Simple HPLC Method for Determining the Glycerol Content of Beer Hong Li,1 Xinglin Han, and Fang Liu, China National Research Institute of Food and Fermentation Industries, Beijing 100015, China; and Gabriella Kun-Farkas and Zsuzsanna Kiss, Department of Brewing and Distilling, Faculty of Food Science, Corvinus University of Budapest, Budapest H-1118, Hungary HPLC columns for the ability to resolve fermentable and nonfermentable sugars, glycerol, and ethanol in wort and beer, and found that the SS-100 Ag+ column performed the best (12). Several researchers are developing several HPLC methods for determining glycerol (1,4). In this study, a simple HPLC method for analyzing the glycerol in beer was posed. Glycerol in beer was separated by a calcium cation exchange column and then detected by a refractive index detector (RID).
ABSTRACT J. Am. Soc. Brew. Chem. 73(4):314-317, 2015 A methodology utilizing HPLC was suggested and validated for determination of the glycerol in beer. The chromatographic separation was carried out on a Waters Sugar-Pak1 (6.5 mm i.d. by 300 mm in length) cations ion exchange column with EDTA calcium disodium salt solution at 0.05 g L–1 as mobile phase and a flow rate of 0.5 mL min–1. Beer (20 µL) filtered by a 0.22-µm membrane syringe filter was directly injected onto the HPLC system for analysis. The validation parameters included linearity, precision, limits of detection and quantitation, and accuracy. The calibration curve was linear (R2 = 0.998) within the calibration range. The precision and recovery was 1.1 and 98.5%, respectively. The limit of detection and limit of quantitation for the glycerol were 2.2 and 7.3 µg/L, respectively. This method was applied to quantify the glycerol in 34 beer samples purchased from the marketplaces in China. The analysis results showed that the glycerol content of the beers varied from 925.2 to 1,502.74 mg/L. Keywords: Beer, Glycerol, HPLC
EXPERIMENTAL Reagents and Chemicals Glycerol (≥99.0%) was supplied by Fluka (Buchs, Switzerland). Anhydrous alcohol and calcium disodium EDTA (≥99.0%) were supplied by Sigma-Aldrich (St. Louis). All other reagents used in this study were of analytical grade. Ultrapure water with a resistivity of not less than 18.2 MΩ·cm was used throughout the study for all experiments.
Glycerol is a colorless, odorless, and viscous liquid. It is a simple polyol compound that is called 1,2,3-propanetriol. Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic nature. Glycerol is sweet-tasting, with a relative sweetness of 0.6 (11). Glycerol in beer is one of the byproducts formed during fermentation of wort by yeast (2,3,8–10, 14,16,17). There are studies showing that the total amount of glycerol produced during fermentation increased with increased osmotic pressure (19), and the higher the wort gravity, the greater the glycerol accumulation (10). Its content in beer is usually higher than a great many other flavor compounds (for example, higher alcohols, esters, and organic acids), and the addition of glycerol to beer above and below the threshold level was found to modify the flavor of the product (20). The effects of glycerol on beer quality lie mainly in two tiers: one is that glycerol can influence beer taste because of its sweetness (20) and the other is that glycerol can make people feel dry in his mouth or give rise to diarrhea after drinking beer due to its hygroscopic nature (15). Therefore, it will be very significant to control the content of glycerol in beer. However, there is not a recommended method for analysis from the American Society of Brewing Chemists or European Brewery Convention (7). Parker and Richardson (20) developed the quantitative determination of the glycerol in beer by gas-liquid chromatography (GC) but this method relates to a complex pretreatment (20). Iverson (8) established another GC analytical procedure for glycerol in beer with a high limit of detection (LOD) (103 mg L–1) (8). Glycerol can also be analyzed via enzymatic methods (5,6,9,18,21,22) or chemical methods (13). McLinn et al (12) evaluated 10 cation-exchange 1
Samples All of the beer samples were purchased from several supermarkets in China. The beer sample was filtered by a 0.22-µm membrane syringe filter before injection onto the HPLC system for analysis. HPLC Analysis HPLC analysis was performed on a Waters 2695 HPLC system (Waters, Milford, MA) equipped with a 2414 Waters RID by setting the detector cell temperature at 40°C and sensitivity at 64. Glycerol was separated at 85°C (controlled by the column oven) on a Waters Sugar-Pak1 (6.5 mm i.d. by 300 mm in length) cation ion exchange column preceded by a guard column (Waters Sep Pak Alumin cartridges) with EDTA calcium disodium salt solution at 0.05 g L–1 as mobile phase, and the flow rate was 0.5 mL min–1. Glycerol was identified by the retention time and was quantified by comparing the peak areas of the samples with those of authentic standards using a 20-µL injection. Validation of the Analytical Method The chromatographic method was validated for linearity, precision, sensitivity, and accuracy. The linearity was determined using different levels of calibration in triplicate and was evaluated by the squared correlation coefficient (R2). Calibration range was defined depending on the content of the glycerol expected in the beer samples. The precision of the method was determined by injecting six individual preparations of samples and by calculating the percent relative standard deviation (%RSD). The sensitivity of the method was determined by establishing the LOD and limit of quantitation (LOQ) for the glycerol based on signal-tonoise ratio (S/N) of 3:1 and 10:1, respectively. LOD and LOQ were determined using standard solutions of the glycerol. The reliability and accuracy of the method was verified by carrying out the recovery studies in triplicate. The accuracy was evaluated at two different concentrations by adding a known amount of the
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http://dx.doi.org/10.1094/ASBCJ-2015-0809-01 © 2015 American Society of Brewing Chemists, Inc.
