SOP: Determination of Sugar Content in Condensed Milk

GIFSQ — Global Institute for Food Safety & Quality

Scope

This method is applied to milk and milk products.

1. Principle

The principle involves clarifying the sample with the help of zinc acetate and potassium ferrocyanide, inverting a part of the sample using hydrochloric acid, and determining the reducing sugars content in both inverted and non-inverted parts of the sample. The sugar content is estimated by determining the volume of the unknown sugar solution required to completely reduce a measured volume of Fehling’s solution. Invert sugar reduces the copper in Fehling’s solution to red insoluble cuprous oxide.

2. Apparatus

a. Volumetric flasks: 100, 250, 500 and 1000 mL capacities

b. Burette: 50 mL, graduated to 0.1 mL

c. Funnels

d. Conical flasks: 250 mL capacities

e. Pipette: 1, 5 and 10 mL capacities

f. Measuring cylinder: 25 and 50 mL capacities

g. Whatman filter paper: Grade 1

h. Watch glass

i. Hot plate

3. Reagents

a. Sodium hydroxide solution: approximately 0.1 N, prepared from sodium hydroxide, analytical reagent grade.

b. Stock solution of invert sugar (Reagent B): weigh accurately 9.5 g of pure sucrose on a watch glass and transfer it to a one litre volumetric flask with 100 mL of water. Add 5 mL of concentrated hydrochloric acid. Allow this to stand for 3 days at 20 to 25°C and then make up to volume with water. (This solution is stable for several months.)

c. Standard solution of invert sugar (Reagent C): neutralise a known aliquot of the stock solution of invert sugar (Reagent B) with sodium hydroxide solution using litmus paper, and dilute with water to a known volume, so that more than 15 mL but less than 50 mL of it shall be required to reduce all the copper in the Fehling’s solution taken for titration. Note the concentration of invert sugar in this solution as mg per 100 mL. Prepare this solution fresh every day.

Note: When 10 mL of Fehling’s solution are taken for titration, a standard invert sugar solution containing 0.12 to 0.30 percent (w/v) of invert sugar is used. Usually 40 mL of stock solution (Reagent B) is diluted to 100 mL to get a titration reading of around 15 mL when titrated against Fehling’s solution.
d. Methylene blue indicator solution: dissolve 0.2 g of methylene blue in water and dilute to 100 mL.

e. Fehling’s solution (Soxhlet modification): prepared by mixing, immediately before use, equal volumes of Solution A and Solution B:

• Solution A: dissolve 34.639 g of copper sulphate (CuSO₄·5H₂O) in water, add 0.5 mL of concentrated sulphuric acid of sp. gr. 1.84, and dilute to 500 mL in a volumetric flask. Filter the solution through prepared asbestos.
• Solution B: dissolve 173 g of Rochelle salt (potassium sodium tartrate — KNaC₄H₄O₆·4H₂O) and 50 g of sodium hydroxide, analytical reagent grade, in water; dilute to 500 mL in a volumetric flask and allow the solution to stand for two days. Filter this solution through prepared asbestos.
  f. Zinc acetate solution: dissolve 21.9 g of crystalline zinc acetate (Zn(CH₃COO)₂·2H₂O) in water and add 3 mL of glacial acetic acid. Make up to 100 mL.

g. Potassium ferrocyanide solution: dissolve 10.6 g of crystalline potassium ferrocyanide and make up to 100 mL with water.

h. Concentrated hydrochloric acid solution (sp. gr. 1.16).

i. Concentrated ammonia solution (sp. gr. 0.88).

j. Dilute ammonia solution: 10 mL of concentrated ammonia solution diluted to 100 mL with water.

k. Dilute acetic acid solution: approximately equivalent to the dilute ammonia solution in strength.

l. Standard dextrose solution: dissolve 0.2 g of pure anhydrous dextrose (previously dried at 100°C and cooled before weighing) in water and make up to 100 mL.

4. Standardization of Fehling’s solution

Pipette accurately 10 mL of each Fehling’s solution (Solution A and B) into a 250 mL Erlenmeyer flask. Add about 10 mL of standard solution of invert sugar (Reagent C) to the flask and heat to boiling. Boil for about 15 seconds and add rapidly further portions of the standard solution of invert sugar until only the faintest perceptible blue colour remains. Then add 2–5 drops of methylene blue solution, continue the heating and addition of standard solution of invert sugar (Reagent C) dropwise until the titration is complete, which is shown by the reduction of the dye (the end point of the titration is blue to red). Do not stir the flask; mixing is achieved by continuous boiling. Note down the volume of standard solution of invert sugar (Reagent C) required to reduce all the copper.

Repeat the titration, adding before heating almost all of the standard solution of invert sugar (Reagent C) needed to reduce all the copper, so that not more than 0.5–1 mL is required later to complete the titration. Heat the mixture to boiling and boil gently for 2 minutes, lowering the flame sufficiently to prevent bumping. Without removing the flask from the flame, add 2–5 drops of methylene blue indicator and complete the titration within a total boiling time of about 3 minutes, by small additions of standard solution of invert sugar (Reagent C), to complete decolourization of the indicator. Note down the volume of standard solution of invert sugar required to reduce all the copper.

