Biomolecules: Identification of an Unknown Sugar
In this lab period, you will determine the identity of an unknown sugar. You
will conduct up to three experiments to collect the data needed to prove the identity
of the unknown. The data that might be obtained includes the time for an osazone
to form, the decomposition point and the specific rotation of the sugar.
Osazone Formation
When one equivalent of an aldehyde or ketone (i.e., compounds that contain
a carbonyl group) reacts with phenylhydrazine, the product is called a
phenylhydrazone. For example, the reaction between benzaldehyde and
phenylhydrazine yields the phenylhydrazone of benzaldehyde as shown in Figure
1.
H
C NN
H
H
C O + H2NN
H
benzaldehyde
phenylhydrazine
+ H2O
phenylhydrazone of benzaldehyde
Figure 1. Preparation of a phenylhydrazone.
When glucose, a pentahydroxyaldehyde or aldohexose, reacts with an excess
of phenylhydrazine, the product is not a phenylhydrazone but a phenylosazone or
simply osazone. An osazone contains two phenylhydrazone groupings. Figure 2
shows the reaction of glucose with an excess of phenylhydrazine.
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H
C O
H
OH
H
HO
H
+ xs H2NN
H
OH
H
OH
CH2OH
D-glucose
H
C NNHPh
C NNHPh
HO
H
H
OH
H
OH
CH2OH
phenylosazone
of D-glucose
phenylhydrazine
Figure 2. Preparation of an osazone from D-glucose.
Mannose, a pentahydroxyaldehyde that differs in configuration from glucose
only at C-2, gives an osazone with exactly the same structure as that of the osazone
of glucose as shown in Figure 3.
H
C O
HO
H
H
HO
H
+ xs H2NN
H
OH
H
OH
CH2OH
D-mannose
H
C NNHPh
C NNHPh
HO
H
H
OH
H
OH
CH2OH
phenylhydrazine
phenylosazone
of D-mannose or
D-glucose
Figure 3. Preparation of an osazone from D-mannose.
How can these reactions help us determine whether we have glucose or
mannose as an unknown? The answer is by timing the reactions. The
osazone from glucose forms in approximately five minutes; whereas, the osazone
from mannose forms in less than one minute. Thus, if we allow our unknown
glucose or mannose to start forming an osazone at exactly the same time we start
osazone formation with authentic samples of glucose and mannose, our unknown
will form its osazone at the same time as one of the known compounds but not the
other. Thus, our sugar forms its osazone simultaneously with one of the known
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sugars. Osazone formation is a chemical property that is useful for the
identification of an unknown sugar.
Decomposition Point
Most sugars do not melt; they decompose. When table sugar or sucrose is
heated, a black color often appears because the sucrose has decomposed. Thus,
when a sugar is heated in a melting point capillary tube, the result in not a melting
range of temperatures, but a temperature at which the sugar appears to decompose.
The temperature at which the sugar decomposes is the decomposition point.
Like melting points, decomposition points are a function of the compound, and
different sugars have different decomposition points. Thus, decomposition points
are useful physical constants for identifying unknown sugars.
Specific Rotation
During the first semester, you learned the meaning of specific rotation. The
specific rotation of a compound is the rotation in a 1-decimeter tube of a
solution made by dissolving one gram of the compound in 100-mL solvent. Thus,
specific rotation is a defined term. We usually do not measure exactly one gram in
100-mL of solvent. We usually calculate the specific rotation, which is a constant.
The symbol for specific rotation is a Greek alpha in square brackets, []. The
observed rotation is simply the Greek alpha, . Thus, the specific rotation is
calculated by equation 1, where c is the concentration in grams per 100 mL and l is
the path length in decimeters. Because [] is a defined term, it has units of degrees
(o) instead of units implied by equation 1.
[]D = /(c  l)
(1)

he specific rotation of sugars cannot be determined immediately after
dissolving them in water. Sugars must be allowed to spend several days in the
aqueous solution before the specific rotation is obtained. To understand why this is
so, we need to understand the term mutarotation. Mutarotation is the
equilibration of the two anomers of the sugar in solution. Glucose exists in cyclic
forms (two anomers) known as -glucopyranose and-glucopyranose, depending
on the configuration at the anomeric carbon atom. These forms equilibrate (come
to a position of equilibrium) in water so that the ratio of alpha to beta forms
remains constant. Thus, the specific rotation of the sugar after it undergoes
mutarotation is a constant, which is the literature value. Figure 4 shows the
equilibria involved in the mutarotation of D-glucose.
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H
C O
CH2OH
O
H
OH
OH
HO
H
H
OH
H
H
OH
OH
CH2OH
H
OH
H
OH
O
H
H
OH
H
H
OH
OH
H
OH
CH2OH
-D-glucopyranose
(-anomer)
62.6%
D-glucose
(open form)
0.002%
-D-glucopyranose
(-anomer)
37.3%
Figure 4. Mutarotation of glucose.
At equilibrium, the  ratio of glucose is about 37/63. The specific rotation
of the mixture is +53o, which is the value calculated from an observed rotation by
equation 1. You will prepare an aqueous solution of your unknown one week in
advance of determining its specific rotation to allow for mutarotation. The specific
rotation is another physical constant that is useful in determining the identity of a
sugar.
Table 1 lists sugars and properties that can be used to identify them. You
will determine the identity of one of the sugars, which will be issued as an
unknown.
Table 1. Sugars.
Decomp.
Approximate
D
Name of Sugar
Temperature [] 20
time for osazone
o
( C)
to form (min.)
