Ref:

SABMiller plc – Group Technical

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Analytical Methods Version:

Unit Standard 3 Version

Date:

Title: Unit 3.15.02 – Sulphur Dioxide Determination By Flow Injection Scheduled

Review:
Analysis (DNTB)

Contents

1 Version & Revision Details.....................................................................................................2

1.1 Version 1.0 ...................................................................................................................2
2 Purpose and Principles ..........................................................................................................3
2.1 Purpose ........................................................................................................................3
2.2 Principles......................................................................................................................3
3 Scope ..................................................................................................................................... 3
4 References............................................................................................................................. 3
5 Definitions and Abbreviations.................................................................................................3
5.1 Definitions.....................................................................................................................3
5.2 Abbreviations................................................................................................................4
6 Method ................................................................................................................................... 4
6.1 Health and Safety.........................................................................................................4
6.2 Equipment ....................................................................................................................5
6.3 Reagents ......................................................................................................................7
6.4 Measurement Procedure............................................................................................10
6.5 Reporting....................................................................................................................13
6.6 Verification..................................................................................................................13
7 Appendices .......................................................................................................................... 14
7.1 Appendix 1: Installation of the Gas Diffusion Membrane on the Total SO2 Cassette.14

POSITION NAME DATE

Originator Analytical Method Developer A. Cameron-Clarke October 2013

Review Analytical Methods Review Team V. Moonsamy February 2014
Approval Group Chief Brewer K. Smart
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1 Version & Revision Details

1.1 Version 1.0

This is the original document Version 1.0.

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2 Purpose and Principles

2.1 Purpose

This method describes the determination of total sulphur dioxide (SO2) in beer by flow injection
analysis (FIA) by means of the colour reaction of SO2 with 5,5’-dithiobis (2-nitrobenzoic acid)
(DTNB or Ellmans reagent) for detection.

2.2 Principles

FIA is a derivative of segmented flow analysis automation without air segmentation, and is based
on the injection by a valve of a liquid sample into a moving, non-segmented, continuous carrier
stream of a suitable liquid. The injected sample forms a zone, which is then transported toward a
detector that continuously records the changes in absorbance, electrode potential, or other
physical parameter resulting from the passage of the sample material through the flow cell. FIA
systems separate each sample from subsequent sample with a carrier reagent. Mixing occurs as
the sample travels through tightly wound Teflon mixing coils. Flow injection injects a sample and
detects it before the next sample is injected.

In this method the sample is hydrolysed with 3.5N HCl at 100°C converting bound SO2 to free. The
total SO2 (bound SO2 which has been freed plus SO2 which was free) passes through a gas
diffusion membrane into a phosphate buffered solution. DTNB reagent is then added, reacting with
the SO2 and producing a strong yellow colour which is measured at 420 nm.

3 Scope

This method is applicable to finished and in-process beer samples, as well as flavoured malt
beverages (FMB) samples and measures SO2 on the range 0 to 25 mg/l.

4 References

Riddles, PW et al Reassessment of Ellman’s reagent, Meth Enzymol: 91, 49-60 (1983)

ISO 3696:1987, Water for analytical laboratory use. Specification and test methods
Instrument operating instructions

5 Definitions and Abbreviations

5.1 Definitions

Bound SO2 – SO2 which is bound to aldehydes and released by treatment with alkali followed by
acid
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Free SO2 – SO2 which is not bound to aldehydes and is free in solution
Total SO2 – Sum of free and SO2 and SO2 bound to aldehydes
Purified water - Water which has been distilled or deionised and is of at least grade 2 as specified
in ISO 3696:1987

5.2 Abbreviations

DTNB – 5, 5’- dithiobis (2- nitrobenzoic acid)

FIA – Flow Injection Analysis
FV – Fermentation Vessel
NaHSO3 – Sodium bisulphite
PPE – Personal protective equipment
SO2 – Sulphur dioxide
SV – Storage Vessel

6 Method

6.1 Health and Safety

6.1.1 Hazards

a) Hydrochloric acid is corrosive.

b) Sodium bisulphite is a mild irritant and may be toxic.
c) Iodine is a serious irritant and highly corrosive.
d) Ethanol is flammable.

