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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

MODULE 1: Materials and Wort

UNIT 1.11: Quality

SECTION 1.11.3: Laboratory Analysis

ABSTRACT: In this section we will review in more detail the topic of

beer quality and the laboratory procedures relevant to measuring the
physical and chemical parameters important in ensuring product
quality consistency. We will deal with the interpretation of analytical
data in simple statistical terms and outline the basic techniques and
methods used to measure some of the more important
characteristics of brewing raw materials and wort composition. The
relevance of ensuring accuracy of data generated and the value of
inter-laboratory collaborative analysis will be described.

LEARNING OUTCOMES: On completion and comprehension of this

unit you will be able to:

1. Understand the basic concepts applied to the interpretation of

analytical data and the use of simple statistics.
2. Describe the relevance of inter-laboratory collaborative analyses.
3. Understand the basic principles of the analytical techniques used
to measure important parameters and characteristics of raw
materials and wort composition.

PREREQUISITE UNDERSTANDING: Section 1.11.1

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

1.11 QUALITY……………………………………………………….…3
1.11.3 LABORATORY ANALYSIS………………………………….….. 3
1.11.3.1 Basic Concepts…………………………………….….. 3
(a) Sampling Error……………………………………………...3
(b) Accuracy and Precision……………………………………3
(c) Repeatability (r95)…………………………………………..4
(d) Reproducibility (R95)……………………………………….4
(e) Specification Ranges (Tolerances)……………………….5
(f) Simple Probability Calculations…………………………...6
1.11.3.2 The Relevance of Inter-Laboratory Collaborative
Checks……………………………………………………8
1.11.3.3 The Basic Principles of Analytical Techniques for : …8
(a) Malt and Adjuncts Hot Water Extract……………………..9
(b) Total Nitrogen……………………………………………….9
(c) Total Soluble Nitrogen……………………………………..9
(d) Moisture……………………………………………………..10
(e) Colour………………………………………………………..10
(f) Free Amino Nitrogen……………………………………….10
(g) S-Methylmethionine and Dimethyl Sulphide………….. 11
(h) Friability…………..……………………………………….....11
(i) Diastatic Power..…………………………………………… 11
(j) Wort Viscosity..…………………………………………….. 11
(k) Hop Resins……..………………………………………….. 12
(l) Hop Essential Oils………………………………………… 12
(m) Biological Oxygen Demand……………………………… 13
(n) Chemical Oxygen Demand……………………………….13
(o) Total Suspended Solids………………………………….. 13
Reference List………………………………………………………14

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

1.11 QUALITY

1.11.3 LABORATORY ANALYSIS

1.11.3.1 BASIC CONCEPTS

(a) Sampling Error

The purpose of sampling is to make some estimate as to the property
of the bulk material without examining the whole of it. A large bulk
will generally require a large sample and for a small bulk
consideration should be given as to whether the analysis requires a
destructive or non-destructive technique. The sample must also be
representative of the bulk as precise laboratory analysis of non-
representative samples may give seriously misleading information.

In-line analysis allows the whole of the bulk to be examined but these
methods are frequently less accurate than laboratory techniques and
are probably best used as a screening process control procedure.

The properties of some materials, such as plastic mouldings, change

after manufacture and such products should be sampled and
examined when their properties have stabilised. Conversely some
materials such as wort deteriorate rapidly after sampling and care
should be taken to ensure they are examined soon after the sample
has been taken.

Bulk beer samples must be homogeneous before a representative

sample can be removed and this is particularly difficult for
fermentation or maturation vessels where yeast growth or
sedimentation may be taking place.

Further details of sampling procedures for specific areas of interest

can be found in the relevant IOB, ASBC and EBC Analytical Method
Manuals.

(b) Accuracy and Precision

Accuracy is defined as the ability of an analytical method to provide

results equal to the true valve.
Precision is a general term to describe the amount of variation in a
method.

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

The differences between accuracy and precision are illustrated in a

'dart board' type of diagram shown in Fig.1 where it can be seen that
the best measurements are both accurate and precise.

* *
* * ** **
* *

A c c ur a t e Pr e c is e a n d a c c u r a t e

*
** * ** **
*

I m p r e c is e a n d in a c c u r a t e Pr e c is e b u t in a c c u r a t e

(c) Repeatability (r95)

Repeatability (r95) can be used as an internal laboratory method
check.
Repeated analysis of the same sample, by the same analyst using
the same equipment at the same time will give results that fall within
the stated range95% of the time, or 19 analyses in 20.

