Full Report on

EXERCISE NO. 8, 9, and 10

GLYCOGEN ISOLATION

PURITY DETERMINATION OF POLYSACCHARIDES

END GROUP DETERMINATION OF POLYSACCHARIDES

Bolandos, Kim Leonard C.

CHEM 161.1 – 2L

1st Semester AY 2018-2019

Groupmate:

CJ Carlos

Pamela Macalagay

Giorgia Escalante

Date performed:

Date submitted: November 20, 2018

Sir Bong Remillion

I. Introduction

Carbohydrates are the most abundant biomolecules on Earth. It constitute 75% by mass of
dry plant material although their abundance in the human body is relatively low. It can be
classified on the basis of molecular size as monosaccharides, oligosaccharides, and
polysaccharides. The exercises used glycogen and amylopectin as samples which are
polysaccharides. Polysaccharides contains many monosaccharide units bonded together by
glycosidic bonds and are often called as glycans (Stoker, 2010).

Glycogen is the main storage polysaccharide found in animal cells. On the other hand,
amylopectin is one of the types of carbohydrates of the starch molecule and is usually found in
plants. Glycogen is a polymer with α-1,-4-linked subunits of glucose and α-1,6-linked branches
like amylopectin. However, glycogen is more extensively branched (every 8 to 10 residues on
average) and more compact than starch. It is especially abundant in the liver wherein in it may
constitute as much as 7% wet weight; it can also be found in the skeletal muscles (Nelson &
Cox, 2008). But the muscle cannot efficiently utilize the glycogen as a glucose source because
the enzyme glucose-6-phosphatase is absent.

Isolation, purification and characterization of glycogen can be done using different

techniques. One involves use of 30% aqueous solution of potassium hydroxide. Since this
method has capability of degrading the glycogen in the sample, newer methods were
developed. Another extraction method involves the use of solvents such as cold, dilute
trichloroacetic acid (TCA), dimethylsulfoxide (DMSO), cold water, aqueous phenol, glycine
buffer and aqueous mercuric chloride (Childress et al., 1970).

After isolation, the purity of the glycogen isolate can be determined using the Nelson’s
method for reducing sugars. The isolate will be subjected to acid hydrolysis to break the
glycosidic bonds and be left with free glucose residues. The hydrolysate will then be treated
with the Nelson’s reagent to measure the amount of free glucose residue in the sample using a
spectrophotometer. The concentration obtained corresponds to the actual glucose
concentration. The theoretical concentration of glycogen sample can be calculated using a
conversion factor based on the molar masses between free glucose residue and glucose residue
in the glycogen. On the other hand, % purity of the isolate is determined by getting the ratio of
actual and theoretical concentrations of glucose residues (Polgase et al, 1952).

End group determination of polysaccharide can be done via periodate oxidation of the
isolate. This reaction requires the presence of a vicinal diol or α-hydroxy carbonyl compound to
form either a carbonyl compound or formic acid. Titrimetric quantitative determination of the
amount of formic acid formed gives an estimate of the number of reducing and non-reducing
end of the polysaccharide.

In this exercise, glycogen was isolated from mussel flesh using 30% KOH and 10% TCA
as extracting solvents and the yield was also determined. The glycogen isolate was further
subjected to purity and end group determination. Also, the purity and end group of a
commercially available amylopectin was determined simultaneously with the glycogen isolate.

II. Materials and Method

A. Glycogen Isolation

1.) Sample preparation

Flesh from the cold mussel samples were separated from the shell while submerged in
water. The flesh was homogenized using a blender. Mass of 40 g of the homogenized flesh
was transferred to an Erlenmeyer flask.

2.) Isolation and Purification

Volume of 7.20 mL of 30% aqueous KOH was added to the 40 g homogenized sample.
The resulting mixture was subjected to boiling water bath with occasional swirling for an
hour or so until the sample was completely digested and no clump was observed. After
subjecting to boiling water bath, it was diluted with 15.0 mL water, and 30 mL 95%
ethanol was added. The resulting mixture was allowed to stand and cool to room
temperature and then was subjected to ice bath for 20 minutes to allow complete
precipitation of glycogen. Collection of the precipitates formed was done by subjecting the
mixture to centrifugation for 5 minutes. The residue (or the glycogen), was dissolved in
6.00 mL cold trichloroacetic acid solution. The resulting solution was subjected again to
centrifugation for 5 minutes. The supernatant was obtained and added with 15.0 mL 95%
ethanol. It was allowed to stand in ice bath for 10 minutes. The precipitate was collected
by means of centrifugation. Extraction with TCA was done twice during the experiment.
The white precipitate obtained was dissolved in 4.00 mL water and reprecipitated by
adding 5 mL 95 % ethanol then 5 mL diethyl ether. The glycogen isolate was allowed to
dry by letting the solvent to evaporate. The mass of the precipitate is the actual mass of
glycogen isolate.

B. Purity Determination of Polysaccharides

1.) Preparation of Reagents

i. Glucose stock solution (20 mM)

Mass of 0.1800g glucose was placed in a 100-mL beaker and was dissolved in 20 mL
distilled water. The solution was transferred quantitatively in 50 mL volumetric flask.
Distilled water was added until reaching the mark.

ii. Glucose solution for standard (2 mM)

Volume of 5.00 mL of the prepared glucose stock solution was diluted to 50.0 mL in a
volumetric flask using distilled water

iii. Nelson’s reagent

 Solution A and solution B were already prepared.

iv. Arsenomolybdate reagent

This reagent was already prepared for the class.

v. Glycogen solution (10 mg/mL)

Mass of 50 mg of glycogen isolate from Exercise 8 was dissolved in 5.00 mL distilled
water.

vi. Amylopectin solution (10 mg/mL)

Mass of 50 mg amylopectin sample was put in a test tube and dissolved with 5.0
mL distilled water. The test tube containing the sample was covered with marble and
the setup was subjected to boiling water bath until amylopectin was dissolved.

