CONTROL OF CHEMICAL LEVELS IN THE CONTROL OF FEEDINGSTUFFS RULES *

ANALYSIS OF CHEMICAL LEVELS

IN THE CONTROL OF FEEDINGSTUFFS

SUBSIDIARY LEGISLATION 437.82

CONTROL OF CHEMICAL LEVELS IN THE

CONTROL OF FEEDINGSTUFFS RULES *

2nd September, 2005

LEGAL NOTICE 309 of 2005.

The title of these rules is the Analysis of Chemical Levels

in the Control of Feedingstuffs Rules.

1. DETERMINATION OF ZINC BACITRACIN

- by diffusion in an agar medium -

1. PURPOSE AND SCOPE

The method is for the determination of zinc bacitracin in feedingstuffs and

premixes. The lower limit of determination is 5 mg/kg (5 ppm) *

2. PRINCIPLE

The sample is extracted at pH 2 with a mixture of methanol/water/hydrochloric

acid, and a sodium sulphide solution. The addition of sodium sulphide is to

precipitate any soluble copper salts that may interfere with the assay. The extract is

brought to pH 6,5, concentrated (where necessary) and diluted. Its antibiotic activity

is determined by measuring the diffusion of zinc bacitracin in an agar medium

inoculated with Micrococcus luteus (flavus). Diffusion is shown by the formation of

zones of inhibition of the microorganism. The diameter of these Zones is taken to be

in direct proportion to the logarithm of the antibiotic concentration over the range of

antibiotic concentrations employed.

3. MICRO-ORGANISM: MICROCOCCUS LUTEUS (FLAVUS) ATCC

10240

3.1.

Maintenance of stock culture

Inoculate tubes containing slopes of culture medium (4.1) with Micrococcusluteus

(flavus) and incubate for 24 hours at 30ºC. Store the culture in a refrigerator at about

4ºC. Reinoculate every two weeks.

Preparation of the bacterial suspension †

3.2.

Harvest the growth from a recently prepared agar slope (3.1) with 2 to 3 ml of

sodium chloride solution (4.3). Use this suspension to inoculate 250 ml of culture

medium (4.1) contained in a Roux flask and incubate for 18 to 20 hours at 30ºC.

Harvest the growth in 25 ml of sodium chloride solution (4.3) and mix. Dilute the

suspension to 1/10 with sodium chloride solution (4.3). The light transmission of the

suspension must be about 75%, measured at 650 nm in a 1 cm cell against sodium

chloride solution (4.3). This suspension may be kept for one week at about 4ºC.

The scope of these regulations is to implement the rules

found under EU Commission Directive 78/633/EEC establishing

EC methods of analysis for the official control of feedingstuffs.

The analyses for official controls of feedingstuffs, as

regards their content of zinc bacitracin, flavophospholipol, iron,

copper, manganese and zinc, shall be carried out in accordance with

the methods described in the Schedule.

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

CULTURE MEDIA AND REAGENTS

Culture medium ‡

4.1.

Meat peptone

6,0 g

Tryptone

4,0 g

Yeast extract

3,0 g

Meat extract

1,5 g

Glucose

1,0 g

Agar

10,0 to 20,0 g

Water

1 000 ml

pH 6,5 to 6,6 (after sterilization).

*1mg feeding stuff grade zinc bacitricin is equivalent to 42 international units (i.u).

†Other methods may be used provided that it has been established that they give similar bacterial

suspensions.

‡Any commercial culture medium of similar composition and giving the same results may be used.

Assay medium *

4.2.

Tryptone

10,0 g

Yeast extract

3,0 g

Meat extract

1,5 g

Glucose

1,0 g

Agar

10,0 to 20,0 g

Tween 80

1 ml

Water

1 000 ml

pH 6,5 (after sterilization).

4.3.

Sodium chloride solution 0,8% (w/v): dissolve 8 g sodium chloride in

water and dilute to 1 000 ml; sterilize.

4.4.

Mixture of methanol/water hydrochloric acid (4.6): 80/17, 5/2, 5 (v/v/v).

4.5.

Phosphate buffer, pH 6,5

Potassium hydrogen phosphate K2HPO4 22,15 g.

Potassium dihydrogen phosphate KH2PO4 27,85 g.

Water to 1 000 ml.

4.6.

Hydrochloric acid (d: 1,18 to 1,19).

4.7.

Hydrochloric acid (0,1 M).

4.8.

Sodium hydroxide 1 M solution.

4.9.

Sodium sulphide about 0,5 M solution.

4.10. Bromocresol purple solution 0,04% (w/v): dissolve 0,1 g of bromocresol

purple in 18,5 ml of 0,01 M sodium hydroxide solution. Make up the volume to 250

ml with water and mix.

4.11.

Standard substance: zinc bacitracin of known activity (in i. u.).

STANDARD SOLUTIONS

Weigh out a quantity of standard zinc bacitracin (4.11) corresponding to 1 050 i.u.

(according to the activity indicated). Add 5 ml of 0,1 M hydrochloric acid (4.7) and

leave to stand for 15 minutes. Add 30 ml of water, adjust the pH to 4,5 with

phosphate buffer (4.5) (about 4 ml), make up to a volume of 50 ml with water and

mix well (1 ml = 21 i.u.). From this solution prepare by successive dilution with

phosphate buffer (4.5) the following solutions

s8 0,42 i.u./ml

s4 0,21 i.u./ml

s2 0,105 i.u./ml

s1 0,0525 i.u./ml

PREPARATION OF DIE EXTRACT AND ASSAY SOLUTIONS

6.1.

Extraction

6.1.1. Premixes and mineral feeds

*Any commercial culture medium of similar composition and giving the same results may be used.

Weigh out a quantity of sample of 2,0 to 5,0 g, add 29,0 ml of the mixture (4.4)

and 1,0 ml of sodium sulphide solution (4.9) and shake briefly. Check that the pH is

about 2. Shake for 10 minutes, add 30 ml of phosphate buffer (4.5), shake for 15

minutes and centrifuge. Take a suitable aliquot of the supernatant solution and adjust

the pH to 6,5 by means of 1 M hydroxide solution (4.8) with a pH-meter or with the

bromocresol purple solution (4.10) as indicator. Dilute with phosphate buffer (4.5) to

obtain an expected zinc bacitracin content of 0,42 i.u./ ml (= u8).

6.1.2. Protein concentrates

Weigh out a quantity of sample of 10,0 g, add 49,0 ml of the mixture (4.4) and 1,0

ml of sodium sulphide solution (4.9) and shake briefly. Check that the pH is about 2.

Shake for 10 minutes. Add 50 ml of phosphate buffer (4.5), shake for 15 minutes and

centrifuge. Take a suitable volume of the supernatant solution and adjust the pH to

6,5 by means of 1 M sodium hydroxide solution (4.8) with a pH-meter or with the

bromocresol purple solution (4.10) as indicator. Evaporate to approximately half

volume in a rotary evaporator at a temperature not exceeding 35ºC.

Dilute with phosphate buffer (4.5) to obtain an expected zinc bacitracin content of

0,42 i.u./ml (= u 8 ).

6.1.3. Other feeds

Weigh out a quantity of sample of 10,0 g (20,0 g for an expected zinc bacitracin

content of 5 mg/kg). Add a mixture of 24,0 ml of the mixture (4.4) and 1,0 ml of

sodium sulphide solution (4.9) and homogenize for 10 minutes. Add 25 ml of

phosphate buffer (4,5), shake for 15 minutes and centrifuge. Take 20 ml of the

supernatant solution and adjust the pH to 6.5 by means of 1 M sodium hydroxide

solution (4.8) with a pHmeter or with the bromocresol purple solution (4.10) as

indicator.

Evaporate to about 4 ml in a rotary evaporator at a temperature not exceeding

35ºC. Dilute the residue with phosphate buffer (4.5) to obtain an expected zinc

bacitracin content of 0,42 i.u./ml (= u 8 ).

6.2.

Assay solutions

From solution u 8 prepare solutions u 4 (expected content: 0,21 i.u./ml), u 2

(expected content: 0,105 i.u./ml) and u1 (expected content: 0,0525 i.u./ l) by means

of successive dilution (1 + 1) with phosphate buffer (4.5).

ASSAY PROCEDURE

7.1.

Inoculation of the assay medium

Inoculate the assay medium (4.2) with the bacterial suspension (3.2) at about

50ºC. By preliminary trials on plates with assay medium (4.2) determine the quantity

of bacterial suspension required to give the largest and clearest zones of inhibition

with the various concentrations of zinc bacitracin.

7.2.

Preparation of the plates

Diffusion through agar is carried out in plates with the four concentrations of the

standard solution (S 8 , S 4 , S 2 and S 1 ) and the four concentrations of the assay

solution (u 8 , u 4 , u 2 and u 1 ). These four concentrations of extract and standard must

necessarily be placed in each plate. To this effect, select plates big enough to allow

at least eight holes with a diameter of 10 to 13 mm and not less than 30 mm between

centres to be made in the agar medium. The test may be carried out on plates

consisting of a sheet of glass with a faced aluminium or plastic ring placed on top,

200 mm in diameter and 20 mm high.

Pour into the plates a quantity of the medium (4.2) inoculated as in point 7.1, to

give a layer about 2 mm thick (60 ml for a plate of 200 mm diameter), Allow to set in

a level position, bore the holes and place in them exactly measured volumes of assay

and standard solutions (between 0,10 and 0,15 ml per hole, according to the

diameter).

Apply each concentration at least four times so that each determination is subject

to an evaluation of 32 zones of inhibition.

7.3.

Incubation

Incubate the plates for 16 to 18 hours at 30 ± 2ºC.

EVALUATION

Measure the diameter of the zones of inhibition to the nearest 0,1 mm. Record the

mean measurements for each concentration on semi-logarithmic graph paper

showing the logarithm of the concentrations in relation to the diameters of the zones

of inhibition. Plot the best fit lines of both the standard solution and the extract, for

example as below

Determine the ‘best fit’ point for the standard lowest level (SL) using the formula

7s 1 + 4s 2 +s 4 - 2s 8

SL =

Determine the ‘best fit’ point for the standard highest level (SH) using the

formula

7s 8 + 4s 4 +s 2 - 2s 1

SH =

Similarly calculate the ‘best fit’ points for the extract lowest level (UL) and the

extract highest level (UH) by substituting u 1 , u 2 , u 4 and u 8 for s 1 , s 2 , s 4 and s 8 in the

above formulae.

Record the calculated SL and SH values on the same graph paper and join them to

give the ‘best fit’ line for the standard solution. Similarly record UL and UH and join

them to give the ‘best fit’ line for the extract.

