Food Chemistry 133 (2012) 352–357
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Monoalkyl carbonates in carbonated alcoholic beverages Marcelo Rabello Rossi, Denis Tadeu Rajh Vidal, Claudimir Lucio do Lago ⇑ Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, CEP 05508-000, São Paulo, SP, Brazil
a r t i c l e
i n f o
Article history: Received 26 September 2011 Received in revised form 22 November 2011 Accepted 16 January 2012 Available online 24 January 2012 Keywords: Monoethyl carbonate Alcoholic beverage Beer Capillary electrophoresis Conductivity detection
a b s t r a c t The presence of monoethyl carbonate (MEC) in beer and sparkling wine is demonstrated for the first time, as well as the formation of this species in drinks prepared with a distilled beverage and a carbonated soft drink. A capillary electrophoresis (CE) equipment with two capacitively coupled contactless conductivity detector (C4D) was used to identify and quantify this species. The concentrations of MEC in samples of lager beer and rum and cola drink were, respectively, 1.2 and 4.1 mmol/l, which agree with the levels of ethanol and CO2 available in these products. Previous results about the kinetics of the reaction suggest that only a small amount of MEC should be formed after the ingredients of a drink are mixed. However, in all three cases (whisky and club soda; rum with cola; gin and tonic water), MEC was quickly formed, which was attributed to the low pH of the drinks. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction A monoalkyl carbonate (MAC) can be thought of as a salt derived from an alkyl carbonic acid (ACA), which is the half-ester of an alcohol and carbonic acid. Independently of the nomenclature and a formal origin, studies about synthesis and properties of such species are mainly conducted in harsh conditions, involving at least low temperature, high pressure, or anhydrous medium. For example, methylcarbonic acid melts at 36 °C followed by decomposition into methanol and carbon dioxide (Gattow & Behrendt, 1972). Their salts are stable at room temperature, but decompose in water (Gattow & Behrendt, 1972; Miller & Case, 1935; Pocker, Davison, & Deits, 1978). Despite this reactivity, the equilibrium constants obtained during studies about hydrolysis suggest that MACs can be formed in aqueous medium from carbonate/bicarbonate and an alcohol (Sauers, Jencks, & Groh, 1975). In fact, monoethyl carbonate (MEC) could be observed by 13C NMR in aqueous/ethanolic medium in a study about the mechanism of bicarbonate activation of hydrogen peroxide (Richardson, Yao, Frank, & Bennett, 2000). In a recent study, we demonstrated the formation of these species in mainly aqueous media by using capillary electrophoresis (CE) with a capacitively coupled contactless conductivity detector (C4D) (Vidal, Nogueira, Saito, & do Lago, 2011). Ionic mobilities, diffusion
⇑ Corresponding author. Tel.: +55 11 3091 3828; fax: +55 11 3091 3781. E-mail addresses: [email protected] (M.R. Rossi), [email protected] (D.T.R. Vidal), [email protected] (C.L. do Lago). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.039
coefficients, and hydrodynamic radii of 11 alcohols were determined using this technique. Scheme 1 shows the set of reactions involved in the formation and decomposition of a MAC in aqueous medium. The reactions are sketched as equilibrium, because all steps are reversible, although the kinetics are not fast for all of them. One can note that the lower the pH, the higher the CO2 production, and thus the low2 er the concentrations of the aqueous (HCO 3 and CO3 ) and alcoholic (MAC) anionic forms. On the other hand, a high pH favours CO2 3 . Thus, there is a pH range within which a MAC can be maintained in appreciable amounts. In the previous study, MACs were detected by capillary electrophoresis from pH 4.5 up to 10.5. Among the MACs, some are of special interest in biochemistry and food chemistry, such as MEC. The equilibrium constant, defined by Eq. (1), is (2.11 ± 0.09) at 20 °C (Vidal et al., 2011), suggests that MEC is the primary form of carbonate in concentrated ethanolic media. Of course, for a low level of alcohol and/or carbonate in water, MEC is not a major constituent, but it should be considered. For example, based on the equilibrium constant, one can anticipate that beer should contain monoethyl carbonate at a level as high as 2.3 mM, considering a concentration of 6% (v/v) of ethanol and 2.5 g/l of total carbonate.
K eq ¼
½CH3 CH2 OCO2 ½H2 O ½CH3 CH2 OH½HCO3
ð1Þ
The present work aims to prove the existence of MEC in some alcoholic beverages and demonstrate that it can be formed or decomposed depending on the way the beverage is prepared before drinking.
M.R. Rossi et al. / Food Chemistry 133 (2012) 352–357
R
OH
O
H
O
+
R
O-
O O
R
H O+
ROH
H+
OO
ROH
CO2 H2O H
H O+
H2O
OH+
O
O-
HO HO
H+
OH O
O
H+ -O
OO
pH
353
respectively, from the injection point. The samples were injected by applying a constant 25 kPa pressure at the sample vial, and the injection volume was controlled by the time of injection. Unless otherwise stated, the injection time was 1 s. The capillary environment was kept at 20 °C. For the experiments with real samples (beer, wine, and drinks), the electropherograms obtained at the second C4D were used for identification and quantitation purposes. For the study about the kinetics at low pH, only the first C4D was used. The electropherograms were filtered with a 0.75-s windowed Gaussian kernel smoothing algorithm after a 3-point windowed median filter (do Lago, Juliano, & Kascheres, 1995). The double filtering was selected to reduce both white noise and some sporadic spikes induced by the high voltage power supply over the detector. The capillary conditioning consisted of three steps: 5-min flushing with NaOH 0.1 mol/l, 5-min flushing with deionized water, and 10-min flushing with the BGE to be used. For the experiments with real samples, a 2-min flushing with BGE was carried out between runs. The between-runs flushing procedure was reduced to 30 s for the low pH experiment. All flushing procedures were carried out at a negative pressure of 73 kPa at the outlet. 2.3. Quantitation of MEC
Scheme 1. CO2 species in equilibrium in a water/alcohol medium.
2. Materials and methods 2.1. Reagents and solutions All the reagents were analytical grade. The solutions were prepared by dissolving or diluting the reagents in 18-MX cm deionized water (Barnstead/Thermolyne, Dubuque, IA, USA). Glyoxylic, oxalic, 2-(N-morpholino)ethanesulfonic (MES) and 2-(N-cyclohexylamino)ethanesulfonic (CHES) acids (Sigma–Aldrich, St. Louis, MO, USA) were obtained as free acids. Sodium salts of acetic (Merck, Darmstadt, Germany), propionic, and butyric acids (Sigma–Aldrich, St. Louis, MO, USA) were used for calibration purposes. The background electrolyte (BGE) was prepared by dissolving a certain mass of the CHES free acid (to 20 mmol/l concentration) in 80% of the final volume with deionized water, adjusting pH to 9.5 with concentrated LiOH (Sigma–Aldrich, St. Louis, MO, USA) solution, and then completing the volume. Concentrated solution of MEC was obtained by shaking 1 g of NaHCO3 (Merck, Darmstadt, Germany) in 10 ml of ethanol (Sigma– Aldrich, St. Louis, MO, USA). After 2 h, the NaHCO3 excess was filtered off and then the solution was kept in a closed reservoir. The beverages were acquired at local market and kept at room temperature. The bottles and cans of the carbonated beverages were opened just before use. Strong cationic resin Amberlit IR 120 (Merck, Darmstadt, Germany) was conditioned by washing with 1 mol/l HCl (Merck, Darmstadt, Germany) solution under stirring, filtered, and washed with deionized water until pH 7 was reached. For capillary conditioning purposes, NaOH (Merck, Darmstadt, Germany) 0.1 mol/l was used.
