Biochemistry Laboratory

Midterm Review Notes

Experiment no. 1 CARBOHYDRATES

Qualitative tests for Carbohydrates

5% of the following carbohydrates solutions were used:
o Monosaccharides Galactose, Glucose, Fructose
o Disaccharides Lactose, Maltose, Sucrose
o Polysaccharides Starch, Dextrin, Cellulose

1) Molischs Test
a. Principle
It is a general test for carbohydrates. The sugars are mixed with -naphtol
(Molisch Reagent). The tube is inclined and concentrated H 2SO4 (sulfuric acid) is
added along the side of the tube, causing the formation of a lower acid layer. The
concentrated acid will dehydrated the sugar allowing it to reach with the alcohol
forming furfural or hydroxymethylfurfural.

b. Positive result
Formation of a purple ring at the interface of the two liquids.

c. Sugars that will give a positive (+) result

All carbohydrates

2) Benedicts Test
a. Principle
Benedicts test is a very sensitive test done under mildly alkaline conditions. It
tests the presence of reducing sugars in a solution. The reagent contains CuSO 4
(cupric sulfate or copper II sulfate), Na2CO3 (sodium carbonate) and sodium
citrate. Benedict's test will detect the presence of aldehydes, and -
hydroxyketones, including those that occur in certain ketoses. When reducing
sugars are heated in the presence of an alkali, they get converted to powerful
reducing compounds known as enediols. Enediols reduce the cupric ions (Cu2+)
present in the Benedict's reagent to cuprous ions (Cu +) which get precipitated as
insoluble red copper (I) oxide or cuprous oxide (Cu2O).

b. Positive result
Formation of a brick-red precipitate. Note that the color may vary from light
green-brick red depending on the concentration of the reducing sugar in the
solution.

c. Sugars that will give a positive (+) result

Monosaccharides - Galactose, Glucose, Fructose
Disaccharides - Maltose, Lactose

3) Fehlings Test
a. Principle
It is a test for reducing sugars. Fehling's test can be used as a generic test for
monosaccharides and other reducing sugars, such as maltose. It will give a
positive result for aldose monosaccharides (due to the oxidisable aldehyde group)
but also for ketose monosaccharides, as they are converted to aldoses by the
base in the reagent. Fehlings test takes advantage of the ready reactivity of
aldehydes by using the weak oxidizing agent cupric (Cu 2+) in an alkaline solution.
In addition to the copper ion, the reagent contains tartrate ions as a complexing

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 Biochemistry Laboratory
Midterm Review Notes

agent to keep the copper ion in the solution. Without the tartrate ions, cupric
hydroxide would precipitate from the basic solution. The tartrate ion is unable to
complex cuprous ion, so the reduction of Cu 2+ to Cu+ by reducing sugars results in
the formation of an orange to red precipitate of Cu2O.

b. Positive results
Formation of an orange to red precipitate. Degree of color will also determine an
estimate on the concentration of the reducing sugar. The darker the color gets,
the higher the concentration of the reducing sugar present in the solution.

c. Sugars that will give a positive (+) result

Monosaccharides Galactose, Glucose, Fructose
Disaccharides Lactose, Maltose

4) Barfoeds test
a. Principle
Barfoeds reagent contains cupric acetate in dilute acetic acid and is used to
distinguish between monosaccharides, disaccharides, and oligosaccharides. The
reagent oxidizes monosaccharides, but not oligosaccharides. Disaccharides are
less easily oxidized but are oxidized if they undergo prolonged heating, causing
hydrolysis of the disaccharides into monosaccharides. The test generally follows
the same principle of Benedicts test but instead of an alkaline solution, the test is
carried out in an acidic solution.

b. Positive results
Formation of a brick-red precipitate. Note that the color may vary according to the
concentration of the sugar.

c. Sugars that will give a positive (+) result

Monosaccharides. Disaccharides did not give a positive results since heating time
is not prolonged.

