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Last name: Liangsiri

First name: Jirapha
Email: jirapha.liangsiri@carlsberg.com
Date: 07.10.2013

BEER ANALYSIS

1. INTRODUCTION

As a producer of consumer products, it is crucial that the final product is produced according to
specification and with a consistent quality. The quality of the product, in our case is beer, can be
assured by systematic and periodic monitoring of raw material, product-in-process, and final
product.
The previous reports described the analysis of raw material (barley, malt, and hop) as well as the
product-in-process (wort). In this report, the following analyses of final beer are explained in
detail.
Apparent extract
Real extract
Alcohol content
Original gravity
Bitterness
Thiobarbiturate Index (TBI)
Ethanol content in alcohol free beer
Vicinal Diketones (VDKs)

2. APPARENT EXTRACT

2.1. PRINCIPLE
Beer gravity is most often measured using hydrometers. However, hydrometers are
calibrated to measure the sugar content of a solution of water. Finished beer contains
alcohol which skews the hydrometer reading because alcohol has lower density than water.
Therefore, a hydrometer reading taken on finished beer shows lower (less extract content)
than the beer actually contains. Apparent extract is the measured hydrometer reading for the
finished beer, usually expressed in degrees Plato.

Before measuring the density to determine the apparent extract, the beer must be
decarbonized in order to get the correct density measurement.
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2.2. MATERIALS, CHEMICALS, AND EQUIPMENTS
Equipment:
Beakers
Fluted filter, diameter 32 cm
Funnel
Density meter

2.3. PROCEDURE
Decarbonation:
Fill 300 500 ml of beer of about 20C into a beaker
Pour beer into another beaker and pour back into the same beaker 20 times
Filtrate the beer through a fluted filter
Measuring density:
Determine density of decarbonized beer

2.4. CALCULATION & RESULT
The apparent extract can be derived from the density with the polynomial formula below:

where:
E
A
= Apparent extract (mass %)
SL
20/20
= Specific gravity of liquid measured at 20C for sample and 20C for water

The measured specific gravity was 1.0068 (1.00501 g/cm
3
). Therefore, the apparent extract
is:

3. REAL EXTRACT, ALCOHOL & ORIGINAL GRAVITY (DISTILLATION METHOD)

3.1. PRINCIPLE
Real extract is the real extract content of the finished beer, accounting for the actual alcohol
content and imperfect nature of hydrometers. The real extract together with the alcohol
content can be determined by a distillation method. After the distillation, the extract and
alcohol are separated. The density of both components can be used to calculate the mass %
of the content.


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3.2. MATERIALS, CHEMICALS, AND EQUIPMENTS
Equipment:
Erlenmeyer flask 500 ml
Analytical scale
Volumetric flask 100 ml
Distillation equipment
Parafilm
Pycnometer or density meter

3.3. PROCEDURE
Weigh 100.0 g ( 0.1 g) of beer into a tared 500 ml flask and then add about 50 ml
of distilled water
Attach the flask to the distillation equipment
Distil the alcohol and trap it in a 100 ml volumetric flask which is filled with 5 ml
of distilled water. The volumetric flask should be covered with Parafilm to prevent
the alcohol to evaporate out.
Make sure that no extract components stick to the hot wall of the flask
Distil until 85-90 ml have been collected, and then stop the distillation.
Make up the content of the 100 ml flask to 100.0 g ( 0.1 g) with distilled water,
close the flask and mix well
Cool down the content of distilling flask to room temperature
Make up the content in the 500 ml flask to 100.0 g ( 0.1 g) with distilled water
Determine SL 20/20C of the distillate (in 100 ml flask) and the residue (in the 500
ml flask) of distillation with the pycnometer or density meter

3.4. CALCULATION & RESULT
The real extract can be derived from the density with the same formula as apparent extract
(see Section 2). The measured specific gravity of the residue was 1.0147 (1.0129 g/cm
3
).
Therefore, the real extract is:

The measured specific gravity of the distillate was 0.9921 (0.99031 g/cm
3
). The alcohol can
be calculated from this density with the polynomial formula below:

where:
A = Alcohol content (mass %)
SL
20/20
= Specific gravity of liquid measured at 20C for sample and 20C for water

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The alcohol content in % mass can be converted to % vol with the following formula.

Thereby the density of beer corresponds to the apparent extract (see Section 2).

The Balling formula is then used for calculating the original extract or original gravity.

4. BITTERNESS (EBC METHOD)

4.1. PRINCIPLE
Bitter substances, mainly iso--acids, are extracted by means of iso-octane from the
acidified sample. The concentration of bitter substances in this extract is then determined
against a reference of pure iso-octane by spectrophotometer at the wavelength of 274 nm.