2.) Preparation of the Calibration Curve

The protocol in Table 9.1 was followed to prepare the standards. The absorbance of the
samples were read at 510 nm. The A510 vs glucose concentration was plotted to
generate a calibration curve.

Table 9.1. Preparation of standard solutions for reducing sugar determination by

Nelson’s method.
Test Tube No.
Reagents Added
1
(mL) 2 3 4 5 6 7 8
(blank)
Glucose (2mM) 0.000 0.050 0.100 0.200 0.400 0.600 0.800 1.000
dH2O 1.000 0.950 0.900 0.800 0.600 0.400 0.200 0.000
Nelson’s Reagent 1.00
Cover the tubes with marbles and place in boiling water bath for 20
minutes. Cool to room temperature.
Arsenomolybdate
1.00
reagent
Mix the contents of the test tubes using a vortex mixer and allow
the tubes to stand for 5 minutes at room temperature.
dH2O 7.00
Mix contents of the tubes on a vortex mixer

3.) Acid Hydrolysis of Glycogen

The protocol given in Table 9.2 was followed.

Table 9.2. Acid hydrolysis of glycogen sample.

Reagents Added Test Tube No.
(mL) 1 2 3 4 5 6 7 8
dH2O 0.200 0.800 0.000
Glycogen solution (10 0.000 0.200
mg/mL)
2 N HCl solution 0.800 0.000 0.800

1.2 N NaOH 1.00 1.00 0.00

Cover tubes with marbles. Allow test tubes no. 1 and 2 to stand in
room temperature. Place tubes 3 to 8 in a boiling water bath.
Starting from test tubes 3, remove the tubes at 5 minutes interval
from the boiling water bath.
Heating time 0 0 5 10 15 20 25 30
1.00 mL of 1.2 N NaOH after the specified heating time
dH2O 8.00
Mix the contents of the test tubes using a vortex mixer.

4.) Acid Hydrolysis of Amylopectin

The protocol given in Table 9.2 was followed using 10 mg/mL amylopectin solution
instead of glycogen.

5.) Determination of the Glucose Content of the hydrolyzed Samples.

From each diluted samples of glycogen and amylopectin, 0.50mL aliquot was
obtained. This was diluted to 1.00mL using distilled water. The tubes were mixed well
and each solution was assayed including the blank with Nelson’s method. The protocol
in Table 9.1 was followed in place of standard glucose solutions. The absorbance was
read at 510 nm using test tube number 1 as the blank for both parts 3 and 4.

C. End Group Determination of Polysaccharides

1.) Preparation of glycogen sample

Two hundred milligrams of glycogen sample was added with 20.0 mL 3% NaCl
and heated in a warm water bath until all solids were dissolved. The glycogen
solution was then transferred to a 50 mL volumetric flask using 2 mL dH2O. Volume
of 10.0 mL 0.350 M NaIO4 was added to the solution. The solution was diluted to 50
mL mark using distilled water. Blank sample was also prepared in a similar manner
but without the glycogen sample. The solution was covered with dark paper and
stored in the refrigerator until the next laboratory period.

2.) Preparation of amylopectin sample

The procedure in C-1 was followed using amylopectin instead of glycogen.

3.) Titration of samples

 Five milliliters aliquot of the periodate mixture was transferred to a 125 mL
Erlenmeyer flask. Then, approximately three drops of ethylene glycol which acts like
the phenolphthalein indicator was added to the mixture. Then it was placed in the
dark for about 15 minutes. It was then titrated with standardized 0.01 N NaOH.
Three trials were prepared for the analysis.

III. Results and Discussion

Glycogen is a polysaccharide containing only glucose units. It serves as the glucose storage
polysaccharide in humans and animals. Its function is similar to that of starch in plants, and is
sometimes referred to as animal starch. The liver and muscle tissues convert the excess glucose
present in the blood to glycogen, which is then stored in these tissues. Some stored glycogen is
hydrolyzed back to glucose whenever glucose blood level drops. These two opposing processes
are called glycogenesis and glycogenolysis, the formation and decomposition of glycogen,
respectively (Stoker, 2010).

Glycogen isolation

In this exercise, glycogen was isolated from mussel flesh using solvent extraction. The first
step involves the addition of 30% hot aqueous KOH to the homogenized flesh with the help of a
blender. This reagent solubilizes the tissue and saponifies fats which in turn remove impurities
such as saponifiable lipids, proteins, and nucleic acids. The glycosidic bond of glycogen is
relatively stable in alkaline condition even at high temperature due to the presence of stable
acetals in their structure (McMurry, 2012). The next step involves the separation of glycogen
from proteins and nucleic acids present through the addition of cold 10% trichloroacetic acid
(TCA) solution. Glycogen is soluble in TCA while proteins are not. Afterwards, 95% ethanol was
added to reprecipitate glycogen. Before the measurement of the isolated product, the
precipitate was submerged in a 5 mL diethyl ether to remove such impurities especially highly
nonpolar impurities such as lipids. Table 8.1 shows the observations during the isolation of
glycogen.

