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BEER BITTERNESS
Bitterness is an important attribute for most beer styles,
whether it be the most prominent flavor or simply added
for balance, bitterness is a fundamental character of beer.
Bitterness in beer predominantly originates from the hops
added to the wort during kettle boil or through chemically
modified hop extracts added to finished beer. Iso--acids
are the primary bittering compounds and are formed in
the brew kettle via a heat-induced isomerization process
and/or through modified hops or hop extracts. Other com-
pounds, including hop oxidation products, also contribute
to the bitterness of beer, especially in dry-hopped beers.
Methods A and D are commonly used for brewery pro-
cess control and provide a reasonable measurement of
overall bitterness. Method D is an automated version of
method A. Both A and D measure both iso--acids and
other compounds that can contribute to overall bitterness.
These methods are prone to erroneously high results if the
beers measured also contain preservatives. Methods C, E,
and F measure iso--acids and are less likely to be af-
fected by other compounds present. Method F is an up-
dated version of method B, which has been archived.
A. BITTERNESS UNITS (BU)
Manual Isooctane Extraction
(IM)
Reagents
(a) 2,2,4-Trimethylpentane (isooctane), CAS No. 540-
84-1, spectrophotometric grade or equivalent.
(b) Hydrochloric acid, CAS No. 7647-01-0, 3M.
(c) Octanol, CAS No. 111-87-5, reagent grade or re-
distilled equivalent. One drop added to 20 mL of
2,2,4-trimethylpentane (reagent a) must not increase
absorbance reading at 275 nm by more than 0.005 in
a 1-cm cell.
(d) Antifoam B silicone emulsion or equivalent (as an al-
ternative to using octanol (reagent c).
Reagent Preparation
(1) Antifoam working solution. Add 1 mL of Antifoam B
silicone emulsion (reagent d) to 9 mL of water and
mix.
Apparatus
(a) Mechanical shaker. A platform type, or a “wrist ac-
tion” type shaker, with extending arm adjusted in a
vertical plane so that tube will be held in a horizontal
position.
(b) Spectrophotometer, capable of measuring at 275 nm.
(c) Cuvettes, quartz, 1-cm.
(d) Centrifuge tubes, 50-mL, with glass stoppers or
screw caps with Teflon lining.
©American Society of Brewing Chemists
(e) Centrifuge that will take 50-mL centrifuge tubes (ap-
paratus d).
(f) Pipettes, 1-, 10-, and 20-mL.
(g) Erlenmeyer flasks, 150-mL.
(h) Transfer pipettes.
Method
Transfer 10.0 mL of chilled carbonated beer (10°C) to a
50-mL centrifuge tube using a pipette that has had a minute
amount of octanol (reagent c) introduced into the tip. As an
alternative to using octanol (reagent c), room-temperature
(approximately 20°C) beer may be degassed prior to pipet-
ting by adding a few drops of antifoam working solution
(reagent 1) to a 150-mL Erlenmeyer flask and adding
approximately 50 mL of beer. Shake the sample to degas,
being careful not to lose any foam. Let the foam completely
collapse, and swirl the sample to rinse any foam from the
sides of the flask prior to pipetting the 10.0 mL of beer to
the centrifuge tube. Add 1 mL of 3M HCl (reagent b) and
20 mL of 2,2,4-trimethylpentane (isooctane) (reagent a).
Stopper or cap the centrifuge tube tightly and place it on a
mechanical shaker for 15 min. The action must be vigorous
enough to mix the layers. Remove the tubes from the
shaker and place upright. Allow the organic layer to sepa-
rate from the aqueous layer for 5 to 10 min. If the emulsion
interface between the layers is greater than one-third the
size of the solvent layer, mechanical techniques should be
employed to help complete the phase separation. The opti-
mum technique depends on the sample and may include
centrifugation, stirring, filtration of the emulsion through
glass wool, or other physical methods. If the emulsion per-
sists, repeat the analysis with a slightly reduced intensity of
shaking. Zero the spectrophotometer at 275 nm with iso-
octane (reagent a) or with the isooctane–octanol blank (20
mL of isooctane [reagent a] plus one drop of octanol [rea-
gent c]), if octanol was used when pipetting. As soon as
possible, transfer sufficient clear upper (isooctane) layer of
the sample to the cuvette of the spectrophotometer. Record
absorbance at 275 nm (see Note).
