PTT 202:
ORGANIC CHEMISTRY FOR BIOTECHNOLOGY
PREPARED BY:
NOR HELYA IMAN KAMALUDIN
helya@unimap.edu.my
Introduction of Carbohydrates
Carbohydrates are aldehydes or ketones of higher
polyhydric alcohols or components that yield these
derivatives on hydrolysis.
Difficulties are encounted in the qualitative and
quantitative analysis of carbohydrates mixtures
because of the structural and chemical similarity of
many compounds.
Analytical specificity may be improved by the
preliminary separation of the components of
the mixture using a chromatographic
technique prior to quantitation.
Chemical methods of carbohydrate analysis
Reaction
with
aromatic
amines
Reduction
methods
Reactions
of the
carbonyl
group
Reactions
with strong
acids and a
phenol
Reduction methods
Reduction of cupric ions (Cu2+) to cuprous ions (Cu+)
• In alkaline solution, it will form yellow cuprous hydroxide, which in turn converted
by the heat of the reaction to insoluble red cuprous oxide (Cu2O).
• Production of a yellow or orange-red precipitate indicates the presence of a reducing
carbohydrate.
• Different carbohydrates will result in the formation of different amounts of cuprous
oxide.
• The methods of measuring cuprous oxide frequents used involves the reduction of
either phosphomolybdic acid or arsenomolybdic acid to lower oxides of molybdenum
by cuprous oxide.
• The intensity of the colored complex produced is related to the concentration of the
reducing substances.
Reduction of ferricyanide ions to ferrocyanide ions
• The color will change from yellow solution to colorless solution by reducing
carbohydrates when heated in alkaline solution.
Disadvantages
• Lack of specificity
• Result in positive error as non-carbohydrate reducing substances present in the
sample.
Reaction with aromatic amines
Various aromatic amines will condense with aldoses and ketoses in glacial
acetic acid to form coloured products
• Maxima absorbance are often characteristic of an individual carbohydrate
or group.
The examples of aromatic amines that have been used
• p-aminobenzoic acid, diphenylamine and p-aminophenol.
These amines has ability to react preferentially with a particular carbohydrate
or class of carbohydrate.
• e.g. p-aminophenol shows some specificity for ketoses compared with
aldoses and is useful for measuring fructose.
These reagents have proved particularly useful for the visualization and
identification of carbohydrates after separation of mixtures by paper or thinlayer chromatography.
Reactions with strong acids and a phenol
Formation of furfural and hydroxymethylfurfural
• Pentoses and hexoses are dehydrated to form furfural and
hydroxymethylfurfural derivatives respectively when heated with a strong acid
(Figure 1).
• The aldehyde groups will condense with a phenolic compound to form a
colored product.
• The reaction forms the basis of some of the oldest qualitative tests for the
detection of carbohydrates, e.g. the Molisch test using concentrated sulphuric
acid and α-naphthol.
Advantage
• Give some degree of specificity through careful choice of both the reaction
conditions and the phenolic compound used which may be possible to produce a
color that is characteristic of a particular carbohydrate or related group.
Disadvantage
• Non-carbohydrate substances may be present in a biological sample that will
decompose on heating and will react in a similar manner to a carbohydrate and
may give a false positive reaction for carbohydrates.
Figure 1: Formation of
furfural and 5hydroxymethylfurfural
Enzymic methods of carbohydrate analysis
Assay of
glucose using
glucose
dehydrogenase
Assay of
glucose using
glucose
oxidase
Assay of
glucose using
hexokinase
Enzymic
methods
Assay of glucose using glucose oxidase
Glucose oxidase enzyme catalyses the oxidation of β-Dglucose by atmospheric oxygen to produce D-gluconolactone,
which is converted to gluconic acid with the production of
hydrogen peroxide.
The oxidation of α-D-glucose occurs at less than 1% the rate
of oxidation the β anomer
• Because of these two forms exist in solution in equilibrium, mutarotation of
the α to the β form must be allowed to reach equilibrium in the sample and
standards for consistent results.
• The inclusion of aldose-1-epimerase (glucomutarotase) in the glucose
oxidase reagent will permit rapid restoration of the α-β equilibrium,
effectively enabling the reaction to go to completion.
Assay of glucose using glucose oxidase
Spectrophotometric
methods
Measurement of
hydrogen peroxide
formed
Electrochemical
methods
Quantitation of
glucose using
glucose oxidase
during the reaction
Measurement of
oxygen consumed
Electrochemical
method
Assay of glucose using glucose oxidase
Measurement of the hydrogen peroxide formed
Spectrophotometric methods
• H202 formed was measured by monitoring the change in color of a
chromogenic oxygen acceptor in the presence of the enzyme
peroxidase.
• Such chromogens are colorless in their reduced form but exhibit
characteristic colors when oxidized.
• Disadvantages: less specific, errors may be introduced in the
second stage of assay reaction by interference of some substances
in the reaction .
Electrochemical methods
• The instruments used for electrochemical measurement of the
H202 produced is glucose analysers especially in an immobilized
form.
