International Federation of Fruit Juice Producers IFU

Fumaric acid
(HPLC)

IFU Analyses Nr. 72 (1998) Page 1 of 3

Principle The content of fumaric acid in fruit juices is determined by high performance liquid chromatography (HPLC).

WORKING INSTRUCTIONS

1. 1.1 1.2. 1.3.

Reagents Double-distilled water or equivalent, suitable for HPLC Solution of sulfuric acid, c (H2SO4) = 0.005 mol/l Fumaric acid, analytical reagent grade

2. 2.1.

Apparatus High performance liquid chromatograph with UV-detector and column oven capable of maintaining 40 C HPLC separation column Aminex HPX - 87H (300 x 7.8 mm with precolumn (e.g. micro-guard cartridge Cation H+, 30 x 4.6 mm) Membrane filters (0.45 m or less), suitable for HPLC

2.2.

2.3.

3.

Determination

3.1.

Preparation of test samples

Fruit juices are diluted 1:20 (concentrates 1 : 100) with water (1.1.) and after membrane filtration (2.3) are used for the HPLC analysis. 3.2. Preparation of standard solution of fumaric acid

A known weight of fumaric acid (1.3) is dissolved in water (1.1) to give a standard solution of known concentration (fumaric acid) approximately = 100 mg/l.

International Federation of Fruit Juice Producers IFU

Fumaric acid
(HPLC)

IFU Analyses Nr. 72 (1998) Page 2 of 3

3.3.

Preparation of calibration solutions

The standard solution (3.2) is diluted with water (1.1) to produce calibration standards with (fumaric acid) in the range of 1 mg/l to 10 mg/l. 3.4. Eluent: Column: Conditions for high performance liquid chromatography Sulfuric acid solution , c(H2SO4) = 0.005 mol/l (1.2) Aminex HPX - 87H (300 x 7.8 mm) with precolumn (e.g. micro-guard cartridge Cation H+, 30 x 4.6 mm) 40C Approximately 0.6 ml/min

Column temperature: Flow:

Note: To avoid build-up of high pressure, the flow should be increased slowly from 0.2 ml/min to 0.6 ml/min during equilibration of the column with eluent (1.2). Detection Wavelength: Injection volume: Retention time for fumaric acid: Run duration: 210 nm Constant volume of approximately 15 l

approximately 14 min. approximately 20 min.

4.

Calculation and Expression of Results

The content of fumaric acid is calculated from the peak area of at least three sample injections, using the determined relationship between peak area and fumaric acid content of the calibration solutions (calibration curve or empirically determined mathematical relationship). Take the dilution factor into account in the calculation. The content of fumaric acid is expressed in mg/l to one place of decimals.

International Federation of Fruit Juice Producers IFU

Fumaric acid
(HPLC)

IFU Analyses Nr. 72 (1998) Page 3 of 3

5.

Precision

Year of collaborative study1996 Number of laboratories 13 Number of samples 3

Sour cherry nectar Sour cherry nectar Apple juice

_ x (mg/l) 14.5 3.4 6.8

sr 0.172 0.165 0.225

r 0.5 0.5 0.6

sR 0.674 0.801 0.682

R 1.9 2.2 1.9

Repeatability (r): Reproducibility (R):

sr = 0.190 sR = 0.725

r = 0.5 mg/l R = 2.0 mg/l

6.

Remarks

The fumaric acid peak is often a rider peak on a matrix peak. In such a case, this should be allowed for when measuring the peak area.