chem 161.1 ex12
TRANSCRIPT
EXERCISE 12
PURITY
DETERMINATION OF
POLYSACCHARIDES
Jovale Vincent V. Tongco
CHEM 161.1 1L
Polysaccharides
Are polymers of
monosaccharides
Formed by three or more
monosaccharides
Polymers of D-Glucose
Common Polysaccharides
CELLULOSE Is a straight chain
polymer of D-glucose
Monomeric units are
connected together
by β-1,4 linkages.
STARCHMixed polysaccharide with
two main components, -
amylose and amylopectin
Adopt a helical structure
Joined together by -1,4 linkages.
Typical chain lengths are approximately 1000 units.
-Amylose
Also linked by -1,4 linkages
Is a highly branched polymer of glucose found in PLANTS.
Branching takes place with -1,6 bonds occurring every 24 to 30 glucose units.
Amylopectin
Counterpart of amylopectin in ANIMALS
Extensive branching that occurs every 8 to 12 glucose units
Highest concentration found in liver and muscle cells.
Glycogen
Purity of Sample
From Nelson’s Assay for glucose
Calculated to be 6.17 µmoles/mg
Percent Purity
% Purity = actual µmoles glucose/mg of glycogen x 100
theoretical µmoles glucose/mg of glycogen
Where:
Actual µmoles glucose = µmoles glucose in the hydrolyzed
sample x DF
Percent Purity
Theoretical µmol glucose = mass of glycogen x 180 x 1 x 1000
mg glycogen 162 180_______ _
mass of glycogen (mg)
Where:
Mass of glycogen is the amount of glycogen used in the
experiment (50 mg)
The Conversions
The molar mass of free glucose is
180 g/mole
The molar mass of glucose residue
is 162 g/mole
The Conversions
The factor 180/162 – converts the mass of glucose residue into mass of free glucose
The factor 1/ 180 – converts amount of free glucose from mg to mmoles
Factor 1000- converts mmoles to moles
Percent Purity
Substituting the amount of isolate:
Theoretical µmol glucose = 50 x 180 x 1 x 1000
mg glycogen 162 180_______ _
50
Theoretical µmol glucose = 6.17
mg glycogen
% purity
The Theoretical µmol glucose/
mg glycogen is the same for
amylopectin and glycogen
since structurally glycogen is
very similar to amylopectin
Standard Curve
Acid Hydrolysis of Glycogen
Back Actual
Acid Hydrolysis of Amylopectin
Back
Dilution Factor
Actual glucose concentrations after
hydrolysis of glycogen and amylopectin
were determined by multiplying
interpolated glucose concentration to the
DF.
DF = (10/0.4)(1/0.5)
= 50
Sample Calculations
Calculating glucose concentration after
hydrolysis:
Use dilution factor (DF=50)
Example:
glucose concn = -0.05165 µmole glucose/ml (1/0.5)(10/0.4)
= -0.78236 µmol glucose/ml glycogen stock
Glycogen
Upon Acid Hydrolysis
Polysaccharide Incubation
timeCorrected
Absorbance
Glucose
conc. after
hydrolysis
Glycogen 90 0.144 0.189189
Amylopectin 90 0.433 0.600284
Actual mol of glucose
Polysaccharide Glucose conc.
after
hydrolysis
DF Actual moles
after hydrolysis
Glycogen 0.189189 50 9.46
Amylopectin 0.600284 50 30.01
Glycogen AmylopectinBack
Actual µmoles glucose/mg of
glycogen
Actual µmoles glucose = mol glucose in
the hydrolyzed sample
For glycogen:
The actual μmoles glucose / mg of glycogen
is 9.46. This is the μmoles glucose in the
hydrolyzate after 90 minutes of heating.
Actual µmoles glucose/mg of
glycogen
For amylopectin:
The actual μmoles glucose / mg of
amylopectin is 30.01. This is the
μmoles glucose in the hydrolyzate
after 90 minutes of heating.
Note:
In the preparation of glycogen/amylopectin
solution 50 mg glycogen/amylopectin and was
dissolved in 5 mL dH20.
The actual μmoles glucose / mg of glycogen
should be divided by 10
50 mg glycogen / 5 mL = 10 mg glycogen.
The actual μmoles glucose / mg of
amylopectin should be divided by 10
50 mg amylopectin / 5 mL = 10 mg
amylopectin
Percent purity
For glycogen isolate
% Purity = 9.46 μmoles glucose / 10 mg glycogen x 100
6.17 μmoles glucose / mg of glycogen
= 15.33 %
Actual Theo
Percent purity
For amylopectin sample:
% Purity = 30.01 μmoles glucose / 10 mg glycogen x 100
6.17 μmoles glucose / mg of glycogen
= 48.64 %
Percent purity
Polysaccharide Percent Purity,
%
Glycogen 15.33
Amylopectin 48.64
The Possible Sources of Errors
Prolong Heating/ Excessive
water evaporation
Limitations of the
spectrophotometric method /
Improper instrument reading
Incomplete hydrolysis
END