Application Gas Chromatography

News
Analysis of Diacetyl and 2,3-pentanedione in Beer Using
Nexis™ GC-2030
No. G316
Among the off-flavors in beer, there are diacetyl and 2,3- „ Chromatogram and Calibration Curves of
pentanedione (collectively known as vicinal diketone: VDK). Standard Samples
More than a certain level of VDK, which is generated during
The chromatogram and calibration curves of standard
fermentation, gives beer its “butterscotch” aroma. The
samples are shown in Fig. 1 and Fig. 2, respectively.
concentration of VDK needs to be controlled to prevent it
from affecting the flavor of the beer. This report presents

Diacetyl

2,3-pentanedione

2,3-hexadione (I.S.)
ダイアセチル

2,3-ペンタンジオン

2,3-ヘキサジオン(I.S.)
the quantitative results for VDK and changes in VDK level
before and after main fermentation in two commercially
available beer samples.
A. Hashimoto, Y. Takemori

„ Reagent Preparation/Quantitative Method 100 ppb

The standard solutions were prepared at concentrations of
25 ppb, 50 ppb and 100 ppb (v/v) by diluting diacetyl and
2,3-pentanedione with water. For preparation of the
internal standard solution, 2,3-hexadione aqueous solution
was added to each standard solution at 50 ppb (v/v). After 50 ppb
pouring 5 mL of each sample into a headspace vial and
sealing the vial, headspace analysis was performed and
calibration curves were prepared. For preparation of the 25 ppb
sample for analysis, the internal standard solution was
added to non-degassed beer samples, as described above.
After 5 mL of each sample was poured into a headspace vial
that was then sealed and heated at 60 °C for 90 minutes 0.0 2.5 5.0 7.5 10.0 min
and subsequently cooled for determination of total
amount of VDK, including precursors*1, these vials were Chromatogram of Standard Samples
placed in the sample tray on HS-20 to perform headspace Area ratio
面積比
analysis and quantification using the prepared calibration 3
0.7
curves. Diacetyl
ダイアセチル
*1 Beer samples were heated in an oven as a pretreatment prior to 0.6 RR22==0.9999
0.9999
analysis because the precursors are converted into VDK by heating.
0.5

„ Instrument Configuration and Analytical Conditions

0.4 2
0.3
The instrument configuration and analytical conditions
are shown in Table 1. 0.2 1

Table 1 Instrument Configuration and Analytical Conditions 0.1

HS-20
0.0
Mode : Loop 0.0 25.0 50.0 75.0 濃度比
Concentration
Oven Temperature : 40 °C Area ratio ratio
Sample Line Temperature : 90 °C 面積比
1.75
Transfer Line Temperature : 95 °C 3
Vial Pressure : 150 kPa 2,3-pentanedione
Vial Heat-retention Time : 40 min
1.50 2,3-ペンタンジオン
Vial Pressurization Time : 1 min 1.25
RR22==0.9978
0.9978
Vial Pressurization : 0.1 min
Equilibrating Time
1.00
Loading Time : 0.5 min 2
Loading Pressurization Time : 0.1 min
0.75
Injection Time : 0.5 min
Needle Flush Time : 5 min
0.50 1
Nexis GC-2030 / ECD-2010 Exceed
0.25
Injection Mode : Split
Split Ratio : 1:20
0.00
Carrier Gas : He 0.0 25.0 50.0 75.0 濃度比
Concentration
Carrier Gas Control : Constant Linear Velocity (40 cm/sec) ratio
Column : SH-Rtx™-624
(60 m × 0.32 mm I.D., 1.80 ʅm) Calibration Curves
Column Temp : 50 °C - 5 °C/min – 120 °C (6 min)
Detector Temp : 130°C
ECD current : 2.5 nA
Detector Gas : N2 15 mL/min
Application No. G316
News

„ Quantitative Results for VDK in Two

2,3-ヘキサジオン(I.S.)
2,3-hexadione (I.S.)
Commercially Available Beer Samples

2,3-ペンタンジオン
2,3-pentanedione
ダイアセチル
The quantitative results for VDK in two commercially
available beer samples are shown in Table 2. The

Diacetyl
representative chromatogram is shown in Fig. 3.
Table 2 Quantitative Results for VDK in Two Brands of Beer
Samples
Beer Diacetyl 2,3-pentanedione
A 18.2 ppb 1.8 ppb
B 39.7 ppb 7.1 ppb

0.0 2.5 5.0 7.5 10.0 12.5 min

Chromatogram of Beer Samples

„ Changes in VDK Level After Main Fermentation
After main fermentation, the diacetyl rest process (a

ダイアセチル

pentanedione

2,3-ヘキサジ
2,3-ペンタン
process in which yeasts are allowed to consume VDK) is

hexadione
オン(I.S.)
Diacetyl
completed, followed by the maturation process. The Day 4 after

ジオン
quantitative results and chromatogram of VDK in beer 主発酵後

2,3-
main

2,3-
4日目
fermentation
samples during the diacetyl rest process are shown in
Table 3 and Fig. 4, respectively. Day 5 after
主発酵後
main
The VDK peak decreases as the duration of main 5日目
fermentation
fermentation increases. This demonstrated the Day 6 after
主発酵後
main
consumption of diacetyl by yeasts and successful 6日目
fermentation
diacetyl rest.
Before
In addition, the quantitative results for VDK in beer bottling
ボトリング前
samples before bottling after the completion of the
maturation process are also shown.
Table 3 Quantitative Results for VDK in Beer Samples After Main 0.0 2.5 5.0 7.5 10.0 12.5 min
Fermentation
Duration after the completion 2,3- Chromatogram of Beer Samples After Main Fermentation and
Diacetyl Before Bottling
of main fermentation pentanedione
Day 4 208.9 ppb 32.1 ppb
Day 5 130.6 ppb 18.6 ppb
Day 6 101.7 ppb 12.0 ppb
Before bottling 39.7 ppb 7.1 ppb

„ Conclusion
VDK that may affect flavor and aroma of beer has
primarily been determined by sensory evaluation.
Meanwhile, headspace analysis using gas
chromatography enables easy quantitative analysis of
even low concentrations of VDK without special
pretreatment such as degassing or concentration.

Appearance of Nexis™ GC-2030 + HS-20

<Reference>
Narihiro Suzuki: Brewer Produces World’s Most Delicious Beer with Wild Yeast; Shinchosha (2019)

<Acknowledgement>
We would like to express our appreciation for the support and cooperation we received from President Narihiro Suzuki and Motoki Sasaki of
Nikenjayamochi Kadoya Honten (Ise Kadoya Microbrewery).

Nexis is a trademark of Shimadzu Corporation in Japan and/or other countries.

Rtx is a trademark or registered trademark of Restek Corporation in the United States and/or other countries.

First Edition: Nov. 2019

For Research Use Only. Not for use in diagnostic procedure.

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