Ref: GLT.QA.AM.

1612
SABMiller plc – Group Technical
Page: 1 of 8

Analytical Method Version: 1.0

Version Date: 1.0

Title: Unit 2.25 Thiobarbituric Acid Index (TBI) Determination Review Date: 20/04/2019
by Spectroscopy

Contents

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

1.1 Version 1 ......................................................................................................................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 ....................................................................................................................4
6.3 Reagents ......................................................................................................................5
6.4 Measurement Procedure..............................................................................................6
Sample blank ......................................................................................................................... 7
6.5 Reporting......................................................................................................................8
6.6 Verification....................................................................................................................8

POSITION NAME SIGNATURE DATE

Originator:

Approver:
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Determination by Spectroscopy

1 Version & Revision Details

1.1 Version 1

This is the original document Version 1.

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Determination by Spectroscopy

2 Purpose and Principles

2.1 Purpose

This document describes a method to determine the heat stress to which a sample has been
subjected by the measurement of lipid peroxidation and other products which result from heating
wort or beer.

2.2 Principles

The thiobarbituric acid index method is a colorimetric assay based on the absorbance of reaction
products of thiobarbituric acid in an acidic medium with a variety of products which result from
heating wort and/or beer, such as 5-hydroxymethy furfural. Values from the analysis can be used
to estimate the level of heat stress a product has undergone during processing (low values = low
thermal stress; high values = high thermal stress).
The reaction gives rise to a yellow chromophore which is measured at 448nm. The
measurement generates a thiobarbituric acid index (TBI) number which is dimensionless and
can be used to estimate the level of heat stress a product has undergone during processing.
By quantifying the effects of heat on wort and beer, adjustments can be made to the process
upstream to reduce the level of heat to which the product is exposed during production.
Reducing the level of heat stress during beer production is believed to have a positive effect
on the flavour stability of the beer produced.

3 Scope

This method can be applied to wort and beer at any stage of production.

4 References

ASBC Methods of Analysis, Thiobarbituric Acid Index 2009, 1-2

Thalacker R. and Bößendörfer “Thiobarbituric acid index (TBI),” Brauwelt International, Helsinki,
2005/I, 35-37

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

5 Definitions and Abbreviations

5.1 Definitions

A448 – Absorbance at 448 nm

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

Thiobarbituric acid index number – A dimensionless number generated from the absorbance of the
coloured product of the reaction between lipid peroxidation products and thiobarbituric acid
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Determination by Spectroscopy

5.2 Abbreviations

PPE – Personal protective equipment

TBA – Thiobarbituric acid
TBI – Thiobarbituric acid index number

6 Method

6.1 Health and Safety

6.1.1 Hazards

a) Glacial acetic acid is corrosive.

b) Thiobarbituric acid is an irritant.
c) Reaction with thiobarbituric acid requires heating.

6.1.2 Safety Requirements

a) General laboratory PPE

b) Safety glasses
c) Chemical resistant gloves
d) Heat resistant gloves

6.1.3 Precautions

a) Use chemical resistant gloves and safety glasses when dispensing glacial acetic acid.
b) When making up solutions with glacial acetic acid work in a fume cupboard.
c) Always add acid to water not water to acid.
d) Wear safety glasses and chemical resistant gloves when working with thiobarbituric acid.
e) Use heat resistant gloves and tongs to handles hot test tubes.

6.2 Equipment

6.2.1 Equipment Requirements

a) Spectrophotometer (visual light capability is sufficient)

b) Water bath, 70°C
c) Analytical Balance, capable of weighing 0.0001g
d) Test tubes (20-25 ml), preferably amber with ground glass stoppers
- Alternately clear glass test tubes may be wrapped with aluminium foil to reduce
light exposure (two per sample required).
- Glass marbles can also be used instead of ground glass stoppers
e) Test tube rack
f) Thermometer, standard, 0 – 100°C
g) Spectrophotometer cuvettes, glass or quartz 1-cm
h) Vortex mixer
i) Auto pipettes, (capable of delivering 5 ml and 10 ml aliquots)
j) Bulb pipettes, Grade A, capable of delivering 5 ml and 10 ml
k) 50 ml and 100 ml volumetric flasks with stoppers
l) 1L Amber reagent bottle
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Determination by Spectroscopy

m) Glass Funnels
n) 250 ml Erlenmeyer flasks
o) Fluted filter paper, 32 cm diameter – Schleicher and Schuell 0860 or Whatman 93V or
similar
p) Refrigerated centrifuge
q) 250 ml centrifuge tubes with caps
r) Glass weighing boat
s) 1000 ml measuring cylinder
t) Ice bath
u) Sonicator
v) Plastic film such as Parafilm or similar

6.2.2 Equipment Preparation

a) Set the spectrophotometer to read in absorbance mode at 448nm.

b) Ensure that the spectrophotometer is turned on at least 30 min before performing
absorbance readings.

6.2.3 Maintenance

Spectrophotometers should be serviced according to manufacturer’s recommendations.

6.2.4 Calibration

None

6.2.5 Storage

None

6.3 Reagents

6.3.1 Reagents Required

a) 100% glacial acetic acid, AR

b) Thiobarbituric acid, AR, molecular weight 144.15

6.3.2 Reagent Preparation

Glacial Acetic Acid Solution, 90%

a) Fill 1000 ml measuring cylinder with 100 ml purified water.

b) Add 100% Acetic acid to 1000 ml mark.
c) Transfer to 1L Amber Schott bottle, cap, invert to mix
d) Store at 4oC
e) Solution is stable for 1 month

Thiobarbituric Acid, 0.02M

a) Weigh off 0.576 g of thiobarbituric acid (AR) or (Molecular weight 144.15) and transfer to
200 ml volumetric flask.
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Determination by Spectroscopy

b) Rinse weighing boat with 90% acetic acid as thiobarbituric acid will stick to glass and add
to flask.
c) Fill flask with approximately 100-150 ml 90% acetic acid and wrap in foil (ensure flask is
completely covered with foil as thiobarbituric acid is light sensitive).
d) Sonicate until dissolved (may take 30 min-45 min).
e) Once dissolved fill up to mark with 90% acetic acid and cool to 20°C.
f) Stopper and invert to mix.
g) Keep flask at 4oC in fridge and wrapped in foil.
h) Prepare freshly each day.

