The analysis of organic acid content of additives, premix, feed, and water.
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1 The analysis of organic acid content of additives, premix, feed, and water. Contents Foreword Introduction Warnings 1. Scope LOD and LOQ 3 2. Normative References 3 3. Definitions Feed (or feeding stuff) 3 4. Principle 3 5. Reagents and Chemicals 4 6. Apparatus 5 7. Sampling Technique 5 8. Procedure Preparation of the test solution Preparation of the test solution for free lactic acid Preparation of feed samples for free lactic acid Preparation of lactic acid concentrates (lactic acid <10 g/100g) Chromatography analysis 7 9. Calculations Limitations Typical Chromatogram 9 1
2 Foreword N/A Introduction N/A Warnings The use of these standards may 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 standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. The dosing of the organic acids is done by using specialised equipment. 1. Scope The method is a HPLC method is based on ion-exclusion chromatography with UV and/or RI detection. The organic acids are extracted with diluted sulphuric acid from the matrix. The method is applicable for the determination of total organic acids and their salts expressed as Malic acid, Citric acid, free Lactic acid, total Lactic acid, Acetic acid, Formic acid, Fumaric acid, Propionic acid, Sorbic acid, and Isobutyric acid. The method is applicable for acid blends, feed, premixtures, water, pure acids and salts. The working range of the acids in the extract is for sorbic and fumaric from 0.5 to 500 mg/l for the UV detector, and from 20 mg/l till 2500 mg/l for the other acid with the UV or RI detector. 2
3 1.1 Limit of detection N/A Analytical method related to authorised feed additives: 1a237a and 1k Normative References N/A 3. Definitions 3.1 Feed, premixtures, acid blends and additive per se. Refer to 1831/2003 Feed additive regulation. 4. Principle The sample is extracted with a diluted sulphuric acid solution. The extract is filtrated or centrifuged, and if necessary a dilution is made with eluent. The organic acids are separated and determined with HPLC ion exclusion chromatography with Refraction-index detection and/or UV detection. In case of pure acids, acid blends or addive per se it can be weight in directly in a volumetric flask and made to volume with diluted sulphuric acid before determining with HPLC ion exclusion chromatography For total lactic acid the sample is extracted with sodium hydroxide and after bringing on ph filtrated or centrifuged before determining with HPLC ion exclusion chromatography. 3
4 5. Chemicals and Reagents 5.1 General All reagents must be of recognised analytical grade. If water is mentioned it must be water grade I according to EN ISO 3696: Sulphuric acid 95-97% 5.3 Sulphuric acid 2M Dilute carefully 111 ml sulphuric acid 95-97% (5.2) in about 700 ml of water. After cooling dilute to 1000 ml and homogenise. 5.4 Sulphuric acid M Dilute 2,5 ml of 2M sulphuric acid (5.3) into 1000 ml water and homogenise. 5.5 Lactic acid 5.6 Formic acid 5.7 Citric acid 5.8 Acetic acid 5.9 Propionic acid 6.0 Butyric acid 6.1 Isobutyric acid 6.2. Malic acid 6.3 Fumaric acid 6.4 Sorbic acid 6.5 Organic acid stock solution I (± mg/l) Accurately weigh approx. 0.6g of Citric acid, Malic acid, Lactic acid, Formic acid, Acetic acid, Propionic acid, Butyric acid and Iso-butyric acid in a 50 ml volumetric flask which contain approximately 25 ml of M sulphuric acid and make to volume with M Sulphuric acid (5.4). 6.6 Organic acid working solutions I ( ± 600, 1200 and 2400 mg/l) Pipette in separate volumetric flasks of 100 ml respectively 5, 10 and 20 ml Organic acid stock solution I (6.5) and dilute with M sulphuric acid (5.4) to volume. Working solutions are stable for 6 months if carefully closed in a flask or bottle. 6.7 Organic acid stock solution II Fumaric acid (± 1000 mg/l) 4
5 Accurately weigh aprrox 50 mg of Fumaric acid and Malic acid in a 100 ml volumetric flask and make to volume with M Sulphuric acid (5.4). 6.8 Organic acid working solutions II (± 50, 100 and 200 mg/l) Pipette in separate volumetric flask of 100 ml respectively 5, 10 and 20 ml Organic acid Stock solution II (6.7) and make to volume with M Sulphuric acid (5.4). Working solutions are stable for 6 months if carefully closed in a flask or bottle. 6.9 Organic acid working solutions Sorbic acid ( ± 200 and 400 mg/l) Accurately with approx 20 and 40 mg Sorbic acid in a 100 ml volumetric flask. Append about 75 ml of diluted sulphuric acid (5.4) and heat the solutions in a water bath of 80ºC till the Sorbic acid is dissolved. After cooling the standard are diluted till volume with M diluted sulphuric acid. This working solutions must prepared daily fresh. 7.0 Sodiumhydroxide 7.1 Sodiumhydroxide 0.1M 7.2 Sodiumhydroxide 10M 6. Apparatus 6.1 HPLC apparatus consistent of: - HPLC pump - UV and/or Refraction index detector - Column heater - Auto sampler 6.2 Balance Balance with a accuracy of 0.1 mg 6.3 Centrifuge ( rpm ca g) 6.4 Water bath with a temperature of 80ºC ± 3ºC. 6.5 Fluted Filter 6.6 Magnetic Stirrer 6.7 Normal Laboratory glassware 7. Sampling Technique A representative laboratory sample of 500 g is taken from a batch (e.g. feed, raw material etc). This is then reduced in mass by a stationary or a rotary riffle splitter to a representative test sample of 100 g which is stored in a refrigerator until the day of analysis. 5
