the analysis of organic acid content of additives, premix

1

Foreword

Introduction

Warnings

1. Scope 2

1.1 LOD and LOQ 3

2. Normative References 3

3. Definitions 3

3.1 Feed (or feeding stuff) 3

4. Principle 3

5. Reagents and Chemicals 4

6. Apparatus 5

7. Sampling Technique 5

8. Procedure 6

8.1 Preparation of the test solution 6

8.2 Preparation of the test solution for free lactic acid 6

8.2.1 Preparation of feed samples for free lactic acid 6

8.2.2 Preparation of lactic acid concentrates (lactic acid <10 g/100g) 6

8.3 Chromatography analysis 7

9. Calculations 7

10. Limitations 8

911. Typical Chromatogram

The analysis of organic acid content of additives,

premix, feed, and water.

Contents

Analytical method related to authorised feed additives: 1a237a and 1k237

2

N/A

N/A

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.

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.

Foreword

Introduction

Warnings

1. Scope


3

N/A

3.1 Feed, premixtures, acid blends and additive per se. Refer to 1831/2003 Feed additive

regulation.

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.

1.1 Limit of detection

2. Normative References

3. Definitions

4. Principle

N/A


4

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 3696:1995.

5.2 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 0.005 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 (± 12000 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 0.005 M sulphuric acid and make to volume with 0.005 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 0.005 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)

5. Chemicals and Reagents


5

Accurately weigh aprrox 50 mg of Fumaric acid and Malic acid in a 100 ml volumetric flask

and make to volume with 0.005 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 0.005 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 0.005 M

diluted sulphuric acid.

This working solutions must prepared daily fresh.

7.0 Sodiumhydroxide

7.1 Sodiumhydroxide 0.1M

7.2 Sodiumhydroxide 10M

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 (14.000 rpm ≈ ca. 17.500g)

6.4 Water bath with a temperature of 80ºC ± 3ºC.

6.5 Fluted Filter

6.6 Magnetic Stirrer

6.7 Normal Laboratory glassware

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.

6. Apparatus

7. Sampling Technique


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.

8.1 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 100.0 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

8.2.1 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 2-3.5 with 2 M sulphuric (5.3) acid and bring to a known volume

(e.g. 100ml)


8.2.2 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 2-3.5 with 2 M sulphuric (5.3) acid and bring to a known volume (e.g. 100ml)


8.3 Recommended conditions Ion-exclusion chromatography analysis

Column : Aminex HPX-87 H (Bioradt)

Eluent : 0.005 M sulphuric acid ( 5.4) filtered before use over a

membrane filter (6.6)

8. Procedure


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.

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.

000.10

***f

m

VC

H

HC st

st

moa =

where:

Coa = content of the organic acid in the test sample (g/100g)

HM = peak area of the test solution

HST = peak area of the organic acid working solution

Cst = 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

10000 = 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:

MWoa

MWsCS oaoa *=

9. Calculation


8

where

Soa = content ot the salt of the organic acid in the test sample

Coa = 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

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.

10. Limitation


9

11.1 Chromatogram of a standard organic acids with RI detection

11. Typical Chromatogram


the analysis of organic acid content of additives, premix

Documents