6.1.2 Safety Requirements

a) General laboratory PPE

b) Safety glasses
c) Surgical mask
d) Surgical and chemical resistant gloves

6.1.3 Precautions

a) Wear safety glasses and chemical resistant gloves when dispensing hydrochloric acid.
b) Always add acid to water NOT water to acid.
c) Wear surgical gloves and safety glasses when working with sodium bisulphite and iodine.
When dispensing the dry powders wear a surgical mask as well.
d) Keep flammable substances away from open flames and other sources of ignition.
e) Ethanol must be stored in a well-ventilated flammables cupboard.
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6.2 Equipment

6.2.1 Equipment Requirements

a) FIA Star 5000 Analyzer unit or similar

 5000-080 Total SO2 Method Cassette: Interference filters, M=420nm and R=720nm
 5027 Sampler
 Teflon Gas Diffusion Membrane: Foss, Part #5000 2875
 Pump Tubing Red/Red (Reagent): Foss, Part #1001 0273
 Pump Tubing Red/Red (Short): Foss, Part #1001 0274
 Pump Tubing White/White: Foss, Part #1001 0271
 Pump Tubing Orange/Orange: Foss, Part #1001 0270
 Pump Tubing Black/Black: Foss, Part #1001 0269
 FIA Lamp: Foss, Part #1001 4924

Note: This method is based on the FIA Star 5000 Analyzer. If other systems are used the
equipment requirements will change according to the manufacturer’s recommendations.

b) Analytical balance capable of weighing to the nearest 0.1 mg

c) Volumetric flasks: 100, 500, 1000 and 2000 ml
d) Bulb pipettes: Class A: 1, 2, 3, 5 and 50 ml
e) Micro burette, 10 ml
f) Sonicator: Ultra Sonic Bath, Model 2510-mth, VWR Part #100501-412 or equivalent
g) Centrifuge tubes: 50 ml
h) Coffee filter: Available commercially (needed for Fermentation Samples)
i) Centrifuge: Sorvall RC 3 BP+ or the equivalent

6.2.2 Equipment Preparation

Start up

a) Check all solutions and fill if low.

b) Connect all lines (Buffer, 3.5N HCl, Colour reagent, Carrier) to reagent water.
c) Latch pump handles.
d) Login to software if using.
e) Run a rinse.
 This will take about 5 minutes.
 This is done only if starting up the instrument. It is not required if the instrument is
already running.
f) All lines should be labelled as to reagent (Buffer, 3.5N HCl, Colour and Carrier (H2O).
g) Switch all lines to appropriate reagent and allow 5-10 minutes to stabilize.
h) Observe reagent baseline(s).
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 Make sure it is a smooth, straight line as displayed by the software.

 Ensure that the Standard Deviation (M-R) is <1.0 mAU as displayed by the
software.
 This step is required before each sample run. See operator manual for
troubleshooting guidelines

6.2.3 Maintenance

Note: Maintenance should be done as recommended by instrument manufacturers. The

following maintenance procedures are based on the recommendations for the FIA Star 5000
system.

Shut down

Daily Cleaning (to be completed after every set of analysis)

a) Connect all lines (Buffer, 3.5N HCL, Colour, Carrier) to Reagent Water
b) Run a rinse cycle command.
c) This will take about 5 minutes.
d) Put pump on standby and ready for start of next set of analysis.

Shutdown

a) Connect all lines (Buffer, 3.5N HCL, Colour and Carrier) to Cleaning Detergent.
b) Run a rinse cycle command.
c) This will take about 5 minutes.
d) Move all lines (Buffer, 3.5N HCL, Colour and Carrier) to the Reagent Water container and
start another Rinse Cycle command.
e) After this cleaning cycle is completed, remove all lines and let the system pump dry.
f) Immediately after system is dry:
 Put pump on standby
 Disconnect all reagent lines
 Release pump latches
 Power off and release pump handles.
g) This shutdown routine is applicable if the instrument will be powered off for more than 24
hours.

