Detection of lactose in dry sample of milk or dry milk -Investigatory Project in Chemistry & Biology

Aim:-Detection of lactose in sample of milk or dry milk using titration method.

Apparatus:- Balance ( Weighing machine), Water bath, Hot plate with magnetic stirrer, burette, burette stand, measuring cylinder, 250 ml volumetric flask, conical flask, funnel, filter paper, pipette.

Requirements:- 

Reagents:-  Lead nitrate, Potassium oxalate, Lactose monohydrate, Disodium hydrogen phosphate, Fehling solution A,Fehling solution B, Methylene blue indicator.

Theory:-There are two methods for the determination of the sugar in milk-

1. titration method and 2. calorimetric method. 

Out of the two procedures,the titration to be the more accurate and easier. The calorimetric method has, however, the advantage that by means of it a very large number of determinations can be made more or less simultaneously.

Theory:-Lactose is a disaccharide sugar synthesized by galactose and glucose subunits and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from lac (gen. lactis), the Latin word for milk, plus the suffix -ose used to name sugars. The compound is a white, water-soluble, non-hygroscopic solid with a mildly sweet taste. It is used in the food industry. Lactose is a disaccharide derived from the condensation of galactose and glucose, which form a β-1→4 glycosidic linkage. Its systematic name is β-D-galactopyranosyl-(1→4)-D-glucose. The glucose can be in either the α-pyranose form or the β-pyranose form, whereas the galactose can only have the β-pyranose form, hence α-lactose and β-lactose refer to the anomeric form of the glucopyranose ring alone. Detection reactions for lactose are the Woehlk and Fearon’s test. Both can be easily used in school experiments to visualize the different lactose content of different dairy products such as whole milk, lactose free milk, yogurt, buttermilk, coffee creamer, sour cream etc.
Lactose is hydrolysed to glucose and galactose, There are two methods for the determination of the sugar in milk-

1. titration method and 2. calorimetric method.
   Out of the these two procedures, the titration to be the more accurate and easier. The calorimetric method has, however, the advantage that by means of it a very large number of determinations can be made more or less simultaneously.
   Step-1- Reagent preparation:-

Step-1- Reagent preparation:-

1. Preparation of Fehling’s solution A-Dissolve 34.64 g of Copper sulphate in 200 ml of water in flask.Shake it to mix copper sulfate. Now add distilled water to it to make volume 500 ml.
2. Preparation of Fehling’s solution B -Dissolve 173 g of Potassiun sodium tartrate  CH3- CH(OH)(COONa)-CH(OH)(COOK).4H2O and 50 g of NaOH  in 200 ml of distilled water and dilute it to 500 ml.Allow to stand  the solution for two days and filter it through fibre glass. 
3. Preparation of Lead nitrate solution:- Take 2 g of it and dissolve in distilled water to make final volume 10 ml
4. Solution of disodium hydrogen phosphate and Potassium oxalate :- Take 0.3 g Potassium oxalate in 10 ml volumetric flask  and add 0.7 g of disodium hydrogen phosphate and  dissolve in distilled water to make final volume 10 ml.

Note- Fehling solution A and B are available with chemistry item supplier/ Shop.

Step-2:-Preparation of standard solution of Lactose:-Take 0.75 g of lactose (dry).Transfer it into 250 ml flask. Add 100 ml hot distilled water.Shake flask to mix lactose in distilled water.Heat the flask at 60 degree celsius for 30 minuts with shaking the solution time to time.Take out flask from water bath and cool the flask to room temperature.Add distilled water to it to make final volume 250 ml.It is standard solution of lactose ( 3 mg / ml)

Step-3-Preparation of sample solution 

Take about 1.8 grams ( say it is 1.885 g of = W) milk powder. For liquid milk or milk product , take 10 grams of sample and for Whey powder, take 1 gram samples should be taken Note the sample weight. transfer the sample into a 250 ml volumetric flask add 100 ml hot distilled water  and dissolve sample in hot water.

Heat the flask in at 60 degree celsius in water bath for 30 minutes and shake/ swirl the flask several times during heating to dissolve sample completely in water.

Cool the flask at room temperature .

Add 4 ml of reagent A in the sample solution present volumetric flask.

Add 5 ml of reagent of reagent b in sample solution / volumetric flask.

Allo to stay flask for 10 minutes.

After 10 minutes milk protein is separated and settle down at the bottom of the flask.

Add distilled water in a volumetric flask and make volume 250 ml.

Now filter the sample solution using whatman filter paper and funnel.

Discard the first 25 ml of filtrate.

Collect the filtrate of the sample solution in a conical flask.

Observations:-

Step-4-Titration of standard solution 

Perform titration of standard lactose solution ( 3mg/ml)

Fill the burette with standard lactose solution with the help of a funnel.

Take 5ml of Fehling solution in a clean conical flask and add 5 ml of fehling solution B and shake to mix.

Now add 15 ml standard lactose solution from burette into mixture of Fehling solution in conical flask and boil content of conical flask using heating plate and colour of contents in conical flask changes blue to bright orange.

Boil the solution for 3 minutes with continuous stirring.

Now add 4 drops of 1% methylene blue.

Add the standard lactose solution from burettee dropwise into conical flask.Try to complete titration within 3 minutes. with continuous stirring.

Stop adding standard lactose solution from burette after appearing bright orange colour in conical flask.

Note the burette reading .

S.No	Initial Reading of Burette	Final Reading of Burette	Difference (V1)
1	0.0 ml	25.9 ml	25.9 ml

Step-5-Titration of sample solution

Fill the burette with filtrate / sample solution with the help of funnel and note down initial reading.

Take 5ml of Fehling solution in clean conical flask and add 5 ml of fehling solution B and shake to mix.

Now add 15 ml filtrate / sample solution from burette into mixture of Fehling solution in conical flask and boil content of conical flask using heating plate and colour of contents in conical flask changes blue to bright orange.

Boil the solution for 3 minutes with continuous stirring.

Now add 4 drops of 1% methylene blue.

Add the filtrate sample solution from burettee 0.5 ml at a time ( or dropwise) into conical flask.Try to complete titration within 3 minutes. with continuous stirring.

Stop adding filtrate from the burette after appearing bright orange colour in conical flask.

Note the final burette reading of sample titration.

S.No	Initial Reading of Burette	Final Reading of Burette	Difference (V1)
1	0.0 ml	25.4 ml	25.4 ml

Step-6- Calculation of Lactose percentage:-

Volume of standard solution (V1) = Final burette reading- initial burette reading = 25.9 ml – 0.0 ml = 25.9 ml

Volume of sample solution (V2) = Final burette reading- initial burette reading = 25.4 ml – 0.0 ml = 25.4 ml

Concentration of standard solution of lactose (C) = 3 mg / ml = 0.003 g/ml

Weight of sample 1.885 g

Final volume of sample solution (V3) = 250 ml

% of lactose = V1 x C x V3 x 100 / V2 x W

% of lactose =25.9 x 0.003 x 250 x 100 /25.4 x 1.885

% of lactose =40.57%

Result: –The % of lactose in given milk sample is 40.57%