The response factor needed for quantitation of MEC was obtained by interpolation in a curve of response factor as a function of the mobility prepared with data for MES, butyrate, propionate, glyoxylate, and acetate, which are monocharged species of mobility similar to MEC. The response factors were obtained from five electropherograms of a standard solution 1.00 mmol/l in each species. Butyrate was used as the internal standard in this calibration as well as for all the real samples except beer. Due to co-migration of butyrate and some other constituent of beer, MES was used as the internal standard in this case. 2.4. Cationic resin treatment Removal of bicarbonate and MEC from a sample was carried out by treatment with cationic resin in H+ form. For 2.0 ml of sample, 0.5 g of resin was added. To improve CO2 removal, the sample was submitted to gentle N2 bubbling for 1 h. 2.5. pH effect study The MEC formation at low pH was evaluated by mixing two reagent solutions just before injection: (1) oxalic acid 14 mmol/l in ethanol/water 1:3 (v/v); and (2) NaHCO3 20 mmol/l and sodium butyrate 2.0 mmol/l in water. The sample was prepared by mixing 500 ll of solution 1 and 500 ll of solution 2 in a 1.5-ml vial. After injection, the vial was kept closed at room temperature until the next injection. However, due to the low complexity of the matrix, baseline resolution was obtained at the first detector, which allowed a greater number of runs during the first 40 min. 3. Results and discussion
2.2. Capillary electrophoresis system 3.1. Quantitation of MEC The development of the capillary electrophoresis equipment is described elsewhere (Saito et al., 2010). Basically, it is composed of a bipolar high-voltage power supply, a thermostatic case, and two C4D detectors positioned along the same capillary (Brito-Neto, da Silva, Blanes, & do Lago, 2005a, 2005b; da Silva & do Lago, 1998; Francisco & do Lago, 2009). Unless otherwise stated, the experiments were carried out with an 80-cm long, 75-lm i.d. silica capillary with the first and second C4D positioned at 10 and 70 cm,
As previously demonstrated (Vidal et al., 2011), the direct calibration of the C4D for quantitation of MACs is not possible, because they decompose along the path since the injection moment until they reach one of the detectors. Thus, the following indirect approach has been used for calibration. Taking into account that the analyte (MEC) is a monocharged species as well as its co-ion in the BGE, the sensitivity of a conduc-
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3.0
2.0
response factor
response factor
2.5
4
EOF mark
2 0 -2 -4
0
10
20
30
40
-5
2
50 -1 -1
mobility / 10 cm V s
1.5 1.0
κ
0.5
6
7
8
time
2
0.0 20
22
24
26
28 -5
2
30
-1 -1
Fig. 1. Response factor as a function of the mobility. Background electrolyte is Li/ CHES 20 mmol/l and the internal standard is butyrate. From the lowest to the highest mobilities, the points correspond to MES, butyrate, propionate, glyoxylate, and acetate 1.0 mmol/l. The lower inset is one of the six electropherograms used in this calibration. The higher inset is a simulated curve based on Eq. (4), spanning a broad range of mobilities, which shows that the linearity is a valid approximation only for a narrow region.
tivity detector (the way the conductivity varies with analyte concentration, dj/dca) can be appropriately described by Brito-Neto et al. (2005b) and Katzmayr, Klampfl, and Buchberger (1999):
ð2Þ
where la, lS, and lO (cm2 V1 s1) are the mobilities of the analyte, its co-ion and its counter-ion in the BGE, respectively, and F is Faraday’s constant (96,485 C mol1). The peak area is given by:
Aa ¼ g a
dj ca dca
ð3Þ
where ga (min) depends on factors that determine the peak shape and its width such as electrodispersion, electric field, and ion mobility. The response factor (ka,is) is the ratio between the sensitivity for the analyte (a) and the internal standard (is). One can note that:
ka;is ¼
g a lis ðla lS Þðla þ lO Þ g is la ðlis lS Þðlis þ lO Þ
4
5
6
7
8
9
10
time / min
mobility / 10 cm V s
dj ðla lS Þðla þ lO Þ ¼ F dca la
3
32
ð4Þ
Eq. (4) suggests that the response factor can be obtained not necessarily for the analyte, but for a species with the same mobility. Thus, one can use a stable species – a carboxylate, for instance – with the same mobility as MEC. In the present study, we used an extension of this idea, where five anionic species with different mobilities in a range comprising of MEC mobility. MES, butyrate, propionate, glyoxylate, and acetate were used to prepare a calibration curve for the response factor (taking butyrate as the internal standard) as a function of the mobility at the same temperature and ionic strength. Fig. 1 shows the calibration curve and an inset with one of the electropherograms. It is worth noting that, as one can deduce from Eq. (4), the response factor is not a linear function of the mobility. This approximation is valid only for a small range of mobilities. Fig. 1 shows that this approximation holds for the three species used for calibration. 3.2. The presence of MEC in beer samples Fig. 2 shows the electropherogram for a sample of lager beer recorded at the second C4D, using the Li/CHES BGE. The first two peaks are the potassium and sodium peaks. Some other cationic species are then observed before the intense system peak
Fig. 2. Electropherogram of a lager beer sample. Peaks at 2.5 and 3.0 min correspond to K+ and Na+, respectively. After the EOF mark, anionic species can be observed. The biggest one, at 8.0 min, corresponds to HCO 3 and other minor species.
(tm = 4.6 min) that marks the electro-osmotic flow (EOF). The anionic species are then recorded from the lowest mobility up to HCO 3, which is the largest peak under this condition. The shape of this peak suggests that other anions are present, but unresolved. The use of a cationic surfactant for reversing EOF is a common approach for anion analysis by capillary electrophoresis. However, in the present case, we opted to make the separation in the counter flow, because of the augmented window of separation. The mobility of EOF is influenced by compounds from the sample that adsorb on the inner wall of the capillary. This behaviour is especially intense for beer samples, and the EOF tends to be reduced after each sample injection. Thus, some cleaning procedure must be adopted to remove the residues from the inner wall. Similarly to EOF, cationic species have a positive mobility and, thus, the peak position is not affected in a great extent. Anions, on the other hand, have their peak position significantly affected by the changes of EOF mobility, and the greater the modulus of the mobility, the greater the instability in the peak position. In the present case, the migration time for HCO 3 is the most inconstant between the several experiments. This behaviour also impairs the identification of the MEC. However, spiking with a concentrated MEC solution is effective for such a task. For the sake of clarity, some electropherograms in the figures were adjusted by correcting the EOF mobility (Ikuta, Yamada, Yoshiyama, & Hirokawa, 2000).
