TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC

METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

1.0 SCOPE

This method is used for the quantitative determination of total sugar in beverages and
food products by volumetric method (The Lane-Eynon Method). Results are
expressed as percentage of sugar in 100g of sample.

2.0 PRINCIPLE
The method involves the inversion of sugars present in food samples with
hydrochloric acid. The sugar present in a specified volume of the hydrolysed solution
is used to reduce copper in the Fehling’s solution previously standardised with
working standard invert sugar solution. The difference in the volume of standard
sugar solution. The difference in the volume of measure of total sugar content of the
sample.

3.0 REFERENCE METHOD

ASEAN Manual of Food Analysis, First Edition 2011

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TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

4.0 APPARATUS / EQUIPMENT

4.1 Hot Plate Magnetic Stirred

4.2 Burette, 25mL

4.3 Analytical Balance
4.4 Fume Hood
4.6 Shaking Water Bath, maintained at 60±5ºC
4.7 Drying oven
4.8 Measuring Cylinder, 100mL
4.9 Erlenmeyer flasks, 250mL
4.10 Beaker 5 mL , 10 ml , 100 ml
4.11 Volumetric flasks, 100mL, 500mL, 1000mL
4.12 Filter Paper

5.0 CHEMICAL / REAGENT

5.1 Hydrochloric Acid, Grade AR

5.2 Sucrose, Grade AR
5.3 Copper ( II ) Sulphate Pentahydrate Solution ( Fehling A )
5.4 Alkaline Tartrate Solution ( Fehling B )
5.5 Sodium Hydroxide
5.6 Working Standard Solution
5.7 Phenolphthalein
5.8 Methylene Blue

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TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

6.0 SAFETY

6.1 Understand the toxicity and safety of the reagents used before starting the
method.

6.2 Use safety devices such as tong or gloves in handling hot containers.

6.3 Always use eye protection during the analysis especially in the titration.

7.0 PREPARATIONS
7.1 Reagent Preparation

7.1.1 Copper (II) Sulphate Pentahydrate Solution (Fehling’s A)

Weight 34.639g of Copper (II) Sulphate Pentahydrate Cu2SO4.5H2O, grade AR, and
dissolve in ultra-pure water. Dilute to 500mL and filter through filter paper.

7.1.2 Alkaline Tartrate Solution (Fehling’s B)

Weight 173g of Potassium Sodium Tartrate Tetrahydrate C4H4KNaO6.4H2O, grade

AR, and 50g Sodium Hydroxide dissolve in ultra-pure water. Dilute to 500mL, let
stand for 2 days and filter through filter paper

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TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

7.1.3 Fehling’s Solution

Prepare by mixing equal volume of reagents in Fehling’s A and Fehling’s B in 1

Litre.

7.1.4 Sodium Hydroxide, AR

Dissolve 20g Sodium Hydroxide in Distilled Water and dilute to 100mL.

7.1.5 Hydrochloric Acid. HCL, 0.5 N

Pipette 4.125mL Concentrated HCL dilute to 100mL.

` 7.2 Standard Preparation

7.2.1 Stock Solution (10 mg/mL)

Dry sucrose in air oven at 100ºC for 1 hour. Weight 5g dried sucrose. Add 2.5 mL
concentrated HCI and dilute with distilled water to 100mL. Store for 3 days at
room temperature, then dilute to 500mL.

7.2.2 Working Standard Solution ( 2.5 mg/ml )

Pipette 125mL stock solution into 500mL volumetric flasks. Add few drops
phenolphthalein, and neutralize with 20% Sodium Hydroxide, NaOH. Dilute to
volume and mix well. Prepare fresh daily.

Note: Acidified 1% invert sugar solution is stable for several months.

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TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

7.3 Quality Control (5%)

Dry sucrose in air oven at 100ºC for 1 hour. Weight 5g dried sucrose and mark
up distilled water in 100 ml volumetric flask

Accurately pipette 10 ml of the solution into 250 ml Erlenmeyer flask, add 5

mL each of reagent Fehling A and Fehling B and 30 ml of distilled water.

Pre-heat electric heater-stirrer so that boiling of solution can start in

approximately 3 minutes. Mix solution, put a magnetic bar into the solution
and place on the pre-heated heater.

While rapidly boiling, add working sugar standard from burette. The volume
used is 0.5 to 1 mL less than that is used in the approximate titration carried
out in approximate titration of sample

Add 3 drops of methylene blue indicator and continue adding standard sugar
solution at intervals of 10 seconds until the indicator is completely
decolourised.

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TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

7.4 Sample Preparation

Thaw the sample to room temperature.

Shake well the liquid sample to take care the fat or other constituent adhering

sample to the wall of the container. If the sample contains lumps or pieces of the

ingredients homogenize in an appropriate blender and mix all samples thoroughly

until the sample become homogenous.

