Compositional analysis of Polysaccharide

A
SEMINAR ON
METHODS FOR COMPOSITIONAL ANALYSIS OF
POLYSACCHARIDES
BY
Dr. ARUIMA KARKUN
ASST. PROFESSOR
G.D. RUNGTA COLLEGE OF SCIENCE & TECHNOLOGY
KOHKA KURUD BHILAI DURG (C.G.) 1

POLYSACCHARIDES
INTRODUCTION
ANALYSIS
ENZYMATIC TREATMENT METHOD
HYDROLYSIS METHOD
ANALYTICAL METHOD
GAS LIQUID CHROMATOGRAPHY
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
NMR METHOD
CONCLUSION
SUMMARY
REFERENCE 2
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POLYSACCHARIDES
Polysaccharides are long carbohydrate molecules of
monosaccharide units joined together by glycosidic
bonds.
 They range in structure from linear to highly
branched.
They may be amorphous or even insoluble in water.
Polysaccharides have a general formula of Cn(H2O)n
where n is usually a large number between 200 and
2500.
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POLYSACCHARIDEs
There are two types of polysaccharides-
(i)-Homopolysaccharides-Contains only one type of
monomer.
 On hydrolysis, they yield only one type of
monosaccharide. Examples:Starch, cellulose,
glycogen
(ii)-Heteropolysaccharides-Contain two or more
types of monomers.
 On hydrolysis, they yield mixture of
monosaccharides. Examples: hyaluronic acid,
chondriotin sulphate, heparin, and mureins.
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POLYSACCHARIDES
Analysis is the process of breaking a complex topic
or substance into smaller parts to gain a better
understanding of it.
The separating of any material or abstract entity into
its constituent elements.
Following methods are used for compositional
analysis of polysaccharides-
-Enzymatic treatment method
-Hydrolysis method
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METHODS FOR COMOSITIONAL ANALYSIS OF
POLYSACCHARIDES
The analysis of oligosaccharides/polysaccharides is
complicated as they contain variety of bondings.
Therefore before proceeding for analysis protein and
lipid moieties are removed by treating with specific
enzymes.
then subjected to stepwise degradation with specific
reagents to reveal the position of glycosidic bonds.
For example-
Exhaustive methylation with methyl iodide
in strongly basic medium yields fully methylated
oligosaccharides. 6
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POLYSACCHARIDES
Which on acid hydrolysis yields monosaccharides
methylated at every hydroxyl group except those
involved in glycosidic bond.
By this treatment the position of bonding can be
ascertained.
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POLYSACCHARIDES
Characterisation of polysaccharides can be done by
hydrolysis with strong acids.
Which yields a mixture of monosaccharides.
These monosaccharides can be converted into
volatile derivatives and analysed by GLC.
Besides acid hydrolysis polysaccharides may be
hydrolysed with specific endoglycosidases to yield
smaller oligosaccharides.
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POLYSACCHARIDES
ANALYTICAL METHODS
Chromatography
Gas liquid chromatography
High performance liquid chromatography
Spectroscopic method
Nuclear magnetic resonance (NMR) spectroscopy
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POLYSACCHARIDES
CHROMATOGRAPHY
Chromatography usually consists of a mobile phase and
a stationary phase.
The mobile phase refers to the mixture of substances
(to be separated ),dissolved in a liquid or a gas.
The various constituents of the mixture travel at
different speeds, causing them to separate.
The separation is based on the interaction between the
mobile and stationary phases.
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POLYSACCHARIDES
GAS LIQUID CHROMATOGRAPHY
 All forms of chromatography involve a stationary
phase and a mobile phase.
In all the other forms of chromatography you will
meet at this level, the mobile phase is a liquid.
In gas-liquid chromatography, the mobile phase is a
gas such as helium and the stationary phase is a high
boiling point liquid absorbed onto a solid.
A FLOW SCHEME OF GAS CHROMATOGRAPHY
INJECTION OF THE SAMPLE
Very small quantities of the sample that you are trying
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POLYSACCHARIDES
to analyse are injected into the machine using a small
syringe.
The injector is contained in an oven whose
temperature can be controlled. It is hot enough so that
all the sample boils and is carried into the column as a
gas by the helium.
HOW TO COLUMN WORKS
There are two main types of column in gas-liquid
chromatography. One of these is a long thin tube
packed with the stationary phase; the other is even
thinner and has the stationary phase bonded to its
inner surface. 12
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POLYSACCHARIDES
The column is typically made of stainless steel and is
between 1 and 4 metres long with an internal
diameter of up to 4 mm.
It is coiled up so that it will fit into a thermostatically
controlled oven.
This is coated with a high boiling liquid - typically a
waxy polymer.
The temperature of the column can be varied from
about 50°C to 250°C.
It is cooler than the injector oven, so that some
components of the mixture may condense at the
beginning of the column. 13
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POLYSACCHARIDES
How separation works on the column
some molecules may dissolve in the liquid stationary
phase Some compounds will be more soluble in the
liquid than others.
The more soluble ones will spend more of their time
absorbed into the stationary phase; the less soluble
ones will spend more of their time in the gas.
The process where a substance divides itself between
two immiscible solvents because it is more soluble in
one than the other is known as partition.
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POLYSACCHARIDES
RETENTION TIME
The time taken for a particular compound to travel
through the column to the detector is known as its
retention time.
This time is measured from the time at which the sample
is injected to the point at which the display shows a
maximum peak height for that compound.
Interpreting the output from the detector-
The output will be recorded as a series of peaks - each
one representing a compound in the mixture passing
through the detector. 15
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POLYSACCHARIDES
Fig.no.1- Instrumentation of gas liquid chromatography. 16

