SABMiller plc – Group Technical Ref: GLT.QA.AM.

1172

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Analytical Methods Version: 1.0

Unit Standard 2
Version Date: 21/10/2014

Title: Unit 2.01 - Measurement of Alpha-Glucan Scheduled Review: 20/01/2018

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................................................................................................................3
6 Method ................................................................................................................................... 4
6.1 Health and Safety.........................................................................................................4
6.2 Equipment ....................................................................................................................4
6.3 Reagents ......................................................................................................................5
6.4 Measurement Procedure..............................................................................................7
6.5 Reporting......................................................................................................................9
6.6 Verification....................................................................................................................9

POSITION NAME DATE

Originator Analytical Methods Developer A Cameron-Clarke August 2013

Review Analytical Methods Review Team V. Moonsamy
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 document describes a method for the spectrophotometric measurement of alpha glucan in
wort and beer.

2.2 Principles

Alpha glucan can negatively influence wort and beer filtration as well as clarity and clarity stability.
Levels ex brewhouse are specific to brewhouse regimes and raw materials used.
Wort levels should be trended and any positive (increasing) trend should be carefully monitored,
especially during filtration.
Alpha glucan can be quantified by formation of a coloured complex in the presence of iodine which
can be measured spectrophotometrically at 530 - 570nm. In this Method the absorbance is read at
570 nm.

3 Scope

This method is applicable to wort and beer at all stages of production.

4 References

Schur F, et al, Brauerei Rundschau, August 1978, p 129 – 144

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

5 Definitions and Abbreviations

5.1 Definitions

Alpha glucan - refers to polysaccharide molecules made up of glucose monomers linked together
by alpha bonds. Starch and starch residues are referred to as alpha glucan.

Purified water - Water which has been distilled or purified and is of at least grade 3 as specified in
ISO 3696:1987

5.2 Abbreviations

PCP - Pentachlorophenol

PPE – Personal protective equipment

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6 Method

6.1 Health and Safety

6.1.1 Hazards

a) Alcohol is highly flammable.

b) Pentachlorophenol (PCP) is extremely toxic.
c) Phosphoric acid is corrosive.

6.1.2 Safety Requirements

a) General laboratory PPE

b) Surgical gloves
c) Safety glasses
d) Flammables cupboard

6.1.3 Precautions

a) Ethanol should be stored in a well-ventilated flammables cupboard and never placed near
open flames or other sources of ignition.
b) Wear surgical gloves when working with PCP and avoid inhalation when weighing out by
working in a fume cupboard.
c) When working with acids wear surgical gloves and safety glasses.

6.2 Equipment

6.2.1 Equipment Requirements

a) Spectrophotometer capable of reading in the visible range, fitted with a 40mm cell holder.
b) Spectrophotometric cell (glass), 40 mm pathway

Notes:
i. A 10mm path length cell may be used and the quantities in Test Method
(Section 6.4.3) reduced by a quarter.
ii. In order to adjust the values to the standard results obtained with a 40mm
path length all absorbances obtained with a 10mm path length cell should be
multiplied by 4.

c) Centrifuge capable of 2000 x g

d) Shaker, such as a circular vibrating shaker
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e) Vortex mixer
f) Top pan balance capable of weighing to 0.005 g
g) Centrifuge tubes with caps, 50 ml
h) Pipettes, graduated straight, 1,2 and 10 ml
i) Measuring cylinders, 10 and 50 ml
j) Glass beaker, 100ml
k) Erlenmeyer flasks, 100, 250 and 500ml
l) Volumetric flask, 1 L
m) Auto-dispenser, 30 ml
n) Rubber-tipped glass rod
o) Tissues
p) McCartney bottles, 30 ml or similar
q) Drying rack.

6.2.2 Equipment Preparation

6.2.3 Maintenance

6.2.4 Calibration

Spectrophotometer is calibrated against purified water in a 40 mm cell at 570 nm.

6.2.5 Storage

6.3 Reagents

6.3.1 Reagents Required

a) Ethanol, 96% AR
b) Pentachlorophenol (PCP)
c) Iodine ampules (0.01M) or
d) Potassium iodide (KIO3), AR and Iodine crystals
e) Phosphoric acid solution 1 M
f) Potassium dihydrogen phosphate solution, 0.1M

6.3.2 Reagent Preparation

0.01M Iodine

If using ampule: prepare as per manufacturer’s instructions.

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Notes:
i. Iodine is light sensitive and must be kept in an amber reagent bottle.
ii. This solution has a shelf life of one month and must be stored in a
refrigerator below 5C.
iii. Use cold.