-
HCO3
MEC
(a)
-
HCO3
MEC
κ
(b)
(c) 8
9
10
time / min Fig. 3. Electropherograms of stout beer samples: (a) pure beer; (b) beer plus concentrated solution of MEC (1:1 v/v); and (c) beer sample after treatment with cationic resin in H+ form. MEC is present in pure beer, which is identified by spiking with a solution containing MEC. The treatment with the cationic resin effectively reduces the amount of MEC by reducing pH and releasing CO2. The changes in migration time for MEC and bicarbonate are attributed to the significant electrodispersion.
M.R. Rossi et al. / Food Chemistry 133 (2012) 352–357
Fig. 3 shows the region of the electropherograms for pure and MEC-spiked samples of a stout beer. Due to the complexity of the matrix, one can argue that another anionic species may account for the peak previously attributed to MEC. This concern is particularly valid for the beer sample, because of the presence of several carboxylic acids, which account for other peaks in that region. Thus, another experiment was carried out to selectively remove MEC. If the sample is acidified, bicarbonate and MEC are converted into CO2 (see Scheme 1), while other carboxylic acids are preserved. However, the simple addition of a strong acid is not convenient, because the increasing ionic strength of the sample results in poorly resolved electropherograms. Thus, a protonated cationic resin was used instead a free acid (Nogueira & do Lago, 2009). As the cations (mainly K+ and Na+, in the present case) are exchanged by hydronium, the pH is reduced, which allows the conversion of MEC, as well as HCO 3 , into CO2. Fig. 3 shows the electropherogram after the treatment of a beer sample with cationic resin. The original position of the bicarbonate peak is occupied by two peaks. It is possible that one of them is the one for bicarbonate, and a residual amount of that ion remains in solution. However, the region for MEC is clear, which endorses the presence of this species in beers. The concentration of MEC in a sample of lager beer (at the second detector position) was (0.67 ± 0.11) mmol/l. The actual concentration in beer is greater, because MEC hydrolyzes during the 8.4 min spent along the column during the migration process. The hydrolysis constant was previously determined as (2.0 ± 0.5) 105 l mol1 s1 (Vidal et al., 2011), which allows calculate the concentration at the sample as (1.2 ± 0.2) mmol/l. The manufacturer of the beer states that it contains 5% of ethanol. Taking into account a concentration of 2.5 g/l for total carbonate in beer (Kuban & Karlberg, 1998), the expected concentration of MEC would be approximately 1.3 mmol/l. Thus, there is a good agreement between the predicted and actual concentration of MEC in beer. Of course, this concentration is slowly reduced over time after the tab of the can is pulled off. 3.3. The presence of MEC in sparkling wine Wine and especially sparkling wine are also candidates to sustain MEC in their composition, because they also have the two ingredients to form MEC: carbon dioxide and ethanol. There is however a shortcoming in this case. There is another species with a mobility similar to MEC under the conditions described to the analysis of the beer samples. Fig. 4 shows the MEC region in the electropherogram. One can note that the significant electrodispersion results in a broad peak that encompasses the whole region.
MEC
(a) (b)
κ (c)
6.4
6.6
6.8
7.0
7.2
7.4
7.6
time / min Fig. 4. Electropherograms of sparkling wine samples: (a) wine; (b) wine plus concentrated solution of MEC (1:1 v/v); and (c) wine sample after treatment with cationic resin in H+ form. The small elevation in (a) at 7.3 min is attributed to MEC, which is increased by spiking (b) and reduced by treatment with the ion exchange resin (c).
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The spiking with MEC and the treatment with cationic resin suggest that MEC may account for the small elevation in the tail of the broad peak (tm = 7.3 min). Peaks in Fig. 4b are smaller, because of dilution. Of course, one can argue that the electrophoretic condition used in this case is far from optimum. However, the point is that MEC is an evanescent species, which restricts the freedom for seeking better electrophoretic conditions. Thus, up until now, this is the best evidence about the existence of MEC in a sparkling wine. 3.4. The presence of MEC in alcoholic drinks Fermentation is a process that yields CO2 and ethanol. Thus, products like beers and wines naturally have the ingredients needed to form MEC. Distilled beverages, on the other hand, are made by a process that augments the level of ethanol, but reduces the CO2 content. However, the preparation of cocktails, like fizz and Collins, reunites the former ingredients. Today, there are more than 200 drink recipes based on a combination of an alcoholic beverage and some sort of carbonated water (Hellmich, 2006). The confirmation of the presence of MEC in all these possible products is out of scope of the present work, but the following experiments aim to give support to the understanding about two points: (1) is the resulting pH of the mixture low enough to prevent the formation of MEC? (2) Taking into account that most of the drinks are prepared just before its consumption, is there time enough to form a significant amount of MEC. The mixture of whisky and club soda was investigated. Whisky is a suitable prototype for ethanol source, because of the high content of alcohol and the low level of anionic interferants. The manufacturer of the club soda states that it contains NaCl, NaHCO3, and CO2. Fig. 5I shows the electropherogram of the two components and the resulting mixture 1:1 (v/v), in which the peak for MEC is evident. Chloride is not observed, because of its high mobility. Another popular combination is rum with cola, which was also prepared at a mixture 1:1 (v/v). Despite the complexity of the matrix of the cola drink when compared to club soda, the MEC peak is evident in the electropherogram of the resulting mixture, as shown in Fig. 5II. For the present conditions, the initial concentration of MEC was (4.1 ± 0.5) mmol/l. This concentration is reduced over time, because CO2 is continuously being lost. Although, to a lesser extent, ethanol is also lost, which also contributes to the diminishing of MEC concentration. The mixture of gin and tonic water 1:1 (v/v) was also investigated. In this case, the source of ethanol also contains some botanical extract, while tonic water, has quinine and a considerable amount of sugars. In spite of the increasing complexity of the matrix, Fig. 5III shows the formation of MEC in a similar amount to the previous cases. In Fig. 5III, one can note that the concentration of bicarbonate in the resulting mixture is smaller than in the original soft drink, which is expected because of dilution. However, this dilution effect is not evident in both other cases. In fact, the concentration of bicarbonate in the rum and cola mixture (Fig. 5II) seems to be greater than in pure cola. However, there are two points to be considered: (1) the release of CO2 continually changes the amount of bicarbonate (and MEC) and (2) dilution augments the pH and the amount of dissolved CO2. Thus, although a precise quantitation is possible (as previously described), the level of MEC in a drink is highly dependent on the nature of the components as well as on the history of its preparation. The apparent pH values for these three recipes were, respectively, 5.6, 2.7, and 3.2. These results suggest that, despite the low pH of the mixture, MEC is formed in these alcoholic drinks. This finding answers the first question stated at the beginning of this section.