Mix the viscous or pasty products with spatula. If the products still contain lumps

or solid particles, homogenize in a blender and stir well until the sample

homogenous.

Grind the dried products to a fine powder using blender and mix well.

7.5 Preparation of analytical sample

7.5.1 For liquid/viscous and pasty products

Weight 10gram transfer to a 250ml volumetric flask and dilute to volume with
water, mix well and filter using filter paper.

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TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

7.5.2 For dried products

Weigh 10gram, in duplicate, test sample containing approximately 30% total

sugar in a 250 mL Erlenmeyer flask.

Add 150 mL water preheated to 60°C ± 5°. Mechanically shake flask for 30
minutes in a water bath maintained at 60°C ± 5°.
Let stand in water bath for another 30 minutes and cool to room temperature.

Transfer to a 250 mL volumetric flask, dilute to volume, mix well and filter
or centrifuge then filter.

Note: Discard the first 25 mL filtrate. Cover funnel during filtration to avoid
evaporation.

7.6 Sugar inversion with acid at room temperature

Pipette 50 mL filtrate into 100 mL volumetric flask.Add 2.5 mL conc. HCL.
Let stand overnight

Add few drops of phenolphthalein and neutralise with 20% NaOH solution.
Add a few drops of dilute HCl (0.5 N HCl) until the red colour disappears.
Dilute to volume with water and mix well

7.7 Sugar inversion with acid at room temperature

Pipette 50 mL filtrate into 100 mL volumetric flask. Add 2.5 mL conc. HCL.
Let stand overnight.

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TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

Add few drops of phenolphthalein and neutralise with 20%NaOH solution.

Add a few drops of dilute HCl (0.5 N HCl) until the red colour disappears.
Dilute to volume with water and mix well.

7.8 Standardisation of Fehling’s solution

Fill burette with working standard solution.
Accurately pipette 5 mL of each reagent in Fehling A and Fehling B into
250ml Erlenmeyer flask.

Mix, and add 30 mL water. Add approximately 19 mL working standard

solution from a burette.Put into the solution a magnetic stirring bar and place
on electric heater-stirrer.Regulate heat so that boiling will begin in
approximately 3 minutes.

Maintain moderate boiling for 2 minutes, reducing heat if necessary to prevent

bumping. Without removing flask from heater, add 3 drops 1% methylene
blue.

Stir moderately and complete titration within 3 minutes by dropwise addition

of working standard solution at intervals of 10 seconds until mixture resumes
bright orange appearance. .Repeat titration 2 times.

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 Note: Maintain continuous evolution of steam while titrating to prevent re-
oxidation of copper by air.

TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC

METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

7.9 Titration of sample

Accurately pipette 5 mL each of reagent Fehling A and Fehling B into 250 mL

Erlenmeyer flask.

Pipette an aliquot of hydrolysed sample solution into the Erlenmeyer flask and
dilute with distilled water to about 50mL.

Pre-heat electric heater-stirrer so that boiling of solution can start in

approximately 3 minutes. Mix solution, put a magnetic bar into the solution
and place on the pre-heated heater.

While rapidly boiling, add working sugar standard from burette. The volume
used is 0.5 to 1 mL less than that is used in the approximate titration carried
out in approximate titration of sample

Add 3 drops of methylene blue indicator and continue adding standard sugar
solution at intervals of 10 seconds until the indicator is completely
decolourised

Note: the titration should be completed within 3 minutes. Volume used

for titration should be within 15 – 50mL. If 10mL of sample make the colour
change before heat, use 5mL of sample. If the colour still changing without
heating try reduce the weight of sample.

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TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

8.0 CALCULATIONS

Total sugar (g per 100 g) =

( F− X ) x I x 250 x 100 x 100

W x 50 x 10 ml

where:

F = volume of standard sugar solution required to reduce 10mL mixed Fehling’s

solution
X = volume of standard sugar solution used in back titration of the sample
I = gram sugar per mL working standard solution.

W = weight of sample

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DOCUMENT CENTRE
TEST INSTRUCTION: DETERMINATION OF TOTAL SUGAR BY VOLUMETRIC
METHOD (THE LANE-EYNON METHOD)

Effective Date:30 September 2020 Instruction No :Kbio-TI-0016 Revision No :2

9.0 ACCEPTANCE OF RESULTS

Accept test results if one or more of the following conditions is satisfied.

a)Duplicate results should not differ by more than 10% of the mean.

b) Recovery test is between 80% to 110%

c) Mean concentration of duplicate results of quality control sample or

certified reference material should be within ± 2 SD in the control chart
based on established acceptance criteria.

Prepared by : Approved by :

_________________________ _______________________
Nor Amira Farhana Abu Bakar Zaiton Binti Abu Baidah
Laboratory Technician Senior Manager

11

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