Determination of Additives in Beverages Using Ascentis® Express Columns

sigma-aldrich.com/analytical reporter 30.3 |20 Food and Beverage AnalysisDetermination of Additives in Beverages Using Ascentis® Express ColumnsOlga Shimelis, Hugh Cramer, and Michael D. Buchananmike.buchanan@sial.comIntroductionBeverages, such as sodas and energy drinks, can include a number of polar ingredients, which are easily soluble in the water matrix of the drinks. These ingredients include sweeteners (sugars and sugar substitutes), caffeine, vitamin supplements, amino acids, organic acids, and plant extracts. Because the analytes are already in solution, there is no need for extensive sample preparation. Dilution followed by direct injection into an HPLC is typically suitable.In this article we present two beverage applications using Ascentis Express HPLC columns. Ascentis Express columns offer faster HPLC on any system. One benefit is their ability to produce the resolution, efficiency, and speed on conventional HPLC systems that is associated with the use of sub-2 micron columns on a UHPLC system, without generating high backpressure. Column chemistries (RP-Amide and HILIC) were selected for this article based on their enhanced performance with polar compounds in comparison to C18.analysis of diet SodaArtificial sweeteners are often used in drinks in specific combinations to mimic the sweetness of natural sugars. They contain less energy than sugar, and are added to maintain the taste without increasing the caloric value. In addition to sweeteners, sodas can contain preservatives, which are added to inhibit microbial growth and to prolong the shelf-life of the drinks. Because artificial sweeteners and preservatives are both considered additives, they are often regulated. Therefore, their identities and concentrations should be determined. Figure 1 shows the HPLC separation of 3 artificial sweeteners (acesulfame, aspartame, and neotame), two preservatives (benzoic acid and sorbic acid), and caffeine. In this case, the RP-Amide column was used. Amide-functional groups in the RP-Amide column provide different selectivity than alkyl-only phases, such as C18. The selectivity differences are most noticeable with polar compounds, like those in this application. The separation of all major components on the Ascentis Express RP-Amide column was achieved in less than 1 minute using a conventional HPLC system with acceptable backpressure (3200 psi/220 bar).analysis of caffeinated energy drinkEnergy drinks contain a variety of marketable ingredients, such as sugars, vitamins, and caffeine. Like sodas, they also typically contain preservatives. The resulting chromatogram from the analysis of an energy drink on an Ascentis Express HILIC column is shown in Figure 2. HILIC chromatography is suitable for the retention and separation of hydrophilic compounds based on differences in their polarities, whether these analytes are acidic, basic, charged, or neutral. Both UV and ELSD were used to detect different types of compounds; the ELSD allowed the detection of the non-UV absorbing sugars. Figure 1. hPlc analysis of artificial Sweeteners, Preservatives, and caffeine in a diet Soda column: Ascentis Express RP-Amide, 3 cm x 4.6 mm I.D., 2.7 µm particles (53921-U) mobile phase: (A) 100 mM ammonium acetate, pH 5.6, titrated with acetic acid; (B) water; (C) acetonitrile gradient: 20% A constant; 5 to 60% C in 1 min; hold at 60% C for 0.1 min flow rate: 3 mL/min column temp.: 40 °C detector: UV at 214 nm injection: 1 µL sample: diet soda 100 - 500 µg/mL in buffer1. Acesulfame2. Benzoic acid3. Sorbic acid4. Caffeine5. Aspartame6. Neotame0 0.2 0.4 0.6 0.8 1.0min152346Figure 2. hPlc analysis of Sugars, Vitamins, Preservatives, and caffeine in an energy drink column: Ascentis Express HILIC, 10 cm x 3.0 mm I.D., 2.7 µm (53970-U) mobile phase: (A) 100 mM ammonium acetate, pH 5.0; (B) water; (C) acetonitrile; (9:1:90, A:B:C) flow rate: 0.6 mL/min pressure: 815 psi column temp.: 35 °C detector: UV, 254 nm or ELSD, 55 °C, 3.5 bar nitrogen injection: 2 µL commercial energy drink diluted 1:9 in acetonitrile1. Caffeine2. Niacinamide (vitamin B3)3. Pyridoxine hydrochloride (vitamin B6)4. Benzoic acid5. Sorbic acid6. Riboflavin (vitamin B2)7. Fructose8. Glucose9. Sucrose10. Taurine0 2 4 6 81 5234678910UV 254 nm detection ELSD detectionmin21Order: 800-325-3010 (U.S.) 814-359-3441 (Global) The conditions used an MS-friendly mobile phase, and yielded rapid analysis (under 2 minutes) and low backpressure (815 psi). Because of these benefits, a HILIC column should always be considered for analysis of beverage components.conclusionThese brief examples demonstrate the application of two Ascentis® Express columns for the analysis of beverages. Two column chemistries, RP-Amide and HILIC, both amenable to the analysis of polar compounds, were successfully used to resolve several beverage components, including sugars, vitamins, sweeteners, preservatives, and caffeine. Ascentis Express columns provided rapid separation using conventional HPLC systems without the major increase in backpressure associated with sub-2 micron columns. Selectivity differences between the two columns can be further employed to alter elution order and compound retention. Featured ProductsDescription Cat. No. Ascentis Express HPLC Columns, (2.7 µm particles)RP-Amide, 3 cm x 4.6 mm I.D. 53921-UHILIC, 10 cm x 3.0 mm I.D. 53970-U Related ProductsDescription Cat. No. Ascentis Express HPLC Columns, 10 cm x 4.6 mm I.D., 2.7 µm particlesC18 53827-UC8 53837-UPhenyl-Hexyl 53352-URP-Amide 53929-UES-Cyano 53491-UF5 53590-UHILIC 53979-UOH5 53776-UAnalytical Standards (Sugars, Vitamins, Sweeteners, Caffeine)Fructose, 1000 mg PHR1002Glucose, 1000 mg PHR1000Sucrose, 1000 mg PHR1001Taurine, 1000 mg PHR1109Riboflavin (Vitamin B2), 1000 mg 47861Nicotinamide, amide derivative of Niacinamide (Vitamin B3), 1000 mg47865-UPyridoxine hydrochloride (Vitamin B6), 1000 mg 47862Acesulfame K, 1000 mg 47134Aspertame, 500 mg 47135Neotame, 100 mg 49777Benzoic acid, 1000 mg 47849Sorbic acid, 1000 mg 47845Caffeine, 100 mg 56396Analytical Reagents and SolventsAcetic acid, eluent additive for LC-MS 49199Ammonium acetate, for HPLC, >99.0% 17836Acetonitrile, LC-MS CHROMASOLV®, >99.9% 34967 Related InformationTo learn more about Sigma-Aldrich solutions for food analysts, visit sigma-aldrich.com/foodDid you know . . .Our Ascentis Express HPLC Columns (JHD) and Ascentis Express HPLC Resource Guide (LRW) brochures fully describe the features and benefits of Ascentis Express HPLC columns?20% Off HPLC AccessoriesSpecify promo code SBV when you order. Offer expires March 29, 2013, and is not valid in Japan. Discount is percent off list price. For more information, visit sigma-aldrich.com/hplc-accessoriesMaximize System Performance with Easy-to-Use HPLC Accessories