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EOF mark
2.5
I
-
HCO3
a
MEC butyrate
MEC
κ
(c) (b)
AMEC / Abutyrate
2.0 1.2
1.4 time / min
1.5
b butyrate
1.0 MEC 1.2
0.5
1.4 time / min
(a) 4
5
6
7
8
9
0.0
10
0
time / min
10
20
30
40
time / min
II -
HCO3
EOF mark
MEC butyrate (c)
κ
(b) (a) 5
6
7
8
9
10
11
time / min -
III
HCO3 EOF mark
MEC (c)
Fig. 6. Relative area of MEC peak as a function of time at pH 4.0. After mixing, the sample is 10 mmol/l in NaHCO3, 7.0 mmol/l in oxalic acid, 1.00 mmol/l in sodium butyrate, and 12.5% (v/v) in ethanol. MEC is readily formed at the beginning (inset a – 0 min), and its amount continuously decreases, because of CO2 release (inset b – 40 min).
behaviour, which makes this hypothesis unlikely, because of the different sources and composition of the ingredients. In fact, an important feature shared among the three drinks is the low pH value, which contrasts to all the previous results that were obtained in alkaline medium. Thus, a new experiment was carried out, using a mixture of ethanol, water, and NaHCO3, and addition of oxalic acid to reduce pH to 4.0. Similarly to what happened when the drinks were prepared, MEC was promptly formed. In fact, due to the release of CO2, the concentration of MEC becomes smaller over the time. Fig. 6 shows the behaviour of the relative area for MEC peak. According to Scheme 1, there are formally two paths to interconvert bicarbonate and MAC: nucleophilic substitution by the solvent onto the carbonate group (the right side branch) and conversion into CO2 (the left side branch). The result suggests that the second one is the faster path.
4. Conclusions
(b)
κ
(a)
5
6
7
8
9
10
time / min Fig. 5. Electropherograms of the components and the resulting drinks: (I) whisky and club soda; (II) rum and cola; (III) gin and tonic water. There are three electropherograms for each experiment: (a) spirits (the source of ethanol), (b) soft drink (the source of CO2), and (c) the resulting 1:1 (v/v) mixture. Butyrate was added in II as internal standard for quantitation purpose.
Taking into account the previous results about the kinetics of MAC formation (Vidal et al., 2011), there is room for doubts about the actual amount of MEC formed until the moment the drink is consumed. However, the electropherograms showed in Fig. 5 were obtained right after mixing the spirit and the carbonate source. This result suggests that catalysis takes place when the drink is prepared, which was not previously observed in the studies about formation of MACs. Being a complex matrix with some natural products, a drink such as gin-and-tonic could contain some remaining enzyme as the catalyst. However, all the three drinks presented the same
Alcoholic fermentation is a process that naturally yields the reagents for the formation of MEC; furthermore, a beverage made by this process has enough time to reach the equilibrium. The experiments carried out in the present study show that neither the pH is low enough to prevent the formation of MEC, nor the release of the pull tab of the can significantly reduces the amount of MEC before the beverage is consumed. MEC was clearly detected in lager and stout beers, and there is evidence that MEC is also present in sparkling wine. In spite of the fact that soda-based alcoholic drinks are prepared just before consumption, MEC was readily detected in different compositions. This is an interesting finding, because the low pH value of the resulting mixture is not enough to significantly eliminate MEC by stimulating CO2 release; furthermore it favours the conversion of bicarbonate into MEC in a time scale compatible with the consumption of the drink. To the best of our knowledge, this is the first time that MEC is detected in any kind of food or beverage. Obviously, since beer has been consumed for millenia, one can anticipate that MEC is not an emerging harmful substance. However, there is a complete lack of information about its role in tasting as well as in ethanol absorption after ingestion. For instance, Ridout, Gould, Nunes, and Hindmarch (2003) studied the effects of carbon dioxide in champagne on psychometric performance and blood–alcohol con-
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centration and concluded that CO2 may accelerate absorption of alcohol. Roberts and Robinson (2007) compared the alcohol absorption from Vodka mixed with still water and carbonated water, and they also concluded that there is a significant difference in absorption rate. In both cases, although detailed explanations were pursued, MEC was not considered, because researchers were not acquainted with the presence of such a dynamically formed species and its possible role in cell membrane permeation, for example. In this sense, the present findings can be useful in new studies about alcohol absorption and its metabolism. Acknowledgements This work was supported by CNPq. We thank Dr. Z.G. Richter for the English revision. References Brito-Neto, J., da Silva, J., Blanes, L., & do Lago, C. (2005a). Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 2. Peak shape, stray capacitance, noise, and actual electronics. Electroanalysis, 17(13), 1207–1214. Brito-Neto, J. G. A., da Silva, J. A. F., Blanes, L., & do Lago, C. L. (2005b). Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 1. Fundamentals. Electroanalysis, 17(13), 1198–1206. da Silva, J., & do Lago, C. (1998). An oscillometric detector for capillary electrophoresis. Analytical Chemistry, 70(20), 4339–4343. do Lago, C. L., Juliano, V. F., & Kascheres, C. (1995). Applying moving median digitalfilter to mass-spectrometry and potentiometric titration. Analytica Chimica Acta, 310(2), 281–288. Francisco, K., & do Lago, C. (2009). A compact and high-resolution version of a capacitively coupled contactless conductivity detector. Electrophoresis, 30(19), 3458–3464. Gattow, G., & Behrendt, W. (1972). Methyl hydrogen carbonate. Angewandte Chemie, International Edition, 11(6), 534–535.
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Hellmich, M. (2006). Ultimate bar book, the comprehensive guide. San Francisco: Chronicle Books LLC. Ikuta, N., Yamada, Y., Yoshiyama, T., & Hirokawa, T. (2000). New method for standardization of electropherograms obtained in capillary zone electrophoresis. Journal of Chromatography A, 894(1–2), 11–17. Katzmayr, M. U., Klampfl, C. W., & Buchberger, W. (1999). Optimization of conductivity detection of low-molecular-mass anions in capillary zone electrophoresis. Journal of Chromatography A, 850(1–2), 355–362. Kuban, P., & Karlberg, B. (1998). On-line coupling of gas diffusion to capillary electrophoresis. Talanta, 45(3), 477–484. Miller, N. F., & Case, L. O. (1935). The kinetics of the alkaline hydrolysis of ethyl carbonate and of potassium ethyl carbonate. Journal of the American Chemical Society, 57(1), 810–814. Nogueira, T., & do Lago, C. (2009). Detection of adulterations in processed coffee with cereals and coffee husks using capillary zone electrophoresis. Journal of Separation Science, 32(20), 3507–3511. Pocker, Y., Davison, B. L., & Deits, T. L. (1978). Decarboxylation of monosubstituted derivatives of carbonic-acid – Comparative studies of water-catalyzed and acidcatalyzed decarboxylation of sodium alkyl carbonates in H2O and D2O. Journal of the American Chemical Society, 100(11), 3564–3567. Richardson, D. E., Yao, H. R., Frank, K. M., & Bennett, D. A. (2000). Equilibria, kinetics, and mechanism in the bicarbonate activation of hydrogen peroxide: Oxidation of sulfides by peroxymonocarbonate. Journal of the American Chemical Society, 122(8), 1729–1739. Ridout, F., Gould, S., Nunes, C., & Hindmarch, I. (2003). The effects of carbon dioxide in champagne on psychometric performance and blood-alcohol concentration. Alcohol and Alcoholism, 38(4), 381–385. Roberts, C., & Robinson, S. P. (2007). Alcohol concentration and carbonation of drinks: The effect on blood alcohol levels. Journal of Forensic and Legal Medicine, 14(7), 398–405. Saito, R., Brito-Neto, J., Lopes, F., Blanes, L., da Costa, E., Vidal, D., et al. (2010). Ionic mobility of the solvated proton and acid–base titration in a four-compartment capillary electrophoresis system. Analytical Methods, 2(2), 164–170. Sauers, C. K., Jencks, W. P., & Groh, S. (1975). Alcohol–bicarbonate–water system – Structure–reactivity studies on equilibria for formation of alkyl monocarbonates and on rates of their decomposition in aqueous alkali. Journal of the American Chemical Society, 97(19), 5546–5553. Vidal, D., Nogueira, T., Saito, R., & do Lago, C. (2011). Investigating the formation and the properties of monoalkyl carbonates in aqueous medium using capillary electrophoresis with capacitively coupled contactless conductivity detection. Electrophoresis, 32(8), 850–856.