Calculations
Calculate bitterness units (BU) of beer with the follow-
ing formula:
BU = absorbance275 × 50
Report bitterness units to nearest one-half unit.
Example
Absorbance of isooctane layer at 275 nm = 0.322
BU = 0.322 × 50 = 16.1
Report BU as 16.0.
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Beer–23
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Note
Notice should be taken of findings that certain preserv-
atives, and possibly some brewing adjuncts or coloring
agents, may contribute to absorbance at the wavelengths
specified in Beer-23A. The possibility of picking up ul-
traviolet-absorbing extraneous substances is greater in the
bitterness unit methods than in the iso--acid methods.
The possible effects of such materials should be checked
before reporting bitterness values.
References
1. American Society of Brewing Chemists. Report of Subcommittee
on Determination of Isohumulones in Beer. Proc. 1967, p. 269;
Proc. 1968, p. 260.
2. Howard, G. A. Estimation of the bitterness of beer. J. Inst. Brew.
74:249, 1968.
3. Rigby, F. L., and Bethune, J. L. J. Inst. Brew. 61:325, 1955.
4. Bishop, L. R. The E.B.C. scale of bitterness. J. Inst. Brew. 73:525,
1967.
5. U.S. Pharmacopeia XVII, p. 1005.
Released 1968; Revised 1975, 2018
Manual Isooctane Extraction:
Reduced Solvent Technique
This method produces results identical to those pro-
duced by Beer-23A, utilizing significantly less solvent
per sample.
Reagents
(a) 2,2,4-Trimethylpentane (isooctane), CAS No. 540-
84-1, spectrophotometric grade or equivalent.
(b) Hydrochloric acid (HCl), CAS No. 7647-01-0, 3M or
6M.
Apparatus
(a) Mechanical shaker, platform or wrist action.
(b) Spectrophotometer, capable of measuring at 275 nm.
(c) Cuvette, quartz, 1- or 10-mm.
(d) Centrifuge tubes with screw caps, 50-mL.
(e) Centrifuge.
(f) Transfer pipettes, 5-mL.
(g) Pipettes, 0.5-, 5.0-, and 10.0-mL.
Method
Transfer 5.0 mL of chilled carbonated beer, using a 5.0-
mL volumetric pipette, to a 50-mL centrifuge tube. Add
0.5 mL of 3M HCl or 0.25 mL of 6M HCl (reagent b) to
the centrifuge tube. Add 10 mL of isooctane (reagent a) to
the centrifuge tube. Prepare a blank using 10 mL of iso-
octane (reagent a) with 0.5 mL of 3M HCl or 0.25 mL of
1
See APPENDIX-1A for calibration of spectrophotometer.
©American Society of Brewing Chemists
6M HCl (reagent b) added to the centrifuge tube. Cap the
centrifuge tubes tightly. Shake two to three times by hand,
and place on a mechanical shaker set to a speed of 8 (or
80% of capacity) for 15 min.
Set the centrifuge speed at 400 × g. Centrifuge the tubes
for 5-min periods to separate the phases (Ref. 3). After the
first centrifugation, tap the centrifuge tube on the counter
to aid in separating the layers and then swirl the tube. Do
this for each sample and the blank. Put the tube back in
the centrifuge for another 5 min.
After the second centrifugation, tap the bottom of the
tube on the counter as before and manually swirl the cen-
trifuge tube a couple of times to help the layers separate.
If after the second centrifugation the layers do not sepa-
rate enough to obtain an approximately 5-mL volume,
centrifuge for an additional 5 min.
Using a transfer pipette, transfer sufficient clear upper
isooctane layer to the cuvette of the spectrophotometer.
Set the instrument to read zero absorbance at 275 nm for
the blank. Record absorbance at 275 nm.
Calculations
Calculate BU of beer using the following formula:
BU = absorbance275 × 50
Report BU to the nearest one-half unit.
Example
absorbance275 = 0.786
BU = 0.786 × 50
BU = 39.3
Report BU as 39.5.