If using potassium iodide:

a) Using a weighing boat, weigh out 4g Potassium Iodide into a 250ml Erlenmeyer flask.
b) Transfer the weighing boat residue to the Erlenmeyer flask with 5ml purified water and swirl
to mix.
c) Weigh out 2.54g Iodine crystals and add to the Erlenmeyer flask.
d) Mix to dissolve.
e) Transfer to a 1 L volumetric flask and make up to volume with purified water.

Notes:
i. Iodine is light sensitive and must be kept in an amber reagent
bottle.
ii. This solution has a shelf life of one month and must be stored in a
refrigerator below 5C.
iii. Use cold.

Phosphate buffer

a) Measure the pH of 250 ml of 0.1M KH2PO4 solution.

b) Adjust pH to 3.5 with 0.1 M phosphoric acid solution.

Pentachlorophenol solution (PCP)

Note: When working with PCP adhere to safety requirements listed under Sections 6.1.2 and
6.1.3.

a) Weigh out 0.33 g PCP into a 100 ml beaker.

b) Add 50 ml ethanol using a measuring cylinder.
c) Heat gently until totally dissolved.

Note: Do NOT use an open flame to heat and avoid over-heating - ethanol is flammable.

d) Transfer to a suitable container and label.

e) Store in a refrigerator at below 5°C - use cold.

6.3.3 Standardisation
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6.3.4 Disposal

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

6.4 Measurement Procedure

6.4.1 Sampling

Adhere to Quality requirements and schedules.

6.4.2 Sample Preparation

Note: Wort and packaged beers must not be filtered. Alpha-glucan will absorb on the filter
paper and be removed from the sample.

Wort

a) If sample can be analysed immediately:

- Mix the sample well in order to re-suspend the solids.
- Pour about 50 ml of sample in a 100 ml beaker and attemperate rapidly to 20 ±
0.5°C.
- Immediately proceed with the analysis as per 6.4.3 below.

b) If the wort has to be stored overnight:

- Pipette 0.1 ml PCP into a McCartney bottle or similar.
- Fill the bottle to the brim with well mixed sample.
- Store in a refrigerator below 5°C.

Note: Prior to the analysis, prepare the sample as in a) above.

Packaged Beer

a) Pour about 200 ml beer in a 500 ml Erlenmeyer flask.

b) Rapidly attemperate to 20 ± 0.5°C.
c) Shake vigorously for one minute, pour it five times from one flask to another and again
shake vigorously for one minute.

Note: Alternately sample can be shaken for 20 min.

6.4.3 Test Method

a) Pipette 7.5 ml prepared sample into a 50 ml centrifuge tube.

b) Dispense 30 ml ethanol into the sample and cap.
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c) Mix the contents for approx. 20 seconds on the vortex mixer or for 2 min on a vibrating
shaker.
d) Centrifuge at force 2000 x g for 10 minutes.
e) Carefully decant the supernatant.

Note: Care must be taken not to lose or disturb the precipitate.

f) Dry the precipitate on a drying rack with the aid of a slow stream of air blown into the tube.

Note: All traces of ethanol must be removed, for it prevents the re-hydration of starch
molecules.

g) Pipette 15 ml phosphate buffer (see Section 6.3.2) onto the dry precipitate.
h) Totally re-dissolve the precipitate by mixing for one minute on a vortex mixer and then for
15 minutes on a mechanical shaker.
i) Centrifuge at force 2000 x g for 10 minutes.
j) Zero the spectrophotometer against purified water in a 40 mm cell at 570 nm.
k) Conduct the blank as follows:
l) Pipette 10 ml phosphate buffer and 0.5 ml iodine solution into a 40 mm cell.
m) Mix well and immediately (between 2-3 seconds) read and record the maximum
absorbance (EJ).

Note: Use a rubber tipped glass rod for mixing sample and iodine.

k) Carefully pipette 10 ml of sample supernatant into a 40 mm cell, read and record the
absorbance (EZ).
l) Pipette 0.5 ml iodine solution into the cell containing the sample.
m) Mix and immediately (between 2-3 seconds) read and record the maximum absorbance
(EH).

Note: Use a rubber tipped glass rod to mix sample and iodine.

6.4.4 Calculations

Residual alpha-glucan:
 E = EH - EJ - (0,952 x EZ)

Where EH = Maximum absorbance of iodated sample

EJ = Maximum absorbance of iodated purified water
EZ = Absorbance of sample supernatant (See Section 6.4.3 g))
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6.5 Reporting

Report results to 2 decimal places.

6.6 Verification

a) Inter Laboratory Error Survey

b) Analytical Methods Unit Standard 0 Verification Routines