The document discusses the analysis of phenolic compounds in whisky using HPLC with fluorescence detection. Ten phenolic compounds were separated within 10 minutes using a C18 column and acetonitrile/water mobile phase. Calibration curves for the standards showed good linearity with R2 > 0.999. Three whisky samples were analyzed and showed differences in their phenolic profiles. The method demonstrated effective separation and quantitation of most phenolic compounds in whisky.

1. Introduction
One of the world’s most popular spirits is
whisky and it comes in many different classes
and types. The character and flavor of these
differing types vary widely due to their varied
chemical composition. Whisky contains a multitude of compounds, which can be
influenced by the variety of grain, the distillation process, and the wood used in
the barrels for the aging process.1
Phenolic compounds contribute bitterness and smokiness to a whisky’s flavor.2
They are more distinct in whiskies produced in Scottish distilleries where barley is
dried using peat fires. During the drying process, the phenolic compounds in the
peat smoke are absorbed by the barley and the flavors are later transferred to the
whisky during the malting process. Additionally, during the maturation of the whisky
in charred barrels, phenolic compounds may be produced in the spirit.1
Analysis of Phenols
in Whisky by HPLC
with FL Detection
A P P L I C A T I O N N O T E
Authors:
Chi Man Ng
Wilhad M. Reuter
PerkinElmer, Inc.
USA
Liquid Chromatography

2. 2
The most important flavor-contributing phenolic compounds
in whisky are phenol, cresols, xyenol and guaiacol. Cresols,
particularly m-cresol, are compounds responsible for the
somewhat medicinal aroma in both Scotch whiskies and
adhesive bandages.1
Guaiacol imparts a slight smoky aroma
and eugenol, more commonly found in cloves, is found in
many whiskies and is partly responsible for their spicy aroma.1, 3
This application focuses on the HPLC separation and quantitation
of ten phenols in three store-bought Scotch whiskies. Method
conditions and performance data, including linearity and
repeatability are presented.
Experimental
Hardware/Software
For all chromatographic separations, a PerkinElmer Altus™
HPLC System was used, including the A-10 Solvent and Sample
Manager, A-10 column heater, integrated vacuum degasser and
an A-10 fluorescence (FL) detector. All instrument control,
analysis and data processing was performed using the Waters®
Empower®
3 Chromatography Data Software (CDS) platform.
Method Parameters
The HPLC method parameters are shown in Table 1.
Solvents, Standards and Samples
All solvents and diluents used were HPLC grade and filtered via
0.45-µm filters.
The phenolic standard set was obtained from Sigma Aldrich®
,
Inc. (Milwaukee, WI), consisting of phenol, o-cresol, m-cresol,
p-cresol, guaiacol, 4-ethylguaiacol, 4-ethylphenol, 2,5-xyenol,
2-ethylphenol and eugenol. A stock 100-ppm standard was
prepared using methanol as diluent. The lower level standards
were then serially prepared from this stock solution.
The whisky samples were purchased at a local store. They were
labeled Whisky1, Whisky2, and Whisky3. Each whisky sample
was then diluted 1:1 with methanol.
Prior to injection, all calibrants and samples were filtered through
0.45-µm filters to remove small particles.
Results and Discussion
Figure 1 shows the chromatographic separation of the 100-ppb
phenolic standard using the conditions described above. The
analysis time was under 10 minutes. Eight of the phenols were
well resolved, while m-cresol and p-cresol co-eluted at 4.28 min.
Table1.HPLCMethodParameters.
HPLC Conditions
Column:
PerkinElmer Brownlee™ SPP 2.7 mm C18 3.0 X 100 mm
(Part# N9308410)
Mobile Phase:
Solvent A:Water
Solvent B:Acetonitrile
Solvent program:
Analysis Time 10 min; 6-minute injection delay time between injections
Flow Rate: 0.5 mL/min. (3500 psi)
Oven Temp.: 25 ºC
Detection (FL): Excitation wavelength: 272 nm; Emission wavelength: 298 nm
Injection Volume: 10 µL
Sampling (Data) Rate: 5 pts./sec
Time
(min)
Flow Rate
(mL/min)
%A %B %C %D
1 Initial 0.5 75.0 25.0 0.0 0.0
2 10.0 0.5 60.0 40.0 0.0 0.0
3 10.1 0.5 75.0 25.0 0.0 0.0
Figure1.Chromatogramofthe100-ppbphenolicstandard.
phenol
guaiacol
m-cresol,p-cresol
o-cresol
3,5-xyenol
4-ethylphenol
4-ethylguaiacol
2-ethylphenol
eugenol
EU
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

