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 PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com 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_01PKI