References
1. American Society of Brewing Chemists. Report of Subcommittee
on Determination of Isohumulones in Beer. Proc. 1967, p. 267,
1968.
2. American Society of Brewing Chemists. Report of Subcommittee
on Reduced Hazardous Solvent Use for Determination of Isohumu-
lone Bitterness Units. Journal 69:276, 2011.
3. Howard, G. A. Estimation of the bitterness of beer. J. Inst. Brew.
74:249, 1968.
4. Rigby, F. L., and Bethune, J. L. J. Inst. Brew. 61:325, 1955.
5. Scientific sounds. ASBC News Lett., p. 13, April 1968.
6. Bishop, L. R. The E.B.C. scale of bitterness. J. Inst. Brew. 73:525,
1967.
7. U.S. Pharmacopeia XVII, p. 1005.
Released 1968; Revised 2018
B. ISO--ACIDS (IAAs)
BY SOLVENT EXTRACTION
This method has been archived and replaced by method F.
doi: 10.1094/ASBCMOA-Beer-23

C. ISO--ACIDS BY SOLID-PHASE
EXTRACTION AND HPLC
The iso--acids in degassed beer are adsorbed onto a
solid-phase extraction (SPE) column and selectively de-
sorbed (Ref. 3). The desorbed iso--acids are analyzed by
high-performance liquid chromatography (HPLC) (Ref.
4).
Reagents
(a) ASBC hop standards, available from ASBCnet.org.
(b) Tetraethylammonium hydroxide, CAS No. 77-98-5,
10% in water.
(c) Water, CAS No. 7732-18-5, HPLC grade.
(d) Methanol, CAS No. 67-56-1, HPLC grade.
(e) Phosphoric acid, CAS No. 13598-36-2, 85% w/w.
(f) Octanol, CAS No. 111-87-5.
Reagent Preparation
(1) Desorbing solvent A, 0.2 mL of phosphoric acid (re-
agent e) in 100 mL of water (reagent c).
(2) Desorbing solvent B, 0.2 mL of phosphoric acid (re-
agent e) in a mixture of 50 mL of water (reagent c)
and 50 mL of methanol (reagent d).
(3) Desorbing solvent C, 0.1 mL of phosphoric acid (re-
agent e) in 100 mL of methanol (reagent d).
Apparatus
(a) Volumetric flasks, 2- and 100-mL.
(b) Volumetric pipets, 20-mL.
(c) Graduated pipets, 1-, 5-, and 10-mL.
(d) C8 SPE octyl column, 500-mg, 3-mL.
(e) Plastic syringe, 30-mL.
(f) High-performance liquid chromatograph, with an ul-
traviolet detector and 20-µL injection loop.
(g) Integrator.
(h) Chromatographic column, Shimadzu, Shim-pack CLC-
ODS, 25 cm × 4.6 mm, or equivalent.
(i) Analytical balance, 0.1-mg capability.
Recommended Instrument Parameters
The HPLC method described in Hops-9D is used for
chromatographic separation and quantitation of the three
iso--acids. This procedure varies from the Hops-9D pro-
cedure by the elimination of the internal standard and the
use of a 20-L sample injection.
Method
Transfer approximately 200 mL of beer to a beaker and
degas by beaker transfer in excess of 20 times. Add one drop
of octanol (reagent f) and make additional transfers to en-
sure foam collapse. Adjust pH to approximately 2.5 by
adding 200 L of phosphoric acid (reagent e) per 100 mL
of beer.
©American Society of Brewing Chemists
Beer–23
Page 3 of 8
Assemble the C8 SPE column equipment and apply the
following adsorption/desorption sequence:
(a) Load 2 mL of methanol (reagent d); discard eluate.
(b) Load 2 mL of water (reagent c); discard eluate.
(c) Load 20 mL of degassed beer using a volumetric pi-
pette; discard eluate.
(d) Load 6 mL of desorbing solvent A (reagent 1); dis-
card eluate.
(e) Load 2 mL of desorbing solvent B (reagent 2); dis-
card eluate.
(f) Collect three successive 0.6-mL aliquots of desorbing
solvent C (reagent 3) in a 2.0-mL volumetric flask.