Note 1: The concentration of the sugar solution should be such that the titre volume should be between 15 and 50 mL, and this can be achieved with 0.2–0.3 g sugar per 100 mL. Alternatively, reagent ‘L’ standard solution of invert sugar can also be used for determination of factor.

5. Procedure — Preparation of the solution

Weigh accurately about 40 g of the well mixed sample and transfer to a 100 mL beaker

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Add 50 mL of hot water at 80 to 90°C; mix and transfer to a 250 mL measuring flask, washing it with successive quantities of distilled water at 60°C until the volume is 120 to 150 mL

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Mix and cool to room temperature, and add 5 mL of the dilute ammonia solution

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Mix and allow to stand for 15 minutes

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Add the exact equivalent of dilute acetic acid to neutralize the ammonia added

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Mix and add 12.5 mL of zinc acetate solution, followed by 12.5 mL of potassium ferrocyanide solution; mix again

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Make up to the 250 mL mark; allow to settle and filter. Mark this solution B-I

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Pipette 50 mL of solution B-I into a 100 mL volumetric flask, add 5 mL of concentrated hydrochloric acid and heat in a water bath at 65–68°C for 5 minutes, rotating the flask for the first 3 minutes

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Cool the solution and neutralize with sodium hydroxide solution. Mark this solution A-I. Make up to 100 mL

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Dilute solution B-I (usually 50 mL of B-I diluted to 100 mL, to obtain a titration reading of around 14 mL when titrated against Fehling’s solution) and solution A-I (usually 15 mL of A-I solution diluted to 100 mL, to obtain a titration reading of around 18 mL), so that the volume of solution required to react with 10 mL Fehling’s solution is between 15 and 50 mL. Mark them B-II and A-II respectively

6. Incremental method of titration

Pour the prepared solution into a 50 mL burette (see Note 6). Pipette 10 mL of mixed Fehling’s solution into a 250 mL conical flask and run in from the burette 15 mL of the solution.

Without further dilution, heat the contents of the flask over a hot plate, and boil. (After the liquid has been boiling for about 15 seconds, it will be possible to judge if the copper is almost fully reduced by the bright red colour imparted to the boiling liquid by the suspended cuprous oxide. When it is judged that nearly all the copper is reduced, add one mL of methylene blue indicator solution (see Note 2). Continue boiling the contents of the flask for one to two minutes from the commencement of ebullition, and then add the prepared solution in small quantities (one mL or less at a time), allowing the liquid to boil for about 10 seconds between successive additions, till the blue colour of the indicator just disappears (see Note 5). In case there appears to be still much unreduced copper, after the mixture of Fehling’s solution with 15 mL of the prepared solution has been boiling for a quarter of a minute, add the prepared solution from the burette in larger increments (more than one mL at a time, according to judgment), and allow the mixture to boil for a quarter of a minute after each addition. Repeat the addition of the prepared solution at intervals of 15 seconds until it is considered safe to add a large increment of the prepared solution. At this stage, continue the boiling for an additional one to two minutes, add one mL of methylene blue indicator solution and complete the titration by adding the prepared solution in small quantities (less than one mL at a time) (see also Note 3).

Note 2: It is advisable not to add the indicator until the neighborhood of the end point has been reached, because the indicator retains its full colour until the end point is almost reached and thus gives no warning to the operator to go slowly.

Note 3: When the operator has had a fair amount of experience with the method, a sufficiently accurate result may often be obtained by a single estimation by the incremental method of titration, but for the utmost degree of accuracy of which the method is capable, a second titration should be carried out by the standard method of titration.

7. Standard method of titration

Pipette 10 mL of Fehling’s solution into a 250 mL conical flask and run in from the burette almost the whole of the prepared solution B-II required to effect reduction of all the copper, so that, if possible, not more than 1 mL shall be required later to complete the titration. Gently boil the contents of the flask for 2 minutes. At the end of 2 minutes of boiling add, without interrupting boiling, one mL of methylene blue indicator solution. While the contents of the flask continue to boil, begin to add the prepared solution (one or two drops at a time) from the burette till the blue colour of the indicator just disappears (see Note 4). The titration should be completed within one minute, so that the contents of the flask boil altogether for 3 minutes without interruption.

Note 4: The indicator is so sensitive that it is possible to determine the end point within one drop of the prepared solution in many cases. The complete decolourization of the methylene blue is usually indicated by the whole reaction liquid, in which the cuprous oxide is continuously generated, becoming bright red or orange in colour. In case of doubt, the flame may be removed from the wire gauze for one or two seconds and the flask held against a sheet of white paper. (A holder of paper, suitably affixed round the neck of the flask, is very convenient for this purpose, as it can be left round the neck of the flask without risk of over-balancing it.) The top edge of the liquid would appear bluish if the indicator is not completely decolorized. It is inadvisable to interrupt the boiling, as the indicator undergoes back oxidation rather rapidly when air is allowed free access into the flask, but there is no danger of this as long as a continuous stream of steam is issuing from the mouth of the flask.

