James D Oliver
email: james.oliver@uws.edu.au

Marianne Gaborieau
email: m.gaborieau@uws.edu.au

Patrice Castignolles
email: p.castignolles@uws.edu.au

solution capillary electrophoresis by photo-oxidation assisted UV detection

Keywords: ethanol detection, pressure mobilization, capillary electrophoresis, photo-oxidation detection, nuclear magnetic resonance spectroscopy

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The determination of ethanol is essential for the analysis of ethanol fermentations and related alcoholic beverages. Bioethanol fermentation of lignocellulosic material is an important process that will hopefully significantly reduce the global demand on fossil fuels. Available methods for the detection and quantification of ethanol in complex matrices are few. Typical methods for the determination of ethanol include Gas Chromatography with Flame Ionization Detection (GC-FID) [1],

High Performance Liquid Chromatography (HPLC) with Refractive Index detection (RI) on a cation exchange resin [2] or High Performance Anion Exchange Chromatography (HPAEC) with Pulsed Amperometric Detection (PAD) [3]. The detection of underivatised ethanol is challenging due to its lack of UV absorption or fluorescence emission. In ethanol fermentation, an analytical method is more advantageous if it can determine both carbohydrates and ethanol. GC methods require derivatization of carbohydrates to make them volatile like ethanol [4], while HPLC [5] and HPAEC have high running costs and, in the case of HPLC, may suffer from poor robustness and recovery [5].

Additionally, no single separation technique can determine simultaneously ethanol and a complex mixture of carbohydrates in a complex matrix such as that of a lignocellulosic fermentation. Some modes of Capillary Electrophoresis (CE) have previously been used in the detection of ethanol.

Ethanol and other solvents have been previously quantified by Micellar ElectroKinetic Capillary chromatography (MEKC) with indirect detection [7] however it requires the use of sodium dodecyl sulfate (SDS) surfactant that may interact with proteins and lipids present in complex samples, such as lignocellulosic fermentations . CE with PAD [8] or indirect UV detection [9] was able to detect ethanol however no quantification was carried out. CE with direct UV detection at high pH is a simple and robust method developed for carbohydrate analysis [10]. The separation has been applied to wide variety of complex matrices including forensic, food, beverage and pharmaceutical samples [6], fruit juices and cognac [10], and complex acid treated plant fiber samples [5,11].

Ethanol was however never determined by this method limiting its application for monitoring ethanol fermentations. Direct UV detection of carbohydrates in CE at high pH was originally suggested to be due to enediolate formation [6,10] but later shown to be due to be a photooxidation reaction [5,6]. This photo-oxidation reaction takes place directly in the detection window [12,13]. Hydroxyl and/or superoxide related radicals may be produced following minimal but sufficient decomposition of water at the low wavelength UV irradiation in the detection window [14], then react with the carbohydrates. Alternatively the carbohydrates may be directly photodecomposed by the irradiation [START_REF] Phillips | Advances in Carbohydrate Chemistry[END_REF]. In both cases the radicals obtained from the carbohydrates decompose through a pathway containing UV-absorbing (250-270 nm) intermediates [13,[START_REF] Laurent | [END_REF] to carboxylate decomposition products [13]. Multiple passes through the detection window in one experiment (reversing the electric field after each pass) revealed that the electrophoretic mobility of the carbohydrates is constant but the peak intensity increases for 6 passes before decreasing [5].

The UV absorption at 250-270 nm is observed with a CE Diode Array Detector (DAD) but not with a classical grate spectrophotometer [12]. Ethanol might also encounter some photo-oxidation under these conditions [17], but it does not lead to any UV absorption. Ethanol undergoes hydrogen abstraction in the presence of some free radicals e.g. in the presence of peroxides [18] or in radical polymerization [19,20]. We hypothesized that ethanol would interfere with the photo-oxidation reaction and hence the detection of the carbohydrate and this interference would lead to a change in direct UV detection in carbohydrates proportionally to the amount of ethanol present. The aim of this study was to investigate a detection method for ethanol with free solution capillary electrophoresis equipment compatible with both pressure mobilization and free solution capillary electrophoresis through its interference with the photo-oxidation reaction, to investigate how ethanol could interfere with the detection of carbohydrates and to apply the detection method to fermentation samples and alcoholic beverages. The long term goal is to develop a separation method that can determine both ethanol and carbohydrates in a complex sample such the fermented lignocellulosic plant fiber.

Materials and Methods

Materials

Water was of MilliQ quality (Millipore, USA). Sodium hydroxide pellets (NaOH) ≥98 %, absolute ethanol ≥99.5 % and magnesium chloride hexahydrate ≥99 % were obtained from Sigma-Aldrich (Australia). Xylitol ≥99 %, sucrose ≥99 % and ammonium sulfate 99 % were obtained from Alfa Aesar (USA). Fused-silica capillaries (50 µm i.d., 360 µm o.d.) were obtained from Polymicro (USA).

Triethylamine ≥99.5 % and tert-butanol ≥99 % was obtained from BDH (UK). Deuterium oxide (D,99.9 %) and fully 13 C-labelled glucose ≥99 % were sourced from Cambridge Isotope Laboratories (USA).