D-glucose (hydrated)
90
+48
4-5
maltose (hydrated)
100
+129
soluble
D-fructose (hydrated)
104
-92
2
D-mannose
132
+14
0.5
D-xylose
145
+19
7
D-Glucose (anhydrous)
146
+53
4-5
L-arabinose
160
+113
10
maltose (anhydrous)
165
+129
soluble
sucrose
185
+66
45
lactose
203
+113
soluble
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Procedure
Preparation of Solution for Specific Rotation
(To be conducted one week before observing the rotation)
1. Obtain an unknown sugar, show the instructor the number of the unknown and
record it in your lab notebook.
2. Keep about 0.3 g of the unknown for next week’s experiments.
3. Tare a 25-mL volumetric flask on an analytical balance that measures to four
decimal places (instrument room).
4. Transfer the remainder of the unknown sugar (about 3 g) to the volumetric flask
and record the mass of the unknown.
5. Add distilled water to the volumetric flask until the water level reaches about 2
cm below the etched mark on the flask.
Water will be added to the mark at the next lab meeting. The sugar will
undergo mutarotation during this time.
6. Stopper the volumetric flask, mark it with your initials, and save it in the
designated location until next week.
Specific Rotation of the Unknown Sugar
1. Retrieve the 25-mL volumetric flask that contains your unknown sugar.
2. Add distilled water to the mark on the flask.
The bottom of the meniscus should appear to touch the etched mark when
the neck of the flask is viewed from the side.
3. Shake the flask to ensure the contents are thoroughly mixed.
4. Carefully fill a clean polarimeter tube with the unknown sugar solution.
The tube diameter is flared at one end. Fill at the narrow end.
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After sealing the tube, gently rotate it so that any air bubbles present collect in
the flared end.
5. Measure the observed rotation  and calculate the specific rotation [] by
equation 1.
Take care to use the correct units of path length and concentration in the
calculations.
Osazone Formation
The key to a good result with this experiment is to prepare the six test
tubes as fast as possible, to ensure the contents of each test tube are thoroughly
mixed, to place all of the test tubes in the water at the same time, and to remove a
test tube only after the osazone has formed in it.
1. Prepare a boiling-water bath by filling a large beaker (i.e., one that can hold six
or seven 6-in test tubes) half full of water and heating the beaker on a hot plate.
2. Place a 0.1-g sample of your unknown and five known sugars in separate 6-in
test tubes marked for identification.
The five known sugars that form osazones are glucose, fructose, mannose,
sucrose, and xylose.
3. Add the following reagents to each test tube in the following order.
a. 4 mL distilled water
b. 0.5-mL saturated sodium bisulfite, NaHSO3, solution
c. 0.3-g solid sodium acetate
4. Mix the contents of each tube thoroughly with a stirring rod. Clean the stirring
rod after each usage by dipping it in a beaker of water and drying it with a paper
towel.
Note: Speed is important in the next three steps. Read them before you do
them and then do them as fast as possible.
6. Ensure the water in your bath is boiling, and the water level in the beaker rises
above the liquid level of the solutions in the test tubes.
5. Add 0.2-g solid phenylhydrazine hydrochloride as quickly as possible to the six
tubes in the following order: (1) mannose, (2) fructose, (3) glucose, (4) xylose, (5)
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sucrose, and (6) your unknown. (The reaction will start as soon as you add this
reagent, so be quick.)
6. Place all six test tubes in the boiling water at the same time and record the start
time to the nearest second. Do not remove any test tube from the water unless you
observe a precipitate—any color change can be regarded as a precipitate)
The five known sugars will form osazones over a 45-min period, starting
with mannose (turns white almost immediately and precipitates in about 30 sec)
and ending with sucrose (yellow precipitate in about 45 min).
7. Constantly monitor the test tubes. As soon as the osazone of mannose forms,
remove its test tube. If your unknown is the next to precipitate, you have mannose
as an unknown. Next, fructose’s osazone will precipitate. Remove its test tube. If
you have fructose as an unknown, your unknown will precipitate following
fructose’s. Remove each known as its osazone forms, leaving your unknown and
the remaining candidates in the hot water. The unknown’s osazone should
precipitate immediately after the authentic sample precipitates.
It is not necessary to wait for the osazones of sugars that precipitate after
your unknown has precipitated. You are finished as soon as you identify your
unknown.
8. Some osazones are water soluble. See Table 1. If you have one of these sugars
as an unknown, you must conduct at least one of the other two experiments to
identify it.
Decomposition Point
1. Using a melting point apparatus, determine the decomposition point of your
unknown sugar.
2. Compare the decomposition temperature with those in Table 1.
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Carbohydrate Questions
Last name__________________________, First name___________________________
Number of Unknown Sugar_______
My unknown is: __________________________
1. The cyclic forms of D-glucose are -D-glucopyranose and -D-glucopyranose.
They differ in configuration at the anomeric carbon atom; therefore, they are called
anomers. What is the general term for stereoisomers that are not enantiomers?
Ans.______________________________________ The general term for two
stereoisomers, like anomers, that differ in only one of several chirality centers is
epimer. Hence, anomers are a special case of epimers.
2. What was the name of the sugar derivative you made in lab? ______________
(i.e., what do you get when you react a sugar with phenylhydrazine?)
3. Draw the structure of a furanose.
4. Draw a Fischer structure of mannose.
5. Draw the structure of -D-glucopyranose in a Haworth projection and in a chair
conformation.
6. Label the anomeric carbon atoms on the structures of problem 5 with a star (*).
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7. How does an aldose differ from a ketose?
8. How does an aldohexose differ from an aldotetrose?
9. Draw structures for L-erythrose and L-threose.
10. Name the seven D-sugars that are diastereomers of D-glucose.
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