4.2. MATERIALS, CHEMICALS, AND EQUIPMENTS
Equipment:
Centrifuge tubes with suitable lids
Spectrophotometer
Quartz cuvette, 1 cm pathlength
Reagent:
Hydrochloric acid, 6N
Iso-octane (2,2,4-trimethyl pentane) for UV spectroscopy. The absorbance of this
solvent must be below 0.010 when measured at 275 nm in a 1 cm cuvette, against a
reference of distilled water


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4.3. PROCEDURE
Pipette exactly 10 ml of degassed beer (for wort: 5 ml + 5 ml water) into tube. Add
0.5 ml of hydrochloric acid followed by 20 ml iso-octane
Close the tube with the cap and check that it is solvent tight
Shake the tube by hand for 2 minutes and let the suspension set for about 30
minutes.
Measure the absorbance of the iso-octane layer in a 1 cm cuvette at 275 nm using
pure iso-octane in the reference tube.

4.4. CALCULATION & RESULT
The bitterness units can be calculated by the following formulas for beer and wort analysis
respectively.





In the practical work, the beer sample was analysed. The measured absorbance and
corresponding bitterness units are shown in Table 1.

Table 1: Results of bitterness analysis
Measurement AbsU IBU Average error allowance
Empty cuvette 0.112
Pure iso-octane 0.065
Beer 1* 0.583 (0.583-0.065)*50 = 25.9 24.7 4% = 24.7 0.988 =
23.71 - 25.69 Beer 2* 0.539 (0.539-0.065)*50 = 23.7
Beer 3* 0.556 (0.556-0.065)*50 = 24.5
*All are from the same beer sample.
The allowed error for bitterness measurement is 4%. Therefore, the first measurement is an
outlier. The bitterness is around 24.5 IBU. In order to get a more accurate result, more
measurements should be done.

5. THIOBARBITURATE INDEX (TBI)

5.1. PRINCIPLE
The TBI or TBZ is considered as a summary parameter of the thermal stress on malt and wort. It
is a classification number, next to hydroxymethylfurfural, which includes a multitude of
Maillard reaction products and other organic compounds.

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The sample is mixed with acetic thiobarbiturate solution, so that the reaction starts. The formed
yellow colour is measured photometrically.

5.2. MATERIALS, CHEMICALS, AND EQUIPMENTS
Equipment:
Spectrophotometer
Glass cuvette, 1 cm pathlength
Water bath, 70C
Brown test tubes with cutting, 20 or 25 ml
Reagent:
Acetic acid, 90%: Dilute 225g of 100% acetic acid with water up to 250g
Thiobarbituric acid, 0.02 mol/l: Dissolve 0.288g of Thiobarbituric acid (M = 144.15
g/mol) in 90% acetic acid using a 100 ml volumetric flask. Warm while dissolving.
After cooling down to 20C, make up to the mark with 90% acetic acid. This reagent
has to be prepared freshly.

5.3. PROCEDURE
Dilute the sample with the suitable dilution rate so that the extinction after reaction
with Thiobarbituric acid is within the range of 0.1 0.5. The recommended dilution
rate are:
Congress wort (pale malt) 4 fold with water
Congress wort (dark malt) 5 fold with water
Worts 10 fold with water
Beers 10 fold with water
Beer (dark strong) 100 fold with water
10 ml of blank solution (in this case is distilled water) is mixed with 5 ml of 90%
acetic acid.
10 ml of each test solution is mixed with 5 ml of thiobarbituric acid. Do this for all
test solutions.
Shake all test tubes well.
Put them in a water bath at 70C for exactly 70 minutes. Avoid direct sunlight and
avoid that the temperature of water decreases more than 1-2C
After the end of the incubation cool the test tubes down to 20C rapidly using cold
tap water or a refrigeration bath
Measure the yellow colour using a 1 cm cuvette and a spectrophotometer at 448 nm
wave length versus water.





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5.4. CALCULATION & RESULT
The Thiobarbiturate number can be calculated from the absorbance measured:



Where:
TBZ = Thiobarbiturate number, dimensionless
EM = Average extinction main value
EB = Average extinction blank value
F = Dilution factor

The results from analysis of dark beer sample and alcohol free beer sample are shown in
Table 2.

Table 2: Result of TBI analysis
Sample no. AbsU Avg. TBZ
1 Water 0.046 0.045
1 Water 0.044
2 Dark beer 0.104 0.1045 (0.1045 0.045)*10*100 =
59.5
2 Dark beer 0.105
3 Alcohol free beer 0.421 0.4205 (0.4205 0.045)*10*10 =
37.6
3 Alcohol free beer 0.420

The dark beer shows a higher TBZ value, which indicates that it has a higher Maillard reaction
products and other organic compounds. The TBZ value can be used as a reference when there is
an adjustment in the recipe of the existing beer.

6. ETHANOL CONTENT IN ALCOHOL FREE BEER

6.1. PRINCIPLE
Ethanol is oxidized to acetaldehyde by nicotinamide-adenine dinucleotide (NAD) in the
presence of enzyme called alcohol dehydrogenase (ADH). The equilibrium of this reaction lies
on the side of ethanol and NAD. It can be completely displaced to the right side at alkaline
conditions and by trapping of the acetaldehyde formed.