Yeast extract were obtained from Oxoid (Australia). Monopotassium phosphate ≥99 % was obtained from Univar (Australia). Vodka (declared alcohol content 37 %) was produced commercially in Australia and purchased locally. The fermentation sample, after dilution, was comprised of 500 mg•L - 1 of each glucose, fructose and yeast extract and of 0.50 mg•L -1 of each MgCl2, (NH4)2SO4 and KH2PO4.

Free Solution Capillary Electrophoresis (CE) and Pressure Mobilization

The instruments were a MDQ P/ACE (Beckman) and a 7100 CE (Agilent) with DADs monitoring at 200 nm and 266 nm with a 10 nm bandwidth. Samples were injected by applying 14 mbar of pressure for 8 s (≈10 nL) followed by mobile phase or background electrolyte (BGE) injected in the same manner.

At the end of a series of injection, the capillary was flushed 1 min with NaOH 1 M, 10 min with water and 10 min with air. Integration was performed using Karat 32 (Beckman) or Chemstation (Agilent) software.

Pressure mobilization

The capillary length was 90 cm with an effective length of 10 cm on the MDQ P/ACE (Beckman) and of 8.5 cm on the 7100 CE (Agilent) instruments. The mobile phase was comprised of 130 mM NaOH unless otherwise specified. Sucrose and xylitol do not reduce in the presence of 130 mM NaOH in water and therefore their solutions were prepared in such medium. The capillary was pre-treated prior to use and between each run by flushing with the mobile phase for 5 min. Pressure mobilization was at 50 mbar unless otherwise specified. For NMR spectroscopy, 1 g•L -1 of 13 C labeled glucose and 2 g•L -1 of ethanol in 130 mM NaOH in D2O was pressure injected continuously at 50 mbar into a 35 cm capillary (26.5 cm effective length) on the 7100 CE instrument with the lamp on; 130 µL was collected, and made up to 580 µL with 130 mM NaOH in D2O.

Free Solution Capillary Electrophoresis (CE)

The capillary length was 90 cm with an 81.5 cm effective length on the 7100 CE (Agilent). The BGE consisted of 130 mM NaOH with 2 g•L -1 of sucrose in the capillary and 130 mM NaOH only, in the inlet and outlet vials. The capillary was pre-treated prior to use by flushing with 1 M NaOH for 20 min followed by water for 5 min then the BGE for 10 min. The BGE containing sucrose was then flushed between injections for 10 min. The electric field was applied for 12 min at 24 kV followed by pressure mobilization at 50 mbar.

NMR

1 H NMR and 13 C NMR spectra were recorded at room temperature on a Bruker DRX300 spectrometer (Bruker, Alexandria, NSW, Australia) operating at 300 MHz for 1 H, equipped with a 5 mm 1 H-13 C dual probe. 1 H NMR spectra were recorded with a 5.3 µs 30 °pulse, a 5 s repetition delay and 800 to 20,480 scans. 13 C NMR spectra were recorded with a 7 µs 90 °pulse, a 3 s repetition delay and 20,358 to 184,320 scans. 1 H and 13 C chemical shift scales were externally calibrated with the resonance of the methyl signal of ethanol in D2O at 1.17 and 17.47 ppm, respectively [21].

3 Results and Discussion:

Photo-oxidation assisted detection of ethanol

Pressure mobilization of sucrose dissolved in 130 mM NaOH led to a Gaussian peak, which intensity decreased when ethanol was added to the sucrose (Figure 1A). Sucrose peak area, height and shape are increasingly affected by ethanol when the ethanol concentration increases. Ethanol interferes with the photo-oxidation reaction of sucrose and suppresses the sucrose signal because of a decrease in concentration of UV absorbing intermediate(s). The signal is monotonically decreasing with the amount of ethanol added. Ethanol disruption occurs in a narrower band than that of the sucrose peak (Figure 1B): ethanol thus suppresses the signal corresponding to the center of the sucrose peak, but not the tail, creating a valley. Considering the Taylor Dispersion Analysis of pressure mobilization [22], this means that ethanol diffuses faster than sucrose in NaOH 130 mM, which is indeed expected from the difference of sizes of the molecules: ethanol diffuses faster than glucose [23], which in turn diffuses faster than sucrose [24]. If sucrose is placed in the mobile phase, then ethanol can be detected indirectly as a negative peak (Figure 1 B). The (negative) peak is then Gaussian confirming that the unusual peak shape in direct detection is due to the difference of diffusion coefficients of the ethanol and the carbohydrate. The peak shape is of importance for determining the ethanol concentration through either the loss in peak area or the loss in peak height (discussed later).

<<Insert Figure 1>> 3

.2 Understanding ethanol interference with carbohydrate photo-oxidation

Reactions of some free radicals with ethanol have been previously extensively studied [19,20,25] especially with ethanol as a transfer agent during radical polymerization. The free radical abstracts a hydrogen from the carbon bearing the alcohol group (α-carbon), and not from the hydroxyl group itself (consistent with the bond dissociation energies, BDE, Table S-1), producing a carbon centered radical [18,26] depicted on Figure 2.

