• Home

chem assignment 11

Name __________________________

Module 11

Post-Lab Assignment

Long Answer (30 points)

In this assignment, you will write a discussion for the experiment performed. You will discuss your data and results in a well-organized paragraph (more than 5 complete sentences). Be sure to explain how you reached your conclusions. Your discussion should cover (in any order):

1) the identity of the unknown sugar;

2) the test results of the unknown analysis compared to each of the knowns;

3) what else these results tell us about the unknown sugar.

chem assignment 11

Experiment 11: CLASSIFICATION
OF CARBOHYDRATES

1

Data Collection and Results Pages Name: __________________________
Date: ___________________

Pre-lab Preparations:

Review the information in your textbook on carbohydrates (sugars). You may also use the
Internet for assistance, if necessary. Allow plenty of time for your pre-lab preparation!

1. Fill in Table 11.1 based on the discussion provided in the Introduction prior to beginning
the lab.

Table 11.1: What the Test Should Tell Us

TEST SELIWANOFF BARFOED BENEDICT
What a Positive
Result Should
Look Like

Type of
Carbohydrate
Expected to
Test Positive

2. Draw the cyclic forms (not the Fischer projections) of glucose, fructose, sucrose, and
lactose in the space below. Label each clearly by name, and identify each as an acetal, ketal,
hemiacetal or hemiketal.

2 EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES

3. Use check marks to classify each of the knowns in Table 11.2 prior to beginning the lab.
You will fill out the last row after you have performed the experiment. (There should be
more than 1 check mark in each row.)

Table 11.2 Carbohydrate Classification

Monosaccharide Disaccharide Aldohexose Ketohexose

Glucose

Fructose

Sucrose

Lactose

Unknown

During the Lab:

4. Fill in Table 11.3 during the lab period as the experiment is performed. (Observations –
What do you see?)

Table 11.3: Actual Observations

SAMPLE SELIWANOFF TEST BARFOED’S TEST BENEDICT’S TEST

glucose

fructose

sucrose

lactose

UNKNOWN

EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES 3

Post-lab Analysis:

5. Fill in Table 11.4 after the experiment has finished. Use your information from above
sections to summarize observations by placing a “+” for a positive result and a “-“ for a
negative result. Determine the identity of the unknown carbohydrate.

Table 11.4: Summary

SAMPLE SELIWANOFF TEST BARFOED’S TEST BENEDICT’S TEST

glucose

fructose

sucrose

lactose

UNKNOWN

CONCLUSION ON IDENTITY OF UNKNOWN: _____________________________

chem assignment 11

Experiment 11: CLASSIFICATION
OF CARBOHYDRATES

1

Purpose: The differences in the molecular structure of some typical carbohydrates are
utilized to distinguish one carbohydrate from another. An unknown carbohydrate is to be
identified based on the three chemical tests described.

Introduction: The term carbohydrate (hydrate of carbon) refers to a class of organic,
biochemical compounds with the general formula Cn(H2O)m, also known as sugars. It was
thus named because heating these compounds produced C and H2O molecules. In actuality,
there are no water molecules in the structure. Instead, the “H2O” is attached to the C atoms
as –H and −OH groups, and these molecules are best considered as either a polyhydroxy
aldehyde (aldose) or polyhydroxy ketone (ketose). D-glucose is an example of an aldose,
and D-ribulose is an example of a ketose. Since glucose contains 6 C atoms, it is an
aldohexose, and ribulose, with 5 C atoms, is a ketopentose.

The structures are shown above as Fischer projections. The location of the hydroxyl group
on the second carbon from the bottom in the Fischer projection determines whether the
structure is the D- or the L-isomer. When the −OH is on the right, it is the D-isomer. When
it is on the left, it is the L-isomer. Thus, both glucose and ribulose shown above are the D-
isomers. In this experiment we will stick with only D-isomers. The Fischer projections are
also useful in identifying a monosaccharide as being an aldose or ketose.

In actuality, these molecules exist in a cyclic form, where they are less easily identified as an
aldose or ketose. For example, D-glucose exists as either β-D-glucose or α-D-glucose.

aldehyde
functional
group

ketone
functional
group

CH2OH

H O

OHH
HOH
OHH
OHH

CH2OH

CH2OH

OHH
OHH

O

D-glucose (an aldohexose) D-ribulose (a ketopentose)

CH2OH

H O

OHH
HOH
OHH
OHH

O
CH2OH

H
H

OH
OH

H OH

H
H

H
O

O
CH2OH

H
H

OH
OH

H OH

H
H

OH
O

CH2OH

H
H

OH OH

H

OH

H OH

H

1

2

3

4

5

6

1
23

4

5

6

D-glucose
in Fischer projection

=

β−D-glucose
in Haworth projection

α−D-glucose
in Haworth projection

β-hyroxy
group

α-hydroxy
group

2 EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES

In this cyclic form, the two anomers (α- and β-isomers) are distinguished by the position of
the hydroxyl group attached to the anomeric C. In the β-anomer, the –OH is up; whereas, in
the α-anomer, the −OH is down, as shown in the Haworth projections below.

O
CH2OH

H
H

OH H

OH

OH

H OH

H

O
CH2OH

H
H

OH OH

H

OH

H OH

H

β−D-glucose α−D-glucose

α-hydroxy
group

β-hyroxy
group

anomeric C

anomeric C

In addition you should be familiar with four more terminologies related to carbohydrates:
hemiacetal, acetal, hemiketal, and ketal. The hemiacetal and acetal are derived from an
aldehyde, and the hemiketal and ketal are derived from a ketone.

R

O

H
R OH

OR’

H

R OR’
OR’

H

R OH
OR’

R”

R OR’
OR’

R”R

O

R”

hemiacetal

+ R’OH + R’OH

acetal

hemiketal

+ R’OH + R’OH

ketal

aldehyde

ketone
The cyclic glucose is a hemiacetal and not a hemiketal because it is derived from an
aldehyde and not a ketone (see the Fischer projection). Furthermore, it is a hemiacetal and
not an acetal. Its anomeric C is bonded to −OH and −OR’ rather than two −OR’ (see
Haworth projection below for D-glucose).

O
CH2OH

H
H

OH H

OH

OH

H OH

H

O
H

H
H

OH OH

CH2OH

H

OH H

OH

D-glucose
a hemiacetal

-OR

-OH

-H

-R’
D-fructopyranose
a hemiketal

-OR

-R’

-OH

-R

anomeric C

The key to recognizing whether a structure is a hemiacetal or hemiketal is to examine the
Haworth projection and locate the O that is part of the ring. Then examine the C atoms
attached to either side of that O. If one of these C atoms has a −OH and −H attached to it,
the molecule is a hemiacetal. If one of these C atoms has a −OH and −R attached to it, the
molecule is a hemiketal. For D-fructopyranose (see Haworth projection shown above), one
of the C atoms is attached to −OR, −OH, −R’ and −R”, and therefore it is considered a
hemiketal. Note that the C on the other side of O in the ring is not attached to an −OH and

EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES 3

therefore is not under consideration. If the ring O is not connected to a C atom with an −OH
group, the molecule is neither a hemiacetal nor a hemiketal.

THE SELIWANOFF’S TEST

In this experiment, you will use the Seliwanoff’s Test to distinguish between an aldose and
a ketose. The reagents consist of resorcinol and concentrated hydrochloric acid. Ketoses
are dehydrated (H2O removed) and then reacted with resorcinol to produce a deep cherry red
color. Formation of this red color indicates the presence of a ketose.

O
CH2OH

H

OH H

OH
OH

CH2OH

H

O
CH2OH

H H

H

O

OH

OH

– 3 H2O

+ HCl

α-D-fructose
(a ketose)

dehydrated fructose

resorcinol

RED DYE

Carbohydrates can be classified as monosaccharides, disaccharides, or polysaccharides.
The monosaccharide (simple sugar) is a single unit of aldose or ketose. The disaccharide is
composed of two monosaccharides bonded together, and the polysaccharide is composed of
a long chain of units of monosaccharides bonded together.

Lactose and sucrose are examples of disaccharides:

Note that since sucrose is made of a glucose and a fructose unit, and fructose is a ketose, this
disaccharide will test positive in the Seliwanoff’s Test.

O
CH2OH

H

OH

H

O
O

CH2OH

H
OH

H H

H

OH

H OH

H

OH

H OH

H

lactose

galactose unit glucose unit

O
CH2OH

H
H

OH

O

H

OH

H OH

H

O
CH2OH

CH2OH
H

OH H

OH
H

sucrose

glucose

fructose

4 EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES

THE BARFOED’S TEST

In this experiment, you will use the Barfoed’s Test to distinguish between monosaccharides
and disaccharides.

The Barfoed reagent contains copper(II) ions in a slightly acidic solution. The solution
oxidizes monosaccharides only. In this reaction, the copper(II) ion is reduced to copper(I)
ion, which appears as the brick red copper(I) oxide, Cu2O. The solution turns various
shades of green, yellow or orange before the red appears. The monosaccharide is acting as
the reducing agent, and is itself oxidized (from aldehyde to carboxylic acid).

CH2OH

H O

OHH
HOH
OHH
OHH

CH2OH

OH O

OHH
HOH
OHH
OHH

Cu2+
blue

Cu+
red

RCHO (aldehyde) ⎯⎯⎯⎯⎯⎯⎯→ RCOOH (carboxylic acid)
+ 2 Cu2+ + 2 H2O + Cu2O + 4 H+

THE BENEDICT’S TEST

The Benedict Test distinguishes between a reducing sugar and a nonreducing sugar. A
reducing sugar is a carbohydrate that, under alkaline condition, forms an aldehyde or ketone
that reacts as a reducing agent. It is similar to the Barfoed Test in that a positive test
involves the reduction of blue Cu2+ to brick red Cu+ in the form of Cu2O. The difference is
that the reagent is alkaline and some disaccharides will give a positive test as well. All
monosaccharides will test positive, but disaccharides with hemiacetal or hemiketals will also
test positive. This is why it is important to be able to distinguish between a hemiacetal and
hemiketal from examining the structures.

Benedict’s reagent can be used to test for the presence of glucose in urine. Glucose found to
be present in urine is an indication of diabetes mellitus. Once a reducing sugar is detected in
urine, further tests have to be undergone in order to ascertain which sugar is present.

You will be given an unknown which is one of the following:
α-D-glucose, α-D-fructose, sucrose or lactose

By doing the three tests on the unknown, alongside the knowns, you will be able to identify
your unknown.

EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES 5

FLOWCHART FOR THE IDENTIFICAITON OF UNKNOWN CARBOHYDRATES

UNKNOWN
positive Barfoed’s Test negative

MONOSACCAHRIDES DISACCHARIDES
Seliwanoff Test Benedict’s Test

positive negative positive negative

KETOSE ALDOSE REDUCING NONREDUCING
SUGAR SUGAR

Equipment/Materials
Test tubes (15), 400-mL beaker, 10-mL graduated cylinder, Seliwanoff reagent, Barfoed’s
reagent, Benedict’s reagent

Procedure
(Using a pen or pencil, record by hand all of your data and results on the Data Collection
and Results Pages.)

Before beginning, complete the Pre-lab Preparations, #1-3, on your Data Collection and
Results Pages. Remember to record the Unknown # of your sample in the table.

Prepare a hot, boiling water bath by filling a 400-mL beaker ½ full of hot tap water and
placing it on a hotplate. Add a couple of boiling chips. Watch the level of the water. It is
likely you have to add more water during the experiment.

THE SELIWANOFF’S TEST
1. Label five test tubes near the top in such a way that the label won’t come off in the water

bath.
G = glucose
F = fructose
S = sucrose
L = lactose
U = unknown

2. Using a 10-mL graduated cylinder, measure 5 mL of the Seliwanoff reagent into one of

the test tubes. Using the level in your first test tube as a guide, fill the other labeled test
tubes with the Seliwanoff reagent.

6 EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES

3. Next add 2 drops of the appropriate carbohydrate solution to each test tube. Mix the
contents by rocking the tube back and forth. Be sure the reagents mix otherwise the test
may not work.

4. Place the test tubes in the beaker of boiling water for 3 full minutes.

5. Record your observations in your notebook and draw a conclusion as to whether your

unknown is a ketose or aldose.

THE BARFOED’S TEST
Check to see whether the hot water bath needs refilling and that it is boiling hot.

6. Label five clean test tubes as before. This time you are to fill the test tubes with the

carbohydrate solutions first. Using a 10-mL graduated cylinder measure 5 mL of
glucose solution in the test tube. With this as the fill guide, fill the other labeled test
tubes with their appropriate carbohydrate solutions.

7. Add 3 mL of Barfoed’s reagent to the first test tube. Using this level as a guide, add 3

mL of the reagent to each of the other labeled test tubes. Mix the contents by rocking
the tube back and forth. Be sure the reagents mix otherwise the test may not work.

8. Place the test tubes into the boiling water bath for 5 full minutes.

9. Record your observations into your lab notebook and draw a conclusion as to whether

your unknown is a monosaccharide or not.

THE BENEDICT’S TEST
Check to see whether the hot water bath needs refilling and that it is boiling hot.

10. Label 5 test tubes as before and fill them as in Step 6 with the appropriate carbohydrate

solution.

11. Using a 10-mL graduated cylinder, measure 4 mL of Benedict’s reagent to the first test

tube and once again use this as a guide to fill the rest. Mix the contents by rocking the
tube back and forth. Be sure the reagents mix otherwise the test may not work.

12. Place the test tubes into the boiling water bath for 3 full minutes.

13. Record your color changes and record your observations in your lab notebook. Draw a

conclusion as to the meaning of these color changes.