Make to volume with desorbing solvent C (reagent 3)
and mix thoroughly. Inject 20 L for HPLC analysis.
Analyze in duplicate.
Calibration
Weigh 20 mg of the calibration standard (reagent a),
and record the mass to the nearest 0.1 mg. Dissolve the
standard in methanol (reagent d) and make to volume in a
100-mL volumetric flask.
Inject 20 L of the calibration solution four times
(twice before and twice after the sample injections). Av-
erage the four response factors.
Calculations
TA std
RF =  % IAA std 
M std (mg)  
 100 
in which
RF = response factor (average of four injections)
TAstd = total area of the three iso--acid peaks in the
calibration assay
Mstd = mass of calibration standard used
% IAAstd = percent purity of iso--acids in the calibra-
tion standard
Because the calibration solution is a 100-mL volume
and the method gives a 10-fold enrichment, calculation of
iso--acids (mg/L) in beer is straightforward.
TA
Iso--acids (mg/L) =
RF
in which
TA = total area of the three iso--acid peaks in the beer
extract (average of duplicate assays)
RF = response factor (average of four injections)
Report results to the nearest 0.1 mg/L.
Example
Mass of calibration standard = 20.7 mg
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Beer–23
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Reported purity of iso--acids in calibration standard =
92.3%
Average total area of calibration standard peaks =
12,770,000
Average total area of sample peaks = 7,527,520
12,770,000
Response factor =  = 668,373
92.3 
 20.7   should be used to define the linear calibration.
 100 
7,527 ,520
Iso--acids (mg/L) = = 11.3
668,373
Precision
Based on a collaborative study (Ref. 2), repeatability
coefficients of variation of 2.0–3.9% and reproducibility
coefficients of variation of 10.3–12.6% can be expected
when iso--acid concentrations in beer are in the range of
10–30 mg/L.
Note
Iso--acid calibration standards will deteriorate over
time. Follow the supplier’s recommendations for storage
of calibration extracts.
References
1. American Society of Brewing Chemists. Report of Subcommittee
on Iso--acids in Beer by Solid-Phase Extraction and High-Perfor-
mance Liquid Chromatography. Journal 50:157, 1992.
2. American Society of Brewing Chemists. Report of Subcommittee
on Iso--acids in Beer by Solid-Phase Extraction and High-Perfor-
mance Liquid Chromatography. Journal 51:173, 1993.
3. Donley, J. R. Solid-phase extraction of hop acids from beer or wort
for subsequent analysis. J. Am. Soc. Brew. Chem. 50:89, 1992.
4. Ono, M., Kakudo, Y., Yamamoto, Y., Nagami, K., and Kumada, J.
Quantitative analysis of hop bittering components and its applica-
tion to hop evaluation. J. Am. Soc. Brew. Chem. 42:167, 1984.
Released 1993; Revised 2018
D. BITTERNESS BY AUTOMATED
FLOW ANALYSIS
An automated flow analyzer is used in determining beer
bitterness by simulating the manual isooctane extraction
technique (Beer-23A). A degassed beer sample is injected
into a carrier stream containing a surfactant, combined with
a 0.6M hydrochloric acid stream and an isooctane stream.
After mixing in a coiled tube, the organic phase is separated
from the flow stream, and the absorbance at 275 nm is de-
termined.
Reagents
(a) Reagent water, greater than 1-M resistivity.
(b) 2,2,4-Trimethylpentane (isooctane), CAS No. 540-
84-1, 99%+ spectrophotometric grade.
©American Society of Brewing Chemists
(c) Brij-35, Aldrich No. 85,836-6 or equivalent.
(d) Hydrochloric acid, CAS No. 7647-01-0, concen-
trated reagent grade (12M).
(e) Antifoam, Antifoam B silicone emulsion or equiva-
lent.
(f) Calibration beers, containing a known beer bitter-
ness unit (BU) level as determined to the nearest 0.1
BU by Beer-23A. At least two different BU levels
Reagent Preparation
(1) Brij-35 stock, 1% (w/v). Dissolve 1.0 g of Brij-35 (re-
agent c) into 100 mL of reagent water (reagent a).