(d) Reproducibility (R95)

Reproducibility can be used as a between laboratories method
check.
Repeated analysis of the same sample by different analysts using
different equipment at different times will give results that fall within
the stated range 95% of the time, or 19 analyses in 20.

R 95 is usually greater than r95..

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

(e) Specification Ranges (Tolerances)

When specification ranges are set, allowance must be made not only
for analytical errors but also for process capability and errors derived
from sampling and averaging. Analytical errors are usually well
documented in the relevant methods manuals and can provide a
conservative guide to the minimum specification ranges required.

To estimate the total errors involved, two approaches are described

in the IOB methods, Statistical Section, one based upon Statistical
Process Control Theory and the other derived from BS4306
(ISO4259).

(It should be noted that BS4306 is restricted to petroleum products

only which are in general homogeneous products in which serious
sampling problems do not arise).

Using Statistical Process Control Theory variations within a process

are usually assumed to be distributed normally about the mean. The
spread of variation is represented by the standard deviation, sigma,
and from statistical theory 99.7% of all the actual values will be in the
range 6 x sigma.

The specification range, the interval between upper (A2) and lower
(A1) must therefore equal or exceeed 6 x sigma. The ratio of the
specification band width to 6 x sigma is termed the process capability
(Cp).

Cp = A1 – A2 (1)
sigma

A Cp value of 2 is usually recommended as the minimum acceptable

value in the construction of specification bands.

Example : For a beer colour target value 10 EBC and R(95) = 0.68
EBC

Since R(95) is 2.8 x standard deviation (where standard deviation is

an estiamte of the true standard deviation – sigma).

Sd = 0.68 = 0.24
2.8

From equation (1) above if Cp = 2

A2 – A1 = (2 x 6 x 0.24) = 2.88 EBC units

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

Thus for a beer colour of target value 10 EBC units the specification
limits are:-

10 +/- 1.44 EBC units

Using BS 4306 specification limits should be expressed as 'not less

than' or 'not greater than'.

Limits may be either:

a double limit, upper and lower
a single limit, upper or lower

The value chosen for the specification limit should take into account
R(95) for the method and for a double limit the specificied range
should not be less than 4 x R(95).

Using the example above the recommended specification band width

would be:

4 x 0.68 = 2.72 EBC units

and the specification limits would be:-

10 ± l.36 EBC units.

(f) Simple Probability Calculations -

Normal Distribution, Standard Deviation, Variance

A normal distribution of values around a mean can be demonstrated
by a symmetrical curve (Fig.2). The spread of the values, and the
curve, is given by the standard deviation (SD).

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

Where :
68% of all observations lie within Mean ± 1 Standard deviation
(M ±1SD)
95% of all observations lie within Mean ± 2 Standard
deviations (M ±2SD)

SD is defined by the formula

SD = (x1 – x) 2 / (n – 1)
where x1 = the individual result
x = the mean result
n = number of results

An example of the calculation of standard deviation is given below:

Beer Colour (xi) (xi - x)2

10.08 0.0004
10.11 0.0001
10.09 0.0001
10.10 0.0000
10.12 0.0004

Total 50.50 0.0010

SD = 0.0010 / 4
= 0.016 EBC

For a distribution curve as shown in Fig.1 approximately 68% of the

values lie within ± 1SO, 95% within ± 2SD and 99.7% within ± 3SD.

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

1.11.3.2 THE RELEVANCE OF INTER-LABORATORY COLLABORATIVE

CHECKS

The purpose of interlaboratory checks is to ascertain whether the

results from a laboratory for a particular analyte are acceptable when
compared with other laboratories using the same or possibly different
methods.

All proficiency schemes should have their own protocol which should
be available for examination.

The major proficiency scheme for the brewing industry is the Brewing
Analytes Proficiency Scheme (BAPS) which is run jointly by the
Laboratory of the Government Chemist (LGC) and the Brewing
Research International (BRI). Samples of a single beer are
distributed every four weeks and participants analyse, on an optional
basis, for analytes that of particular relevance or interest.

A microbiological development of BAPS is also available.

Other relevant inter-laboratory check schemes are the Distilling

Analytes Proficiency Scheme (DAPS) and the Independent
Laboratories Collaborative Scheme (ILCS). Pauls Malt Limited run a
scheme for malt analysis.

