15
Experimental: Materials:
Figure 2. Free and total carbohydrates present in an instant coffee sample with conditions described in the Experimental section. Peaks:1) mannitol 2) fucose 3) sucrose 4) arabinose 5) galactose 6) glucose 7) xylose 8) mannose 9) fructose
The beverage samples were purchased from a local market. Carbohydrate standards were purchased from Sigma-Aldrich (St. Louis, MO), Fisher Chemical (Pittsburgh, PA), or Pfanstiehl (Waukegan, IL). The honey samples included a store- bought clover honey. The ‘Beekeeper’ and ‘New Zealand Manuka’ honeys were generous gifts of Kevin Thayer (beekeeper) and James Johnstone (from New Zealand). All water used for sample preparation and chromatography was deionised (DI) and had a resistivity of 18 mΩ-cm or higher. Samples that were fi ltered used a 0.2 µm Nalgene PES syringe fi lter (Thermo Scientifi c, Waltham, MA).
Equipment:
Figure 3: Analysis of energy drinks for glucose, fructose, and sucrose. Conditions are described in the Experimental section.
Figure 4: Analysis of fruit juices for glucose, fructose, and sucrose. Conditions are described in the Experimental section.
of common beverages can be assayed with a simple sample dilution and fi ltration prior to analysis. The recently introduced Thermo Scientifi c Dionex CarboPac PA210- 4 µm Fast column was used to profi le the small carbohydrates in honey in the third application.
Samples were analysed on a Thermo Scientifi c Dionex Integrion system (Thermo Scientifi c, Sunnyvale CA) or a Thermo Scientifi c Dionex ICS-5000+ system along with a Thermo Scientifi c Dionex AS-AP autosampler. Both systems have eluent generation at backpressures up to 5000 psi, temperature control, an electrochemical detector, and an electrochemical cell.
The Dionex ICS-5000+ system and the Dionex Integrion system included isocratic pumps with eluent degas. For 0.4 µL injections the autosampler was equipped with an injection valve that had a 0.4 µL internal injection loop.
Sample Preparation:
Instant coffee samples were prepared for free and total carbohydrates as described in AOAC Method 995.13 [4]. The free carbohydrate samples were prepared by dissolving 300 mg of instant coffee in 100 mL of DI water. The solution was treated with a Thermo Scientifi c Dionex OnGuard II RP cartridge as directed by the cartridge instructions. The fi ltrate was passed through a 0.2 µm syringe fi lter prior to injection. For total carbohydrates, 300 mg of instant coffee was dissolved in 50 mL of 1.0 M HCl and placed in a boiling water bath for 2.5 h, swirling every 30 min. After cooling to room temperature under tap water, the solution was diluted to 100 mL with DI water and fi ltered through folded fi lter paper (qualitative, fast). The sample was then treated with a Dionex OnGuard II Ag/H cartridge to remove chloride ion, as directed by the cartridge instructions, and passed through a 0.2 µm syringe fi lter prior to injection. Functional drinks and fruit juice samples were diluted 1 to 10,000 with DI water and fi ltered as described above. Honey samples were diluted 1 to 1000 (w/w) with DI water.
Chromatography Conditions:
All eluents were delivered using an electrolytic eluent generator. Prepared coffee samples (0.4 µL) were separated on a Thermo Scientifi c Dionex CarboPac SA10-4 µm column (4 x 250 mm) preceded by its guard column (4 x 50 mm) using 1 mM potassium hydroxide. The fl ow rate was 1.5 mL/min and the column temperature was 40ºC. Carbohydrates were detected by PAD using a disposable gold on PTFE working electrode with a 0.062” gasket, Ag/AgCl reference electrode, and a four-potential waveform [5]. Prepared functional beverage and fruit juice samples (10 µL) were separated on a Thermo Scientifi c Dionex CarboPac PA20 column (3 x 150 mm) preceded by its guard column (3 x 30 mm) using 33 mM potassium hydroxide. The fl ow rate was 0.5 mL/min and the column temperature was 30ºC. Detection conditions were the same as for coffee except that a 0.002” gasket was used. Diluted honey samples (10 µL) were separated on a Dionex CarboPac PA210-4 µm Fast column (4 x 150 mm) preceded by its guard column (4 x 50 mm) using a 48 mM potassium hydroxide. The fl ow rate was 0.8 mL/min and the column temperature was 30ºC. Detection conditions were the same as for coffee except that a 0.002” gasket was used.
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