(2) Brij-35 carrier solution, 0.005% (v/v). Pipet 5 mL of
the Brij stock (reagent 1) into a 1,000-mL volumetric
flask and dilute to volume with reagent water (rea-
gent a).
(3) Hydrochloric acid, 0.6M, with Brij-35, 0.005% (v/v).
Add 200 mL of reagent water (reagent a) to a 1,000-
mL volumetric flask. Add 49.6 mL of concentrated
12M reagent grade hydrochloric acid (reagent d) and
mix. Add 5 mL of Brij stock (reagent 1) to the flask
and mix by swirling. Bring to volume with reagent
water (reagent a).
Apparatus
(a) Automated flow analyzer, configured and optimized
for bitterness determination.
(b) Ultraviolet detector with flow cell, capable of 275-nm
detection with a bandpass of about 6 nm.
(c) Ultrasonic bath.
(d) Eye dropper.
(e) Erlenmeyer flasks, 250- or 500-mL.
(f) Pipette, 5-mL.
(g) Graduated cylinder, 50-mL.
(h) Volumetric flasks, 100- and 1,000-mL.
(i) Volumetric pipets, 5-mL.
(j) Balance, capable of 0.1 g.
Recommended Instrument Parameters
(a) Isooctane flow rate, 2.0 mL/min or greater.
(b) Carrier flow rate, 0.4 mL/min or less.
(c) 0.6M HCl flow rate, 0.4 mL/min.
(d) Sample loop volume, 200 L.
(e) Mixing coil length, 6 m.
Calibration
Use the Brij carrier solution (reagent 2) as calibration
standard number 1 for a 0 BU calibration point. Place two
drops of antifoam (reagent e) into an Erlenmeyer flask
and slowly pour about 150 mL of a calibration beer (rea-
gent f) into this flask. Swirl the flask several times to mix
and slowly remove some carbonation. Touch the edge of
the flask briefly (1 or 2 s) into the ultrasonic bath to re-
move large volumes of carbonation. Repeat above step to
doi: 10.1094/ASBCMOA-Beer-23

remove enough carbonation to prevent foaming up the
sides of the flask. Swirl the contents to remove any foam
from the sides of the flask if necessary. Place the flask in
the ultrasonic bath for 5 min. Label this flask calibration
standard number 2 or 3 based on its position within the
calibration plot. Repeat the degassing procedure for the
other calibration beer and label accordingly.
Inject the calibration standards in triplicate. Calibrate
the instrument using the “known” BU values determined
by Beer-23A for each of the standards and their respec-
tive average peak heights obtained from the analyzer.
Method
Degas beer samples (see Notes) as described under the
calibration section. Inject each sample in triplicate, and
record the average peak height for calculation of BU, or
record the BU value calculated by the analyzer.
Calculations
The bitterness results are predicted from the linear re-
lationship of beer BU and the absorbance intensity at 275
nm (based on peak height) of the bittering components
extracted into the isooctane phase. A least-squares fit of
this relationship generates the slope and intercept used to
calculate the sample bitterness from the sample peak
height. Report the values obtained to the nearest 0.1 BU:
BU = (slope × peak height) – intercept
Example
Slope = 1.088 × 10–4
Intercept = –8.397 × 10–2
Peak height of beer injection = 118,186 units
BU = [(1.088 × 10–4) × 118,186] – (–8.397 × 10–2)
= 12.8
Precision
Based on a collaborative study (Ref. 2), repeatability
coefficients of variation within a single laboratory of 0.7–
1.5% and reproducibility coefficients of variation com-
paring two or more laboratories of 4.4–6.2% can be ex-
pected.
Notes
1. A collaborative study (Ref. 2) indicated that signifi-
cantly lower BU results were obtained with this method
compared with Beer-23A when beers containing tetra-
hydro-iso--acids were analyzed. This problem was not
encountered when analyzers with optimized extraction
systems (Ref. 3) and grating monochrometers were used.
2. The collaborative studies (Refs. 1,2) did not investi-
gate performance of the method on hexahydro-reduced
hop constituents or on mixtures of the various bittering
constituents.