Weekly Cleaning

a) Pump Reagent Water through all lines for 5 minutes.

b) Place all lines into a beaker of Cleaning detergent.
c) Perform the 2 cleaning cycles to clean out all ports of the valve with Cleaning Detergent
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d) Flush Cleaning Detergent out of the system by pumping Reagent Water for 20 minutes.
e) Once this is completed, he instrument is ready to be started up using the start-up sequence
in Section 6.2.2.

Routine Maintenance

a) Other routine maintenance can now be performed after the Weekly Cleaning routine. (See
Operator’s Manual).
b) Diffusion membrane and pump tubes should be replaced monthly (see Appendix 1).

Notes:
i. A ruptured membrane gives no proper peak. In addition, the background
absorbance will be several 100mAU higher than usual.
ii. The best indicator for pump tube replacement is to observe the flow at start-up.
If flow fluctuation is visible the tubes should be replaced.
iii. Regularly scheduled replacements of membranes and tubes will minimize the
chance of problems developing.

6.2.4 Calibration

a) Calibrate the instrument prior to each sample run by running the 5 standards prepared as in
Table 1, Section 6.3.2.
 Slope must be 0.998000 or greater.
 If the instrument does not keep records of calibration, use an Excel spread sheet to
keep a log of all calibration data as well as results of calibration standards analysed
as unknown.
b) Insert a sample of the original 15 ppm calibration standards at random intervals in the
sample list to be run as unknown.
c) This 15 ppm standard must be +/- 0.5 ppm of the original calibrated value.
d) If not, recalibrate and re-run the list.

6.2.5 Storage

6.3 Reagents

6.3.1 Reagents Required

a) Sodium (Mono) Phosphate Monohydrate: NaH2PO4 • H2O

b) Di-Sodium Phosphate Heptahydrate: Na2HPO4 • 7 H2O
c) Ethanol: >95 % (do not use denatured)
d) DNTB
e) Sodium Bisulphite (NaHSO3)
f) SCABA Detergent or similar
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g) Hydrochloric Acid (Concentrated)

h) Sodium Thiosulfate 0.1N ampule

Note: This solution must be replaced by the expiry date given on the bottle and replaced
accordingly. If the solution becomes cloudy before the expiry date, it must be replaced
immediately.

i) Iodine Solution 0.1N ampule

j) Starch Indicator ( Vitex or similar)1% Solution
k) Sodium Hydroxide 40% w/w

Notes:
i. Starch indicator can also be purchased as powder and made up as a 1%
solution with Reagent Water.
ii. Sodium hydroxide can also be purchased as pellets and made up as a 40%
(w/w) solution with reagent water.

6.3.2 Reagent Preparation

Reagent water

a) Water used for the reagents and standards must be degassed before use. This applies
especially to water produced by reverse osmosis or ion exchange equipment which may be
high in gas content.
b) Using freshly prepared (i.e. by ion exchange/reverse osmosis etc.) purified water, boil for
15 min to degas.
c) Cool down to room temperature.
d) Use this water for all standards and reagent preparation.

Phosphate Buffer, pH 8.4

a) Dissolve 1.00g monosodium phosphate monohydrate and 66.66g di-sodium phosphate

heptahydrate in 1500 ml Reagent Water in a 2000 ml volumetric flask.
b) Add 100 ml 96% ethanol.
c) Make up to volume with Reagent Water.
d) Filter through a 0.45 μm filter before use.
e) Solution is stable for 1 month.

DTNB Reagent

a) Weigh 0.760g DNTB into a 1000 ml volumetric flask.

b) Add 100 ml 96% ethanol and completely dissolve.
c) Make up to the mark with Phosphate Buffer, pH 8.4.
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d)Filter through a 0.45 μm filter, then allow the reagent to stabilize for 2 days at room
temperature prior to use.
e) Solution is stable for 1 month.