6 On the day of analysis the test sample is reduced in a particle size (with a sieve size of 1.0 mm) by a grinding mill, if possible. Do not grind intensively as this will cause evaporation of (volatile) organic acids due to the high temperature produced during intensive grinding. For liquid acids as well as acid/-salts fully dissolved in water, take a representative sample; additional treatments like grinding is not needed. 8. Procedure 8.1 Preparation of the test solution Accurately weigh approx 10 g, or less test sample depending of the expected concentration of the acids, and extract with ml 0.005M sulphuric acid (5.4). by shaking or magnetic stirring for at least 30 minutes. In case of pure acids, acid blends or addive per se, and acid/-salts dissolved in water, it can be weighed in directly in a volumetric flask and made to volume with 0.005M sulphuric acid. Check the ph of the solutions, this must be between 2 and 3.5, if needed bring on ph with 2 M sulphuric acid or 0.1 M sodium hydroxide solution. Centrifuge or filtrate (6.5) the test solution before HPLC analysis. N.B. determination of sorbic acid needs a extra step, heating till 80ºC during 30 minutes in a water bath, before shaking. 8.2 Preparation of the test solution for total lactic acid Preparation of feed, premix, water and additive samples for total lactic acid (lactic acid <10g/100g) Accurately weigh approx 10 g test sample, or less depending of the expected concentration of the lactic acid in a beaker glass. Add about 40 ml 0.1 M sodium hydroxide (7.1) and stir the solution for 30 minutes. ph should be >11, if lower add some more NaOH and stir again. Bring the solution on ph with 2 M sulphuric (5.3) acid and bring to a known volume (e.g. 100ml) Centrifuge or filtrate (6.5) the test solution before HPLC analysis Preparation of total lactic acid concentrates (lactic acid >10 g/100g) Accurately weigh in a amount of the test sample depending of the expected concentration of the lactic acid in a beaker glass of 100 ml. Add about 20 ml water and 2 ml 10 M NaOH (7.2) and stir for 10 minutes. ph should be > 13, if lower add some more NaOH and stir again. Bring the solution on a temperature above the 65 C in a magnetron. After cooling bring the solution on ph with 2 M sulphuric (5.3) acid and bring to a known volume (e.g. 100ml) Centrifuge or filtrate (6.5) the test solution before HPLC analysis. 8.3 Recommended conditions Ion-exclusion chromatography analysis Column Eluent : Aminex HPX-87 H (Bioradt) : M sulphuric acid ( 5.4) filtered before use over a membrane filter (6.6) 6
7 Eluent flow rate : 0.7 ml/min Injection volume : 30 µl Oven temperature : 25ºC ( for the most organic acids, see annex A table 1) Run time : 30 minutes (except Benzoic and sorbic acid) Detector : UV ( at 217 nm) or Refraction-index Typical retention times see table in Annex A 8.3 Procedure The HPLC system must be stabilised during at least 1 hour. Inject first a couple of standards till a reproducibility signal, and baseline are obtained. Next inject the three working standard solutions then a maximum of 8 sample solutions again the three standard solutions again 8 sample solutions and so on. The sample concentration must be within the linear range of the calibration. 9. Calculation The concentration of the acid in the test sample is calculated using the following formula in which the area of the test solution is compared with the area of the calibration solutions by means of linear regression. Coa = H H m st * C st V f * * m where: C oa H M H ST C st = content of the organic acid in the test sample (g/100g) = peak area of the test solution = peak area of the organic acid working solution = concentration of the organic acid in the test solution (mg/l) V = volume of sample extract (ml). m = weight of test portion (g). f = dilution factor = calculation factor from mg/kg to % The concentration of organic acids are in general stated in mg/kg and/or can be calculated in g/100g (%). If the acid is to be expressed as the corresponding salt the following formula should be applied: Soa = C * oa MWs MWoa 7
8 where Soa = content ot the salt of the organic acid in the test sample C oa = content of the organic acid in the test sample (g/100g) MWs = Molecular weight of the salt of the organic acid MWoa= Molecular weight of the organic acid 10. Limitation The Phosphoric acid can disturb the citric acid content when using the RI detection. In this case the solution is to use UV detection. Fumaric acid has a high UV absorbance, and can disturb the quantification of the adjacent peaks. RI detection is a better solution, for this problem Glycerine has almost the same retention time as lactic acid on RI detection. In this case use UV detection, or use a higher temperature of the column, for a better separation. 8
9 11. Typical Chromatogram 11.1 Chromatogram of a standard organic acids with RI detection 9
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