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Monoalkyl carbonates in carbonated alcoholic beverages Marcelo Rabello Rossi, Denis Tadeu Rajh Vidal, Claudimir Lucio do Lago ⇑ Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, CEP 05508-000, São Paulo, SP, Brazil
a r t i c l e
i n f o
Article history: Received 26 September 2011 Received in revised form 22 November 2011 Accepted 16 January 2012 Available online 24 January 2012 Keywords: Monoethyl carbonate Alcoholic beverage Beer Capillary electrophoresis Conductivity detection
a b s t r a c t The presence of monoethyl carbonate (MEC) in beer and sparkling wine is demonstrated for the first time, as well as the formation of this species in drinks prepared with a distilled beverage and a carbonated soft drink. A capillary electrophoresis (CE) equipment with two capacitively coupled contactless conductivity detector (C4D) was used to identify and quantify this species. The concentrations of MEC in samples of lager beer and rum and cola drink were, respectively, 1.2 and 4.1 mmol/l, which agree with the levels of ethanol and CO2 available in these products. Previous results about the kinetics of the reaction suggest that only a small amount of MEC should be formed after the ingredients of a drink are mixed. However, in all three cases (whisky and club soda; rum with cola; gin and tonic water), MEC was quickly formed, which was attributed to the low pH of the drinks. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction A monoalkyl carbonate (MAC) can be thought of as a salt derived from an alkyl carbonic acid (ACA), which is the half-ester of an alcohol and carbonic acid. Independently of the nomenclature and a formal origin, studies about synthesis and properties of such species are mainly conducted in harsh conditions, involving at least low temperature, high pressure, or anhydrous medium. For example, methylcarbonic acid melts at 36 °C followed by decomposition into methanol and carbon dioxide (Gattow & Behrendt, 1972). Their salts are stable at room temperature, but decompose in water (Gattow & Behrendt, 1972; Miller & Case, 1935; Pocker, Davison, & Deits, 1978). Despite this reactivity, the equilibrium constants obtained during studies about hydrolysis suggest that MACs can be formed in aqueous medium from carbonate/bicarbonate and an alcohol (Sauers, Jencks, & Groh, 1975). In fact, monoethyl carbonate (MEC) could be observed by 13C NMR in aqueous/ethanolic medium in a study about the mechanism of bicarbonate activation of hydrogen peroxide (Richardson, Yao, Frank, & Bennett, 2000). In a recent study, we demonstrated the formation of these species in mainly aqueous media by using capillary electrophoresis (CE) with a capacitively coupled contactless conductivity detector (C4D) (Vidal, Nogueira, Saito, & do Lago, 2011). Ionic mobilities, diffusion
⇑ Corresponding author. Tel.: +55 11 3091 3828; fax: +55 11 3091 3781. E-mail addresses: [email protected] (M.R. Rossi), [email protected] (D.T.R. Vidal), [email protected] (C.L. do Lago). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.039
coefficients, and hydrodynamic radii of 11 alcohols were determined using this technique. Scheme 1 shows the set of reactions involved in the formation and decomposition of a MAC in aqueous medium. The reactions are sketched as equilibrium, because all steps are reversible, although the kinetics are not fast for all of them. One can note that the lower the pH, the higher the CO2 production, and thus the low2 er the concentrations of the aqueous (HCO 3 and CO3 ) and alcoholic (MAC) anionic forms. On the other hand, a high pH favours CO2 3 . Thus, there is a pH range within which a MAC can be maintained in appreciable amounts. In the previous study, MACs were detected by capillary electrophoresis from pH 4.5 up to 10.5. Among the MACs, some are of special interest in biochemistry and food chemistry, such as MEC. The equilibrium constant, defined by Eq. (1), is (2.11 ± 0.09) at 20 °C (Vidal et al., 2011), suggests that MEC is the primary form of carbonate in concentrated ethanolic media. Of course, for a low level of alcohol and/or carbonate in water, MEC is not a major constituent, but it should be considered. For example, based on the equilibrium constant, one can anticipate that beer should contain monoethyl carbonate at a level as high as 2.3 mM, considering a concentration of 6% (v/v) of ethanol and 2.5 g/l of total carbonate.
K eq ¼
½CH3 CH2 OCO2 ½H2 O ½CH3 CH2 OH½HCO3
ð1Þ
The present work aims to prove the existence of MEC in some alcoholic beverages and demonstrate that it can be formed or decomposed depending on the way the beverage is prepared before drinking.
M.R. Rossi et al. / Food Chemistry 133 (2012) 352–357
R
OH
O
H
O
+
R
O-
O O
R
H O+
ROH
H+
OO
ROH
CO2 H2O H
H O+
H2O
OH+
O
O-
HO HO
H+
OH O
O
H+ -O
OO
pH
353
respectively, from the injection point. The samples were injected by applying a constant 25 kPa pressure at the sample vial, and the injection volume was controlled by the time of injection. Unless otherwise stated, the injection time was 1 s. The capillary environment was kept at 20 °C. For the experiments with real samples (beer, wine, and drinks), the electropherograms obtained at the second C4D were used for identification and quantitation purposes. For the study about the kinetics at low pH, only the first C4D was used. The electropherograms were filtered with a 0.75-s windowed Gaussian kernel smoothing algorithm after a 3-point windowed median filter (do Lago, Juliano, & Kascheres, 1995). The double filtering was selected to reduce both white noise and some sporadic spikes induced by the high voltage power supply over the detector. The capillary conditioning consisted of three steps: 5-min flushing with NaOH 0.1 mol/l, 5-min flushing with deionized water, and 10-min flushing with the BGE to be used. For the experiments with real samples, a 2-min flushing with BGE was carried out between runs. The between-runs flushing procedure was reduced to 30 s for the low pH experiment. All flushing procedures were carried out at a negative pressure of 73 kPa at the outlet. 2.3. Quantitation of MEC
Scheme 1. CO2 species in equilibrium in a water/alcohol medium.
