29.1.14
, 5% CH3COOH elutes
AOAC Official Method 979.08
Benzoate, Caffeine,
and Saccharin in Soda Beverages
Liquid Chromatographic Method
First Action 1979
Final Action 1984
A. Principle
Saccharin, benzoate, and caffeine are simultaneously deter-
mined in soda beverages by isocratic LC, using CH3COOH solu-
tion as mobile phase with UV detection at 254 nm. Artificial colors
and sorbates may interfere. Beverage blanks containing color or
sorbate should be run to assure non-interference by these compounds.
In absence of beverage blanks, analyze test samples with 2 different
mobile phases with different amounts of isopropanol and/or
CH3COOH or by method of standard additions. Adjustment of mobile
phase (% acid and/or % isopropanol) can resolve interfering peaks.
B. Apparatus and Reagents
(a) Liquid chromatograph.—Equipped with suitable injection
device, solvent delivery system, UV detector, 10 mV strip chart re-
corder or equivalent electronic integrator and µBondapak C18 col-
umn, 300 × 3.9 (id) mm (Waters Corp.) or equivalent. Operating
conditions: flow rate 2 mL/min; injection volume 10 µL; detector
wavelength 254 nm; temperature ambient.
(b) Mobile phase.—20% CH3COOH (v/v) buffered to pH 3.0
with saturated sodium acetate solution. Modify with 0–2%
isopropanol to obtain baseline resolution and retention times of stan-
dards from mixed standard solution in ca 10 min. Solution is stable
2–3 days. Degas prior to use. (Alternatively, lower CH3COOH con-
centration may be used, and, for some columns, may be necessary to
obtain retention and/or resolution. Lower CH3COOH concentration
solutions give longer retention times, e.g.
compounds in 35 min.)
(c) Standard solutions.—Prepare individual standard solutions
from compounds of known purity to give following concentrations
in H2O: sodium saccharin, 0.50 mg/mL; caffeine, 0.050 mg/mL; and
sodium benzoate, 0.50 mg/mL. Use these solutions to determine
sensitivity for detector response and retention times of individual
standards.
(d) Mixed standard solution.—Prepare solution containing
0.50 mg/mL sodium saccharin, 0.50 mg/mL sodium benzoate, and
0.050 mg/mL caffeine in H2O. Use this solution to optimize LC con-
ditions for complete resolution of the 3 compounds and to quantitate.
C. Preparation of Test Sample
(a) Carbonated beverages.—Decarbonate by agitation or ultra-
sonic treatment. If free of particulate matter, inject directly.
(b) Beverages containing particulate matter.—Filter through a
suitable membrane filter (0.45 µm), discarding first few mL filtrate.
If large amount of particulate matter is present, centrifuge prior to
filtering. Inject filtered solution directly.
D. Determination
Inject known volume (ca 10 µL) of mixed standard solution in du-
plicate. Peak heights should agree within ≤2.5%. Inject known vol-
ume (ca 10 µL) of prepared test sample in duplicate. Measure peak
heights of standard and test sample components.
Compound, % = C′ × (H/H′) × (V′/V) × 0.1
where C′ = concentration of standard in mg/mL; H and H′ = average
peak height of test sample and standard, respectively; V and V′ =
volume injected in µL of test sample and standard, respectively.
Reference: JAOAC 62, 1011(1979).
CAS-58-08-2 (caffeine)
CAS-128-44-9 (saccharin sodium)
CAS-532-32-1 (sodium benzoate)
© 2000 AOAC INTERNATIONAL