METHODS OF ANALYSIS FOR THE OFFICIAL CONTROL OF FEEDING-STUFFS RULES *

METHODS OF ANALYSIS FOR

THE OFFICIAL CONTROL OF FEEDING-STUFFS

SUBSIDIARY LEGISLATION 437.69

METHODS OF ANALYSIS FOR THE OFFICIAL

CONTROL OF FEEDING-STUFFS RULES *

20th May, 2005

LEGAL NOTICE 141 of 2005.

(1) The title of these rules is the Methods of Analysis for

the Official Control of Feeding-Stuffs Rules.

(2) The scope of these rules is to implement the provisions

found under European Union Council Directive 71/250/EEC on the

methods of analysis for the official control of feeding-stuffs.

METHODS OF ANALYSIS OF THE COMPONENTS OF FEEDING STUFFS

1. GENERAL PROVISIONS ON METHODS OF ANALYSIS FOR

FEEDINGSTUFFS

A.

PREPARATION OF SAMPLES FOR ANALYSIS

1. PURPOSE

The procedures described below concern the preparation for analysis of final

samples, sent to the control laboratories after sampling in accordance with the

provisions laid down by First Commission Directive 76/371/EEC of 1 March 1976

establishing Community methods of sampling for the official control of feeding

stuffs.

These samples must be prepared in such a way that the amounts weighed out, as

provided for in the methods of analysis, are homogeneous and representative of the

final samples.

2. PRECAUTIONS TO BE TAKEN

All the necessary operations must be performed in such a way as to avoid as far as

possible contamination of the sample and changes of its composition. Grinding,

mixing and sieving should be carried out as quickly as possible with minimal

exposure of the sample to the air and light. Mills and grinders likely to appreciably

heat th e sam ple should not be used . Manu al gri nding is recomm ended for

feedingstuffs which are particularly sensitive to heat. Care should also be taken to

ensure that the apparatus itself is not a source of contamination of trace elements.

If the preparation cannot be carried out without significant changes in the moisture

content of the sample, determine the moisture content before and after preparation

according to the method laid down in Part 1 of the Annex to Second Commission

Directive 71/393/EEC of 18 November 1971 establishing Community methods of

analysis for the official control of feedingstuffs, as amended by Commission

Directive 73/47/EEC of 5 December 1972.

3. PROCEDURE

Mix thoroughly the final sample either mechanically or manually. Divide the

sample into two equal portions (the quartering method should be used where

applicable). Keep one of the portions in a suitable clean, dry container, fitted with an

air-tight stopper, and prepare the other portion or a representative part of it, of at

least 100 g, as indicated below.

3.1. Feeding stuffs which can be ground as such Unless otherwise specified in

the methods of analysis, sieve the whole sample through a sieve with a square mesh

of 1 mm side (in accordance with recommendation ISO R565) after grinding, if

necessary. Avoid any over-grinding. Mix the sieved sample and collect it in a

suitable clean, dry container fitted with an air-tight stopper. Mix again, immediately

before weighing out the amount for analysis.

3.2. Feeding Stuffs which can be ground after drying Unless otherwise specified

in the methods of analysis, dry the sample to bring its moisture content down to a

level of 8 to 12%, according to the preliminary drying procedure described under

point 4.3 of the method of determination of moisture mentioned in section 2 above.

Then proceed as indicated in section 3.1.

3.3. Liquid or semi-liquid feeding stuffs

Collect the sample in a suitable clean, dry container, fitted with an air-tight

stopper. Mix thoroughly immediately before weighing out the amount for analysis.

3.4. Other feeding stuffs

Samples which cannot be prepared according to one of the above procedures

should be treated by any other procedure which ensures that the amounts weighed

out for the analysis are homogeneous and representative of the final samples.

For the purpose of these rules -

"Act" means the Veterinary Services Act;

"the Community" means the European Community as established

under the Treaty establishing the European Community;

"the Commission" means the European Commission;

"competent authority" means the Veterinary Services within the

territory of Malta as provided under article 2 of the Act;

"Member State" means a State which is a Member within the

European Community.

(1) The competent authority shall require that analyses for

official controls of feeding-stuffs within its jurisdiction as regards

their levels of hydrocyanic acid, calcium, carbonates, crude ash,

ash insoluble in HCl chlorine from chlorides, lactose, potassium,

sodium, sugars and urea, and the estimation of the urease activity

of products derived from soya be carried out using the methods set

out in the Schedule.

(2) The general rules contained in Part 1 of the Schedule shall

apply to the methods of analysis adopted pursuant to Council

Directive 70/373/EEC.

(3) As regards undesirable substances within the meaning of

Directive 2002/32/EC of the European Parliament and of the

Council including dioxins and dioxin-like PCBs, point C(3) of the

first part of Directive 71/250/EEC of the Council of the European

Union shall apply.

*These rules have been repealed by Legal Notice 37 of 2025.

Storage of samples

Samples must be stored at a temperature that will not alter their composition.

Samples intended for the analysis of vitamins or substances which are particularly

sensitive to light should be stored in brown glass containers.

B.

PROVISIONS RELATING TO REAGENTS AND APPARATUS USED IN

METHODS OF ANALYSIS

1. Unless otherwise specified in the methods of analysis, all analytical reagents

must be analytically pure (a.p.). When determining trace elements, the purity of the

reagents must be checked by a blank test. Depending upon the results obtained,

further purification of the reagents may be required.

2. Any operation involving preparation of solutions, dilution, rinsing or

washing, mentioned in the methods of analysis without indication as to the nature of

the solvent or diluent employed, implies that water must be used. As a general rule,

water should be demineralized or distilled. In particular cases, which are indicated in

the methods of analysis, it must be submitted to special procedures of purification.

3. In view of the equipment normally found in control laboratories, only those

instruments and apparatus which are special or require specific usage are referred to

in the methods of analysis. They must be clean, especially when very small amounts

of substances have to be determined.

C.

APPLICATION OF METHODS OF ANALYSIS AND EXPRESSION OF

THE RESULTS

1. In general a single method of analysis is established for the determination of

each substance in feedingstuffs. Where several methods are given, the particular

method used by the control laboratory must be indicated on the analysis report.

2. The result given in the analysis report shall be the average value obtained

from at least two determinations, carried out on separate portions of the sample, and

of satisfactory repeatability.

This result shall be expressed in the manner laid down in the method of analysis to

an appropriate number of significant figures and shall be corrected, if necessary, to

the moisture content of the final sample prior to preparation.

3. As regards undesirable substances within the meaning of Directive 2002/32/

EC, including dioxins and dioxin-like PCBs, a product intended for animal feed shall

be considered as non-compliant with the established maximum content, if the

analytical result is deemed to exceed the maximum content taking into account

expanded measurement uncertainty and correction for recovery. The analysed

concentration corrected for recovery and the expanded measurement uncertainty

subtracted from the analytical result is used to assess compliance. This procedure is

only applicable in cases where the method of analysis enables the estimation of

measurement uncertainty and correction for recovery (eg. Not possible in case of

microscopic analysis).

The analytical result shall be reported as follows (in so far the used method of

analysis enables to estimate the measurement uncertainty and recovery rate)

(a) corrected or uncorrected for recovery, the manner of reporting and the

level of recovery being indicated;

(b) as "x +/- U", whereby x is the analytical result and u is the expanded

measurement uncertainty, using a coverage factor of 2 which gives a

level of confidence of approximately 95%.

2. DETERMINATION OF HYDROCYANIC ACID

1. PURPOSE AND SCOPE

This method makes it possible to determine the level of hydrocyanic acid, free and

combined in the form of glycosides, in feeding-stuffs and in particular in products

derived from flax seed, manioc flour and certain species of beans.

