Analysis of Dietetic
Soft Drinks
Varian Application Note
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.
Number 23
Chromatographic Conditions
Analytical MCH-5 N-Cap 150 x 4.6mm
o
Column Thermostatted to 30 C
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.
 Figure 2 shows the separation for a diet soft
Figure 1 shows a typical separation for the
drink containing all four compounds found in diet
standard mixture.
beverages.

Figure 1. Standards for the Gradient Analysis of

Soft Drinks
S=saccharin, A=aspartame, C=caffeine,
B=benzoic acid
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 Diet Cola Diet Cola Caffeine

mg/354ml X Y Free Diet
Cola
Saccharin 69.1 62.2 69.9
Aspartame 30.4 28.5 32.0
Caffeine 43.2 33.8 0
Benzoic 56.0 30.5 59.0
Acid

Table 1. Concentration of Analytes in Diet Soft

Drinks

These data represent typical results.

For further information, contact your local Varian Sales Office.
LC23:0795