BIOCHEMISTRY LABORATORY

CARBOHYDRATES (4)
JAILOUISE A. PEREZ
BACHELOR OF SCIENCE IN NURSING

CARBOHYDRATES a. The suffix “ose” refers to a molecule as a

Introduction and Etymology
 Carbohydrates are also known saccharides.
 Etymology of Saccharides: comes from the greek word
sákkharon. (meaning, “sweet”)
 The word carbohydrates come from the two words
carbo and hydrates, which means hydrated carbon
molecules.
 General Formula: CH2O
 H2O is the hydrated part
 Carbohydrates taste sweet in its monosaccharide form,
but don't taste sweet in their complex form.
 Main Function: Provides energy to the consumer's
body throughout the day and acts as the consumer's
primary source of energy.
3 Classification of Carbohydrates
1. Sugar Units (based on sugar units)
a. Monosaccharides- one sugar unit. (simple
carbohydrates)
i. simple sugar, most common of which
is glucose
ii. typically contain three to seven
carbon atoms
iii. formula: CH2O
b. Disaccharides- two sugar units. (simple
carbohydrates)
i. A dehydration reaction, also known
as a condensation reaction or
dehydration synthesis, occurs when
two monosaccharides join together.
ii. Common disaccharide:
1. Lactose – disaccharide
consisting of glucose and
galactose and is found
naturally in milk.
2. Maltose - or malt sugar, is a
disaccharide made up of
two glucose molecules.
3. Sucrose – The most
common disaccharide is
sucrose (table sugar),
which is made of glucose
and fructose.
c. Polysaccharides- many sugar units. (complex
carbohydrates)
i. A polysaccharide is a long chain of
monosaccharides linked together by
glycosidic bonds.
ii. Polysaccharides are important in
living organisms and include starch,
glycogen, cellulose, and chitin
2. Number of Atoms (based on the number of atoms
present in a molecule of a monosaccharide)
a. Triose - 3
b. Tetrose - 4
c. Pentose - 5
d. Hexose - 6 (e.g. glucose)
e. Heptose – 7
3. Functional Groups (base don the functional groups)
monosaccharide.
b. Aldose
i. contains an aldehyde group.
ii. aldoses are also called polyhydroxy
aldehydes.
c. Ketose
i. contain ketones.
ii. ketoses are also called polyhydroxy
ketones.
Starch
 It is a mixture of two polysaccharides, amylose and
amylopectin
 used to store sugars in plants (both polymers of
glucose).
 Plants can synthesize glucose using light energy
gathered during photosynthesis.
 Excess glucose is stored as starch in various plant
parts, including roots and seeds, beyond the plant's
immediate energy needs.
 The starch in the grains provides food for the embryo
as it germinates, and humans and animals will break it
down into glucose monomers using digestive enzymes.
Isolation of Starch
1. Aim
a. To isolate starch from potatoes
2. Procedure
a. Scrap the clean surface of potatoes with a
knife and cut into pieces and place them in a
mixture and stir for one to two minutes.
b. The grinding process is repeated three to four
times until we get a homogenous suspension.
c. Then strain through fine cheesecloth int
another beaker.
d. Allow standing for a few minutes and then
noting the weight deposition of starch at the
bottom.
e. Pour the supernatant fluid and fill the beaker
with water containing the starch in it.
f. Stir well and again allow the starch to settle
down.
g. Repeat four to five times by separating starch
that can be washed thoroughly.
h. Collect the white deposited starch on to watch
glass.
i. Dry it in an oven and then weigh the sample
gravimetrically.
Qualitative Tests for Carbohydrates
Bradfoed’s Test
 Barfoed’s test is a chemical test used to detect the
presence of monosaccharides, which detects reducing
monosaccharides in the presence of disaccharides.
 Principle
o This reaction can be used for disaccharides,
but the reaction would be very slow. It is
based on the reduction of cupric (II) acetate to
cuprous (I) oxide (Cu2O), which forms a brick-
red precipitate. Disaccharides may react, but
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 the reaction is much slower because they
have to get hydrolyzed first and then react
with the reagent cupric acetate to produce
cuprous oxide.
o Monosaccharides usually react in about 1-2
minute while the reducing disaccharides take
much longer time between 7-12 minutes to
react with the reagent.