2. PRINCIPLE

The sample is suspended in water. The hydrocyanic acid is released by the action

of enzymes, entrained by steam distillation and collected in a specific volume of

acidified silver nitrate solution.

The silver cyanide is separated by filtration and the excess silver nitrate is titrated

with a solution of ammonium thiocyanate.

3. REAGENTS

3.1. A suspension of sweet almonds: crush twenty blanched sweet almonds in

100 ml of water at 37 to 40ºC. Check that there is no hydrocyanic acid in 10 ml of

the suspension using sodium picrote paper or by carrying out a blank test as

described in the last paragraph of 5.

3.2. 10% solution (w/v) of sodium acetate, neutral to phenolphthalein.

3.3. Anti-foaming emulsion (e.g. silicone).

3.4. Nitric acid, d: 1ꞏ40.

3.5. Silver nitrate solution: 0ꞏ02 N.

3.6. Ammonium thiocyanate solution: 0ꞏ02 N.

3.7. Saturated solution of ammonium ferric sulphate.

3.8. Ammonia, d: 0ꞏ958.

4. APPARATUS

4.1. Oven with thermostat set at 38ºC.

4.2. Apparatus for distillation by entrainment in steam fitted with a condenser

with a curved extension piece.

4.3. 1000 ml flat-bottomed flasks with ground-glass stoppers.

4.4. Oil bath.

4.5. Burette graduated in 1/20 ml.

PROCEDURE

Weigh 20 g of the sample to the nearest 5 mg, place in 1 litre flat-bottomed flask

and add 50 ml of water and 10 ml of sweet almond suspension (3.1.). Stopper the

flask and transfer to the oven for sixteen hours at 38ºC. Next, cool to room

temperature and add 80 ml of water, 10 ml of sodium acetate solution (3.2) and a

drop of anti-foaming emulsion (3.3).

Connect the flask to the steam distillation apparatus and place in an oil bath which

has first been brought to a temperature slightly above 100ºC. Distil 200 to 300 ml of

liquid by passing a powerful current of steam through the flask and gently heating

the oil-bath. Collect the distillate in an Erlenmeyer flask protected from the light and

containing exactly 50 ml of silver nitrate solution 0ꞏ02 N (3.5) and 1 ml of nitric acid

(3.4). Make sure that the condenser's extension piece is immersed in the silver nitrate

solution.

Transfer the contents of the Erlenmeyer flask to a 500 ml volumetric flask, make

up to volume with water, stir and filter. Remove 250 ml of the filtrate, add

approximately 1 ml solution of ammonium ferric sulphate (3.7) and back-titrate the

excess silver nitrate with the solution of ammonium thiocyanate 0ꞏ02 N (3.6) taken

from the burette graduated in 1/20 ml.

A blank test may, if required, be carried out by applying the same procedure to 10

ml of sweet almond suspension (3.1), omitting the sample to be analysed.

CALCULATION OF RESULTS

If the blank test indicates that silver nitrate solution 0.02 N has been consumed,

subtract the value of this from the volume consumed by the distillate of the sample. 1

ml of AgNO 3 0ꞏ02 N corresponds to 0ꞏ54 mg of HCN. Express the result as a

percentage of the sample.

OBSERVATIONS

If the sample contains a large quantity of sulphides (e.g. beans), a black

precipitate of silver sulphide is formed which is filtered together with the silver

cyanide deposit. The formation of this precipitate causes a loss of silver nitrate

solution 0ꞏ02 N, the volume of which must be subtracted from the volume used to

calculate the HCN content. To do this, proceed as follows

Treat the deposit left on the filter with 50 ml of ammonia (3.8) in order to dissolve

the silver cyanide. Wash the residue in dilute ammonia and then determine its silver

content. Convert the value obtained into ml of silver nitrate solution 0ꞏ02 N.

The sample’s HCN content may also be determined by titrating the acidified

ammoniacal filtrate with nitric acid.

3. DETERMINATION OF CALCIUM

1. PURPOSE AND SCOPE

The method makes it possible to determine the total calcium content of feeding-

stuffs.

2. PRINCIPLE

The calcium is ashed, the ash treated with hydrochloric acid and the calcium

precipitated as calcium oxalate. Dissolve the precipitate in sulphuric acid and titrate

the oxalic acid which forms with a solution of potassium permangante.

3. REAGENTS

3.6. Hydrochloric acid A.R., d: 1ꞏ14

3.2. Nitric acid A.R., d: 1ꞏ40

3.3. Sulphuric acid A.R., d: 1ꞏ13

3.4. Ammonia A.R., d: 0ꞏ98

3.5. Cold saturated solution of ammonium oxalate A.R.

3.6. 30% solution (w/v) of citric acid A.R.

3.7. 5% solution (w/v) of ammonium chloride A.R.

3.8. 0ꞏ04% solution (w/v) of bromo-cresol green

3.9. Solution of potassium permanganate 0ꞏ1 N

4. APPARATUS

4.1. Electric muffle-furnace with air circulation and thermostat.

4.2. Platinum, silica or porcelain crucibles for ashing.

4.3. Glass filter crucibles of G4 porosity.

5. PROCEDURE

Weigh approximately 5 g of the sample (or more if necessary) to the nearest mg,

calcine at 550ºC and transfer the ash to a 250 ml beaker.

Add 40 ml of hydrochloric acid (3.1), 60 ml of water and a few drops of nitric acid

(3.2). Bring to the boil and keep at boiling point for thirty minutes. Cool and transfer

the solution to a 250 ml volumetric flask. Rinse, bring the volume up to the mark

with water, homogenise and filter.

Using a pipette, remove to a 250 ml beaker an aliquot amount containing 10 to 40

mg of calcium according to the assumed calcium content. Add 1 ml of citric acid

solution (3.6) and 5 ml of ammonium chloride solution (3.7).

Make the volume up to approximately 100 ml with water. Bring to the boil, add

eight to ten drops of bromo-cresol green solution (3.8) and 30 ml of a warm solution

of ammonium oxalate (3.5). If a precipitate forms, dissolve it by adding a few drops

of hydrochloric acid (3.1).

Neutralise very slowly with ammonia (3.4), stirring continuously until a pH

reading of 4.4 to 4.6 is obtained (i.e. when the indicator changes colour).

Place the beaker in a boiling water bath and keep there for thirty minutes to allow

the precipitate which has formed to settle. Remove the beaker from the water bath.

Leave it to stand for an hour and filter through a G 4 filter crucible.

Wash the beaker and the crucible with water until the excess ammonium oxalate is

completely removed (the absence of chloride in the washing water indicates that they

have been sufficiently washed).

Dissolve the precipitate on the filter in 50 ml of warm sulphuric acid (3.3). Rinse

the crucible with warm water and make the filtrate up to approximately 100 ml.

Bring the temperature up to 70 - 80ºC and titrate drop by drop with a solution of

potassium permanganate (3.9) until a pink colour is obtained which lasts for one

minute.

6. CALCULATION OF RESULTS

1 ml of potassium permanganate 0ꞏ1 N corresponds to 2ꞏ004 mg of calcium.

Express the result obtained as a percentage of the sample.

7. OBSERVATIONS

7.1. For very low contents of calcium proceed as follows{ filter the calcium

oxalate precipitate through an ash-free filter paper. After washing, dry the filter and

ash at 550ºC in a platinum crucible. Redissolve the residue in a few drops of

sulphuric acid (3.3), evaporate until dry, calcine again at 550ºC and weigh. If W is

the weight of the calcium sulphate obtained, the calcium content of the aliquot

amount taken as a sample = W × 0ꞏ2944.

