Biochemistry Laboratory

P2. Laboratory Exam. Part 1

Instructions: Research and complete the table below with information that are essential for further
understanding of Carbohydrates and Proteins. This will be the part one of your examination in laboratory,
the second half or practical exam will be an oral recitation regarding this topic so make sure that you are
familiar with these tests. The oral examination will be on April 25, 2021 at 1 PM.
Note: This is a group examination, indicate which group member is assigned for each tests. Deadline for this
research activity will be on April 25, 2021 at 5 PM.

TEST FOR CARBOHYDRATES

I. General test for carbohydrates
- ABRERA, VANESSA MAE
Test Principle Reagent End-product Interfence
Molish test Concentrated sulphuric Molish 5- hydroxymethyl Positive result:
acid hydrolyses glycosidic reagent furfural (bottom purple
Use/ Purpose: bonds to give the - reaction) and ring on the
-specific for monosaccharaides, alphanaphthol furfural junction
carbohydrate which are then in 95% (top reaction)
s dehydrated to furfural ethanol Negative result:
and its derivatives. No purple ring on
H2so4 the junction

Anthrone test Carbohydrate gets -Test solution: The formation of a Positive Test:
Use/ Purpose: dehydrated upon 5 % Glucose, 5 greenish color in a Presence of
-Used to detect reaction with % Sucrose, 5 solution indicates carbohydrates
carbohydrate in a concentrated sulfuric % Starch the presence of gives GREEN color.
given solution acid to form furfural. carbohydrates.
Furfural then reacts with -Anthrone
anthrone to form bluish reagent: 0.2 %
green color. anthrone in
conc. H2SO4

II. Test For Carbohydrates Based On Their Ability To Form Furfural And Its Derivatives
- CANTA, ALLEJAH JANE F.
Test Principle Reagent End-product Interference
Seliwanoff’s test  The reagent of this  S The test reagent POSITIVE RESULT:
 To test consists of eliwanoff’s dehydrates Ketoses present;
detect the resorcinol and reagent: add ketohexoses to form 5- cherry red-colored;
presence of concentrated HCl. 0.05% resorcinol hydroxymethylfurfural complex formed
ketohexose  The acid hydrolysis (m- . 5-
s in a given of polysaccharides and hydroxybenzene) hydroxymethylfurfural NEGATIVE RESULT:
sample. oligosaccharides yields in 3 N HCl. further reacts with Ketoses absent
 To simpler sugars. Dissolve 50 mg resorcinol present in
distinguish  Ketoses are more resorcinol in 33 the test reagent to
ketoses rapidly dehydrated than ml concentrated produce a red product
from aldoses. HCl and make it within two minutes
aldoses.  Ketoses undergo 100 ml with (reaction not shown).
dehydration in the water. Aldohexoses react to
presence of  T form the same
concentrated acid to est sample product, but do so
yield 5-hydroxymethyl  D more slowly.
furfural. istilled water
 The dehydrated
ketose reacts with two
equivalents of resorcinol
in a series of
condensation reactions
to produce a complex
(not a precipitate),
termed xanthenoid,
with deep cherry red
 color.
 Aldoses may react
slightly to produce a
faint pink to cherry red
color if the test is
prolonged.
 The product and
reaction time of the
oxidation reaction helps
to distinguish
between carbohydrates
.
 Other carbohydrates
like sucrose and inulin
also give a positive
result for this test as
these are hydrolyzed by
acid to give fructose.

