KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY

COLLEGE OF DAIRY SCIENCE AND TECHNOLOGY, MANNUTHY

PRACTICAL RECORD
MILK CARBOHYDRATES, MINERALS AND WATER
SOLUBLE VITAMINS
(DC 512)

Submitted by,
 INDEX

Sl.N DATE CONTENT PAGE SIGN.

o. No.

Determination Of Lactose Content In Milk By Lane

1
Eynon Method (Volumetric Method)

2 Estimation Of Lactose In Milk By Picric Acid

Colorimetric Method

3 Estimation Of Lactose Content By Munson Walker

Method (Gravimetric)

4 Estimation Of Lactose In Milk By Polarimetric

Method

5
Determination Of Calcium Content In Milk By
Precipitation Method
Exp. No:
Date:

DETERMINATION OF LACTOSE CONTENT IN MILK BY LANE EYNON

METHOD (VOLUMETRIC METHOD)

Aim

To find the lactose content in milk, skim milk and whey by Lane Eynon method

Principle

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 (CuSO 4,) 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 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, Cu 2O 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 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
2. Acetic acid solution: 10% (w /v; aqueous) solution.
3. Methylene blue indicator: 1 % (w /v; aqueous) solution.
4. Standard lactose solution (0.5 %)

Procedure

A. Preparation of milk filtrate

1. Pipette 25 ml of the prepared sample of milk into a 250 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 3.25 ml acetic acid (10%). Mix well and make the final volume with
distilled water up to mark and mix again.
3. After 30 min filter the content of the flask through a Whatman filter paper Grade 42.

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.
2. Fill up a burette with the standard lactose solution
3. 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.
4. Add 3 to 4 drops of methylene blue indicator without removing from the flame.
5. Titrate the content of the flask against 0.5 % standard lactose solution from the burette
until the blue colour disappears and the bright brick-red colour of precipitated Cu 20
appears. Let V1 ml be the titer for this experiment.

C. Determination in milk

1. Pipette 5 ml of Fehling's solution A and 5 ml of Fehling's solution Busing two separate

pipettes in a 250 mI Erlenmeyer flask. Fill up a burette with the prepared milk filtrate
2. Titrate the solution following the same procedure as mentioned above.

Calculation

Let

V1= volume in ml, of standard lactose solution taken to reduce 10 ml of Fehling's solution;

V2 = volume in ml, of prepared milk filtrate taken to reduce 10 ml of Fehling's solution.

1 ml of standard lactose solution contain = 0.005 g of lactose

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

Lactose present in 100 ml of milk = V1 x 0.005 x 100 x 250
V2 25
% lactose in milk = 5xV1/ V2
Observation

Burette reading(ml)
Volume of Fehling Volume of
Sample Sl.No
Solution (A+B) ml Initial Final filtrate

Standard
Lactose
solution
(0.5 %)

Milk filtrate

Curd filtrate

Whey filtrate

Result

Inference

Exp. No:
Date:
 ESTIMATION OF LACTOSE IN MILK BY PICRIC ACID COLORIMETRIC
METHOD

Aim

To determine the lactose content in milk by picric acid method

Principle

The picric acid method is based upon the fact that reducing sugars, in alkaline solution,
(when heated) reduce picric acid which is yellow in color, to picramic acid which is a deep
mahogany red. In this method a known quantity of milk is diluted to a specific volume of
saturated picric acid. Well mixed solution is filtered and measured volume of clear filtrate is
mixed with sodium carbonate in a standard joint tube placed in a boiling water bath for
development of color. The intensity of color is proportional to lactose content.

Apparatus

Water bath, standard joint tubes, spectrometer, glass wares

Reagents

Saturated picric acid solution, 25% sodium carbonate, 0.5% lactose solution

Procedure

Preparation of filtrate

1. Take 1 ml of well mixed sample in a 25 ml volumetric flask

2. Add picric acid to make the volume up to the mark
3. Shake well and keep for few minutes
4. Filter through Whatman filter paper
5. Collect the filtrate

Preparation of standard curve

1. Take 0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1ml,1.2ml, standard lactose solution in separate
standard joint tubes
2. Make the volume of each tubes to 2ml with picric acid
3. Add 1ml of Na2CO3 solution in each tube
4. Take 2ml of milk filtrate in other tube and add 1ml of Na2CO3 solution
5. Place them in boiling water bath for 20 min
6. After boiling make the volume of each of the test tube to 20 ml by adding distilled water
7. Measure the optical density
8. Plot readings in a graph

Observation

Concentration of Standard lactose solution : 5 mg/ml

Lactose Absorbance
concentration(mg)
0
1
2
3
4
5
6

Skim milk

Whey

Result
Inference

Exp. No:
Date:

ESTIMATION OF LACTOSE CONTENT BY MUNSON WALKER METHOD

(GRAVIMETRIC)
Aim
To determine lactose content in milk and curd by Munson Walker gravimetric method

Principle
Carbohydrates are oxidized in the presence of heat and an excess of copper sulphate and
alkaline tartrate under carefully controlled conditions which leads to the formation of a
copper oxide precipitate
Reducing sugar + Cu2+ + Base Oxidized sugar + Cu2O
The amount of precipitate formed is directly related to concentration of reducing sugar in the
initial sample. The concentration of precipitate present can be determined gravimetrically.