The BAPS scheme uses robust statistics in which the mean is

replaced by the median and the standard deviation by the Median of
Absolute Differences (MAD) from the sample median. This is then
multiplied by 1.483 to make it equivalent (MAD) to the standard
deviation in the normal distribution.

Results are reported in terms of Z - scores

Z score = X1 - assigned value

established standard deviation.

The proficiency of a laboratory is judged by the absolute value of its

2 – score using the following criteria -

Z<2 = Satisfactory

2< Z <3 Questionable

Z> 3 = Unsatisfactory

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

1.11.3.3 ANALYTICAL TECHNIQUES

The principles of analytical techniques for malt, adjuncts, wort (where

relevant)

(a) Malt and Adjuncts Hot Water Extract

Malt is milled to produce a grist.

Soluble material within the grist is extracted with hot water during
mashing.
At the end of mashing, insoluble material is removed by filtration
leaving a clear wort.
The specific gravity of the clear wort is determined at 20°C and from
this value the Hot Water Extract is calculated.

For adjuncts the grist is composed of a mixture of unmalted adjunct

and an ale malt control.

(b) Total Nitrogen

Two methods are in common use, one using the Dumas principle
and the other using the Kjeldahl procedure.

Dumas
Samples are pyrolysed at 950 ºC in high purity oxygen. The
combustion gases are collected and equilibrated to constant
temperature and pressure. A fixed volume of the combustion gases
is then sampled and carried in a stream of high purity helium, through
a series of absorbents to remove moisture, oxygen and carbon
dioxide. Nitrous oxides are reduced over hot copper and the
resultant nitrogen is measured with a thermal conductivity detector.

Kjeldahl
Nitrogenous compounds are digested with hot sulphuric acid in the
presence of catalysts to give ammonium sulphate.

The digest is made alkaline with sodium hydroxide solution and

released ammonia is distilled into an excess of boric acid solution.
The ammonia is titrated with standard acid.

(c) Total Soluble Nitrogen

A standard laboratory wort is prepared as in IOB Method 2.4 (Malt,
Hot Water Extract).
An aliquot of the wort is evaporated almost to dryness in the
presence of a few drops of concentrated sulphuric acid.

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

The total nitrogen content of the almost dry residue is determined as

described in IOB Method 1.5 (Barley, Total Nitrogen Content).

Malt moisture content is determined as described in IOB Method 2.3

(Malt, Moisture Content).

The total soluble nitrogen content of the malt is calculated and

expressed on a dry weight basis to two decimal places.

(d) Moisture
The sample is milled to form a fine grist.
The grist is dried in an oven that has been previously standardised.
The moisture content of the malt is calculated from the loss in mass
during drying.

(e) Colour
The EBC has established a series of comparison standards using
coloured glasses ranging in intensity from 2 to 27 EBC colour units.
At the lower end of the scale the colour match is with the yellower
pale worts; at the upper end of the scale the colour match is with the
redder dark worts and caramels. If the colour match is not good a
cell of more suitable path length must be chosen in order to obtain
the best match possible.

Every person entrusted with the visual measurement of colour must

be known to be free from colour blindness. To ensure this, it is
necessary to test all such persons by means of a book of test charts
by Ishihara (Tests for Colour Blindness, published by Chapman &
Hall, 11 New Fetter Lane, London EC4P 4EE).

Filtered wort is prepared from the test malt using IOB Method 2.4
(Malt, Hot Water Extract).
The colour of light from a standard source, following transmission
through the test wort, is matched to that of one of a series of
calibrated colour glasses.

(f) Free Alpha-Amino Nitrogen

The sample and a standard solution are heated in the presence of
ninhydrin at pH 6.7 and the absorbances at 570 nm are measured
against a reagent blank.

For beers a correction for the colour of the sample is applied.

It is recommended that the wort produced during the course of malt

analysis is diluted 2 ml in 100 ml.

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

(g) S-Methylmethionine
Free malt DMS may be measured by mixing ground malt with water
in a vial sealed with a septum. This is then incubated at 35°C for 30
minutes to volatilise any DMS but avoid conversion of precursor. A
headspace sample is analysed using a gas chromatograph with a
flame ionisation detector.

Total malt DMS may be measured by chemically degrading any

precursor to DMS using a caustic solution and measured by
incubating at 50°C then injecting the headspace. Incubation at 50°C
is used in order to overcome mixing problems caused by the high
viscosity of the matrix thus facilitating headspace equilibrium.