5) Seliwanoffs Test
a. Principle
This is used to differentiate ketohexoses from aldohexoses. Ketohexoses reacts
faster with the solution containing hydrochloric acid and resorcinol than
aldohexoses. Acid hydrolysis of poly and oligosaccharides yields Prolonged
heating should be avoided.

b. Positive result
Formation of a bright cherry red solution
(A light pink solution is produced by aldoses, this signifies a negative result)

c. Sugars that will give a positive (+) result

Fructose, Sucrose

6) Iodine Test
a. Principle
Iodine on its own (small non-polar molecule) is insoluble in water. Therefore
Potassium triiodide solution - Iodine dissolved in potassium iodide solution - is
used as a reagent in the test. To be more specific, potassium iodide dissociates,
and then the Iodide ion reacts reversibly with the Iodine to yield the the triiodide

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 Biochemistry Laboratory
Midterm Review Notes

ion. A further reaction between a triiodide ion and an iodine molecule yields the
pentaiodide ion. The triiodide and pentaiodide ions formed are linear and slip
inside the helix of the amylose (form of starch). The starch-iodide complex is
formed as charge is transferred between the starch and iodide ion. The transfer of
charge between the starch and the iodide ion changes the spacing between the
energy levels/ orbitals. This change results in the starch-iodide complex absorbing
light at a different resulting in an intense purple color.

b. Positive result
Deep purple color or bluish black color. Upon cooling, the color intensifies and
addition of Na2S2O3 (sodium thiosulfate) will cause the colored solution to change
to a colorless solution.

c. Sugars that will give a positive (+) result

Starch, dextrins and cellulose

7) Osazone Formation
a. Principle
When sugar is added to phenhylhydrazine and NaAc (sodium acetate) and then
heated, a yellow precipitate is formed. The product formed may then be compared
with standards using different melting points to identify the composition of the
precipitate. Another portion of the precipitate may be examined under the
microscope to reveal the formation of a distinctive crystalline structure.
Gluccosazones are fine yellow needles aggregated like bundles of hay. Note that
glucose, maltose, fructose and mannose form the same osazone crystals.
Glucose and fructose differ only in their structure at the 1st two carbon atoms, C 1
and C2. In osazone formation the two carbon atoms participates in the reaction
and during osazone formation the structural dissimilarities at C 1 and C2
disappears.

b. Positive result
Formation of a yellow precipitate and upon microscopic examination, presence of
fine needle like structures.

c. Sugars that will give a positive (+) result

All reducing sugars

8) Hydrolysis of Sucrose
a. Principle
Sucrose, in its unhydrolyzed state, does not normally react with Benedicts and
Seliwanoffs reagent since it is not a reducing sugar. But when sucrose is
subjected to an acidic solution it will yield glucose and fructose as end products. It
is then testes for reducing sugars (Benedicts test) and test for ketohexoses
(Seliwanoffs test).

b. Positive results
i. Benedicts test for hydrolyzed sucrose orange to brick red precipitate
ii. Seliwanoffs test for hydrolyzed deep pink to bright cherry red solution

9) Isolation of Polysaccharide from Chicken Liver

a. Principle

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 Biochemistry Laboratory
Midterm Review Notes

In most animals, glycogena polysaccharide, is stored in the liver as a repeating

chain on glucose unit linked by -1,4 glycosidic bonds in its main chain and -1,6
glycosidic bonds in its branches. The isolation of glycogen from the chicken liver is
attained by using the mechanism of precipitation. It is then tested using the
Benedicts test, Seliwanoffs test and iodine.

b. Results
Benedicts test positive (+)
Seliwanoffs test negative (-)
Iodine test negative (-)

Experiment no. 2 PROTEINS AND AMINO ACIDS

Color Reactions
1) Biuret Reaction
a. Principle
This test will give a positive result for compounds containing 2 or more peptide
bonds. The amino group present in the peptide bond will react with the cupric
ions in the reagent to form a Biuret complex.

b. Positive result
Formation of a distinctive purple solution.

c. Amino acids that will give a positive (+) result

Albumin and casein

2) Xanthoproteic test
a. Principle
Nitration of amino acids that contain benzene ring will yield the product
nitrobenzene.

b. Positive result
Formation of a yellow to orange coloration.

c. Amino acids that will give a positive (+) result

Tyrosine, tryptophan and phenylalanine

3) Millons Test
a. Principle
The presence of phenol group in amino acid tyrosine is nitrated by a solution of
mercuric and mercurous nitrates in concentrated nitric acid.

b. Positive results
A white precipitate will form turning brick red on prolonged heating due to the
formation of a mercury complex of nitrophenyl derivatives. Addition of NaNO 2
turns the precipitate darker pink or red.

