample is assumed
to be equal to the total mass of monosaccharides
present.
• The concentration of insoluble and soluble dietary
fiber can also be determined by this method using
similar separation steps as the total, insoluble and
soluble gravimetric method mentioned above.
• However, it does not provide information about the
lignin content.