7.2. If the sample consists solely of mineral substances, dissolve in hydrochloric

acid without ashing it first. In the case of products such as calcium-aluminium

phosphate which are difficult to dissolve in acid, melt as follows by an alkaline

process before dissolving: mix the sample to be analysed thoroughly in a platinum

crucible with a mixture five times its weight, consisting of equal amounts of

potassium carbonate and sodium carbonate. Heat carefully until the mixture is

completely melted. Cool, and dissolve in hydrochloric acid.

7.3. If the magnesium content of the sample is high, precipitate the calcium

oxalate a second time.

4. DETERMINATION OF CARBONATES

1. PURPOSE AND SCOPE

T h i s m e t h o d m a k e s i t p o s s i b l e t o d e t e r m i n e t h e a m o u n t o f c a r b o na t e s ,

conventionally expressed as calcium carbonate, in most feeding-stuffs.

However in certain cases (for example, with iron carbonate) a special method must

be used.

2. PRINCIPLE

The carbonates are decomposed in hydrochloric acid[ the carbon dioxide released

is collected in a graduated tube, and its volume compared with that released under

the same conditions by a known quantity of calcium carbonate A.R.

3. REAGENTS

3.1. Hydrochloric acid, d: 1ꞏ10.

3.2. Calcium carbonate, A.R.

3.3. Sulphuric acid, approximately 0ꞏ1 N, coloured with methyl red.

4. APPARATUS

Scheibler-Dietrich apparatus (see diagram) or equivalent apparatus.

5. PROCEDURE

According to the sample's carbonate content, weigh a portion of the sample as

shown below

0ꞏ5 g

for products containing from 50 to 100% of carbonates, expressed as

calcium carbonate;

1 g

for products containing from 40 to 50% of carbonates, expressed as

calcium carbonate;

2 to 3 g

for other products.

Place the portion of the sample in the special flask (4) of the apparatus, fitted with

a small tube of unbreakable material containing 10 ml of hydrochloric acid (3.1), and

connect the flask to the apparatus. Turn the three-way cock (5) so that the tube (1)

connects with the outside. Using the mobile tube (2), which is filled with coloured

sulphuric acid (3.3) and connected to the graduated tube (1), bring the level of the

liquid up to the zero mark. Turn the cock (5) in order to connect up tubes (1) and (3)

and check that the level is at zero.

Run the hydrochloric acid (3.1) slowly over the portion of the sample, tilting the

flask (4). Make the pressure equal by lowering the tube (2). Shake the flask (4) until

the release of carbon dioxide has stopped completely.

Restore pressure by bringing the liquid back to the same level in tubes (1) and (2).

After a few minutes, when the volume of gas has become constant, take the reading.

Carry out a control test in the same conditions on 0ꞏ5 g of calcium carbonate (3.2).

6. CALCULATION OF RESULTS

The content in grammes of carbonates, expressed as calcium carbonate, as a

percentage of the sample, is calculated by using the formula

V x 100

T x 2W

where

V = ml of CO 2 released by the portion of the sample.

T = ml of CO 2 released by 0ꞏ5 g of CaCO 3 A.R.

W = weight, in grammes, of the portion of the sample.

7. OBSERVATIONS

7.1. When the portion of the sample weighs more than 2 g, first place 15 ml of

distilled water in the flask (4) and mix before beginning the test. Use the same

volume of water for the control test.

7.2. If the apparatus used has a different volume from that of the Scheibler-

Dietrich apparatus, the portions taken from the sample and from the control

substance and the calculation of the results must be adapted accordingly.

5. DETERMINATION OF CRUDE ASH

1. PURPOSE AND SCOPE

This method makes it possible to determine the crude ash content of feedingstuffs.

2. PRINCIPLE

The sample is ashed at 550ºC; the residue is weighed.

3. REAGENTS

20% solution (w/v) of ammonium nitrate.

4. APPARATUS

4.1. Hot-plate.

4.2. Electric muffle-furnace with thermostat.

4.3. Crucibles for ashing made of platinum or an alloy of platinum and gold

(10% Pt, 90 Au), either rectangular (60 × 40 × 25 mm) or circular (diameter: 60 to 75

mm, height: 20 to 25 mm).

5. PROCEDURE

Weigh out to the nearest mg approximately 5 g of the sample (2ꞏ5 in the case of

products which have a tendency to swell) and place in a crucible for ashing which

has first been calcined and tared. Place the crucible on the hot-plate and heat

gradually until the substance carbonises. Put the crucible into the muffle-furnace set

at 550ºC ± 5ºC. Keep at this temperature until white, light grey or reddish ash is

obtained which appears to be free from carbonaceous particles. Place the crucible in

a desiccator, leave to cool and weigh immediately.

6. CALCULATION OF RESULTS

Calculate the weight of the residue by deducting the tare.

Express the result as a percentage of the sample.

7. OBSERVATIONS

7.1. The ash of substances which are difficult to ash must be subjected to an

initial ashing of at least three hours, cooled and then a few drops of 20% solution of

ammonium nitrate added to it (carefully, to avoid dispersal of the ash or the

formation of lumps). Continue calcining after drying in the oven. Repeat the

operation as necessary until ashing is complete.

7.2. In the case of substances resistant to the treatment described under 7.1,

proceed as follows: after ashing for three hours, place the ash in warm water and

filter through a small, ash-free filter. Ash the filter and its contents in the original

crucible. Place the filtrate in the cooled crucible, evaporate until dry, ash and weigh.

7.3. In the case of oils and fats, weigh accurately a sample of approximately 25 g

in a suitably sized crucible. Carbonise by setting light to the substance with a strip of

ash-free filter paper. After combustion, moisten with as little water as possible. Dry

and ash as described under 5.

6. DETERMINATION

OF

ASH

WHICH

IS

INSOLUBLE

IN

HYDROCHOLORIC ACID

1. PURPOSE AND SCOPE

This method makes it possible to determine the level in feeding-stuffs of mineral

substances which are insoluble in hydrochloric acid. Two methods can be used,

depending on the nature of the sample.

1.1. Method A: applicable to straight organic feeding-stuffs and to most

compound feeding-stuffs;

1.2. Method B: applicable to mineral compounds and mixtures and to compound

feeding-stuffs whose content in substances insoluble in hydrochloric acid, as

determined by Method A, is greater than 1%.

2. PRINCIPLE

2.1. Method A: the sample is ashed, the ash boiled in hydrochloric acid and the

insoluble residue filtered and weighed.

2.2. Method B: the sample is treated with hydrochloric acid. The solution is

filtered, the residue ashed and the ash thus obtained treated in accordance with

Method A.

3. REAGENTS

3.1. Hydrochloric acid 3 N.

3.2. 20% solution (w/v) of trichloroacetic acid.

3.3. 1% solution (w/v) of trichloroacetic acid.

4. APPARATUS

4.1. Hot plate.

4.2. Electric muffle-furnace with thermostat.

4.3. Crucibles for ashing made of platinum or an alloy of platinum and gold

(10% Pt, 90% Au), either rectangular (60 × 40 × 25 mm) or circular (diameter 60 to

75 mm, height: 20 to 25 mm).

5. PROCEDURE

5.1. Method A

Ash the sample using the method described for the determination of crude ash.

Ash obtained from that analysis may also be used. Place the ash in a 250 to 400 ml

beaker using 75 ml of hydrochloric acid 3 N (3.1). Bring slowly to the boil and boil

gently for fifteen minutes. Filter the warm solution through an ash-free filter paper

and wash the residue with warm water until the acid reaction is no longer visible.