S-Methylmethionine is obtained by subtracting the value for Free

malt DMS from the Total malt DMS value.

(h) Friability
Whole malt corns are fragmented by the mechanical action of the
Friabilimeter's revolving drum.
Small corn fragments pass through the mesh of the drum whereas
larger fragments are retained.
The mass of large fragments remaining after 8 minutes is determined
and from this the friability is calculated.

(i) Diastatic Power

Malt is extracted according to IOB Method 2.6 (Malt, Cold Water
Extract).
An aliquot of the extract is allowed to hydrolyse a buffered starch
solution.
The amount of reducing sugars in the starch hydrolysate is measured
by titration with Fehling's solution.

The Diastatic Power (DP) is calculated from the titre.

(j) Wort Viscosity

Viscosity is that property of a fluid defined by the resistance offered
to a shearing force under laminar flow conditions.

The viscosity of a fluid is determined by measuring the time taken for

a fixed volume at 20° to flow through a capillary under a reproducible
head. The viscosity is then calculated either by comparing the flow
time with that of water or, if the viscometer constants are known, by
use of the appropriate factor.

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

A laboratory wort is freshly prepared by IOB Method 2.4 (Malt, Hot

Water Extract).
The viscosity of the wort is measured within 60 min from the start of
filtration by IOB Method 8.2 (Beer, Viscosity).

(k) Hop Resins

Lead Conductance Value

Bitter substances are extracted with toluene from hops, hop powders
and hop pellets. A two phase mixture of toluene and salt solution is
used for hop extracts. An aliquot of the toluene extract is diluted with
methanol and the bitter substances in the resulting solution are
determined by conductimetric titration with lead acetate solution.

High Performance Liquid Chromatography

In the case of hops and hop pellets, a sample is macerated in the

presence of butyl acetate. The extract thus made is then diluted to a
suitable concentration with methanol.

In the case of hop extracts, these are diluted to a suitable

concentration with methanol.

When the samples re in the correct form, they are analysed by high
performance liquid chromatography (HPLC) fitted with a Nucleosil
C18 column and an ultra violet (UV) detector. Calibration is by an
external standard.

(l) Hop Essential Oils

Steam Distillation and GLC

The hops, in whichever form, are introduced into a flask containing

water. This is then boiled, and the hop oils collected in a modified
Dean & Stark receiver. These oils are then analysed by gas
chromatography with split/splitless injection and flame ionisation
detection.

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

(m) Biological Oxygen Demand (BOD)

Biological Oxygen Demand is defined as the mass of dissolved
oxygen required by a specific volume of liquid for the process of
biochemical oxidation under prescribed conditions, after 5 days at
20°C, in the dark.

Biochemical oxidation of the organic matter in the water is primarily

brought about by the action of heterotrophic bacteria.

In practice a volume of sample is placed in a bottle and the bottle

topped up with dilution water. The dissolved oxygen is measured
and the stopper replaced. After incubation for 5 days the DO is
measured again. The BOD value is the difference the two DO values
after making a correction for the volume taken.

Dissolved oxygen may be measured chemically or by using a DO

probe.

(n) Chemical Oxygen Demand COD)

Chemical Oxygen Demand measures the oxygen requirement of a
water sample by oxidation in a standard, arbitrary manner with
sulphuric acid and potassium dichromate.
The residual dichromate is determined titrimetrically. Colorimetric
methods, based on the colour changes in the reaction, are available
commercially.

(o) Total Suspended Solids

Suspended matter is removed from a measured volume of sample by
filtration under reduced pressure through a pre-treated, pre-weighed,
glass-fibre filter paper and determined gravimetrically after washing
and drying at 105C to constant weight. Volatile material and ash in
the suspended matter may be determined by ignition at 500°C.

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Dipl. Brew. Module 1: Unit 1.11 – Quality – Section 1.11.3

Reference List

(1) Institute of Brewing Analytical Methods.

Available from the Institute of Brewing and Distilling,
33 Clarges Street, London, W1Y 8EE

(2) Analytical Methods - American Society of Brewing Chemists.

(3) Analytica EBC.

(4) Oakland, J.S and Followell, R.F., Statistical Process Control,

2nd Edn, Heinemann Newnes 1990.

(5) Statistics for Analytical Chemistry,

J C Miller and J N Miller, Wiley and Sons 1986

© The Institute of Brewing and Distilling, September 2008 (Version 2009)