Introduction Methods of analysis

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