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