POLYSACCHARIDES
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
In general, the method involves a liquid sample being
passed over a solid adsorbent material packed into a
column using a flow of liquid solvent.
The separation can be greatly improved by applying high
pressure in the range of 5000-10000 psi, hence this
technique is also referred to as high pressure liquid
chromatography.
FLOW SCHEME FOR HPLC
Injection of the sample
Injection of the sample is entirely automated, pressure
involved in sample injection. 17
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POLYSACCHARIDES
Retention time
The time taken for a particular compound to travel
through the column to the detector is known as its
retention time.
This time is measured from the time at which the
sample is injected to the point at which the display
shows a maximum peak height for that compound.
Different compounds have different retention times.
The detector
There are several ways of detecting when a
substance has passed through the column.
A common method which is easy to explain
uses ultra-violet absorption. 18
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POLYSACCHARIDES
Interpreting the output from the detector
The output will be recorded as a series of peaks - each
one representing a compound in the mixture passing
through the detector and absorbing UV light.
The peaks as a way of measuring the quantities of the
compounds present.
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POLYSACCHARIDES
Fig.no.2-Instrumentation of high performance liquid chromatography . 20
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POLYSACCHARIDES
Fig.no.3-The separation of several amino acids by HPLC 21
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METHODS FOR COMPOSITIONAL ANALYIS OF
POLYSACCHARIDES
NMR SPECTROSCOPY
Nuclear magnetic resonance spectroscopy, commonly
referred to as NMR, has become the preeminent
technique for determining the structure of organic
compounds.
A method is presented for the detailed and accurate
quantitative determination of the monomeric
composition of polysaccharides.
In NMR molecular sample usually dissolve in a liquid
solvent, is place in a magnetic field and the absorption
of radio frequency by certain nuclei is measured.
 All nuclei possess positive charge which causes
nuclei to act as tiny magnet.
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POLYSACCHARIDES
The nuclei when placed in a magnetic field the spins orient
themselves with or opposed to the external magnetic field.
Protons on different atoms and in different molecular
enviroment absorb energy of different level.
The NMR instrument is designed to measure the energy
difference between the nuclear spin states.
Fig.no.4-Spinning of nuclei in magnetic field. 23
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POLYSACCHARIDES
The magnetic moment of the lower energy +1/2 state is
aligned with the external field, but that of the higher
energy -1/2 spin state is opposed to the external field.
Note that the arrow representing the external field points
North.
Strong magnetic fields are necessary for NMR
spectroscopy.
NMR experiment are not limited to the study of protons.
 Resonance signal from the other atomic nuclei
including C13 , P31 can be detected. 24
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POLYSACCHARIDES
C13 NMR technique have been specially valuable for
the study of carbohydrates, amino acids, fatty acid
structures.
P31 spectra of phosphorylated biomolecules display a
peak for each type of phosphorus.
For example-
The catabolism of glucose by glycolysis in
erythrocytes has been monitored by C13 NMR and the
movement of ATP in phosphoryl group transfer processes
has been studied by P31 NMR.
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POLYSACCHARIDES
Fig.no.5- C13 NMR spectra of sucrose. 26

METHODS FOR COMPOSITIONAL ANALYSIS
OF POLYSACCHARIDES
Fig.no.6- P31 NMR spectra of human forearm muscle showing the effect of exercise. 27

POLYSACCHARIDES
A-before exercise
B&C-during 19 minutes of exercise
D-5-6 minutes after C
Peak as signments-1-β phosphorus of ATP
2-α phosphorus of ATP, 3-γ phosphorus of ATP
4-phosphocreatine, 5- inorganic phosphate
Phosphocreatine is used as a major source of energy
during exercise.
It is hydrolysed to creatine and inorganic phosphate.
The level of ATP remains relatively constant during
exercise because it is produced and used at about the
same rate. 28
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METHODS FOR COMPOSITIONAANALYSIS OF
POLYSACCHARIDES
Polysaccharides or complex carbohydrates are
generally very large molecular weight molecule also
composed of monosaccharide chains.
Polysaccharides are complex as they contain variety
of bondings.
Polysaccharides have a general formula of Cn(H2O)n
where n is usually a large number between 200 and
2500.
Analysis is the process of breaking a complex topic
or substance into smaller parts to gain a better
understanding of it. 29
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POLYSACCHARIDES
The analysis of oligosaccharides/polysaccharides is
complicated as they contain variety of bondings.
Therefore before proceeding for analysis protein and
lipid moieties are removed by treating with specific
enzymes.
Characterisation of polysaccharides can be done by
hydrolysis with strong acids.
Following analytical methods are used for the analysis-
Chromatography
Gas liquid chromatography
High performance liquid chromatography
Nuclear magnetic resonance spectroscopy 30
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POLYSACCHARIDES
C.B.Powar 2006 Biochemistry
& G.R.Chatwal
Michael M.Cox 2008 Principles of
David L.Nelson Biochemistry
U. Satyanarayana 2010 Biochemistry
Rodney Boyer Third edition Modern
experimental
biochemistry
Some contents from net-
www.chemguide.co.uk
www.novapublishers.com
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Compositional analysis of Polysaccharide

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Compositional analysis of Polysaccharide