 (CH3COO)2Cu + 2H2O → 2CH3COOH + Cu(OH)2
 Cu(OH)2 → CuO + H2O
 D-glucose + 2CuO →
 D-gluconic acid + Cu2O
 Materials and Procedure
o Materials:
 Barfoed’s reagent - the reagent used
in this test. It is prepared by adding
0.33 molar solution of neutral cupric
(II) acetate to 1% acetic acid
solution.
 test solution: 5 % Glucose, 5 %
Sucrose, 5 % Maltose, 5 % Lactose,
5 % Starch
 Test tubes
 Test tube stand
 Pipette
 Water bath
 Procedure:
o Take 1 ml of a sample solution placed in a
clean, dry test tube. The concentration of
disaccharides sample (if used) should not
exceed 1% (w/v).
o Take 1 ml of distilled water in another tube as
control.
o Add 2-3 drops of Barfoed’s reagent to all test
tubes.
o The solution is then heated in a boiling water
bath for two minutes and allowed to cool. The
boiling should not be done for more than 2
minutes as the disaccharides might hydrolyze
into monosaccharides and give a positive
result.
o Record color and record the time required to
develop a red precipitate.
 Result Interpretation of Barfoed’s Test
o The presence of red precipitate detects the
presence of reducing monosaccharides in the
sample.
o If the color appears within the first few
minutes, the sample contains reducing
monosaccharides.
o if the color appears later than the first 3
minutes, the sample is of reducing
disaccharides.
 Limitation of Barfoed’s Test
o This test cannot be used to detect sugar in
urine as urine contains Clions, which might
interfere with the reaction.
o If a higher concentration of disaccharides is
present in a sample, it might give a positive
result.
 Difference between Barfoed’s test and Benedict’s test
o The Barfoed’s reagent is similar to Benedict’s
reagent except that the Ph is lower (around
4.5), and heating time is reduced to two
minutes.
o Benedict’s test would determine if the sample
is a reducing sugar, and Barfoed’s test would
determine if it is a monosaccharide or
disaccharide.
Seliwanoff’s Test
 Seliwanoff’s test is used to differentiate between
sugars that have a ketone group (ketose) and sugars
that have an aldehyde group (aldoses). This is a timed
color reaction specific to ketohexoses.
 Principle
o The reagent of this test consists of resorcinol
and concentrated HCl.
o The acid hydrolysis of polysaccharides and
oligosaccharides yields simpler sugars.
o Ketoses are more rapidly dehydrated than
aldoses.
o Ketoses undergo dehydration in the presence
of concentrated acid to yield 5-hydroxymethyl
furfural.
o The hydrated ketoses react 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.
o Aldoses may react slightly to produce a faint
pink to cherry red color if the test is
prolonged.
o The product and reaction time of oxidation
reaction helps to distinguish between
carbohydrates.
o Other carbohydrates like sucrose and inulin
also give a positive result for this test as these
are hydrolyzed by acid to give fructose.
 Materials and Procedure
o Materials:
 Test solution: 5% glucose, 5%
sucrose, 5% frusctose
 Seliwanoff’s reagent - add 0.05%
resorcinol (m-hydroxybenzene) in 3
NHCl. Dissolve 50mg resorcinol in
33ml concentrated HCl and make it
100ml with water.
 Dry test tubes
 Test tube stand
 Water bath
 Pipettes
o Procedure:
 Take 2 clean test tubes and add 1 ml
of test sample in one test tube and
take 1 ml of distilled water in another
tube as control.
 Add 2 ml of Seliwanoff’s reagents in
both test tubes.
 Keep the test tubes in a water bath
for 1 minute.
 Look for the development of the red
color and note it down.
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 Result Interpretation of Seliwanoff’s Test o Different precipitates were formed depending
o The formation of the cherry red-colored upon the sugar concentration in urine
complex indicates a positive result which  Result Interpretation of Benedict’s Test
means that the given sample contains
ketoses.
o The absence of such color or the appearance
of the color after a prolonged period of time
indicates a negative result which means that
the test sample does not have ketosis.
 Uses of Seliwanoff’s test
o Seliwanoff’s color reaction is used in the
method of the colorimetric determination of
fructose in fermentation media.
o A modified version of this test can be used for
the determination of the concentration of
ketoses in a given sample.
