Biochem lab 8.

Place all the tubes in a warm water bath

(60 degrees Celsius)
Midterm
9. Remove all the tubes once a change is
Fehling‘s Test observed in some of the samples.

- to detect reducing sugar in a given 10. Note the changes and record your
solution observations.

PRINCIPLE

• Fehling’s test is one of the sensitive test

for detection of reducing sugars.

• Fehling’s reagents

Fehling’s solution A = aqueous copper sulphate

Fehling’s solution B = alkaline sodium

potassium tartarate ( Rochelle salt) — chelating RESULTS (before heating)
agent

*These two solution are mixed in equal amount

before test.*

Procedure:

1. In a 10mL vial, put 2mL of Fehling’s A

solution

2. Add 2mL of Fehling‘s B solution and

mix.

3. Note and record the color of the

reulting reagent. This serves as the Positive Fehling’s Test = Reddish brown ppt
Fehling’s reagent. Negative Fehling’s Test = No reddish brown ppt

4. Divide the Fehling’s reagent into four Fructose = negative

test tubes of 1 mL (15 drops) each. Sucrose = negative
5. Label the tubes as follows: glucose, Glucose = negative
sucrose, fructose, and lactose.
Lactose = negative
6. Add into each labeled test tubes 1 mL
(15 drops) of the 10% sugar solution RESULTS (after heating)
(glucose, sucrose, fructose, lactose)
respectively and mix thoroughly.

7. Note any changes.

 b. Place 1 mL of Benedict’s reagent

c. Place 1 mL each of glucose, sucrose,

lactose, and fructose into 4 of the test
tubes

d. Note any changes. If none, heat in a

water bath.

e. Remove from bath. Allow to cool.

Record observations.

f. Place 1 mL of random urine into the last

test tube.
Positive Fehling’s Test = Reddish brown ppt
g. Note any changes. If none, heat in
Negative Fehling’s Test = No reddish brown ppt
water bath for 5 minutes.
Fructose = positive
h. Allow to cool. Record observations.
Sucrose = negative
Principles
Glucose = positive
• A reducing sugar is any sugar with a
Lactose = positive hemiacetal

WHY DID SUCROSE TESTED NEGATIVE FOR • Reducing sugars have the property of
transferring hydrogen (electrons) to
FEHLING’S TEST?
other compounds.
Sucrose is a non reducing sugar.
• LEORA – GEROA
Fructose, Lactose and Glucose are reducing
sugars.

Benedict’s Test

• Test for simple carbohydrates

• Identifies reducing sugars

(monosaccharides and some
disaccharides) with a free aldehyde
functional group

• Reagent is copper (II) sulfate (CuSO4)

• These sugars are in equilibrium with its
• Positive result ranges from green to red open-chain aldehyde
to brown

• Can test for traces of sugar in urine

Procedure

a. Prepare 5 test tubes

 • As the concentration of reducing sugar
increases, the nearer the final color is to
brick-red and the greater the
precipitate formed.

Results

 Fructose does not have an aldehyde,

but a ketone, but still gives a positive
result for the Benedict’s test. An
exception to the rule because the
carbon adjacent to the carbon with the
ketone group has a hydroxyl group. The
ketone will be in equilibrium with an
aldehyde through tautomerization. A
tautomer is an isomer wherein only the
electrons and protons are located
differently.
 Sucrose is technically a non-reducing
sugar. But why does it give a positive
result? At first, the result of sucrose was
negative, but after heating, it was
broken down into glucose and fructose,
thus giving a positive result.

Barfoed’s Test

CARBOHYDRATES

• Aldehydes readily become oxidized to

 Aldehydes (-CHO) & Ketones (-CO)
carboxylic acids in the presence of an
constitute the major groups in
oxidant
carbohydrates
• The aldehyde in the sugars reduces
 are mainly divided into
copper (II) to copper (I), which forms
monosaccharides, disaccharides and
copper (I) oxide, a reddish-brown
polysaccharides.
substance
  The commonly occurring
monosaccharides includes glucose,
fructose, galactose, ribose, etc.