Dry the filter containing the residue and ash in a tared crucible at a temperature of

not less than 550ºC and not more than 700ºC. Cool in a desiccator and weigh.

5.2. Method B

Weigh 5 g of the sample to the nearest mg and place in a 250 to 400 ml beaker.

Add 25 ml of water and 25 ml of hydrochloric acid 3 N (3.1) successively, mix and

wait for effervescence to cease. Add a further 50 ml of hydrochloric acid 3 N (3.1).

Wait for any release of gas to cease then place the beaker in a boiling water bath and

keep it there for thirty minutes or longer, if necessary, in order to hydrolyse

thoroughly any starch which may be present.

Filter while warm through an ash-free filter and wash the filter in 50 ml of warm

water (see observation 7). Place the filter containing the residue in a crucible for

ashing, dry and ash at a temperature of not less than 550ºC and not more than 700ºC.

Place the ash in a 250 to 400 ml beaker using 75 ml of hydrochloric acid 3 N (3.1);

continue as described in the second subparagraph of 5.1.

6. CALCULATION OF RESULTS

Calculate the weight of the residue by deducting the tare. Express the result as a

percentage of the sample.

7. OBSERVATION

If filtration proves difficult recommence the analysis, replacing the 50 ml of

hydrochloric acid 3 N (3.1) by 50 ml of 20% trichloroacetic acid (3.2) and washing

the filter in a warm solution of 1% trichloroacetic acid (3.3).

7. DETERMINATION OF CHLORINE FROM CHLORIDES

1. PURPOSE AND SCOPE

This method makes it possible to determine the amount of chlorine in chlorides

which are soluble in water, conventionally expressed as sodium chloride. It is

applicable to all feeding-stuffs.

2. PRINCIPLE

The chlorides are dissolved in water. If the product contains organic matter it is

clarified. The solution is slightly acidified with nitric acid and the chlorides

precipitated in the form of silver chloride by means of a solution of silver nitrate.

The excess silver nitrate is titrated with a solution of ammonium thiocyanate, by

Volhard’s method.

3. REAGENTS

3.1. Solution of ammonium thiocyanate 0.1 N.

3.2. Solution of silver nitrate 0.1 N.

3.3. Saturated solution of ammonium ferric sulphate.

3.4. Nitric acid, d: 1.38.

3.5. Diethyl ether A.R.

3.6. Acetone A.R.

3.7. Carrez I solution: dissolve in water 21.9 g of zinc acetate, Zn (CH 3 COO) 2 ꞏ

2H 2 O and 3 g of glacial acetic acid. Make up to 100 ml with water.

3.8. Carrez II solution{ dissolve in water 10ꞏ6 g of potassium ferrocynaide K 4 Fe

(CN) 63 ꞏ 3H 2 O. Make up to 100 ml with water.

3.9. Active carbon A.R., free from chlorides and not absorbing them.

4. APPARATUS

Mixer (tumbler): approximately 35 to 40 rpm.

5. PROCEDURE

5.1. Preparation of the solution

According to the nature of the sample, prepare a solution as shown under 5.1.1,

5.1.2 or 5.1.3.

At the same time carry out a blank test omitting the sample to be analysed.

5.1.1. Samples free from organic matter

Weigh to the nearest mg a sample of not more than 10 g and

containing not more than 3 g of chlorine in the form of chlorides.

Place with 400 ml of water in a 500 ml volumetric flask at

approximately 20ºC. Mix for thirty minutes in the tumbler, bring up

to volume, homogenise and filter.

5.1.2. Samples containing organic matter, excluding the products listed

under 5.1.3.

Weigh approximately 5 g of the sample to the nearest mg and place

with1 g of active carbon in a 500 ml volumetric flask. Add 400 ml of

water at approximately 20ºC and 5 ml of Carrez solution I (3.7), stir

then add 5 ml of Carrez solution II (3.8). Mix for thirty minutes in the

tumbler, bring up to volume, homogenise and filter.

5.1.3. Cooked feeding-stuffs, flax cakes and flour, products rich in flax flour

and other products rich in mucilage or in colloidal substances (for

example, dextrinated starch)

Prepare the solution as described under 5.1.2 but do not filter. Decant

(if necessary centrifuge), remove 100 ml of the supernatant liquid and

transfer to a 200 ml measuring flask. Mix with acetone (3.6) and

bring up to volume with this solvent, homogenise and filter.

5.2. Titration

Using a pipette, transfer to an Erlenmayer flask from 25 ml to 100 ml of the

filtrate (according to the assumed chlorine content) obtained as described under

5.1.1, 5.1.2 or 5.1.3. The aliquot portion must not contain more than 150 mg of

chlorine (C l). Dilute if necessary to not less than 50 ml with water, add 5 ml of nitric

acid (3.4.), 20 ml of saturated solution of ammonium ferric sulphate (3.3) and two

drops of ammonium thiocyanate solution (3.1) transferred by means of a burette

filled up to the zero mark. Using a burette, transfer the silver nitrate solution (3.2) in

such a way that an excess of 5 ml is obtained. Add 5 ml of diethyl ether (3.5) and

shake hard to coagulate the precipitate.

Titrate the excess silver nitrate with the ammonium thiocyanate solution (3.1)

until the reddish-brown tint has lasted for one minute.

6. CALCULATION OF RESULTS

The amount of chlorine (w), expressed as sodium chloride, present in the volume

of filtrate taken for titration is calculated by using the following formula

W = 5ꞏ845 (V 1 - V 2 ) mg

where

V 1 = ml of silver nitrate solution Cꞏ1 N added

V 2 = ml of ammonium thiocyanate solution 0ꞏ1 N used for titration.

If the blank test indicates that silver nitrate solution 0ꞏ1 N has been consumed

deduct this value from the volume (V 1 - V 2 ).

7. OBSERVATIONS

7.1. Titration may also be carried out by potentiometry;

7.2. In the case of products which are very rich in oils and fats, first de-fat with

diethyl ether or light petroleum;

7.3. In the case of fish-meal, titration may be carried out by Mohr’s method.

DETERMINATION OF LACTOSE

1. PURPOSE AND SCOPE

This method makes it possible to determine the level of lactose in feedingstuffs

containing more than 0ꞏ5% of lactose.

2. PRINCIPLE

The sugars are dissolved in water. The solution is subjected to fermentation by the

yeast Saccharomyces cerevisiae which leaves the lactose intact. After clarification

and filtration the lactose content of the filtrate is determined by the Luff-Schoorl

method.

3. REAGENTS

3.1. Suspension of Saccharomyces cerevisiae{ suspend 25 g of fresh yeast in 100

ml of water. The suspension will keep for a maximum period of one week in a

refrigerator.

3.2. Carrez solution I: dissolve in water 21ꞏ9 g of zinc acetate Zn (CH 3 COO) 2 ꞏ

2H 2 O and 3 g of glacial acetic acid. Make up to 100 ml with water.

3.3. Carrez solution II: dissolve in water 10ꞏ6 g of potassium ferrocyanide K 4 Fe

(CN) 6 ꞏ 3H 2 O. Make up to 100 ml with water.

3.4. Luff-Schoorl reagent

Stirring carefully, pour the citric acid solution (3.4.2) into the sodium carbonate

solution (3.4.2). Add the copper sulphate solution (3.4.1) and make up to 1 litre with

water. Leave to settle overnight and filter. Check the normality of the reagent thus

obtained (Cu 0ꞏ1 N; Na 2 CO 3 2N).