Reagents

Fehling’s solution, 0.5N NaOH

Procedure

1. Weigh accurately 25g of prepared sample

2. Dilute with 400ml distilled water in a 500ml graduated flask
3. Add 10 ml of Fehling’s solution A (CuSO4.5H2O) and 8.8ml of NaOH solution
4. Fill up the mark using distilled water
5. Mix thoroughly and filter
6. Add 25 ml of the diluted solution into the conical flask containing solutions A and B
7. Mix it with 50ml of prepared filtrate
8. Heat the contents of the conical flask to boil for two minutes
9. Trial should be run out with 50ml each of reagent and distilled water
10. Filter the hot solution
11. Wash the precipitate of cuprous oxide with water at 60°C
12. Dry the filter and weigh

Observations

Weight (g)
Sl No Sample
Filter Paper + Dish Filter Paper + Dish Dried Sample
 + Dried Sample
1 Milk

2 Curd

3 Blank

Weight of Cu2O = Weight of Dried Sample - Weight of blank (dried)

Result

Inference

Exp. No:
Date:
 ESTIMATION OF LACTOSE IN MILK BY POLARIMETRIC METHOD

Aim

To determine lactose content in given sample of milk by Polarimetry

Principle

Certain organic substances (notably those containing asymmetric carbon atoms) possess the
property of rotating the plane of polarized light. Such substances are called as optically active
substances. A compound is considered to be optically active if linearly polarized light is
rotated when passing through it. The amount of optical rotation is determined by the
molecular structure and concentration of chiral molecules in the substance. Ordinary light
propagates itself through spherical waves in all directions in space, but when such light is
allowed to pass through specially designed prism, like Nichol's prism, (prepared from
particular substance like naturally occurring crystalline calcium fluoride, calcite etc.) the
emerging light travels in one plane only. Such light is called "plane polarized light". Solution
of substances like lactose, glucose, etc., when placed in the path of plane polarized light;
rotate the plane of polarized light and such compounds are known as "optically active". The
extent of change (rotation) in the plane of the polarized light depends on factors like the
specific nature of the optically active compound, its concentration in the solution and
temperature of the solution etc. The quantitative relationship between these variables is given
by the following equation:

Lactose concentration = 100 * α * C * 100

L * [α]20

Where

α = the observed angular rotation in degree;

L = length of tube in millimeters

C = concentration of the compound in solution in g/100 ml

[α] 20 = the specific rotation at 20°C using D line of sodium (589.3 nm)

Reagents
 Mercuric iodide solution: Dissolve 32.3g of potassium iodide and 13.5g mercuric chloride
in 200ml glacial acetic acid and add 640ml distilled water
 Phosphotungstic acid - 5%(w/v) in distilled water

Procedure

 Weigh accurately 65.8g milk into 100ml volumetric flask

 Add 30ml of mercuric iodide solution
 Add phosphotungstic acid to the mark
 Shake well and keep for 15 min
 Filter through Whatman filter paper
 Collect filtrate and warm to 20˚C
 Illuminate polarimeter with sodium lamp
 At first tubes are completely filled with distilled water with air being trapped only in the
air trap provided in the tube
 Put the tube in proper position
 Polarize tube at maximum illumination point
 Adjust the scale to zero position
 Transfer the filtrate to 200mm tube to reduce the error in reading
 Place the tube in position and polarize
 Measure the amount and direction of rotation from the scale

Result

Inference

Exp. No:
Date:
 DETERMINATION OF CALCIUM CONTENT IN MILK BY PRECIPITATION
METHOD
Aim

To determine the calcium content in milk by Precipitation method

Principle

Determination of calcium involves first preliminary treatment to convert it to soluble form.

The method depends on precipitation of calcium as oxalate , which is then dissolved in
Sulfuric acid and liberated oxalic acid is titrated with standard potassium permanganate
solution.

Reagents

 Hydrochloric acid –1:9 by volume.

 Ammonium hydroxide solution – 50 per cent (v/v)
 Dilute ammonium hydroxide solution – 2 per cent (v/v)
 Ammonium oxalate solution – Saturated
 Concentrated sulphuric acid -1:4 by volume
 Standard potassium permanganate solution – 0.1 N

Procedure

1. Ashing and extraction –

a. Accurately weigh about 10 g of milk into a silica dish. Char carefully and continue
the ashing in a muffle furnace at a temperature not above 450ºC until the ash is
white or almost so.
b. Cool the ash, moisten with a few ml of concentrated hydrochloric acid (7-8ml) drop
by drop, warm gently to dissolve and transfer to a 100 ml volumetric flask.
c. Wash the dish with distilled water and transfer the washing to the flask. Make up the
volume to the mark.
2. Precipitation –
a. Transfer a 20 ml of the solution prepared as in (1) to a 250 ml beaker
b. Add dropwise diluted (1:1) Ammonium hydroxide and heat to 60-70C.Add to hot
solution , saturated solution of ammonium oxalate dropwise till precipitate formed,
heat to boiling and allow to stand for three hours or more.
c. Filter the solution through whatman filter paper(42) and add 20 ml of hot water
into precipitate to wash it and decant the clear solution through filter. Add
sufficient hot HCl dropwise to the precipitate adhering to the filter paper and take
the washing into the beaker containing bulk of ppt. Wash the filter paper 5-6 times
with hot water and collect the washing in the original beaker. Dissolve the ppt in
beaker by adding HCl if required. Reprecipitate the CaC 2O4 from this solution by
 adding NH4OH and (NH4)2C2O4 as mentioned above and filter. Wash the ppt with
hot water until chloride free.
3. Titration -
a. Wash the filter paper with hot water and few ml of dilute sulphuric acid, heat to
80 to 90ºC and titrate with the 0. 1N Potassium Permanganate solution
maintaining high temperature until the permanent pink colour persists for atleast
30 seconds.

Calculation

Calcium percent by weight = 1.002* V/W

Where,
V = Volume in ml of 0.1 N KMnO4
W = Weight in g of sample taken for test

Result

Inference