 Limitation of Seliwanoff’s test o If the color upon boiling is changed into green,
o The high concentration of glucose or other then there would be 0.1 to 0.5 percent sugar
sugar may interfere by producing similar in solution.
colored compounds with Seliwanoff’s reagent. o If it changes color to yellow, then 0.5 to 1
o Prolonged boiling can transform glucose to percent sugar is present.
fructose by the catalytic action of acid and o If it changes to orange, then it means that 1 to
form cherry red-complex giving a false- 1.5 percent sugar is present.
positive result. o If color changes to red,then 1.5 to 2.0 percent
o This test is a generalized test and does not
sugar is present.
distinguish between specific ketoses, and a o If color changes to brick red,it means that
separate test is required for the particular
more than 2 percent sugar is present in
ketose sugar identification.
solution.
 Positive Benedict’s Test: Formation of a reddish
Benedict’s Test
precipitate within three minutes. Reducing sugars
 Benedict's Test is used to determine whether or not a
present. Example: Glucose
substance contains simple carbohydrates. Benedict's
 Negative Benedict’s Test: No color change (Remains
test detects reducing sugars with free ketone or
Blue). Reducing sugars absent. Example: Sucrose.
aldehyde functional groups (monosaccharides and
some disaccharides). The presence of glucose in urine
Fehling’s Test
can be detected using Benedict's solution.
 Fehling’s Solution (deep blue colored) is used to
 Principle
determine the presence of reducing sugars and
o A reducing sugar is converted to an enediol
aldehydes. Perform this test with fructose, glucose,
when it is heated in the presence of an alkali
maltose and sucrose.
(which is a relatively powerful reducing
 Objective: to detect reducing sugar in a given solution.
agent). When reducing sugars are present in
the analyte, Benedict's reagent's cupric ions  Principle
(Cu2+) are reduced to cuprous ions (Cu+). o Fehling’s test is one of the sensitive tests for
These cuprous ions react with the reaction detection of reducing sugars. Fehling’s
mixture to form copper(I) oxide, which reagents consist of two solution Fehling’s
precipitates as a brick-red compound. solution A and solution B. Fehling’s solution A
 Materials and Procedure is an aqueous copper sulphate and Fehling’s
o Materials: solution B is alkaline sodium potassium
tartrate (Rochelle salt). Rochelle salts (sodium
 Test tube
potassium tartrate) present in the reagent acts
 Test tube holder
as the chelating agent in this reaction.These
 Pipette
two solutions are mixed in equal amounts
 Urine sample
before test.
 Burner
o On heating an aldehyde or reducing sugar
 Procedure:
with Fehling’s solution give reddish brown
o Using a pipette, accurately take 5ml of
precipitate. Formation of red precipitate of
Benedict’s reagent and slowly transfer it to
cuprous oxide denotes the presence of
test tube
reducing sugar.
o Take 5 ml of freshly collected urine by pipette
 Materials and Procedure
and add it to test tube with Benedict’s reagent o Reagents:
o Test tube should be held securely with the
 7g Cu(II) , sulfate pentahydrate,
test tube holder to heat it on the burner for 2 distilled water
minutes  35g potassium sodium tartrate
o On heating the sample, the presence of sugar  10g sodium hydroxide
is indicated in the sample of urine when a  Procedure:
green precipitate (traces of reducing sugar) is o Prepare 2 aqueous solutions called Fehling’s
apparent
solution A and B
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 o Mix both solutions
o Prepare 3 test tubes for glucose, sucrose, and
a mixture of sucrose and citric acid.
o Put distilled water into the test tubes to
dissolve
o Fill the beaker with boiling hot water and put
the test tube that contains sucrose and citric
acid for 2-3 minutes
o When it is cool down add the Fehling's
reagent to the test tubes.
o Prepare another beaker with boiling hot water
and heat the test tubes for 2-3 minutes
o Observe the results.
 Result Interpretation of Fehling’s Test
o Positive Fehling’s Test: brown, reddish-brown
and dark blue.
o Negative Fehling’s Test: no change in colors
or still blue.
Molisch’s Test
 Molisch’s test is a chemical test which is used to check
for the presence of carbohydrates in a given analyte.
