BS EN 17298 pdf free download

BS EN 17298-2019 pdf free download.Animal feeding stuffs — Methods of sampling and analysis — Determination of benzoic and sorbic acid by High Performance Liquid Chromatography (HPLC).
4 Principle The sample is extracted with a mixture of sodium acetate buffer and methanol. The extract is filtered or centrifuged and – if necessary – diluted. The amount of organic acids extracted from the sample is determined with high-performance liquid chromatography (HPLC), applying a RP 18 column, in conjunction with diode array detection (DAD) or ultraviolet detection (UV). The quantification is carried out by an external calibration. WARNING — The use of this European Standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this European Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
5 Reagents
5.1 Water, grade 2 in accordance with EN ISO 3696.
5.2 Benzoic acid, p.a., minimum 99 % purity.
5.3 Sorbic acid, p.a., minimum 99 % purity.
5.4 Acetic acid, p.a., w = 100 %.
5.5 Methanol, gradient grade, w ≥ 99,8 %.
5.6 Sodium acetate trihydrate, p.a.
5.7 Sodium acetate solution, c = 1 mol/l.
Dissolve 13,6 g sodium acetate trihydrate (5.6) with approximately 80 ml water (5.1) in a 100 ml volumetric flask, mix and fill up to the mark with water (5.1).
The maximum storage time is 3 months at room temperature.
5.8 Acetic acid solution, c = 1 mol/l.
Dissolve 6,00 g acetic acid (5.4) with approximately 80 ml water (5.1) in a 100 ml volumetric flask, mix and fill up to the mark with water (5.1).
The maximum storage time is 3 months at room temperature.
5.9 Acetate buffer, c = 0,1 mol/l.
Mix 50,0 ml sodium acetate solution (5.7) and 50,0 ml acetic acid solution (5.8) in a 1 000 ml volumetric flask and add approximately 700 ml water (5.1). Adjust the pH with acetic acid solution (5.8) to 4,6. Fill up to the mark with water (5.1).
The maximum storage time is 6 months at room temperature.
NOTE A pH of 4,6 is the best range for an optimal HPLC separation regarding the referred conditions.
5.10 HPLC Mobile phase
Mix acetate buffer (5.9) and methanol (5.5) in proportion of 75:25 (v/v) by means of graduated cylinder. Filter for HPLC use.
The maximum storage time is 6 months at room temperature.
5.11 Extraction solution
Mix acetate buffer (5.9) and methanol (5.5) in proportion of 60:40 (v/v) by means of graduated cylinder.
The maximum storage time is 6 months at room temperature.
5.12 Standard stock solution, app. 500 mg/l.
Weigh 125 mg benzoic acid (5.2) and 125 mg sorbic acid (5.3) into the same 250 ml volumetric flask.
Dissolve with approximately 150 ml extraction solution (5.11), dissolve 5 min in an ultrasonic bath and fill to the mark with extraction solution (5.11).
The maximum storage time is 2 months at 4 °C.
NOTE 1 The shelf life of the stock solution is limited because of esterification reactions.
NOTE 2 If available, single acid standard solutions with adequate and required specifications can be used.
Determine the exact concentration of the stock solution using the reference standard purity value provided by the supplier according to Formula (1).
Apparatus
Usual laboratory equipment and, in particular, the following:
6.1 Laboratory grinder, capable of grinding to a particle size of less than or equal to 1,0 mm.
6.2 Analytical balance, suitable to accurately weigh between 0 g and 10 g with an accuracy of 0,1 mg.
6.3 Ultrasonic bath
6.4 pH meter capable of being read to at least one place of decimals.
6.5 Pipettes (electronic or manual) in the range 100 μl to 5 000 μl.
6.6 Centrifuge, approx. 5 000 × g.
6.7 Membrane filter for HPLC use, 0,45 µm (for example: Ready-to-use filter unit with a hydrophilic, low protein-binding membrane made of regenerated cellulose).
6.8 Folded filter, pore size 4 µm to 7 µm (ash free paper filter).
6.9 HPLC system (pump, autosampler, column oven) with DAD/UV detector
6.10 HPLC column, RP 18 column, e.g. Nucleosil 120 C18, 5 µm, 250 mm x 4,0 mm.BS EN 17298 pdf download.