Table 8.1. The observation

Action Taken Observatios
Mussel Flesh Slimy light brown with shells
Homogenized flesh Brown, viscous fishy mixture
Addition of 10% hot KOH solution Color become lighter
After heating the mixture Dark brown solution with less viscous
Addition of 95 % alcohol Color become lighter
After cooling in an ice bath Precipitates settled at the bottom
Centrifugation ---
Precipitate Brown powder
Supernatant Brown liquid
Dissolution of 10% TCA Green brown mixture
Centrifugation ---
Precipitate Dark brown
 Supernatant Light brown
Addition of 95 % ethanol A clear solution with a distinct ppt at the
bottom
Standing in an ice bath Precipitates formed with brown colored
Centrifugation ---
Precipitate ---
Supernatant ---
Dissolution of precipitate ---
Addition of 95 % ethanol Visible light brown precipitate
Addition of the diethyl ether The same appearance of light brown
precipitate
Glycogen Isolate Lighter solid precipitate

Table 8.2. Determination of the yield of glycogen isolate.

Parameters Values
Mussel flesh, g 40.04
Mass of the isolated sample, g 0.48
Yield, g glycogen / 10 g mussel flesh 0.1198801199
Percent yield (actual/theo x 100), % 14.97004494

The yield of glycogen during isolation can be affected by time and temperature. At higher
temperature and at longer period of time of extractions, the sample or microorganisms in the
sample may produce enzymes that break down glycogen molecule into glucose units. Hence,
lower yield of isolation since the glycogen molecule would be degraded. At lower temperature,
the activity of these enzymes decreases, favoring the isolation of glycogen that would help to
increase the yield (Levine et al, 1953).

Glycogen is relatively more stable to alkali hydrolysis than other biomolecules like
proteins and lipids. Proteins are degraded into salts of free amino acids during alkaline
hydrolysis. Some amino acids (e.g., arginine, asparagine, glutamine, and serine) are completely
destroyed while others are racemized (such as structurally modified from a left-handed
configuration to a mixture of left-handed and right handed molecules). The temperature
conditions and alkali concentrations of this process destroy the protein coats of viruses and the
peptide bonds of prions. On the other hand, all of the ester bonds of lipids, as well as the sterol
esters and phospholipids of cell secretions and cell membranes, hydrolyze with the consumption
of the alkali, producing the sodium and potassium salts of fatty acids, namely soaps. Amide
groups in glycolipids, another cell membrane constituent, are also hydrolyzed, with consumption
of the alkali. Polyunsaturated fatty acids and carotenoids (pigments) undergo molecular
rearrangements and are thus destroyed (Thacker, 2004).

As of carbohydrates, these represent the cell and tissue constituents most slowly affected
by alkaline hydrolysis. Both glycogen and starch are immediately solubilized, but the breakdown
of these polymers requires much longer treatment than is required for large intracellular and
extracellular polymers. Once broken down, the constituent monosaccharides (such as glucose,
galactose, and mannose) are rapidly destroyed by the hot aqueous alkaline solution.
Significantly, large carbohydrate molecules such as cellulose are resistant to alkaline hydrolysis
digestion (Thacker, 2004).

The amount of glycogen in an animal varies greatly due to different factors. Diet greatly
affects the amount of glycogen in an animal cell. If the animal starves, the glycogen levels in its
body decreases. On the other hand, carbohydrate-rich diet increases the amount of glycogen.
Another factor is the stress that an organism experiences and how often it exercises. The more
an organism experiences stress or performs physical activities, the lower the glycogen levels are
and the more relaxed and rested it is, the higher the glycogen levels are. The environmental
temperature is also a factor. Prolonged chilling causes shivering of muscles which lower
glycogen level. Hormones acting in an organism also affect glycogen levels in the body. For
instance, higher levels of insulin in the body increases glycogen level while higher level of
epinephrine and glucagon which triggers glycogenolysis that decreases the level of glycogen. As
the organism gets older, there is also a decrease in the level of glycogen in the body. A
phenomenon called acidosis decreases amount of glycogen by rapidly breaking down glycogen
(Adeva-Andany et al., 2016).

Glycogen storage diseases (GSD) are usually inherited. A person acquiring these diseases
has an absence of an enzyme related to glycogen metabolism and it usually affects the vital
organs. Most are diagnosed in childhood. Symptoms include weakness, tiredness and low blood
sugar levels.

Von Gierke’s disease (GSD type I)has defective glucose-6-phosphatase enzyme in the
glycogen metabolism. Organs affected are the liver and kidney. In this kind of disease the
glycogen is in normal structure but in increased amounts than normal. Symptoms of this
disease include massive enlargement of liver, severe hypoglycemia, and ketosis
(Anastasopoulou, 2017).

Pompe’s disease (GSD type II) has a defective α-1, 4-glucosidase. The enzymes affected
normally catalyze reactions that ultimately convert glycogen compounds to monosaccharides, of
which glucose is the predominant component. This results in glycogen accumulation in tissues,
especially muscles, and impairs their ability to function normally. This disease affects all organs.
Glycogen is in normal structure, but there is a massive increase in its amount. Clinical features
include cardiorespiratory failure which causes death usually before age 2 (Anastasopoulou,
2017).

