Biomolecules: Identifcation 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 specifc rotation of the sugar.
Osazone Formation
hen one e!uivalent of an aldehyde or ketone "i.e., compounds that contain a carbonyl
group# reacts with phenylhydrazine, the product is called a phenylhydrazone. $or example, the
reaction between benzaldehyde and phenylhydrazine yields the phenylhydrazone of
benzaldehyde as shown in $igure %.
C O
H
+ H
2
NN

phenylhydrazine
H
C NN
H
H
+ H
2
O
benzaldehyde
phenylhydrazone of benzaldehyde
Figure 1. &reparation of a phenylhydrazone.
hen glucose, a pentahydroxyaldehyde or aldohexose, reacts with an excess of
phenylhydrazine, the product is not a phenylhydrazone but a phenylosazone or simply osazone.
'n osazone contains two phenylhydrazone groupings. $igure ( shows the reaction of glucose
with an excess of phenylhydrazine.
C O
OH H
H HO
OH H
OH H
CH
2
OH
H
+
H
2
NN
H
xs
C NNHPh
C NNHPh
H HO
OH H
OH H
CH
2
OH
H
D-glucose
phenylhydrazine
phenylosazone
of D-glucose
Figure 2. &reparation of an osazone from )*glucose.
+annose, a pentahydroxyaldehyde that di,ers in confguration from glucose only at -*(,
gives an osazone with exactly the same structure as that of the osazone of glucose as shown in
$igure ..
Lab 14 1
C O
H HO
H HO
OH H
OH H
CH
2
OH
H
+
H
2
NN
H
xs
C NNHPh
C NNHPh
H HO
OH H
OH H
CH
2
OH
H
D-mannose
phenylhydrazine
phenylosazone
of D-mannose or
D-glucose
Figure 3. &reparation of an osazone from )*mannose.
/ow can these reactions help us determine whether we have glucose or mannose as an
unknown0 The answer is by timing the reactions. The
osazone from glucose forms in approximately fve minutes1 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 sugars. 2sazone formation is a chemical property that is useful for the identifcation
of an unknown sugar.
Decomposition Point
+ost sugars do not melt1 they decompose. hen 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. 3ike melting points, decomposition points are a function of the
compound, and di,erent sugars have di,erent decomposition points. Thus, decomposition points
are useful physical constants for identifying unknown sugars.
Specifc Rotation
)uring the frst semester, you learned the meaning of specifc rotation. The specifc
rotation of a compound is the rotation in a %*decimeter tube of a solution made by dissolving
one gram of the compound in %44*m3 solvent. Thus, specifc rotation is a defned term. e
usually do not measure exactly one gram in %44*m3 of solvent. e usually calculate the specifc
rotation, which is a constant. The symbol for specifc rotation is a 5reek alpha in s!uare brackets,
6 7. The observed rotation is simply the 5reek alpha, . Thus, the specific rotation is calculated
by e!uation %, where c is the concentration in grams per %44 m3 and l is the path length in
decimeters. 8ecause 6 7 is a defined term, it has units of degrees "
o
# instead of units implied by
e!uation %.
6 7
)
9 :" c l# "%#
he specific rotation of sugars cannot be determined immediately after dissolving them in
water. ;ugars must be allowed to spend several days in the a!ueous solution before the specifc
rotation is obtained. To understand why this is so, we need to understand the term mutarotation.
Lab 14 2
Mutarotation is the e!uilibration of the two anomers of the sugar in solution. 5lucose exists in
cyclic forms "two anomers# known as *glucopyranose and *glucopyranose, depending on the
confguration at the anomeric carbon atom. These forms e!uilibrate "come to a position of
e!uilibrium# in water so that the ratio of alpha to beta forms remains constant. Thus, the specifc
rotation of the sugar after it undergoes mutarotation is a constant, which is the literature value.
$igure < shows the e!uilibria involved in the mutarotation of )*glucose.
