Full Report on

Exercise 7
ISOLATION OF GLYCOGEN
and
Exercise 8
DETERMINATION OF GLYCOGEN PURITY

Angelica F. Costales
CHEM 160.1 – 2L
Midyear, 2015

Groupmates:
Alyssa Beth S. Cruz
Angel Marie H. Dargantes
Jeremy Rizza D.R. Gonzales

Laboratory Instructor:
Prof. Bong Carlo Remillion

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 INTRODUCTION

In order for our organ systems to function well, energy is needed to be supplied to the body.
The energy comes in the form of carbohydrate that is an important component of the cell
membrane. Not only does it provides energy to the body, but also serve as energy storage.

Carbohydrates are organic molecules specifically carbon compounds that have aldehyde
(C-H=O) or ketone (C=O) groups. According to Stoker (2006), carbohydrates can be classified
broadly as monosaccharides and compound carbohydrates wherein the former includes simple
sugars that can’t be hydrolyzed further into smaller carbohydrates and the latter which includes
double sugars (disaccharides), few sugars (oligosaccharides), or of many sugars (polysaccharides).
Polysaccharides can either be storage or structural polysaccharides. The storage polysaccharide is
the form of carbohydrate for energy storage such as starch and glycogen. Compared to other
saccharides, these high compact polymers have a different physical and chemical properties. In
plants, polysaccharides are called starch, while in humans and animals, they are called glycogen.

Glycogens are polymers of glucose that contain glycosidic bonds. These molecules are
resistant to hydrolytic activity of OH at increased temperature. While other interactions such as
peptide bonds in proteins, ester bonds in lipids, and phosphodiester bonds in ribonucleic acids are
hydrolyzed using an alkaline pH at an elevated temperature. This way, glycogen can be isolated
and purified by addition of ethanol in a relatively high temperature.

The isolation of glycogen is necessary for conducting researches that would relate to the
availability of glycogen in the liver or other parts of the body in order to cure diseases or disorders
that would affect the glycogen metabolism and thus, dysfunction other internal organ systems such
as the heart, lungs, kidney, brain, and etc.

One way of isolating glycogen is through the use of the reagents 30% hot aqueous
potassium hydroxide (KOH), 10 % trichloroacetic acid (TCA), 95% ethanol and the washing
solvents, diethyl ether and absolute ethanol. The KOH serves as the alkaline solution that
hydrolyzes proteins, lipids and ribonucleic acids especially in high temperature. While as for the
TCA, it was mentioned in the study conducted by Roe, et.al. (1960), that alkaline residue digestion
after the four homogenizations with trichloroacetic acid solution, followed by alchoholic
precipitation, yielded an additional 45% of glycogen to the initial 57% total glycogen brought by
thorough grinding of the sample with 5% TCA. This explains the effectivity of the TCA in
extracting the glycogen from the other residues. As for the 95% ethanol, it precipitates out the
glycogen isolate that is washed by polar and non-polar solvents, absolute ethanol and diethyl ether,
respectively, to remove the traces of H2O and hasten the dehydration of the sample. Afterwards,
actual yield would be needed to calculate in order to know the amount or mass of glycogen per
mass of the sample.

Although isolation isn’t finished after precipitation. It has still has to undergo test for
purity. For carbohydrates, one way to test for an isolated samples purity is through the Nelson’s
assay and afterwards reading the absorbance at 510 nm with a spectrophotometer. The Nelson’s
assay tests for the presence of reducing sugars, such as glucose, maltose and lactose. It
quantitatively determines the amount of, for example, glucose colorimetrically. Since the isolated

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glycogen is not a reducing sugar, it needs to be hydrolyzed to break the glycosidic bonds and
release the glucose. This acid hydrolysis is done through heating the glycogen isolate samples with
alkaline copper reagent to form a rust colored precipitate. Then the amount of cuprous oxide
produced will then be determined by the addition of arsenomolybdic acid (reduced to
arsenomolybdous acid). Afterwards, an intense blue color will be observed. Another test for
reducing sugars is 3,5-dinitrosalicylic acid (DNS) which is about 10 times less sensitive than
Nelson’s assay. It is the method for determination of xylanase activity which dominates in
laboratories around the globe (Bailey, et.al., 1992).

