Feedingstuffs Rules, 2005

L.N. 309 of 2005

VETERINARY SERVICES ACT

(CAP. 437)

Analysis of Chemical Levels in the Control of

Feedingstuffs Rules, 2005

IN exercise of the powers conferred by article 25 of the Veterinary

Services Act, the Minister for Rural Affairs and the Environment has

made the following rules>–

The title of these rules is the Analysis of Chemical Levels in

the Control of Feedingstuffs Rules, 2005.

.

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) 1

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 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: MICROCOCCUS LUTEUS (FLAVUS) ATCC

10240

3.1. Maintenance of stock culture

Inoculate tubescontaining slopes of culture medium (4.1) with Micrococcusluteus( flavus ) and incubate for

24 hours at 30 ºC. Store the culture in a refrigerator at about 4 ºC. Reinoculate every two weeks.

3.2. Preparation of the bacterial suspension a

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 30 ºC. 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 4 ºC.

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 to these Rules.

CULTURE MEDIA AND REAGENTS

4.1. Culture medium b

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)

a

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

suspensions

b

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

4.2. Assay medium b

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).

(1) 1 mg feedingstuff grade zinc bacitracin is equivalent to 42 international units (i.u.).

(a) Other methods may be used provided that it has been established that they give similar bacterial

suspensions.

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

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

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 35 ºC.

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 pH-

meter or with the bromocresol purple solution (4.10) as indicator.

Evaporate to about 4 ml in a rotary evaporator at a temperature not exceeding 35 ºC. 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 50 ºC. 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 u1). 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 ± 2 ºC.

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

7 s 1 + 4 s 2 + s 4 − 2 s 8

SL =

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

7 s 8 + 4 s 4 + s 2 − 2 s 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

5 s 1 + 2 s 2 − s 4 5 s 2 + 2 s 4 − s 8

( a ' ) SL =

or

5 s + 2 s 2 − s 1 5 s 8 + 2 s 4 − s 2

( b ' ) SH = 4

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 37 ºC,

store in a refrigerator at about 4 ºC and reinoculate every month onto agar slopes.

3.2. Preparation of the bacterial suspension a

Set aside two tubes containing the stock culture (3.1) and reinoculate them weekly. Incubate for 24 hoursat

37 ºC and store in a refrigerator at about 4 ºC.

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 37 ºC. Make a suspension of the growth in the sodium chloride solution

(4.3). The light 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

4.1. Culture medium b

Meat peptone

6·0 g

Tryptone

4·0 g

Yeast extract

3·0 g

Meat extract

1·5 g

Glucose

1·0 g

Agar

15·0 g

Water

1 000 ml

pH 6·5 (after sterilization)

4.2. Assay medium

4.2.1. Base layer c

Meat peptone

6·0 g

Yeast extract

3·0 g

Meat extract

1·5 g

Agar

10·0 g

Water

1 000 ml

pH 6·5 (after sterilization)

4.2.2. Seed layer

As for point 4.1, with the addition of 2·0 g of silicone anti-foaming emulsion. d

4.3. Sodium chloride solution 0·4 % (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.

a

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

b

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).

c

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).

d

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 1·5 % (w/v): dissolve 1·5 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 6·6 to 8·1).

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 µ g flavophospholipol per millilitre. Stored in stoppered flasks at 4

ºC 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

0·2 µg/ml

0·1 µg/ml

0·05 µg/ml

0·025µg/ml

7. PREPARATION OF THE EXTRACT

7.1. Extraction

7.1.1. Concentrates, premixes and mineral feeds

Weigh out a quantity of sample of 2·0 to 5·0 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 8·1 to 8·2 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 8·1 to 8·2 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 5·0 to 30·0 g containing at least 30 µ g of flavophospholipol.

