Determination of Additives in Carbonated Beverages
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- Adele Quinn
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1 pplication Note 9 Determination of dditives in Carbonated everages Introduction The soft drink industry is one of the largest in the world, with revenue from sales of carbonated soft drinks totaling billions of dollars annually. While the recipes for these beverages remain proprietary, critical components used as preservatives and sweeteners are subject to analysis for quality control, and must be identified on the beverage s label. dditives such as benzoate and sorbate function as preservatives. Citrate is used as both a preservative and a flavor agent. Caffeine is an integral part of the overall profile of most colas. spartame, acesulfame, and saccharin are three common artificial sweeteners which function as sugar substitutes. Many methods have been developed for determining sweeteners and additives in drink formulations. - In this pplication Note, we separate caffeine, sorbate, benzoate, citrate, aspartame, acesulfame and saccharin (structures are shown in Figure ) in carbonated drinks in a single run using the cclaim Mixed-Mode WX- column. This column features a new mixedmode silica-based packing material that incorporates both hydrophobic and weak anion-exchange properties. Unlike traditional reversed-phase substrates, the new packing features an alkyl longchain with an ionizable H C N H C N CH Caffeine CH N N S cesulfame N K + S Saccharin Figure. Structures of the seven analytes. C enzoate terminus. The column demonstrates great potential for separating a wide variety of samples that contain a mixture of anionic and neutral compounds, including food, beverage, pharmaceutical, and chemical samples. The cclaim column s mixed-mode capability provides determination of the compounds of interest in many samples in a single injection, rather than two separate injections on two different types of columns. NH H spartame H NH H NH Na + H C Sorbate K + H Citrate CH pplication Note 9
2 nalyte Table. Preparation of Mixed Stock Standard Solution Concentration of Stock Standard Volume of dded Stock Standard (ml) Volume of Mixed Stock Standard (ml) Concentration of Each Standard 0 Caffeine 00. spartame cesulfame Saccharin (diluted with water) 0 Sorbate enzoate Citrate Equipment UltiMate 000 HPLC system HPG 00 pump with SRD 00 Solvent Rack w/degasser TCC 000 Thermostatted Column Compartment WPS 000TSL utosampler VWD 00 UV/Vis detector Chromeleon.80 SP Chromatography Workstation Reagents and Standards Water, Milli-Q Gradient 0 Methanol (CH H) and acetonitrile (CH CN), HPLC grade, Fisher Methanesulfonic acid (MS), > 99.%, ldrich Potassium dihydrogen phosphate (KH P ), analytical grade, SCRC, China Caffeine, analytical grade, SCRC, China Sorbate, analytical grade, SCRC, China enzoate, analytical grade, SCRC, China Citrate, analytical grade, SCRC, China spartame, analytical grade, Niutang Chemical Co. Ltd, China cesulfame, analytical grade, Supelco/Sigma ldrich United States Saccharin, analytical grade, lfa esar China Preparation of Standards Stock standard solutions The concentrations of stock standard solutions were 00 mg/l for aspartame, 000 mg/l for acesulfame, saccharin, sorbate and benzoate, 00 mg/l for caffeine, and 00 mg/l for citrate. Mixed stock standard solution The mixed stock standard solution was prepared according to the procedure specified in Table. Mixed working standard solutions The mixed stock standard solution was diluted with mobile phase solution to prepare the mixed working standard solutions used for calibration. The concentrations of each analyte in the mixed working standard solutions are shown in Table. Determination of dditives in Carbonated everages