 The two monosaccharides combine

together to form disaccharides which
include sucrose, lactose and maltose.

Monosaccharides

Disaccharides

Polysaccharides
Objectives:
◉ To detect reducing sugar
◉ To distinguish monosaccharides from
◉ The reaction will be negative in the
reducing disaccharides
Reducing sugars
 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
Principles:
◉ Barfoed’s test is used for distinguishing
monosaccharides from reducing
disaccharides.
◉ A red copper(II) oxide precipitate is
formed will indicates the presence of
reducing sugar.
presence of disaccharide sugars
because they are weaker reducing
agents.
○ like sucrose and trehalose have
glycosidic bonds between
their anomeric carbons and
thus cannot convert to an open-
chain form with an aldehyde
group; they are stuck in the
cyclic form
■ is combined with the
test solution and boiled.

◉ Monosaccharides usually react in about ◉ This test is specific for monosaccharides

1-2 minutes ○ Due to the weakly acidic nature
◉ Reducing disaccharides take much of Barfoed's reagent, it is
longer time between 7-12 minutes to reduced only by
react with the reagent. monosaccharides

◉ Brick red color is obtained in this test

which is due to formation of Cuprous
oxide.

◉ The reaction with disaccharides is

slower because disaccharides have to
get hydrolyzed first and then react with
the reagent Cupric acetate (Barfoed’s
Reagent) to produce Cuprous oxide

Barfoed’s test

◉ Barfoed's test is used to detect the

presence of monosaccharide (reducing)
sugars in solution.

◉ Barfoed's reagent

○ Ethanoic (acetic) acid

○ copper(II) acetate
Procedure
1. Prepare 4 test tubes and label as
follows: “Glucose”, “Sucrose”,
“Fructose”, “Lactose”
Glucose Sucrose Fructose Lactose
1 mL (15 drops) Barfoed’s Reagent
1 mL (15 drops) of each test sugars
Mix for a minute
4. Note any changes. When no changes
occurs, place the tube in a warm water
bath (60 degree Celsius) for not more
than 3 minutes
5. Remove all the tubes once a change is
observed in some samples. Note any
changes and record
Standard results
◉ Positive Barfoed’s test:
○ development of brick red color
ppt within 3-5 minutes
◉ Negative Barfoed’s test:
○ absence of red color
Positive Barfoed’s test:
Glucose
Fructose
Negative Barfoed’s test:
Lactose
Sucrose
◉ Brick Red precipitation within 2-3 min.
boiling in case of a monosaccharide,
while 7-12 min for disaccharide.
◉ This delay in the development of the
color of in case of disaccharides is due
to slow reduction of disaccharide
during the reaction.
SELIWANOFF’S TEST
- Seliwanoff’s test is a compound test
which separates aldose and ketose
sugars. Ketoses are differentiated from
aldoses by means of their
ketone/aldehyde usefulness. If the
sugar contains a ketone bunch, it is a
ketose. If the sugar contains an
aldehyde bunch, it is an aldose. This test
is much like Bial’s test. This test
depends on the rule that, when heated,
 ketones are more quickly dried out than 3. Add 1 mL (15 drops) of each of test
aldoses. It is named after Theodor sugars (glucose, sucrose, fructose)
Seliwanoff, the scientific expert, who respectively and mix thoroughly for a
performed the test. minute.

