DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
UNIVERSITY OF DHAKA
Determination of lactose content of milk by Benedict’s Method
Principle
Benedict reagent for the estimation of reducing sugars contains potassium thiocyanate
as well as coppersulfate, and in the presence of the former a white precipitate of
cuprous thiocynate is formed in the reduction instead of the usual red precipitate of
cuprous oxide. The small amount of potassium ferecyanide also aids in keeping
cuprousoxide in solution. As the precipitate formed is readily observed. The alkali used
is sodium carbonate, which has the advance over hydroxides of trying less likely to
cause destruction of small amounts of sugar. The solution also has the great advantage
of being stable for an indefinite length of time.
Reagent
1.
2.
3.
4.
Sodium tungstate
Sulfuric acid
Standard lactose solution
Benedict’s quantitative solution
:
:
:
:
10.0% solution
0.75 N
0.5% (freshly prepared)
Dissolve 200.0g Na-citrarte, 100.0g anhydrous
Na3CO3 and 125.0g Potassium thiocyanate in
600 ml water with the aid of gentle heating.
Filter, cool and add 18.0g of copper sulfate
dissolved in 100 ml water. Pour slowly, stirring
continuously. Add 5.0 ml of 5.0% potassium
ferrocyanide solution and make upto a litre with
water. Filter, if not clear.
Procedure
Pipette 10.0 ml of milk into a 100.0 ml volumetric flask and add 5 ml of 10% Ntungstate solution and 5 ml of 0.75 N sulfuric acid solution. Mix well stand for 5.0 min
and make upto the mark with water. Mix well and filter (Different proportions of milk
may be used, if necessary keeping the reagent content fixed). In the case of Cow’s
milk it is suggested to take 20.0 ml milk and 12.0 ml of the reagent due to different
proportion of lactose and protein. Take the clear filtrate into the burette.
Pipette 25.0 ml of Benedict’s and solution in a conical flask and add freshly prepared
10 ml of 40 Na2Co3 solution. Introduce a few pieces of pumice stone to reduce
bumping. Bring it to boioling and while boiling titrate the solution with the milk titrate
in the burette. The end point is reached when blue colour has completely disappeared
and only white ppt. remains. Take three readings and determine the mean volume.
Repeat the experiment with freshly prepared standard lactose solution.
CALCULATION:
Gram of lactose per 100.0 ml of milk =
No. of times the milk is diluted
Ml of milk required for titration
Where
A = g of lactose required to titrate 25.0 ml of Benedict’s solution
= Volume of standard lactose
mukesh/
14.05.07
x
0.5
100
X A 100