Acid hydrolysis of glycogen, based on literatures, requires heating for 30 minutes with the
reagents HCl solution, distilled water and 1.2 N NaOH solution. Thus, if experiment set-up has
the same condition as mentioned above, the concentration of glucose can be obtained to compute
for the actual content of glucose per mass of glycogen. When this is obtained, the percentage of
the purity can now be solved since the equation for percent purity is written below.

% purity = Actual µmol glucose / mg glycogen x 100%

Theoretical µmol glucose / mg glycogen

Thus, the aim of this exercise is to familiarize students with proper glycogen isolation and
acid hydrolysis techniques and for them to gain knowledge in computing for the actual yield of the
glycogen and determining the percent purity of the hydrolyzed glycogen isolate.

METHODOLOGY
A. Isolation of Glycogen
Sample Preparation
Fresh clams were used in the experiment. The flesh were separated from the shell and were
placed in an ice bath. After the flesh was cooled, it was homogenized. 121.247 g of the
homogenized flesh was weighed and transferred into a 125-mL Erlenmeyer flask.

Isolation of the Glycogen

Afterwards, 21.60 mL of hot 30% aqueous KOH was added to the mixture and was heated
in a boiling water bath for one hour while stirring once in a while. Distilled water (15.0 mL) was
then added to the mixture and was transferred to a 250 mL Erlenmeyer flask. In order to precipitate
the glycogen from the mixture, 30.0 mL 95% ethanol was added and swirled. It was allowed to
stand and cool to room temperature by placing it in an ice bath for 20 minutes. After the particles
had settled down, the mixture was transferred into centrifuge tubes and was centrifuged for 5
minutes in order for the precipitate to be collected. It was important for the speed to be half the
maximum rpm and that the mass of the tubes’ within 0.1 g of each other. The supernatant was
discarded and the collected glycogen precipitate was dissolved in a 6.00 mL of cold 10% TCA
solution. It was then centrifuged for the complete removal of glycogen from the tissues by
removing the precipitate and retaining the supernatant. Glycogen from the supernatant was then
recovered by adding 15 mL of 95% ethanol. The mixture was left in an ice bath for 10 minutes to
stand and precipitate was collected after performing centrifugation for 5 minutes. Since the
glycogen precipitate still wasn’t purely white, 12 mL of 10% TCA solution was added and later
on, the mixture was centrifuged to retain the supernatant. It was then on added with 30.0 mL of

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95% etOH, to recover the glycogen from the supernatant and was stood in an ice bath for 10
minutes again. After the particles settled down, centrifugation was performed in order to collect
the precipitate. The dirty white colored precipitate was dissolved I 10 mL absolute ethanol and
8mL distilled water in order to reprecipitate. It was stood in an ice bath and then the precipitate
was recovered by centrifugation. 8 mL of absolute ethanol was added to wash the collected
precipitate and it was allowed to stand in room temperature for some minutes. Glycogen precipitate
was again washed with 4.0 mL diethyl ether and was allowed to dry in a pre-weighed watchglass.
After it has dried, the weight of the isolate was determined.

B. Determination of Glycogen Purity

Preparation of a Standard Curve
Glucose served as the standard for the preparation of the standard curve. It was prepared
by diluting a 20 mM glucose solution into 2.0 mM that served as the stock solution. Seven (7) test
tubes were prepared, and each was added with different concentration of glucose solution. The
amount of glucose and distilled water (in mL) used were as follows, respectively: 1 – 0.0,1.0; 2 –
0.1,0.90; 3- 0.2, 0.80; 4 – 0.4, 0.6; 5 – 0.6, 0.4; 6 – 0.8, 0.2; 7 – 1.0, 0.0.

Acid hydrolysis of Glycogen

Meanwhile, hydrolysis of glycogen was performed by weighing 50 mg of glycogen isolate
in a 50 mL beaker. It was then diluted by adding 5.0 mL distilled water to have a concentration of
10 mg/mL that would serve as the stock glycogen solution. Eight test tubes were utilized with
varying concentration of the glycogen. Test tube 1 had 0.4 mL dH2O and no glycogen solution.
Test tube 2 had 0.60 distilled water with 0.40 mL glycogen solution. The rest of the test tubes (3-
8) had no dH2O about 0.40 mL glycogen solution was added to each. Afterwards, different amounts
of 2 N HCl solution was added to each test tubes. 0.60 mL of 2 N HCl was measured and added to
test tube 1, 0.60 mL was added to test tubes 3-8, while test tube 2 didn’t contain 2 N HCl. Each of
the test tubes 1 and 2 also contained 1.00 mL of 1.2 N NaOH solution. These test tubes were
allowed to stand in room temperature while the tubes 3-8 was dispersed in a boiling water bath.
The tubes in the boiling water bath were removed at 5-minute intervals which started from 5
minutes after boiling. Then, reaction was terminated for each of the test tubes 3-8 by adding 1.0
mL of 1.2 N NaOH after the specified heating time. All the test tubes were then diluted to 8.0 mL
distilled water and were mixed using a vortex mixer. A 0.5 mL aliquot from each of the diluted
samples was obtained and diluted again to a final volume of 1.0 mL by using distilled water and
was mixed using a vortex mixer.

Nelson’s Assay
Then Nelson’s reagent was prepared freshly by mixing 50 mL of reagent with 2 mL reagent
B. Reagent A was prepared by weighing 12.5 g of Na 2CO3, 12.5 g sodium potassium tartrate and
100 g NA2SO4 and diluting them in a 500 mL distilled water. While the reagent b was prepared by
dissolving 7.5 g of CuSO4 in a 100 mL beaker with 50 mL distilled water and with the addition of
1 drop of concentrated H2SO4.

Standard solution and samples were assayed simultaneously to obtain accurate results. For
all the test tubes of the standard and the sample, 1.0 mL freshly prepared Nelson’s reagent was
added. The tubes were placed in a boiling water bath and were covered with marbles for 20
minutes. It was cooled to room temperature afterwards and 1.0 mL arsenomolybdate reagent was

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added to each. The contents of the tubes were mixed using a vortex mixer and were allowed to
stand for 5 minutes at room temperature. Then, 7.0 mL distilled water was added to each of the
test tubes and again mixed by the vortex mixer.

Spectrophotometry
The standards’ and samples’ absorbance were read at 510 nm. The standard curve was
graphed by plotting the absorbance at 510 nm versus the glucose concentration in µmol/mL.
Meanwhile, the content of the glucose in the hydrolyzed glycogen was obtained through
interpolating its absorbance values on the ẋ of the standard curve.

RESULTS AND SAMPLE CALCULATIONS

Table 7.1 Stepwise observation of glycogen isolation from clam flesh.

Steps
1. Separation of flesh from shell
2. Homogenization of flesh mixture
3. Saponification by heating with
aqueous KOH
4. Swirling during boiling water bath
5. After boiling for 1 hour
6. Addition of water
7. After addition of 95% etOH
8. After stirring
9. After standing in ice bath
10. After centrifugation
Supernatant
Precipitate
11. Addition of 10% TCA
12. After centrifugation
Observations
Dark brown color
Viscous, turbid, light brown mixtures with
particles
Viscous, turbid formation of three layers
(cream, yellow, cream), heterogeneous
mixture
Viscous, turbid transition of middle layer
from yellow to dark brown, heterogeneous
mixture
Opaque, turbid dark orange and brown layers,
heterogeneous mixture with precipitate
Opaque, turbid dark orange and brown layers,
heterogeneous mixture with precipitate
Thin or smooth, dark brown homogeneous
mixture with precipitate
Creamy, cloudy, brown homogeneous
mixture without ppt
Smooth, darker brown with creamy lower
layer, heterogeneous mixture
Heterogeneous mixture
Smooth, brown homogeneous mixture –
discarded
Viscous, creamy, dirty white homogeneous
mixture
Viscous, creamy, dirty white homogeneous
mixture
Heterogeneous mixture

