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Experiment 5 Benedict - S Test and Fehling - S Test

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428 views6 pages

Experiment 5 Benedict - S Test and Fehling - S Test

...

Uploaded by

vixen.heartless
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© © All Rights Reserved
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Experiment No.

5
BENEDICT’S TEST and FEHLING’S TEST:
TEST FOR REDUCING OR NON-REDUCING CARBOHYDRATES (SUGARS)

I. OBJECTIVES:
At the end of the activity, the students are expected to:
1. distinguish between the reducing and non-reducing sugars using the
two different tests:
a) Benedict’s test
b) Fehling’s test
2. determine the positive test results for reducing sugars.

II. APPARATUS/MATERIALS:
Test tubes in a rack
Test tube brush
Test tube holder
Laboratory Hot plate
Beaker (500 mL)
Graduated cylinder (10 mL)
Droppers (to be provided by the group)

III. CHEMICALS:
1% of the following solutions:
Glucose, Fructose, Lactose, Sucrose, Starch
Benedict’s reagent
Fehling’s A reagent and Fehling’s B reagent

IV. DISCUSSIONS:
A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone
group. The enolization of sugars under alkaline conditions is an important
consideration in reduction tests. The ability of a sugar to reduce alkaline test
reagents depends on the availability of an aldehyde or keto group for reduction
reactions. A number of sugars especially disaccharides or polysaccharides have
glycosidic linkages which involve bonding a carbohydrate (sugar) molecule to
another one, and hence there is no reducing group on the sugar; like in the case of
sucrose, glycogen, starch and dextrin. In the case of reducing sugars, the presence
of alkali causes extensive enolization especially at high pH and temperature. This
leads to a higher susceptibility to oxidation reactions than at neutral or acidic pH.
These sugars, therefore, become potential agents capable of reducing Cu +2 to Cu+,
Ag+ to Ag and so forth. Most commonly used tests for detection of reducing sugars
are Fehling’s Test, Benedict’s Test and Barfoed’s Test (Barfoed’s test is separated).

Reducing and Nonreducing Properties of Carbohydrates:


1. Aldoses (polyhydroxy aldehydes).
All aldoses are reducing sugars because they contain free aldehyde
functional groups. The aldehydes are oxidized by mild oxidizing agents (e.g.,

31
Benedict’s or Fehling’s reagent) to the corresponding carboxylates. For
example: R–CHO + 2Cu+2 NaOH → R–COO–Na+ + Cu2O(s) (from Fehling’s
reagent) (red precipitate)
2. Ketoses (polyhydroxy ketones).
All ketoses are reducing sugars because they have a ketone functional
group next to an alcohol functional group.

3. Hemiacetal functional group (potential aldehydes).


Carbohydrates with hemiacetal functional groups can reduce mild
oxidizing agents such as Fehling’s reagent because hemiacetals can easily
form aldehydes through mutarotation.

FEHLING’S TEST:
Fehling's test is used to determine if a reducing sugar is present in a sample
using a reagent called the Fehling's reagent. The reagent is an alkaline solution,
containing a copper tartrate complex with Cu 2+ ions. When the reagent reacts with
the aldehyde group of a reducing sugar, the Cu 2+ ions are reduced to Cu+ ions,
forming a red precipitate of cuprous oxide. The chemical reaction of the key
functional groups can be viewed below:

Preparation of Fehling's solution A:


Dissolve 35g of Cu2SO4.7H2O in water and make up to 500ml
Preparation of Fehling's solution B:
Dissolve 120 g of KOH and 173 g of Sodium Potassium Tartrate
(Rochelle salt) in water and make up to 500 ml
Fehling’s reagent: Equal volumes of Fehling A and Fehling B are mixed
to form a deep blue solution.