Acetaldehyde is quantitatively oxidized to acetic acid in the presence of aldehyde
dehydrogenase (Al-DH).
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In order to measure the alcohol content, the produced NADH is measured by means of its light
absorbance at 340 nm.

6.2. MATERIALS, CHEMICALS, AND EQUIPMENTS
Equipment:
Spectrophotometer
Cuvette, 1 cm pathlength
Pipettes

Reagent:
Buffer solution
ALDH tablets
ADH solution

6.3. PROCEDURE
Pipette 3 ml of buffer solution in all cuvettes.
Add 1 tablet of ALDH in all cuvettes, stir and dissolve the tablet into the solution.
Pipette 0.1 ml of distilled water into 2 cuvettes (at least) for blank samples.
Pipette 0.1 ml of beer sample into other 2 cuvettes (at least) for main sample. If
there are more than one beer to analyse, do the same procedure for other beers (at
least 2 cuvettes each).
Mix the solutions together and wait for 3 minutes.
After 3 minutes, measure the absorbance of the samples. Record the measurement
as A
1
.
After the measurement, add 0.05 ml of ADH to activate the second reaction.
Wait for approx. 5-10 minutes for the reaction to complete and then measure the
absorbances. Record the measurement as A
2
.

6.4. CALCULATION & RESULT
According to the general equation for calculating the concentration in reactions in which
the amount of NADH formed is stoichiometric to half the amount of substrate.






where:
c = concentration
V = final volume (ml)
v = sample volume (ml)
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MW = molecular weight of the substance to be assayed (g/mol)
D = light path (cm)
= extinction coefficient of NADH at 340 nm = 6.3 (l*mmol
-1
*cm
-1
)
A = Absorbance difference = (A
2
-A
1
)
sample
(A
2
-A
1
)
blank


For the performed analysis, the formula for alcohol concentration can be filled in as:






If the sample has been diluted during the preparation, the result must be multiplied by the
dilution factor F.

The results from spectrophotometer and the calculation for concentration are shown in
Table 3 and 4.

Table 3: Results of ethanol analysis (part 1)
Sample
no.
A
1
A
2
A
2
A
1
A (1) C (g/l) =
(1)*0.1152
1 Blank 0.201 0.262 0.061
1 Blank 0.200 0.260 0.060
2 Standard beer 0.210 0.759 0.549 0.489 0.0562
2 Standard beer 0.197 0.771 0.574 0.514 0.0591
3 Alcohol free beer 0.408 4.000 N/A*
3 Alcohol free beer 0.430 4.000 N/A*
4 0.0% ABV beer 0.387 0.483 0.096 0.036 0.0041
4 0.0% ABV beer 0.390 0.488 0.098 0.038 0.0044
*Not applicable for further analysis because the sample were not diluted and it was out of
range for the spectrophotometer.

Table 4: Results of ethanol analysis (part 2)
Sample Avg. concentration
(g/l)

Concentration
(g/100 ml)
Alcohol (% vol)
Standard beer 0.0576 0.00576

0.0% ABV beer 0.00425 0.000425

(1)
Density of alcohol = 0.789 g/100 ml
(2)
Standard beer were diluted with 10 fold
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7. VICINAL DIKETONES (PHOTOMETRIC METHOD)

7.1. PRINCIPLE
The vicinal diketones (VDK) are distilled from the beer. The distillate is mixed with a solution
of o-phenylenediamine to form a dericate of quinoxaline. After acidification, the amount of
reaction products is measured by spectrophotometer at the wavelength of 335 nm.

7.2. MATERIALS, CHEMICALS, AND EQUIPMENTS
Equipment:
Distillation apparatus
Kjeldahl flask
Graduated flask, 25 ml
Erlenmeyer flasks, 15 ml
Spectrophotometer
Cuvette, 2 cm pathlength
Reagent:
Hydrochloric acid, 4N
1,2-phenylenediamine, 1% in 4N HCl

7.3. PROCEDURE
Fill in 100g of non-degassed beer with some drops of antifoam oil into a Kjeldahl
flask
Distillate about 25 ml (2 minutes) into a 25ml graduated flask
Fill this flask up to 25 ml with distilled water
Take out 2 times 10 ml of the distillate into 2 of 15 ml Erlenmeyer flasks. One flask
for blank sample and another one for main sample.
Add 0.5ml of 1,2-phenylenediamine solution into the flask of main value
Cover both flask and leave them in the dark for 30 minutes
After 30 minutes, take the flasks out and add 2.0 ml and 2.5 ml of HCl into main
sample and blank sample respectively.
Measure the extinction at 335 nm in a 2 cm cuvette within 20 minutes.

7.4. CALCULATION & RESULT
The VDK can be calculated according to the following formula:



where:
VDK = Vicinal diketones (mg/kg)
EM = Average extinction main value
EB = Average extinction blank value
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The measured absorbances of blank and main value at 335 nm are 0.094 and 0.165
respectively. Therefore, the VDK of the beer sample is: