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An automated flow-injection extraction method for determination of bittering compounds in beer
Analytica Chimica Acta, 187 (1986) 339-342 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Short Communication
AN AUTOMATED FLOW-INJECTION EXTRACTION METHOD FOR DETERMINATION OF BITTERING COMPOUNDS IN BEER
YLVA SAHLESTRGM Department of Analytical Stockholm (Sweden)
Chemistry,
Royal Institute
of Technology,
S-100 44
SIGRID TWENGSTRGM and BO KARLBERG* Bifok AB, Box 124, S-l 91 22 Sollentuna
(Sweden)
(Received 11th April 1986)
Summary. The manual standard liquid-liquid extraction method for determining bitterness in beer is adapted for a flow-injection extraction system. With the flow-injection method, the separate solvent blank extraction required in the batch procedure is unnecessary. The injected sample volume is 100 11, the sampling frequency is about 60 h-l and the consumption of iso-octane is only about 1 ml/sample.
The bittering compounds in beer consist mainly of iso+acids, which have been identified as three pairs of diastereoisomeric compounds, namely isohumulone, isocohumulone and isoadhumulone. These are converted from the a-acids in hops during the wort boiling process. The complete structure of all bitter-tasting compounds is not yet fully understood [l] . This has been the main reason for the difficulties in finding relevant analytical methods. Consequently, a variety of methods is applied: e-acids can be measured conductometrically or gravimetrically as their corresponding lead salts, and polarimetric assay has been suggested, but this measurement does not include racemic a-acids [ 11. All these methods are very sensitive to interfering compounds. Liquid chromatography has long been regarded as an attractive way of solving the selectivity problems and much research has been devoted to the development of a fast and reliable routine method primarily for iso-eacids [Z-4]. However, there remain unsolved problems involving selection of an internal standard, a relevant reference compound, resolution speed and column packing material. A modification of a method developed by Moltke and Meilgaard [ 51 in 1955 is now widely used for routine purposes. Degassed beer is extracted after acidification with iso-octane in a 1:2 (v/v) ratio. After centrifugation, the absorbance of the organic phase is measured at 275 nm against pure iso-octane. This absorbance value is used to define an empirical entity called a Bittering Unit: BU = 50 X absorbance. The BU was introduced by the EBC Analysis Committee [6] in order to avoid orbitrary assumptions about the chemical structure and nature of the bittering 0003-2670/86/$03.50
0 1986 Elsevier Science Publishers B.V.
340
compounds in beer. This communication presents an automation of this extraction method based on a flow-injection extraction unit described recently [ 71 . Experimental Reagents and solvents. All reagents and solvents were of analytical grade. Distilled water was used throughout. Iso-octane (Merck) was used as the extraction solvent and distilled water was poured through before use. The carrier was 0.1 M hydrochloric acid. Iso-octanol was added to the beer samples as an anti-foaming agent. Beer samples. All beer samples were supplied from Pripps AB (Sweden); they included a variety of dark and light beers as well as worts and unfiltered green beer. Apparatus. A FIAstar 5020 unit (Tecator, Sweden) wasused together with the extraction manifold described earlier [7]. The flow scheme is shown in Fig. 1. The organic solvent (iso-octane) was introduced via a displacement bottle. A Pye-Unicam SP6-550 U.V. spectrophotometer with a flow cell (8-~1 internal volume, lo-mm light path) was used as detector (275 nm). A recorder was coupled to the spectrophotometer and all peak values were automatically recorded and displayed on the FIAstar unit. The injection volume was 100 ~1. The separating membrane was of PTFE (l.O+m pore size) and the separated organic phase was rejoined with the aqueous stream to a common waste outlet (see Fig. 1). All measurements were made at room temperature. Procedure. Pump 1 in the FIAstar unit was reserved for expelling the organic solvent from the displacement bottle and pump 2 for propelling aqueous carrier into the extraction unit. On starting up the system, pump 1 was activated first and pump 2 was started when the system had been filled with organic solvent. On closing down, the reversed procedure was applied. The beer samples were degassed in an ultrasonic bath for a couple of minutes in order to eliminate gas bubbles. When necessary, water droplets or other impurities in the flow cell were rinsed out with ethanol introduced via a special gauge in the extraction unit [ 71. Manual batch extractions were done simultaneously with the flow-injection extractions in order to compare the two methods. Thus, 10 ml of degassed beer was pipetted into a flask, followed by addition of 1 ml of 3 M
Fig. 1. Flow-injection manifold for the extraction of bittering compounds in beer. C, 0.1 M HCl at 1.0 ml min-I; org, iso-octane at 0.5 ml min-I. Coil lengths: (1) 50 cm; (2) 200 cm; (3) 50 cm.