In the absence of any interference the lines should be parallel. For practical

purposes the lines can be considered parallel if the values (SH-SL) and (UH-UL) do

not differ by more than 10% from their mean value.

If the lines are found to be non-parallel either u1 and s1 or u8 and s8 may be

discarded and SL, SH, UL and UH calculated, using the alternative formulae, to give

alternative ‘best fit’ lines

5s 1 + 2s 2 - s 4

5s 2 + 2s 4 - s 8

(a) SL =

or

5s 4 + 2s 2 - s 1

5s 8 + 2s 4 - s 2

(b) SH =

or

and similarly for UL and UH. The same criteria of parallelism should be satisfied.

The fact that the result has been calculated from three levels should be noted on the

final report.

When the lines are considered as being parallel, calculate the logarithm (log A) of

the relative activity (A) by means of one of the following formulae, depending upon

whether three or four levels have been used for the assessment of parallelism.

For four levels

(c) log A =

For three levels

(d) log A =

or

(d) log A =

Activity of sample extract = activity of relevant standard × A (u 8 = s 8 × A)

If the relative activity is found to be outside the range of 0,5 to 2,0, then repeat the

assay making appropriate adjustments to the extract concentrations or, if this is not

possible, to the standard solutions.

When the relative activity cannot be brought into the required range, any result

obtained must be considered as approximate and this should be noted on the final

report.

When the lines are considered as not being parallel, repeat the determination. If

parallelism is still not achieved, a satisfactory determination Has not been obtained.

Express the result in milligrams of zinc bacitracin per kilogram of feedingstuff.

REPEATABILITY

The difference between the results of two determinations carried out on the same

sample by the same analyst should not exceed

-

2 mg/kg, in absolute value, for contents of zinc bacitracin up to 10 mg/kg,

-

20% related to the highest value for contents from 10 to 25 mg/kg,

-

5 mg/kg, in absolute value, for contents of 25 to 50 mg/kg,

-

10% related to the highest value for contents above 50 mg/kg.

2. DETERMINATION OF FLAVOPHOSPHOLIPOL BY DIFFUSION IN

AN AGAR MEDIUM

1. PURPOSE AND SCOPE

The method is for the determination of flavophospholipol in feedingstuffs,

concentrates and premixes. The lower limit of determination is 1 mg/kg (1 ppm).

2. PRINCIPLE

The sample is extracted with diluted methanol by heating under reflux. After

centrifuging, the extract is purified (where necessary) by treatment with ion

exchange resins and diluted. Its antibiotic activity is determined by measuring the

diffusion of flavophospholipol in an agar medium inoculated with Staphylococcus

aureus. Diffusion is shown by the formation of zones of inhibition of the micro-

organism. The diameter of these zones is taken to be in direct proportion to the

logarithm of the antibiotic concentration over the range of antibiotic concentrations

employed.

3. MICRO-ORGANISM: STAPHYLOCOCCUS AUREUS ATCC 6538 P

3.1. Maintenance of stock culture

Inoculate Staphylococcusaureus onto agar slopes of culture medium (4.1).

Incubate for 24 hours at 37ºC, store in a refrigerator at about 4ºC and reinoculate

every month onto agar slopes.

Preparation of the bacterial suspension *

3.2.

Set aside two tubes containing the stock culture (3.1) and reinoculate them

weekly. Incubate for 24 hours at 37ºC and store in a refrigerator at about 4ºC.

24 hours before the assay, inoculate with this growth two to four tubes containing

slopes of culture medium (4.1). Incubate for 16 to 18 hours at 37ºC. Make a

su spensi on of th e growt h in the sodi um chl oride so lu tio n (4.3). T he l igh t

transmission of the suspension must be about 40 %, measured at 578 nm in a 1 cm

cell against sodium chloride solution (4.3).

4. CULTURE MEDIA AND REAGENTS

Culture medium †

4.1.

Meat peptone

6ꞏ0 g

Tryptone

4ꞏ0 g

Yeast extract

3ꞏ0 g

Meat extract

1ꞏ5 g

Glucose

1ꞏ0 g

Agar

15ꞏ0 g

Water

1 000 ml

pH 6ꞏ5 (after sterilization)

4.2.

Assay medium

4.2.1. Base layer ‡

Meat peptone

6ꞏ0 g

Yeast extract

3ꞏ0 g

Meat extract

1ꞏ5 g

Agar

10ꞏ0 g

Water

1 000 ml

pH 6ꞏ5 (after sterilization)

4.2.2. Seed layer

As for point 4.1, with the addition of 2ꞏ0 g of silicone anti-foaming emulsion. §

4.3.

Sodium chloride solution 0ꞏ4 % (w/v): dissolve 4 g of sodium choride a.p.

in water and dilute to 1 000 ml; sterilize.

4.4.

Methanol, pure.

4.5.

Methanol 50 % (v/v): dilute 500 ml of methanol (4.4) with 500 ml of water.

*Other methods may be used provided it has been established that they give similar bacterial suspensions.

†Any commercial culture medium of similar composition and giving the same results may be used, e.g.

oxoid antibiotic medium 1 (CM 327) with an addition of oxoid agar No 3 (L 13).

‡Any commercial culture medium of similar composition and giving the same results may be used, e.g.

oxoid antibiotic medium 2 (CM 335) with an addition of oxoid agar No 3 (L 13).

§e.g. SE 2 from Wacker Chemie GmbH, Munich.

4.6.

Methanol 80% (v/v): dilute 800 ml of methanol (4.4) with 200 ml of water.

4.7.

Tris(hydroxymethyl) aminomethane a.p.

4.8.

Potassium chloride methanolic solution 1ꞏ5% (w/v): dissolve 1ꞏ5 g of

potassium chloride a.p. in 20 ml of water, make up the volume to m 100 ml with

methanol (4.4).

4.9.

Cation exchanger: Dowex 50 W×8, 20 to 50 mesh, Na form (cat. Serva No

41600) or equivalent.

4.10. Anion exchanger: Dowex 1×2, 50 to 100 mesh, Cl form (cat. Serva No

41010) or equivalent. Before use, keep for 12 to 14 hours in 80% methanol (4.6).

4.11.

Glass wool.

4.12.

pH indicator paper (pH 6ꞏ6 to 8ꞏ1).

4.13.

Ascorbic acid.

4.14.

Standard substance: flavophospholipol of known activity.

5. APPARATUS

5.1.

Glass tube for chromatography, internal diameter: 9 mm, length: 150 to

200 mm, fitted with a stopcock at the narrowed part of the lower end and a ground-

glass joint (to connect with the dropping funnel (5.2)) at the upper end.

5.2.

Dropping funnel 250 ml, fitted with a stopcock and a ground-glass joint.

5.3.

250 ml conical flask with ground-glass joint.

5.4.

Reflux condenser with ground-glass joint.

6. STANDARD SOLUTIONS

Dissolve an accurately weighed quantity of the standard substance (4.14) in 50%

methanol (4.5) and dilute to give a stock solution containing 100 µg

flavophospholipol per millilitre. Stored in stoppered flasks at 4ºC this solution is

stable for up to two months. From this stock solution prepare by successive dilution

with 50% methanol (4.5) the following solutions

0ꞏ2 µg/ml

0ꞏ1 µg/ml

0ꞏ05 µg/ml

0ꞏ025 µg/ml

7. PREPARATION OF THE EXTRACT

7.1.

Extraction

7.1.1. Concentrates, premixes and mineral feeds

Weigh out a quantity of sample of 2ꞏ0 to 5ꞏ0 g and add about 150 mg of ascorbic

acid (4.13). Homogenize with 150 ml of 50% methanol (4.5) in a conical flask (5.3)

and adjust the pH to 8ꞏ1 to 8ꞏ2 with about 400 mg of tris(hydroxymethyl)

aminomethane (4.7). Check the pH with indicator paper (4.12). Allow to stand for 15

minutes, then readjust the pH to 8ꞏ1 to 8ꞏ2 with tris(hydroxymethyl) aminomethane

(4.7) and boil for 10 minutes under reflux (5.4) with constant stirring. Allow to cool,

centrifuge the mixture and decant the extract.

7.1.2. Other feeds

Weigh out a quantity of sample of 5ꞏ0 to 30ꞏ0 g containing at least 30 µg of

flavophospholipol. Homegenize with 150 ml of 50% methanol (4.5) in a conical

flask (5.3) and adjust the pH to 8ꞏ1 to 8ꞏ2 with about 400 mg of tris(hydroxymethyl)

aminomethane (4.7). Check the pH with indicator paper (4.12). Allow to stand for 15

minutes, then readjust the pH to 8ꞏ1 to 8ꞏ2 with tris(hydroxymethyl)aminomethane

(4.7) and boil for 10 minutes under reflux (5.4) with constant stirring. Allow to cool,

centrifuge the mixture and decant the extract.

7.2.

Purification (this step may be omitted for concentrates, premixes and

mineral feeds)

Mix 110 ml of the extract with 11 g of the cation exchanger (4.9), boil for one

minute under reflux (5.4) with constant stirring. Separate the cation exchanger by

centrifugation or filtration. Mix 100 ml of the extract with 150 ml of methanol (4.4)

and store the solution for 12 to 15 hours at 4ºC. Filter off the flocculent mass while

cold.

Insert a glass wool plug (4.11) at the bottom end of a glass tube (5.1), pour into the

tube 5 ml of the anion exchanger (4.10) and wash the column with 100 ml of 80%

methanol (4.6). Using the funnel (5.2), transfer to the column a volume of filtrate of

at least 100 ml which is expected to contain 16 µg of flavophospholipol (200 ml for

a 30 g sample of feedingstuff at 1 ppm). Where necessary, before application to the

column, dilute the filtrate with 80% m ethanol (4.6) to obtain an expected

flavophospholipol content of 16 µg/100 ml. Adjust the flow rate to about 2 ml/

minute. Discard the effluent. Then wash the column with 50 ml of 80% methanol

(4.6) and discard the effluent. Elute the flavophospholipol with the methanolic

solution of potassium chloride (4.8) keeping the flow rate to about 2 ml/minute.

Collect 50 ml of the eluate in a graduated flask, add 30 ml of water and mix. This

solution should have a flavophospholipol content of 0ꞏ2 µg/ml (= U 8 ).

7.3.

Assay solutions

Where necessary (i.e. when the purification step has been omitted), dilute the

extract obtained in point 7.1.1 with 50% methanol (4.5) to obtain an expected

flavophospholipol content of 0ꞏ2 µg/ml (= U 8 ). From solution U 8 prepare solutions

U 4 (expected content: 0ꞏ1 µg/ml), U 2 (expected content: 0ꞏ05 µg/ml) and U 1

(expected content: 0ꞏ025 µg/ml) by means of successive dilution (1 + 1) with 50%

methanol (4.5).

