REDUCING TEST FOR CARBOHYDRATES
Biochemistry lab
TEST BASED ON THE REDUCING PROPERTIES OF SUGARS
REDUCING SUGARS
• It must have a free aldehyde or ketone group
• Common dietary monosaccharides: glucose,
galactose and fructose
REDUCING TEST FOR CARBOHYDRATES
BENEDICT’S TEST
• It is a very sensitive test done under mildly alkaline
conditions
• Benedict’s reagent: composed of CuSo4, Na2CO3,
and sodium citrate
• Positive result: change of color of benedict’s reagent
into brick red precipitate
• Also used to test for the presence of glucose in urine
IMPORTANT NOTES
• Positive result: brick red precipitate
• CuSO4: copper (II) sulfate
• Na2CO3: sodium carbonate
• Most commonly used laboratory test for routine test
PRINCIPLE:
• when a benedict’s solution and simple carbohydrates
are heated, the solution changes to orange/brick red.
This reaction is caused by the reducing property of
simple carbohydrates. The copper (II) ions in the
benedict’s solution are reduced to copper (I) ions,
which causes the color change.
IMPORTANT NOTES
• copper (CU) ions has the charge of +2 and +1.
• CU+2 will be reduced to CU+1 which causes the color
change
• Aldose(Glucose) + benedict’s solution(cupric ion or
Cu(citrate)2), then it will be subjected to heat, and the
benedict’s reagent will have a cuprous oxide(Cu2O(s)
which is +1 which is solid and gives off a brick red
precipitate
• Cupric ion is the higher; +2
INDICATION:
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IMPORTANT NOTES
• Blue solution: color of benedict solution (no other
reagent added)
• Green/yellow precipitate: traces of reducing sugar
• Orange red precipitate: moderate reducing sugar
present
• Brick red: large amount of reducing sugar
• Sample: urine
• Aim: to detect the presence or reducing sugar in urine
PROCEDURE:
• using a pipette, accurately take 5 ml of Benedict’s
reagent and slowly transfer it to the test tube.
• Take 5 ml of freshly collected urine by pipette and add
it to the test tube with benedict’s reagent.
• Place the test tube in boiling water bath for 2-3
minutes
• Observe the color of the solution and note whether a
precipitate was formed.
• Avoid prolonged heating
• Record your results
IMPORTANT NOTES
• Urine is freshly collected (maximum of 4-6 hours)
• Urine must NOT have a glucose content because it is
the indication of diabetes
BARFOED’S TEST
• Barfoed’s reagent – contains cupric acetate in dilute
acetic acid
• It oxidizes monosaccharides but not oligosaccharides
• Disaccharides are less oxidized but are oxidized if
they undergo prolonged heating which causes
hydrolysis of the disaccharides into monosaccharides
and produces a positive result.
• It is used to distinguish between monosaccharides,
disacchardies and oligosaccharides.
• Positive result: brick red precipitate
• Unlike benedict’s test, the test is carried out under
acidic rather than basic medium.

IMPORTANT NOTES
• Almost same with benedict’s test but the only
difference is the barfoed is in acidic medium while
benedict’s test is in basic medium.
• Cuprous oxide gives the brick red color
• Barfoed’s test is a copper reduction test
PROCEDURE:
• Place 1 ml of each 3% solutions of glucose, xylose,
fructose and lactose and starch in separately labeled
test tubes.
• Add 3 ml of barfoed’s reagent in each test tube
• Place the test tube in boiling water bath for 2-3
minutes
• Observe the color of the solution and note whether a
precipitate was formed.
• Avoid prolonged heating
• Record your results
IMPORTANT NOTES
• Set a timer
• Faster result: monosaccharide is present
• Slower result: disaccharide is present
• Remain blue if carbohydrate is absent
• Positive result: brick red precipitate
TOLLEN’S TEST
• Also known as Silver mirror test
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• Sugars with aldehyde groups are capable reducing
tollen’s reagent.
• Tollen’s reagent: ammoniacal solution of silver
• Positive result: gray to black precipitate (silver mirror)
IMPORTANT NOTES
• Active component is the ammoniacal silver nitrate
PROCEDURE:
• Place 5 drops of 3% solutions of glucose, xylose, and
sucrose in separate test tubes.
• add 2 ml of Tollen’s reagent into each test tube
preparation: Add 1 drop of NaOH solution to 6 ml of
5% AgNO3. Add dilute NH4OH (1 ml concentrated
NH4OH + 5 ml water) until the brown precipitate of
silver oxide that forms just dissolves. This reagent
must be prepared fresh and not stored since it
decomposed when left standing and yields an
explosive decomposition product. Discard all leftover
materials.
• Boil for about 5 minutes. Note and record your
observations.
IMPORTANT NOTES
• Tollen’s reagent: 1 drop of Naoh + 6 ml6 ml of 5%
AgNO3 + diluted NH4OH
• Diluted NH4OH: 1 ml concentrated NH4OH + 5ml
water)
• Add NaoH (sodium hydroxide) solution drop wise to
get the precipitate of silver oxide.
• Add 2 drops of ammonium hydroxide solution while
shaking the mixture and the previously formed
precipitate dissolves.