This test is named after Czech-Austrian botanist Hans
Molisch, who is credited with its discovery. Molisch’s
test involves the addition of Molisch’s reagent (a
solution of ∝-naphthol in ethanol) to the analyte and
the subsequent addition of a few drops of concentrated
H2SO4 (sulphuric acid) to the mixture.
 The formation of a purple or a purplish-red ring at the
point of contact between the H2SO4 and the analyte +
Molisch’s reagent mixture confirms the presence of
carbohydrates in the analyte. An image detailing a
positive result for Molisch’s test is provided below.
 A positive reaction for Molisch’s test is given by almost
all carbohydrates (exceptions include tetroses &
trioses). It can be noted that even some glycoproteins
and nucleic acids give positive results for this test
(since they tend to undergo hydrolysis when exposed
to strong mineral acids and form monosaccharides).
 Principle
o In Molisch’s test, the carbohydrate (if present)
undergoes dehydration upon the introduction
of concentrated hydrochloric or sulphuric acid,
resulting in the formation of an aldehyde. This
aldehyde undergoes condensation along with
two phenol-type molecules (such as ∝-
naphthol, resorcinol, and thymol), resulting in
the formation of a purple or reddish-purple
coloured complex.
o A illustration detailing the reactions
undergone by D-glucose when it is subjected
to Molisch’s Test is provided above
 Materials and Procedure
o Reagents:
 Molisch reagent: Dissolve 3.75 g of
α-naphthol in 25 ml of Ethanol 99%.
This reagent should be prepared
fresh.
 Concentrated sulphuric acid
 Test sample
o Materials:
 Test tubes
 Test tube stand
 Pipette
 Distilled water
 Procedure:
o Take 2 ml of each distilled water and test
sugar solutions in four test tubes separately.
o Add two drops of Molisch reagent to each
tube.
o Hold the test tube in an inclined position and
gently add 1 ml concentrated H2SO4 along
the wall of the test tube. Do not mix the acid
with the solution. A black ring may form if
concentrated acid is not added slowly as the
heat generated from the reaction can char the
carbohydrates.
o Observe the test tube for the formation of a
purple-colored ring at the layer between the
solution and the acid.
 Result Interpretation of Molisch’s Test
o The formation of the purple colored ring
occurs at the interface between the sulphuric
acid and the test solution.
o The sulphuric acid remains above the test
solution as the acid is denser than the test
solution.
o The absence of color indicates a negative
result.
Iodine’s Test
 The iodine test can help to distinguish starch from
monosaccharides, disaccharides, and other
polysaccharides. Starch is a carbohydrate in the form
of a polysaccharide. It is found in abundance in
potatoes and cereals (oats, barley, rice, wheat).
Amylose (10-20%) and Amylopectin (10-20%) are
monomeric units found in natural starch (80-90
percent). D-glucose subunits are present in both
monomers. However, the D-glucose unit is positioned
differently in Amylose and Amylopectin.
 Principle
o Amylose reacts with starch to generate a
blue-black colored complex with the iodine.
Iodine is found inside the spiral or helical
structure of Amylose, forming a charge
transfer (CT) combination with it. As a result,
for this test, iodine in water (Lugol's iodine) is
used, which is an aqueous solution of
molecular iodine (I) and potassium iodide (KI).
The IKI solution is another name for this:
I + KI = IKI solution
 Iodine purpose in Lugol’s iodine test
o The iodine molecule is water insoluble. In
order to make the laboratory reagent,
potassium iodide is used. Iodide ions arise
when potassium iodide dissociates. In
solution, iodide ions combine to generate the
triiodide ion (I3–), which then forms polyiodide
ions (In–). Triiodide ion chemistry is
responsible for the production of polyiodide
ions in general. Polyiodide ions can be
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 triiodide (I3–), pentaiodide (I5–), or
heptaiodide (I7–) and are negatively charged.