<<Insert Figure 2>> To investigate this mechanism and ensure that it is not due an impurity in ethanol, interference from ethanol, methanol, iso-propanol, tert-butanol and triethylamine with sucrose photo-oxidation was compared. The interference is also observed for these four other compounds confirming that the detection is not due to an impurity present in ethanol. The transfer coefficient to ethanol, methanol and iso-propanol has been determined previously in the radical polymerization of alkyl acrylates [25]. The transfer coefficient to isopropanol is 3 times higher than the one to ethanol and 29 times higher than the one to methanol. This trend indicates a higher reactivity of alcohols toward the acrylate carbon-centered radicals when the alkyl group (on the alcohol) increases in size. The BDE [START_REF] Luo | Handbook of bond dissociation energies in organic compounds[END_REF] indicate the same trend (Table S-1), increasing from isopropanol, to ethanol and methanol, with tert-butanol having the highest BDE of all while the BDE of ethanol and methanol have a lot closer values. Triethylamine has a slightly lower BDE than isopropanol. The BDEs of the alcohols and amines and their relative ability to interfere with the photo-oxidation of carbohydrates were compared. At the lower alcohol/amine concentration, ethanol has the highest inference and tertbutanol and methanol have the lowest. Although methanol has a more favorable BDE, tert-butanol has more hydrogens to abstract. Iso-propanol has the highest peak difference, however not significantly different from that of ethanol (Figure 3). The trend in the inhibition of photo-oxidation is quantitatively but also qualitatively different from that in transfer in polymerization and that of the BDEs, therefore the hydrogen abstraction on the ethanol is likely not a critical step in the kinetics of the reaction.

<<Insert Figure 3>> To investigate if the change in signal was due to difference of Refractive Index (RI), the concentration of the analyte by the analytes RI [START_REF] Lide | [END_REF] was plotted against the relative difference in peak height. If the change in signal was due to a difference in RI, the relationship would have been linear, but this is not the case as seen on Figure S-1. A control injection of pure ethanol also shows that RI does not play a significant role in the observed interference (Figure S-2). Interference with the photo-oxidation of sucrose may be due to ethanol reacting with the hydroxide/superoxide radical initiator or the carbon or oxygen centered radical of the sucrose during photo-oxidation (Figure 5). To determine whether sucrose and/or ethanol were consumed or regenerated after passing the detection window, a multiple passing experiment was performed similar to what has been done previously [5] but using pressure mobilization and in the presence of ethanol. Sucrose was passed in front of the detection window, then the pressure was reversed and the sucrose passed again. This was done to a total of 28 passes for sucrose with and without ethanol. Sucrose on its own showed results similar to the previous passing experiment [5] with a buildup of UV absorbing species in the first five passes of the detection window followed by a decrease (Figure 4A). This change of the signal intensity with the number of passes would not happen if the detection was due to differences in refractive index and thus confirms that the detection of ethanol is due to photo-oxidation and not to a difference in refractive index.

In the presence of 250 mg•L -1 and 1 g•L -1 ethanol the highest peak is observed at the 7th pass and the peak height is lower than with sucrose. The difference in peak height decreases in parallel, with the number of passes and then becomes constant, within the (large) experimental error after 10 passes (Figure 4B). The decrease of the peak height difference may indicate a faster consumption of ethanol than sucrose over time. As expected, the peak height difference is smaller with 250 mg•L -1 than 1 g•L -1 ethanol, but the behavior, decrease of the peak height differences for the first 10 passes and plateau is similar. The multiple passes of sucrose in the detection window showed that interference of ethanol is at a maximum on its first pass.

<<Insert Figure 4>> <<Insert Figure 5>> The role of ethanol was further investigated by 1 H and 13 C NMR. As NMR experiments require a deuterated solvent, it was first checked that the same interference of ethanol was observed in D2O as in H2O. Injections were carried out with 130 mM NaOH in H2O as well as in D2O: the relative peak height difference between sucrose peaks with and without ethanol in D2O is only slightly larger in D2O (Table S-2). This indicates that either the reactivities of the radicals •OH and •OD toward carbohydrate are not significantly different or more likely that water is not a reactant in the ethanol interference with the photo-oxidation reaction, the carbohydrate UV irradiation being directly responsible for the hydrogen abstraction and free radical formation. The differing values between the D2O and H2O can be attributed to the change in peak tailing (Figure S-4) due to the higher viscosity of D2O. Assuming that the photo-oxidation reaction as well as the ethanol interference are similar in D2O and H2O, a sample of 1 g•L -1 glucose ( 13 C fully labelled) in D2O was continuously pressure injected at 50 mbar for 94.5 hours (after 89 h, pressure was increased to 100 mbar to offset backpressure from the outlet vial), as previously performed [13], but in the presence of 2 g•L -1 ethanol. The sample was consecutively analyzed by 1 H (Figure 6 and Figure S-5) and 13 C NMR (Figure 7 and Figure S-6). To determine whether ethanol was consumed or regenerated after passing the detection window, the area of the 1 H NMR signal areas of the methyl group of ethanol were compared for the sample, control as well as fresh control (Figure 6). The sum of total area of the normalized peaks was respectively 30 for the sample, 69 for the control and 191 for the fresh control. The loss of ethanol in the control shows that ethanol underwent thermal decomposition over the period of 8 days (at 4°C). Taking temperature and time into account (Table S-3) ca 17 % of the ethanol was consumed by the photo-oxidation reaction. This is consistent with the multiple pass experiment and a faster consumption of ethanol than carbohydrate. Ethanol can be directly photooxidized to some acetate (Figure S-7) and aldehydes [17] and in high alkaline conditions used in this work acetaldehyde (ethanal) would further react to form sodium acetate. The radical formed from ethanol abstraction reaction (Figure 2) might also interfere with the photo-oxidation of glucose.