The solution’s pH should be approximately 9ꞏ4.

3.4.1. Copper sulphate solution: dissolve 25 g of copper sulphate A.R. Cu

SO 4 5H 2 O, free from iron, in 100 ml of water.

3.4.2. Citric acid solution: dissolve 50 g of citric acid A.R. C 6 H 8 O 7 ꞏ H 2 O in

50 ml of water.

3.4.3. Sodium carbonate solution: dissolve 143ꞏ8 g of anhydrous sodium

carbonate A.R. in approximately 300 ml of warm water. Leave to

cool.

3.5. Granulated pumice stone boiled in hydrochloric acid, washed in water and

dried.

3.6. 30% solution (w/v) of sodium iodide.

3.7. Sulphuric acid 6 N.

3.8. Solution of sodium thiosulphate 0ꞏ1 N.

3.9. Starch solution{ add a mixture of 5 g of soluble starch in 30 ml of water to 1

litre of boiling water. Boil for three minutes, leave to cool, and if necessary add 10

mg of mercuric iodide as a preservative.

4. APPARATUS

Water bath with thermostat set at 38-40ºC.

5. PROCEDURE

Weigh 1 g of the sample to the nearest mg and place this portion of the sample in a

100 ml volumetric flask. Add 25 to 30 ml of water. Place the flask in a boiling water

bath for thirty minutes and then cool to approximately 35ºC. Add 5 ml of yeast

suspension (3ꞏ1) and homogenise. Leave the flask to stand for two hours in a water

bath, at a temperature of 38-40ºC. Cool to approximately 20ºC.

Add 2ꞏ5 ml of Carrez solution I (3.2) and stir for thirty seconds, then add 2ꞏ5 ml of

Carrez solution II (3.3) and again stir for thirty seconds. Make up to 100 ml with

water, mix and filter. Using a pipette, remove an amount of filtrate which does not

exceed 25 ml and which preferably contains from 40 to 80 mg of lactose and transfer

it to a 300 ml Erlenmeyer flask. If necessary, make up to 25 ml with water.

Carry out a blank test in the same way with 5 ml of yeast suspension (3.1).

Determine the lactose content according to Luff-Schoorl, as follows: add exactly

25 ml of Luff-Schoorl reagent (3.4) and two granules of pumice stone (3.5). Stir by

hand-while heating over a free flame of medium height and bring the liquid to the

boil in approximately two minutes. Place the Erlenmeyer immediately on an

asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which

a flame has been lit. The flame shall be regulated in such a way that only the base of

the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for

exactly ten minutes. Cool immediately in cold water and after approximately five

minutes titrate as follows

Add 10 ml of potassium iodide solution (3.6) and immediately afterwards

(carefully, because of the risk of abundant foaming) add 25 ml of sulphuric acid 6 N

(3.7). Titrate with sodium thiosulphate solution 0ꞏ1 N (3.8) until a dull yellow colour

appears, add the starch indicator (3.9) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-

Schoorl reagent (3.4) and 25 ml of water, after adding 10 ml of potassium iodide

solution (3.6) and 25 ml of sulphuric acid 6 N (3.7) without boiling.

6. CALCULATION OF RESULTS

Using the attached table, establish the amount of lactose in mg which corresponds

to the difference between the results of the two titrations, expressed in ml of sodium

thiosulphate 0ꞏ1 N.

Express the result as parts of anhydrous lactose as a percentage of the sample.

7. OBSERVATION

For products containing more than 40% of fermentable sugar, use more than 5 ml

of yeast suspension (3.1).

Table of values for 25 ml of Luff-Schoorl reagent

ml of Na 2 S 2 O 3 0ꞏ1 N, two minutes’ heating, ten minutes’ boiling

Na 2 S 2 O 3 0ꞏ1

Glucose, fructose

Lactose

Maltose

Na 2 S 2 O 3

N

invert sugars

G 12 H 22 O 11

C 12 H 22 O 11

0ꞏ1 N

ml

mg

difference

mg

difference

mg

difference

ml

2.4

2.4

3.6

3.7

3.9

3.9

4.8

2.4

7.3

3.7

7.8

3.9

7.2

2.5

11. 0 3.7 11.7 3.9 9.7 2.5

14. 7 3.7

15. 6 4.0 12.2 2.5

18. 4 3.7

19. 6 3.9 14.7 2.5

22. 1 3.7

23. 5 4.0 17.2 2.6

25. 8 3.7

27. 5 4.0 19.8 2.6

29. 5 3.7

31. 5 4.0 22.4 2.6

33. 2 3.8

35. 5 4.0 25.0 2.6

37. 0 3.8

39. 5 4.0 25.0 2.4 37.0 3.8 39.5 4.0 30.3 2.7

44. 6 3.8

47. 5 4.1 33.0 2.7

48. 4 3.8

51. 6 4.1 35.7 2.8

52. 2 3.8

55. 7 4.1 38.5 2.8

56. 0 3.9

59. 8 4.1 41.3 2.9 59.9 3.9

9

4.1

Na 2 S 2 O 3 0ꞏ1

Glucose, fructose

Lactose

Maltose

Na 2 S 2 O 3

N

invert sugars

G 12 H 22 O 11

C 12 H 22 O 11

0ꞏ1 N

ml

mg

difference

mg

difference

mg

difference

ml

44.2

2.9

63.8

3.9

68. 0 4.2 47.1 2.9 67.7 4.0

72. 2 4.3 50.0 3.0 71.7 4.0

76. 5 4.4 53.0 3.0 75.7 4.1

80. 9 4.5 56.0 3.1 79.8 4.1

85. 4 4.6 59.1 3.1 83.9 4.1

90. 0 4.6 62.2 88.0

6

10. DETERMINATION OF POTASSIUM

1. PURPOSE AND SCOPE

This method makes it possible to determine the level of potassium in feeding-

stuffs.

2. PRINCIPLE

The sample is ashed and the ash dissolved in hydrochloric acid. The potassium

content of the solution is determined by flame photometry in the presence of caesium

chloride and aluminium nitrate. The addition of these substances largely eliminates

interference from disturbing elements.

3. REAGENTS

3.1. Hydrochloric acid A.R., d: 1.12.

3.2. Caesium chloride A.R.

3.3. Aluminium nitrate Al (NO 3 ) 3 ꞏ 9 H 2 O, general purpose reagent.

3.4. Potassium chloride A.R., anhydrous.

3.5. Loading agent{ dissolve in water 50 g of caesium chloride (3.2) and 250 g of

aluminium nitrate (3.3), make up to 1 litre with water and homogenise. Store in

plastic bottles.

3.6. Standard solution of potassium: dissolve in water 1ꞏ907 g of potassium

chloride (3.4), add 5 ml of hydrochloric acid (3.1), make up to 1 litre with water and

homogenise. Store in plastic bottles. 1 ml of this solution contains 1ꞏ00 mg of

potassium

4. APPARATUS

4.1. Platinum, silica or porcelain crucibles for ashing, provided if necessary with

lids.

4.2. Electric muffle-furnace with thermostat.

4.3. Flame photometer.

5. PROCEDURE

5.1. Analysis of sample

As a general rule, weigh out 10 g of the sample to the nearest 10 mg, place in a

crucible and ash at 450ºC for three hours. After cooling transfer the ash

quantitatively to a 500 ml graduated flask, using 250 to 300 ml of water and then 50

ml of hydrochloric acid (3.1). When all release of carbon dioxide has ceased, heat

the solution and keep at a temperature of about 90ºC for two hours, stirring

occasionally. After cooling to room temperature, make up to the mark with water,

shake and filter. Transfer to a 100 ml graduated flask an aliquot part of the filtrate

containing a maximum of 1ꞏ0 mg of potassium, add 10ꞏ0 ml of loading agent (3.5),

make up to the mark with water and homogenise. In the case of higher levels of

potassium dilute the solution to be analysed in suitable proportions before adding the

loading agent.