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 Supernatant
Precipitate
13. Addition of 95% etOH
14. After standing in ice bath
15. Transfer into centrifuge tubes
16. After centrifugation
Supernatant
Precipitate
17. After addition of 12 mL 10% TCA
18. After centrifugation
Supernatant
Precipitate
19. After addition of 95% etOH
20. Stand in ice bath
21. After centrifugation
Supernatant
Precipitate
22. After dissolved in 10 mL abs etOH
and water
23. Stand in ice bath
24. After centrifugation
Supernatant
Precipitate
25. Addition of 8 mL abs etOH
26. After standing in room temperature
27. After addition with diethyl ether
28. After drying in a watchglass
Table 7.2. Percent yield of glycogen from clam flesh.
Mass of watchglass
Mass of watchglass + glycogen
Opaque, formation of 4 layers (Merky brown,
dark brown, merky brown, off-white)
heterogeneous mixture
Opaque, brown, homogeneous mixture –
discarded
Opaque, brown homogeneous mixture
Opaque, formation of 3 layers (dark brown,
light brown, creamy brown), heterogeneous
mixture with precipitate
Opaque, creamy brown homogeneous mixture
Heterogeneous mixture
Opaque, creamy, dirty white homogeneous
mixture –discarded
Opaque, light creamy homogeneous mixture –
collected
Light creamy homogeneous mixture
Heterogeneous mixture
Thin, merky brown, liquid, dirty white
heterogeneous mixture – collected
Thin, solid, yellowish homogeneous
precipitate- discarded
Thick, creamy, brown, homogeneous mixture
Curdy heterogeneous mixture of very light
yellow and clear layers with precipitate
Heterogeneous mixture
Thin, translucent, homogeneous mixture –
discarded
Dirty white precipitate – collected
Translucent white (slightly yellowish)
heterogeneous mixture with ppt
Clear white liquid with clear creamy
heterogeneous mixture
Heterogeneous mixture
Clear homogeneous liquid – discarded
Dirty white precipitate – collected
Thin, white or milky, homogenenous mixture
Clear supernatant with milky or white
precipitate, heterogeneous mixture
Clear layer, white precipitate, heterogeneous
mixture
White powder crystals
33.831 g
35.040 g
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 Mass of glycogen 1.209 g
Mass of clam flesh 121.247 g
Actual yield 0.997 %
Theoretical yield 2%
% Yield 49.85 %

Standard Curve of Glucose

1
Absorbance at 510 nm

0.8

0.6

0.4

0.2

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Glucose concentration (µmol/mL)

Figure 8.1.Graph of the glucose concentration (µmol/mL) over the absorbance at 510 nm.
Table 8.1. Absorbance of standard glucose solutions
Test tube no. Glucose concentration A510
(µmol/mL)
1 0 0.000
2 0.2 0.172
3 0.4 0.286
4 0.8 0.558
5 1.2 0.797
6* 1.6 1.191
7* 2.0 1.180
8 - -

LR Values:
r = 0.998609822
slope =0.656077586 mL/µmol
y-intercept = 0.021439655

Note: * - not included in the graph in Figure 8.1

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 Concentration of glucose in the hydrolyzed sample
0.5

0.4
Absorbance at 510 nm
0.3

0.2

0.1

0
-1.63 1.558 0.957 5.301 11.627 17.342 33.27 29.002
-0.1
Glucose concentration (µmol/mL)

Figure 8.2. Graph of the glucose concentration (µmol/mL) from the hydrolyzed glycogen over
the corrected absorbance at 510 nm.

Table 8.2. Acid hydrolysis of glycogen.

Test tube no. Incubation time A510 Corrected Glucose
min) absorbance Concentration
(µmol/mL)
1 (blank) 0 0.010 0 -1.63
2 0 0.009 -0.001 1.558
3 5 0.055 0.034 0.957
4 10 0.101 0.091 5.301
5 15 0.184 0.174 11.627
6 20 0.259 0.249 17.342
7 25 0.468 0.458 33.270
8 30 0.412 0.402 29.002
SAMPLE CALCULATIONS
Calculations for actual yield
Mass in grams of the actual yield (g)
= mass of glycogen
mass of clam
= 1.209 g
121.247 g
= 0.00997 grams
Percentage of the actual yield (%)
= mass of glycogen x 100
Mass of clam
= 1.209 g x 100
121.247 g
= 0.997 %

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Calculation for theoretical yield
Theoretical yield of glycogen (in %) = 2%
In grams = 2% / 100
= 0.02 grams

Calculation for Percent yield

= actual yield x 100
Theoretical yield
= 0.997 % x 100
2%
= 0.4985 x 100
= 49.85%