2. Materials and methods 2.1. Reagents and solutions All the reagents were analytical grade. The solutions were prepared by dissolving or diluting the reagents in 18-MX cm deionized water (Barnstead/Thermolyne, Dubuque, IA, USA). Glyoxylic, oxalic, 2-(N-morpholino)ethanesulfonic (MES) and 2-(N-cyclohexylamino)ethanesulfonic (CHES) acids (Sigma–Aldrich, St. Louis, MO, USA) were obtained as free acids. Sodium salts of acetic (Merck, Darmstadt, Germany), propionic, and butyric acids (Sigma–Aldrich, St. Louis, MO, USA) were used for calibration purposes. The background electrolyte (BGE) was prepared by dissolving a certain mass of the CHES free acid (to 20 mmol/l concentration) in 80% of the final volume with deionized water, adjusting pH to 9.5 with concentrated LiOH (Sigma–Aldrich, St. Louis, MO, USA) solution, and then completing the volume. Concentrated solution of MEC was obtained by shaking 1 g of NaHCO3 (Merck, Darmstadt, Germany) in 10 ml of ethanol (Sigma– Aldrich, St. Louis, MO, USA). After 2 h, the NaHCO3 excess was filtered off and then the solution was kept in a closed reservoir. The beverages were acquired at local market and kept at room temperature. The bottles and cans of the carbonated beverages were opened just before use. Strong cationic resin Amberlit IR 120 (Merck, Darmstadt, Germany) was conditioned by washing with 1 mol/l HCl (Merck, Darmstadt, Germany) solution under stirring, filtered, and washed with deionized water until pH 7 was reached. For capillary conditioning purposes, NaOH (Merck, Darmstadt, Germany) 0.1 mol/l was used.
The response factor needed for quantitation of MEC was obtained by interpolation in a curve of response factor as a function of the mobility prepared with data for MES, butyrate, propionate, glyoxylate, and acetate, which are monocharged species of mobility similar to MEC. The response factors were obtained from five electropherograms of a standard solution 1.00 mmol/l in each species. Butyrate was used as the internal standard in this calibration as well as for all the real samples except beer. Due to co-migration of butyrate and some other constituent of beer, MES was used as the internal standard in this case. 2.4. Cationic resin treatment Removal of bicarbonate and MEC from a sample was carried out by treatment with cationic resin in H+ form. For 2.0 ml of sample, 0.5 g of resin was added. To improve CO2 removal, the sample was submitted to gentle N2 bubbling for 1 h. 2.5. pH effect study The MEC formation at low pH was evaluated by mixing two reagent solutions just before injection: (1) oxalic acid 14 mmol/l in ethanol/water 1:3 (v/v); and (2) NaHCO3 20 mmol/l and sodium butyrate 2.0 mmol/l in water. The sample was prepared by mixing 500 ll of solution 1 and 500 ll of solution 2 in a 1.5-ml vial. After injection, the vial was kept closed at room temperature until the next injection. However, due to the low complexity of the matrix, baseline resolution was obtained at the first detector, which allowed a greater number of runs during the first 40 min. 3. Results and discussion
2.2. Capillary electrophoresis system 3.1. Quantitation of MEC The development of the capillary electrophoresis equipment is described elsewhere (Saito et al., 2010). Basically, it is composed of a bipolar high-voltage power supply, a thermostatic case, and two C4D detectors positioned along the same capillary (Brito-Neto, da Silva, Blanes, & do Lago, 2005a, 2005b; da Silva & do Lago, 1998; Francisco & do Lago, 2009). Unless otherwise stated, the experiments were carried out with an 80-cm long, 75-lm i.d. silica capillary with the first and second C4D positioned at 10 and 70 cm,
As previously demonstrated (Vidal et al., 2011), the direct calibration of the C4D for quantitation of MACs is not possible, because they decompose along the path since the injection moment until they reach one of the detectors. Thus, the following indirect approach has been used for calibration. Taking into account that the analyte (MEC) is a monocharged species as well as its co-ion in the BGE, the sensitivity of a conduc-
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3.0
2.0
response factor
response factor
2.5
4
EOF mark
2 0 -2 -4
0
10
20
30
40
-5
2
50 -1 -1
mobility / 10 cm V s
1.5 1.0
κ
0.5
6
7
8
time
2
0.0 20
22
24
26
28 -5
2
30
-1 -1
Fig. 1. Response factor as a function of the mobility. Background electrolyte is Li/ CHES 20 mmol/l and the internal standard is butyrate. From the lowest to the highest mobilities, the points correspond to MES, butyrate, propionate, glyoxylate, and acetate 1.0 mmol/l. The lower inset is one of the six electropherograms used in this calibration. The higher inset is a simulated curve based on Eq. (4), spanning a broad range of mobilities, which shows that the linearity is a valid approximation only for a narrow region.
tivity detector (the way the conductivity varies with analyte concentration, dj/dca) can be appropriately described by Brito-Neto et al. (2005b) and Katzmayr, Klampfl, and Buchberger (1999):
ð2Þ
where la, lS, and lO (cm2 V1 s1) are the mobilities of the analyte, its co-ion and its counter-ion in the BGE, respectively, and F is Faraday’s constant (96,485 C mol1). The peak area is given by:
Aa ¼ g a
dj ca dca
ð3Þ
where ga (min) depends on factors that determine the peak shape and its width such as electrodispersion, electric field, and ion mobility. The response factor (ka,is) is the ratio between the sensitivity for the analyte (a) and the internal standard (is). One can note that:
ka;is ¼
g a lis ðla lS Þðla þ lO Þ g is la ðlis lS Þðlis þ lO Þ
4
5
6
7
8
9
10
time / min
mobility / 10 cm V s
dj ðla lS Þðla þ lO Þ ¼ F dca la
3
32
ð4Þ
Eq. (4) suggests that the response factor can be obtained not necessarily for the analyte, but for a species with the same mobility. Thus, one can use a stable species – a carboxylate, for instance – with the same mobility as MEC. In the present study, we used an extension of this idea, where five anionic species with different mobilities in a range comprising of MEC mobility. MES, butyrate, propionate, glyoxylate, and acetate were used to prepare a calibration curve for the response factor (taking butyrate as the internal standard) as a function of the mobility at the same temperature and ionic strength. Fig. 1 shows the calibration curve and an inset with one of the electropherograms. It is worth noting that, as one can deduce from Eq. (4), the response factor is not a linear function of the mobility. This approximation is valid only for a small range of mobilities. Fig. 1 shows that this approximation holds for the three species used for calibration. 3.2. The presence of MEC in beer samples Fig. 2 shows the electropherogram for a sample of lager beer recorded at the second C4D, using the Li/CHES BGE. The first two peaks are the potassium and sodium peaks. Some other cationic species are then observed before the intense system peak
Fig. 2. Electropherogram of a lager beer sample. Peaks at 2.5 and 3.0 min correspond to K+ and Na+, respectively. After the EOF mark, anionic species can be observed. The biggest one, at 8.0 min, corresponds to HCO 3 and other minor species.