©American Society of Brewing Chemists
Beer–23
Page 5 of 8
References
1. American Society of Brewing Chemists. Report of Subcommittee
on Beer Bitterness by Automated Flow Analysis. Journal 52:182,
1994.
2. American Society of Brewing Chemists. Report of Subcommittee
on Bitterness in Beer by Automated Flow Analysis. Journal 53:216,
1995.
3. Sakuma, S., Kikuchi, C., Kowaka, M., and Mawatari, M. A flow-
injection analyzer for determining the bitterness of wort and beer.
J. Am. Soc. Brew. Chem. 51:51, 1993.
4. Switala, K. J., and Schick, K. G. Automated bitterness determina-
tion of beer utilizing a micro phase separator and flow injection
analysis. J. Am. Soc. Brew. Chem. 48:18, 1990.
Released 1995; Revised 2018
E. ISO--ACIDS IN BEER
BY HPLC
Iso--acids (IAA) in wort and degassed beer are de-
termined by high-performance liquid chromatography
(HPLC) using direct injection. In a previous HPLC method
(Beer-23C), IAA were absorbed onto a solid-phase ex-
traction column and selectively desorbed prior to injec-
tion. The current method requires less sample preparation
and eliminates the use of solid-phase extraction.
Reagents
(a) Reagent water (18-M resistivity or greater).
(b) Methanol, CAS No. 67-56-1, HPLC grade.
(c) Phosphoric acid (H3 PO4 ), CAS No. 7664-38-2, 85%,
analytical grade.
(d) Ethylenediaminetetraacetic acid (EDTA), CAS No.
6381-92-6, 0.1M, Na2 or Na4, analytical grade.1
(e) ASBC hop standards, available from ASBCnet.org.
(f) Antifoam FG-10.2
Reagent Preparation
(1) Acidic methanol. Add 0.5 mL of phosphoric acid
(reagent c) to 1 L of methanol (reagent b).
(2) Mobile phase solution. Mix 750 mL of methanol (re-
agent b), 240 mL of reagent water (reagent a), 10 mL
of phosphoric acid (reagent c), and 1 mL of 0.1M
EDTA (reagent d).
(3) 10% Antifoam FG-10 solution. Add 10 mL of anti-
foam (reagent f) to a 100-mL volumetric flask and
bring to volume with reagent water (reagent a)
Apparatus
(a) Balance, accurate to 0.1 mg.
(b) Ultrasonic bath.
(c) Volumetric flasks, 100-mL.
(d) HPLC, with a column heater and ultraviolet detector.
1
This(g)
is optional; however, the addition of EDTA will sharpen the peaks in
some HPLC systems.
2
The use of a 10% solution of antifoam FG-10 during degassing by soni-
cation is optional.
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Beer–23
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(e) Analytical column, C18, 250 × 4 mm (Nucleosil 5 or
equivalent), or 125 × 4 mm (Nucleodur 100-5 C18 ec
or equivalent).
(f) Precolumn, optional.
(g) Syringe filters, 0.45 µm, polytetrafluoroethylene
(PTFE).
(h) Aluminum foil.
Recommended Instrument Parameters
HPLC Conditions. These are recommendations and
can be modified for desired separation (i.e., UPLC).
Column temperature: 35°C
Detector setting: 270 nm
Flow rate: 1.0–1.4 mL/min
Injection volume: 20 µL
Calibration
Standard Stock Solution. Weigh 30–33 mg of IAA
standard into a 100-mL clear-glass volumetric flask with
accuracy to 0.1 mg. Dissolve the standard in 40 mL of
acidic methanol (reagent 1) with sonication. Make up to
volume. Protect the standard from light by wrapping the
flask in aluminum foil. Store the solution in a freezer.
Calibration Standard Solutions. Calibration standard
solutions are made by diluting the stock IAA standard so-
lution with acidic methanol (reagent 1). Calibration solu-
tions are used to produce a four-point linear calibration
curve, where IAA (mg/L) is plotted against the sum of the
IAA peak areas.