Linear regression values of standard curve

r = 0.998609822
slope =0.656077586 mL/µmol
y-intercept = 0.021439655

Calculation for glucose concentration

Dilution factor = (10 mL/0.4 mL) (1mL/0.5 mL)
= 50
Corrected absorbance = absorbance – 0.010
µmol/mL glucose = (corrected absorbance)ẋ * DF

For test tube 1:

µmol/mL glucose = (0.01 – 0.01)ẋ * 50
= (0)ẋ * 50
= - 1.63 µmol/mL
For test tube 2:
µmol/mL glucose = (0.009 – 0.01)ẋ * 50
= (-0.001)ẋ * 50
= 1.558 µmol/mL
For test tube 3:
µmol/mL glucose = (0.044 – 0.01)ẋ * 50
= (0.034)ẋ * 50
= 0.957 µmol/mL
For test tube 4:
µmol/mL glucose = (0.101 – 0.01)ẋ * 50
= (0.091)ẋ * 50
= 5.301 µmol/mL
For test tube 5:
µmol/mL glucose = (0.184– 0.01)ẋ * 50
= (0.174)ẋ * 50
= 11.627 µmol/mL
For test tube 6:
µmol/mL glucose = (0.259 – 0.01)ẋ * 50

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 = (0.249)ẋ * 50
= 17.342 µmol/mL
For test tube 7:
µmol/mL glucose = (0.468– 0.01)ẋ * 50
= (0.458)ẋ * 50
= 33.270 µmol/mL
For test tube 8:
µmol/mL glucose = (0.412 – 0.01)ẋ * 50
= (0.402)ẋ * 50
= 29.003 µmol/mL

Calculation for actual glucose content

Actual glucose content = glucose content of test tube 8

Actual µmol/mL glucose = (0.412 – 0.01)ẋ * 50

= (0.402)ẋ * 50
= 29.003 µmol/mL

Calculation for actual µmol glucose per mg of glycogen

Actual µmol glucose per mg of glycogen
=`actual glucose content
Glycogen stock solution

= 29.003 µmol/mL
10 mg/mL

= 2.900 µmol/mg

Calculation for Percent purity

Theoretical µmol glucose / mg glycogen = (Total mass of glucose in glycogen (mg) x
180/162 x 1mmol/180 mg x 1000µmol/1 mmol)
Mass of glycogen (mg)

= 50 mg x 180/162 x 1mmol/180 mg x 1000 µmol/1 mmol

50 mg

= 6.17 µmol/mg

% purity = Actual µmol glucose / mg of glycogen x 100

Theoretical µmol glucose / mg glycogen

= 2.900 µmol/mg x 100

6.17 µmol/mg

= 47.006 %

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 DISCUSSION

Glycogen is the storage form of carbohydrates in animals. They are highly branched
polysaccharides composed of α-D-glucopyranose units linked by α-1,4 and α-1,6 glycosidic bonds.
These bonds are resistant to hydrolytic activity of –OH at elevated temperature. While as for the
peptide bonds (in proteins), ester bonds (in lipids) and phosphodiester bonds (in nucleic acids)
hydrolysis occurs at elevated temperature in alkaline pH. Thus, in order to isolate glycogen, the
unwanted biomolecules have to be removed through use of alkali solution and other necessary
reagents in high temperature.

In the first part of the isolation process, 30% hot aqueous KOH was added. This was in
order to solubilize the tissues as it served as a hot concentrated alkali and also to saponify fats/lipids
present (Eichenberger, 2012). While the 10% TCA added was used to extract or separate glycogen
from the proteins and nucleic acids. After centrifugation, to precipitate glycogen from the mixture,
95% ethanol was used. In the final part of the process, traces of water are needed to be removed
by the addition of absolute ethanol and diethyl ether. The difference between these two is that
absolute ethanol is more polar, making the water residues attach to these molecules in order to
dehydrate the glycogen. While diethyl ether hastens the precipitation process since it is a volatile
solvent.

