Carbohydrates—Sugars
Carbohydrates are polyhydroxy aldehydes, polyhydroxy ketones, or compounds which can be
hydrolyzed to them. Carbohydrates include sugars, starches, and cellulose. In this experiment, different
reactions are used to distinguish different classes of sugars, and also, glucose pentaacetate, a derivative of
glucose, is synthesized.
Sugars are water-soluble carbohydrates. A sugar which cannot be hydrolytically converted into
simpler carbohydrates is called a monosaccharide. A sugar, which can be hydrolyzed into two equivalents
of monosaccharide, is called a disaccharide. Depending on the functional group it bears, a
monosaccharide can be classified as an aldose, if it contains an aldehyde group, or a ketose, if it contains a
keto group. Depending on the number or carbons it has, a monosaccharide can also be classified as: e.g., a
triose (three carbons), a tetrose (four carbons), a pentose (five carbons), a hexose (six carbons), etc. For
example arabinose, bearing an aldehyde terminating group on a five carbon chain, is an aldopentose, and
fructose, containing a keto group on a chain of six carbons, is a ketohexose.
CHO
CH 2 OH
HO
OH
O
H
OH
OH
HO
H
H
OH
CH 2 OH
CH 2 OH
D-Arabinose
D-Fructose
Disaccharides are made up of two monosaccharide units bound through glycoside linkage to each
other. Sucrose, table sugar, is a disaccharide which, on hydrolysis, gives two different monosaccharides:
glucose and fructose.
HOH2 C
Sucrose
O
HO
OH
OH
O OH
HOH2 C
CH 2OH
O
OH
Test to distinguish monosaccharides from disaccharides—Barfoed’s test
The reagent is cupric acetate in acetic solution. Monosaccharides react with this reagent within 5
minutes to give a red precipitate, cuprous oxide. Disaccharides, however, take a longer time to react since
the aldehyde function is masked as an acetal.
R
O
C
H
+
2 Cu2+
blue
R
O
C
OH
+
Cu 2O
red ppt
Test to distinguish ketoses from aldoses-Seliwanoff’s test
Under acidic conditions, ketoses dehydrate very rapidly to give furfural which then reacts with
resorcinol (1,3-dihydroxybenzene) to give colored product (Equation 1).
CH 2 OH
O
R
H
OH
H
H
OH
H
OH
H
HO
O
C H 2 OH
H
OH
OH
R
H+
H
-H 2 O
H
HO
O
CH O
H
OH
H
H+
O
R
CHO
-2 H 2 O
CH 2 OH
R = H ; fu rfu ra l
R = C H 2 OH ; 5 -h yd ro xy me thy lfu rfu ral
R = H ; k e to p en to se
R = C H 2 OH ; k eto h ex os e
HO
O
R
CHO
OH
resorcinol
red colored product
However, under the same conditions, the formation of furfural from aldoses is slow, presumably since a
β-elimination is required in the dehydration to furfural (Equation 2).
CHO
CH O
H C OH
H C OH
H C OH
-H 2 O
H C OH
C O
CH 2
H C
! -El im
H C OH
CH O
C H 2 OH
H C OH
H C OH
H C OH
H C OH
CH 2 OH
CH 2 OH
R
H
O
H
HO
CHO
H+
OH
R
O
CH O
-2 H 2 O
R = H ; a ld o pe n to s e
R = C H 2 OH ; al d oh e xo se
Test to distinguish pentoses from hexoses-Bial’s test
The reagent for the Bial’s test is a solution of orcinol 5-methyl-1, 3-benzenediol (5methylresorcinol) and ferric chloride in concentrated hydrochloric acid. As shown in equations (1) and
(2), under acidic conditions, pentoses undergo dehydration to give oxo-2, 4-cyclopentadiene-2carboxaldehyde (furfural) while hexoses give oxo-2, 4-cyclopentadiene-5-hydromethyl-2-carboxaldehyde
(5-hydroxymethylfurfural). Furfural reacts with orcinol in the presence of ferric chloride to give a blue-togreen color. 5-Hydroxymethylfurfural reacts with orcinol in the presence of ferric chloride to give a
brown-to-grey color.
Test to distinguish reducing sugars from non-reducing sugars-Benedict’s test or Fehling’s test
Aldehydes and α-hydroxy ketones reduce Benedict’s solution (an alkaline solution of cupric ion
complexed with citrate ion), or Fehling’s solution (an alkaline solution or cupric ion complexed with
tartrate ion).
