Documente Academic
Documente Profesional
Documente Cultură
2011-35493, 2011-85007
Biochem 34.1 HEJ, Sir Marvin Pelovello
I. ABSTRACT
Glycogen is a vital carbohydrate that functions as the storage form of energy found in animal cells. This experiment
generally aims to learn the techniques and understand the principles in isolating glycogen. Specifically, its purpose
is to explain the principle behind using cold precipitation for isolating glycogen and to confirm the presence of
carbohydrates using qualitative tests. Crude glycogen was efficiently purified and extracted from chicken liver by
performing methods of homogenization, centrifugation, and cold precipitation by ethanol. Neutralized hydrolyzate
was then produced from half of the extracted crude sample through acid hydrolysis with heat. The basis of the
Molisch test is the condensation reaction between furfural compound and -naphthol wherein a purple ring
formation that was observed in the samples confirms the presence of carbohydrates. The hydrolyzate theoretically
gives a positive result, formation of red precipitate, for the detection of reducing sugars by Benedicts test since
ideally its components are unbound glucose which is a reducing sugar. Reaction with phenylhydrazine results to the
formation of osazone crystals or a yellow solution, the positive outcome for the Osazone test, which was obtained,
confirming the presence of glucose. Barfoeds test and Seliwanoffs test can be done to improve characterization of
carbohydrates.
II. KEYWORDS: glycogen, carbohydrates, Molisch test, Benedicts test, Osazone test
III. INTRODUCTION
Carbohydrates are the most abundant principles in isolating glycogen. Specifically, the
biomolecules on earth. Each year, photosynthesis experiment aims to explain the principles behind
converts more than 100 billion metric tons of carbon using cold precipitation for the isolation and to
dioxide and water into cellulose and other plant confirm the presence of carbohydrates using
products. Certain carbohydrates, such as sugar and qualitative tests.
starch, are dietary staples in most parts of the world,
and the oxidation of carbohydrates is the central IV. EXPERIMENTAL
energy-yielding pathway in most non-photosynthetic Isolation of glycogen started with the
cells (Nelson & Cox, 2012). separation of the desired compound from the
supernatant fraction of chicken liver. The chicken
Originally, carbohydrates are referred to as liver was washed and pat dried before obtaining 20
compounds containing Cn(H2O)n. This formula is only grams of the sample. A small volume of 7.4 pH
true for simple sugars, or monosaccharides. Other phosphate buffer was added. The sample was
types of carbohydrates, oligosaccharides and minced finely and placed in the homogenizer with
polysaccharides, are based on monosaccharide units 150 mL of the homogenizing solution (7.4 pH
and have slightly different general formulas phosphate buffer). The sample was homogenized to
(Campbell & Farrell, 2013). Generally, carbohydrates even and smooth consistency and was then
are macromolecules that are made up of polymers of transferred to falcon tubes. The falcon tubes were
polyhydroxy aldehydes or ketones linked together by centrifuged at 3000 rpm for 10 minutes. The
glycosidic bonds. They are called aldoses or ketoses, precipitate was discarded while 15 mL of the
depending on the nature of the carbonyl group supernatant was collected in a test tube. One mL of
present. They are called trioses, pentoses, depending 10% acetic acid was added to the test tube, covered
on the number of carbons in the molecule. with a marble, and placed in a boiling water bath for
five minutes. The supernatant was transferred to
Isolation techniques for carbohydrates are falcon tubes, cooled, and then centrifuged at 3000
easier to perform due to the weak interactions rpm for five minutes. The precipitate was discarded
involved as compared to other biomolecules. They and the supernatant was transferred to a test tube
are water-soluble and do not denature readily. and cooled to about 10oC in the refrigerator. Absolute
ethanol was added to fill half to a third of the test
The general objective of the experiment is to tube. Appearance of white, flocculent precipitate was
be able to learn the techniques and understand the observed. The solution is placed in the refrigerator for
Table 1. Visible results obtained in performing Based on Table 1, positive results are given by
Molisch test on crude sample, NH sample, (+) 1% the crude and neutralized hydrolyzate test
glucose, (-) distilled H2O. (Asterisk mark, *, indicates compounds. This confirms that the extracted crude
theoretical result) sample is a carbohydrate, and the hydrolyzed state is
Test Compounds composed of carbohydrates. However, this test
Molisch
(+) 1% (-) distilled doesnt really confirm if the isolated carbohydrate is
Test crude NH
glucose H2O glycogen because this test is positive for all types of
Visible Purple Purple Purple No carbohydrates. Moreover, this is nonspecific for
result ring solution ring* discoloration* carbohydrates since this will give a positive for
glycoproteins, glycolipids, and nucleic acids as well.
