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Figure 1: Sugars in a flavored rum sample.There is nothing better than sitting down on a Friday night with friends at the pub or bar, sipping some coconut-flavored rum. As chemists, we don’t just savor those drinks, we want to know why it tastes so good. So, what do chemists do for fun? You guessed it—analyze our favorite alcoholic beverages for sugar content.

Mono- and di-saccharide sugar determinations are often used in the food and beverage industry to ensure the quality of a formulated product, to maintain or select for desired sweetness, and to characterize and confirm the source of the carbohydrates. Sugars are added to a desired sweetness by the addition of sucrose refined from sugar beets and sugar cane or high fructose corn syrup (HFC) where ~50 percent of the glucose is converted to the sweeter fructose. In the U.S., corn syrup is more commonly used as a sweetener because of its availability and lower cost. Similarly, cane sugar is commonly used in tropical regions where it is plentiful, whereas beat sugar is more common in Europe. The growing public concerns that sugar consumption may be related to increased obesity and diabetes rates has led to more detailed product labeling and, therefore, an increased demand for carbohydrate analysis.Figure 2: Sugars in a scotch liqueur sample.

Carbohydrate determinations can be challenging because they are poor chromophores and are therefore problematic to detect by UV absorption without lengthy and costly derivitization. Refractive index (RI) detection is non-specific with poor sensitivity, and therefore susceptible to chromatographic interferences. In contrast, electrochemical detection used in high performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is specific by directly detecting analytes based on their oxidation potentials at high pH without the need for derivitization.

The latest advancement in integrated ion chromatography (IC) instrumentation, the Thermo Scientific Dionex Integrion HPIC system, can operate continuously up to 5,000 psi for both 4 mm and 2 mm i.d. column formats with automated eluent generation. These higher pressures allow the analyst to take full advantage of the high efficiency and fast separations offered by smaller particle size separation columns. This article discusses the analytical method, the advantages and the results.Figure 3: Sugars in a rice wine sample.

Instrument and method
The Dionex Integrion HPIC system is a compact, fully integrated IC system design with separate compartments for pump, column heater with injection valve and detector with suppressor to provide faster column oven equilibration. The instrument has new IC PEEK Viper fittings to minimize void volume problems to improve chromatography and ensure accurate reporting, radio frequency tracking of consumables for GMP compliance, and independent tablet control.

Glucose, fructose and sucrose in diluted alcoholic samples are ionized by the high pH eluent, separated at 0.5 mL/min using electrolytically generated 35 mM KOH on a Thermo Scientific Dionex CarboPac PA20 (3 × 250 mm) column. The sugars are detected by PAD using a disposable gold working electrode (Au on PTFE), Ag/AgCl reference electrode, and the High Carbohydrate Analysis kit. The analysis is facilitated by the Dionex Integrion HPIC system, which is designed with a metal-free flow path. The method conditions are shown in each chromatogram.

Various locally purchased wine, whiskey, Scotch and rum samples were analyzed. The samples were diluted with deionized water prior to analysis: 100-fold (flavored, liqueurs, wines) and 5-fold (distilled alcohols). The rice wine and red wine samples were also filtered (syringe filter, 0.2 µm, nylon) and treated with Dionex OnGuard RP cartridges.

Method discussion
Both the Dionex CarboPac SA10-4µm and CarboPac PA20 columns are suitable for fast mono- and di-saccharide separations. However, the Dionex CarboPac PA20 column was selected for this application because the column selectivity fully resolves ethanol from the sugar peaks of interest. The Dionex CarboPac PA20 contains 6.5 µm-diameter nonporous beads covered with functionalized latex bead resin. The elution order is glucose, then fructose and sucrose.

Inconsistencies in manual eluent preparation occur as a result of carbon dioxide absorption, variation between preparations or by slight variations from the SOP. Eluent generation eliminates these variations by electrolytically generating the eluent inline. The results are an accurate and precise delivery of eluent through programming without environmental contamination, saving time normally spent on manual eluent preparation. Additionally, less pump maintenance is needed because the pump is exposed only to water.