Homegenize with 150 ml of 50 % methanol (4.5) in a conical flask (5.3) and adjust the pH to 8·1 to 8·2

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 8·1 to 8·2 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 4 ºC. 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 µ g 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 % methanol (4.6) to obtain an

expected flavophospholipol content of 16 µ g/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 0·2 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 0·2 µ g/ml (= U 8 ). From

solution U 8 prepare solutions U4 (expected content: 0·1 µg/ml), U 2 (expected content: 0·05 µ g/ml) and U 1

(expected content: 0·025 µ g/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 50 ºC. 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 1·5 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 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.

8.3. Incubation

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

9. 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

( a ) SL =

( b ) SH = 8

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

by substituting U1, U2, U4 and U8 for S1, S2, S4 and S8 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 S or U8 and S8 may be discarded and SL, SH,

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

( a ' ) SL =

jew

( b ' ) SH =

jew

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 =

jew

(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: 0·5 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:1·19).

3.2. Hydrochloric acid a.p. (6 N).

3.3. Hydrochloric acid a.p. (0·5 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: 1·84).

3.6. Hydrogen peroxide a.p. (approximately 100 volumesof 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

5.1. 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 0·2 mg in a quartz or platinum crucible (4.3) (see Note

(b)), dry in an oven at 105 ºC 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 475 ºC 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 CO2formation). 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 0·5 N).

(ii) If the residue in the filter appears black (carbon), put it back in the furnace and ash again at 450 to 475

ºC. 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 0·5 N).

Notes

(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 550 ºC 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 150 ºC. 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 0·5 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 475 ºC.

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 0·5 N and (in the cases of iron, manganese and zinc) a lanthanum chloride

concentration equivalent to 0·1 % 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

µ g Fe/ml

working

standard

solution

(3.7.1)

(1 ml =

100 µg Fe)

+ ml 6 N

0.5

HCl (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

µ g Cu/ml 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

µ g Mn/ml

0.1

0.2

0.4

0.36

0.8

1.0

µ g Mn/ml

ml working

standard

solution

(3.9.1)

(1 ml = 10 µ

g 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

ml working

standard

solution

(3.10.1)

(1 ml = 10

µg Zn)

+ ml 6 N

0.5

HCl (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 0·5 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 0·5 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: 248·3 nm

Cu: 324·8 nm

Mn: 279·5 nm

Zn: 213·8 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

The presence of large quantities of phosphates may interfere with the determination of iron, manganese and

zinc. Such interference must be corrected by addition of lanthanum chloride solution (3.11). If, however, in

Ca + Mg

2 , addition of lanthanum chloride solution (3.11) to the solution for

the sample the weight ratio

P

analysis and to the calibration solutions may be omitted.

Mitbug] fl-Istamperija tal-Gvern - Printed at the Government Printing Press

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: żingu bacitracin ta’ attivita` magħrufa (f’ i.u).

5. SOLUZZJONIJIET STANDARD

Iżen kwantita` standard Zinc bacitracin (4.11) li tikkorrispondi għal 1050 i.u. (skond l-attivita` 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 ipprepara 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

b

Kull culture medium kummerċjali li għandu komposizjoni simili u li jagħti l-istess riżultati jista’ jintuża.

6 PREPARAZZJONI TA’ SOLUZZJONIJIET TA’ ESTRATT U SOLUZZJONIJIET TA’ ASSAĠĠ

6.1 Estrazzjoni

6.1.1 Taħlitiet minn qabel u ikel minerali

Iżen kwantita` ta’ 2.0 sa 5.0 g mil-kampjun u żid 29ml tat-taħlita (4.4) u 1.0ml soluzzjoni tas-sodium

sulphide (4.9) u ixxekja. Ara li l-pH hi madwar 2. Ixxekja għal 10 minuti, żid 30ml phosphate buffer

(4.5), ixxekja 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 kwantita` tal-kampjun ta’ 10.0g, żid 49ml mit-taħlita (4.4) u 1ml soluzzjoni tas-sodium sulphide

(4.9) u ixxekja. Ara li jkollok pH ta’ xi 2. Ixxekja għal 10 minuti. Id 50ml phosphate buffer (4.5),

ixxekja 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’

temperature 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 kwantita` 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) ixxekja għal 15-il minuta u uża

centrifuge. Ħu 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 u1 (kontenut mistenni: 0.0525 i.u./ml) permezz tad-dilwizzjoni suċċessiva (1+1) bil-

phosphate buffer (4.5).

