Environmentally Friendly Method for the Analysis of Caffeine and Benzoic Acid in Soft Drinks

Abstract

A simple analysis of caffeine and benzoic acid in soft drinks using the compact Axcend Focus LC® is demonstrated. Linear regression of the calibration data gave R2 values of 0.9993 and 0.9998 for caffeine and benzoic acid, respectively. Analysis of a commercial soft drink gave 228 ppm caffeine (RSD 1.1%) and 184 ppm benzoic acid (RSD 1.8%). Solvent usage was estimated to be approximately 28 μL per analysis.

Introduction

The determination of benzoic acid and caffeine in soft drinks is routinely done to ensure that products aresafe and meet label claims as well as health and safetyrequirements as set by the FDA. The average caffeinecontent in caffeinated soft drinks is approximately 100-300 ppm, while energy drinks can contain over 600ppm. Caffeine-free drinks are guaranteed to containno caffeine. The ability to rapidly analyze caffeine indrinks using a small portable instrument with minimalmobile phase solvent (~ 28 μL) consisting of relativelyharmless components (i.e., water, methanol, and a littlephosphoric acid) is significant.

Experimental

Preparation of standard calibration solutions. HPLC grade water (Millipore-Sigma, #270733) was used for standard preparation. Caffeine was purchased from Spectrum Chemical, and sodium benzoate was from Sigma-Aldrich (#109169). A standard solution was prepared by dissolving caffeine in HPLC grade water and diluting to a concentration of 10 mg/mL. Likewise,a standard solution of 10 mg/mL of sodium benzoate in water was also prepared (equivalent to 8.47 mg/mL benzoic acid). A stock solution (1000 ppm each)of caffeine and benzoic acid was then prepared by combining 0.300 mL of the caffeine standard solution with 0.354 mL of the sodium benzoate standard solution and diluting to 3.00 mL with 1% H3PO4 (Sigma-Aldrich, #49685) in water. The resultant mixture was subsequently diluted to prepare a series of 10, 50, 100,150, 200, and 250 ppm standard calibration solutions

Preparation of soft drink samples. Before chromatographic analysis, carbonated samples were degassed by sonicating for approximately 5 min and then filtered through a syringe filter (Kinesis KX Syringe Filter, Regenerated Cellulose, 13 mm dia., 0.22 μm pore size). Each sample was loaded into the injection loop with a 25-μL syringe (Hamilton #1702SN 22’S/2”/AS) after rinsing 3 or 4 times with HPLC grade water.

HPLC analysis. HPLC analysis was accomplishedusing an Axcend Focus LC equipped with a cartridgecontaining a standard 10 cm x 0.150 mm i.d. capillarycolumn packed with 1.7 μm dp C18 stationaryphase, an on-column UV-absorption detector with255 nm LED light source, and an injection valve with200 nL loop. The binary mobile phase componentswere (A) 3% methanol + 0.1% formic acid in waterand (B) methanol with 0.1% formic acid. The pumpswere operated at a combined flow rate of 1.7 μL/min. A 4-min equilibration time at the beginningof each analysis was applied once the instrumentreached 5,000 psi during an initial pressurizationstep. The solvent program was 3% B during the 4-minequilibration followed by a linear gradient to 96% B in6 min (held constant for another 2 min). A summary ofthe HPLC analysis conditions is given in Table 1. Underthese conditions, caffeine and benzoic acid eluted at5.1 and 6.6 min, respectively.

Table 1. Summary of HPLC Analysis Conditions.

Results

Calibration curves. Figure 1 shows a chromatogram of the 50-ppm caffeine and benzoic acid standard calibration solution. The rise in baseline at the end of the chromatogram resulted from a short rinse with 96% acetonitrile to remove any residual compounds in the chromatographic system. Figure 2 shows
calibration plots for both caffeine and benzoic acid from HPLC analyses of the standard calibration
solutions. Linear regression of the calibration data gave R2 values of 0.9993 and 0.9998 for caffeine and
benzoic acid, respectively.

Figure 1. Chromatogram of calibration standard solution containing 50 ppm each cafeine and benzoic acid.

Analysis of commercial soft drink

Table 2 lists the experimental data obtained from 6 repeat analyses. Figure 3 shows a chromatogram obtained from one of these runs of the commercial soft drink sample.

Table 2. Results Obtained from 6 Repeat Analyses of a Commercial Soft Drink.

Conclusion

The portable, compact Axcend Focus LC can easily analyze commercial soft drinks for caffeine and benzoic acid
with excellent accuracy, linearity, and repeatability. Analysis costs are significantly reduced because of the very
small volumes of mobile phase solvents required for each analysis. The Axcend Focus LC can likewise be applied
to the analysis of samples from a wide range of industries including chemical manufacturing, consumer products,
cosmetics, foods, beverages, pharmaceuticals, environmental contaminants, petrochemicals, and many
others, often with analysis facilitated by the small size of the instrument.