Bial’s orcinol This test is based on the  test reagent: The reaction works in POSITIVE:
test principle that under 5 % Glucose, 5 a way that pentose is Formation of blue
hydrolysis pentosans are % Ribose, 5 % dehydrated by the color. (e.g ribose
Bial’s test is hydrolyzed into pentoses. Fructose reagent and a furfural sugar)
useful in Further, pentoses are  Bial’s reagent form is formed. Ocinol
distinguishing dehydrated to yield furfural, then reacts with this NEGATIVE:  
 Water bath
pentoses sugar which in turn condense with furfural which then Formation of any
from hexoses orcinol to form a blue-green  Dry test reacts with iron to give other color
sugars. precipitate. In the presence tubes a bluish colored indicates negative
Pentosses ( such of hexoses, hydroxyfurfural  Pipettes product and the test. Hexose sugar
as ribose sugar) is formed instead of furfural presence of pentose is ( glucose, fructose)
form furfural in which upon condensation detected. A positive generally gives
acidic medium with orcinol forms a muddy result in indicated green, red or
which condense brown colored precipitate. when a bluish color brown color
with orcinol in The intensity of the appears in the product.
presence of precipitation is directly solution. Remember
ferric ion to give proportional to the that only bluish color
blue green concentration of the indicates a positive
colored complex pentoses in the sample. The test. If some other
which is soluble intensity of the color color appears then the
in butyl alcohol. developed depends on the result is negative. And
concentration of HCl, ferric this was an easy
chloride, orcinol, and the experiment to perform
duration of boiling. The and check the
concentration of the sugars presence of pentose.
is determined by measuring
the absorbance of 620 nm
wavelength in a
spectrophotometer or in a
red filter colorimeter.
Mucic acid test  Monosaccharides  Mucic acid Mucic acid test is POSITIVE:
 To upon treating with reagent: a test that is highly Crystal present
detect the potent oxidizing agents concentrated specific and is used for
presence of like nitric acid yield nitric acid the detection of the NEGATIVE:
galactose saccharic acids  Test sample presence of galactose No crystal present
and lactose (dicarboxylic acids). (1%) and lactose. It is also
in a given  Nitric acid has the  Distilled termed galactaric
sample. capacity to oxidize both water acid that is named
 To aldehyde and primary after the product of
distinguish alcoholic groups present the reaction.
between at C1 and C6
the respectively of galactose
galactose to yield an insoluble
containing precipitate (rod-shaped
saccharides crystals) of mucic acid
and other under higher
sugars. temperature.
 Lactose also yields a
mucic acid, due to the
hydrolysis of the
 glycosidic bond between
the glucose and
galactose subunits of
the carbohydrate.
 Other
monosaccharides like
glucose also have a
similar structure;
however, the resultant
precipitate formed in
glucose is water-soluble
under room
temperature.

Biochemistry Laboratory
III. Test For Carbohydrates Based On The Reducing Property Of Sugars
-CASIS, JAN MICHAEL F.

a. Reducing sugars - is any sugar that is capable of acting as a reducing agent because it has a free
aldehyde group or a free ketone group. All monosaccharides are reducing sugars, along with some
disaccharides, some oligosaccharides, and some polysaccharides.
b. Non- Reducing Sugars- Sugars that can be oxidised by mild oxidising agents are called reducing
sugars. A non-reducing sugar is a sugar that is NOT oxidised by mild oxidising agents. All common
monosaccharides are reducing sugars. The disaccharides maltose and lactose are reducing sugars.