These polyiodide ions operate as charge
donors and bind to Amylose. Lugol's iodine
solution is brown. Electrons absorb light
energy and are stimulated to a higher energy
level in the charge transfer complex of
polyiodide ions and Amylose. A blue-black
color is seen by the human eye as the
complementary color to the light energy
received by the charge transfer complex.
o In short, the benchtop iodine color is brown in
color. The polyiodides (I3–, I5–, I7–) are
colorless, and the amylose-iodide complex is
blue-black.
o The iodine starch test's principle underpins all
iodometric titrations that use the starch
indicator. The intensity of the blue color
reduces as the temperature rises and when
water-miscible solvents such as isopropyl
alcohol, ethanol, and others are present. The
amylose-iodine combination dissociates when
the temperature rises. The helical structure of
the amylose-iodine complex is rebuilt when
the temperature falls, resulting in the
regeneration of the blue-black color complex.
 Materials and Procedure
o Materials:
 Iodine solution or Lugol’s reagent*
 Test tubes
 Test tube stand
 Water bath
 Vortex mixer
 Dropper
o Procedure:
 Take two test tubes and label your
test tubes as- test sample and
control sample
 Take a small sample (solid
sample:500 mg -1000mg; liquid: 1ml)
in a clean and dried test tube labeled
as a test sample.
 Take 1ml of the purified water in the
clean and dried test tube labeled as
the control sample.
 Add 2-3 drops of Lugol’s iodine
solution to both the test tubes and
mix it thoroughly on a vortex mixer.
 Observe the color that develops in
both the test tubes.
 The test tubes should then be
heated on a water bath until the color
disappears.
 Allow the test tubes to cool down
completely and observe the color in
both the test tubes.
 Result Interpretation of Iodine’s Test
o Based on the observation in the color change
of the samples, the following may be
concluded as the iodine test for starch results:
o The appearance of blue-black color indicates
the presence of the starch in the sample, i.e.,
a positive iodine test.
o The brown color or no change in the color of
the sample indicates the absence of the
starch in the given sample, i.e., a negative
iodine test.
Bial’s Test
 This test is based on the idea that pentosans are
degraded into pentoses during hydrolysis. Pentoses
are also dehydrated to produce furfural, which then
condenses with orcinol to produce a blue-green
precipitate. In the presence of hexoses, hydroxyfurfural
is generated instead of furfural, which forms a murky
brown colored precipitate when combined with orcinol.
The concentration of pentoses in the sample is directly
proportional to the intensity of the precipitation. The
color intensity is determined by the concentrations of
HCl, ferric chloride, orcinol, and the time spent boiling.
The concentration of sugars is estimated by using a
spectrophotometer or a red filter colorimeter to
measure the absorbance of 620 nm wavelength.
 Principle
o Conc. HCI acts on pentoses to form furfurals.
o This condenses with orcinol to give green
color compounds.
o Bial's test is useful in distinguishing pentoses
sugar from hexoses sugars. Pentose form
furfural in acidic medium which condense with
orcinol in presence of ferric ion to give blue-
green colored complex which is soluble in
butyl alcohol.
 Materials and Procedure
o Materials:
o Equipment:
 UV Spectrophotometer
 Vortex mixer
 Mantle heater/Water Bath.
o Chemical/Reagents:
 Bial’s Reagent
 Ribose sugar
 Other carbohydrates if desired
 Sample
o Glassware and other equipments:
 Test tubes, Test tube stand,
Pipettes,
 Beaker, Ice Test tube caps, Tissue
paper, Wash bottle
 Procedure:
o Pipette out different volumes (50 μl, 100 μl,
and so on) of ribose solution from the
supplied stock solution (200μg /ml) into a
series of test tubes and make up the volume
to 1 mL with distilled water.
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 o Take a tube labeled as one as blank
containing 1ml of just distilled water and the
rest of the tubes labeled 2 to 9 for
construction of a standard curve. Tubes 10-15
are for the unknown samples.
o Add 5 ml of the bial’s reagent to each tube
and mix well by vortexing.
o Cool the tubes.
o Cover the tubes with caps on top and
incubate at 90°C for 17 minutes or boiling
water bath for 10 minutes.
o Cool the tubes to room temperature and
measure the optical density of the solutions at
620 nm against a blank.
o Prepare a standard curve of absorbance
against ribose concentration.
o Determine the amount of ribose in the
unknown sample by plotting a standard curve
of A620 on the Y-axis and concentration of
Ribose on the X-axis.
 Result Interpretation of Bial’s Test
o The presence of a blue-green complex
indicates the presence of pentoses in the
sample.
o Using the graph, the concentration of ribose
sugar in the sample can be determined. A
similar interpretation can be made for RNA
detection as well.