8. ASSAY PROCEDURE

8.1.

Inoculation of the assay medium

Inoculate the assay medium (4.2.2) with the bacterial suspension (3.2) at about

50ºC. By preliminary trials on plates with assay medium (4.2.2) determine the

quantity of bacterial suspension required to give the largest and clearest zones of

inhibition with the various concentrations of flavophospholipol (about 30 ml/litre).

8.2.

Preparation of the plates

Diffusion through agar is carried out in plates with the four concentrations of the

standard solution (S 8 , S 4 , S 2 , S 1 ) and the four concentrations of the assay solution

(U 8 , U 4 , U 2 , U 1 ). These four concentrations of extract and standard must necessarily

be placed in each plate. To this effect, select plates big enough to allow at least eight

holes with a diameter of 10 to 13 mm and not less than 30 mm between centres to be

made in the agar medium. The test may be carried out on plates consisting of a sheet

of glass with a faced aluminum or plastic ring placed on top, 200 mm in diameter

and 20 mm high. Pour into the plates a quantity of the medium (4.2.1) to give a layer

about 1ꞏ5 mm thick (45 ml for a plate of 200 mm diameter). Allow to set in a level

position and then over-layer with a quantity of the medium (4.2.2) inoculated as in

point 8.1 to give a layer 1 mm thick (30 ml for a plate of 200 mm diameter). Allow to

set again in a level position, bore the holes and place in them exactly measured

volumes of assay and standard solutions (between 0ꞏ10 and 0ꞏ15 ml per hole,

according to the diameter).

Apply each concentration at least four times so that each determination is subject

to an evaluation of 32 zones of inhibition.

8.3.

Incubation

Incubate the plates for 16 to 18 hours at 28 to 30ºC.

9. EVALUATION

Measure the diameter of the zones of inhibition to the nearest 0ꞏ1 mm. Record the

mean measurements for each concentration on semi-logarithmic graph paper

showing the logarithm of the concentrations in relation to the diameters of the zones

of inhibition. Plot the best fit lines of both the standard solution and the extract, for

example as below. Determine the ‘best fit’ point for the standard lowest level (SL)

using the formula

(a) SL =

(b) SH =

Similarly calculate the ‘best fit’ points for the extract lowest level (UL) and the

extract highest level (UH) by substituting U 1 , U 2 , U 4 and U 8 for S 1 , S 2 , S 4 and S 8 in

the above formulae.

Record the calculated SL and SH values on the same graph paper and join them to

give the ‘best fit’ line for the standard solution. Similarly record UL and UH and join

them to give the ‘best fit’ line for the extract.

In the absence of any interference the lines should be parallel. For practical

purposes the lines can be considered parallel if the values (SH-SL) and (UH-UL) do

not differ by more than 10% from their mean value. If the lines are found to be non-

parallel, either U 1 and S or U 8 and S 8 may be discarded and SL, SH, UL and UH

calculated, using the alternative formulae, to give alternative ‘best fit’ lines

(a) SL =

or

(b) SH =

or

and similarly for UL and UH. The alternative best fit lines should be checked for

parallelism as before. The fact that the result has been calculated from three levels

should be noted on the final report. When the lines are considered as being parallel,

calculate the logarithm of the relative activity (log. A) by means of one of the

following formulae.

For four levels

(c) log A =

For three levels

(d) log A =

or

(d) log A =

Real activity = supposed activity × relative activity.

When the lines are considered as not being parallel, repeat the determination. If

parallelism is still not achieved, calculate the logarithm of the relative activity (log.

A) by means of formula (c). The result obtained must however be considered as

approximate and this should be noted in the final report.

REPEATABILITY

The difference between the results of two determinations carried out on the same

sample by the same analyst should not exceed: 0ꞏ5 mg/kg, in absolute value, for

contents of flavophospholipol from 1 and up to 2 mg/kg

25% related to the highest value for contents greater than 2 and up to 10 mg/kg;

20% related to the highest value for contents greater than 10 and up to 25 mg/kg;

5 mg/kg, in absolute value, for contents greater than 25 and up to 50 mg/kg;

10% related to the highest value for contents above 50 mg/kg.

3. DETERMINATION OF THE TRACE ELEMENTS IRON, COOPER,

MANGANESE AND ZINC

1. PURPOSE AND SCOPE

The method makes it possible to determine the trace elements iron, copper,

manganese and zinc in feedingstuffs. The lower limits of determination are

iron (Fe)

20 mg/kg

copper (Cu)

10 mg/kg

manganese (Mn)

20 mg/kg

zinc (Zn)

20 mg/kg

2. PRINCIPLE

The sample is brought into solution in hydrochloric acid after destruction of

organic matter, if any. The elements iron, copper, manganese and zinc are

determined, after appropriate dilution, by atomic absorption spectrometry.

3. REAGENTS

Introductory comments

For preparation of the reagents and analytical solutions use water free from the

cations to be determined, obtained either by double distilling water in a borosilicate

glass or quartz still or by double treatment on ion exchange resin.

The reagents must be of at least analytical grade (a.p.). Freedom from the element

to be determined must be checked in a blank experiment. If necessary, the reagents

must be further purified.

In place of the standard solutions described below, commercial standard solutions

may be used provided that they are guaranteed and have been checked before use.

3.1.

Hydrochloric acid a.p. (d:1ꞏ19).

3.2.

Hydrochloric acid a.p. (6 N).

3.3.

Hydrochloric acid a.p. (0ꞏ5 N).

3.4.

Hydrofluoric acid 38 to 40% (v/v) having an iron content of less than 1 mg

Fe/litre and a residue after evaporation of less than 10 mg (as sulphate)/litre.

3.5.

Sulphuric acid a.p. (d: 1ꞏ84).

3.6.

Hydrogen peroxide a.p. (approximately 100 volumes of oxygen (30% by

weight)).

3.7.

Standard iron solution (1 000 µg Fe/ml) prepared as follows: dissolve 1 g

of iron wire a.p. in 200 ml of 6 N hydrochloric acid (3.2), add 16 ml of hydrogen

peroxide (3.6) and make up to one litre with water.

3.7.1. Working standard iron solution (100 µg Fe/ml) prepared by diluting the

standard solution (3.7) 1 + 9 with water.

3.8.

Standard copper solution (1 000 µg Cu/ml) prepared as follows: dissolve 1

g of copper in powder form (a.p.) in 25 ml of 6 N hydrochloric acid (3.2), add 5 ml

of hydrogen peroxide (3.6) and make up to one litre with water.

3.8.1. Working standard copper solution (10 µg Cu/ml) prepared by diluting the

standard solution (3.8) 1 + 9 with water and then diluting the resulting solution 1 + 9

with water.

3.9.

Standard manganese solution (1 000 µg Mn/ml) prepared as follows

dissolve 1 g of manganese in powder form (a.p.) in 25 ml of 6 N hydrochloric acid

(3.2) and make up to one litre with water.

3.9.1. Working standard manganese solution (10 µg Mn/ml) prepared by diluting

the standard solution (3.9) 1 + 9 with water and then diluting the resulting solution 1

+ 9 with water.

3.10. Standard zinc solution (1 000 µg Zn/ml) prepared as follows: dissolve 1 g

of zinc in strip or leaf form (a.p.) in 25 ml of 6 N hydrochloric acid (3.2) and make

up to one litre with water.

3.10.1. Working standard zinc solution (10 µg Zn/ml) prepared by diluting the

standard solution (3.10) 1 + 9 with water and then diluting the resulting solution 1 +

9 with water.

3.11. Lanthanum choride solution prepared as follows: dissolve 12 g of

lanthanum oxide in 150 ml of water, add 100 ml of 6 N hydrochloric acid (3.2) and

make up to one litre with water.

4. APPARATUS

4.1.

Muffle furnace with temperature regulation and recorder.

4.2.

Glassware must be of resistant borosilicate type and it is recommended to

use apparatus which is reserved exclusively for trace element determinations.

4.3.

Platinum crucible and (optional) quartz crucible.

4.4.

Atomic absorption spectrophotometer meeting the requirements of the

method with regard to sensitivity and precision in the required range.

5. PROCEDURE

Samples containing organic matter

5.1.1. Ashing and preparation of the solution for analysis *

(i)

Place 5 to 10 g of sample weighed to the nearest 0ꞏ2 mg in a quartz or

platinum crucible (4.3) (see Note (b)), dry in an oven at 105ºC and

introduce the crucible into the cold muffle furnace (4.1). Close the

furnace (see Note (c)) and gradually raise the temperature to 450 to

475ºC over about 90 minutes. Maintain this temperature for 4 to 16

hours (e.g. overnight) to remove carbonaceous material and then open

the furnace and allow to cool (see Note (d)).

Wash the crucible out with a total of about 5 ml of hydrochloric acid

(3.1) and add the latter slowly and carefully to the beaker (there may be

a vigorous reaction due to CO2 formation). Add hydrochloric acid (3.1)

drop wise with agitation until all effervescence has stopped. Evaporate

to dryness, occasionally stirring with a glass rod.

Next add 15 ml of 6 N hydrochloric acid (3.2) to the residue followed

by about 120 ml of water. Stir with the glass rod, which should be left in

the beaker, and cover the beaker with a watch glass. Bring gently to the

boil and maintain at boiling point until no more ash can be seen to

dissolve. Filter on ash-free filter paper and collect the filtrate in a 250

ml volumetric flask. Wash the beaker and filter with 5 ml of hot 6 N

hydrochloric acid (3.2) and twice with boiling water. Fill the volumetric

flask up to the mark with water (HCl concentration about 0ꞏ5 N).

(ii) If the residue in the filter appears black (carbon), put it back in the

furnace and ash again at 450 to 475ºC. This ashing, which only requires

a few hours (about three to five hours), is complete when the ash

appears white or nearly white. Dissolve the residue with about 2 ml of

hydrochloric acid (3.1), evaporate to dryness and add 5 ml of 6 N

hydrochloric acid (3.2). Heat, filter the solution into the volumetric

flask and make up to the mark with water (HCl concentration about 0ꞏ5

N).

(a) In determining trace elements it is important to be alert to the risks of

contamination, particularly by zinc, copper and iron. For this reason, the equipment

used in preparing the samples must be free of these metals.

To reduce the general risk of contamination, work in a dust-free atmosphere with

scrupulously clean equipment and carefully washed glassware. The determination of

zinc is particularly sensitive to many types of contamination, e.g. from glassware,

reagents, dust, etc.