Numerous possible interferences of the ethanol with glucose photo-oxidation are consistent with the NMR results; they are represented on Figure 5.

<<Insert Figure 6>> The photo-oxidation of glucose under these conditions has been studied previously [13,29]. The possible products of the interference that are not consistent with the NMR results are represented on Figure S-8. At point (a) of the reaction on Figure 5, glucose would form a carbon centered radical from hydrogen abstraction by a hydroxyl radical formed by the splitting of water or more likely by direct photo-irradiation [START_REF] Phillips | Advances in Carbohydrate Chemistry[END_REF]. The glucose carbon-centered radical is a tertiary radical that is thus unlikely to abstract a hydrogen from ethanol regenerating glucose and forming a secondary radical on ethanol. Abstraction of hydrogen from ethanol might also occur on the free radicals (b) following the ring opening step which was described previously [13]. A glucose would be regenerated in an altered open form containing an aldehyde functional group and thus more prone to thermal degradation. Similar to this, ethanol might interfere with the production of free radicals formed by the low-UV irradiation of water (Figure S-9) but this is unlikely as D2O would give a different interference. The carbon centered free radical of glyceraldehyde (c) (2,3-dihydroxypropanal) might also abstract the same hydrogen from ethanol displacing the equilibrium from the semidiones previously identified as possible UV absorbing intermediates [13]. Glyceraldehyde can be oxidized to form glyceric, glycolic, acetic and methanoic acid as well as carbon dioxide (d) [30]. Hydrogen abstraction from ethanol would most likely occur from oxygen centered radicals (e) rather than carbon centered radicals. The resulting product, (Z)-prop-1-ene-1,2,3-triol (f), was not observed in <<Insert Figure 7>> The ethanol was not 13 C labelled in this experiment but it is detected with a signal-to-noise ratio of 43 for the fresh control and 6.5 for the sample for the shift at 18 ppm. Any products of reactions with ethanol representing less than 20 % of the initial ethanol should thus not be detected (Table S- 4). In previous work [13] glucose did not significantly thermally degrade on a month scale in 130 mM NaOD in D2O. In the control experiment of this work some glucose was degraded thermally by the alkaline conditions in D2O in the presence of the ethanol after 14 days. Ethanol might form transient hemiacetal and acetal leading to higher proportion of open glucose making its aldehyde functional group more prone to thermal degradation. The products from such degradation have the same functional groups, carboxylates, as that from the photo-oxidation as shown by identical shifts at 170, 180 as well as similar massifs between 60 and 80 ppm (Figure 7). The photo-oxidation by the CE's deuterium lamp sped up the formation of carboxylates (≈ 170 and 180 ppm) and produced other end products (≈ 20 and 40 ppm) seen previously [13]. The formation of glycolate and methanoate, as discussed previously [13], as well as glycerate and malonate (Table S- S-5). Ethanol is still present in the NMR spectra after UV irradiation, thus ethanol is not completely photo-oxidized to its end products acetic acid and its aldehyde [17].

Previous studies have investigated the potential end products (D-Glucose penta-acetate, D-Gluconic acid, D-Glucuronic acid and D-Glucosaccharic acid) [31] and UV absorbing intermediates (Asorbic acid, (Z)-3-hydroxyacrylaldehyde, 2-keto-gluconic acid and 4-deoxy-5-keto-3,6-manno saccharolactone) [START_REF] Laurent | [END_REF] of the photo-oxidation of glucose solutions in the presence of oxygen, however none of these are observed in our 13 C NMR spectrum (Table S-5 and S-6).

Quantification of ethanol by pressure mobilization

Before application to samples analysis, the effect of carbohydrate concentration, residence time in the detection window and ethanol concentration were determined.

Effect of sucrose concentration and residence time on ethanol determination.

To determine the effect of residence time in the detection window, the difference in peak height from ethanol interference was measured with pressure mobilization at pressures of 10, 50 and 100 mbar. As shown previously [12], pressure mobilization at 10 mbar creates the highest signal due to the prolonged residence time in the detection window (Figure S-10) allowing more time for photooxidation to take place. However this also results in the longest analysis time (17 min for 10 cm). The differences in peak height between sucrose with and without ethanol (Table S-7) are not significantly different at 10 and 50 mbar, and using 50 mbar instead of 10 mbar reduces analysis time from 17 to 4.6 min.

The effect of sucrose concentration on the difference in peak height from ethanol interference was studied in the range of 0.25 to 8 g•L -1 sucrose in 130 mM NaOH spiked with 1 g•L -1 ethanol (Figure 8).

