1.5 Appendix: method procedure summaries
Analysis of soft drinks
2
Sample preparation Accurately weigh 10 g of sample into a 25 mL beaker and adjust to pH 7.0 with
0.1 molL sodium hydroxide.
Extraction Transfer neutralised sample to centrifuge tube. Rinse beaker and pH electrode with
2 × 5 mL portions of water and transfer washings to centrifuge tube. Add 5 mL 0.1 molL cetylpyridinium chloride in water, mix and add 10 mL of water-
saturated n-butanol. Shake vigorously for 10 min on mechanical shaker. Centrifuge at 1000 g for 5 min and transfer upper organic layer to a 25 mL volumetric flask
using a Pasteur pipette. Repeat the procedure with three 5 mL portions of water- saturated n-butanol.
Make the combined n-butanol extracts up to 25 mL with water-saturated n
-butanol. Accurately dilute an aliquot of the filtrate with an equal volume of mobile phase 1 L + 1 L dilution of mobile phase A and solution B. Mix and filter
a portion through a filter. Quantitative determination: HPLC
Load 20 µL of sample extract onto column and use gradient linear elution to
achieve optimum separation. Column
Spherisorb C8, 250 × 4.6 mm, 5 µm
Guard column packed with 40
µm reverse phase material e.g. Perisorb RP8 30–40
µm Mobile phase
60 Solution B and 40 Solution A linear gradient to 80 Solution B and 20 Solution A after 20 min
Flow rate 1.5 mLmin
Detector 430 nm
Solution A Phosphate buffer and water are diluted 50 mL + 850 mL, and
this solution is de-gassed. To the de-gassed solution, 50 mL of cetylpyridinium chloride solution is added and the final
solution made to 1 L in a volumetric flask. The solution is de- gassed before the addition of cetylpyridinium chloride
solution to avoid frothing.
Solution B Cetylpyridinium chloride solution is diluted 50 mL to 1 L
with a 1 L + 1 L dilution of acetonitrile and methanol.
Analysis of beverages
3
Sample preparation The samples were prepared as follows:
1 Quantitative determination by direct preparation using calibration graphs:
5 mL of the sample was transferred to a 25 mL flask and diluted with deionised water to the mark.
2 Quantitative determination by standard addition: to 5 mL of the beverage
sample were added different amounts 2, 4, 6, 8 mgL of the dye to determine and proceed as before.
Analysis of beverages The samples were filtered through a Millipore filter before being injected into the
chromatographic system and all the experiments were carried out in duplicate.
HPLC conditions Column
Nova-Pak C18 Mobile phase
Eluent A Methanol
Eluent B NaH
2
PO
4
Na
2
HPO
4
buffer solution 0.1 M pH=7
Gradient profile t
initial 20 eluent A, 80 eluent B
t
1
2 min 100 eluent A
t
2
4 min 100 eluent A
t 5 min
20 eluent A, 80 eluent B Flow rate
2 mLmin Injection volume
20 µL
Detection 520 nm