Assay of glucose using glucose oxidase
Measurement of the oxygen consumed
The initial oxidation of glucose can be monitored and this is most
easily achieved by measuring the amount of oxygen consumed
during the reaction.
An electrochemical method using a polographic Clark oxygen
electrode has been used and the first oxygen electrode to be
described for the measurement of glucose in contained
soluble glucose oxidase held between cuprophane membrane.
Recent modifications have been resulted in the
incorporation of a variety of forms of immobilized
glucose oxidase into the electrode.
Assay of glucose using glucose dehydrogenase
Glucose can be measured using bacterial glucose
dehydrogenase
• Catalyses the dehydrogenation of β-D-glucose to Dgluconolactone
• The hydrogen being transferred to NAD+ or NADP+.
• The method involves measuring the increase in
absorbance at 340 nm caused by the production of
NADH using either a kinetic or fixed time assay
technique.
• A mutarotase should be included in the assay to
accelerate the conversion of a α to the β form.
Assay of glucose using hexokinase
Measuring of glucose using hexokinase enzyme
• Catalyses the phosphorylation of glucose to produce glucose-6phosphate with adenosine triphosphate (ATP) as the phosphate
donor and magnesium ions as an activator.
• The rate of formation of glucose-6-phosphate can be linked to the
reduction of NADP by the enzyme glucose-6-phosphate
dehydrogenase.
• The indicator reaction can be monitored spectrophotometrically at
340 nm as below:
Assay of glucose using hexokinase
Characteristic of hexokinase enzyme
• Not specific for glucose only
• Capable of converting some other hexoses to their
corresponding 6-phosphate derivatives.
• Additionally, the specificity of the enzyme may vary
slightly depending on its source.
• For example, yeast hexokinase will catalyse the
phosphorylation of a number of hexoses as well as glucose
including D-mannose, D-fructose, D-glucosamine.
• This provides an assay system for mannose or fructose if
the initial reaction is linked to a suitable second reaction
(Figure 1)
Mannose, fructose
and glucose may all
be
assayed
independently
using hexokinase
and the appropriate
additional enzymes.
All the reactions
can be monitored
in absorbance at
340 nm as NADP+
is
reduced
to
NADPH.
Figure 1: Assay systems for mannose, fructose and glucose using hexokinase.
Separation and identification of
carbohydrate mixtures
Gas-liquid
chromatography
High
performance
liquid
chromatography
Paper and thinlayer
chromatography
Techniques
of separation
and
identification
Paper and thin-layer chromatography
Both ascending and descending paper
chromatography techniques have been used,
when thin-layer supports are employed
As the number of carbohydrates present in the sample is
often small, careful choice of solvent will generally make it
unnecessary to perform the two-dimensional separations.
Thus, the separation of carbohydrate mixtures
depends on the solvent systems and
locating reagents.
Paper and thin-layer chromatography
Solvent systems
• Several monophasic solvent systems are useful for the separation of
carbohydrate mixtures as listed in Table 1.
• The smallest solute molecules, the fastest of mobility.
• Thus, pentoses have higher RF values than hexoses, followed by
disaccharides and oligosaccharides.
Locating reagents
• A variety of reagents can be used for visualization of the separated
components.
• Useful to run duplicate chromatograms and use a different stain on each
one to assist in identification of unknown spots.
• The actual reagent composition may be modified either in terms of
concentration of components or by substitution of one chemical for another
similar one.
• Such variations in reagent composition may be advantageous in promoting
the production of characteristic colors for different carbohydrates.
Table 1: Some
monophasic
solvents for thinlayer
chromatography of
carbohydrates
Gas-liquid chromatography
This technique is necessary to identify or to quantitate one or more
carbohydrates especially when they are present in small amounts.
GLC can be carried out using volatile derivatives of carbohydrates such
as O-trimethylsilyl (TMS) derivatives which probably used most
frequently due to simple preparation and have been successfully applied
to wide variety of compounds.
The choice of stationary phase will depend upon the
nature of the carbohydrates to be separated.
For example; OV-17 stationary phase useful for isometric
separations while OV-1 useful for wider range of carbohydrates.
Figure 2: Separation of equimolar concentrations of methyglycosides by GLC.
The analysis was performed on an OV-1 stationary phase using a
temperature gradient from 120 to 220˚C
High performance liquid chromatography
Function
• For separation and quantitation of carbohydrate mixtures.
• This method does not necessitate the formation of a volatile derivative as in
GLC
• Techniques used: Partition and Ion-exchange Techniques.
Partition chromatography
• Is usually performed in the reverse phase mode using a chemically bonded
stationary phase and acetonitrile (80:20) in 0.1 mol/l acetic acid as the mobile
phase.
Ion-exchange chromatography
• Anion and cation exchange resins can be used.
• Carbohydrates form anionic complexes in alkaline borate buffers and
quaternary ammonium anion-exchange resins in hdroxyl form can be used for
their separation.
• Sulphonated cationic exchange resins with metal counter-ions are also useful
for carbohydrate analysis.
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