Note 5: It should be observed that with both incremental and standard methods of titration, the flask containing the reaction mixture is left on the wire gauze over the flame throughout the titration; it may be removed for a few seconds to ascertain if the end point is reached.

Note 6: In adding sugar solution to the reaction mixture, the burette may be held in hand over the flask. The burette may be fitted with a small outlet tube bent twice at right angles, so that the body of the burette can be kept out of the steam while adding sugar solution. Burettes with glass taps are unsuitable for this work, as the taps become heated by the steam and are liable to jam.

Repeat the titration using the solution A-II.

8. Calculation

Original Reducing Sugars % (ORS) in condensed milk = (W₁ ÷ V₁) × 250 × (100 ÷ W₂) × (1 ÷ 1000) × F₁

Total Reducing Sugars % (TRS) in condensed milk = (W₁ ÷ W₂) × 25 × [2F₂ ÷ V₂]

Sucrose % = TRS − ORS

Note: If standardization of Fehling’s solution is carried out by Lactose Solution (Reagent L) instead of the standard solution of invert sugar prepared under acidic conditions at room temperature, multiply the Sucrose % obtained in the above formula by a factor 0.95 for calculating sucrose content.

Where,
– W₁ = weight in mg of sucrose corresponding to 10 mL of Fehling’s solution
– W₂ = weight in g of the material taken for the determination
– F₂ = dilution factor for solution A-II from A-I
– V₂ = volume in mL of solution A-II corresponding to 10 mL of Fehling’s solution
– F₁ = dilution factor for the solution B-II from B-I
– V₁ = volume in mL of solution B-II corresponding to 10 mL of Fehling’s solution

Derivation of the above formulae (as given in the source)

1. Standardization of Fehling’s solution using standard reducing sugar. Let 1 mL of standard solution of invert sugar (Reagent B) contain M mg of total invert sugar.

2. Lactose (original reducing sugar in the SCM sample). Let V₁ = mL of diluted sample filtrate (B-I) required for the titration of 10 mL of Fehling’s solution, and V = mL of the standard solution of invert sugar (Reagent C) required for the titration of 10 mL of Fehling’s solution. Now 10 mL of Fehling’s solution = V mL of standard reducing sugar solution = V₁ mL of diluted sample filtrate. Thus V₁ mL of diluted sample filtrate ≡ V mL of standard reducing sugar solution ≡ V × M mg of standard reducing sugar, or W₁ mg of standard reducing sugar. Therefore, 100 mL of sample filtrate contains (W₁ ÷ V₁) × 100 mg of original reducing sugar. Since 100 mL of this sample filtrate was obtained from 50 mL of B₁ solution, 250 mL of B₁ sample filtrate will contain (W₁ ÷ V₁) × (100 ÷ 50) × 250 mg of original reducing sugar. Since 250 mL of sample filtrate was obtained from 40 g (say W₂ g) of SCM: Lactose (g) present in 100 g of SCM (say C1) = (W₁ ÷ V₁) × (100 ÷ 50) × 250 × (100 ÷ W₂) × (1 ÷ 1000) g.

3. Total reducing sugar (lactose + hydrolyzed sucrose). Let V₂ = mL of diluted sample filtrate (A-I) required for the titration of 10 mL of Fehling’s solution, and V = mL of the standard solution of invert sugar (Reagent B) required for the titration of 10 mL of Fehling’s solution. Now 10 mL of Fehling’s solution = V mL of standard reducing sugar solution = V₂ mL of diluted sample filtrate. Thus V₂ mL of diluted sample filtrate ≡ V × M mg, or W₁ mg of standard reducing sugar. Therefore, 100 mL of sample filtrate (A-II) contains (W₁ ÷ V₂) × 100 mg of total reducing sugar. Since 100 mL of this sample filtrate was obtained from 15 mL of A-I solution, 100 mL of A-I sample filtrate will contain (W₁ ÷ V₂) × (100 ÷ 15) mg. Since 100 mL of this sample filtrate was obtained from 50 mL of B-I solution, 100 mL of B-I sample filtrate will contain (W₁ ÷ V₂) × (100 ÷ 15) × (100 ÷ 50) mg, and 250 mL of B-I sample filtrate will contain (W₁ ÷ V₂) × (100 ÷ 15) × (100 ÷ 50) × 250 mg of total reducing sugar. Since 250 mL of sample filtrate was obtained from 40 g (say W₂ g) of SCM: Total reducing sugars (g) present in 100 g of SCM (say C2) = (W₁ ÷ V₂) × (100 ÷ 15) × (100 ÷ 50) × 250 × (100 ÷ W₂) × (1 ÷ 1000). This calculation is on the assumption that 15 mL of A-I solution was diluted to 100 mL.

Total sucrose (%) in the SCM = C2 − C1.

References

Manual of Methods of Analysis of Foods, Milk and Milk Products, FSSAI, 2016, Method 9.4.1.