Identification of an Unknown Saccharide
Introduction
The purpose of this portion of the qualitative analysis project is to characterize an
unknown saccharide by measuring its melting point, observing its behavior under
mild oxidizing conditions, and observing its optical rotation. Your unknown will be
among those found in the table found in this handout. Once you have identified the
unknown, you will provide its cyclic and open chain structures (if any), as well as
classify it in several ways: reducing or non-reducing, monosaccharide or
disaccharide, aldose or ketose, pentose or hexose. The reaction scheme shown here
demonstrates the types of structures you will be encountering in this experiment.
(R) H
O
O
O
H (R)
OH
OH
H (R)
CH2 OH
!-form
open chain
form
"-form
Melting Point
You will determine the melting point in the usual way. Many carbohydrates undergo
decomposition at the melting point, so darkening and/or gas formation may be
observed. In addition, the melting point range may be wider than that for other pure
substances. You may wish to take the melting point two or three times get a good
idea of the melting range. Make sure you do not over-fill the melting point tube.
Oxidation Tests
Your saccharide unknown can be classified as a reducing sugar, or a non-reducing
sugar. Saccharides that give a positive Tollens test are classified as reducing, while
those that do not are classified as non-reducing. A reaction with Tollens reagent
signifies that the saccharide forms an aldehyde in equilibrium with several other
possible species under the conditions of the test (basic aqueous solution). You
should perform the test as it is described in your lab text and compare to at least
one positive control and one negative control.
Be aware that extended heating of a non-reducing glycoside in aqueous solution may
eventually hydrolyze it, forming one or more reducing sugars. This will give a falsepositive test.
Optical Rotation
As shown in the scheme above, if a saccharide is a reducing sugar, it exists in
hemiacetal or hemiketal and can open up to form a hydroxy aldehyde or hydroxy
ketone. The process of ring opening and closing is called mutarotation because of
the inherent change in optical rotation that is observed as such a process occurs. A
single open chain form can produce both α- and β-cyclic forms. In addition, both
six-membered rings (pyranoses) and five-membered rings (furanoses) can form,
each with their own α- and β-cyclic forms. If your saccharide can mutarotate, you
will observe an initial optical rotation that corresponds to the predominant form
found in the solid unknown. As the different forms equilibrate through the open
chain form, the optical rotation will change and reach a steady value after one or two
days.
For example, pure α-D-glucose has a specific rotation of 112.2˚ when initially
measured, but eventually the specific rotation eventually changes to 52.7˚ due the
presence of β- D-glucose in the equilibrium mixture. Since β-D-glucose is known to
have a specific rotation of 18.7˚, the equilibrium mixture contains approximately
36% α-anomer and 64% β-anomer.
The theory and procedures for determination of optical rotation, a, and specific
rotation, [α]D20, are found in your lab text in Technique 14 (p. 143-147). Note that
“α” is used both to symbolize optical rotation and to represent one of the saccharide
anomers.
Polarimetry Procedure
Make sure you “zero” the polarimeter by filling a tube with deionized water and then
calibrate the polarimeter by checking a 5.0% or 10.0% solution of sucrose. Practice
making careful polarimeter readings; the two sides of the screen will be of even
darkness not only at the correct rotation, but also at increments of 90˚ from the
correct rotation. Be sure to record both the sign and the magnitude of the rotation.
It is suggested to take several readings to calculate the average observed rotation.
Accurately weigh and precisely record the mass of approximately 1.5 grams (record
actual mass to 3 decimals) of your unknown in a 25 mL volumetric flask. Add
enough deionized water to make exactly 25 mL of solution. You must now work
quickly. Make sure the sample is dissolved before you continue. Rinse a clean
polarimeter cell with a few mL’s of your solution then quickly fill the cell and take
your readings. Note if the readings are moving in a certain direction or remaining
constant. Pour the solution back into the volumetric flask and add a few drops of
concentrated aqueous ammonia to the sugar solution. Wait a few moments for it to
equilibrate before recording the equilibrium optical rotation. Or, carefully pour the
solution back into the volumetric flask and stopper the flask. Store it until the
following lab period and determine the optical rotation again at that time.
Convert your experimental rotations, a, into specific rotations, [α]D20. Show your
work. Determine the identity of your unknown based on the sign and value of your
specific rotations, as well as the direction of the mutarotation (if any).
Dr. Kline and Dr. Anderson
Revised May 10, 2005
Carbohydrate
Melting Point
(°C)
[α ]D20
(initially)
[α ]D20
(equilibrium)
α-D-glucose hydrate
83
102
47.9
α-D-ribose
88-92
-23.1
-23.1
α-L-ribose
88-92
20.3
20.7
methyl β-D-glucopyranose
104
-34
N/A
Β-D-maltose hydrate
119-121
111.7
130.4
β-D-fructose
119-122
-132.2
-89.5
β-D-mannose
132
-17
14.2
α-D-mannose
133-140
29.3
14.2
D-mannose
133-140
N/A
14.2
β-D-glucose (dextrose)
148
18.7
52.7
α-D-glucose (dextrose)
153-156
112.2
52.7
α-D-xylose
156-158
93.6
18.8
β-L-arabinose
160-163
190.6
104.5
α-D-galactose
168-170
150.7
80.2
α-L-galactose
168-170
-150.7
-80.2
α-L-sorbose
171-173
-43.7
-43.4
D-sucrose
160-186
66.4
N/A
α-D-lactose hydrate
219
85
52.6
β-D-lactose (anhydrous)
252
34.9
55.4
(α, β-mixture)