*Green fodder (fresh or dried) is liable to contain large amounts of vegetable silica, which may retain trace

elements and must be removed. For samples of these feedingstuffs, therefore, the following modified

procedure must be followed.

Carry out operation 5.1.1 (i) as far as the filtration. Wash the filter paper containing the insoluble residue

twice with boiling water and place it in a platinum crucible (4.3).

Ignite in the muffle furnace (4.1) at a temperature below 550ºC until all carbonaceous material has

completely disappeared. Allow to cool, add a few drops of water followed by 10 to 15 ml of hydrofluoric

acid (3.4) and evaporate to dryness at about 150ºC. If any silica remains in the residue, redissolve it in a few

millilitres of hydrofluoric acid (3.4) and evaporate to dryness. Add five drops of sulphuric acid (3.5) and

heat until no more white fumes are given off. After the addition of 5 ml of 6 N hydrochloric acid (3.2) and

about 30 ml of water, heat, filter the solution into the 250 ml volumetric flask and make up to the mark with

water (HCl concentration about 0ꞏ5 N). Proceed then with the determination from point 5.1.3.

(b) The weight of sample to be ashed is calculated from the approximate trace

element content of the feedingstuff in relation to the sensitivity of the

spectrophotometer used. For certain feedingstuffs low in trace elements it may be

necessary to start with a 10 to 20 g sample and make up the final solution to only 100

ml.

(c) Ashing must be carried out in a closed furnace without injection of air or

oxygen.

(d)

The temperature indicated by the pyrometer must not exceed 475ºC.

5.1.2. Spectrophotometric determination

5.1.2.1. Preparation of calibration solutions

For each of the elementsto be determined, prepare from the working standard

solutions given in points 3.7.1, 3.8.1, 3.9.1 and 3.10.1 a range of calibration

solutions, each calibration solution having an HCl concentration of about 0ꞏ5 N and

(in the cases of iron, manganese and zinc) a lanthanum chloride concentration

equivalent to 0ꞏ1% La (w/v).

The trace element concentrations selected must lie within the range of sensitivity

of the spectrophotometer used. The tables below show, by way of example, the

compositions of typical ranges of calibration solutions; depending, however, on the

type and sensitivity of spectrophotometer used it may be necessary to select other

concentrations.

Iron

µg Fe/ml

0.5

µg Fe/ml working standard

solution (3.7.1)

(1 ml = 100 µg Fe) + ml 6 N HCl

0.5

(3.2)

+ 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml with water.

Copper

µg Cu/ml

0.1

0.2

0.4

0.6

0.8

0.1

µg Cu/ml working standard

solution (3.8.1)

(1 ml = 10 µg Cu) + ml 6 N HCl

(3.2)

Make up to 100 ml with water.

Manganese

µg Mn/ml

0.1

0.2

0.4

0.36

0.8

1.0

µg Mn/ml working standard

solution (3.9.1)

(1 ml = 10 µg Mn) + ml 6 N HCl

(3.2)

+ 10 ml lanthanum chloride solution (3.11) and make up to 100 ml with water.

Zinc

µg Zn/ml

0.05

0.1

0.2

0.4

0.6

0.8

µg Zn/ml working standard

solution (3.10.1)

(1 ml = 10 µg Zn) + ml 6 N HCl

0.5

(3.2)

+ 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml with water.

5.1.2.2. Preparation of solution for analysis

For the determination of copper, the solution prepared from point 5.1.1 can

normally be used directly. If necessary to bring its concentration within the range of

the calibration solutions, an aliquot portion may be pipetted into a 100 ml volumetric

flask and made up to the mark with 0ꞏ5 N hydrochloric acid (3.3).

For the determination of iron, manganese and zinc, pipette an aliquot portion of

the solution prepared from point 5.1.1 into a 100 ml volumetric flask, add 10 ml of

lanthanum chloride solution (3.11) and make up to the mark with 0ꞏ5 N hydrochloric

acid (3.3) (see also point 8 ‘Observation’).

5.1.2.3. Blank experiment

The blank experiment must include all the prescribed steps of the procedure

except that the sample material is omitted. The calibration solution ‘0’ must not be

used as the blank.

5.1.2.4. Measurement of the atomic absorption

Measure the atomic absorption of the calibration solutions and of the solution to

be analyzed using an oxidizing air-acetylene flame at the following wavelengths

Fe

248ꞏ3 nm

Cu

324ꞏ8 nm

Mn: 279ꞏ5 nm

Zn

213ꞏ8 nm

Carry out each measurement four times.

5.2.

Mineral feedingstuffs

If the sample contains no organic matter, prior ashing is unnecessary. Proceed as

described in point 5.1.1 (i) starting from the second paragraph. Evaporation with

hydrofluoric acid may be omitted.

6. CALCULATION OF RESULTS

Using a calibration curve, calculate the trace element concentration in the solution

to be analyzed and express the result in milligrams of trace element per kilogram of

sample (ppm).

7. REPEATABILITY

The difference between the results of zwo parallel determinations carried out on

the same sample by the same analyst should not exceed

-

5' mg/kg, in absolute value, for contents of the trace element concerned up

to 50 mg/kg;

-

10% of the higher result for contents of the trace element concerned from 50

and up to 100 mg/kg;

-

10 mg/kg, in absolute value, for contents of the trace element concerned

from 100 and up to 200 mg/kg;

-

5% of the higher result for contents of the trace element concerned above

200 mg/kg.

8. OBSERVATION

T h e p r e s e n c e o f l a rg e q u a n t i t i e s o f p h o s p h a t e s m a y i n t e r f e r e w i t h t h e

determination of iron, manganese and zinc. Such interference must be corrected by

addition of lanthanum chloride solution (3.11). If, however, in the sample the weight

ratio

Ca + Mg

2,

P

addition of lanthanum chloride solution (3.11) to the solution for analysis and to the

calibration solutions may be omitted.

15g

Potassium dihydrogen phosphate KH2PO4

85g

Ilma sa

1000ml

4.6

Hydrochloric acid (d:1.18 sa 1.19)

4.7

Hydrochloric acid (0.1M)

4.8

Soluzzjoni ta’ sodium hydroxide 1M

4.9

Soluzzjoni ta’ Sodium Sulphide ta’ xi 0.5M

4.10

Soluzzjoni ta’ bromocresol purple 0.04% (w/v): ħoll 0.1g bromocresol

purple f’18.5 ml soluzzjoni ta’ sodium hydroxide 0.1M. Żid sal-marka ta’ 250ml bl-

ilma u ħallat.

4.11

Standard substance: zingu bacitracin ta’ attività magħrufa (f’i.u).

5. SOLUZZJONIJIET STANDARD

Iżen kwantità standard Zinc bacitracin (4.11) li tikkorrispondi għal 1050 i.u.

(skond l-attività indikata). Żid 5ml 0.1M hydrochloric acid (4.7) u ħallih joqgħod

15-il minuta. Żid 30ml ilma, adatta l-pH sa 4.5 bil-phosphate buffer (4.5) (xi 4ml),

żid sal-marka ta’ 50ml bl-ilma u ħallat sew (1ml = 21 i.u.)

Minn din is-soluzzjoni pprepara b’diluzzjoni suċċessiva bil-phosphate buffer (4.5)

is-soluzzjonijiet li ġejjin

0.42 i.u./ml

0.21 i.u./ml

0.105 i.u./ml

0.0525 i.u./ml

PREPARAZZJONI TA’

SOLUZZJONIJIET

TA’

ESTRATT

U

SOLUZZJONIJIET TA’ ASSAĠĠ

*Kull culture medium kummer`jali li għandu komposizzjoni simili u li jagħti l-istess riżultati jista’

jintuża.

6.1

Estrazzjoni

6.1.1

Taħlitiet minn qabel u ikel minerali

Iżen kwantità ta’ 2.0 sa 5.0 g mill-kampjun u żid 29ml tat-taħlita (4.4) u 1.0ml

soluzzjoni tas-sodium sulphide (4.9) u ixxejkja. Ara li l-pH hi madwar 2. Ixxejkja

għal 10 minuti, żid 30ml phosphate buffer (4.5), ixxejkja għal 15-il minuta u uża

centrifuge. Ħu aliquot kemm hemm bżonn mis-supernatant solution u adatta l-pH

fuq 6.5 permezz ta’ 1M soluzzjoni tal-hydroxide (4.8) li jkollha pH-meter jew bis-

soluzzjoni tal-bromocresol purple (4.10) bħala indikatur. Ħallat bil-phosphate buffer

(4.5) biex ikollok kontenut mistenni ta’ Zinc bacitracin i.u/ml (= u 8 ).

6.1.2

Koncentrati ta’ proteini

Iżen kwantità tal-kampjun ta’ 10.0g, żid 49ml mit-taħlita (4.4) u 1ml soluzzjoni

tas-sodium sulphide (4.9) u ixxejkja. Ara li jkollok pH ta’ xi 2. Ixxejkja għal 10

minuti. Żid 50ml phosphate buffer (4.5), ixxejkja għal 15-il minuta u uża centrifuge.

Ħu volum adattat mis-supernatant solution u adatta l-pH sa 6.5 5 permezz ta’ 1M

soluzzjoni tal-hydroxide (4.8) li jkollha pH-meter jew bis-soluzzjoni tal-bromocresol

purple (4.10) bħala indikatur. Evapora qisu nofs is-soluzzjoni f’rotary evaporator

f’temperatura ta’ mhux iktar minn 35 grad Celsius.

Ħallat bil-phosphate buffer (4.5) biex ikollok kontenut mistenni ta’ zinc bacitracin

i.u/ml (= u 8 ).

6.1.3

Feeds oħra

Iżen kwantità tal-kampjun ta’ 10.0g (20g fil-każ ta’ kontenut mistenni ta’ zinc

bacitracin ta’ 5mg/kg). Żid taħlita ta’ 24ml mit-taħlita (4.4) u 1.0ml soluzzjoni ta’

sodium sulphide (4.9) u omoġenizza għal 10 minuti. Żid 25ml phosphate buffer (4.5)

ixxejkja għal 15-il minuta u uża centrifuge. Hu 20ml mis-supernatant solution u

adatta l-pH sa 6.5 5 permezz ta’ 1M soluzzjoni tal-hydroxide (4.8) li jkollha pH-

meter jew bis-soluzzjoni tal-bromocresol purple (4.10) bħala indikatur. Evapora qisu

nofs is-soluzzjoni f ’rotary evaporator f ’temperatura ta’ mhux iktar minn 35 grad

Celsius. Ħallat bil-phosphate buffer (4.5) biex ikollok kontenut mistenni ta’ zinc

bacitracin i.u/ml (= u 8 ).

6.2

Soluzzjonijiet ta’ l-assaġġ

Mis-soluzzjoni u 8 ipprepara soluzzjoni u 4 (kontenut mistenni: 0.21 i.u./ml), u 2

(kontenut mistenni: 0.105 i.u/ml) u u 1 (kontenut mistenni: 0.0525 i.u./ml) permezz

tad-dilwizzjoni suċċessiva (1+1) bil-phosphate buffer (4.5).

7. PROCEDURA TA’ L-ASSAĠĠ

7.1.

Tilqim tal-medju ta’ l-assaġġ

Laqqam il-medju ta’ l-assaġġ (4.2) bil-bacterial suspension (3.2) f ’madwar 50

grad celsius. Permezz ta’ testijiet preliminari fuq plates bil-medju ddetermina l-

kwantità ta’ bacterial suspension meħtieġa biex ikollok iż-żoni ta’ inibizzjoni kemm

jista’ jkun ċari u kbar mad-diversi konċentrazzjonijiet ta’ zinc bacitracin.

7.2

Preparazzjoni tal-plates

Id-diffużjoni fuq l-agar ssir fuq plates bl-4 konċentrazzjonijiet tas-standard

solution (S 8 , S 4 , S 2 u S 1 ) u l-4 konċentrazzjonijiet ta’ l-assay solution (u 8 , u 4 , u 2 u

u 1 ). L-4 konċentrazzjonijiet tas-standard solution u ta’ l-estratt iridu jitpoġġew fil-

plates. Għalhekk għandhom jintgħażlu plates li huma kbar biżżejjed biex ikunu

jistgħu isiru mill-inqas 8 toqob fl-agar medium b’djametru ta’ 10 sa 13mm u mhux

inqas minn 30mm bejn iċ-ċentri. Ikkalkula l-kwantità ta’ kultura medium imlaqqam

(7.1) li hemm bżonn biex ikun hemm għata uniformi ta’ madwar 2 mm ħxuna. It-test

jista’ jsir fuq plates li jikkonsisti f ’lastra tal-ħġieġ b’ħolqa tal-aluminju jew tal-

plastik fil-wiċċ, b’djametru ta’ 200mm u għoli ta’ 20mm.

Ferra ġol-plates kwantità tal-medium (4.2) imlaqqma kif jidher f’7.1, biex ikollok

strat ta’ xi 2mm ħxuna (60mm għal plate b’djametru ta’200mm). Ħallih joqgħod

f ’pożizzjoni illivellata, agħmel it-toqob u poġġi fihom volum preċiż ta’ assay u

stan dard sol ut ion s (bej n 0 .1 0 u 0 .15m l , skon d id-dj am etru). App li ka kul l

konċentrazzjoni mill-inqas erba’ darbiet biex kull determinazzjoni tkun soġġetta

għal evalwazzjoni ta’ 32 inhibition zones.

7.3

Inkubazzjoni

Żomm il-plates inkubati minn 16 sa 18-il siegħa fi 28 sa 30ºC.

8. EVALWAZZJONI

Kejjel id-djametru taż-żoni ta’ inibizzjoni sa l-eqreb 0.1mm. Żomm rendikont tal-

miżuri medji ta’ kull konċentrazzjoni fuq semi-logarithmic graph paper bil-

logarithm tal-konċentrazzjonijiet relatati mad-djametri taż-żoni ta’ inibizzjoni.

Ipplottja il-best fit lines ta’ kemm is-standard solution kif ukoll l-estratt, per

eżempju

Determina il-‘best fit’ point għal standard lowest level (SL) permezz tal-formula

7s 1 + 4s 2 +s 4 - 2s 8

SL =

Determina il-‘best fit’ point għal standard highest level (SH) permezz tal-formula

7s 8 + 4s 4 +s 2 - 2s 1

SH =

F’mod simili ikkalkula l-‘best fit’ points għal extract lowest level (UL) u l-extract

highest level (UH) billi tissostitwixxi u 1 , u 2 , u 4 u u 8 għal s 1 , s 2 , s 4 u s 8 fil-formoli

ta’ fuq.

Żomm rendikont tal-valuri kalkolati SL u SH fuq l-istess graph paper u

għaqqadhom biex ikollok il-‘best fit’ line għal standard solution. Żomm rendikont

ta’ UL u UH u għaqqadhom biex ikollok il-‘best fit’ line għall-estratt.

Jekk ma jkunx hemm interferenza l-linji għandhom ikunu paralleli. Għal skopijiet

prattiċi l-linji għandhom jiġu kkonsidrati paralleli jekk il-valuri (SH-SL) u (UH-UL)

ma fihomx differenza ta’ iktar minn 10% tal-valur tagħhom medju.

Jekk il-lini ma jkunux paralleli, u 1 , s 1 , u 8 jew s 8 jista’ jintrema u SL, SH, UL u UH

kalkolati permezz ta’ dawn il-formuli alternattivi, biex ikollok ‘best fit’ lines

alternattivi

5s 1 + 2s 2 - s 4

5s 2 + 2s 4 - s 8

(a) SL =

jew

5s 4 + 2s 2 - s 1

5s 8 + 2s 4 - s 2

(b) SH =

jew

U l-istess ghal UL u UH. L istess kriterji ta’ paralleliżmu għandhom jiġu

sodisfatti. Il-fatt li r-riżultat ikun ġie kalkolat fuq tliet livelli għandu jiġi annotat fuq

ir-rapport finali.

Meta l-linji huma kunsidrati bħala paralleli, ikkalkola l-logarithm ta’ l-attività

relattiva (log A) permezz ta’ xi waħda minn dawn il-formoli, skond jekk ikunux ġew

użati tlieta jew erba’ livelli fl-istima tal-paralleliżmu.

Għal erba’ livelli

(`) log A =

Għal tliet livelli

(d) log A =

jew

(d) log A =

Attività ta’ kampjun estratt = attività li tidher attività ta’ standard rilevanti × A (u 8

= s 8 × A).

Jekk l-attività relattiva tkun barra l-medda ta’ bejn 0.5 sa 2.0, irrepeti l-

adattamenti tal-proċedura biex issir l-assay sal-konċentrazzjonijiet eżatti, jew jekk

mhux possibbli, sa standard solutions. Meta l-attività relattiva ma tistax tinġieb fil-

medda meħtieġa, kull riżultat irid jigi kkunsidrat approssimat u dan għandu jitniżżel

fir-rapport finali.

Meta l-linji huma kkunsidrati mhux paralleli, irrepeti d-determinazzjoni. Jekk il-

paralleliżmu ma jirriżultax, id-determinazzjoni għandha tiġi kkunsidrata

insodisfaċenti.

Esprimi r-riżultat f’milligrammi ta’ virginiamycin għal kull kilogramma ta’ għalf.

9. RIPETIZZJONIJIET

Id-differenza bejn ir-riżultati ta’ żewġ determinazzjonijiet paralleli li jsiru fuq l-

istess kampjun mill-istess analista ma għandhomx jeċċedu

-

2mg/kg, valur relattiv, fil-każ ta’ kontenut ta’ virginiamycin sa 10mg/kg;

-

20% relatat ma’ l-ogħla valur fil-każ ta’ kontenut ta’ 10 sa 25mg/kg,

-

5mg/kg, valur assolut, fil-każ ta’ kontenut minn 25 sa 50mg/kg,

-

10% relatat ma’ l-ogħla valur fil-każ ta’ kontenut ta’ iktar minn 50mg/kg.

2. DETERMINAZZJONI TA’ FLAVOPHOSPHOLIPOL BID-DIFFUŻJONI

F’AGAR MEDJA

1. GĦAN U SKOP

Dan il-metodu jiddetermina l-flavophopholipol fl-għalf, konċentrati u taħlilitet

minn qabel. Il-lower limit of determination hu 1mg/kg (1ppm).

2. PRINĊIPJU

Il-kampjun jiġi estratt bil-methanol imħallat billi jissaħħan f ’reflux. Wara is-

centrifuging, l-estratt jiġi ppurifikat (fejn neċessarju) permezz ta’ trattament b’ion

exchange resins u jiġi mħallat. L-attività antibiotika tiegħu tiġi determinata billi

t i t ke j j e l i d - d i ff u ż j o n i t a l -f l a v o p h o s p h o l i p ol f ’ a g a r m e d i um i m l a q qa m b l -

staphylococcus aureus. Id-diffużjoni tidher bil-formazzjoni ta’ żoni ta’ inibizzjoni

tal-mikro-organiżmu. Id-djametru ta’ dawn iż-żoni hu ikkunsidrat direttament

proporzjonali mal-logarithm tal-konċentrazzjoni antibiotika fuq il-medda ta’

konċentrazzjonijiet antibiotiċi użati.

3. MIKRO-ORGANIŻMU: STAPHYLOCOCCUS AUREUS ATCC 6538P

3.1.

Manutenzjoni tas-stock culture

Laqqam is-staphylococcus aureus fuq agar slopes ta’ kultura medium (4.1).

Inkubahom għal 24 siegħa f ’temperatura ta’ 37 grad Celsius, żomm fi friġġ

f’temperatura ta’ 4 gradi Celsius u erġa’ laqqam kull xahar fuq agar slopes.

Preparazzjoni tal-bacterial suspension *

3.2.

Warrab żewġ tubi bis-stick kultura (3.1) u laqqamhom kull ġimgħa. Inkubahom

għal 24 siegħa f ’temperatura ta’ 37 grad Celsius u żommhom fi friġġ f’madwar 4

gradi.

24 siegħa qabel l-assay laqqam b’din il-kultivazzjoni 2 sa 4 tubi li fihom slopes ta’

kultura medium (4.1). Inkubahom minn 16 sa 18-il siegħa f’temperatura ta’ 37 grad.

Agħmel sospensjoni tal-kultivazzjoni f ’soluzzjoni tas-sodium chloride (4.3). It-

trasmissjoni tad-dawl tas-soluzzjoni għandha tkun madwar 40% imkejla fuq 578nm

f’ċellula tal-1cm f’soluzzjoni tas-sodium chloride.

4. CULTURE MEDIA U REAĠENTI

Culture medium #

4.1.

Meat peptone

6ꞏ0 g

Tryptone

4ꞏ0 g

Estratt tal-ħmira

3ꞏ0 g

Estratt tal-laħam

1ꞏ5 g

Glukożju

1ꞏ0 g

Agar

15ꞏ0 g

Ilma

1 000 ml

pH 6ꞏ5 (wara s-sterilizzazjoni)

4.2.