Principle 4. Note any changes. When no change is

observed, place the tube in a warm
• Seliwanoff Test is a test to separate
water bath (60 ◦C).
keto sugars structure Aldo sugars. Keto
sugars get dried out in the nearness of 5. Remove ALL tubes once a change is
concentrated acids to yield furfurals or observed in some of the samples.
their subsidiaries which react with
6. Note any changes and record your
resorcinol in Seliwanoff reagent to yield
observation.
a cherry-red hue complex as the
positive result. A cherry-red shading Results
shows the nearness of ketohexoses.
Ketopentoses yield blue-green series TEST OBSERVATION
while aldoses and disaccharides give no
shading change. GLUCOSE NO CHANGES AT
ALL
• With this test, fructose will bring about
cherry red shading, while the aldose
SUCROSE BROWN
sugars like glucose will give a negative
PRECIPITATE
result with no red shading showing up
in the series. In any case, if the warming
is not as it is expected (which is more FRUCTOSE CHANGE IN
than 5 minutes), aldose sugar will COLOR (LIGHT
create a pink shading. While the sucrose PINK/ LIGHT
(a blend of fructose and glucose) will PURPLE)
create a cherry red shading as a result
Positive Seliwanoff’s test: Fructose and Sucrose
of the fructose in it.
Negative Seliwanoff’s test: Glucose

BIAL’S ORCINOL TEST

Procedures:

A. Prepare 3 test tubes and label as follows:

PROCEDURE
1. Prepare 3 tubes and label as follows:
“glucose,” “sucrose,” and “fructose”
2. Place 1 mL (15 drops) of Seliwanoff’s
reagent into each tube.
“glucose,” “fructose,” “xylose”
B. Place 1 mL (15 drops) of Bial’s orcinol reagent
into each tube
C. Add 1 mL (15 drops) of each of the test
sugars (glucose, fructose, xylose) respectively
and mix thoroughly for a minute
D. Note any changes. When no change is
observed, place the tube in warm water bath
(60°C).
F. Remove ALL tubes once a change is observed
in some of the samples.
G. Note any changes and record your
observations.
Bial’s Orcinol Test:
Reagent:
-Ferric acid (H2FeO4), Hydrochloric acid (HCl),
and Orcinol (C7H8O2)
Pentose form furfural in acidic medium which
condense with orcinol in presence of ferric ion
to give blue green colored complex.
Result interpretation:
Positive: formation of blue color (e.g. Ribose)
Negative: formation of any other color indicates
negative test.
Glucose (C6H12O6): Negative
Fructose (C6H12O6): Negative
Xylose (C5H10O5):
MUCIC ACID TEST
- Specific test to identify the presence of
the sugar galactose
- Galactose is either independently
present in solutions or obtained by the
hydrolysis of lactose.
Principle:
 Oxidation of most monosaccharides by
nitric acid yields soluble dicarboxylic
acids. However, oxidation of galactose
yields an insoluble mucic acid. Lactose
will also yield a mucic acid, due to
hydrolysis of the glycosidic linkage
between its glucose and galactose
subunits. Being insoluble,
galactosaccharic acid crystals separate
out.
Components
Nitric acid - able to oxidize the terminal
groups of aldoses, but leaves the secondary
hydroxyl groups unchanged.
Dicarboxylic acid - formed from galactose is
called mucic acid and is insoluble in cold
aqueous solution.
Equation
HC=O-(CHOH)4-COOH (aq) + 2 HNO3 (aq)
--> HOC=O-(CHOH)4-COOH (s) + H2O (l) + 2
NO2 (g)
Sample equation
Galactose reacts with HNO3:
CH2OH-(CHOH)4-CHO + HNO3 ----> CH2OH-
(CHOH)4-CHO-NO3 + H+
Results
When galactose is oxidized by nitric acid,
INSOLUBLE MUCIC ACID is formed.
Glucose: give similar acids but they are
water-soluble under conditions of the test.
Lactose: disaccharide; the nitric acid
catalyzes and oxidizes sugars to form
crystals.
The nitric acid first catalyzes hydrolysis of
lactose to glucose and galactose. Then the
nitric acid oxidizes both of those sugars to
the tetrahydroxy acids. Mucic acid
crystallizes out, which is a positive test.

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