341
hydrochloric acid and 20 ml of iso-octane. The flask was shaken vigorously for 10 min on a wrist action shaker. Worts, green beers and beer samples from fermentation tanks were then centrifuged. The extract was transferred to a lo-mm quartz cell and measured against water-equilibrated A-octane at 275 nm. Calculutions. In order to compare the two methods, all values were individually related to one sample (Pripps Bla III). This product was intermittently used as an in-house standard at Pripps to calibrate the method. Results and discussion The results of the comparative evaluation of the automatic and manual methods are presented in Table 1. Samples from 22 products were tested: the first seven were sampled from the manufacturing process and the rest TABLE 1 Comparison between batch and flow-injection extraction (f.i.e.) methods for the determination of bitterness in a variety of beer and malt samples Sample No.
Batch
F.i.e. Type
A -Pk
A sample 1
Asame
4,
4,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Unfiltered bulk product As1 Incompletely fermented malt As3 AS3 As3 As3 LighP Darka Lighta LighP Darkb Lightb Lightb Lightb Darkb LightC DarkC LightC LightC LightC LightC (standard)
Asampd
Batch/ f.i.e.
0.351
0.98
0.500
1.02
1.04
0.359 0.386
1.0 1.08
0.509 0.585
1.03 1.19
1.03 1.10
0.401 0.626 0.389 0.107 0.260 0.227 0.234 0.250 0.246 0.215 0.496 0.307 0.357 0.409 0.366 0.321 0.260 0.349 0.359
1.12 1.74 1.08 0.30 0.72 0.63 0.65 0.70 0.69 0.60 1.38 0.86 0.99 1.14 1.02 0.89 0 72 0.97 1.0
0.562 0.903 0.540 0.152 0.371 0.320 0.334 0.351 0.342 0.308 0.640 0.417 0.516 0.525 0.504 0.449 0.361 0.475 0.492
1.14 1.84 1.10 0.31 0.75 0.65 0.68 0.71 0.70 0.63 1.30 0.85 1.05 1.07 1.02 0.91 0.73 0.97 1.0
1.02 1.06 1.02 1.03 1.04 1.03 1.05 1.01 1.01 1.05 0.94 0.99 1.06 0.94 1.0 1.02 1.01 1.0 1.0
a1.7% ethanol. b2.7% ethanol. c4.1% ethanol.
342
from marketed beer products. Sample 22 was used as a standard (Pripps Bla III). The correlation equation was calculated as y = 1.011 x + 0.007, where y respresents the f.i.e. values and 3c the batch values. The repeatability was 1.7% when one sample was injected 25 times. For each flow-injection set-up, a specific BU/f.i.e. factor could be estimated by running parallel determinations by the two methods: BU/f.i.e. factor = Asample (batch) X 50/A,,,pl,
(f.i.e.)
The average BU/f.i.e. factor was 70 for the results presented in Table 1 with an r.s.d. of 3.3%. By using different sample volumes the factor value could be varied within certain limits. The phase-volume ratio also influenced the BU/f.i.e. factor, thus offering a second possibility for its variation. In a separate experiment, the two methods were compared for identical phasevolume ratios. The flow-injection values were about 30% lower than corresponding batch values when the injected volume was 100 ~1. This is in excellent agreement with earlier findings for caffeine [ 71. In conclusion, the flow-injection method offers the following advantages: (a) blanking is automatic as the solvent background absorbance is automatically taken as the baseline; (b) less than 200 /.11of sample is used per analysis; (c) the sample throughput is large, at about 60 h-l ; (d) consumption of organic solvent is low, at about 1 ml per determination; (e) reproducible sample handling is ensured with respect to time and volume; and (f) washingup is minimal. The authors are indebted to Dr. Glaes-Goran Johansson and Prof. Folke Ingman for valuable discussions. REFERENCES 1 J. S. Hough, D. E. Briggs, R. Stevens and T. W. Young, Malting and Brewing Science, 2nd edn., London, 1982. 2 M. Verzele, C. Dewaele and M. van Kerrebroeck, J. Chromatogr., 244 (1982) 321. 3 E. J. Knudson and K. J. Siebert, J. Am. Sot. Brew. Chem., 41, 2 (1983) 51. 4 M. Verzele and M. de Potter, J. Chromatogr., 166 (1978) 320. 5 A. B. Moltke and M. Meilgaard, Brygmesteren, 12 (1955) 65. 6 Analytica-EBC, 3rd edn., 1975. 7 Y. Sahlestrbm and B. Karlberg, Anal. Chim. Acta, 179 (1986) 315.