Assay medium

4.2.1. Base layer +

Meat peptone

6ꞏ0 g

Estratt tal-ħmira

3ꞏ0 g

Estratt tal-laħam

1ꞏ5 g

Agar

10ꞏ0 g

Ilma

1 000 ml

pH 6ꞏ5 (wara sterilization)

4.2.2. Seed layer

Bħal punt 4.1, biż-żieda ta’ 2ꞏ0 g silicone anti-foaming emulsion. ¢

4.3.

Sodium chloride solution 0ꞏ4 % (w/v): ħoll 4 g sodium choride a.p. fl-ilma

u ħallat sa 1 000 ml; sterilizza.

*Jistghu jintużaw metodi oħra sakemm ikun stabilit li jagħtu bacterial suspensions simili.

#Kull culture medium b’kompożizzjoni simili u li jagħti l-istess riżultati jista’ jintuża, eż. oxoid

antibiotic medium 1 (CM 327) b’żieda ta’ oxoid agar No 3 (L 13).

+Kull culture medium b’komposizzjoni simili u li jagħti l-istess riżultati jista’ jintuża, eż oxoid

antibiotic medium 2 (CM 335) b’żieda ta’ oxoid agar No 3 (L 13).

¢eż. SE 2 minn Wacker Chemie GmbH, Munich.

4.4.

Methanol, pur.

4.5.

Methanol 50% (v/v): ħallat 500 ml methanol (4.4) ma’ 500 ml ilma.

4.6.

Methanol 80% (v/v): ħallat 800 ml methanol (4.4) ma’ 200 ilma.

4.7.

Tris(hydroxymethyl) aminomethane a.p.

4.8.

Potassium chloride methanolic solution 1ꞏ5% (w/v): ħoll ꞏ5 g potassium

chloride a.p. f’20 ml ilma, żid sal-marka ta’ 100 ml bil-methanol (4.4).

4.9.

Cation exchanger: Dowex 50 W×8, 20 to 50 mesh, Na form (cat. Serva No

41600) jew ekwivalenti.

4.10. Anion exchanger: Dowex 1×2, 50 to 100 mesh, Cl form (cat. Serva No

41010) jew ekwivalenti. Qabel ma tużah, żommu għal 12 sa 14 siegħa f ’80%

methanol (4.6).

4.11.

Glass wool.

4.12.

pH indicator paper (pH 6ꞏ6 to 8ꞏ1).

4.13.

Ascorbic acid.

4.14.

Standard substance: flavophospholipol ta’ attività magħrufa.

5. APPARAT

5.1.

Tubu tal-ħġieġ għal chromatography, djametru intern: 9 mm, tul: 150 sa

200 mm, iffittjat b’stopcock fil-parti dejqa tat-tarf ta’ isfel u ground-glass joint (biex

jitqabbad mad- dropping funnel (5.2)) fit-tarf ta’ fuq.

5.2.

Dropping funnel 250 ml, iffittjat b’stopcock u ground-glass joint.

5.3.

250 ml conical flask b’ground-glass joint.

5.4.

Reflux condenser b’ground-glass joint

6. SOLUZZJONIJIET STANDARD

Ħoll kwantità eżatta ta’ standard substance (4.14) f ’50% methanol (4.5) u ħoll

biex ikollok stock solution b’kontenut ta’ 100µg flavophospholipol għal kull

millilitru. Jekk jinżamm f’stoppered flasks f’temperatura ta’ 4 gradi Celsius din is-

soluzzjoni stabbli sa xahrejn.

Minn din is-stock solution ipprepara b’taħlita suċċessiva ma’ 50% methanol (4.5)

is-soluzzjonijiet segwenti

0ꞏ2 µg/ml

0ꞏ1 µg/ml

0ꞏ05 µg/ml

0ꞏ025µg/ml

7. PREPARAZZJONI TA’ L-ESTRATT

7.1

Estrattazjoni

7.1.1

Kon`entrati, taħlitiet li jsiru minn qabel u tmigħ minerali

Iżen 2.0 sa 5.0 g mil-kampjun u żid 150mg ascorbic acid (4.13). Omoġenizza

b’150ml 50% methanol (4.5) f ’conical flask (5.3) u adatta l-pH fuq 8.1 sa 8.2

b’madwar 400mg tris(hydroxymethyl)aminomethane (4.7). Aqra l-pH b’indicator

paper (4.12). Ħallih joqgħod għal 15-il minuta, mbagħad erġa’ adatta l-pH fuq 8.1 sa

8.2 bit-tris(hydroxymethyl)aminomethane (4.7) u għallih għal għaxar minuti taħt

reflux (5.4) filwaqt li tħawdu bla waqfien. Ħallih jiksaħ, għaddih minn centrifuge u

poġġih f’decanter.

7.1.2

Feeds oħra

Iżen 5ꞏ0 sa 30ꞏ0 g mil-kampjun b’kontenut ta’ mill-inqas 30 µg flavophospholipol.

Omoġenizza b’150ml 50% methanol (4.5) f’conical flask (5.3) u adatta l-pH fuq 8.1

sa 8.2 b’madwar 400mg tris(hydroxymethyl)aminomethane (4.7). Aqra l-pH

b’indicator paper (4.12). Ħallih joqgħod għal 15-il minuta, mbagħad erga’ adatta l-

pH fuq 8.1 sa 8.2 bit-tris(hydroxymethyl)aminomethane (4.7) u għallih għal għaxar

minuti taħt reflux (5.4) filwaqt li tħawdu bla waqfien. Ħallih jiksaħ, għaddih minn

centrifuge u poġġih f’decanter

7.2

Purifikazzjoni (din il-fażi tista’ titħalla barra fil-każ ta’ kon`entrati,

taħlitiet li jsiru minn qabel u tmigħ minerali)

Ħallat 110ml mill-estratt ma’ 11g cation exchanger (4.9), għallih għal minuta taħt

re f l u x ( 5 . 4 ) f i l w a q t l i t ħ a w d u b l a w a q f i e n . I s s e p a r a l - c a t i o n e x c h a n g e r

b’centrifugation jew filtrazzjoni. Ħallat 100ml mill-estratt ma’ 150ml ethanol (4.4) u

żomm is-soluzzjoni għal 12 sa 15-il siegħa f’temperatura ta’ 4 gradi Celsius. Neħħi

bil-filtrazzjoni il-flocculent mass meta tkun kiesħa.

Daħħal glass wool plug (4.11) fit-tarf ta’ isfel ta’ glass tube (5.1), ferra fit-tube

5ml anion exchanger (4.10) u aħsel it-tubu b’100ml 80% methanol (4.6). Permezz

tal-funnel (5.2) ittrasferixxi fil-kolonna volum tal-filtrat ta’ mill-inqas 100ml li hi

mistennija li jkolha 16 µg flavophosphlipol (200ml għal kampjun ta’ għalf ta’ 30g

fuq 1ppm). Fejn neċessarju, qabel l-applikazzjoni fil-kolonna, ħallat il-filtrat b’80%

methanol (4.6) biex ikollok il-mistenni kontenut ta’ flavophosphlipol ta’ 16 µg/

100ml. Adatta l-flow rate għal xi 2ml/minuta. Armi l-effluent. Imbagħad aħsel il-

kolonna b’80% methanol (4.6) u armi l-effluent.

Naddaf il-flavophospholipol bis-soluzzjoni metanolika tal-potassium chloride

(4.8) u żomm il-flow rate madwar 2ml/minuta. Iġbor 50ml mill-ħasil f ’graduated

flask, żid 30ml ilma u ħallat. Din is-soluzzjoni għandha jkollha kontenut ta’

flavophospholipol ta’ 0.2 µg/ml (= U 8 ).

7.3

Soluzzjonijiet ta’ l-Assay

Fejn neċessarju (meta l-fażi tal-purifikazzjoni tkun tħalliet barra) ħallat l-estratt

miksub fil-punt 7.1.1 b’50% methanol (4.5) biex jagħtik il-mistenni kontenut ta’

flavophosphlipol ta’ 0.2 µg/ml (=U 8 ).

Mis-soluzzjoni U 8 ipprepara soluzzjoni U 4 (kontenut mistenni: 0.1 µg/ml), U 2

(kontenut mistenni: 0.5 µg/ml) u U 1 (kontenut mistenni: 0.025 µg/ml) permezz tad-

diluzzjoni suċċessiva (1+1) b’50% methanol (4.5).

8. PROĊEDURA TA’ L-ASSAĠĠ

8.1

Tilqim ta’ l-assay medium

Laqqam l-assay medium (4.2.2) bil-bacterial suspension (3.2) f’madwar 50 grad

celsius. Permezz ta’ testijiet preliminari fuq plates bil-medium (4.2.2) iddetermina l-

kwantità ta’ bacterial suspension meħtieġa biex ikollok iż-żoni ta’ inibizzjoni kemm

jista’ jkun ċari u kbar mad-diversi konċentrazzjonijiet ta’ flavophospholipol (xi

30ml/litru).

8.2

Preparazzjoni tal-plates

Id-diffużjoni fuq l-agar ssir fuq plates bl-4 konċentrazzjonijiet tas-standard

solution (S 8 , S 4 , S 2 , S 1 ) u l-4 konċentrazzjonijiet ta’ l-assay solution (U 8 , U 4 , U 2 ,

U 1 ). L-4 konċentrazzjonijiet tas-standard solution u ta’ l-estratt iridu jitpoġġew fil-

plates. Għalhekk għandhom jintgħażlu plates li huma kbar biżżejjed biex ikunu

jistgħu jsiru mill-inqas 8 toqob fl-agar medium b’djametru ta’ 10 sa 13mm u mhux

inqas minn 30mm bejn iċ-ċentri. It-test jista’ jsir fuq plates li jikkonsistu f’lastra tal-

ħġieġ b’ħolqa ta’ l-aluminju jew tal-plastik fil-wiċċ, b’djametru ta’ 200mm u għoli

ta’ 20mm.

Ferra ġol-plates kwantità tal-medium (4.2.1) imlaqqma kif jidher f ’7.1, biex

ikollok strat ta’ xi 1.5mm ħxuna (45ml għal plate b’djametru ta’ 200mm). Ħallih

joqgħod f ’pożizzjoni livellata, u agħmel strat ieħor fuqu b’kwantità tal-medium

(4.2.2) imlaqqam bħal fil-punt 8.1 biex ikollok strat ta’ 1mm (30ml għal plate ta’

40mm). Erġa ħallih joqgħod f ’pożizzjoni livellata, agħmel it-toqob u poġġi fihom

volumi preċiżi ta’ assay u standard solutions (bejn 0.10 u 0.15ml, skond id-

djametru).

