UV/VIS Application Note

Iso-alpha and Alpha Acids of Beer

According to ASBC
Introduction
In beer, one of the major ingredients is hops If the test beer is cloudy, the first step is to clarify the
which contain components called alpha acids. beer: transfer approximately 100 mL of beer in
centrifuge tubes and centrifuge at 3000 rpm for 15
During the brewing process, a big part of these
minutes. Don’t filter the sample: Some alpha and
alpha acids is transformed to iso-alpha acids iso-alpha acids may get lost during filtration.
which are bitter components of beer. Place 100 ml of beer, into the 200 mL Erlenmeyer
This application determines the concentration of flask, cover the flask loosely with aluminum foil or
both acids by an extraction method and using other inert material and place the bottle in an
UV/VIS spectroscopy at two distinct wavelengths ultrasonic bath containing enough water to cover the
level of beer in the Erlenmeyer and sonicate for 10
in the UV region.
min.
Pipette 50 mL of the decarbonated beer in a
Materials and Method centrifuge tube. Add 3.0 mL of 6N HCl and 25.0 mL
Samples and Reagents of iso-octane.
 Beer of choice Close the centrifuge tube and put it into the shaking
 Hydrochloric acid, 6N machine set at its highest shaking intensity for 30
 Hydrochloric acid, 4N minutes.
 Iso-Octane (2,2,4-trimethylpentane) Centrifuge then for 5 minutes at 3000 rpm to
 Anhydrous Sodium Sulfate separate the organic phase from the aqueous one.
 Methanol, 6N Remove the aqueous phase (on the bottom of the
 Sodium Hydroxide, 6N, carbonate-free tube) by pipetting out of the tube. Then, add to the
iso-octane phase as much sodium sulfate as needed
Instruments and Accessories in order to obtain a clear solution after a short
manual shaking.
 UV Excellence instrument (UV7, UV5, UV5Bio
Pipette 10 mL of this solution in a 25 mL volumetric
or UV5Nano) flask. Add 10.0 mL of acidic methanol and mix the
 10 mm standard cuvettes (may be solution by turning the flask upside down 100 times.
disposable cuvettes) Pipette 5.0 mL of the supernatant solution into a new
 Centrifuge and centrifuge tubes 100 mL with 25 mL volumetric flask and fill the flask up to the
solvent proof seal mark with alkaline methanol and mix it.
 Shaking machine Fill a cuvette with the sample solution and one with
 Ultrasonic bath the blank solution.
 200 ml wide mouthed Erlenmeyer flask
 Aluminum foil Measurement Parameters
Method: Fixed wavelength
Measurement Procedure Path length: 1cm
Samples and Reagent Preparation Measurement duration: 3s
Alkaline methanol: Take 0.2 mL of 6N NaOH and No of wavelengths: 2
fill up to100 mL with methanol Wavelength selection n°1: 255 nm (A1)
Acidic methanol: Mix 64 mL of methanol and 36 Background correction: 1-Point, 450 nm
mL of 4N HCl Wavelength selection n°2: 360 nm (A2)
Blank solution: Mix 5.0 mL of Iso-octane and 20 Background correction: 1-Point, 450 nm
mL of alkaline methanol. Calculation R1: %Iso-Alpha acids (mg/L)
A1*48.5 - A2*26.5
Calculation R1: %Alpha acids (mg/L)
A2*80

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 Results References
Mean values of six measurements are reported in the
UV/VIS Application Note

following table:  MEBAK, “Brautechnische Methoden”, Band II,

1993
Std
Result Srel  Decarbonation of beer:
Sample Parameter Dev
(mg/L) (%) http://analytica-ebc.com/
(mg/L)
Iso-alpha
30.1 0.164 0.55
acids
Beer 1 Further information
Alpha
1.6 0.064 3.97
acids www.mt.com/uv-vis
Iso-alpha
19.4 0.418 2.15
acids
Beer 2
Alpha
1.1 0.143 13.55
acids
Iso-alpha
12.6 0.137 1.08
acids
Beer 3
Alpha
0.5 0.046 9.11
acids

Precisions
The following remarks are helpful to reach a better
precision during the procedure:

 Add up to three spatulas of sodium sulfate to

consider the solution is clear. Except in some
special cases it is enough.
A white and cloudy precipitate can be seen
during this step which will not disappear with
the sodium sulfate, even if the solution
becomes saturated. Moreover, the liquid can
be trapped in this precipitate. To release it,
just mix the precipitate with the tip of a
pipette. There is normally enough clear
solution to take without problem 10 mL of it
for the next step.

 If only 50mL centrifuge tubes are available,

take two 50 mL tubes and split the requested
amount in two equal parts. Join the two
parts together before adding the sodium
sulfate.

 The acidic methanol, alkaline methanol and

the blank solution have to be made freshly

 The blank solution and the sample solution

at the final step are both two-phase
solutions. In both cases, use the lower phase
of the solution.
If some drops of the upper phase get into the
cuvette, they will normally float on top and
don’t affect the measurement. Just make sure
that these drops don’t remain in a dispersed
form in the methanol phase. In this case,
they can affect the measurement.
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