R
O
C
H
+
2 Cu2+
blue
R
O
C
OH
+
Cu 2O
red ppt
Sugars containing these groups are known as reducing sugars. All monosaccharides are reducing
sugars. Disaccharides containing a free hemi-acetal group are also mild reducing agents. Most
disaccharides are reducing sugars (an important exception is sucrose).
O
OH
H OH 2 C
Fre e He m i-A ce tal
O
HO
Su c ros e
OH
O OH
C H 2 OH
OH
O
H OH 2 C
OH
Acetylation of glucose-preparation of glucose pentaacetate
In solution, sugars exist in more than one form. Often, the open straight chain form and a cyclic
hemiacetal form (six-membered ring or five-membered ring) are the major isomers present. In forming
the cyclic isomer, the carbonyl carbon becomes a chiral center. As a result, two stereoisomeric
configurations (anomers) are formed, the α-anomer and the β-anomer. The anomer in which the -OH
group at C-1 is below the ring (down) in the Haworth projection, is called α. The anomer in which the OH group at C-1 is above the ring (up) in the Haworth projection is called β.
6 CH 2 OH
1 CHO
H
HO
H
H
5
OH
H
OH
OH
6 CH 2 OH
4
O
OH
OH
3
1
2
OH
Haworth Projection of
!-D-glucopyranose
OH
6 CH 2 OH
5
4
D-Glucose
O
OH
OH
OH
3
1
Haworth Projection of
"-D-glucopyranose
2
OH
Glucose pentaacetate can be prepared by acetylation of glucose with acetic anhydride. In the
presence of an acidic catalyst (for example, zinc chloride), the preferred product is the α-anomer. In the
presence of a basic catalyst (such as sodium acetate), the β-anomer predominates.
1 CHO
H
OH
HO
H
H
H
OH
OH
6 CH 2 OH
D-Glucose
6 CH2 OAc
(CH 3 CO)2 O
Catalyst
5
4
AcO
O
OAc
3
OAc
1
2
OAc
H 3C
O
C
O
O
C
CH3
Acetic Anhydride (Ac 2 O)
O
C CH 3
Ac
Experimental Procedure: (Timing: one 3 h period, do part I while heating the reaction mixture in part II)
Put on your safety goggles
I. Test for Sugars:
Prepare a beaker of boiling water for this experiment. Perform the following tests for the following
1% sugar solutions: arabinose; fructose; glucose; lactose; maltose; sucrose, and an unknown obtained
from your instructor.
A. Barfoed’s Test
Place 1 mL of Barfoed’s reagent1 and 10 drops of each 1% sugar solutions in separate, labeled test
tubes. Heat the tubes in boiling water for 5 min. Remove the tubes and observe the results. The presence
of a red precipitate (cuprous oxide) indicates a positive test for monosaccharides (then do tests B, D, E
for these sugars). Disaccharides give negative test (then do tests B and C for these sugars).
B. Benedict’s Test
Place 1 ml of 1% sugar solutions in labeled test tubes and add 5 ml of Benedict’s reagent4. Heat the
tubes in boiling water for several min. A red precipitate is a positive test for reducing sugar.
C. Hydrolysis of a Disaccharide
Place 10 ml of the above 1% disaccharide solution in a large test tube. Add 1 mL of 3 N HCl, and
heat the solution in boiling water for 10 min. Carefully neutralize the solution with 3 N NaOH to give the
hydrolysis products (presumably monosaccharides). Then use the product of this step to do tests D and
E.
D. Seliwanoff’s Test
Place 10 drops of 1% sugar solutions in labeled test tubes and add 2 mL of Seliwanoff’s reagent2.
Heat the tubes in boiling water for 2 min. Ketohexoses give red solutions and ketopentoses give bluegreen solutions. Aldoses provide no color.
E. Bial’s Test
Place 10 drops of 1% sugar solutions in labeled test tubes. Add 2 mL of Bial’s reagent3 and heat the
tubes in boiling water until a color appears. Pentoses give a blue-to-green color solution. Hexoses give a
brown-to-grey color solution. If the color is not distinct, add 4 mL water and 1 mL of 1-butanol. Shake
the tube vigorously and observe the result. The colored product should be in the 1-butonal layer.
F. Unknown Sugar Sample
Depending on the results for the known sugars and comparing them to the results for the unknown,
determine which sugar the unknown is.
Flow Chart Form of Directions:
Perform
Test
A
(Barfoed’s)
on
all
sugars
If
sugar
tests
positive
(red
precipitate)
perform
the
following:
Perform
Tests
B
(Benedict’s),
D
(Seliwanoff’s),
E
(Bial’s)
If
sugar
tests
negative
follow
these
steps:
Perform
Test
B
(Benedict’s)
on
original
1%
sugar
solution.
Do
Step
C
(Hydrolisis
of
a
Disaccharide)
and
use
product
for
the
following:
Perform
Test
D
(Seliwanoff’s).
Perform
Test
E
(Bial’s).
Results from Part I:
Sugar solution
A. Barfoed’s
test
B. Benedict’s
test
C. Bial’s test
D. Seliwanoff’s Conclusion
test
(e.g.
aldopentose)
1.
2.
3.
4.
5.
6.
7.
Note
1
2
3
4
Barfoed’s reagent: Mix 33.3 g of cupric acetate with 5 mL of acetic acid, and then dilute to 500
mL.
Seliwanoff’s reagent: Dissolve 0.25 g of resorcinol in 500 mL of 6 N hydrochloric acid (1 volume
of concentrated HCl and 1 volume of distilled water).
Bial’s reagent: Dissolve 1.5 g of orcinol in 500 mL of concentrated hydrochloric acid, and then
add 2 mL of 10% aqueous ferric chloride.
Benedict’s reagent: Dissolve 88 g of hydrated sodium citrate and 50 g of anhydrous sodium
carbonate in 400 mL of distilled water with heating. Add 8.8 g of cupric sulfate crystals dissolved
in 50 mL of distilled water. Dilute the mixture to 500 mL. The solution should be clear, if not
filter it.
II. Acetylation of Glucose (do part 1 or 2)
CAUTION: Acetic anhydride is a lachrymator and should be measured under the hood on a
bicarbonate tray. Cap the bottle immediately after use. All glassware should be dry.
1.
Preparation of α-Glucose Pentaacetate
In a porcelain mortar, grind a small amount of anhydrous ZnCl2.5 Place 0.5 g of the powdered zinc
chloride in a 100 mL round-bottomed flask. Add 14 mL (15 g, 0.15 mol) of acetic anhydride. Attach an
air-cooled condenser to the flask, and heat gently. Slowly add 2.7 g (0.015 mol) of powdered anhydrous
glucose. Since the reaction is vigorous, do not add all the glucose in one portion. Remove the condenser
before addition, and replace the condenser and swirl the reaction mixture between additions. After all the
glucose is added, put the condenser in place, and heat the mixture for one hour. Then, slowly pour the
reaction mixture, with vigorous stirring, into a beaker containing 150 mL of crushed ice. Stir until all the
ice has melted, and the oil which separates solidifies. Collect the product, α-glucose pentaacetate, by
suction filtration. The product can be recrystallized form about 15 ml of methanol. (α-glucose
pentaacetate has mp 100° C)
2.
Preparation of β-Glucose Pentaacetate
In a porcelain mortar, grind and mix 2.7 g (0.015 mol) of anhydrous glucose and 1.5 g of sodium
acetate. Transfer the mixture to a 100 mL round-bottomed flask, and add 14 mL (15 g, 0.15 mol) of acetic
anhydride. Attach a condenser to it, and heat the mixture on a steam bath for one hour. Swirl the reaction
mixture frequently during the heating period. Then, slowly pour the mixture, with vigorous stirring, into a
beaker containing 150 mL of crushed ice. Stir until all the ice has melted. Collect the product with suction
filtration. The product, β-glucose pentaacetate, can be recrystallized from about 15 mL of methanol. (βglucose pentaacetate mp 134° C).
Note: Zinc chloride reacts with moisture in the atmosphere. Minimize its exposure to atmosphere
by carrying this out quickly.
Questions for Report:
1. How useful are the following reagents in classifying an unknown sugar? Barfoed’s reagent;
Seliwanoff’s reagent; Bial’s reagent; Benedict’s reagent.
2. What is a reducing sugar?
3. Define and illustrate:
a. Disaccharide
b. Monosaccharide
c. Ketohexose
d. Anomer
4. What is acetylation? Name two acetylating reagents.
5. In the preparation of glucose pentaacetate, the warm reaction mixture was poured, with stirring,
into 125 ml of ice in a beaker. What is the purpose of this step?