Short Communication
AN AUTOMATED FLOW-INJECTION EXTRACTION METHOD FOR DETERMINATION OF BITTERING COMPOUNDS IN BEER
YLVA SAHLESTRGM Department of Analytical Stockholm (Sweden)
Chemistry,
Royal Institute
of Technology,
S-100 44
SIGRID TWENGSTRGM and BO KARLBERG* Bifok AB, Box 124, S-l 91 22 Sollentuna
(Sweden)
(Received 11th April 1986)
Summary. The manual standard liquid-liquid extraction method for determining bitterness in beer is adapted for a flow-injection extraction system. With the flow-injection method, the separate solvent blank extraction required in the batch procedure is unnecessary. The injected sample volume is 100 11, the sampling frequency is about 60 h-l and the consumption of iso-octane is only about 1 ml/sample.
The bittering compounds in beer consist mainly of iso+acids, which have been identified as three pairs of diastereoisomeric compounds, namely isohumulone, isocohumulone and isoadhumulone. These are converted from the a-acids in hops during the wort boiling process. The complete structure of all bitter-tasting compounds is not yet fully understood [l] . This has been the main reason for the difficulties in finding relevant analytical methods. Consequently, a variety of methods is applied: e-acids can be measured conductometrically or gravimetrically as their corresponding lead salts, and polarimetric assay has been suggested, but this measurement does not include racemic a-acids [ 11. All these methods are very sensitive to interfering compounds. Liquid chromatography has long been regarded as an attractive way of solving the selectivity problems and much research has been devoted to the development of a fast and reliable routine method primarily for iso-eacids [Z-4]. However, there remain unsolved problems involving selection of an internal standard, a relevant reference compound, resolution speed and column packing material. A modification of a method developed by Moltke and Meilgaard [ 51 in 1955 is now widely used for routine purposes. Degassed beer is extracted after acidification with iso-octane in a 1:2 (v/v) ratio. After centrifugation, the absorbance of the organic phase is measured at 275 nm against pure iso-octane. This absorbance value is used to define an empirical entity called a Bittering Unit: BU = 50 X absorbance. The BU was introduced by the EBC Analysis Committee [6] in order to avoid orbitrary assumptions about the chemical structure and nature of the bittering 0003-2670/86/$03.50
0 1986 Elsevier Science Publishers B.V.
340
compounds in beer. This communication presents an automation of this extraction method based on a flow-injection extraction unit described recently [ 71 . Experimental Reagents and solvents. All reagents and solvents were of analytical grade. Distilled water was used throughout. Iso-octane (Merck) was used as the extraction solvent and distilled water was poured through before use. The carrier was 0.1 M hydrochloric acid. Iso-octanol was added to the beer samples as an anti-foaming agent. Beer samples. All beer samples were supplied from Pripps AB (Sweden); they included a variety of dark and light beers as well as worts and unfiltered green beer. Apparatus. A FIAstar 5020 unit (Tecator, Sweden) wasused together with the extraction manifold described earlier [7]. The flow scheme is shown in Fig. 1. The organic solvent (iso-octane) was introduced via a displacement bottle. A Pye-Unicam SP6-550 U.V. spectrophotometer with a flow cell (8-~1 internal volume, lo-mm light path) was used as detector (275 nm). A recorder was coupled to the spectrophotometer and all peak values were automatically recorded and displayed on the FIAstar unit. The injection volume was 100 ~1. The separating membrane was of PTFE (l.O+m pore size) and the separated organic phase was rejoined with the aqueous stream to a common waste outlet (see Fig. 1). All measurements were made at room temperature. Procedure. Pump 1 in the FIAstar unit was reserved for expelling the organic solvent from the displacement bottle and pump 2 for propelling aqueous carrier into the extraction unit. On starting up the system, pump 1 was activated first and pump 2 was started when the system had been filled with organic solvent. On closing down, the reversed procedure was applied. The beer samples were degassed in an ultrasonic bath for a couple of minutes in order to eliminate gas bubbles. When necessary, water droplets or other impurities in the flow cell were rinsed out with ethanol introduced via a special gauge in the extraction unit [ 71. Manual batch extractions were done simultaneously with the flow-injection extractions in order to compare the two methods. Thus, 10 ml of degassed beer was pipetted into a flask, followed by addition of 1 ml of 3 M
Fig. 1. Flow-injection manifold for the extraction of bittering compounds in beer. C, 0.1 M HCl at 1.0 ml min-I; org, iso-octane at 0.5 ml min-I. Coil lengths: (1) 50 cm; (2) 200 cm; (3) 50 cm.
