Analysis of Phenols in Whisky by HPLC with FL Detection

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
with FL Detection
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
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
Table 1. HPLC Method Parameters.
HPLC Conditions
Experimental
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
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
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
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.
phenol
300.00
o-cresol
400.00
200.00
100.00
eugenol
EU
500.00
4-ethylguaiacol
600.00
4-ethylphenol
700.00
3,5-xyenol
m-cresol, p-cresol
800.00
2-ethylphenol
guaiacol
900.00
Time
(min)
Mobile Phase:
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.
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.
PerkinElmer Brownlee™ SPP 2.7 mm C18 3.0 X 100 mm
(Part# N9308410)
Solvent A: Water
Solvent B: Acetonitrile
Solvent program:
Column:
0.00
0.00
1.00
2.00
Figure 1. Chromatogram of the 100-ppb phenolic standard.
2
3.00
4.00
5.00
Minutes
6.00
7.00
8.00
9.00
10.00
4.0x106
2.0x106
0.0
-2.0x106
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Concentration (ppb)
70.00
80.00
90.00
100.00
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
6x10
1.6x107
700.00
A r ea
1.4x107
1.0x107
A r ea
600.00
7 7
1.2x10
4x10
7 6
8.0x10
2x10
EU
500.00
6.0x106
4.0x106
0
2.0x106
400.00
0.0
0.00
-2.0x106
300.00
20.00
0.00
10.00
40.00
20.00
60.00
30.00
100.00
40.00
50.00
60.00
Concentration (ppb)
C alibration P lot
200.00
80.00
Concentration (ppb)
120.00
70.00
140.00
80.00
90.00
160.00
100.00
C alibration P lot
8x107
4x107
100.00
6x107
3x107
0.00
1.00
2.00
3.00
A r ea
A r ea
0.00
4x107
4.00
2x107
5.00
Minutes
7
2x107
6.00
7.00
8.00
9.00
10.00
1x10
Figure 2. 0Overlay of 10 replicates of the 100-ppb phenolic standard.
0
0.00
20.00
40.00
80.00
100.00
120.00 mix,
140.00
160.00in concentration
From a serial
dilution
series
of60.00the phenolic
standard
ranging
from
5 to
100 ppb,
calibration
set was140.00
0.00
20.00
40.00
60.00 a 5-level
80.00
100.00
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
each Plevel).
R2 coefficients > 0.999 (n = 3 atC alibration
lot
4x107
C alibration P lot
C alibration P lot
C alibration P lot
7
3x10
1.8x107
8x107
phenol
1.6x107
2.5x107
A r ea
7
2x10
1.4x107
6x107
2.0x107
1.2x107
7
1x10
1.0x107
7
1.5x10
4x107
A r ea
A r ea
A r ea
guaiacol
8.0x106
0 6
6.0x10
1.0x107
2x107
4.0x106
2.0x106 0.00
5.0x106
20.00
40.00
60.00
80.00
Concentration (ppb)
0.0
100.00
120.00
140.00
0
0.0
R2 = 0.99999
-2.0x106
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Concentration (ppb)
70.00
80.00
90.00
-5.0x106
100.00
R2 = 0.99927
0.00
0.00
20.00
10.00
20.00
40.00
30.00
4x107
2.0x107
o-cresol
120.00
80.00
90.00
140.00
100.00
160.00
110.00
3,5-xyenol
3x107
1.5x107
2.0x107
1.5x107
2x107
1.0x107
A r ea
A r ea
A r ea
80.00
100.00
50.00
60.00
70.00
Concentration (ppb)
Concentration (ppb)
C alibration P lot
C alibration P lot
C alibration P lot
2.5x107
60.00
40.00
1.0x107
1x107
5.0x106
5.0x106
0.0
0
0.0
R2 = 0.99977
-5.0x106
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Concentration (ppb)
80.00
90.00
100.00
R2 = 0.99976
0.00
0.00
110.00
20.00
10.00
20.00
40.00
30.00
60.00
80.00
40.00
50.00
60.00
Concentration (ppb)
Concentration (ppb)
100.00
70.00
80.00
120.00
90.00
140.00
100.00
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
A r e aA r e a
2.5x107
1.0x107
4x107
2.0x107
5.0x106
C alibration P lot
6
8x10
1.5x107
A r ea
2x107
1.0x107
6x106
0.0
0.00
0.00
10.00
20.00
20.00
30.00
40.00
40.00
50.00
60.00
Concentration (ppb)
60.00
80.00
100.00
70.00
80.00
120.00
90.00
140.00
100.00
160.00
A r ea
0
5.0x106
4x106
0.0
3
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.
guaiacol
900.00
Whisky1
800.00
200.00
100.00
2-ethylphenol
eugenol
phenol
300.00
4-ethylguaiacol
400.00
o-cresol
EU
500.00
4-ethylphenol
600.00
3,5-xyenol
m-cresol, p-cresol
700.00
0.00
0.00
1.00
2.00
3.00
4.00
5.00
Minutes
6.00
7.00
8.00
9.00
10.00
guaiacol
900.00
Whisky2
800.00
200.00
100.00
2-ethylphenol
eugenol
phenol
300.00
4-ethylguaiacol
400.00
o-cresol
EU
500.00
4-ethylphenol
600.00
3,5-xyenol
m-cresol, p-cresol
700.00
0.00
0.00
1.00
2.00
3.00
4.00
5.00
Minutes
6.00
7.00
8.00
9.00
10.00
guaiacol
900.00
Whisky3
800.00
200.00
100.00
2-ethylphenol
eugenol
phenol
300.00
4-ethylguaiacol
400.00
o-cresol
EU
500.00
4-ethylphenol
600.00
3,5-xyenol
m-cresol, p-cresol
700.00
0.00
0.00
1.00
2.00
3.00
4.00
5.00
Minutes
6.00
Figure 4. Chromatograms of Whisky1, Whisky2, and Whisky3 (blue) overlaid with 100-ppb phenolic std. (black).
4
7.00
8.00
9.00
10.00
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.
Conclusion
Table 2. Quantitative Results for Whisky Samples.
The phenolic compounds in the three whisky samples were easily
detectable and, apart from the two co-eluters and 4-ethylphenol,
easily quantitated.
Phenols
phenol
Whisky1 (ppb)
Whisky2 (ppb)
Whisky3 (ppb)
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
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.
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].
* Not detected
** Not available: m-cresol and p-cresol coeluted
*** Not available: 4-ethylphenol eluted as part of a doublet
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