3 Table. Concentrations of Mixed Working Standard Solutions nalyte Concentration # # # # # # # # 8 # 9* Caffeine spartame cesulfame Saccharin Sorbate enzoate Citrate * This additional standard is prepared by adding 0 µl of the 00 mg/l caffeine stock standard to 90 µl of mobile phase. Sample Preparation Six bottled carbonated drinks were purchased from a local supermarket. Prior to injection, the samples were filtered though a 0.-µm filter and diluted with mobile phase. Chromatographic Conditions Column: cclaim Mixed-Mode WX-, µm,. 0 mm (P/N = 098) Column Temp.: 0 C Mobile Phase: 0 mm KH P /CH CN (% : %, v/v, ph.0, adjusted with MS) Flow rate. ml/min Detection: bsorbance at 0 nm Results and Discussion ptimized chromatographic conditions Separations with the cclaim Mixed-Mode WX- column can be optimized by changing the following conditions of the mobile phase: ) concentration of organic solvent, ) ionic strength (buffer concentration), and ) ph. The effects of changing these parameters are demonstrated by recording the retention time changes of a polar compound (-hydroxybenozic acid) and nonpolar compound (butylbenzene) with controlled changes of the mobile phase. Retention time for the nonpolar compound was almost unchanged when the buffer concentration changed from 00 to 0 mm. It altered only slightly when ph was changed from to., but increased significantly when the organic solvent concentration was decreased from 0% to %. Retention time of the polar compound increased significantly when the buffer concentration was changed from 00 to 0 mm. Retention time decreased significantly when ph was changed from to., and decreased slightly when the organic solvent concentration was decreased from 0% to %. Using these guidelines, the chromatographic conditions were optimized pplication Note 9
4 0 Column: cclaim Mixed-Mode WX- (µ,. 0 mm) Eluent: % 0 mm KH P (ph.0) : % CH CN Peaks:. Caffeine mg/l. spartame. Sorbate 0. enzoate 0. Citrate 0. cesulfame 0. Saccharin Table. Reproducibility of Retention Times and Peak reas nalyte RT RSD peak RSD Caffeine spartame Sorbate enzoate Citrate cesulfame Saccharin This table shows seven injections of mixed standard # (Table ) Figure. Chromatogram of mixed stock standard. to obtain separation and baseline resolution of seven analytes in carbonated beverages (Figure ). Reproducibility, linearity and detection limits Prior to sample analysis method reproducibility was demonstrated by making seven replicate injections of mixed stock standard solution. Table summarizes the retention time and peak area precision data. Calibration linearity for each of the seven compounds was determined by making replicate injections of a mixed standard prepared at eight different concentrations. The external standard method was used to calculate the calibration curve and to quantify each of the compounds in the six samples tested. Table shows the calibration data. The single-sided Student s t-distribution was used to estimate method detection limits (MDL). These data are also reported in Table. Sample analysis Six different soft drinks were tested: one lemonlime soda, one orange soda, two colas, and two diet colas. The diet colas contained no added sugar. Figures -8 show chromatograms of each sample and the same sample spiked with a mixed standard. Results, amounts Table. Calibration Data and MDLs nalyte Equations r RSD MDL Caffeine = 0.00C spartame = 0.0C Sorbate = 0.00C enzoate = 0.089C Citrate = 0.008C cesulfame = 0.09C Saccharin = 0.C Note: The single-sided Student s t test method (at the 99% confidence limit) was used to determine MDL, where the standard deviation (SD) of the peak area of seven injections is multiplied by. (at n = ) to yield the MDL Determination of dditives in Carbonated everages
