Analysis of Phenols in Whisky by HPLC with FL Detection
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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.
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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](https://image.slidesharecdn.com/01226601analysisofphenolsinwhiskybyhplcwithfldetection-150720135002-lva1-app6891/85/Analysis-of-Phenols-in-Whisky-by-HPLC-with-FL-Detection-5-320.jpg)
Analysis of Phenols in Whisky by HPLC with FL Detection
- 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
- 5. For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Copyright ©2015, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 012266_01 PKI PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com 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