C O
OH H
H HO
OH H
OH H
CH
2
OH
H
D-glucose
(open form)
0.002%
H
OH
OH
H
H
OH H
OH
CH
2
OH
O
H
OH
H
OH
H
OH H
OH
CH
2
OH
O
-D-glucopyranose
(-anomer)
62.6%
-D-glucopyranose
(-anomer)
37.3%
Figure 4. +utarotation of glucose.
't e!uilibrium, the ratio of glucose is about .=:>.. The specific rotation of the
mixture is ?@.
o
, which is the value calculated from an observed rotation by e!uation %. You will
prepare an a!ueous solution of your unknown one week in advance of determining its specifc
rotation to allow for mutarotation. The specifc rotation is another physical constant that is useful
in determining the identity of a sugar.
Table % lists sugars and properties that can be used to identify them. You will determine
the identity of one of the sugars, which will be issued as an unknown.
Tale 1. ;ugars.
Aame of ;ugar
)ecomp.
Temperatu
re
"
o
-#
6 7
)
(4
'pproximate
time for osazone
to form "min.#
)*glucose "hydrated# B4 ?<C <*@
maltose "hydrated# %44 ?%(B soluble
)*fructose "hydrated# %4< *B( (
)*mannose %.( ?%< 4.@
)*xylose %<@ ?%B =
)*5lucose "anhydrous# %<> ?@. <*@
3*arabinose %>4 ?%%. %4
maltose "anhydrous# %>@ ?%(B soluble
sucrose %C@ ?>> <@
lactose (4. ?%%. soluble
!rocedure
Preparation of Solution for Specifc Rotation
"To e conducted one week efore oser#ing t$e rotation%
Lab 14 3
%. 2btain an unknown sugar, show the instructor the number of the unknown and record it in
your lab notebook.
(. Deep about 4.. g of the unknown for next weekEs experiments.
.. Tare a (@*m3 volumetric Fask on an analytical balance that measures to four decimal places
"instrument room#.
<. Transfer the remainder of the unknown sugar "about . g# to the volumetric Fask and record
the mass of the unknown.
@. 'dd distilled water to the volumetric Fask until the water level reaches about ( cm below the
etched mark on the Fask.
Water will be added to the mark at the net lab meetin!" #he su!ar will under!o
mutarotation durin! this time"
>. ;topper the volumetric Fask, mark it with your initials, and save it in the designated location
until next week.
Specifc Rotation of the $nknown Su!ar
%. Getrieve the (@*m3 volumetric Fask that contains your unknown sugar.
(. 'dd distilled water to the mark on the Fask.
#he bottom of the meniscus should appear to touch the etched mark when the neck of
the %ask is &iewed from the side"
.. ;hake the Fask to ensure the contents are thoroughly mixed.
<. -arefully fll a clean polarimeter tube with the unknown sugar solution.
#he tube diameter is %ared at one end" Fill at the narrow end"
'fter sealin! the tube( !ently rotate it so that any air bubbles present collect in the %ared
end"
@. +easure the observed rotation and calculate the specific rotation 6 7 by e!uation %.
#ake care to use the correct units of path len!th and concentration in the calculations"
Osazone Formation
#he key to a !ood result with this eperiment is to prepare the si test tubes as fast as
possible( to ensure the contents of each test tube are thorou!hly mied( to place all of the
test tubes in the water at the same time( and to remo&e a test tube only after the osazone has
formed in it"
%. &repare a boiling*water bath by flling a large beaker "i.e., one that can hold six or seven >*in
test tubes# half full of water and heating the beaker on a hot plate.
Lab 14 4
(. &lace a 4.%*g sample of your unknown and fve known sugars in separate >*in test tubes
marked for identifcation.
