lane eynon

2.
Determination of lactose content in milk by Lane-

Eynon method (volumetric method)
Lactose is the characteristic carbohydrate in milk (approximately 48-

50 g/L in cow milk) of all the mammals. Milk contains only trace amounts of
other sugars, including glucose (50 mg/L), fructose, glucosamine,
galactosamine etc. Lactose is a disaccharide consists of glucose and galactose
moieties linked through β, 1-4 glycosidic bond. The hemiacetal group of the
glucose moiety is potentially free i.e. lactose is a reducing sugar and thus it is
capable of reducing appropriate oxidizing agents
Lane-Eynon method for the estimation of lactose is based on the

reducing property of lactose present in milk. Reducing sugars, are able to
function as reducing agents because of free aldehyde group present in the
molecule. The reducing properties of these sugars are usually observed by
their ability to reduce metal ions notably copper, iron or silver in alkaline
solution. This very property of sugars has been used in this method using
Fehling solution. Fehling solution is a mixture of Fehling A (CuSO4) and
Fehling solution B (alkaline sodium-potassium tartrate). When CuSO4 is
made alkaline (during mixing of Fehling A and Fehling B), the Cu(OH)2 gets
precipitated but in the presence of sodium-potassium tartrate, it forms a
soluble complex with copper compound and prevents the precipitation of
Cu(OH)2 . The complex is soluble and behaves/reacts as if it is alkaline
Cu(OH)2 solution. The Fehling solution when heated gives rise to cupric
oxide (CuO) which in turn reacts with reducing sugar and gets reduced to
cuprous oxide (Cu2O), brick-red precipitates, resulting into the oxidation of
sugars to corresponding acids.
For lactose estimation by this method, milk sample is treated with

acetic acid to precipitate protein and fat. The filtrate so obtained is treated
with alkaline copper sulfate solution (Fehling’s reagent) while heating.
During the reaction, Cu2O precipitates are formed which are brick red in
colour. The end point of the titration can be detected using methylene blue as
an indicator, which on reduction, changes colour of the solution from blue to
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colourless. At this stage complete precipitation of Cu2O occurs in the form of
brick-red precipitation.
Reaction
Reagents
1. Fehling's Solution: It is freshly prepared by mixing equal volumes of

the following two solutions, termed as Fehling's A and Fehling's B.
Fehing's A: Dissolve 34.639 g CuSO4.5H2O in sufficient distilled water,
add a few drops of 1 N sulphuric acid to prevent formation of cloudy
appearance or turbidity (use a minimum quantity of acid). Dilute the
solution with distilled water to 500 ml. If necessary, filter through
prepared asbestos, on Gooch crucible. Fehling’s B: Dissolve 173 g
Rochelle salt (sodium potassium tartrate; KNaC4H4O6.4H2O) in distilled
water. Add to it 50 g of sodium hydroxide and dilute the mixture to 500
ml with distilled water. Allow to stand in a rubber or polyethene
stoppered container for two days and filter through prepared asbestos.
2. Acetic acid solution: 10% (w/v; aqueous) solution.
3. Methylene blue indicator: 1% (w/v; aqueous) solution.
4. Standard lactose solution: Weigh accurately 5 g of this dried lactose and
dissolve in freshly boiled and cooled distilled water. Transfer the solution
quantitatively to a 1 L volumetric flask and dilute up to mark with
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distilled water. Mix well and store it in a cool place. One ml of this
solution contains 5 mg lactose.
Procedure
A. Preparation of milk filtrate
1. Pipette 10 ml of the prepared sample of milk into a 100 ml volumetric

flask.
2. Add 30 to 40 ml distilled water and the content of the flask are warmed
to 40 to 45°C, immediately add 1.5 ml acetic acid (10%). Mix well and
make the final volume with distilled water up to mark and mix again.
Keep the flask undisturbed for 30 min.
3. After 30 min filter the content of the flask through a Whatman filter
paper Grade 42. Discard the first few ml of the filtrate and collect the rest
in a clean dry Erlenmeyer flask fitted with stopper.
B. Standardization of Fehling's solution
1. Pipette 5 ml of Fehling’s solution A and 5 ml of Fehling’s solution B using