The largest difference in peak height is observed at 2 g•L -1 sucrose. At 4 g•L -1 sucrose the difference in peak height decreases and the difference in area even decreases to zero (see Figure S-11). The difference in peak height above 4 g•L -1 sucrose is thus likely only due to peak broadening but not to the photo-oxidation process. Overloading of the capillary is shown by peak tailing especially at 8 g•L -1 likely due to viscous fingering. To avoid tailing the difference in viscosity of the sample and the mobile phase needs to be kept minimal. Above 2 g•L -1 sucrose, the free radical concentration formed during the photo-oxidation may be too high and induce more termination reactions: the ethanol is then not playing a role any more. This is similar to the increase of the detection sensitivity adding a photo-initiator only up to a certain photo-initiator concentration in previous work [13]. To analyze samples which do not contain photo-oxidizing carbohydrates, carbohydrates need to be added but no more than 2 g•L -1 should be added as it provides optimal peak height as well as the largest difference in peak height making the detection more sensitive. The analysis of samples already containing photo-oxidizing carbohydrates is a challenge with the pressure mobilization method. The total sugar concentration could be controlled, possibly by dilution into a known concentration of carbohydrates where the concentration in the sample is negligible compared to that of the added sucrose. CE is a preferable alternative over pressure mobilization in this case as it can separate the carbohydrates, as well as other compounds, from ethanol (discussed later). Using separation by CE, the ethanol band is free of carbohydrates and ethanol can be detected if a known amount, such as 2 g•L -1 sucrose, of carbohydrate is added to the BGE at the time the ethanol reaches the detection window to obtain indirect detection of ethanol as on Figure 1-B. The effect of sucrose concentration on the signal-to-noise ratio (S/N) was also considered (Figure S-13) and S/N was found to be optimal between 2 and 8 g•L -1 sucrose.

<<Insert Figure 8>>

Performances of the quantification of ethanol method

Using pressure mobilization, ethanol can be quantified by either the peak area or peak height. The precisions of both methods were compared (Table 1). The RSD values are of the order of 2-10 % above 125 mg•mL -1 of ethanol at this stage since no internal standard is used. Quantification by peak height is more precise than that by peak area. This may be due to the irregular peak shape and some pressure variation during the injection. Slight variation in pressure results in variation in the width of the ethanol band: the peak tail is affected by these variations, thus the peak area, while the peak height is not influenced. As expected, the RSD values increase when the ethanol concentration decreases.

The limit of detection (LOD) and the limit of quantification (LOQ) were also compared in BGEs containing sucrose and xylitol (same molar concentration) with 130 mM NaOH (Table 2). There is in fact a range of concentration in which ethanol can be detected: too low of an ethanol concentration leads to no interference of the photo-oxidation, while too high of an ethanol concentration lead to complete inhibition of the photo-oxidation. The lower LOD is taken as the concentration for which S/N is equal to 3 [13,32]; where S/N was determined as the ratio of the signal taken as the difference of the peak heights between the sucrose only injection and the sucrose and ethanol injection, and of the noise taken as the sum of the noises of each signal. The lower LOQ is defined for the same reason as the ethanol concentration for which S/N is equal to 10, with S/N determined as above. The upper LOQ is related to the blank injection of pure ethanol and defined as the ethanol concentration for which the peak has a S/N equal to the sum of 10 and of the S/N of pure ethanol, with the signal taken as the one of the ethanol injections (the lowest point in the middle valley of the peak, Figure 1A, in the presence of sucrose). Sucrose and xylitol were chosen as they are stable in basic media.

<<Insert Table 1>> <<Insert Table 2>> A linear fit, determined by best empirical fit, can be achieved when the relative difference in peak height is plotted over the cube root of the ethanol concentration. A cubic fit is likely due to the reaction not being first order. The standard curve obtained with the MDQ maintained a similar cubic previously observed for carbohydrates [12]. The lamp usage time may play a role and the MDQ instrument had a more recent lamp (990 h) than the 7100 CE one (1950 h).

Pressure mobilization was applied to the determination of ethanol in a "real" well-characterized sample to benchmark the method: vodka was chosen since as it has a considerable ethanol concentration, no carbohydrates and minimal interfering compounds. The declared ethanol concentration was 37 %, the sample was diluted by 1:600 with a solution of 2 g•L -1 sucrose in 130 mM NaOH. Quantification of the ethanol content was found identical to the one determined by the vodka manufacturer (to 2 significant digits) indicating that trace amounts of other compounds [33] did not have an impact on the quantification of ethanol by photo-oxidation interference. To measure ethanol in samples containing both carbohydrates and ethanol, a CE method was developed as follows.