Cori’s disease (GSD type III) has a defective amylo-1, 6- glucosidase which is a glycogen
debranching enzyme. The organs affected are the muscles and liver. There is an increase in the
amount of glycogen and shorter outer branches. Symptoms include swollen abdomen, low
blood sugars on fasting and growth delayed during childhood (Anastasopoulou, 2017).

Andersen’s disease (GSD type IV) has a deficient glycogen branching enzyme. The organs
affected are the liver and spleen. Glycogen in the body are in normal amount, but have very
long branches. In the perinatal variant usually symptoms become apparent in the first few
months of a baby's life. Such signs typically include failure to thrive - slow growth and failure to
gain weight at the expected rate. There may be an abnormally enlarged liver and spleen
(Cincinnati Children's Hospital Medical Center, 2018).

McArdle’s disease (GSD type V) has a defective phosphorylase. The affected organ is the
muscle. Glycogen is in normal structure, but there is a moderate increase in its amount in the
body. McArdle disease is a rare metabolic disorder which causes muscle pain in everyday
activities and exercise. If activity is prolonged despite the pain then muscle damage ensues with
the risk of muscle breakdown and kidney failure (Cincinnati Children's Hospital Medical Center,
2018).

Purity Determination of Polysaccharides

Purity determination of the isolated glycogen was done using acid hydrolysis and Nelson’s
assay for the reducing sugar. Acid hydrolysis cleaves glycosidic bonds resulting to free glucose
units which can be easily analyzed colorimetrically by the Nelson’s assay. Acid hydrolysis was
done at varied time interval using the same amounts of glycogen.

Table 9.1. Data for the standard calibration curve.

Standard Glucose Solution, Corrected ABS at 510 nm

μmol/mL
0 0
0.1 0.029
0.2 0.055
0.4 0.124
0.8 0.221
1.2 0.372
1.6 0.440
2.0 0.589

0.7
0.6 y = 0.29x + 0.0004
0.5 R² = 0.9957
Absorbance

0.4
0.3
0.2
0.1
0
0 0.5 1 1.5 2 2.5
Concentration, μmol/mL

Figure 9.1. Calibration Curve for the Standard Glucose Solution.

Table 9.2. Linear regression analysis of the standard glucose solution.

 Parameters Values
y-int 4.079074253 x 10-4
Slope 0.2899582128
r 0.997826544

Table 9.3. Data for the Nelson’s Assay of Glycogen Hydrolysates.

Test Tube # ABS at 510 Corrected Interpolated []. Dilution Actual conc.,
nm ABS μmol/mL Factor μmol/mL
1 0.051 0 0 100 0
2 0.069 0.018 0.06067113052 6.067113
3 0.077 0.026 0.08826131299 8.826131
4 0.090 0.039 0.1330953595 13.30954
5 0.076 0.025 0.08481254019 8.481254
6 0.092 0.041 0.1399929051 13.99929
7 0.101 0.05 0.1710318604 17.10319
8 0.107 0.056 0.1917244973 19.17245

Table 9.4. Data for the Nelson’s Assay of Amylopectin Hydrolysates.

Test Tube # ABS at 510 Corrected Interpolated []. Dilution Actual conc.,
nm ABS μmol/mL Factor μmol/mL
1 0.046 0 0 100 0
2 0.070 0.024 0.08136376738 8.136377
3 0.067 0.021 0.07101744895 7.101745
4 0.084 0.038 0.1296465867 12.96466
5 0.118 0.072 0.2469048622 24.69049
6 0.096 0.05 0.1710318604 17.10319
7 0.092 0.046 0.1572367692 15.72368
8 0.120 0.074 0.2538024078 25.38024

30

25

20
μmol/mL

15
Glycogen
10 Amylopectin

0
0 10 20 30 40
min
 Figure 9.2. The comparison of the plot for the glycogen and amylopectin.

Both glycogen and amylopectin sample were subjected to acid hydrolysis to yield its
monosaccharide unit, glucose. The actual glucose contents of each sample were determined by
Nelson’s method since it requires a reducing sugar and all monosaccharides such as glucose are
reducing sugars. Using the absorbance values of the hydrolyzed samples, concentration of
glucose of each sample was determined using calibration curve established as shown on Table
9.1. Table 9.3 and 9.4 shows the absorbance values and glucose concentration by interpolation
of hydrolyzed glycogen and amylopectin, respectively.

As seen on Figure 9.2, somehow amylopectin has a higher actual concentration than the
glycogen with the curve presented as the heating time increases. This could suggest that the
amylopectin, upon acid hydrolysis have yielded a higher amount of glucose residue or a
reducing sugar compared to glycogen.

Table 9.5. Data on the percent purity of glycogen and amylopectin.

Parameters Value
Glycogen Amylopectin
Hydrolysis time, min 30 30
Abs 510 0.056 0.074
DF * 100
Actual glucose content, 19.17244973 25.38024078
μmol/mL
Actual μmol glucose/ mg 1.917244973 2.538024078
sample
Theoretical μmol glucose/ 6.17 6.17
mg smple
% Purity 31.07366245 41.13491212

To calculate for the actual μmol glucose/mg of glycogen and amylopectin, both test
tubes 8 were considered since it has the highest glucose concentration and is within range of
the calibration curve. It was assumed that hydrolysis of the samples is complete, thus these
values are represented on table 9.5. Moreover, data for the calculation of percent purity was
summarized in Table 9.5 wherein glycogen and amylopectin samples have 31.07366245% and
41.13491212% purity, respectively. Errors may be due to the incomplete hydrolysis of glycogen
and amylopectin since the assumption is a complete hydrolysis of the sample which will give a
low absorbance reading and lower concentration of glucose in the samples. Also, prolong
heating and limitations of the spectrophotometer might contribute to the errors during the
experiment.