Test Principle Reagents End-product Interferences

Benedict’s test When Benedict's solution One litre of Benedict's Benedict's False positive
and simple carbohydrates reagent can be reagent starts reactions may also
are heated, the solution prepared by mixing out aqua-blue. be obtained if
changes to orange red/ 17.3 grams of copper As it is heated certain drugs are
brick red. ... The copper sulfate pentahydrate in the presence present, e.g.
(II) ions in the Benedict's (CuSO4. 5H2O), 100 of reducing salicylates,
solution are reduced to grams of sodium sugars, it turns penicillin,
Copper (I) ions, which carbonate (Na2CO3), yellow to streptomycin,
causes the color change. and 173 grams of orange. In isoniazid, and p-
sodium citrate in general, blue to aminosalicyclic
distilled water blue-green or acid.
(required quantity). yellow-green is
negative, Chemicals present
yellowish to in a concentrated
bright yellow is urine which may
a moderate reduce Benedict’s
positive, and reaction include
bright orange is creatinine, urate,
a very strong and ascorbic acid
positive. (reduction is
slight).
Barfoed’s test Monosaccharides usually Barfoed's reagent Positive reducing
react in about 1-2 minute consists of a 0.33 Barfoed’s test: disaccharides also
while the reducing molar solution of development of give positive
disaccharides take much copper (II) acetate in brick red color barfoed test on
longer time between 7-12 1% acetic acid solution. ppt within 3-5 prolong heating
minutes to react with the The reagent does not minutes
reagent. Brick red color is keep well and it is Negative
obtained in this test therefore advisable to Barfoed’s test:
which is due to formation make it up when it is absence of red
of cuprous oxide actually required. color
Nylander’s The test works on the When Nylander's a chemical test In the experiment
test principle that when a reagent, which consists used for the solution that
subnitrate of bismuth of bismuth nitrate, detecting the yield a positive
 comes in contact with potassium sodium presence of result was glucose,
grape sugar in a boiling tartrate and potassium reducing galactose,
alkaline solution, it hydroxide, is added to sugars. When maltose, fructose,
reduces to black metallic a solution with Nylander's and lactose while
bismuth. For the test, boil reducing sugars, a reagent, which sucrose, glycogen
10 cubic centimeters of black precipitate of consists of and starch was
urine with 1 cubic metallic bismuth is bismuth nitrate, negative indicated
centimeter of the reagent formed. potassium by a clear solution.
for two minutes. sodium tartrate
and potassium
hydroxide, is
added to a
solution with
reducing
sugars, a black
precipitate of
metallic
bismuth is
formed
Tollen’s test The Tollen's reagent is the Tollens' reagent is an a chemical test Like acetaldehyde
alkaline solution of silver alkaline solution of used to and formic acid
nitrate (AgNO3) mixed ammoniacal silver differentiate phenylhydro-
with liquid ammonia nitrate and is used to reducing sugars oxylamine is a
(NH3), which results in test for aldehydes. from non- powerful reducing
the formation of a Silver ions in the reducing agent Thus it
complex. The aqueous presence of hydroxide sugars. This test reduces
solution of silver nitrate ions come out of is also called ammonical silver
forms a silver aqua solution as a brown the silver mirror nitrate (Tollens
complex where the water precipitate of silver(I) test based on reagent ) and
acts as a ligand. oxide, Ag2O(s). This the end Fehling s solution
precipitate dissolves in product of this Nitrobenzene fails
aqueous ammonia, test. to do so .
forming the
diamminesilver(I) ion,
[Ag(NH3)2]+.

TEST FOR AMINO ACIDS

General Tests for Amino acid
- BRIONES, CHERUBIN FAITH T.

Test Principle Reagent Results

Biuret Test Biuret test is a general  Copper sulfate Biuret test positive:
Use: Purpose: A Biuret test for compounds (CuSO40  color changes to
test is a chemical test having a peptide bond.  Sodium purple
used to determine the Biuret is a compound hydroxide (NaOH)  all peptides and
presence of a peptide formed by heating urea protein give the
bond in a substance. to 180° C. When biuret is  Sodium
potassium test positive
treated with dilute
copper sulfate in alkaline tartarate  Histidine is the
(commonly known only amino acid
condition, a purple
colored compound is as Rochelle salt) that give biuret
test positive.
formed. This is the basis
of biuret test widely
Biuret test negative:
used for identification of
proteins and amino
 No color change
acids.

The principle of biuret

test is conveniently used
to detect the presence of
proteins in biological
 fluids.

Xanthoproteic Test Xanthoproteic test is  Concentrated Positive result:

Use/Purpose: used to detect amino Nitric acid The appearance of a dark
This test is used to acids containing  Original solution yellow or orange-colored
differentiate aromatic an aromatic nucleus  Aqueous solution represents
amino acids which give (tyrosine, tryptophan ammonia a positive test. This
positive result from and phenylalanine) in a indicates the presence of
other amino acids protein solution which aromatic groups in the
gives yellow color nitro proteins
derivatives on heating and amino acids. 
with conc. HNO3. The
aromatic benzene ring Negative result:
undergoes nitration to The absence of a dark
give yellow colored yellow or orange-colored
product. Phenylalanine solution represents
gives negative or weakly a negative test.
positive reaction though
this amino acid contains
aromatic nucleus
because it is difficult to
nitrate under normal
condition. On adding
alkali to these  nitro
derivative salts, the color
change from yellow to
orange
Ninhydrin Test The amino group  Dissolve 0.35g of Positive result:
Use/Purpose: belonging to a free ninhydrin in 100 ml The presence of a
The ninhydrin test is a amino acid undergoes a ethanol (iso- purple-colored complex
chemical test which is chemical reaction with propanol or 1:1 in the tube represents a
used to check whether a ninhydrin, which mixture of positive result and
given analyte contains behaves as an oxidizing acetone/butanol indicates the presence of
amines or α-amino agent. When exposed to may be used instead amino acid in the
acids.  ninhydrin, the amino of ethanol). sample.
acid undergoes oxidative  Diluent solvent
deamination, resulting in (for the quantitative Negative result:
the liberation of CO2, test): Mix equal The absence of the
NH3, and an aldehyde volumes of water complex in the tube
along with hydrindantin and n-propanol. represents a negative
(which is a reduced form  Standard solution result and indicates the
of ninhydrin). (1% protein solution) lack of amino acids in the
Now, the ammonia goes  Sample solution sample.
on to react with another
ninhydrin molecule to
form diketohydrin (which
is also known as
Ruhemann’s complex).
This complex is
responsible for the deep
blue colour. When the
analyte contains Imino-
acids like proline, a
yellow coloured complex
is formed. When
asparagine is used, the
colour of the resulting
complex is brown.

