A P P L I C AT I O N N O T E

Liquid Chromatography

Authors:
Chi Man Ng
Wilhad M. Reuter
PerkinElmer, Inc.
USA

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

900.00
guaiacol

800.00
m-cresol, p-cresol

700.00

600.00
2-ethylphenol

500.00
EU

400.00
3,5-xyenol
o-cresol

4-ethylphenol

4-ethylguaiacol

300.00
phenol

eugenol

200.00

100.00

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes

Figure 1. Chromatogram of the 100-ppb phenolic standard.

2
 4.0x106

2.0x106

0.0

-2.0x106

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Concentration (ppb)

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%.
C alibration P lot

8x107
C alibration P lot
1.8x107
7
1.6x107
6x10
700.00
1.4x107

7 7
1.2x10
4x10

A r ea
600.00 1.0x107

A r ea
7 6
8.0x10
2x10

6.0x106
500.00
4.0x106
0
2.0x106
400.00
EU

0.0
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00
-2.0x106 Concentration (ppb)
300.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Concentration (ppb)
C alibration P lot C alibration P lot
200.00
8x107
4x107

100.00
6x107
3x107

0.00
4x107
A r ea

2x107

A r ea
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
2x107 Minutes
7 1x10

Figure 2. 0Overlay of 10 replicates of the 100-ppb phenolic standard. 0

From a serial
0.00 dilution
20.00 series
40.00 of60.00the phenolic
80.00 standard
100.00 120.00 mix, ranging
140.00 160.00in concentration
0.00 from
20.00 5 to 100 ppb,
40.00 60.00 a 5-level 80.00 calibration
100.00 set was140.00
120.00
Concentration (ppb) Concentration (ppb)

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 atC alibration
each Plevel).
lot

4x107

C alibration P lot C alibration P lot

7
C alibration P lot
1.8x107
3x10
8x107
1.6x107 phenol guaiacol
2.5x107
7
A r ea

1.4x107
2x10
6x107
1.2x107 2.0x107

7
1.0x107
1x10
7
4x107
1.5x10
A r ea
A r ea

8.0x106
A r ea

0 6
6.0x10 1.0x107
2x107
4.0x106
5.0x106
2.0x106 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00
Concentration (ppb) 0
0.0
0.0
-2.0x106
R2 = 0.99999 R2 = 0.99927
-5.0x106
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00
Concentration (ppb) Concentration (ppb)
Concentration (ppb)

C alibration P lot
C alibration P lot C alibration P lot

4x107
2.5x107 o-cresol 2.0x107
3,5-xyenol

2.0x107 3x107
1.5x107

1.5x107
2x107
A r ea
A r ea

A r ea

1.0x107
1.0x107

1x107
5.0x106
5.0x106

0
0.0 0.0
R2 = 0.99977 R2 = 0.99976
-5.0x106
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Concentration (ppb)
Concentration (ppb) Concentration (ppb)

Figure 3. Four representative linearity results of the 5-level calibration set of the phenolic standard mix.
C alibration P lot

C alibration P lot
2.0x107
8x107
C alibration P lot
1.5x107
6x107
2.5x107
A r e aA r e a

1.0x107 C alibration P lot

4x107 2.0x107

5.0x106 6
1.5x107
8x10
2x107
A r ea

1.0x107
0.0 6x106

0
5.0x106
3
A r ea

4x106
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Concentration (ppb)
0.0
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00
 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.

900.00

guaiacol
800.00
Whisky1

700.00

m-cresol, p-cresol
600.00

2-ethylphenol
500.00
EU

400.00

3,5-xyenol
o-cresol
300.00

4-ethylphenol

4-ethylguaiacol
phenol

eugenol
200.00

100.00

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes

900.00
guaiacol

800.00 Whisky2

700.00
m-cresol, p-cresol

600.00

2-ethylphenol
500.00
EU

400.00
3,5-xyenol
o-cresol

300.00
4-ethylphenol

4-ethylguaiacol
phenol

eugenol
200.00

100.00

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes

900.00
guaiacol

800.00 Whisky3

700.00
m-cresol, p-cresol

600.00
2-ethylphenol

500.00
EU

400.00
3,5-xyenol
o-cresol

300.00
4-ethylphenol

4-ethylguaiacol
phenol

eugenol

200.00

100.00

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes

Figure 4. Chromatograms of Whisky1, Whisky2, and Whisky3 (blue) overlaid with 100-ppb phenolic std. (black).

4
 Considering the above and based on the standard calibration Conclusion
plots, the quantitative results for each whisky sample are shown in This work has demonstrated the applicability of this method for
Table 2. This was based on the average of three replicates per the effective analysis of phenols using a PerkinElmer Altus HPLC
injection. Compared to one another, each whisky sample had System with FL detection. The results exhibited very good retention
differing concentrations of each of the phenols. Overall, the results time repeatability as well as excellent linearity over the tested
show Whisky1 had significantly higher eugenol concentration concentration ranges. All the phenolic compounds were well
compared to Whisky2 and Whisky3, and more than double the separated in under ten minutes, except for m-cresol and p-cresol,
total amount of phenol of Whisky3. which are typically difficult to resolve from one another.
Table 2. Quantitative Results for Whisky Samples. The phenolic compounds in the three whisky samples were easily
Phenols Whisky1 (ppb) Whisky2 (ppb) Whisky3 (ppb) detectable and, apart from the two co-eluters and 4-ethylphenol,
phenol 16 120 63 easily quantitated.
guaiacol 158 106 103
References
m-cresol ND* NA** NA**
1. [Online]. http://www.compoundchem.com/2015/03/31/whisky/.
p-cresol ND* NA** NA**
[Accessed 20 May 2015].
o-cresol 47 113 67
3,5-xyenol 43 46 41 2. M. Lehtonen. Chromatographia. 16, 201, 1982.
4-ethylphenol NA*** NA*** NA*** 3. [Online]. http://whiskyscience.blogspot.com/2011/201/peat.html.
4-ethylguaiacol 112 69 25 [Accessed 18 May 2015].
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

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