Method
Dilute wort 50% by volume with acidic methanol (rea-
gent 1). Beer can be degassed by adding one drop of 10%
antifoam (reagent 3) and then sonicating for 10 min. After
the foam has settled, a 50% by volume dilution is per-
formed with acidic methanol (reagent 1). If wort or beer
is highly hopped, a dilution other than 50% will have to
be made so the diluted sample is <30 IBU. Both wort and
beer should be filtered through 0.45-µm PTFE syringe fil-
ters prior to injection.
Fig. 1. Typical HPLC chromatogram.
©American Society of Brewing Chemists
Calculations
The stock standard concentration is calculated as fol-
lows:
Cstd = Mstd/0.1 L
in which
Cstd = concentration of stock standard solution (mg/L)
Mstd = weight of individual IAA standard (mg)
IAA concentration in beer or wort is calculated by de-
termining a response factor (RF) from the four-point linear
calibration curve. The curve is prepared by plotting IAA
calibration standard solutions (mg/L) on the x axis versus
the sum of the areas of the IAA peaks on the y axis. The
inverse of the slope is the RF used to calculate the IAA
concentration present in the beer or wort:
RF = 1/slope
IAA (mg/L) = (sum of IAA peak areas) × RF
Example
Sum of IAA peak areas = 7,331
Slope of calibration curve = 248.58
RF = 1/248.58
IAA (mg/L) = (7,331) (1/248.58) = 29.5 mg/L
A typical HPLC chromatogram is shown in Figure 1.
Precision
Based on a collaborative study, repeatability and re-
producibility coefficients of variation of 1.5–11.1 and
9.7–16.6%, respectively, can be expected when IAA con-
centrations in wort are in the range of 6–66 mg/L. Re-
peatability and reproducibility coefficients of variation
of 3.15–3.20 and 6.2–11.6%, respectively, can be ex-
pected when IAA concentrations in beer are in the range
of 16–48 mg/L.
Notes
1. It is recommended that only clear-glass vials and
volumetric flasks be used. Certain brown glassware has
been found to induce instability in solutions of DCHA-
Iso standard.
2. Because the standard is in acidic methanol, it is sta-
ble for at least 1 month if protected from light and evapo-
ration.
References
1. American Society of Brewing Chemists. Report of Subcommittee
on Iso-alpha-acids in Beer and Wort by High Performance Liquid
Chromatography. Journal 69:288, 2011.
2. European Brewery Convention. Analytica, 7th ed. Section 7 Hops,
Method 7.9, Iso-alpha-acids and reduced iso-alpha-acids in hop
products by HPLC, 2008.
Released 2011; Revised 2018
doi: 10.1094/ASBCMOA-Beer-23

F. ISO-α-ACIDS BY SOLVENT
EXTRACTION
This is a spectrophotometric method for determination
of iso--acids (IAA) in beer.
Reagents
(a) 2,2,4-Trimethylpentane (isooctane), CAS No. 540-
84-1, spectrophotometric grade or equivalent.
(b) Methanol, CAS No. 67-56-1, reagent grade, with an
absorbance of not more than 0.04 at 260 nm in a 1-cm
cell, compared with freshly redistilled water from an
all-glass still.
(c) Hydrochloric acid, CAS No. 7647-01-0, 4M and 6M.
(d) Sodium hydroxide, CAS No. 1310-73-2, 1.5M.
Reagent Preparation
(1) Acidic methanol. In a 250-mL beaker, add 32 mL of
4M HCl (reagent c) to 68 mL of methanol (reagent
b) and swirl to mix.
(2) Alkaline methanol. Just before use, add 1.0 mL of
1.5M sodium hydroxide (reagent d) to 500 mL of
methanol (reagent b) in a 600-mL beaker, and mix
well with a glass stirring rod.
Apparatus
(a) Mechanical shaker. A platform type or a “wrist ac-
tion” type shaker, with extending arm adjusted in a
vertical plane so that the tube will be held in a hori-
zontal position, or equivalent.
(b) Spectrophotometer, capable of measuring at 255 nm.
(c) Quartz cuvette, 1-cm.
(d) Centrifuge tubes, 50-mL with Teflon-lined screw
caps.
(e) Centrifuge, capable of taking 50-mL tubes.
(f) Separatory funnels, 125-mL.