Applika kull konċentrazzjoni mill-inqas erba’ darbiet biex kull determinazzjoni

tkun soġġetta għal evalwazzjoni ta’ 32 inhibition zones.

8.3

Inkubazzjoni

Żomm il-plates inkubati minn 16 sa 18-il siegħa f’28 sa 30 grad celsius.

9. EVALWAZZJONI

Kejjel id-djametru taż-żoni ta’ inibizzjoni sa l-eqreb 0.1mm. Żomm rendikont tal-

miżuri medji ta’ kull konċentrazzjoni fuq semi-logarithmic graph paper bil-

logarithm tal-konċentrazzjonijiet relatati mad-djametri taż-żoni ta’ inibizzjoni.

Ipplottja il-best fit lines ta’ kemm is-standard solution kif ukoll l-estratt, per

eżempju

Iddetermina il-‘best fit’ point għas-standard lowest level (SL) permezz tal-

formula

(a) SL =

Iddetermina il-‘best fit’ point għas-standard highest level (SH) permezz tal-

formula

(b) SH =

F’mod simili kkalkula il-‘best fit’ points għall-extract lowest level (UL) u l-

extract highest level (UH) billi tissostitwixxi U 1 , U 2 , U 4 u U 8 għal S 1 , S 2 , S 4 u S 8 fil-

formuli ta’ fuq.

Żomm rendikont tal-valuri kkalkulati SL u SH fuq l-istess graph paper u

għaqqadhom biex ikollok il-‘best fit’ line għas-standard solution. Żomm rendikont

ta’ UL u UH u għaqqadhom biex ikollok il-‘best fit’ line għall-estratt.

Jekk ma jkunx hemm interferenza l-linji għandhom ikunu paralleli. Għal skopijiet

prattiċi l-linji għandhom jiġu kkonsidrati paralleli jekk il-valuri (SH-SL) u (UH-UL)

ma fihomx differenza ta’ iktar minn 10% tal-valur tagħhom medju.

Jekk il-linji ma jkunux paralleli, u 1 , s 1 , u 8 jew s 8 jista’ jintrema u SL, SH, UL u

UH ikkalkulati permezz ta’ dawn il-formuli alternattivi, biex ikollok ‘best fit’ lines

alternattivi

(a) SL =

jew

(b) SH =

jew

U l-istess għal UL u UH. L-istess kriterji ta’ paralleliżmu għandhom jiġu

sodisfatti. Il-fatt li r-riżultat ikun ġie kkalkulat fuq tliet livelli għandu jiġi annotat

fuq ir-rapport finali.

Meta l-linji huma kkunsidrati paralleli, ikkalkula il-logarithm ta’ l-attività

relattiva (log A) permezz ta’ waħda minn dawn il-formuli, skond jekk tliet jew erba’

livelli ġew użati fl-istima tal-paralleliżmu.

Għal erba livelli

(`) log A =

Għal tliet livelli

(d) log A =

jew

(d) log A =

Attività Reali = Attività supposta × rattività reali.

Meta l-linji huma kkunsidrati mhux paralleli, irrepeti d-determinazzjoni. Jekk il-

paralleliżmu ma jirriżultax, id-determinazzjoni ikkalkula l-logarithm tar-relative

activity (log A) permezz tal-formula (`). Ir-riżultat għandu jiġi kkunsidrat

approssimat u dan għandu jitniżżel fir-rapport finali.

10. RIPETIZZJONI

Id-differenza bejn ir-riżultati ta’ żewġ determinazzjonijiet paralleli li jsiru fuq l-

istess kampjun mill-istess analista ma għandhomx jeċċedu

-

0.5mg/kg, valur assolut, fil-każ ta’ kontenut ta’ flavophosphlipol minn 1 sa

2mg/kg;

-

25% relatat ma’ l-ogħla valur fil-każ ta’ kontenut ta’ aktar minn 2 sa 10mg/

kg;

-

20% relatat ma’ l-ogħla valur għal kontenut ta’ aktar minn 10 sa 25mg/kg;

-

5mg/kg, valur assolut, fil-każ ta’ kontenut minn 25 sa 50mg/kg;

-

10% relatat ma’ l-ogħla valur fil-każ ta’ kontenut ta’ iktar minn 50mg/kg.

3. DETERMINAZZJONI TAT-TRACE ELEMENTS ĦADID, RAM,

MANGANIŻ U ŻINGU

1. Għan u Skop

Dan il-metodu jiddetermina t-trace elements ħadid, ram, manganiż u żingu fl-

għalf. Il-limiti baxxi għad-determinazzjoni huma

Ħadid (Fe)

20mg/kg

Ram (Cu)

10mg/kg

Manganiż (Mn)

20mg/kg

Żingu (Zn)

20mg/kg

2. Prinċipju

Il-kampjun jitħallat mal-hydrochloric acid wara li jinqered, jekk preżenti, il-

materjal organiku. L-elementi iron, ram, manganiż u żingu jiġu determinati, wara t-

taħlit li hemm bżonn, permezz ta’ atomic absorption spectrometry.

3. REAĠENTI

Kummenti introduttorji

Għall-preparazzjoni tar-reaġenti u soluzzjonijiet analitiċi uża ilma mingħajr

cations biex jiġi determinat, miksub jew billi tagħmel double sitilling ta’ l-ilma

f ’borosilicate glass jew quartz still jew bit-trattament doppju fuq ion exchange

resin.

Ir-reaġenti għandhom ikunu ta’ mill-inqas grad analitiku (a.p). Għandek tara li ma

jkunx hemm l-element li jrid jiġi determinat permezz ta’ blank experiment. Jekk

neċessarju, ir-reaġenti għandhom jiġu purifikati aktar.

Minflok standard solutions deskritti hawn taħt, jistgħu jintużaw standard

solutions kummerċjali sakemm dawn ikunu garantiti u ċekkjati qabel ma jintużaw.

3.1.

Hydrochloric acid a.p. (d:1ꞏ19).

3.2.

Hydrochloric acid a.p. (6 N).

3.3.

Hydrochloric acid a.p. (0ꞏ5 N).

3.4.

Hydrofluoric acid 38 sa 40% (v/v) b’kontenut ta’ ħadid ta’ inqas minn 1

mg Fe/litre u residwu wara l-evaporazzjoni ta’ inqas minn 10 mg (bħala sulphate)/

litru.

3.5.

Sulphuric acid a.p. (d: 1ꞏ84).

3.6.

Hydrogen peroxide a.p. (madwar 100 volumi ossiġenu (30% meta

jintiżen)).

3.7.

Soluzzjoni b’working standard tal-ħadid (1 000 µg Fe/ml) ippreparata kif

ġej: ħoll 1 g ta’ wire tal-ħadid a.p. f’200 ml 6 N hydrochloric acid (3.2), żid 16 ml

hydrogen peroxide (3.6) żid sal-marka ta’ litru bl-ilma.

3.7.1. Soluzzjoni b’working standard tal-ħadid (100 µg Fe/ml) ippreparata billi

titħallat standard solution (3.7) 1 + 9 bl-ilma.

3.8.

Soluzzjoni standard tar-ram (1 000 µg Cu/ml) ippreparata kif ġej: ħoll 1 g

trab tar-ram (a.p.) f’25 ml 6 N hydrochloric acid (3.2), żid 5 ml hydrogen peroxide

(3.6) żid sal-marka ta’ litru bl-ilma.

3.8.1. Soluzzjoni b’working standard ta’ ram (10 µg Cu/ml) ippreparata billi

titħallat standard solution (3.7) 1 + 9 bl-ilma u mbagħad tħallat is-soluzzjoni

riżultanti 1 + 9 bl-ilma.

3.9.

Soluzzjoni standard ta’ manganiż (1 000 µg Mn/ml) ippreparata kif ġej

ħoll 1 g of trab tal-manganiż (a.p.) f ’25 ml 6 N hydrochloric acid (3.2) u żid sal-

marka ta’ litru bl-ilma.

3.9.1. Soluzzjoni b’working standard ta’ manganiż (10 µg Mn/ml) ippreparata

billi titħallat is-standard solution (3.9) 1 + 9 bl-ilma u mbagħad tħallat is-soluzzjoni

riżultanti 1 + 9 bl-ilma.

3.10. Soluzzjoni standard taż-żingu (1 000 µg Zn/ml) ) ippreparata kif ġej: ħoll

1 g żingu fil-forma ta’ strippa jew werqa (a.p.) f’25 ml 6 N hydrochloric acid (3.2),

u żid sal-marka ta’ litru bl-ilma.

3.10.1. Soluzzjoni b’working standard taż-żingu (10 µg Zn/ml) ippreparata billi

titħallat is-standard solution (3.10) 1 + 9 bl-ilma u mbagħad tħallat is-soluzzjoni

riżultanti 1 + 9 bl-ilma.

3.11. Soluzzjoni ta’ lanthanum choride ippreparata kif ġej: ħoll 12g lanthanum

oxide f’150ml ilma, żid 100ml 6 N hydrochloric acid (3.2) u żid sal-marka ta’ litru

4. APPARAT

Muffle furnace b’regolazzjoni tat-temperatura u recorder.

L-oġġetti tal-ħġieġ iridu jkunu tat-tip borosilicate reżistenti u huwa

rakkomandat li jintuża apparat li hu apposta għad-determinazzjoni tat-trace

Crucible tal-platinum u (mhux essenzjali) crucible tal-quartz.

Atomic absorption spectrophotometer li jissodisfa r-rekwiżiti tal-metodu li

għandu x’jaqsam mas-sensitività u l-preċiżjoni fil-medda meħtieġa.

5. PROĊEDURA

Kampjuni b’kontenut ta’ materjal organiku

Ashing u preparazzjoni tas-soluzzjoni għall-analiżi *

(i)

Poġġi 5 sa 10g mill-kampjun sa l-eqreb 0.2mg fi crucible tal-quartz jew

tal-platinum (4.3) (ara n-nota (b), nixfu f’forn fuq temperatura ta’ 105

gradi u daħħal il-crucible fil-cold muffle furnace (4.1). Agħlaq il-forn

(ara n-nota (`) u ogħlli t-temperatura gradwalment to 450 grad sa 475

grad għal xi 90-il minuta. Żomm din it-temperatura għal 4 sa 16-il

siegħa (eż għal lejl) biex tneħħi materjal karboniku u mbagħad iftaħ il-

forn u ħallih jiksah (ara n-nota (d).

Aħsel il-crucible b’total ta’ madwar 5ml hydrochloric acid (3.1) u żid

dan ta’ l-aħħar bil-mod u attentament f’beaker (jista’ jkun hemm

reazzjoni qawwija minħabba l-formazzjoni ta’ CO2). Żid hydrochloric

acid (3.1) fil-forma ta’ qtar u aġitah sakemm tieqaf l-effervexxenza.