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Simple HPLC Method for Determining Glycerol Content glycerol standard to the beer sample and by calculating the recovery of the glycerol from the %RSD and percent recovery. Statistical Analysis Glycerol content is shown as the mean of at least three measurements in duplicate experiments. Statistical techniques employed were simple descriptive statistics, regression analysis, and Pearson correlation analysis. Statistical analyses were conducted through the statistical software package SPSS. The statistical significance level was set at P = 0.05. RESULTS AND DISCUSSION Chromatographic Separation of the Glycerol by the HPLC Method Typical chromatograms showing the elution profile of the glycerol in a beer, a spiked beer, and a standard solution (containing sugars) are shown in Figure 1. Glycerol in both a beer and a standard solution were well separated according to the method (shown in Experimental section). The retention time of glycerol was
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10.91 min, where there was no evident interference from the fructose, glucose, maltose, sucrose, maltotriose, and ethanol in a beer or wort sample. Therefore, the resolution for the glycerol in beer samples was acceptable. Validation of the HPLC Method A glycerol calibration curve was constructed with six points (0, 175.7, 878.5, 1,757, 2,635.5, and 3,514 mg/L), in triplicate, using the external standard method. The chromatographic peak area showed a significant positive correlation with the concentration of the glycerol. The results of the regression analysis and calibration range are shown in Table I. The calibration curves were linear over the concentration ranges studied. It can be observed that the regression equation had an ideal coefficient of determination (R2 = 0.998), indicating an excellent fit of the glycerol content to the linear model within the range studied. The repeatability of the method, expressed as RSD for a total of six replicates, was tested for a beer sample. The resulting RSD was below 1.5%.
Fig. 1. Chromatograms obtained from analysis of a standard solution containing A, common sugars; B, a beer sample; and C, a spiked beer sample by the HPLC method.
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The LOD and LOQ for the glycerol were calculated based on the S/N. The LOD and LOQ were 2.2 and 7.3 µg/L, respectively (Table I). The LOD and LOQ value are in a sufficiently low range to determine the glycerol in normal beers. Accuracy of the method was evaluated by percent recovery of the glycerol. Recovery of the analytical method was studied by spiking a known amount of glycerol to a beer and then analyzing both spiked and nonspiked samples in triplicate. The recovery of the glycerol was 98.5% (Table II).
TABLE I Calibration Parameters, Repeatability, and Limit of Detection for the Glycerola Analyte
Glycerol
Range (mg/L) Linearity (R2) Repeatability (RSD %) (n = 6) LOD (mg/L) LOQ (mg/L)
≈0–3,514 0.998 1.1 2.2 7.3
a
CONCLUSIONS
RSD = relative standard deviation; LOD = limit of detection; and LOQ = limit of quantitation. TABLE II Recovery Rate for the Glycerol Standard Added to a Beer (n = 3)
Analyte Glycerol
Application of the HPLC Method to Marketplace Beer Samples Glycerol contents in 34 beer samples purchased in domestic markets were analyzed. All of the beers except for Weissbier (imported from Belgium) were produced in China. The detailed results are listed in Table III. Glycerol content in beer ranged from 925.2 to 1,502.74 mg/L (Table III). Here, the Weissbier, of which original extract was maximal, had the maximum glycerol content. The mean content of glycerol in beer was 1,172.63 mg/L. The main advantages of the method for analyzing glycerol content in beer developed here were that the operation was simple, without special pretreatment, and that the precision was good. The method can be used for routine analysis.
A methodology for the determination of the glycerol in beer by HPLC was proposed and validated in this study. The proposed method offers the following advantages: the sample can be directly injected without time-consuming sample preparation; and it exhibits excellent precision, accuracy, and detection limits, with good recoveries (98.5%), for the determination of the glycerol in beer. The chromatographic method yielded a very satisfactory separation of the standards and, under the same conditions, the separation of the glycerol in real samples. Thus, this procedure can be used to determine the glycerol content in various beer samples.
Concentration added (mg/L)
Concentration found (mg/L)
Recovery (%)
ACKNOWLEDGEMENTS
0 878.50
1,013.65 1,878.97
… 98.5
We thank the Ministry of Science and Technology of China under grant number 2014DFG31770 for providing financial support.
TABLE III Glycerol Content of Beers Beer 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Beer type Light-colored Light-colored Light-colored Light-colored Light-colored Weissbier Light-colored Light-colored Light-colored Light-colored, nonpasteurized Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored Light-colored, crystallized Light-colored Light-colored, nonpasteurized Light-colored Light-colored Light-colored, crystallized Light-colored Light-colored Light-colored, crystallized
Original extract (°P)
Ethanol (% v/v)
8.8 9 9 9.3 8.4 11.6 8.2 8.2 10 8.9 8 9 9.1 7.4 8 8 8 8.9 8.9 8.2 9.2 9 9.1 8.1 9.1 8.1 8.1 8.2 8 8 7.3 7.1 7 7.2
3.9 3.8 3.9 3.8 3.5 5.04 3.5 3.5 4.2 3.5 3.4 3.7 3.8 3.1 3.6 3.5 3.6 4 4 3.9 3.6 3.9 4 3.5 4 3.5 3.5 3.6 3.5 3.5 3.1 3 3 3.2
Glycerol (mg L–1) 1,042.75 1,042.95 1,046.42 1,078.28 945.95 1,502.74 956.56 951 1,186.5 985.36 925.2 967.47 1,005.54 1,058.3 1,169.46 1,199.55 1,226.71 1,288.946 1,297.951 1,210.028 1,379.31 1,387.972 1,298.501 1,370.032 1,311.05 1,243.85 1,234.712 1,367.776 1,241.833 1,236.349 1,154.002 1,144.92 1,158.887 1,252.475
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