3.5N HCl

a) Carefully add 145 ml of concentrated HCl to 500 ml Reagent Water.

b) Mix well.
c) Solution is stable for 1 month.

Stock Standard Solution, 500 mg/l SO2:

a) Add 100 ml 96% ethanol to 500 ml Reagent Water in a 1000 ml volumetric flask.
b) Based on the sulphite standardisation assay (see Section 6.3.3) add the proper amount of
NaHSO3 powder to this solution to achieve 500 mg/l SO2 and dissolve.
c) Make up to the mark with Reagent Water and mix well.
d) Store at 4°C in amber reagent bottle.
e) Bring to room temperature before use.
f) Solution is stable for 5 days.

Note: For a sample calculation see Section 6.4.4.

Working Standards

a) Working Standards are made up by diluting Stock Standard Solution as indicated in Table 1
to 100 ml in volumetric flasks with Reagent Water.
b) Prepare working standards daily.

Table 1 Working Standard Preparation

Volume of Stock
Concentration of SO2
Standard Solution to be
in Working Standard
made up to 100ml
(mg/l)
(ml)
0.0 0
1.0 5
2.0 10
3.0 15
5.0 25
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Cleaning Detergent

a) In a 2 L volumetric flask, add 200 ml of 40% w/w Sodium Hydroxide, 200 mL of 96%
ethanol (do not use denatured) and 20 ml of SCABA Detergent Concentrate.
b) Make up to the mark with Reagent Water.
c) Mix well.

6.3.3 Standardisation

Note: In order to prepare accurate calibration standards, the exact SO2 content of
Sodium Bisulphite reagent must be determined before making the stock standard solution.

Determination of per cent SO2 in Na HSO3

a) Using a 50 ml bulb pipette, dispense 50 ml of 0.1N iodine solution into a 100-ml beaker.
b) Weigh 220 mg NaHSO3, record the exact weight, and add to the iodine solution.
c) Cap the beaker with parafilm and mix the solution gently using a magnetic stirrer and allow
to stand at room temperature in a dark area for a minimum of 5 minutes and no longer than
10 minutes.
d) Carefully add 1 ml of concentrated HCl and 5 ml of starch indicator.
e) Mix and titrate the excess iodine by carefully adding 0.1N sodium thiosulfate standard using
a 10 ml micro burette until 1 drop of the titrant changes the colour of the mixture from blue
to colourless.
f) Record the volume of 0.1N sodium thiosulfate used; this is equivalent to the volume of
excess iodine solution.
g) Calculate the exact per cent of SO2 in NaHSO3 (see Section 6.4.4.1).

6.3.4 Disposal

All aqueous waste may be disposed of down the sink with plentiful flushing with water.

6.4 Measurement Procedure

6.4.1 Sampling

Notes:
i. SO2 is extremely volatile (boiling point -10°C) so sampling cold is critical.
ii. Samples should be kept cold and stoppered as much as possible between
operations.
iii. Sampling coils and collection tubes must be pre-chilled in ice water.
iv. Loss of carbon dioxide during and after sampling must be avoided as this
results in loss SO2.
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a) Adhere to regional QA requirements and scheduling.

b) Working on crushed ice, connect a sample coil to the tank sample cock and thoroughly
flush the line and coil with sample.
c) Slowly fill the centrifuge tube to the brim with sample to avoid loss of CO2.
d) Immediately cap the tube.

Note: Keep the sample in crushed ice.

6.4.2 Sample Preparation

Note: Degassing and filtration can result in loss of SO2.

a) Samples must be degassed and devoid of particulate matter before the analysis.
b) Prepare a control (Reference) beer with known SO2 values to be run at the beginning and
at the end of each sample list.
c) Do not add any tributyl phosphate defoamer.