341
hydrochloric acid and 20 ml of iso-octane. The flask was shaken vigorously for 10 min on a wrist action shaker. Worts, green beers and beer samples from fermentation tanks were then centrifuged. The extract was transferred to a lo-mm quartz cell and measured against water-equilibrated A-octane at 275 nm. Calculutions. In order to compare the two methods, all values were individually related to one sample (Pripps Bla III). This product was intermittently used as an in-house standard at Pripps to calibrate the method. Results and discussion The results of the comparative evaluation of the automatic and manual methods are presented in Table 1. Samples from 22 products were tested: the first seven were sampled from the manufacturing process and the rest TABLE 1 Comparison between batch and flow-injection extraction (f.i.e.) methods for the determination of bitterness in a variety of beer and malt samples Sample No.
Batch
F.i.e. Type
A -Pk
A sample 1
Asame
4,
4,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Unfiltered bulk product As1 Incompletely fermented malt As3 AS3 As3 As3 LighP Darka Lighta LighP Darkb Lightb Lightb Lightb Darkb LightC DarkC LightC LightC LightC LightC (standard)
Asampd
Batch/ f.i.e.
0.351
0.98
0.500
1.02
1.04
0.359 0.386
1.0 1.08
0.509 0.585
1.03 1.19
1.03 1.10
0.401 0.626 0.389 0.107 0.260 0.227 0.234 0.250 0.246 0.215 0.496 0.307 0.357 0.409 0.366 0.321 0.260 0.349 0.359
1.12 1.74 1.08 0.30 0.72 0.63 0.65 0.70 0.69 0.60 1.38 0.86 0.99 1.14 1.02 0.89 0 72 0.97 1.0
0.562 0.903 0.540 0.152 0.371 0.320 0.334 0.351 0.342 0.308 0.640 0.417 0.516 0.525 0.504 0.449 0.361 0.475 0.492
1.14 1.84 1.10 0.31 0.75 0.65 0.68 0.71 0.70 0.63 1.30 0.85 1.05 1.07 1.02 0.91 0.73 0.97 1.0
1.02 1.06 1.02 1.03 1.04 1.03 1.05 1.01 1.01 1.05 0.94 0.99 1.06 0.94 1.0 1.02 1.01 1.0 1.0
a1.7% ethanol. b2.7% ethanol. c4.1% ethanol.
342
from marketed beer products. Sample 22 was used as a standard (Pripps Bla III). The correlation equation was calculated as y = 1.011 x + 0.007, where y respresents the f.i.e. values and 3c the batch values. The repeatability was 1.7% when one sample was injected 25 times. For each flow-injection set-up, a specific BU/f.i.e. factor could be estimated by running parallel determinations by the two methods: BU/f.i.e. factor = Asample (batch) X 50/A,,,pl,
(f.i.e.)
The average BU/f.i.e. factor was 70 for the results presented in Table 1 with an r.s.d. of 3.3%. By using different sample volumes the factor value could be varied within certain limits. The phase-volume ratio also influenced the BU/f.i.e. factor, thus offering a second possibility for its variation. In a separate experiment, the two methods were compared for identical phasevolume ratios. The flow-injection values were about 30% lower than corresponding batch values when the injected volume was 100 ~1. This is in excellent agreement with earlier findings for caffeine [ 71. In conclusion, the flow-injection method offers the following advantages: (a) blanking is automatic as the solvent background absorbance is automatically taken as the baseline; (b) less than 200 /.11of sample is used per analysis; (c) the sample throughput is large, at about 60 h-l ; (d) consumption of organic solvent is low, at about 1 ml per determination; (e) reproducible sample handling is ensured with respect to time and volume; and (f) washingup is minimal. The authors are indebted to Dr. Glaes-Goran Johansson and Prof. Folke Ingman for valuable discussions. REFERENCES 1 J. S. Hough, D. E. Briggs, R. Stevens and T. W. Young, Malting and Brewing Science, 2nd edn., London, 1982. 2 M. Verzele, C. Dewaele and M. van Kerrebroeck, J. Chromatogr., 244 (1982) 321. 3 E. J. Knudson and K. J. Siebert, J. Am. Sot. Brew. Chem., 41, 2 (1983) 51. 4 M. Verzele and M. de Potter, J. Chromatogr., 166 (1978) 320. 5 A. B. Moltke and M. Meilgaard, Brygmesteren, 12 (1955) 65. 6 Analytica-EBC, 3rd edn., 1975. 7 Y. Sahlestrbm and B. Karlberg, Anal. Chim. Acta, 179 (1986) 315.