Evapora sakemm jinxef u kultant ħawwad b’glass rod.

Żid 15ml 6N hydrochloric acid (3.2) mar-residwu u mbagħad xi 120ml

ilma. Ħawwad bil-glass rod li għandha tkun fil-beaker, u għatti l-beaker

b’watch-glass. Agħllih bil-mod u żommu fuq il-boiling point sakemm

ma tarax iktar ash jinħall. Iffiltrah fuq ash-free filter paper u iġbor il-

filtrat f’250ml volumetric flask. Aħsel il-beaker u ffiltrah b’5ml 6N

hydrochloric acid jaħraq (3.2) u darbtejn bl-ilma jagħli. Imla l-

volumetric flask sal-marka bl-ilma (konċentrazzjoni ta’ HCL xi 0.5N).

(ii) Jekk ir-residwu fil-filter jidher iswed (carbon) erġa’ poġġih fil-forn u

erġa’ ġibu għal irmied f’450 sa 475 grad Celsius. Dan l-ashing, li

*Għalf aħdar (frisk jew niexef) jista’ jkollu ammonti kbar ta’ vegetable silica li jista’ jżomm trace

elements u jrid jitneħħa. Fil-każ ta’ kampjuni ta’ l-ghalf, għalhekk, din il-pro`edura immodifikata

Imxi ma’ l-operazzjoni 1.1 (i) sal-filtrazzjoni. Aħsel il-filter paper li fiha r-residwu insolubli darbtejn

bl-ilma jagħli u poġġih fi crucible tal-platinum (4.3).

Saħħnu fil-muffle furnace (4.1) f’temperatura taħt il-550 gradi Celsius sakemm il-materjal karboniku

kollu jitlaq. Ħallih jiksaħ, żid ftit qtar ilma u 10 sa 15ml hydrofluoric acid (3.4) u evapora sakemm

tinxef f’madwar 150 gradi Celsius. Jekk tibqa’ xi silica fir-residwu, erġa’ ħollha fi ftit millilitri

hydrofluoric acid (3.4) u evaporah sakemm jinxef. Żid ħames qatriet sulphuric acid u saħħan sakemm

ma joħroġx iktar duħħan abjad. Wara li żżid 5ml 6 N hydrochloric acid (3.2) u xi 30ml ilma, saħħan,

iffiltra s-soluzzjoni ġol-250ml volumetric flask u żid sal-marka bl-ilma (kon`entrazzjoni ta’ HCL

madwar 0.5N). Ippro`edi bid-determinazzjoni mill-punt 5.1.3.

jeħtieġ biss ftit sigħat (xi 3 sa 5 sigħat), hu lest meta l-ash jidher abjad

jew kważi abjad. Ħoll ir-residwu ma’ xi 2ml hydrochloric acid (3.1),

nixxef sakemm jinxef u żid 5ml 6N hydrochloric acid (3.2). Saħħan u

iffiltra s-soluzzjoni f’volumetric flask u żid sal-marka bl-ilma

(konċentrazzjoni tal-HCL xi 0.5N).

Noti

(a) Meta tiddetermina xi trace elements hu importanti li toqgħod attent għar-

riskju ta’ kontaminazzjoni, speċjalment biż-żingu, ram u ħadid. Għal din ir-raġuni t-

tagħmir użat fil-preparazzjoni tal-kampjuni ma għandux ikollu minn dawn il-metalli.

Biex jitnaqqas ir-riskju ġenerali ta’ kontaminazzjoni, aħdem f’atmosfera mingħajr

trab b’tagħmir nadif ħafna u apparat tal-ħġieġ maħsul attentament. Id-

determinazzjoni taż-żingu hija partikolarment sensittiva għal ħafna tipi ta’

kontaminazzjoni, eż mill-ħġieġ, reaġenti, trab, eċċ.

(b) Il-piż tal-kampjun li ser jgħaddi mill-proċess ta’ ashing jiġi kalkulat mill-

a p p ro x i m a t e t r a c e e l e m e n t c o n t e n t f l - g h a l f i r r e l a t m a s - s e n s i t i v i t à t a ’

spectrophotometer użat. Fil-każ ta’ xi għalf li għandu kontenut baxx ta’ trace

elements, jista’ jkun neċessarju li tibda b’kampjun ta’ 10 sa 20g u żżid is-soluzzjoni

finali sa 100ml biss.

(`)

L-ashing irid isir fi closed furnace fejn ma jkunx hemm arja jew ossiġenu.

(d) It-temperatura indikata mill-pyrometer ma għandhiex teċċedi 475 grad

Celsius.

5.1.2

Spectrophotometric determination

5.1.2.1 Preparazzjoni tal-calibration solutions

Għal kull element li jrid jiġi determinat, ipprepara minn standard working

solutions mogħtija fil-punti 3.7.1, 3.8.1, 3.9.1, u 3.10.1 medda ta’ calibration

curves, b’kull calibration solution b’konċentrazzjoni ta’ HCL ta’ xi 0.5N u (fil-każ

tal-ħadid, manganiż u żingu) konċentrazzjoni ta’ lanthanum chloride ekwivalenti

għal 0.1% La (w/v). Il-konċentrazzjonijiet ta’ trace elements magħżula għandhom

ikunu fil-medda ta’ sensittività tas-spectrophotometer użat. It-tabelli hawn taħt juru,

bħala eżempju, il-kompożizzjoni tal-meddiet tipiċi ta’ calibration solutions; iżda

skond it-tip u s-sensittività ta’ spectrophotometer użat jista’ jkun neċessarju li

jintgħażlu konċentrazzjonijiet oħra.

Ħadid

µg Fe/ml

0.5

µg Fe/ml soluzzjonijiet standard

li jaħdmu (3.7.1)

(1 ml=100 µg Fe) +ml 6N HCI

0.5

(3.2)

+ 10 ml ta’ soluzzjoni ta’ lanthanum chloride (3.11) u ħallat ma’ 100 ml ta’ ilma.

Bronz

µg Cu/ml

0.1

0.2

0.4

0.6

0.8

0.1

µg Cu/ml soluzzjonijiet standard

li jaħdmu (3.8.1)

(1 ml = 100 µg Cu) +ml 6N HCI

(3.2)

Ħallat ma’ 100 ml ta’ ilma .

Manganiż

µg Mn/ml

0.1

0.2

0.4

0.36

0.8

1.0

µg Mn/ml soluzzjonijiet

standard li jaħdmu (3.9.1)

(1 ml=100 µg Mn) + ml 6N HCI

(3.2)

+ 10 ml ta’ soluzzjoni ta’ lanthanum chloride (3.11) u ħallat ma’ 100 ml ta’ ilma.

Żingu

µg Zn/ml

0.05

0.1

0.2

0.4

0.6

0.8

µg Zn/ml soluzzjonijiet standard

li jaħdmu (3.10.1)

(1 ml = 100 µg Zn) + ml 6N HCI

0.5

(3.2)

+ 10 ml ta’ soluzzjoni ta’ lanthanum chloride (3.11) u ħallat m’a 100 ml ta’ ilma.

5.1.2.2 Preparazzjoni tas-soluzzjoni ta ’l-analiżi

Għad-determinazzjoni tar-ram, is-soluzzjoni ppreparata mill-punt 5.1.1 tista’

normalment tintuża direttament. Jekk hu neċessarju biex tinġieb il-konċentrazzjoni

fil-medda tal-calibration solutions, tista’ tiġi ppipettjata porzjon ta’ aliquot f’100ml

volumetric flask, żid 10ml soluzzjoni tal-lanthanum chloride (3.11) u żid sal-marka

b’0.5N hydrochloric acid (3.3) (ara wkoll il-punt 8 ‘osservazzjoni’).

5.1.2.3 Blank experiment

Il-blank experiment għandu jinkludi l-fażijiet preskritti kollha tal-proċedura ħlief

li l-materjal tal-kampjun irid jitħalla barra.

Il-calibration solution "0" m’għandhiex tintuża bħala l-blank.

5.1.2.4 Tkejjil ta’ l-atomic absorption

Kejjel l-atomic absorption tal-calibration solutions u tas-soluzzjoni li ser tiġi

analizzata b’oxidizing air-acetylene flame fuq il-wavelengths li ġejjin

Fe

248.3nm

Cu

324.8nm

Mn

279.5nm

Zn

213.8nm

Kejjel kull wieħed erba’ darbiet.

5.2

Għalf minerali

Jekk il-kampjun ma fihx materjal organiku, mhux neċessarju li jkun hemm ashing

qabel. Kompli kif deskritt fil-ppunt 5.1.1 (i) mil-bidu tat-tieni paragrafu. L-

evaporazzjoni bil-hydrofluoric acid tista’ titħalla barra.

6. KALKOLAZZJONI TAR-RIŻULTATI

Permezz ta’ calibration curve, ikkalkula l-kalkolazzjoni tat-trace elements fis-

soluzzjoni li għandha tiġi analizzata u esprimi r-riżultat f ’milligrammi ta’ trace

element għal kull kilogramma tal-kampjum (ppm).

7. RIPETIZZJONI

ANALIŻI TA’ LIVELLI KIMIĊI

FIL-KONTROLL TA’ L-GĦALF

Id-differenza bejn ir-riżultati ta’ żewġ determinazzjonijiet paralleli meħuda mill-

istess kampjun mill-istess analista m’għandhomx jeċċedu

5mg/kg, f’valur assolut, fil-każ ta’ kontenut tat-trace element ikkonċernat

ta’ mhux iktar minn 50mg/kg;

10% mill-ogħla riżultat fil-każ ta’ kontenut tat-trace element minn 50 sa

100mg/kg;

10mg/kg, f’valur assolut, fil-każ ta’ kontenut tat-trace element ikkonċernat

minn 100 sa 200mg/kg;

5% mill-ogħla riżultat fil-każ ta’ kontenut tat-trace element ta’ aktar minn

200mg/kg.

8. OSSERVAZZJONI

Il-preżenza ta’ kwantitajiet kbar ta’ phosphates tista’ tkun tikkawża interferenza

mad-determinazzjoni ta’ ħadid, manganiż u żingu. Din l-interferenza trid tiġi

korretta permezz ta’ żieda ta’ soluzzjoni ta’ lanthanum chloride (3.11). Madankollu,

jekk fil-kampjun il-weight ratio

Ca + Mg

2,

P

iż-żieda tas-soluzzjoni ta’lanthanum chloride (3.11) mas-soluzzjoni għall-analiżi u

mal-calibration solutions tista’ titħalla barra.