carbohyd.

doc
CARBOHYDRATES

Two percent solutions of several mono- and di- saccharides have been prepared for you. Use
these test solutions (T.S.), and any unknown sugar solutions in the test procedures below. Record your
results as instructed on the accompanying Laboratory Report Form.
1 mL of the T.S. Place the test tube in a hot
THE MOLISCH TEST. All carbohydrates react water bath for about 5 minutes. Allow the tube
with Molisch's reagent (15% α-naphthol in EtOH) to cool. Note any change in the color of the
to give a purple color. The reaction is very solutions, and/or the formation of any precipi-
complex, but the test is a reliable indication for tates.
the presence of carbohydrates.
In a small (13 X 100) test tube (t.t.) mix RED TETRAZOLIUM
thoroughly 1 mL of the T.S. and 2 drops of Red tetrazolium (RT) is a nearly color-
Molisch's reagent. less, water-soluble substance that oxidizes
Place 1 mL of conc H2SO4 in a second aldoses and ketoses, as well as other α-ketols,
t.t. Hold the second t.t. at an angle of and thereby is reduced. The reduced form is a
approximately 30o; and very carefully pour the water-insoluble, intensely colored pigment, a
T.S. solution down the side of the second t.t. diformazan.
onto the H2SO4, so that two separate layers are
formed. Observe any color change at the
junction of the two liquids. 2H
N N Cl N N + HCl
N N N N
BENEDICT'S OR FEHLING'S TEST FOR
REDUCING SUGARS. The reagent is a
complex of copper(II) ions in base, resulting in Red Tetrazolium (RT) RT - Diformazan
an appealing blue-colored solution. ALL mono-
saccharides, whether aldoses or ketoses, give Red tetrazolium affords a highly sensitive test for
positive results with Benedict's or Fehling's reducing sugars, and it distinguishes between α-
solutions. Hence, they are referred to as ketols and simple aldehydes more sharply than
"reducing sugars" since they reduce the blue Fehling's and Tollens' tests, two older tests that
copper(II) ion to yield the insoluble orange-red were used for this purpose.
copper(I) oxide, Cu2O.

Combine 1 mL of Benedict's solution and Procedure

 Put one drop of each of the T.S.'s in
clean, marked test tubes, and to each test tube,
add 1/2 mL (10 drops) of a 0.5% aqueous Procedure
solution of red tetrazolium and 1 drop of 3M In labeled 10 X 75 mm test tubes, place in each,
NaOH soln. Put the tubes into a beaker of hot 1 mL of the T.S. Place 1 mL of distiled water
water and note the time at which each tube into another t.t. to serve as a reference or control
develops color. solution.
BARFOED'S TEST To each tube add 1 mL of Bial's reagent
Barfoed's test is used to distinguish between and a boiling chip. Place in a boiling water bath
reducing monosaccharides and disaccharides. and note the color that develops. In tubes that
The reagent is copper(II) acetate in acetic acid. have weak or no color, the product of the
Procedure: reaction can be concentrated by diluting the
Label a 10 X 75 mm test tube for each T.S. you mixture with 2 mLs of water, adding 0.5 mL of
are going to use. Place 1 mL (20 drops) of the cyclohexanol, and then extracting the colored
T.S. into the test tube, add 0.5 mL (10 drops) of product into the alcohol layer by shaking vigor-
Barfoed's reagent and mix. Place the test tubes ously.
into a hot-water bath for 15 minutes. Note the
time it takes for a red precipitate to appear. SELIWANOV'S TEST
Seliwanov's test is used to distinguish
BIAL'S TEST between aldoses and ketoses. The reagent
Bial's test is used to distinguish between pent- consists of 1,3-dihydroxybenzene (resorcinol) in
oses and hexoses. The reagent consists of 3,5- aqueous hydrochloric acid.
dihydroxytoluene and iron(III) chloride in Procedure
hydrochloric acid. To 10 X 75 mm test tubes, add 1 mL of Seliwa-
nov's reagent. To each t.t. add 5 drops of a T.S.
Mix and heat the tubes in a boiling water bath for
2 minutes. The intensity of the color is
proportional to the α-ketol concentration.
.The Hydrolysis of Sucrose to yield Each of you will take your turn reading
“Invert Sugar” the polarimeter. In your Laboratory Report

Your instructor will prepare a sucrose (table Form, record the values of observed rotation,

sugar) solution. When it is prepared, the α, and the TIME at which you read the

disaccharide will begin to undergo hydrolysis polarimeter. Using the formula provided,

to yield equal amounts of glucose and calculate the specific rotation, [α] , and record
fructose. As the hydrolysis proceeds, the it in acceptable form.

optical rotation of the sucrose solution will

decrease, and ultimately cross from positive
(dextrorotatory) values to negative
(levorotatory) values; i.e. the rotation will
have been inverted. Hence, the hydrolyzed
sucrose is known is “invert sugar”.

________________________

OPTICAL ROTATION
A polarimeter has been set up, and a
polarimeter cell containing a sugar
solution has been prepared. When
directed by your instructor, go to the
polarimeter and take a reading of the
rotation of the sugar solution. Your
instructor will help you read the
vernier scale (see illustration). Write
down the pertinent data; the time;
and record your calculated value on
the Laboratory Report Form.

carbohyd-lr.doc
LABORATORY REPORT FORM ON CARBOHYDRATES
 NAME: Section: Date:

1. Complete the CYCLIC structures of the mono- and di- saccharides you used by placing their
OH groups (ONLY) in their proper positions.

glucose fructose galactose

CH2OH CH2OH
CH2OH OH
O O O
OH OH

H CH2OH

sucrose maltose lactose

CH2OH
CH2OH
O
CH2OH CH2OH O
OH
O O
OH CH2OH O
O O
O
CH2OH
O

H CH2OH

2. Complete the chart below indicating how each sugar responded for the tests performed.
A "+" indicates a positive test. LEAVE IT EMPTY FOR A NEGATIVE TEST!

Name of Sugar Molisch Benedict's RT Barfoed's

Glucose
Fructose
Galactose
Lactose
Maltose
Sucrose
Unknown ( )
Unknown ( )
Unknown ( ) O

C H
3. Complete the H C OH equation below for the reaction of glucose with Benedict's solution. Write
the structure for 2+ the oxidation product of glucose; and the formula
for the orange- HO C H Cu(citrate) (OH)2 red precipitate.
H C OH

H C OH

CH2OH
4. Shown below is the equation for the formation of the osazone of D-glucose AND of D-mannose:

CH O
CH O CH NNH
HO C H
H C OH C NNH
3 H2N NH 3 H2N NH
HO C H
HO C H HO C H
H C OH
H C OH H C OH
H C OH
H C OH H C OH
CH2OH
CH2OH CH2OH
D-glucose D-mannose

NOTE: that carbons 1 and 2 are involved. Thus, aldoses which differ only at carbon number 2
(they are called epimers) will form identical osazones.
Refer to the Table of Aldoses in your text, and list BY NAMES (not necessarily their structures),
the FOUR EPIMERIC PAIRS of tne D-aldohexoses which will form identical osazones.

5. Calculate the specific rotation: [ α] = α / (concentration X cell length in decimeters)

Time of observation: (The clock on the wall occasionally works.)

Observed rotation: α = (What you read at the polarimeter)
Temperature: (Look at your thermometer for the room temp.)
Solvent: (usually, 15% sulfuric acid)
Cell Length: (Unless told otherwise, it should be 2 decimeters.)
Concentration: gm/mL ( usually, 15.5 gm in 50 mLs)
Record your value for specific rotation:

[ α]=