End Group Determination of Polysaccharides

In the end group determination of polysaccharides, periodate oxidation was performed.

The presence of vicinal diol or an α-hydroxy carbonyl group is a requirement for periodate
oxidation. Using periodate, vicinal diols are cleaved consuming one mole of the oxidizing agent
and producing two carbonyls.
 On the other hand, oxidation of HIO4 with α-hydroxy carbonyls produces an aldehyde and
formic acid. Oxidation with this molecule consumes one mole of HIO4.

A chain containing three neighboring hydroxyl group yields two aldehyde groups and one
formic acid. The middle hydroxyl group is oxidized in two steps. In this oxidation two moles of
oxidizing agent is consumed.

Glycogen isolate and amylopectin were dissolved using 3 % NaCl and the mixtures were
heated gently on a warm water bath followed by the addition of NaIO4 in a volumetric flask.
The solution was kept in the dark for 1 week. Periodate oxidation may occur upon cleavage of
the bond between two adjacent carbons which carry carbonyl, hydroxyl, or amino group.
Sodium periodate (NaIO4) is generally used because of its suitable solubility. Oxidation of the
carbohydrates is accompanied by reductions of I0-4 to I0-3 and the oxidation state of each
functional group in the carbohydrate is increased by at least one level (alcohol > aldehyde/
ketone > acids > CO2). Free sugars which are hemiacetals or hemiketals react as if they existed
entirely in their straight chain form (Chemgaroo, 2016).

The amount of formic acid produced was used to calculate the degree of polymerization of
polysaccharide in this experiment. Methods that may be used to determine the formic acid
produced are: a.) direct titration with alkali, b.) liberation and titration of iodine from solution of
iodide, c.) manomeric determination of carbon dioxide liberated from a bicarbonate buffer, and
d.) spectroscopic determination of formic acid. In this experiment, direct titration was used with
standard 0.01 N NaOH as titrant. One variation of the titration is the used of ethylene glycol as
an indicator. Since the periodate ion requires a vicinal diol, ethylene glycol has a structure with
this requirement. This would ensure that the titration of alkali would correspond to the amount
of formic acid present in the solution. The ethylene glycol would consume the excess periodate
ion present in the solution.
Table 10.1. Standardization of the 0.01 NaOH.

Parameters Values
T1 T2 T3
Mass KHP, g 0.0489 0.0585 0.0481
MW KHP, g/mol 204.22
Vinitial , mL 0.00
V final , mL 25.9 32.8 28.5
V used , mL 25.9 32.8 28.5
NaOH Concentration, 9.245083185 x10^-3 8.733408018 x10^-3 8.264221419 x10^-3
N
Average 8.747570874 x10^-3
Concentration, N

Table 10.2. Results of titration of the glycogen and amylopectin sample with the standardized
NaOH.

Sample Trial Volume, mL

Initial Final Used
Blank 1 0 0.1 0.1
2 0.1 0.1
3 0.1 0.1
Glycogen 1 2.9 2.9
2 2.7 2.7
3 2.7 2.7
Amylopectin 1 0.8 0.8
2 0.7 0.7
3 0.7 0.7

Tale 10.3. Data for the determination of the number of glucose per segment of glycogen.

Parameters Trial 1 Trial 2 Trial 3 Average

No. of non- 2.44932E-05 2.27437E-05 2.27437E-05 2.33269E-05
reducing ends
(NA), mol
No. of Segments 4.89864E-05 4.54874E-05 4.54874E-05 4.66537E-05
(SA), mol
Total moles of 3.836254623 x10-4
glucose in
glycogen, mol
Ave. glucose 8
units/segment

Tale 10.4. Data for the determination of the number of glucose per segment of amylopectin.
 Parameters Trial 1 Trial 2 Trial 3 Average
No. of non- 6.1233E-06 5.24854E-06 5.24854E-06 5.54013E-06
reducing ends
(NA), mol
No. of Segments 1.22466E-05 1.04971E-05 1.04971E-05 1.10803E-05
(SA), mol
Total moles of 5.077961878 x10-4
glucose in
amylopectin,
mol
Ave. glucose 46
units/segment

Table 10.5. Comparison of the degree of branching of glycogen to amylopectin.

Sample Trial % Branching Average %

Branching
Glycogen 1 6.384664838 6.080635661
2 5.928621073
3 5.928621073
Amylopectin 1 1.205857812 1.091013836
2 1.033591848
3 1.033591848

Table 10.6. Values per glycogen sample.

Parameter Average
No. of glucose 5061.728395 ~ 5062
units per
glycogen
molecule
Total mole of 7.578942061 x10-8
glycogen in the
sample
No. of non- 308
reducing ends
per molecule
(NM)
No. of segments 616
per molecule
(SM)

No. of glucose 8
per segment

Table 10.7. Values per amylopectin sample.

 Parameter Average
No. of glucose 6172.839506 ~ 6173
units per
amylopectin
molecule
Total mole of 8.226982424 x10-8
amylopectin in
the sample
No. of non- 67
reducing ends
per molecule
(NM)
No. of segments 135
per molecule
(SM)

No. of glucose 46
per segment

To determine the degree of branching, the amount of formic acid produced after oxidation
was measured using titration with the standardized NaOH. Based on the calculated values after
titration, it can be observed that the glycogen has more reducing ends compared to
amylopectin. Due to this, the amount of glucose per molecule is lower for glycogen than
amylopectin. Also, the number of glucose per segment for the glycogen is lower with 8 than the
amylopectin with a value of 46. Based on these observations, it can be concluded that glycogen
is more branched than amylopectin. This can be justified based on the computed degree of
branching for glycogen and amylopectin which are 6.080635661% and 1.091013836%,
respectively.