Quantification of ethanol in real samples by CE

As mentioned previously, samples containing both carbohydrates and ethanol require separation before ethanol determination. CE was used to separate ethanol in a standard containing glucose and fructose. A BGE of 130 mM NaOH was placed in the inlet and outlet vials and the capillary was flushed with a BGE containing 130 mM NaOH with 2 g•L -1 of sucrose. Sucrose, as a disaccharide, has an apparent mobility lower than the EOF marker but higher than the glucose and fructose, and thus should completely migrate past the detection window before glucose and fructose migrate past the detection window. Ethanol, due to its higher pKa than that of sucrose, should pass the detection window whilst sucrose is still in the background. As a result, ethanol can be detected by interference with the photo-oxidation of sucrose only, while glucose and fructose can be detected directly by photo-oxidation (Figure 9). When the electric field was applied for the entire duration of the separation, the detection of the ethanol peak was not repeatable due to a large amount of noise . When the electric field was stopped after 12 min and 50 mbar pressure applied, the ethanol peak was repeatable (Figure 9B) and separated sufficiently from the carbohydrates. The carbohydrates were incompletely separated from each other in comparison to separations seen previously in similar conditions [5,13]. In this case, a reinjection in conditions optimal to the separation of carbohydrates should be done separately. As expected there was a drop in the baseline at 26-27 min indicating that the sucrose has completely migrated out of the capillary. As in pressure mobilization, the extent of interference was proportional to the amount of ethanol added (Figure 9-B).

<<Insert Figure 9>> The maximum increase in temperature inside the capillary due to Joule heating was only 1.9 °C as calculated previously [13,34]. The LOD, and LOQ and recovery were determined (Table 2) and are similar to the one obtained by pressure mobilization only on simpler samples. The LOD of the CE method (34.4 mg•L -1 ) can compete with the sensitivity of an optical alcohol meter (LOD < 1580 mg•L -1 [35]) for the ability to determine carbohydrates with the same equipment. The S/N was similar with BGEs containing either 2 g•L -1 or 0.5 g•L -1 sucrose (Figure S -19). A diluted fermentation sample spiked with 500 mg•L -1 of ethanol was used to test the recovery of the method. The sample showed 110 %.

The precision may be improved with an internal standard as discussed in previous work [5]. The accuracy and robustness of the method are however demonstrated using this complex matrix without any filtration being required. Vials for online fermentation monitoring by CE were recently developed and tested on acids [36]. The CE separation presented in this work will allow the method to be extended to the quantification of both ethanol and carbohydrates in complex mixtures for a comprehensive online monitoring of ethanol fermentations.

Conclusions

Ethanol can be detected and quantified by interference with carbohydrate photo-oxidation.

Methanol, propanol and triethylamine are also shown to be detected by this method. The presence of ethanol during photo-oxidation did not lead to the observation by 1 H or 13 C NMR of any endproducts that were not seen previously in the absence of ethanol. The photo-oxidation might be due to direct UV irradiation of the carbohydrate leading to the formation of free radicals and then UV absorbing intermediate. Ethanol might react with oxygen centered radicals along this reaction pathway and partially suppress the UV absorption. This detection can be utilized with pressure mobilization for a simple and fast detection of ethanol with a capillary electrophoresis instrument or an even simpler set-up. When sucrose is used as the photo-oxidizing carbohydrate in pressure mobilization, the optimal concentration is 2 g•L -1 . This detection has a LOD and LOQ of 34.9 and 117 mg•L -1 respectively. The quantitative recovery (100%)

was measured with a sample of vodka. CE was used to determine ethanol in spiked fermentation samples containing glucose and fructose. Ethanol in CE can be detected as an indirect peak when sucrose is placed in the BGE in the capillary. A fermentation sample spiked with ethanol showed 110 % recovery, showing that the robustness of the CE with direct UV detection of carbohydrates also applies to ethanol quantification. The ability of CE with direct UV detection to monitor both complex mixtures of carbohydrates, as shown by previous research, and now to monitor ethanol, makes it highly promising method to monitor ethanol fermentations online. The method can be applied to a number of compounds, for example antioxidants, taking advantage of the robustness of the method. BGE in outlet and inlet was 130 mM NaOH, BGE in capillary was 130 mM NaOH + 2 g•L -1 of sucrose (black, S/N = 37) and 130 mM NaOH + 0.5 g•L -1 of sucrose (red, S/N = 36). Migration was by electric field (24 kV) for 12 min followed by pressure mobilization at 50 mbar. Current was 160 µA.

Figures:

Mechanism of the photo-oxidation and of the interference with ethanol

Performed on 7100 CE instrument.

13 C

 13 NMR (Figure S-8) (alkene would produce signals between130 and 160 ppm), but it may rather be simply an intermediate. Alternative end products such as malondialdehyde would still be formed then oxidized to carboxylates (g).

  5) would fit the shifts observed in the 13 C NMR. Glucose, in its cyclic form, is observed in the control sample through signals at 98 and 102 ppm. The loss of these signals indicates complete degradation of glucose either by photooxidation or alkaline degradation. The photo-oxidation of glucose in the presence of ethanol did not lead to the identification of any new end products thus ruling out combination reactions of ethanol based radicals with glucose based radicals leading to larger unique end products (Figure S-8), however 2 ethanol base radicals could form butan-2,3-diol (Figure S-8) that cannot be ruled out as an end product (Table

  fit when the standard was injected on 4 different days over a month were analyzed together. A combination of the standard curves obtained by the MDQ and 7100 has a correlation coefficient of 0.98 (Figure S-14) and thus indicate no significant difference between the standard curves obtained from the MDQ or from the 7100. The curve for xylitol and sucrose are similar (Figure S-15) and may suggest a universal calibration with all photo-oxidizing carbohydrates. Sucrose leads to more repeatable peak heights than xylitol as the photo-oxidizing even though a more regular peak shape is obtained with xylitol in the presence of ethanol (Figure S-16). The S/N was also compared between the 7100 CE and the MDQ instruments (Figure S-17). The latter instrument was found to be more sensitive for ethanol detection, by 40 % (S/N of 149 for the 7100 CE and of 209 for the MDQ), as was