FV Samples

Notes:
i. Never filter samples through filter paper.
ii. In removing the sample from the centrifuge care must be taken not
to disturb the sediment.
iii. After centrifugation keep the sample cold in a beaker with crushed
ice.

a) Centrifuge 40 ml of each sample at 2000 x g for 20 minutes.

b)Decant the supernatant with no yeast transfer, then filter the appropriate volume with a 45
micron filter into the sample cup before analysis. Sonication is not required.

Aging Sample, Bright Beer Samples and Packaged Product:

a) Decant 100 ml of beer into a 500 ml Erlenmeyer flask.

b) Sonicate uncovered for 15 minutes.
c) All of the samples must be run within 1 hour of completion or sample preparation.
d) Any samples that resulted in SO2 values that exceed the calibration range (maximum 25
ppm) should be diluted and re-analysed.

6.4.3 Test Method

a) Create sample list using the software Sample List Browser and run samples.
 The sample list should be less than 10 samples per run.
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b) Insert a sample of the original 15 ppm calibration standards at random intervals in the list to
be run as unknown.
 This 15 ppm standard must be +/- 0.5 ppm of the original calibrated value.
 If not, recalibrate and re-run the list.
c) A control (Reference) beer sample should also be analysed at the beginning and end of the
run to check for continuity.
d) All samples should be analysed within 1 hour after sample preparation is completed.
e) The calibration standard, analysed as unknown data, should be logged and kept for
f) historical tracking.

6.4.4 Calculations

6.4.4.1 SO2 content of Stock Standard Solution

Note: The assay of SO2 content in NaHSO3, which is used as the primary standard for the
calibration curve for SO2 is determined by iodometric titration shown by the equations
below.

1. HSO3 - (aq) + H2O + I2 (aq) (excess)  2I- + SO4 2- + 3H+ + 2I- (aq)

a) The volume of 0.1N thiosulphate used to back titrate excess iodine is equivalent to the
excess volume of iodine present (T ml).
b) Each ml of 0.1N iodine consumed is equivalent to 3.203 mg of SO2.
Excess Iodine volume = T ml
Volume used = (50 – T) ml
Amount of SO2 in stock solution = 3.203 x (50 –T) mg
Therefore % SO2 content in NaHSO3 = Amount of SO2 in stock solution x 100
220mg (amount NaHSO3 used in assay, Section 6.3.3)

Example

From sulphite assay: 5.21 ml of excess 0.1N iodine was found from titration with 0.1N Sodium
Thiosulfate.
This means 50.00–5.21, or 44.79 mL of 0.1N iodine reacted with sulphite.
This is equivalent to 44.79 x 3.203 of SO2 = 143.5 mg
SO2 Content (%) = 143 x 100
220
= 65.21 %

6.4.4.2 Amount of NaHSO3 to be used in the preparation of the Stock Standard Solution

To prepare Stock Standard Solution of 500 mg/l of SO2:

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From assay of NaHSO3, calculated SO2 content = Y % (Section 6.4.4.1)

SO2 required in Stock Standard Solution = 500mg/l

Amount of NaHSO3 to be added/l = 500 x 100 mg

Y

This is the weight of NaHSO3 to be used to make a 1L volume of stock standard

solution with the concentration of 500 mg/L SO2.

6.5 Reporting

Report results to the nearest mg/l

6.6 Verification

a) Inter Laboratory Error Verification

b) Reference control (where available)
c) Analytical Methods Unit Standard 0 Verification Routines
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7 Appendices

7.1 Appendix 1: Installation of the Gas Diffusion Membrane on the Total SO2 Cassette

a) Open the pack and carefully pull out the membrane.

b) Pump air through the system.
c) Remove the 6 screws from the gas diffusion unit.
d) Lift the upper Plexiglas block and remove the old gas diffusion membrane.
e) Inspect and rinse the channels in both upper and lower blocks.
f) Place the new gas diffusion membrane on the lower Plexiglas block.
g) Put the upper Plexiglas block back on.
h) Tighten the 6 screws.
i) Pump water to check that there is no leakage.