3. 3
Figure 2 shows the overlay of 10 replicate 100-ppb phenolic standard injections, demonstrating exceptional reproducibility. The retention
time (RT) %RSD for eugenol was 0.11%.
From a serial dilution series of the phenolic standard mix, ranging in concentration from 5 to 100 ppb, a 5-level calibration set was
generated. Figure 3 shows the calibration results for four representative phenols. All nine phenols followed a linear (1st
order) fit and had
R2
coefficients > 0.999 (n = 3 at each level).
Figure2.Overlayof10replicatesofthe100-ppbphenolicstandard.
EU
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
phenol
o-cresol
R2
= 0.99977 R2
= 0.99976
Figure3.Fourrepresentativelinearityresultsofthe5-levelcalibrationsetofthephenolicstandardmix.
Calibration Plot
Area
-2.0x106
0.0
2.0x106
4.0x106
6.0x106
8.0x106
1.0x107
1.2x107
1.4x107
1.6x107
1.8x107
Concentration (ppb)
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Calibration Plot
Area
0
2x107
4x107
6x107
8x107
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00
R2
= 0.99999
-2.0x106
0.0
2.0x106
4.0x106
Concentration (ppb)
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Calibration Plot
Area
0
2x107
4x107
6x107
8x107
Concentration (ppb)
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00
Calibration Plot
Area
0
1x107
2x107
3x107
4x107
Concentration (ppb)
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00
Calibration Plot
Area
-5.0x106
0.0
5.0x106
1.0x107
1.5x107
2.0x107
2.5x107
Concentration (ppb)
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00
Calibration Plot
Area
0.0
5.0x106
1.0x107
1.5x107
2.0x107
Concentration (ppb)
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Calibration Plot
Area
0
2x107
4x107
6x107
8x107
Concentration (ppb)
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00
Calibration Plot
Area
0
1x107
2x107
3x107
4x107
Concentration (ppb)
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00
Calibration Plot
Area
-5.0x106
0.0
5.0x106
1.0x107
1.5x107
2.0x107
2.5x107
Concentration (ppb)
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00
Calibration Plot
Area
0.0
5.0x106
1.0x107
1.5x107
2.0x107
Concentration (ppb)
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Calibration Plot
Area
4x106
6x106
8x106
3,5-xyenol
Calibration Plot
Area
-2.0x106
0.0
2.0x106
4.0x106
6.0x106
8.0x106
1.0x107
1.2x107
1.4x107
1.6x107
1.8x107
Concentration (ppb)
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Calibration Plot
Area
0
2x107
4x107
6x107
8x107
Concentration (ppb)
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00
Calibration Plot
Area
0
1x107
2x107
3x107
4x107
Concentration (ppb)
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00
Calibration Plot
Area
0.0
5.0x106
1.0x107
1.5x107
2.0x107
2.5x107
guaiacol
R2
= 0.99927

4. 4
Using the same chromatographic conditions, three whisky samples
were analyzed: Whisky1, Whisky2, and Whisky3. The results are
shown in Figure 4. Comparing the chromatograms of these
whisky samples with the 100-ppb phenolic standard, it can be
observed that the samples contained a majority of the phenols.
For Whisky1, there was no peak corresponding to the RT of
m-cresol and p-cresol, though an unknown component eluted
nearby at 4.20 min. For Whisky2 and Whisky3, while a peak
corresponding to m-cresol and p-cresol was detected, as these
two phenols coeluted, individual quantitation was not possible.
Also, for all three whisky samples, the peak at 7.38 min,
corresponding to 4-ethylphenol, appeared as part of a doublet
and, therefore, could not be quantitated. The unknown peak in
the doublet is likely attributable to the sample matrix.
Figure4.ChromatogramsofWhisky1,Whisky2,andWhisky3(blue)overlaidwith100-ppbphenolicstd.(black).
phenol
guaiacol
m-cresol,p-cresol
o-cresol
3,5-xyenol
4-ethylphenol
4-ethylguaiacol
2-ethylphenol
eugenol
EU
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
phenol
guaiacol
m-cresol,p-cresol
o-cresol
3,5-xyenol
4-ethylphenol
4-ethylguaiacol
2-ethylphenol
eugenol
EU
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
phenol
guaiacol
m-cresol,p-cresol
o-cresol
3,5-xyenol
4-ethylphenol
4-ethylguaiacol
2-ethylphenol
eugenol
EU
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Whisky2
Whisky1
Whisky3

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Conclusion
This work has demonstrated the applicability of this method for
the effective analysis of phenols using a PerkinElmer Altus HPLC
System with FL detection. The results exhibited very good retention
time repeatability as well as excellent linearity over the tested
concentration ranges. All the phenolic compounds were well
separated in under ten minutes, except for m-cresol and p-cresol,
which are typically difficult to resolve from one another.
The phenolic compounds in the three whisky samples were easily
detectable and, apart from the two co-eluters and 4-ethylphenol,
easily quantitated.
References
1. [Online]. http://www.compoundchem.com/2015/03/31/whisky/.
[Accessed 20 May 2015].
2. M. Lehtonen. Chromatographia. 16, 201, 1982.
3. [Online]. http://whiskyscience.blogspot.com/2011/201/peat.html.
[Accessed 18 May 2015].
Considering the above and based on the standard calibration
plots, the quantitative results for each whisky sample are shown in
Table 2. This was based on the average of three replicates per
injection. Compared to one another, each whisky sample had
differing concentrations of each of the phenols. Overall, the results
show Whisky1 had significantly higher eugenol concentration
compared to Whisky2 and Whisky3, and more than double the
total amount of phenol of Whisky3.
Table2.QuantitativeResultsforWhiskySamples.
Phenols Whisky1 (ppb) Whisky2 (ppb) Whisky3 (ppb)
phenol 16 120 63
guaiacol 158 106 103
m-cresol ND* NA** NA**
p-cresol ND* NA** NA**
o-cresol 47 113 67
3,5-xyenol 43 46 41
4-ethylphenol NA*** NA*** NA***
4-ethylguaiacol 112 69 25
2-ethylphenol 21 20 9
eugenol 325 79 21
Total Phenols 722 553 329
* Not detected
** Not available: m-cresol and p-cresol coeluted
***Not available: 4-ethylphenol eluted as part of a doublet