Figure 1 :

 1 Figure 1: Pressure mobilization at 50 mbar: (A) of 2 g•L -1 sucrose in 130 mM NaOH not spiked (solid line) or spiked with ethanol at 250 mg•L -1 (dotted line), 1 g•L -1 (dashed line) and 2 g•L -1 (dotteddashed line), with NaOH 130 mM as the mobile phase. (B) of 2 g•L -1 sucrose in 130 mM NaOH (with 130 mM NaOH as the mobile phase, dotted line) and of 1 g•L -1 ethanol in 2 g•L -1 sucrose and 130 mM NaOH (with 130 mM NaOH with 2 g•L -1 sucrose as the mobile phase, solid line). Performed on MDQ

Figure 2 :

 2 Figure 2: Hydrogen abstraction from ethanol by a free radical R•. Adapted from [18].

Figure 3 :

 3 Figure 3: Interference of alcohols and triethylamine at 5 mM (white) and 44 mM (striped) with the photo-oxidation of 2 g•L -1 sucrose during pressure mobilization. Relative difference in peak height

Figure 4 :

 4 Figure 4: Possible reaction scheme for the interference of ethanol with glucose photo-oxidation.

Figure 5 :

 5 Figure 5: Peak heights in the pressure mobilization of 2 g•L -1 sucrose (black square), 2 g•L -1 sucrose and 250 mg•L -1 ethanol (circle) and 2 g•L -1 sucrose and 1 g•L -1 ethanol (cross) in 130 mM NaOH

Figure 6 : 1 H

 61 Figure 6: 1 H NMR of 2 g•L -1 ethanol in the presence of 1 g•L -1 fully labelled 13 C glucose continuously

Figure 7 :

 7 Figure 7: 13 C NMR of 1 g•L -1 fully labelled 13 C glucose in the presence of 2 g•L -1 ethanol continuously

Figure 8 :

 8 Figure 8: Sucrose peak height (solid line), sucrose spiked with 1 g•L -1 ethanol peak height (dotted

Figure 9 :

 9 Figure 9: Detection of ethanol and carbohydrates via CE (A) and detection of varying concentrations

Figure S- 4 :

 4 Figure S-4: Pressure mobilization of sucrose in 130 mM NaOH in H2O (solid line) and 130 mM NaOD

1 H

 1 Figure S-5: 1 H NMR of 1 g•L -1 fully labelled 13 C glucose in the presence of 2 g•L -1 ethanol continuously

Figure S- 6 :

 6 Figure S-6: 13 C NMR of 1 g•L -1 fully labelled 13 C glucose in the presence of 2 g•L -1 ethanol continuously

Figure S- 7 :

 7 Figure S-7: Oxidation of ethanol radical to acetic acid (a-d) adapted from [5] and to butan-2,3-diol

Figure S- 8 :

 8 Figure S-8: Possible but unobserved products of glucose photo-oxidation in the presence of ethanol.

Figure S- 9 :

 9 Figure S-9: Possible interference of water derived radicals by ethanol.

Figure S- 10 :

 10 Figure S-10: Effect of residence time in the window on peak height of sucrose 2 g•L -1 (squares on dotted line) and sucrose spiked with 1 g•L -1 ethanol (squares on solid line) in pressure mobilization at

Figure S- 11 :

 11 Figure S-11: Peak areas of sucrose (solid line) and sucrose spiked with 1 g•L -1 ethanol (dotted line), as

Figure S- 12 :

 12 Figure S-12: Sucrose peak at 500 mg•L -1 (black solid), 1000 mg•L -1 (black dotted), 2000 mg•L -1 (red solid) 4000 mg•L -1 (red dotted) and 8000 mg•L -1 (blue solid) without ethanol (A) with 1000 mg•L -1

Figure S- 13 :

 13 Figure S-13: Effect of sucrose concentration on the signal to noise ratio (S/N).

Fig S- 15 :

 15 Fig S-15: Calibration curve of ethanol concentration against difference in peak height for sucrose

Figure S- 16 :

 16 Figure S-16: pressure mobilization of 5.8 mM sucrose (black) and xylitol (red) in the presence of 1 g•L - 1 ethanol. Performed on MDQ instrument.