(tm = 4.6 min) that marks the electro-osmotic flow (EOF). The anionic species are then recorded from the lowest mobility up to HCO 3, which is the largest peak under this condition. The shape of this peak suggests that other anions are present, but unresolved. The use of a cationic surfactant for reversing EOF is a common approach for anion analysis by capillary electrophoresis. However, in the present case, we opted to make the separation in the counter flow, because of the augmented window of separation. The mobility of EOF is influenced by compounds from the sample that adsorb on the inner wall of the capillary. This behaviour is especially intense for beer samples, and the EOF tends to be reduced after each sample injection. Thus, some cleaning procedure must be adopted to remove the residues from the inner wall. Similarly to EOF, cationic species have a positive mobility and, thus, the peak position is not affected in a great extent. Anions, on the other hand, have their peak position significantly affected by the changes of EOF mobility, and the greater the modulus of the mobility, the greater the instability in the peak position. In the present case, the migration time for HCO 3 is the most inconstant between the several experiments. This behaviour also impairs the identification of the MEC. However, spiking with a concentrated MEC solution is effective for such a task. For the sake of clarity, some electropherograms in the figures were adjusted by correcting the EOF mobility (Ikuta, Yamada, Yoshiyama, & Hirokawa, 2000).
-
HCO3
MEC
(a)
-
HCO3
MEC
κ
(b)
(c) 8
9
10
time / min Fig. 3. Electropherograms of stout beer samples: (a) pure beer; (b) beer plus concentrated solution of MEC (1:1 v/v); and (c) beer sample after treatment with cationic resin in H+ form. MEC is present in pure beer, which is identified by spiking with a solution containing MEC. The treatment with the cationic resin effectively reduces the amount of MEC by reducing pH and releasing CO2. The changes in migration time for MEC and bicarbonate are attributed to the significant electrodispersion.
M.R. Rossi et al. / Food Chemistry 133 (2012) 352–357
Fig. 3 shows the region of the electropherograms for pure and MEC-spiked samples of a stout beer. Due to the complexity of the matrix, one can argue that another anionic species may account for the peak previously attributed to MEC. This concern is particularly valid for the beer sample, because of the presence of several carboxylic acids, which account for other peaks in that region. Thus, another experiment was carried out to selectively remove MEC. If the sample is acidified, bicarbonate and MEC are converted into CO2 (see Scheme 1), while other carboxylic acids are preserved. However, the simple addition of a strong acid is not convenient, because the increasing ionic strength of the sample results in poorly resolved electropherograms. Thus, a protonated cationic resin was used instead a free acid (Nogueira & do Lago, 2009). As the cations (mainly K+ and Na+, in the present case) are exchanged by hydronium, the pH is reduced, which allows the conversion of MEC, as well as HCO 3 , into CO2. Fig. 3 shows the electropherogram after the treatment of a beer sample with cationic resin. The original position of the bicarbonate peak is occupied by two peaks. It is possible that one of them is the one for bicarbonate, and a residual amount of that ion remains in solution. However, the region for MEC is clear, which endorses the presence of this species in beers. The concentration of MEC in a sample of lager beer (at the second detector position) was (0.67 ± 0.11) mmol/l. The actual concentration in beer is greater, because MEC hydrolyzes during the 8.4 min spent along the column during the migration process. The hydrolysis constant was previously determined as (2.0 ± 0.5) 105 l mol1 s1 (Vidal et al., 2011), which allows calculate the concentration at the sample as (1.2 ± 0.2) mmol/l. The manufacturer of the beer states that it contains 5% of ethanol. Taking into account a concentration of 2.5 g/l for total carbonate in beer (Kuban & Karlberg, 1998), the expected concentration of MEC would be approximately 1.3 mmol/l. Thus, there is a good agreement between the predicted and actual concentration of MEC in beer. Of course, this concentration is slowly reduced over time after the tab of the can is pulled off. 3.3. The presence of MEC in sparkling wine Wine and especially sparkling wine are also candidates to sustain MEC in their composition, because they also have the two ingredients to form MEC: carbon dioxide and ethanol. There is however a shortcoming in this case. There is another species with a mobility similar to MEC under the conditions described to the analysis of the beer samples. Fig. 4 shows the MEC region in the electropherogram. One can note that the significant electrodispersion results in a broad peak that encompasses the whole region.
MEC
(a) (b)
κ (c)
6.4
6.6
6.8
7.0
7.2
7.4
7.6
time / min Fig. 4. Electropherograms of sparkling wine samples: (a) wine; (b) wine plus concentrated solution of MEC (1:1 v/v); and (c) wine sample after treatment with cationic resin in H+ form. The small elevation in (a) at 7.3 min is attributed to MEC, which is increased by spiking (b) and reduced by treatment with the ion exchange resin (c).
355
The spiking with MEC and the treatment with cationic resin suggest that MEC may account for the small elevation in the tail of the broad peak (tm = 7.3 min). Peaks in Fig. 4b are smaller, because of dilution. Of course, one can argue that the electrophoretic condition used in this case is far from optimum. However, the point is that MEC is an evanescent species, which restricts the freedom for seeking better electrophoretic conditions. Thus, up until now, this is the best evidence about the existence of MEC in a sparkling wine. 3.4. The presence of MEC in alcoholic drinks Fermentation is a process that yields CO2 and ethanol. Thus, products like beers and wines naturally have the ingredients needed to form MEC. Distilled beverages, on the other hand, are made by a process that augments the level of ethanol, but reduces the CO2 content. However, the preparation of cocktails, like fizz and Collins, reunites the former ingredients. Today, there are more than 200 drink recipes based on a combination of an alcoholic beverage and some sort of carbonated water (Hellmich, 2006). The confirmation of the presence of MEC in all these possible products is out of scope of the present work, but the following experiments aim to give support to the understanding about two points: (1) is the resulting pH of the mixture low enough to prevent the formation of MEC? (2) Taking into account that most of the drinks are prepared just before its consumption, is there time enough to form a significant amount of MEC. The mixture of whisky and club soda was investigated. Whisky is a suitable prototype for ethanol source, because of the high content of alcohol and the low level of anionic interferants. The manufacturer of the club soda states that it contains NaCl, NaHCO3, and CO2. Fig. 5I shows the electropherogram of the two components and the resulting mixture 1:1 (v/v), in which the peak for MEC is evident. Chloride is not observed, because of its high mobility. Another popular combination is rum with cola, which was also prepared at a mixture 1:1 (v/v). Despite the complexity of the matrix of the cola drink when compared to club soda, the MEC peak is evident in the electropherogram of the resulting mixture, as shown in Fig. 5II. For the present conditions, the initial concentration of MEC was (4.1 ± 0.5) mmol/l. This concentration is reduced over time, because CO2 is continuously being lost. Although, to a lesser extent, ethanol is also lost, which also contributes to the diminishing of MEC concentration. The mixture of gin and tonic water 1:1 (v/v) was also investigated. In this case, the source of ethanol also contains some botanical extract, while tonic water, has quinine and a considerable amount of sugars. In spite of the increasing complexity of the matrix, Fig. 5III shows the formation of MEC in a similar amount to the previous cases. In Fig. 5III, one can note that the concentration of bicarbonate in the resulting mixture is smaller than in the original soft drink, which is expected because of dilution. However, this dilution effect is not evident in both other cases. In fact, the concentration of bicarbonate in the rum and cola mixture (Fig. 5II) seems to be greater than in pure cola. However, there are two points to be considered: (1) the release of CO2 continually changes the amount of bicarbonate (and MEC) and (2) dilution augments the pH and the amount of dissolved CO2. Thus, although a precise quantitation is possible (as previously described), the level of MEC in a drink is highly dependent on the nature of the components as well as on the history of its preparation. The apparent pH values for these three recipes were, respectively, 5.6, 2.7, and 3.2. These results suggest that, despite the low pH of the mixture, MEC is formed in these alcoholic drinks. This finding answers the first question stated at the beginning of this section.