c. Amino acids that will give a positive (+) result

Tyrosine

4) Hopkins Cole
a. Principle

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 Biochemistry Laboratory
Midterm Review Notes

The aldehyde present in the reagent will react with the indole ring of tryptophan
forming a colored complex.

b. Positive results
Formation of a blue and violet colored ring.

c. Amino acids that will give a positive (+) result

Trytophan

5) Paulys Test
a. Principle
When proteins containing either tyrosine or histidine are reacted with diazotized
sulfanilic acid under alkaline conditions, a red color is formed by a coupling
reaction.

b. Positive result
Formation of a yellow to red solution.

c. Amino acids that will give a positive (+) result

Imidazole group of Tyrosine and Histidine

6) Lead Acetate Reaction

a. Principle
This test is specific for sulfur-containing amino acids like cysteine and methionine.
The sulfhydryl or disulfide groups are converted to inorganic sulfide, Na 2S, in
strongly alkaline solution. This will then react with lead acetate to form a colored
precipitate.

b. Positive result
Formation of a brownish-black precipitate of lead sulfide.

c. Amino acids that will give a positive (+) result

Cysteine, cystine and methionine

7) Hellers Ring test

a. Principle
Heller's test is a chemical test that shows that strong acids cause the
denaturation of precipitated proteins. Concentrated nitric acid is added to a
protein solution from the side of the test tube to form two layers. It is a common
procedure to test for the presence of proteins in urine.

b. Positive result
Formation of a white ring at the junction of the two liquids.

c. Amino acids that will give a positive (+) result

Albumin

Amphoteric Character of Proteins

Due to the presence of free carboxylic and free amino groups at the end of protein it can
react with acids and bases.

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 Biochemistry Laboratory
Midterm Review Notes

In acidic medium protein carries positive charges at amino group and in alkaline medium
it carries negative charges at carboxylic group.

Denaturation of Proteins by Heat and Extreme pH

Temperatures and pH below and beyond the proteins threshold will cause the protein to
either loss their function or be totally denatured.

Precipitation of Alcohol
Albumin is generally insoluble in ethanol, thus forming a white precipitate.

Precipitation of Proteins by Cations (Heavy Metal Salts)

Heavy metal salts, such as CuSO4 act to denature proteins in much the same manner as
acids and bases. Heavy metal salts usually contain metals with high atomic weights.
Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal
salt with a protein usually leads to an insoluble metal protein salt. Heavy metals may
also disrupt disulfide bonds because of their high affinity and attraction for sulfur and will
also lead to the denaturation of proteins.

The metal ion will dissociate and bind to a part of the protein. An advantage of this type
of precipitation is that they have great precipitating power in a dilute solution.

Precipitation of Protein by Anions (Alkaloidal reagents)

Alkaloidal reagents (e.g. tannate & trichloroacetate) are high molecular weight anions.
The negative charge of these anions counteracts the positive charge of the amino group
in proteins giving a precipitate.

Experiment no. 3 ENZYMES

1) Enzyme Action
o Catalase is an enzyme responsible for the decomposition of hydrogen peroxide (H 2O2)
to produce water and oxygen as shown in the balanced equation below:
2 H2O2 2 H2O + O2

o When the glowing splinter was places into the mouth of the test tube the glow
became brighter. This signifies that there is continuous liberation of oxygen from the
decomposition of H2O2.

o Catalase is present in tears, saliva, blood, kidney, muscles, and brain in the human
body. This wide-spread distribution is due to the importance of catalase in the
decomposition of H2O2, which is considered toxic to most living tissues.

2) Protein Nature of Enzymes

o Catalase was tested for Biuret reaction.
o It tested positive (formation of purple solution) since the catalase is a complex
protein therefore having 2 or more peptide bonds.

3) Enzymes as Catalyst

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 Biochemistry Laboratory
Midterm Review Notes

Saliva contains an enzyme called amylase which breaks down starch in multiple units of
glucose. Thus, a combination of saliva and starch will give positive results to tests for
reducing sugars and iodine test.

Since amylase is a glycoside hydrolase acting specifically on -1,4 glycosidic bonds it

cannot hydrolyze sucrose into glucose and fructose since sucrose has an -1,1 glycosidic
bond. Therefore, a combination of sucrose and starch will not give positive results to
tests for reducing sugars and iodine test.

-END-

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