Figure S- 18 :

 18 Figure S-18: CE of ethanol when the electric field (24 kV) was applied for the entire separation.

Figure S- 19 :

 19 Figure S-19: Detection of 1 g•L -1 ethanol via CE by interference with the photo-oxidation of sucrose.

Table S -

 S 2: Comparison of the effects of ethanol inhibited photo-oxidation of sucrose in 130 mM

	NaOH in H2O and in D2O. Values are normalized by migration time. Examples of the corresponding
	elugrams are shown on Figure S-2.
	Concentration of ethanol in 2 g•L -1 sucrose in (mg•L -1 ) 130 mM NaOH	Sucrose (x 10 4 ) peak area	RSD (%)	Relative area (%) difference in peak	RSD (%)	Sucrose (mAU) peak height	RSD (%)	Relative height (%) difference in peak	RSD (%)
				130 mM NaOH in H2O
	2000	0.38	6.5	71	3.1	0.13	0.084	82	1.7
	1500	0.47	2.6	64	6.5	0.18	1.1	75	2.3
	1000	0.64	2.4	51	11	0.23	1.8	68	2.9
	500	0.85	4.7	35	18	0.34	4.0	53	3.6
	250	0.98	6.8	25	22	0.43	5.9	40	3.1
	0	1.3	14	-	-	0.72	8.0	-	-
				130 mM NaOH in D2O
	2000	0.35	5.0	68	2.0	0.05	4.9	88	≥ 0.1
	250	0.97	3.3	12	10	0.32	3.7	20	12
	0	1.1	13	-	-	0.40	4.9	-	-
	RSD = Relative standard deviation

Table S -

 S 3: Calculation of rate of ethanol thermal decomposition obtained from the integration of assuming that the decomposition reaction is 3 times slower at 4 °C than 20 °C.# calculated as the loss of peak area in the control multiplied by the ratio of time spent at 20 °C by the control and the sample.

	Figure 4
	Time	Time	Total		Total	Loss in	Rate of	Peak area	Peak
	spent	spent	equivalent	loss	peak area	thermal	loss by	area loss
	at	at	time at	in	due to	decom-	photo-	by
	4 °C	20 °C	20 °C *	peak	thermal	position at	oxidation	photo-
					area	decom-	20 °C		oxidation
						position	(loss/hour)	(%)
	Control 317	3	108		122	122	1.13	0	0
	Sample 0	118	118		161	161 #	1.13	28	17
	Signal to			Signal to
	Noise ratio	Number of	Noise ratio
	(S/N) of ethanol signal at 18 ppm in fresh control	scans of fresh control (undiluted)	(S/N) of ethanol signal at 18 ppm in	Dilution factor of sample	Number of scans of sample	𝐸 = (	3 𝐶 × √ 𝐴 𝐷 𝐵	) × 100
	(A)	(B)		sample		(C)	(D)
	43		75776		6.5	4.46	184320		20 %
	NB: this calculation assumes that all signals of interest are fully relaxed between scans in the 13 C
	NMR experiment.

*

Table S-4: Estimate of the minimal concentration (E) of end products that, resulting from decomposition of ethanol, could be detected by

13

C NMR.

Table S -

 S 5: Predicted13 C shifts of potential end products of carbohydrate photo-oxidation in the presence of oxygen. Prediction done with ChemDraw Ultra 12. Bold, underlined chemical shifts are not observed in the13 C NMR spectrum (Figure4)

	D-Glucose penta-acetate [3]	D-Gluconic acid [3]	D-Glucuronic acid [3]	D-Glucosaccharic acid [3]	Malonic acid (Malonate)	Glyceric acid (Glycerate)	Butan-2,3-diol
			200.8
	170	176	176	176	169	176
	94.9		88.3
	74.7-69.0	73.1-64.4	71.3-67.3	73.4-68.3	45.2	64.4-71.5	75.3
	21.0, 20.7						18.6

Table S -

 S 6: Predicted13 C shifts of potential UV absorbing intermediates from carbohydrate photooxidation as studied by[4]. Prediction done with ChemDraw Ultra 12. Bold, underlined chemical shifts are not observed in the13 C NMR spectrum (Figure4)

	Asorbic acid	(Z)-3-hydroxyacrylaldehyde (reductone)	2-keto-gluconic acid (diol form)	4-deoxy-5-keto-3,6-manno saccharolactone
		191.1		175
		180.2		173
	167		165	167
	162
	119		137
	116	105.3	72.6-63.4	117
	67.4			76.5, 75.9

Table S -

 S 7: Effect of pressure on the peak height (n=5) of sucrose with and without ethanol (Figure S-

	5).
	Pressure	2000 mg•L -1 sucrose, A	2000 mg•L -1 sucrose spiked with 1000 mg•L -1 ethanol, B	Difference (A-B)	(Difference in peak height) x (sucrose velocity)
	(mbar)	Peak height (mAU)	SD	Peak height (mAU)	SD	Peak (mAU) height	SDs Sum of	Value	SD
	100	1.62	0.0425	0.858	0.0835	0.764	0.126	1.3	0.21
	50	6.12	0.0327	3.80	0.156	2.32	0.189	1.1	0.09
	10	15.4	0.393	12.9	0.555	2.54	0.948	0.56	0.21

Acknowledgements

The authors wish to acknowledge Dr Yohann Guillaneuf (Aix-Marseille University) for discussions and David Fania and Dr Kamali Kannangara for 13 C glucose.

6 References

Tables

 Table 1:Comparison of peak height and peak area in the pressure mobilization of xylitol and sucrose (2 g•L -1 ) in 130 mM NaOH for the quantification of ethanol (n=5). Peak height was measured to the lowest valley point (Figure 1A). Performed on MDQ instrument.

Concentration

of ethanol (mg

Supplementary information for

Ethanol determination using pressure mobilization and free solution capillary electrophoresis by photo-oxidation assisted ultraviolet detection James D. Oliver, 1 Marianne Gaborieau, 2