(g) Beaker, 250- and 600-mL.
(h) Volumetric flasks, 25-mL, glass-stoppered.
(i) Transfer pipettes.
(j) Volumetric pipettes, 2-, 5-, 10-, and 15-mL.
(k) Graduated cylinder, 10-mL.
(l) Volumetric flask, 10-mL.
(m) Glass stirring rod.
Method
Decant approximately 100 mL of chilled beer (50°F)
into a 250-mL beaker and decarbonate by swirling in
order to pipette the volumes as accurately as possible.
Transfer 5.0 mL of the chilled decarbonated beer to a 50-
mL centrifuge tube using a 5.0-mL volumetric pipette.
Add 2.0 mL of 6M HCl (reagent c) and 15.0 mL of iso-
octane (reagent a). Cap the centrifuge tubes tightly and
place on a mechanical shaker set to a speed of 8 (or 80%
of capacity) for 30 min. The action must be vigorous. Set
©American Society of Brewing Chemists
Beer–23
Page 7 of 8
the centrifuge speed at 400 × g. Centrifuge the tubes for
two 5-min intervals to separate the phases. After the first
centrifugation, tap the centrifuge tube on the counter to
aid in separating the layers, and then swirl the tube. Re-
peat for each sample. Return to centrifuge for another 5
min. At least 10 mL of the upper isooctane layer is needed
for transfer. If required, centrifuge the tubes a third time
or long enough to separate the phases.
Set up a second set of centrifuge tubes and separatory
funnels for each sample. Pipette 10 mL of the acidic
methanol solution (reagent 1) into each tube. As soon as
possible, accurately transfer 10 mL of the upper, clear
isooctane layer using a plastic transfer pipette to a 10-
mL graduated cylinder. Caution: when pipetting the 10-
mL aliquot of the upper isooctane layer, do not aspirate
the emulsion layer or globules. Transfer the 10-mL iso-
octane layer to the 50-mL centrifuge tube, separatory
funnel, or stoppered graduated cylinder containing 10.0
mL of acidic methanol solution (reagent 1). Cap the cen-
trifuge tubes, separatory funnels, or stoppered graduated
cylinders tightly and invert 100 times at a rate that will
allow the contents to pass from one end of the container
to the other about once each second. Carefully vent the
pressure from the extraction vessel. Allow the phases to
separate. Prepare alkaline methanol (reagent 2) as de-
scribed above.
As soon as possible, transfer 5.0 mL of the upper layer
to a 25-mL volumetric flask and bring to volume with
alkaline methanol (reagent 2). Invert 30 times and mix
well. Read the absorbance at 255 nm using the spectro-
photometer set to read zero absorbance for a reagent blank
consisting of 5.0 mL of isooctane made to 25 mL with
alkaline methanol (reagent 2).
Calculations
Calculate the IAA of beer by the following formula:
IAA = [(absorbance255 × 96.15) + 0.4] × 3
Report IAA (mg/L) to the nearest half unit.
Example
Absorbance255 = 0.221
IAA = [(0.221 × 96.15) + 0.4] × 3 = 64.9
Report IAA as 65.0.
References
1. American Society of Brewing Chemists. Report of the Subcommit-
tee on Determination of Isohumulones in Beer. Proc. 1967:267,
1968.
2. American Society of Brewing Chemists. Report of the Subcommit-
tee on Determination of Bitterness Units and Iso-alpha-acid Levels
in Dry-Hopped Beers Using the Iso-alpha-acids Spectrophotometric
Method. Journal 68:213, 2010.
3. Rigby, F. L., and Bethune, J. L. J. Inst. Brew. 61:325, 1955.
doi: 10.1094/ASBCMOA-Beer-23
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4. Bishop, L. R. The E.B.C. scale of bitterness, J. Inst. Brew. 73:525,

1967.
5. Howard, G. A. Estimation of the bitterness of beer, J. Inst. Brew.
74:249, 1968.
6. Scientific sounds. ASBC News Lett., p. 13, April 1968.
7. U.S. Pharmacopeia XVII, p. 1005.

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©American Society of Brewing Chemists doi: 10.1094/ASBCMOA-Beer-23