Test for Amino Acids

 - CANILANG, KENNETH

Test Principle Reagent Results

Millon’s test Millon’s test is based on Millon’s reagent: Positive result: A

the principle of Millon’s reagent consists positive result in the
nitrification of the of mercuric nitrate and Millon’s test is
phenol group in tyrosine, mercurous nitrate demonstrated by the
which then forms dissolved in nitric acid formation of a red or
complexes with heavy and distilled water. pink colored
metals like mercury. The Preparation of Millon’s precipitate. This
reagent used for the test reagent: Dissolve 160 indicates the presence
is called Millon’s reagent, grams of mercuric of tyrosine or tyrosine
and it consists of nitrate and 160 grams of containing protein.
mercuric nitrate and mercurous nitrate in 400
mercurous nitrate that is ml concentrate nitric Negative result: A
dissolved in acid solution. The negative result in the
concentrated nitric acid. reagent is then made to Millon’s test is
In the test, the phenol 1000 ml by the addition demonstrated by the
group on the tyrosine of 600ml distilled water. absence of colored
molecule is nitrated by The formula can be precipitate in the test
the nitric acid present in adjusted to suit the tube. This indicates the
the reagent. The nitrated performance absence of tyrosine or
tyrosine then combines parameters. tyrosine-containing
with the mercury ions in protein.
the solution to form a
red-colored precipitate
or solution. In some
proteins containing
tyrosine, the initial
reaction between
mercuric nitrate results
in a white or yellow
colored precipitate. After
the addition of nitric acid
and heating, however,
the residue turns red in
color. Both of these
results are considered
positive results and
indicate the presence of
tyrosine in the solution.
Lead Acetate Test or The test is based on the ● 2% lead acetate Positive test:
Sulfur Test principle of detection of solution in water A positive test in the
sulfur in a solution by the ● 40% NaOH Lead sulfide test is
degradation of the S-H or ● Sample represented by the
S-S group in amino acids formation of black
under strongly alkaline precipitate at the
conditions. Amino acids bottom of the test
like cysteine and cystine tube. This indicates the
release sulfur in the presence of cysteine or
presence of strong cystine in the solution.
alkaline conditions at a
high temperature. The Negative test:
sulfur then combines A negative result in the
with the alkali (NaOH) to Lead sulfide test is
form Na2 The Na2S thus represented by the
formed reacts with lead absence of black
acetate to form lead residue in the test
sulfide, which results in a tube. This indicates the
black residue. For the absence of cysteine or
reaction to take place, cystine.
free sulfur ions should be
 present in the medium.
Sakaguchi Test Sakaguchi test is based ● Sakaguchi reagent: 1% Positive result:
on the principle of 1-naphthol in alcohol A positive result on the
reaction between 1- with a few drops of 10% Sakaguchi’s test is
naphthol and the sodium hypobromite demonstrated by the
guanidinium groups in solution of bromine formation of red color.
arginine, in the presence water. This indicates the
of an oxidizing agent. ● 40% NaOH presence of an arginine
The exact mechanism of ● Sample (0.1% of or guanidinium
the reaction is not yet arginine or 0.1% of compound.
known; however, the creatine)
reaction results in the Negative result:
formation of a red- A negative result in the
colored complex due to Sakaguchi’s test is
the formation of an demonstrated by the
indole-like structure. The absence of red color.
Sakaguchi reagent This indicates an
consists of sodium absence of arginine or
hypobromite and 1- a guanidinium
naphthol. The sodium compound.
hypobromite acts as an
oxidizing agent that
facilitates the hydrogen
bonding between two
arginine molecules.

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