Figure 10.1. Structure of amylopectin (Source:

https://www.smartkitchen.com/resources/amylopectin)
 Figure 10.2. Structure of glycogen (Source: http://sphweb.bumc.bu.edu/otlt/MPH-
Modules/PH/PH709_BasicCellBiology/PH709_BasicCellBIology_print.html)

Theoretically, the glycogen is more branched with branching occurred at every 8-12
glucose residues while amylopectin, even with the same glycosidic bond present in the structure
with glycogen, it is less branched since the branching only occurred every 24-30 glucose
residues (Nelson and Cox,2008).

IV. Summary and Conclusion

Carbohydrates found in nature are mostly polysaccharides. Each polysaccharide differs

from another in the identity of recurring monomer units, the length of their chains, types of
bonds linking the units, and the degree of branching. The most important polysaccharides are
glycogen in animal cells and starch in plant cells. Since glycogen and starch have many
exposed hydroxyl groups available to hydrogen bond with water, they are heavily hydrated.

In one of the exercise, glycogen was isolated from mussel flesh using 30 % hot aqueous
KOH. Further purification was done by extraction of the precipitate with cold 10%
trichloroacetic acid. The obtained percent yield was 14.97004494% with respective to the
mass of the theoretical value if a muscle tissue must weight 2% of it. We could say that there
is an incomplete precipitation of glycogen.

Afterwards, the purity of the glycogen isolate and commercially available amylopectin was
determined by subjecting the samples to acid hydrolysis followed by determination of amount of
glucose formed using the Nelson’s method. The purity of glycogen and amylopectin samples
were 31.07366245% and 41.13491212% purity, respectively. Errors may be due to the
incomplete hydrolysis of glycogen and amylopectin which will give a low absorbance reading
and lower concentration of glucose in the samples.

The end group determination of polysaccharides was done by subjecting the samples to
peroxide oxidation. The amount of formic acid produced was used to calculate the degree of
polymerization of polysaccharide in this experiment. In this experiment, direct titration was
used with standard 0.01 N NaOH as titrant to determine the amount of formic acid produced.
Based on the calculated values after titration, it was observed that the glycogen has more
reducing ends compared to amylopectin. Due to this, the amount of glucose per molecule is
lower for glycogen than amylopectin. Based on these observations, it can be concluded that
glycogen is more branched than amylopectin. This can be justified based on the computed
degree of branching for glycogen and amylopectin which are 6.080635661% and
1.091013836%, respectively.

V. Sample Calculation

A. Isolation of Glycogen

1.) Yield of isolated glycogen

𝑔 𝑖𝑠𝑜𝑙𝑎𝑡𝑒𝑑 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛 0.48

𝑦𝑖𝑒𝑙𝑑 (%) = 𝑥10 = 𝑥10
𝑔 𝑜𝑓 ℎ𝑜𝑚𝑒𝑔𝑒𝑛𝑖𝑧𝑒𝑑 𝑚𝑢𝑠𝑠𝑒𝑙𝑠 40.04
= 0.1198801199 g glycogen/ 10 g mussel flesh

𝑎𝑐𝑡𝑢𝑎𝑙 % 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛 0.1198801199

𝑦𝑖𝑒𝑙𝑑 (%) = 𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 % 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛 𝑥100 = 2 % 𝑥 40.04
x 100

𝑦𝑖𝑒𝑙𝑑 (%) = 14.97004494 %

B. Purity Determination of polysaccharides

1.) Actual glucose content (µmol/mL) for glycogen:

Equation of the line from the calibration curve: y = 0.29x + 0.000408

*Glycogen sample:
x= (𝐴510)(𝑥̂)(𝐷𝐹)
𝑥 = 0.056(𝑥
̂)(100) = 19.17244973 µmol/mL

*Amylopectin sample
x= (𝐴510)(𝑥̂)(𝐷𝐹)
𝑥 = 0.074(𝑥
̂)(100) = 25.38024078 µmol/mL

2.) Actual µmol glucose/mg glycogen

*Glycogen sample (test tube 8)

 𝑎𝑐𝑡𝑢𝑎𝑙 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 19.17244973µ𝑚𝑜𝑙/𝑚𝐿 𝜇𝑚𝑜𝑙𝑔𝑙𝑢𝑐𝑜𝑠𝑒
= = 1.917244973
𝑐𝑜𝑛𝑐. 𝑠𝑡𝑜𝑐𝑘 10 𝑚𝑔/𝑚𝐿 𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛

*Amylopectin sample (test tube 8)

𝑎𝑐𝑡𝑢𝑎𝑙 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 25.38024078µ𝑚𝑜𝑙/𝑚𝐿 𝜇𝑚𝑜𝑙𝑔𝑙𝑢𝑐𝑜𝑠𝑒

= = 2.538024078
𝑐𝑜𝑛𝑐. 𝑠𝑡𝑜𝑐𝑘 10 𝑚𝑔/𝑚𝐿 𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛

3.) Percent purity of glycogen

*Glycogen sample
𝐴𝑐𝑡𝑢𝑎𝑙 µ𝑚𝑜𝑙 𝑔𝑙𝑢𝑐𝑜𝑠𝑒/𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑝𝑢𝑟𝑖𝑡𝑦 = 𝑥100
𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 µ𝑚𝑜𝑙 𝑔𝑙𝑢𝑐𝑜𝑠𝑒/𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛

1.917244973𝑔𝑙𝑢𝑐𝑜𝑠𝑒/𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑝𝑢𝑟𝑖𝑡𝑦 = 𝑥100 = 31.07366245%
6.17 µ𝑚𝑜𝑙 𝑔𝑙𝑢𝑐𝑜𝑠𝑒/𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛

*Amylopectin sample
𝐴𝑐𝑡𝑢𝑎𝑙 µ𝑚𝑜𝑙 𝑔𝑙𝑢𝑐𝑜𝑠𝑒/𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑝𝑢𝑟𝑖𝑡𝑦 = 𝑥100
𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 µ𝑚𝑜𝑙 𝑔𝑙𝑢𝑐𝑜𝑠𝑒/𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛

𝑔𝑙𝑢𝑐𝑜𝑠𝑒
2.538024078 𝑚𝑔
𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑝𝑢𝑟𝑖𝑡𝑦 = 𝑔𝑙𝑢𝑐𝑜𝑠𝑒
𝑥100 = 41.13491212%
6.17 µ𝑚𝑜𝑙 𝑚𝑔 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛

C. End group determination of polysaccharides

1.) Standardization of 0.01 N NaOH

𝑚 1
𝑁𝑁𝑎𝑂𝐻 = ( ) ( )
𝐸𝑊 𝐾𝐻𝑃 𝑉 𝑁𝑎𝑂𝐻

𝟎.𝟎𝟒𝟖𝟗 𝟏
𝑁𝑁𝑎𝑂𝐻 = (𝟐𝟎𝟒.𝟐𝟐) (𝟎.𝟎𝟐𝟓𝟗) = 9.245083185 x10-3 N

𝑁𝑁𝑎𝑂𝐻 = 𝑀𝑁𝑎𝑂𝐻 == 9.245083185 x10-3 M

2.) No. of non-reducing ends (NA) = Total moles formic acid

NA = MNaOH(VNaOH – Vblank)

*For Glycogen Sample

1𝐿
NA = ( 9.245083185 x10-3) (2.9 mL – 0.1 mL) ( )
1000 𝑚𝐿
 NA = 2.44932 x10-5mol

*For Amylopectin Sample

1𝐿
NA = (0.009001250094 M) (0.8 mL – 0.1 mL) (1000 𝑚𝐿)
NA = 6.1233 x10-6mol

3.) Number of segments (SA)

SA = 2NA – 1 = 2NA
*For Glycogen Sample

SA = 2(2.44932 x10-5mol)
SA = 4.89864 x10-5mol

*For Amylopectin Sample

SA = 2(6.1233 x10-6mol)

SA = 1.22466 x10-5 mol

4.) Total moles of glucose in glycogen, mol

%𝑝𝑢𝑟𝑖𝑡𝑦 𝑥 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑝𝑜𝑙𝑦𝑠𝑎𝑐𝑐ℎ𝑎𝑟𝑖𝑑𝑒

Total mols of glucose =
𝑀𝑀 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒

*For Glycogen sample:

31.07366245 𝑔
( )(200 𝑚𝑔)(1 𝑚𝑔)
Total mols of glucose = 100 1000
162 𝑔/𝑚𝑜𝑙

Total mols of glucose = 3.836254623 x 10-4

*For Amylopectin Sample:

41.13491212 𝑔
( )(200 𝑚𝑔)(1 𝑚𝑔)
Total mols of glucose = 100 1000
162 𝑔/𝑚𝑜𝑙

Total mols of glucose = 5.077961878 x10-4

5.) Average glucose per segment

𝑡𝑜𝑡𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑖𝑛 𝑔𝑙𝑦𝑐𝑜𝑔𝑒𝑛 𝑠𝑎𝑚𝑝𝑙𝑒

Ave. glucose per segment =
2𝑁𝐴

*For Glycogen sample:

3.836254623 x 10−4
Ave. glucose per segment =
4.466537 x 10−5
 Ave. glucose per segment = 8.22283 ~ 8 glucose per segment

*For Amylopectin Sample:

5.077961878 x10−4
Ave. glucose per segment =
1.10803x 10−5

Ave. glucose per segment = 45.82874 ~ 46 glucose per segment

6.) Degree of branching

Number of branch points = NA

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑏𝑟𝑎𝑛𝑐ℎ 𝑝𝑜𝑖𝑛𝑡𝑠

%branching = 𝑥 100
𝑡𝑜𝑡𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒

*For Glycogen Sample:

2.44932 x 10−5
%branching = 𝑥 100
3.836254623 x 10−4

%branching = 6.384664838 %

*For Amylopectin Sample:

6.1233 x10−6
%branching = 𝑥 100
5.077961878 x10−4

%branching = 1.205857812 %
7.) Glucose per molecule

𝑀𝑊𝑝𝑜𝑙𝑦𝑠𝑎𝑐𝑐ℎ𝑎𝑟𝑖𝑑𝑒
Glucose per molecule =
𝑀𝑊𝑔𝑙𝑢𝑐𝑜𝑠𝑒𝑟𝑒𝑠𝑖𝑑𝑢𝑒

*For Glycogen sample:

8.2 𝑥 105 𝑔/𝑚𝑜𝑙

Glucose per molecule =
162 𝑔/𝑚𝑜𝑙
Glucose per molecule = 5061.728395 ~ 5062 glucose/molecule

*For Amylopectin Sample:

1 𝑥 106 𝑔/𝑚𝑜𝑙
Glucose per molecule =
162 𝑔/𝑚𝑜𝑙
Glucose per molecule = 6172.839506 ~ 6173 glucose/molecule
8.) Total moles of polysaccharide in the sample
% 𝒑𝒖𝒓𝒊𝒕𝒚∗𝒎𝒂𝒔𝒔 𝒐𝒇 𝒑𝒐𝒍𝒚𝒔𝒂𝒄𝒄𝒉𝒂𝒓𝒊𝒅𝒆
Total moles of polysaccharide in the sample = 𝑴𝑴 𝒐𝒇 𝒑[𝒐𝒍𝒚𝒔𝒂𝒄𝒄𝒉𝒂𝒓𝒊𝒅𝒆

*For Glycogen sample:

31.07366245 200
( )𝑥( )
Total moles of glycogen in the sample = 100
5
1000
= 7.578942061 x 10-8mol
8.2 𝑥 10 𝑔/𝑚𝑜𝑙

*For Amylopectin Sample:

41.13491212 200
( )𝑥( )
Total moles of glycogen in the sample = 100
6
1000
=8.226982424 x 10-8mol
1 𝑥 10 𝑔/𝑚𝑜𝑙

9.) Number of non-reducing ends per molecule (NM)

𝑡𝑜𝑡𝑎𝑙 𝑚𝑜𝑙𝑠 𝑜𝑓 𝑓𝑜𝑟𝑚𝑖𝑐 𝑎𝑐𝑖𝑑 𝑓𝑜𝑟𝑚𝑒𝑑

NM = 𝑡𝑜𝑡𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑝𝑜𝑙𝑦𝑠𝑎𝑐𝑐ℎ𝑎𝑟𝑖𝑑𝑒

*For Glycogen Sample:

2.33269 x 10−5 mol

NM = = 307.7857016 ~308 glucose units/segment
7.578942061 x 10−8 mol

*For Amylopectin sample:

5.54013 x 10−6 mol

NM = 8.226982424 x 10−8 mol
= 67.34097284~ 67 glucose units/segment

10.)Number of segments per molecule (SM)

SM = 2NM

*For Glycogen sample:

SM = 2(308glucose units/segment)= 616 segments/molecule

*For Amylopectin Sample:

SM = 2(67glucose units/segment)= 135 segments/molecule

11.)Number of glucose per segment

𝑔𝑙𝑢𝑐𝑜𝑠𝑒𝑝𝑒𝑟𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒
Number of glucose per segment = 𝑆𝑀

*For Glycogen sample:

 𝑔𝑙𝑢𝑐𝑜𝑠𝑒
5062
Number of glucose per segment = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒
𝑠𝑒𝑔𝑚𝑒𝑛𝑡𝑠
616
𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒

Number of glucose per segment= 8.217532468 ~ 8 𝑔𝑙𝑢𝑐𝑜𝑠𝑒/𝑠𝑒𝑔𝑚𝑒𝑛𝑡

*For Amylopectin sample:

𝑔𝑙𝑢𝑐𝑜𝑠𝑒
6173
Number of glucose per segment = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒
𝑠𝑒𝑔𝑚𝑒𝑛𝑡𝑠
135
𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒
Number of glucose per segment= 45.72592593 ~ 46 𝑔𝑙𝑢𝑐𝑜𝑠𝑒/𝑠𝑒𝑔𝑚𝑒𝑛𝑡

VI. References

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Ameneiros-Rodríguez. 2016.Glycogen metabolismin humans. BBA Clin 5: 85–100.

 Anastasopoulou, C. 2017. Glycogen storage diseases types I-VII. Retrieved

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Modules/PH/PH709_BasicCellBiology/PH709_BasicCellBIology_print.html

 Chemgaroo. 2016. Oxidative cleavage of 1,2-diols. Retrieved from

http://www.chemgapedia.de/vsengine/vlu/vsc/en/ch/2/vlu/oxidation_reduktion/ox_s
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 Childress, C.C., B. Sacktor, W. Grossman, and E. Bueding. 1970. Isolation, ultrastructure, and
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 Cincinnati Children's Hospital Medical Center. 2018. Glycogen Storage Disease (GSD).
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 Levine, S. et al. 1953. Glycogen of enteric bacteria. J.Bacteriol 66(6): 664-670.

 McMurry, J. 2012. Organic Chemistry. 8th ed. Belmont, CA: Brooks/Cole Cengage Learning.

 Nelson, D. L. and M. M. Cox. 2008. Lehninger Principles of Biochemistry. 5thed.

New York: W.H. Freeman and Company.

 Polgase, W.J., E.L. Smith, and F.H. Tyler. 1952. Studies on human glycogen. J. Biol. Chem.
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 Stoker, H.S. 2010. General, Organic, and Biological Chemistry. 5th ed. USA: Brooks/Cole
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 Structure of Amylopectin. Smart Kitchen. Retrieved from
https://www.smartkitchen.com/resources/amylopectin

 Thacker, H.L. 2004. Alkaline hydrolysis. Retrieved on 30 April 2018 from

https://krex.k-state.edu/dspace/bitstream/handle/2097/662/Chapter6.pdf.