In a carbohydrate application using PAD, the waveform is 0.5 sec in which a series of four-potentials (versus Ag/AgCl) are applied for a defined time, resulting in two cycles of the waveform each second. The charge from the potential is allowed to dissipate for 0.20 sec before the carbohydrate response is measured from 0.2 to 0.4 sec. Then the electrode is automatically cleaned and prepared for detection by each waveform cycle, thereby providing a reliable and consistent response.

Sample analysis
Simple sugars were determined in the diluted alcoholic-based samples by HPAE-PAD and facilitated by the Dionex Integrion HPIC system. Three examples are shown in Figures 1 through 3. The results for the analyzed samples are summarized in Table 1. Figure 1 shows the separation of glucose, fructose and sucrose in 100-fold diluted spicy flavored rum sample using 35 mM KOH at 0.5 mL/min on the Dionex CarboPac PA20 and detection by PAD. A small void peak is present ~1 min. The rum sample is a blend of spices and sugar added to a distilled product from the fermentation of sugar cane. The diluted spicy rum sample has similar concentrations of glucose and fructose, ~300 mg/L with a larger sucrose peak eluting last, at ~1 g/L. The total sugar concentrations are >160 g/L.

The second chromatogram (Figure 2) is a diluted Scotch liqueur sample. A liqueur is a distilled alcohol that is blended with sugar, spices, fruit or nuts followed by a short aging period. Liqueurs generally are a sweet, slightly viscous and aromatic beverage. Figure 2 shows the chromatogram of the diluted liqueur sample, containing similar amounts of the three sugars: 0.20 to 0.30 g/L. The undiluted liqueur contains ~70 g/L of sugars.

In Figure 3, the chromatogram of diluted rice wine has only a glucose peak plus the void volume peak. Rice wine is generated from fermented rice; as a result the sweetness is due solely to glucose, ~22 g/L (undiluted).

Table 1 summarizes the total sugar concentrations in the analyzed samples corrected for dilution. The distilled Scotch and whiskey samples have low sugar concentrations, <0.5 g/L of total glucose and fructose. The American bourbon whiskey also contains sucrose. The two flavored rum, liqueur and two wine samples have high sugar concentrations from 22 to 188 g/L. Although sucrose was not detected in the coconut rum sample, this sample had the highest sugar content (188 g/L).

It is important to note that the flavored spicy rum, Scotch liqueur and the sparkling wine samples contain sucrose, which is the disaccharide of glucose and fructose. Therefore, if the sucrose were completely converted to glucose and fructose as expected during digestion, the flavored rum, liqueur and sparkling wine samples would contain about 80, 20 and 17 g/L, respectively, of additional glucose and fructose.
 

  Glucose* (g/L) Fructose* (g/L) Sucrose* (g/L) Total Sugar (g/L)
Table 1: Summarized calculated total sugar results.
Bourbon, American Whiskey 0.085 0.045 0.110

0.24**

Canadian Whiskey .200 .145 -- .35
Flavored Rum, Berry 51.0 64.0 -- 105
Flavored Rum, Coconut 80.0 108.0 -- 188
Flavored Rum, Spicy 30.0 28.0 108 166**
Irish Whiskey 0.210 0.135 -- 0.35
Rice Wine 22.0 0.6 -- 22.6

Conclusion
Electrochemical detection methods, such as HPAE-PAD, are selective and sensitive methods to directly determine carbohydrate concentrations without toxic and costly derivitization agents.
Here we demonstrated mg/L to g/L concentrations of simple sugars in alcoholic beverages using a High Carbohydrate Analysis kit to extend the detection linear range.
So, which alcoholic beverage should you drink if you are avoiding dietary sugars? Based on these results, the Irish and Scotch whiskeys have the lowest results. If your pallet likes a little sugar, rice wine would be a good choice.   

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