Tollen’s Test
 Tollens test also known as the Silver Mirror Test is a
qualitative biochemical test used to distinguish
between aldehydes and ketones which are collectively
known as carbonyl compounds and contain the
Carbonyl functional group in their structure.
 Principle
o Tollens’ reagent oxidizes an aldehyde into the
corresponding carboxylic acid.
o The reaction is accompanied by the reduction
of silver ions in Tollens’ reagent into metallic
silver, which forms a mirror on the test tube
o Ketones are not oxidized by Tollens’ reagent,
so the treatment of a ketone with Tollens’
reagent in a glass test tube does not result in
a silver mirror
 Materials and Procedure
o Materials:
o Chemicals/Reagents:
 0.1 M AgNO3
 Conc. HNO3
 Conc. NH4OH
 0.8 M KOH
 0.5 M Dextrose
o Glasswares and other equipments:
 Large test tube/rubber stopper
 Beaker
 Deionized water
 Water bath
 Procedure:
o Clean the test tube to be used by rinsing with
concentrated nitric acid and washing well with
hot water.
o Prepare Tollen’s reagent as follows: Add 50
mL of 0.1 M AgNO3 to the beaker and add
NH4OH to this. A brown precipitate will form.
Continue adding NH4OH until the solution
becomes clear. To this, add 25 mL of 0.8 M
KOH. Again, add NH4OH until the solution
becomes clear.
o To perform demonstration, add 5 mL of
dextrose solution to the test tube and to this
add 25 mL of Tollen’s reagent. The solution
will turn yellow and brown then become
cloudy and dark before silver begins to form
on the inside of the test tube. This should take
a couple of minutes.
o Remove the contents from the test tube and
rinse the tube with water. The tube with a
“silver mirror” can now be observed and yield
results.
 Result Interpretation of Tollen’s Test
o The formation of a dark grey precipitate or
silver mirror on the bottom and sides of the
test tube indicates a positive result, which
means that the given sample contains
reducing sugars/ aldoses.
o The absence of such precipitate indicates a
negative result, which means that the test
sample doesn’t have reducing sugars/
aldoses/α-hydroxy ketoses.
Osazone’s Test
 Developed by German Chemist Emil Fischer, Osazone
test is a chemical test used to detect reducing
sugars.This test even allows the differentiation of
different reducing sugars on the basis of the time of
appearance of the complex.This test is also termed
Phenyl hydrazine test based on the reagent used for
this test.
 Objective:
o To detect reducing sugars.
o To differentiate reducing sugars from non-
reducing sugars.
o To distinguish different reducing sugars
between each other.
 Principle
o The reagent for this test consists of
phenylhydrazine in acetate buffer.
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 o Carbohydrates with free or potentially free
carbonyl groups react with phenylhydrazine to
form osazone.
o The condensation-oxidation-condensation
reaction between three molecules of
phenylhydrazine and carbon one and two of
aldoses and ketoses yields 1, 2-
diphenyhydrazone, which is known as
osazone.
o Osazone appears as yellow-colored crystals
of characteristic shape, solubility, melting
point, and time of formation. Osazone is
different for different sugars.
 Materials and Procedure
o Materials:
o Chemical/Reagents:
 0.5 g of phenylhydrazine
hydrochloride
 0.1 g of sodium acetate.
 Glacial acetic acid
 Test sample
o Glasswares and other equipment:
 Test tubes
 Test tube stand
 Pipettes
 Vortex
 Water bath
 Microscope
o Procedure:
 Take 5 ml of test solution in a clean,
dry test tube.
 Add 0.3 g of osazone mixture and
five drops of glacial acetic acid to the
test tube.
 Mix it well and warm the test tube
gently in the water bath if required to
dissolve all the elements.
 Keep the test tube in boiling water
and observe the formation of crystals
at various time points.
 Observe the shape of the crystal
under low magnification under a
microscope.
 Result Interpretation of Osazone’s Test
o Based on the shape and structure of the
crystals and their time of appearance,
different sugars can be identified.
o The following is a chart for the identification of
reducing sugar through this test:

 Thus, through this table, different sugars can be

identified on the basis of the structure of the crystal
and the time of appearance.