32
BENEDICT’S TEST:
Benedict’s test is used to test for simple carbohydrates. The Benedict’s
test identifies reducing sugars (monosaccharides and some disaccharides),
which have free ketone or aldehyde functional groups. Benedict’s solution
can be used to test for the presence of glucose in urine.
Some sugars such as glucose are
called reducing sugars because they are
capable of transferring hydrogens
(electrons) to other compounds, a process
called reduction. When reducing sugars
are mixed with Benedicts reagent and
heated, a reduction reaction causes the
Benedicts reagent to change color. The
color varies from green to dark red (brick) or
rusty-brown, depending on the amount of and type of sugar.
When Benedict’s solution and simple carbohydrates are heated, the
solution changes to orange red/ brick red. This reaction is caused by the
reducing property of simple carbohydrates. The copper (II) ions in the
Benedict’s solution are reduced to Copper (I) ions, which causes the color
change.

The red copper(I) oxide formed is insoluble in water and is precipitated


out of solution. This accounts for the precipitate formed. As the concentration
of reducing sugar increases, the nearer the final color is to brick-red and the
greater the precipitate formed. Sometimes a brick red solid, copper oxide,
precipitates out of the solution and collects at the bottom of the test tube.
Benedict’s solution is a deep-blue alkaline solution used to test for the
presence of the aldehyde functional group, – CHO. It is composed of
anhydrous sodium carbonate, sodium citrate, and copper (II) sulfate
pentahydrate.

33
V. PROCEDURES:
A. Fehling’s Test:
1. Boil about 250 mL water in your 500 mL beaker. This is your hot water
bath.
2. Label your test tubes from 1 – 6 as follows:

Test tube Substance Test tube Substance


# #
1 Glucose 4 Sucrose
2 Fructose 5 Starch
3 Lactose 6 Distilled H2O
(negative control)

3. Place approximately 4 mL of Fehling’s solution (2 mL Fehling’s solution A +


2 mL of Fehling’s solution B) into each of six labeled test tubes.
4. Add 1 mL (20 drops) of each of the following carbohydrates to the
corresponding test tubes as shown in the table above. Test tube #6
Distilled water serve as the negative control.
5. Place the 6 test tubes in a boiling water bath for about 2-5 minutes.
6. Record your observation in table 5.1 in RS #5.

B. Benedict’s Test:
1. Boil about 250 mL water in your 500 mL beaker. This is your hot water
bath.
2. Label your test tubes from 1 – 6 as follows:

Test tube Substance Test tube Substance


# #
1 Glucose 4 Sucrose
2 Fructose 5 Starch
3 Lactose 6 Distilled H2O
(negative control)

3. Place approximately 1 mL (20 drops) of each of the carbohydrates to the


corresponding test tubes as indicated in the table above. Test tube #6
Distilled water serve as the negative control.
4. Add 2 mL of Benedict’s reagent to each 6 test tubes.
5. Place the 6 test tubes in a boiling water bath for about 2-5 minutes.
6. Record your observation in table 5.1 of RS #5.
7. Basing on your observation identify which one is reducing sugar and non-
reducing sugar by placing a tick (✓) mark in the appropriate box in table
5.2 of RS #5.

34
Experiment No. 5
BENEDICT’S TEST and FEHLING’S TEST:
TEST FOR REDUCING OR NON-REDUCING CARBOHYDRATES (SUGARS)

REPORT SHEET #5

Name: ________________________________ Date Submitted: ______________


Group #: ______ Section: ______ Instructors Signature: ________

Table 5.1 Observations

Test Substanc Observations


tube # e Fehling’s Test Benedict’s Test

1 Glucose

2 Fructose

3 Lactose

4 Sucrose

5 Starch

Distilled
6 H2O
(negativ

35
e
control)

Table 5.2 Reducing Sugar and Non-Reducing Sugars


Carbohydrates/Sugar Reducing Sugar Non-reducing Sugar
Glucose
Fructose
Lactose
Sucrose
Starch

QUESTIONS:

1. Among the 5 carbohydrates tested, which of them is/are a non-reducing sugar?

2. Explain why this/these sugar are non-reducing sugars?

3. What are the composition of Fehling’s reagent?

4. What are the composition of Benedicts reagent?

CONCLUSION:

36

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