two separate pipettes in a 250 ml Erlenmeyer flask. Fill up a burette with
the standard lactose solution and connect the burette end with an offset
tube to keep the burette tube out of steam.
2. Heat the content of the flask to boiling over burner or heater and
maintain moderate boiling for 2 min. To prevent bumping add some
inert boiling chips. Add 3 to 4 drops of methylene blue indicator without
removing from the flame. Titrate the content of the flask against standard
lactose solution from the burette until the blue colour disappears and the
bright brick-red colour of precipitated Cu2O appears (at the end point the
Cu2O suddenly settles down giving a clear supernatant). Note the
volume of lactose solution required for the standardization of Fehling’s
solution. After this preliminary titration, further titration or titrations
should be carried out, adding practically the whole of the standard
lactose solution volume (one ml less than required as observed in first
titration) required before commencing the heating and continuing the
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titration as before. The titration must be completed within 3 min from the
commencement of boiling. Let V1 ml be the titer for this experiment.
Note:
Carefully note the first disappearance of blue colour. Once missed, it is difficult to
ascertain the end point. Maintain the boiling at a uniform rate during the titration.
C. Determination in milk
Here also the titration is taken up in two stages. In the first stage, an
approximate estimation is made, while in the second stage more accurate
result is obtained.
Stage 1
1. Pipette 5 ml of Fehling’s solution A and 5 ml of Fehling’s solution B using

two separate pipettes in a 250 ml Erlenmeyer flask. Fill up a burette with
the prepared milk filtrate and connect the burette end with an offset tube
to keep the burette tube out of steam.
2. Add 10 ml of the milk filtrate from the burette into flask containing
Fehling’s solution and heat to boiling on wire gauge over burner. Boil for
about 15 sec and rapidly add further amount of lactose filtrate until only
faint perceptible blue colour remains. Add 3-4 drops of methylene blue
indicator and complete the titration to the first disappearance of blue
colour by adding lactose filtrate drop-wise.
Stage 2
Repeat the experiment of Stage 1, but add almost the entire prepared milk
filtrate (one ml less than required as observed in first titration) required to
reduce all copper, as determined by Stage 1, and complete the titration as
described under standardization of Fehling's solution. Let V2 ml be the titer
for this experiment.
Calculation
V1
% lactose in milk = 5x
V2
Where
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V1 = volume in ml, of standard lactose solution taken to reduce 10 ml of
Fehling’s solution; and
V2 = volume in ml, of prepared milk filtrate taken to reduce 10 ml of

Fehling’s solution.
Note:
1 ml of standard lactose solution contain = 0.005 g of lactose
Let V1 ml = amount of standard lactose solution taken to reduce 10 ml of Fehling’s

solution (5 ml each of Fehling’s A and B solution)
Let V2 ml = amount of milk filtrate solution taken to reduce 10 ml of Fehling’s

solution (5 ml each of Fehling’s 1 and 2 solution)
10 ml of Fehling’s solution = V1 ml of standard lactose solution
= V2 ml of milk filtrate
V2 ml of milk filtrate ≡ V1 ml of standard lactose solution
= V1 x 0.005 g of lactose
100 ml of milk filtrate =

V1 x 0.005
x 100
V2
Since 100 ml of milk filtrate is obtained from 10 ml of milk
V1 x 0.005 100
Lactose present in 100 ml of milk = x 100 x
V2 10
V1
% lactose in milk = 5x
V2
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2.

Determination of lactose content in milk by Lane-

Lactose is the characteristic carbohydrate in milk (approximately 48-

Lane-Eynon method for the estimation of lactose is based on the

For lactose estimation by this method, milk sample is treated with

1. Fehling's Solution: It is freshly prepared by mixing equal volumes of

1. Pipette 10 ml of the prepared sample of milk into a 100 ml volumetric

B. Standardization of Fehling's solution

1. Pipette 5 ml of Fehling’s solution A and 5 ml of Fehling’s solution B using

1. Pipette 5 ml of Fehling’s solution A and 5 ml of Fehling’s solution B using

V2 = volume in ml, of prepared milk filtrate taken to reduce 10 ml of

Let V1 ml = amount of standard lactose solution taken to reduce 10 ml of Fehling’s

Let V2 ml = amount of milk filtrate solution taken to reduce 10 ml of Fehling’s

10 ml of Fehling’s solution = V1 ml of standard lactose solution

V2 ml of milk filtrate ≡ V1 ml of standard lactose solution

100 ml of milk filtrate =

Since 100 ml of milk filtrate is obtained from 10 ml of milk

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