Notes:
i. 200ml is enough for approximately 40 samples.
ii. As thiobarbituric acid dissolves, the solution will have a faint yellow tint. If
the solution turns dark yellow, discard.
iii. During sonication the foil may disintegrate. If so, rewrap the flask with foil if
too much of the surface area is exposed to light.

6.3.3 Standardisation

None

6.3.1 Disposal

All solutions can be flushed down the sink with copious flushing with water.

6.4 Measurement Procedure

6.4.1 Sampling

a) Sample according to regional Quality requirements and scheduling.

b) Wort should be sampled half way through cooling.

6.4.2 Sample Preparation

Wort

a) After collection wort can be kept for a maximum of 24 hours at 4°C before analysis.
b) Centrifuge at 50C at 2000 x g for 10 minutes to remove any particulate matter as this will
affect the absorbance.
 Alternately wort may be filtered through filter paper with Kieselguhr.
c) Make a 10 x dilution by pipetting 5ml sample into a 50ml volumetric flask using a bulb
pipette; make up to mark with purified water. Stopper and invert flask to mix.

Note: Diluted samples should yield absorbance values of between 0.10 to 0.50 units. If
absorbance is outside this range adjust dilution as required and repeat assay.

d) If not analysed the same day, freeze the samples.

e) Samples must be analysed within 5 days.

Note: Before analysing all frozen samples must be completely thawed, mixed gently, and re-
centrifuged at 5°C for 10 min at 2000 x g.
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Determination by Spectroscopy

Package Product:

a) Cold packaged product samples are degassed by filtration through folded filter paper or by
placing on shaker for 20 min.
b) Discard the first 30ml and collect the next 10 to 15 ml.
c) Using a bulb pipette make a 10 x dilution by pipetting 5 ml of the filtered sample into a 50
ml volumetric flask and make up to mark with purified water. Stopper and invert to mix.
d) If not analysed the same day, freeze the samples.
e) Samples must be analysed within 5 days.

Note: Before analysing all frozen samples must be completely thawed, mixed gently, and re-
centrifuged at 5°C for 10 min at 2000 x g.

Sample blanks

a) Sample blanks must be prepared for each brand being analysed.

b) Select wort or packaged sample of same brand as samples being analysed to be used as
blanks.
c) Make a 10 x dilution by pipetting 10 ml of sample to be used as blank into a 100ml
volumetric flask using a bulb pipette and make up to the mark with purified water.
d) Approximately 100ml diluted sample will be required per assay.

6.4.3 Test Method

Note: If not using amber test tubes cover in test tubes with foil before beginning pipetting.

6.4.3.1 Preparation of tubes

Reagent blank

a) Pipette 10ml of purified water into a test tube in duplicate using a bulb pipette.
b) Add 5 ml of 0.02 M thiobarbituric acid solution, stopper or cover with plastic film and vortex.
c) When heating remove the film (if used) and cover tube with a marble.
d) Proceed as for main sample.

Sample blank

a) For each brand, pipette 10ml of 10 x diluted sample blank into a test tube using a bulb
pipette (prepare duplicate sample blanks)
b) Using an auto pipette add 5 ml of 90% acetic acid solution, stopper or cover with plastic film
and vortex.
c) When heating remove the film (if used) and cover tube with a marble.
d) Proceed as for main sample

Samples

a) Using a bulb pipette dispense in duplicate 10 ml of 10 x diluted sample into a test tube.
b) Add 5 ml of 0.02 M thiobarbituric acid solution, stopper or cover with plastic film and vortex.
c) When heating remove the film (if used) and cover tube with a marble.
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Determination by Spectroscopy

d) Place blanks and samples test tubes in test tube rack, adding one capped test tube
containing 15 ml water (to be used as a temperature check) and place entire test tube rack
into 70oC water bath.

Notes:
i. Avoid direct sunlight
ii. Ensure that the temperature in the bath decreases only briefly by no more
than 1-2°C when the test tubes are introduced.
.
e) Heat the samples for 60 min.
f) At the end of 60 min, cool the samples to 20°C quickly by placing the entire rack into a
water and ice bath.
g) Place thermometer in the test tube of water, and pull out the rack when the temperature
reaches 20°C.

6.4.3.2 Measurement

a) Zero the instrument at 448 nm with purified water.

b) Starting with blanks, read the absorbance of each test tube at 448 nm.

6.4.4 Calculations

Calculate the corrected absorbance as follows:

Corrected Absorbance = Sample A448 – (Sample Blank A448 + Reagent Blank A448)

Calculate TBI follows:

TBI = (Corrected absorbance) × 10 × F

F = dilution factor

Note: Cold wort (after wort cooling) ≤ 60 for lagers. Expect higher values with brews
containing dark malts.

Example

Sample A448 = 0.475

Reagent blank A448 = 0.012

Sample blank A448 = 0.024

TBI = 0.475 – (0.024 + 0.012) x 10 x 10

= 43.9

6.5 Reporting

Report TBI to the nearest whole number

6.6 Verification

a) Reference control (where available)

b) Inter Laboratory Error Survey (where available for this parameter)