After the drying of the precipitate, the mass of glycogen was found out to be 1.209 grams,
having an actual yield of 0.997% and a percent yield of 49.85%. This would mean that 0.997% of
the mass of the clam samples are glycogen since the actual yield is equivalent to the mass of
glycogen over the mass of fresh clam sample. However, the percent yield measures the efficiency
of the reaction or process and since the percent yield achieved was only 49.85%, this means that
the glycogen in the clam samples weren’t fully recovered. This may have been due to experimental
errors brought by mismeasurement and/or human errors during handling of the mixtures.

Once the glycogen was isolated, its purity will be determined by measuring it absorbance
through spectrophotometry after conducting the nelson’s assay. The relevance of using nelson’s
assay is that is serves as a test for presence of reducing sugars. Although glycogen is not a reducing
sugar, therefore, it needs to be hydrolyzed first to obtain its monomer units which are glucose-
glucose. To be able to dissociate the glycosidic bonds of the glycogen, the sample should be
subjected to a strong acid, which is HCl, in an elevated or high temperature (Driskell, 2012). Thus,
when glycosidic bonds are broken, the monomer units which are reducing sugars (glucose) can
now be tested with the Nelson’s assay. Here, when the sugar is heated with alkaline copper reagent,
a rust colored Cu2O is formed. And its amount formed will be determined colorimetrically by the
addition of arsenomolybdic acid. Then, reduction of the arsenomolybdic acid to arsenomolybdous
acid occurs by the Cu+. An intense blue color is achieved afterwards and is measured
colorimetrically. As intensity of the color increases, the number of reducing sugars also increases.

The standard curve shown in Figure 8.2 shows an increasing trend with the glucose
concentration (µmol/mL) over the absorbance values read at 510 nm. Although three absorbance
weren’t included in the graph since its values are greater than 1.

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 In Table 8.2, shown is the corrected absorbance readings of the samples obtained by
subtracting 0.010, which is the absorbance value of test tube 1 in the samples. These values were
used to determine the glucose concentration after hydrolysis of glycogen (µmol/mL) of the diluted
solutions. The corrected absorbance readings of the samples were interpolated from the standard
curve’s ẋ. Acid hydrolysis of glycogen occurs after 30 minutes in boiling water bath with HCl.
Test tube 8, which was subjected to the same condition previously mentioned, became the basis of
the actual glucose content of the completely hydrolyzed sample. Its concentration was found out
to be 29.002 µmol/mL and from this, the actual µmol of glucose per mg of glycogen was computed
to be 2.90 µmol/mg.

Aside from the relevant information above, the percent purity of the glycogen sample was
found to be 47.006% pure. This was obtained by dividing the actulal µmol of glucose per mg of
glycogen by the theoretical µmol of glucose per mg of glycogen. Thus, the isolated glycogen from
the clam is not entirely pure and that contaminants such as protein and other residues are present
in the isolated sample. To correct the statement above, though the actual yield is 0.997%, we can’t
really say that this value is equivalent to the percent of glycogen in the mass of the clam samples
since the isolation process weren’t efficient and that the isolated glycogen is only 47.006% pure.
This may be due to experimental errors that happen in almost all research studies including the
isolation of biomolecule that causes the impurities.

There are different types of glycogen storage diseases that affect the human body’s
glycogen metabolism. The GSD II or also called as acid maltase deficiency or Pompe disease is
caused by an accumulation of glycogen in the lysozyme due to the deficiency of lysosomal
enzyme, α-1,4-glucosidase. It affects the human body such that progressive muscle weakness or
myopathy is felt throughout the body and thus affects various body tissues specifically the heart,
skeletal muscles, liver and nervous system. Another kind of GSD is the hepatorenal glycogenosis
(GSD Type 1). It is a genetic disease that results from a deficiency in the glucose-6-phosphatase
enzyme which causes the inability of the liver to produce free glucose from glycogen and from
gluconeogenesis. Thus, it causes sever hypoglycemia in man due to the affected two principal
metabolic mechanisms that supplies glucose to the rest of the body during fasting, creating an
increased stored glycogen in liver and kidneys. Though it can be treated by frequent or continuous
feedings of cornstarch or other carbohydrates.