7. PROĊEDURA 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 (4.1) iddetermina l-kwantita` ta’ bacterial

suspension meħtieġa biex ikollok iz-zoni 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 and

S 1 ) u l-4 konċentrazzjonijiet ta’ l-assay solution ( u 8 , u 4 , u 2 and 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-kwantita` 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 kwantita` 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’pożiżżjoni illivellata, agħmel it-

toqob u poġġi fihom volum preċiż 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.

7.3 Inkubazzjoni

Żomm il-plates inkubati minn 16 sa 18-il siegħa fi 28 sa 30 0 C.

8. EVALWAZZJONI

Kejjel id-djametru taz-zoni 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 taz-zoni 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

7 s 1 + 4 s 2 + s 4 − 2 s 8

SL =

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

7 s 8 + 4 s 4 + s 2 − 2 s 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 il-linji għandhom ikunu paralleli. Għal skopijiet prattiċi il-linji

għandhom jiġu ikkonsidrati 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, u1, s1, u8 jew s8 jista’ jintrema u SL, SH, UL u UH kalkolati permezz

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

5 s 1 + 2 s 2 − s 4 5 s 2 + 2 s 4 − s 8

( a ' ) SL =

or

5 s 4 + 2 s 2 − s 1 5 s 8 + 2 s 4 − s 2

( b ' ) SH =

or

U l-istess għal UL u UH. L- istess kriterji ta’ paralleliżmu għandhom jiġu sodisfatti. Il-fatt li r-riżultat

ikun ġie kalkolat fuq tlett livelli għandu jiġi annotat fuq ir-rapport finali.

Meta l-linji huma kunsidratibħala paralleli, ikkalkola l-logarithm ta’ l-attivita` 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

(c) log A=

Għal tlett livelli

( d ) log A =

jew

( d ' ) log A =

Attività ta’ kampjun estratt= attivita li tidher * attivita ta’ standard rilevanti * A

(u 8 =s 8 X A)

Jekk l-attivita` 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-

attivita` relattiva ma tistax tinċieb fil-medda meħtieġa, kull riżultat irid jiġi ikkunsidrat approssimat u

dan għandu jitniżel fir-rapport finali.

Meta l-linji huma ikkunsidrati mhux paralleli, irrepeti d-determinazzjoni. Jekk il-paralleliżmu ma

jirriżultax, id-determinazzjoni għandha tiġi ikkunsidrata insodisfaċenti.

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

9. REPETIZZJONIJIET

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 mal-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 mal-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 teħlilitet 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 ippurifikat (fejn neċessarju) permezz ta’ trattament b’ ion exchange resins u jiġi mħallat. L-

attivita` antibiotika tiegħu tiġi determinata billi titkejjel id-diffużjoni tal-flavophospholipol f’agar

medium imlaqqam bl-staphylococcus aureus. Id-diffużjoni tidher bil-formazzjoni ta’ zoni ta’

inibizzjoni tal-mikro-organiżmu. Id-djametru ta’ dawn iz-zoni hu ikkunsidrat direttement 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 kultura

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.

3.2. Preparazzjoni tal-bacterial suspension a

Warrab żewġ tubi bis-stick kultura (3.1) u laqqanhom kull ġimgħa. Inkubahom għal 24 siegħa f’

temperature 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 suspensjoni 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-1ċm f’soluzzjoni tas-sodium chloride.

