Page 194 - Analytical method for food addtives

E297: Fumaric acid and its salts 131

10.5 Appendix: method procedure summaries

Analysis of apple juice 1
Fumaric acid was determined using C18 reverse phase column (25 or 30 cm long)
packed with 5 µm spherical particles, under the following conditions: ambient
temperature, mobile phase of 0.01 M KH PO in 0.75 % phosphoric acid at
2 4
0.8 mL/min, and a UV detector at 214 (or 210) nm. Samples were analysed by
diluting 5.00 g juice with 5.00 g water, mixing thoroughly, filtering through a
0.45 µm filter and injecting 20 µL (or standard loop).

Analysis of lager beers 13

Organic acid determination by HPLC

Sample preparation
Lager beers were degassed in a rotary evaporator at a temperature below 30 ºC for
30 min. Samples were decolourised with activated carbon (2 g/100 mL of beer)
and percolated through filter paper (Whatman 1; 11 µm). Propionic acid (5 mL)
was added to the filtered solution (internal standard solution 5 mg/mL H O) and
2
then neutralised with 1 N NaOH to pH values of about 7.50–8.00.

Anionic resin adsorption
A chromatographic column (1 cm i.d. × 25 cm) filled with Amberlite Resin IRA
400 (anionic) or Dower 1X2 (c. 10 mL) was wet with 20 mL of 0.1 N NaOH and
washed with distilled water to pH 7.0. After neutralisation, the sample was slowly
filtered through this column in order to exchange all the organic acids. Afterwards,
the sugar fractions were removed by rinsing with distilled water (c. 25 mL) and the
organic acids were released from the anionic resin c. 25 mL of 0.1 N HCl and
washed with distilled water until they reached a volume of 50 mL. The treated
sample was filtered at 0.4 µm and was ready for HPLC analysis.

HPLC conditions
Column Alltima C18 Alltech (4.6 mm i.d. × 250 mm) with Hamilton
C18 precolumn

Mobile phase H O–H PO –MeOH (94:50:1)
2 3 4
Flow rate 0.8 mL/min at ambient temperature
Injection volume 100 µL
Detection Refractive index detector (RI-3 varian differential)