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M.R. Rossi et al. / Food Chemistry 133 (2012) 352–357
EOF mark
2.5
I
-
HCO3
a
MEC butyrate
MEC
κ
(c) (b)
AMEC / Abutyrate
2.0 1.2
1.4 time / min
1.5
b butyrate
1.0 MEC 1.2
0.5
1.4 time / min
(a) 4
5
6
7
8
9
0.0
10
0
time / min
10
20
30
40
time / min
II -
HCO3
EOF mark
MEC butyrate (c)
κ
(b) (a) 5
6
7
8
9
10
11
time / min -
III
HCO3 EOF mark
MEC (c)
Fig. 6. Relative area of MEC peak as a function of time at pH 4.0. After mixing, the sample is 10 mmol/l in NaHCO3, 7.0 mmol/l in oxalic acid, 1.00 mmol/l in sodium butyrate, and 12.5% (v/v) in ethanol. MEC is readily formed at the beginning (inset a – 0 min), and its amount continuously decreases, because of CO2 release (inset b – 40 min).
behaviour, which makes this hypothesis unlikely, because of the different sources and composition of the ingredients. In fact, an important feature shared among the three drinks is the low pH value, which contrasts to all the previous results that were obtained in alkaline medium. Thus, a new experiment was carried out, using a mixture of ethanol, water, and NaHCO3, and addition of oxalic acid to reduce pH to 4.0. Similarly to what happened when the drinks were prepared, MEC was promptly formed. In fact, due to the release of CO2, the concentration of MEC becomes smaller over the time. Fig. 6 shows the behaviour of the relative area for MEC peak. According to Scheme 1, there are formally two paths to interconvert bicarbonate and MAC: nucleophilic substitution by the solvent onto the carbonate group (the right side branch) and conversion into CO2 (the left side branch). The result suggests that the second one is the faster path.
4. Conclusions
(b)
κ
(a)
5
6
7
8
9
10
time / min Fig. 5. Electropherograms of the components and the resulting drinks: (I) whisky and club soda; (II) rum and cola; (III) gin and tonic water. There are three electropherograms for each experiment: (a) spirits (the source of ethanol), (b) soft drink (the source of CO2), and (c) the resulting 1:1 (v/v) mixture. Butyrate was added in II as internal standard for quantitation purpose.
Taking into account the previous results about the kinetics of MAC formation (Vidal et al., 2011), there is room for doubts about the actual amount of MEC formed until the moment the drink is consumed. However, the electropherograms showed in Fig. 5 were obtained right after mixing the spirit and the carbonate source. This result suggests that catalysis takes place when the drink is prepared, which was not previously observed in the studies about formation of MACs. Being a complex matrix with some natural products, a drink such as gin-and-tonic could contain some remaining enzyme as the catalyst. However, all the three drinks presented the same
Alcoholic fermentation is a process that naturally yields the reagents for the formation of MEC; furthermore, a beverage made by this process has enough time to reach the equilibrium. The experiments carried out in the present study show that neither the pH is low enough to prevent the formation of MEC, nor the release of the pull tab of the can significantly reduces the amount of MEC before the beverage is consumed. MEC was clearly detected in lager and stout beers, and there is evidence that MEC is also present in sparkling wine. In spite of the fact that soda-based alcoholic drinks are prepared just before consumption, MEC was readily detected in different compositions. This is an interesting finding, because the low pH value of the resulting mixture is not enough to significantly eliminate MEC by stimulating CO2 release; furthermore it favours the conversion of bicarbonate into MEC in a time scale compatible with the consumption of the drink. To the best of our knowledge, this is the first time that MEC is detected in any kind of food or beverage. Obviously, since beer has been consumed for millenia, one can anticipate that MEC is not an emerging harmful substance. However, there is a complete lack of information about its role in tasting as well as in ethanol absorption after ingestion. For instance, Ridout, Gould, Nunes, and Hindmarch (2003) studied the effects of carbon dioxide in champagne on psychometric performance and blood–alcohol con-
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centration and concluded that CO2 may accelerate absorption of alcohol. Roberts and Robinson (2007) compared the alcohol absorption from Vodka mixed with still water and carbonated water, and they also concluded that there is a significant difference in absorption rate. In both cases, although detailed explanations were pursued, MEC was not considered, because researchers were not acquainted with the presence of such a dynamically formed species and its possible role in cell membrane permeation, for example. In this sense, the present findings can be useful in new studies about alcohol absorption and its metabolism. Acknowledgements This work was supported by CNPq. We thank Dr. Z.G. Richter for the English revision. References Brito-Neto, J., da Silva, J., Blanes, L., & do Lago, C. (2005a). Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 2. Peak shape, stray capacitance, noise, and actual electronics. Electroanalysis, 17(13), 1207–1214. Brito-Neto, J. G. A., da Silva, J. A. F., Blanes, L., & do Lago, C. L. (2005b). Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 1. Fundamentals. Electroanalysis, 17(13), 1198–1206. da Silva, J., & do Lago, C. (1998). An oscillometric detector for capillary electrophoresis. Analytical Chemistry, 70(20), 4339–4343. do Lago, C. L., Juliano, V. F., & Kascheres, C. (1995). Applying moving median digitalfilter to mass-spectrometry and potentiometric titration. Analytica Chimica Acta, 310(2), 281–288. Francisco, K., & do Lago, C. (2009). A compact and high-resolution version of a capacitively coupled contactless conductivity detector. Electrophoresis, 30(19), 3458–3464. Gattow, G., & Behrendt, W. (1972). Methyl hydrogen carbonate. Angewandte Chemie, International Edition, 11(6), 534–535.
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