The table below is given as a guide for a sample of about 10 g.

Assumed potassium content

Dilution factor

Aliquot part in ml of the

of the sample (% K)

solution

up to 0ꞏ1

-

0ꞏ1 to 0ꞏ5

-

0ꞏ5 to 1ꞏ0

-

1ꞏ0 to 5ꞏ0

1: 10

5ꞏ0 to 10ꞏ0

1: 10

10ꞏ0 to 20ꞏ0

1: 20

Measure by flame photometry at a wavelength of 768 nm. Calculate the result by

means of a calibration curve.

5.2. Calibration curve

Place exactly 10 ml of the standard solution (3.6) in a 250 ml graduated flask,

make up to the mark with water and homogenise. Place in 100 ml graduated flasks

exactly 5, 10, 15, 20 and 25 ml of this solution, corresponding respectively to

amounts of potassium of 0ꞏ2, 0ꞏ4, 0ꞏ6, 0ꞏ8 and 1ꞏ0 mg. Complete the series with a

blank flask containing no standard solution. Add 10 ml of loading agent (3.5) to each

flask, make up to the mark with water and homogenise. Carry out the measurements

as indicated in 5.1. The calibration curve is generally linear up to a potassium

concentration of 1 mg in 100 ml of solution.

6. CALCULATION OF RESULTS

Express the result as a percentage of the sample.

7. OBSERVATIONS

It is not always necessary to add loading agent (3.5) in order to eliminate the

interference of disturbing elements.

11. DETERMINATION OF SODIUM

1. PURPOSE AND SCOPE

This method makes it possible to determine the level of sodium in feedings stuffs.

2. PRINCIPLE

The sample is ashed and the ash dissolved in hydrochloric acid. The sodium

content of the solution is determined by flame photometry in the presence of caesium

chloride and aluminium nitrate. The addition of these substances largely eliminates

interference from disturbing elements.

3. REAGENTS

3.1. Hydrochloric acid A.R., d: 1.12.

3.2. Caesium chloride A.R.

3.3. Aluminium nitrate Al (NO 3 ) 3 ꞏ 9 H 2 O, general purpose reagent

3.4. Sodium chloride A.R., anhydrous.

3.5. Loading agent: dissolve in water 50 g of caesium chloride (3.2) and 250 g of

aluminium nitrate (3.3), make up to 1 litre with water and homogenise. Store in

plastic bottles.

3.6. Standard solution of sodium: dissolve in water 2ꞏ542 g of sodium chloride

(3.4), add 5 ml of hydrochloric acid (3.1), make up to 1 litre with water and

homogenise Store in plastic bottles, 1 ml of this solution contains 1ꞏ00 mg of

sodium.

4. APPARATUS

4.1. Platinum, silica or porcelain crucibles for ashing, provided if necessary with

lids.

4.2. Electric muffle-furnace with thermostat.

4.3. Flame photometer.

5. PROCEDURE

5.1. Analysis of sample

As a general rule, weigh out 10 g of the sample to the nearest 10 mg, place in a

crucible (4.2) and ash at 450ºC for three hours. Avoid overheating (ignition). After

cooling transfer the ash quantitatively to a 500 ml graduated flask, using 250 to 300

ml of water and then 50 ml of hydrochloric acid (3.1). When all release of carbon

dioxide has ceased, heat the solution and keep at a temperature of about 90ºC for two

hours, stirring occasionally. After cooling to room temperature, make up to the mark

with water, shake and filter. Transfer to a 100 ml graduated flask an aliquot part of

the filtrate containing a maximum of 1ꞏ0 mg of sodium, add 10ꞏ0 ml of loading agent

(3.5), make up to the mark with water and homogenise. In the case of higher levels

of sodium, dilute the solution to be analysed in suitable proportions before adding

the loading agent.

The table below is given as a guide for a sample of about 10 g.

Assumed sodium content of

Dilution factor

Aliquot part in ml of the

the sample (% K)

solution

up to 0ꞏ1

-

0ꞏ1 to 0ꞏ5

-

0ꞏ5 to 1ꞏ0

-

1ꞏ0 to 5ꞏ0

1: 10

5ꞏ0 to 10ꞏ0

1: 10

10ꞏ0 to 20ꞏ0

1: 20

Measure by flame photometry at a wavelength of 589 nm. Calculate the result by

means of a calibration curve.

5.2. Calibration curve

Place exactly 10 ml of the standard solution (3.6) in a 250 ml graduated flask,

make up to the mark with water and homogenise. Place in 100 ml graduated flasks

exactly 5, 10, 15, 20 and 25 ml of this solution, corresponding respectively to

amounts of sodium of 0ꞏ2, 0ꞏ4, 0ꞏ6, 0ꞏ8 and 1ꞏ0 mg. Complete the series with a blank

flask containing no standard solution. Add 10 ml of loading agent (3.5) to each flask,

make up to the mark with water and homogenise. Carry out the measurements as

in di cat ed in 5 .1. T he cal ib rati on c urve is g eneral ly l in ear up t o a so di um

concentration of 1 mg in 100 ml of solution.

6. CALCULATION OF RESULTS

Express the result as a percentage of the sample.

7. Observations

7.1. For products containing more than 4% of sodium, it is preferable to ash the

substance for two hours in a crucible with a lid. After cooling, add water, bring the

ash into suspension by means of a platinum wire, dry and ash again for two hours in

the crucible with a lid.

7.2. If the sample consists solely of mineral substances, dissolve without prior

ashing.

12. DETERMINATION OF SUGAR

1. PURPOSE AND SCOPE

This method makes it possible to determine the amount of reducing sugars and

total sugars after inversion, expressed as glucose or where appropriate as sucrose,

converting by the factor 0ꞏ95. It is applicable to compound feeding-stuffs. Special

methods are provided for other feeding-stuffs. Where necessary, lactose should be

measured separately and taken into account when calculating the results.

2. PRINCIPLE

The sugars are extracted in dilute ethanol[ the solution is clarified with Carrez

solutions I and II. After eliminating the ethanol, the quantities before and after

inversion are determined by the Luff-Schoorl method.

3. REAGENTS

3.1. 40% ethanol (v/v) d: 0ꞏ948 at 20ºC, neutralised to phenolphthalein.

3.2. Carrez solution I: dissolve in water 21ꞏ9 g of zinc acetate Zn (CH 3 COO) 2 ꞏ

2H 2 O and 3 g of glacial acetic acid. Make up to 100 ml with water.

3.3. Carrez solution II: dissolve in water 10ꞏ6 g of potassium ferrocyanide K 42 Fe

(CN) 6 ꞏ 3H 2 O. Make up to 100 ml with water.

3.4.‘0ꞏ1% solution (w/v) of methyl orange.

3.5. Hydrochloric acid 4 N.

3.6. Hydrochloric acid 0ꞏ1 N.

3.7. Sodium hydroxide solution 0ꞏ1 N.

3.8. Luff-Schoorl reagent

Stirring carefully, pour the citric acid solution (3.8.2) into the sodium carbonate

solution (3.8.3). Add the copper sulphate solution (3.8.1) and make up to 1 litre with

water. Leave to settle overnight and filter. Check the normality of the reagent thus

obtained (Cu 0ꞏ1 N; Na 2 CO 3 2 N).