Benedicts test is specific and highly sensitive The obtained result (see Table 2) for the crude
to reducing sugars. This makes it useful to sample under Benedicts test corresponds to the
distinguish between reducing and non-reducing negative control. This confirms that glycogen is a
sugars. Reducing sugars are able to reduce solutions non-reducing sugar despite having a reducing end.
of various metallic ions. The general principle behind The presence of a reducing end may not be sufficient
the Benedicts test is that in weak alkaline solution to be detected by the test. For the hydrolyzate, it is a
aided with heat, the reducing sugars reduce cupric negative outcome incorrect result. Theoretical
(II) ions to green/yellow/orange/red precipitate of result for the hydrolyzate shows a positive result
cuprous (I) ions, while the sugars themselves are since glucose which is a reducing sugar is present.
oxidized to sugar acids. Oxidation of reducing sugars Possible cause for the incorrect result is the
is done in the free aldehyde functional groups of incomplete release of glucose monomers from the
aldoses (glucose, mannose, etc.). This test also glycogen.
detects if the aldehyde group in the sugar is unbound
(free) or bound. On the other hand, oxidation is also Table 3. Visible results observed and time of crystal
possible for ketoses, sugars with ketone functional appearance recorded in performing Osazone test on
group. Fructose, a ketose with -hydroxymethyl crude sample, NH sample, (+) 1% glucose, (+) 1%
ketone group, gives a positive result for Benedicts arabinose, (-) distilled H2O. (Asterisk mark, *,
test. This is due to the fact that under high pH indicates theoretical result)
(alkaline), fructose is converted to isomers of glucose Test compounds
Osazone (-)
and mannose which are aldoses and thus exhibits (+) 1% (+) 1%
Test crude NH distilled
reducing properties (Garcia, et al., n.d.). arabinose
glucose H2O
Visible Yellow Yellow Yellow Yellow Clear
Benedicts reagent is composed of CuSO4,
result solution* solution solution* solution solution*
sodium carbonate, and sodium citrate. These 4 mins 1 min
compounds in the presence of heat and reducing Time of 4-5 10
& 30 & 15 -
sugars carry out the following reactions.
appearance
secs secs
mins* mins*
Osazone test is the last qualitative test After performing the Osazone test, the
performed in the experiment. It is named as such neutralized hydrolyzate was observed as a yellow-
since this detects and identifies reducing sugars like colored solution (prefer to Table 3). This is similar to
monosaccharides and disaccharides based on the the obtained result for positive controls- 1%
formation of osazone and its formation time. Sugar arabinose, 1% glucose (theoretical). This confirms
osazones are yellow crystalline compounds the presence of reducing sugar which is glucose in
characteristic to every reducing sugars, therefore are the sample. It can be assumed that hydrolysis of
seen in various shapes and forms (under the glycogen was successful and release of glucose as
microscope). Formation of these crystals under monomeric units was effectively executed. It is
certain conditions, either hot or cold, further observed that the time of appearance of osazone
identifies the reducing sugar. Generally, crystals for the crude sample is within the theoretical
monosaccharides give crystals on heating and all range of the positive control (1% glucose) which
disaccharides give crystal on cooling (IMDCBiochem, verifies that glucose is present in glycogen. The time
2010). In addition, the crystal formation time of appearance under NH is theoretically similar to the
determines the reducing sugar in the sample. For 1% glucose positive control since it is expected that
instance, glucosazone is formed by glucose within 4- the reducing sugar in the hydrolyzate is ideally
5 minutes of heating. glucose.
The reagent used in this test is It is to be noted that the samples were not
phenylhydrazine reagent that is made up of viewed under the microscope, thus there are no are
phenylhydrazine and sodium acetate diluted in water. images procured in the experiment for Osazone test.
Sodium acetate provides a constant pH in the Instead, theoretical image is shown below displaying
solution. The mechanism of this test (see Figure 7) the crystal (glucosazone) formed by glucose in
includes the reaction of carbonyl group of the Osazone test.
reducing carbohydrate with phenylhydrazine under
boiling temperature forming phenylhydrazone. Then,
this resulting product reacts with another two
molecules of phenylhydrazine producing the
insoluble osazone crystals. Formation of these
osazone crystals suggests a positive result for
Osazone test (Nigam & Ayyagari, 2007). False
negative results will be produced if the added
phenylhydrazine reagent is insufficient or the heat is
not at boiling temperature causing for an incomplete
reaction to occur. Figure 8. Needle- shaped crystals of glucosazone viewed under the
microscope. (Chhabra, 2014)