#he f&e known su!ars that form osazones are !lucose( fructose( mannose( sucrose( and
ylose"
.. 'dd the following reagents to each test tube in the following order.
a. < m3 distilled water
b. 4.@*m3 saturated sodium bisulfte, Aa/;2., solution
c. 4..*g solid sodium acetate
<. &i' the contents of each tube thorou!hly with a stirring rod. -lean the stirring rod after each
usage by dipping it in a beaker of water and drying it with a paper towel.
(ote: S)eed is im)ortant in t$e ne't t$ree ste)s. *ead t$em efore +ou do t$em and
t$en do t$em as fast as )ossile.
>. Hnsure the water in your bath is boiling, and the water level in the beaker rises above the
li!uid level of the solutions in the test tubes.
@. 'dd 4.(*g solid phenylhydrazine hydrochloride as !uickly as possible to the six tubes in the
following orderI "%# mannose, "(# fructose, ".# glucose, "<# xylose, "@# sucrose, and "># your
unknown. "The reaction will start as soon as you add this reagent, so be !uick.#
>. &lace all six test tubes in the boiling water at the same time and record the start time to the
nearest second. )o not remove any test tube from the water unless you observe a precipitateJ
any color change can be regarded as a precipitate#
#he f&e known su!ars will form osazones o&er a )*+min period( startin! with mannose
,turns white almost immediately and precipitates in about -. sec/ and endin! with sucrose
,yellow precipitate in about )* min/"
=. -onstantly monitor the test tubes. 's soon as the osazone of mannose forms, remove its test
tube. If your unknown is the next to precipitate, you have mannose as an unknown. Aext,
fructoseEs osazone will precipitate. Gemove its test tube. If you have fructose as an unknown,
your unknown will precipitate following fructoseEs. Gemove each known as its osazone forms,
leaving your unknown and the remaining candidates in the hot water. The unknownEs osazone
should precipitate immediately after the authentic sample precipitates.
0t is not necessary to wait for the osazones of su!ars that precipitate after your
unknown has precipitated" 1ou are fnished as soon as you identify your unknown"
C. ;ome osazones are water soluble. ;ee Table %. If you have one of these sugars as an unknown,
you must conduct at least one of the other two experiments to identify it.
Decomposition Point
%. Ksing a melting point apparatus, determine the decomposition point of your unknown sugar.
(. -ompare the decomposition temperature with those in Table %.
Lab 14 5
,aro$+drate -uestions
;tu AoLLL ;ecLLLL3ast nameLLLLLLLLLLLLLLLLLLLLLLLLLL, $irst LLLLLLLLLLLLLLLLLLLLL
Aumber of Knknown ;ugarLLLLLLL +y unknown isI LLLLLLLLLLLLLLLLLLLLLLLLLL
"In the multiple*choice !uestions, put an M for all answers that are correct.#
%. The * and * forms of )*glucopyranose may be described asI
LL'. anomers
LL8. epimers
LL-. enantiomers
LL). diastereomers
(. hat is the generic name of the sugar derivative you made in lab0
"i.e., what do you get when you react a sugar with phenylhydrazine0#
'ns.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL
.. ' furanose is a cyclic form of a sugar that contains a furan*like structure. $uran isI
LL'. a fve*member ring.
LL8. a cyclic ether.
LL-. a fve*member lactone.
LL). an ester.
<. )raw a $ischer structure of glucose.
@. )raw the structure of * )*glucopyranose in a chair conformation.
>. &ut a star "N# on the anomeric carbon atom on your structure of problem @.
=. ' ketotetrose containsI
LL'. an aldehyde
LL8. a ketone
LL-. four carbon atoms.
LL). two primary alcohols.
C. 'n aldopentose containsI
LL'. an aldehyde
LL8. a ketone
LL-. a primary alcohol.
LL). fve carbon atoms.
B. )raw structure of 3*erythrose in a sawhorse proOection.
%4. Aame the seven )*sugars that are diastereomers of )*glucose.
Lab 14 6
%.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL
(.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL
..LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL
<.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL
@.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL
>.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL
=.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL
Lab 14 7