The solution’s pH should be approximately 9ꞏ4.

3.8.1. Copper sulphate solution: dissolve 25 g of copper sulphate A.R., Cu

SO 4 ꞏ 5H 2 O, free from iron, in 100 ml of water.

3.8.2. Citric acid solution: dissolve 50 g of citric acid A.R., C 6 H 8 O 7 H 2 O in

50 ml of water.

3.8.3. Sodium carbonate solution: dissolve 143ꞏ8 g of anhydrous sodium

carbonate A.R. in approximately 300 ml of warm water. Leave to

cool.

3.9. Sodium thiosulphate solution 0ꞏ1 N

3.10. Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1

litre of boiling water. Boil for three minutes, leave to cool and if necessary add 10

mg of mercuric iodide as a preservative.

3.11. Sulphuric acid 6 N.

3.12. 30% solution (w/v) of potassium iodide.

3.13. Granulated pumice stone boiled in hydrochloric acid, washed in water and

dried.

3.14. 3-methylbutan-l-ol.

4. APPARATUS

Mixer (tumbler): approximately 35 to 40 rpm.

5. PROCEDURE

5.1. Extraction of sample

Weigh 2.5 g of the sample to the nearest mg and place in a 250 ml volumetric

flask. Add 200 ml of ethanol (3.1) and mix in the tumbler for one hour. Add 5 ml of

Carrez solution I (3.2) and stir for one minute. Add 5 ml of Carrez solution II (3.3)

and again stir for one minute. Make up to volume with ethanol (3.1), homogenise

and filter. Remove 200 ml of the filtrate and evaporate to approximately half volume

in order to eliminate most of the ethanol. Transfer the evaporation residue

quantitatively to a 200 ml volumetric flask using warm water, cool, bring up to

volume with water, homogenise and filter if necessary. This solution will be used to

determine the amount of reducing sugars and, after inversion, of total sugars.

5.2. Determination of reducing sugars

Using a pipette, remove not more than 25 ml of the solution containing less than

60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with

distilled water and determine the content of reducing sugars by the Luff-Schoorl

method. The result is expressed as the percentage content of glucose in the sample.

5.3. Determination of total sugars after inversion

Using a pipette take 50 ml of the solution and transfer to a 100 ml volumetric

flask. Add a few drops of methyl orange solution (3.4) then, carefully and stirring

continuously, add hydrochloric acid 4 N (3.5) until the liquid turns a definite red.

Add 15 ml of hydrochloric acid 0ꞏ1 N (3.6), immerse the flask in a fast boiling water

bath and keep there for thirty minutes. Cool rapidly to approximately 20ºC and add

15 ml of sodium hydroxide solution 0ꞏ1 N (3.7). Make up to 100 ml with water and

homogenise. Remove not more than 25 ml containing less than 60 mg of reducing

sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and

determine the content of reducing sugars by the Luff-Schoorl method. The result is

expressed as the percentage of glucose or, where appropriate, sucrose, by

multiplying by the factor 0ꞏ95.

5.4. Titration by the Luff-Schoorl method

Using a pipette, take 25 ml of Luff-Schoorl reagent (3.8) and transfer to a 300 ml

Erlenmeyer flask; add exactly 25 ml of the clarified sugar solution. Add 2 granules

of pumice stone (3.13), heat, stirring by hand, over a free flame of medium height

and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer

immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in

diameter under which a flame has been lit. The flame shall be regulated in such a

way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the

Erlenmeyer flask, Boil for exactly ten minutes. Cool immediately in cold water and

after approximately five minutes titrate as follows

Add 10 ml of potassium iodide solution (3.12) and immediately afterwards

(carefully, because of the risk of abundant foaming), add 25 ml of sulphuric acid 6 N

(3.11). Titrate with sodium thiosulphate solution 0ꞏ1 N (3.9) until a dull yellow

colour appears, add the starch indicator (3.10) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-

Schoorl reagent (3.8) and 25 ml of water, after adding 10 ml of potassium iodide

solution (3.12) and 25 ml of sulphuric acid 6 N (3.11) without boiling.

6. CALCULATION OF RESULTS

Using the table establish the amount of glucose in mg which corresponds to the

difference between the values of the two titrations, expressed in mg of sodium

thiosulphate 0ꞏ1 N.

Express the result as a percentage of the sample.

7. SPECIAL PROCEDURES

7.1. In the case of feeding-stuffs which are rich in molasses and other feeding-

stuffs which are not particularly homogeneous, weigh out 20 g and place with 500 ml

of water in a 1 litre volumetric flask. Mix for one hour in the tumbler. Clarify using

Carrez 1 (3.2) and II (3.3) reagents as described under 5.1, this time however using

four times the quantities of each reagent. Bring up to volume with 80% ethanol (v/v).

Homogenise and filter. Eliminate the ethanol as described under 5.1. If there is no

dextrinised starch, bring up to volume with distilled water.

7.2. In the case of molasses and straight feeding-stuffs which are rich in sugar

and almost starch-free (carobs, dried beetroot cossettes etc.), weigh out 5 g, place in

a 250 ml volumetric flask, add 200 ml of distilled water and mix in the tumbler for

one hour, or more if necessary. Clarify using Carrez I (3.2) and II (3.3) reagents as

described under 5.1. Bring up to volume with cold water, homogenise and filter. In

order to determine the amount of total sugars, continue as described under 5.3.

8. OBSERVATIONS

8.1. In order to prevent foaming it is advisable to add (irrespective of the

volume) approximately 1 ml of 3-methylbutan-l-ol (3.14) before boiling with Luff-

Schoorl reagent.

8.2. The difference between the content of total sugars after inversion, expressed

as glucose, and the content of reducing sugars, expressed as glucose, multiplied by

0ꞏ95, gives the percentage content of sucrose.

8.3. In order to determine the content of reducing sugars, excluding lactose, two

methods may be adopted

8.3.1. for an approximate calculation, multiply by 0ꞏ675 the lactose content

established by a different method of analysis and subtract the result

obtained from the content of reducing sugars.

8.3.2. For an accurate calculation of reducing sugars, excluding lactose, the

same sample must be used for the two final determinations. One of

the analyses is carried out on part of the solution obtained under 5.1,

the other on part of the solution obtained during the determination of

lactose by the method laid down for that purpose (after fermenting

the other types of sugar and clarifying).

In both cases the amount of sugar present is determined by the Luff-Schoorl

method and calculated in mg of glucose. One of the values is subtracted from the

other and the difference is expressed as a percentage of the sample.

Example

The two volumes taken correspond, for each determination, to a sample of 250

mg.

In the first case 17 ml of sodium thiosulphate solution 0ꞏ1 N corresponding to

44ꞏ2 mg of glucose is consumed; in the second, 11 ml, corresponding to 27ꞏ6 mg of

glucose.

The difference is 16ꞏ6 mg of glucose.

