Analysis of Dietetic Soft Drinks

Varian Application NoteNumber 23

Ron Lewis Varian Chromatography Systems

Key Words: Aspartame, Caffeine, Benzoic Acid, Saccharin, Beverages

Introduction One of the many ingredients contained in soft drink beverages is the stimulant caffeine. Caffeine is the best known member of a class of compounds - the xanthines –that act as stimulants on the central nervous system. Extensive use of caffeine as a stimulant dates back to the early 1500’s when European explorers, having discovered their stimulating effects, brought back coffee, tea and kola nuts from distant voyages. The popularity of caffeinated drinks has continued to the present day. Given this popularity, an easy and rapid method of analyzing beverages for caffeine content is desirable. This is complicated by the presence of other UV absorbing compounds that are present in diet soft drinks. Benzoic acid is commonly used as a preservative. Either aspartame or saccharin are used as sweeteners. An analytical method that separates these compounds with minimal sample preparation is desirable.

Experimental Instrumentation

For chromatographic separation a ternary gradient pump was used. Detection was performed with a variable wavelength UV/VIS detector.

Chromatographic Conditions Analytical Column

MCH-5 N-Cap 150 x 4.6mm Thermostatted to 30oC

Mobile Phase 90% 0.01M KH2PO4 ,pH=2.5/10% Methanol to 60% Buffer/40% Methanol in 25 minutes

Flow Rate 1.3 ml/min

Detection 256 nm Sample Preparation

Samples of soft drinks were directly injected with no cleanup prior to injection. Results Both methanol and acetonitrile were evaluated for use as the strong solvent for this analysis. Methanol was the preferred solvent since aspartame was clearly resolved from and eluted prior to the caffeine peak. Even though aspartame was resolved from caffeine when acetonitrile was used, it eluted after the acetonitrile and analysis time was longer than the methanol gradient. Benzoic acid had sufficient retention at pH 2.5 to remove it from the vicinity of all the other components. If a pH in the range of 4.5 to 6.0 was used, benzoic acid eluted earlier and interfered with the other components of interest.

These data represent typical results. For further information, contact your local Varian Sales Office.

LC23:0795

Figure 1 shows a typical separation for the standard mixture.

Figure 1. Standards for the Gradient Analysis of Soft Drinks S=saccharin, A=aspartame, C=caffeine, B=benzoic acid

Figure 2 shows the separation for a diet soft drink containing all four compounds found in diet beverages.

Figure 2. Gradient Analysis of Soft Drinks

S=saccharin, A=aspartame, C=caffeine, B=benzoic acid

Table 1 gives the results for the analyses of three different diet cola drinks. Analytes in mg/354ml

Diet Cola X

Diet Cola Y

Caffeine Free Diet

Cola

Saccharin 69.1 62.2 69.9

Aspartame 30.4 28.5 32.0

Caffeine 43.2 33.8 0

Benzoic Acid

56.0 30.5 59.0

Table 1. Concentration of Analytes in Diet Soft Drinks