5 Column: cclaim Mixed-Mode WX- (µ,. 0 mm) Eluent: % 0 mm KH P (ph.0) : % CH CN 0 Peaks: (Spike concentration, mg/l). Caffeine.. spartame 0. Sorbate.. enzoate.. Citrate 00. cesulfame.. Saccharin 0 00 Column: cclaim Mixed-Mode WX- (µ,. 0 mm) Eluent: % 0 mm KH P (ph.0) : % CH CN Peaks: (Spike concentration, mg/l). Caffeine.. spartame 0. Sorbate.. enzoate.. Citrate 00. cesulfame.. Saccharin Figure. verlay of chromatograms of a lemon-lime carbonated beverage, diluted ten-fold () and the same sample spiked (). 00 Column: cclaim Mixed-Mode WX- (µ,. 0 mm) Eluent: % 0 mm KH P (ph.0) : % CH CN Peaks: (Spike concentration, mg/l). Caffeine.. spartame 0. Sorbate.. enzoate.. Citrate 00. cesulfame.. Saccharin 0 Figure. verlay of chromatograms of diet cola #, () diluted three-fold and () the same sample spiked Column: cclaim Mixed-Mode WX- (µ,. 0 mm) Eluent: % 0 mm KH P (ph.0) : % CH CN Peaks: (Spike concentration, mg/l). Caffeine.. spartame 0. Sorbate.. enzoate.. Citrate 00. cesulfame.. Saccharin 0 Figure. verlay of chromatograms of cola #, () diluted fivefold and () the same sample spiked Figure. verlay of chromatograms of cola #, () diluted threefold and () the same sample spiked. pplication Note 9
6 00 Column: cclaim Mixed-Mode WX- (µ,. 0 mm) Eluent: % 0 mm KH P (ph.0) : % CH CN Peaks: (Spike concentration, mg/l). Caffeine.. spartame 0. Sorbate.. enzoate.. Citrate 00. cesulfame.. Saccharin 0 Column: cclaim Mixed-Mode WX- (µ,. 0 mm) Eluent: % 0 mm KH P (ph.0): % CH CN 0 Peaks: (Spike concentration, mg/l). Caffeine.. spartame 0. Sorbate.. enzoate.. Citrate 00. cesulfame.. Saccharin Figure. verlay of chromatograms of carbonated beverage diet cola #, () diluted three-fold and the same sample () spiked of each additive per beverage, and recovery data are summarized in Table. Samples show an unidentified peak eluting at approximately. min, however, this peak was not present in either of the diet colas tested (samples and ). Tests showed the peak to correspond to fructose (Figure 9). Figure 8. verlay of chromatograms of () orange soda sample, diluted ten-fold and () the same sample spiked. 0 8 Column: cclaim Mixed-Mode WX- (µ,. 0 mm) Eluent: % 0 mm KH P (ph.0) : % CH CN Fructose E D C Figure 9. verlay of chromatograms of () lemon-lime carbonated beverage diluted ten-fold, () cola #, diluted three-fold, (C)cola #, diluted five-fold, (D) orange soda, diluted ten-fold, and (E) single standard of fructose, %, w/w. Determination of dditives in Carbonated everages
7 Table. nalysis Results for the Carbonated Drinks nalyte Lemon-Lime Soda (Diluted 0-fold) Cola # (Diluted -fold) dded dded Caffeine ND spartame ND ND 0.0. Sorbate ND enzoate ND Citrate cesulfame ND..9 0 ND.. 0 Saccharin ND ND nalyte Diet Cola # (Diluted -fold) Cola # (Diluted -fold) (mg/l dded (mg/l dded Caffeine spartame ND ND Sorbate enzoate ND.. 8 Citrate cesulfame. 9 8 ND.. 08 Saccharin ND ND nalyte Diet Cola # (Diluted -fold) range Soda (Diluted 0-fold) (mg/l dded (mg/l dded Caffeine... 0 ND.. 0 spartame ND Sorbate ND.. 99 ND.. 9 enzoate Citrate cesulfame... 0 ND..8 0 Saccharin ND ND Notes:. ne sample and one spiked sample were prepared, with injections made for each.. ND= not detected. = measured value of sample diluted fold. = measured value of spiked sample measured value of sample pplication Note 9
8 REFERENCES. N.M.M. Coelho, L.M. Coelho, E.S. de Lima,. Pastor and M. de la Guardia, Talanta, Saad, Md.F. ari, M.I. Saleh, K. hmad and M.K.M. Talib, J. Chromatogr., M. S. García-Falcón and J. Simal-Gándara, Food Control, T. Pérez-Ruiz, C. Martínez-Lozano, V. Tomás and J. Martín, J. Chromatogr., User Guide for cclaim Mixed-Mode WX Columns, Dionex Corporation LPN 0.. X. Liu and C. Pohl, New Multimode Stationary Phase: Characterization, Chromatographic Evaluation and pplication, Dionex Corporation. LPN cclaim, UltiMate, and Chromeleon are registered trademarks of Dionex Corporation MilliQ is a registered trademark of Millipore Corporation Passion. Power. Productivity. Dionex Corporation North merica Europe sia Pacific 8 Titan Way U.S. (8) 9-00 ustria () enelux () () 9 P.. ox 0 Canada (90) 8-90 Denmark () France () Germany (9) 99 0 Sunnyvale, 8 C Determination of dditives Ireland () in Carbonated 00 Italy (9) 0 everages Switzerland () 0 99 South merica United Kingdom () 9 razil () 0 (08) ustralia () 90 China (8) 8 8 India (9) Japan (8) 88 Korea (8) 80 Singapore () Taiwan (88) 8 LPN 9 /0 00 Dionex Corporation
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