The content of reducing sugars (excluding lactose), calculated as glucose, is

therefore

4 x 16 x 6

= 6.64%

Table of values for 25 ml of Luff Schoorl reagent

Na 2 S 2 O 3 0ꞏ1

Glucose, fructose

Lactose

Maltose

Na 2 S 2 O 3

N

invert sugars

G 12 H 22 O 11

C 12 H 22 O 11

0ꞏ1 N

ml

mg

difference

mg

difference

mg

difference

ml

2.4

2.4

3.6

3.7

3.9

3.9

4.8

2.4

7.3

3.7

7.8

3.9

7.2

2.5

11.0

3.7

11.7

3.9

9.7

2.5

14.7

3.7

15.6

4.0

12.2

2.5

18.4

3.7

19.6

3.9

14.7

2.5

22.1

3.7

23.5

4.0

17.2

2.6

25.8

3.7

27.5

4.0

19.8

2.6

29.5

3.7

31.5

4.0

22.4

2.6

33.2

3.8

35.5

4.0

25.0

2.6

37.0

3.8

39.5

4.0

27.6

2.7

40.8

3.8

43.5

4.0

30.3

2.7

44.6

3.8

47.5

4.1

33.0

2.7

48.4

3.8

51.6

4.1

35.7

2.8

52.2

3.8

55.7

4.1

38.5

2.8

56.0

3.9

59.8

4.1

41.3

2.9

59.9

3.9

63.9

4.1

44.2

2.9

63.8

3.9

68.0

4.2

47.1

2.9

67.7

4.0

72.2

4.3

50.0

3.0

71.7

4.0

76.5

4.4

53.0

3.0

75.7

4.1

80.9

4.5

56.0

3.1

79.8

4.1

85.4

4.6

59.1

3.1

83.9

4.1

90.0

4.6

62.2

88.0

94.6

14. DETERMINATION OF UREA

1. PURPOSE AND SCOPE

This method makes it possible to determine the level of urea in feedingstuffs.

2. PRINCIPLE

The sample is suspended in water with a clarifying agent. The suspension is

filtered. The urea content of the filtrate is determined after the addition of 4-

dimethylaminobenzaldehyde (4-DMAB) by measuring the optical density at a

wavelength of 420 nm.

3. REAGENTS

3.1. Solution of 4-dimethylaminobenzaldehyde{ dissolve 1ꞏ6 g of 4-DMAB A.R.

in 100 ml of 96% ethanol and add 10 ml of hydrochloric acid A.R. (d: 1ꞏ19). This

reagent keeps for a maximum period of two weeks.

3.2. Carrez solution I: dissolve in water 21ꞏ9 g of zinc acetate Zn (CH 3 COO) 2 ꞏ

2H 2 O and 3 g of glacial acetic acid. Make up to 100 ml with water.

3.3. Carrez solution II: dissolve in water. 10ꞏ6 g of potassium ferrocyanide, K 4

Fe (CN) 6 ꞏ 3H 2 O.

Make up to 100 ml with water.

3.4. Active carbon A.R. which does not absorb urea (to be checked).

3.5. 0ꞏ1% solution (w/v) of urea A.R.

4. APPARATUS

4.1. Mixer (tumbler): approximately 35 to 40 rpm.

4.2. Test tubes: 160 × 16 mm with ground-glass stoppers.

4.3. A spectrophotometer.

5. PROCEDURE

5.1. Analysis of sample

Weigh out 2 g of the sample to the nearest mg and place with 1 g of active carbon

(3.4) in a 500 ml volumetric flask. Add 400 ml of water and 5 ml of Carrez solutions

I (3.2) and II (3.3). Mix for thirty minutes in the tumbler. Make up to volume with

water, shake and filter.

Remove 5 ml of the transparent colourless filtrates, place in test tubes with

ground-glass stoppers, add 5 ml of 4-DMAB solution (3.1) and mix. Place the tubes

in a hot-water bath at 20ºC. After fifteen minutes measure the optical density of the

sample solution with the spectrophotometer at 420 nm. Compare with the blank test

solution of the reagents.

5.2. Calibration Curve

Remove volumes of 1, 2, 4, 5 and 10 ml of the urea solution (3.5), place in 100 ml

volumetric flasks and make up the volume with water. Remove 5 ml from each

solution, add 5 ml of 4-DMAB solution (3.1) to each of them, homogenise and

measure the optical density as shown above in comparison with a control solution

containing 5 ml of 4-DMAB and 5 ml of water free from urea. Plot the calibration

curve.

6. CALCULATION OF RESULTS

Determine the amount of urea in the sample using the calibration curve.

Express the result as a percentage of the sample.

7. OBSERVATIONS

7.1. In the case of contents of urea exceeding 3%, reduce the sample to 1 g or

dilute the original solution so that there are not more than 50 mg of urea in 500 ml.

7.2. In the case of low contents of urea, increase the sample as long as the filtrate

remains transparent and colourless.

7.3. If the sample contains simple nitrogenous compounds such as amino acids,

the optical density should be measured at 435 nm.

16. ESTIMATION OF THE UREASE ACTIVITY OF PRODUCTS

DERIVED FROM SOYA

1. PURPOSE AND SCOPE

This test makes it possible estimate the urease activity of products derived from

soya and to show whether these products have been cooked for a sufficient length of

time.

2. PRINCIPLE

Urease activity is estimated by the amount of ammoniacal nitrogen liberated per 1

g of product per minute at 30ºC from a solution of urea.

3. REAGENTS

3.1. Hydrochloric acid 0ꞏ1 N

3.2.

Solution of sodium hydroxide 0ꞏ1 N.

3.3. Loading agent of phosphate 0ꞏ05 M, containing, per 1000 ml, 4ꞏ45 g of

disodium phosphate (Na 2 HPO 4 ꞏ 2H 2 O) and 3ꞏ40 g of monopotassium phosphate

(KH 2 PO 4 ).

3.4. Freshly-prepared urea loading agent containing 30ꞏ0 g of urea per 1000 ml

of loading agent (3.3); pH 6ꞏ9-7ꞏ0.

4. APPARATUS

4.1. Potentiometric titration apparatus or high sensitivity pH-meter (0ꞏ02 pH)

with magnetic stirrer.

4.2. Water bath fitted with thermostat set at 30ºC exactly.

4.3. Test-tubes with ground-glass stoppers, 150 × 18 mm.

5. PROCEDURE

Crush about 10 g of the sample (for example in a coffee mill) so that it passes

through a sieve with a mesh of 0ꞏ2 mm. Weigh out 0ꞏ2 g of the crushed sample to the

nearest mg, place in a test tube with a ground-glass stopper and add 10 ml of urea

loading agent (3.4). Stopper immediately and shake vigorously. Place the tube in a

water bath set at 30ºC exactly and keep there for exactly thirty minutes.

Immediately add 10 ml of 0ꞏ1 N hydrochloric acid (3.1), cool rapidly to 20ºC and

transfer the contents of the tube quantitatively to a titration vessel, rinsing twice with

5 ml of water. Using a glass electrode (4.1) titrate immediately and rapidly to pH 4ꞏ7

with the 0ꞏ1 N sodium hydroxide solution (3.2) by electrometry.

Carry out a blank test as follows

Quickly place a sample of 0ꞏ2 g weighed to the nearest mg in a test tube with a

ground-glass stopper, add 10 ml of 0ꞏ1 N hydrochloric acid (3.1) and then 10 ml of

urea loading agent (3.4). Cool the tube immediately in ice-water and leave there for

thirty minutes. Under the conditions indicated above, transfer the contents of the

tube to the titration vessel using the 0ꞏ1 N sodium hydroxide solution (3.2) up to pH

4ꞏ7.

6. CALCULATION

The urease activity is calculated by using the formula

mg N

.4 (b – a)

AT 30°C =

g min

30 . E

where

a = ml of 0ꞏ1N sodium hydroxide solution consumed by the sample,

b = ml of 0ꞏ1N sodium hydroxide solution consumed in the blank test,

E = weight of sample in g.

7. OBSERVATION

7.1. This method is suitable for a urease activity of up to 1 mg of N`g`min at

30ºC. For more active products, the size of the sample can be reduced to 50 mg.

7.2. Products containing more than 10% of crude fatty substance must first be

defatted cold.