SUMMARY AND CONCLUSION

The isolation of glycogen involved the addition of necessary reagents which resulted to the
change of color from viscous brown heterogeneous mixture to creamy brown to dirty white to
milky white then finally to a white precipitate. The transition of these colors was due to the heat
and addition of 30% aqueous KOH, 10% TCA, 95% ethanol, diethyl ether and absolute ethanol.
After drying the white precipitate, its mass was found out to be 1.209 grams. The actual yield of
isolated glycogen is 0.997%. This amount is the actual produced product after isolation. Having a
theoretical yield of 2%, its percent yield was calculated to be 49.85% which means that the
experiment didn’t produce as much glycogen isolate as expected due to experimental and
unavoidable errors.

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 Meanwhile, the values of the standard curve, having glucose as the standard, presented an
increasing trend as shown in figure 8.1.

The absorbance of the hydrolyzed glycogen isolate subjected to increasing incubation time
was found out to be also increasing. Interpolating these absorbances to the standard curve resulted
to the concentration of glucose from the hydrolyzed glycogen isolate for the test tubes 1, 2, 3, 4,
5, 6, 7, and 8, which were -1.63, 1.558, 0.957, 5.301, 11.627, 17.342, 33.270, 29.002, respectively.
When plotted over the respective absorbance, the graph was increasing. Thus, as incubation time
increases, absorbance increases too, as well as the glucose concentration obtained.

For the actual glucose content of the hydrolyzed glycogen isolate, 29.003 µmol/mL was
the concentration. This was based on the test tube 8 which was subjected to the hydrolysis
conditions of glycogen. Meanwhile, 2.900 µmol was found out to be the actual content of glucose
per milligram of the glycogen isolate. Since the theoretical µmol of gluose per milligram of
glycogen is 6.17 µmol/mg, when the actual µmol glucose per mg of glycogen is divided by this
value, a purity of 47.006% is achieved. This would mean that the isolated glycogen wasn’t pure
enough. It still had contaminants and other residues possibly proteins, nucleotides or low molecular
weight oligonucleotides.

Thus, the conduction of these experiments resulted to achieving the isolate and its actual
yield, and also determining the purity of the glycogen isolate through acid hydrolysis and Nelson’s
assay. Although it can be concluded that the isolation process wasn’t 100% efficient since the
glucose from the hydrolyzed glycogen isolate isn’t pure.

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 LITERATURE CITED

Bailey, M.L., et. al. 1992. Interlaboratory testing of methods for assay of xylanase activity. Journal
of biotechnology. Pp. 257-270. Retrieved on July 14, 2015 at
http://www.sciencedirect.com/science/article/pii/016816569290074J

Driskell, J. 2012. Carbohydrates. Retrieved on July 13, 2015 at http://www.scribd.com/doc/9090

9780/Lab-Report-5-BMB#scribd

Eichenberger, E., et. al. 2012. Handbook of Experimental Pharmacology. Springer Science &
Business Media. Pp 105-106. Retrieved on July 13, 2015 at
https://books.google.com.ph/books?id=hpv6CAAAQBAJ&pg=PA104&lpg=PA104&dq
=addition+of+hot+30+KOH+in+glycogen+isolation&source=bl&ots=kMg27yn_f4&sig=
1qP1S3KjxYAoxfI7qSYpOeZqCiA&hl=en&sa=X&ei=F1OjVdu6Ls7voASgl4DACQ&v
ed=0CBsQ6AEwADgK#v=onepage&q=addition%20of%20hot%2030%20KOH%20in%
20glycogen%20isolation&f=false

Roe, J.H., et.al. 1960. Complete removal of glycogen from tissues by extraction with
trichloroacetic acid solution. The Journal of Biological Chemistry. Page 1. Retrieved on
July 14, 2015 at http://www.jbc.org/content/236/5/1244.full.pdf

Stoker, H.S., 2006. General, Organic and Biological Chemistry. Cengage Learning. Pp 551-552.
Retrieved on July 14, 2015 at
https://books.google.com.ph/books?id=6vy8MZeIcowC&pg=PA552&lpg=PA552&dq=
who+classified+carbohydrates&source=bl&ots=fFE52-
2_Ow&sig=PxbK1aV3v15AW40Bcsqs5HqnZjA&hl=en&sa=X&ved=0CEkQ6AEwCDg
UahUKEwiEw42o2NrGAhWOo4gKHXtjB0A#v=onepage&q=who%20classified%20car
bohydrates&f=false

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