4. CULTURE MEDIA U REAĠENTI

4.1. Culture medium b

Meat peptone

6·0 g

Tryptone

4·0 g

Estratt tal-ħmira

3·0 g

Estratt tal-laħam

1·5 g

Glukożju

1·0 g

Agar

15·0 g

1 000 ml

pH 6·5 (wara s-sterilizzazjoni)

4.2. Assay medium

4.2.1. Base layer c

Meat peptone

6·0 g

Estratt tal-ħmira

3·0 g

Estratt tal-laħam

1·5 g

Agar

10·0 g

1 000 ml

pH 6·5 (after sterilization)

4.2.2. Seed layer

Jistgħu jintużaw metodi oħra sakemm ikun stability li jagħtu bacterial suspensions simili.

Kull culture medium b’komposizzjoni 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).

Bħal punt 4.1, biż-żieda ta’ 2·0 g silicone anti-foaming emulsion. d

4.3. Sodium chloride solution 0·4 % (w/v): ħoll 4 g sodium choride a.p. fl-ilma u ħallat sa 1 000

ml; sterilizza.

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 1·5 % (w/v): ħoll·5 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 6·6 to 8·1).

4.13. Ascorbic acid.

4.14. Standard substance: flavophospholipol ta’ attivita` magħrufa.

5. APPARAT

5.1. Tubu tal-ħġieġ għal chromatography, djametru internali: 9 mm, tul: 150 sa 200 mm, iffitjat b’

stopcock fil-parti dejqa tat-tarf t’isfel u ground-glass joint (biex jitqabbad mad- dropping funnel

(5.2)) fit-tarf ta’ fuq.

5.2. Dropping funnel 250 ml, iffitjat 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 kwantita` 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

0·2 µg/ml

0·1 µg/ml

d

eż. SE 2 minn Wacker Chemie GmbH, Munich.

0·05 µg/ml

0·025µg/ml

7. PREPARAZZJONI TA’ L-ESTRATT

7.1 Estrattazjoni

7.1.1 Konċentrati,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 jiksah, għaddih

minn centrifuge u poġġih f’decanter.

7.1.2 Feeds oħra

Iżen 5.0 sa 30.0 g mil-kampjun b’kontenut ta’ mill-inqas 30ug 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 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 jiksah, għaddih

minn centrifuge u poġġih f’decanter

7.2 Purifikazzjoni (din il-fażi tista’ titħalla barra fil-każ ta’ konċentrati, taħlitiet li jsiru minn qabel u

tmigħ minerali)

Ħallat 110ml mill-estratt ma’ 11g cation exchanger(4.9), għallih għal minuta taħt reflux(5.4) filwaqt li

tħawdu bla waqfien. Issepara l-cation exchanger 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 16ug 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’ 16µg/100ml. Adatta l-

flow rate għal xi 2ml/minuta. Armi l-effluent . Imbagħad aħsel il-kolonna b’80% methanol (4.6) u armi

l-effluwent.

Naddaf il-flavophospholipol bis-soluzzjoni metanolika talpotassium chloride (4.8) u żomm il-flow rate

madwar 2ml/minuta. Iġbor 50ml mil-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 f’punt 7.1.1

b’50% methanol (4.5) biex itik il-mistenni kontenut ta’ flavophosphlipol ta’ 0.2µg/ml (=U 8 ).

Mis-soluzzjoni U 8 ipprepara soluzzjoni U4 (kontenut mistenni: 0.1 µg./ml), U2 (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-kwantita` ta’ bacterial

suspension meħtieġa biex ikollok iz-zoni 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 fit-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. 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 kwantita` 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’pożiżżjoni livellata, u

agħmel strat ieħor fuqu b’kwantita` tal-medium (4.2.2) imlaqqam bħal f’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 taz-zoni 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 taz-zoni 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 ikkalkula 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 ikkalkulati 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 il-linji għandhom ikunu paralleli. Għal skopijiet prattiċi il-linji

għandhom jiġu ikkonsidrati 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, u1, s1, u8 jew s8 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 ikkalkulat fuq tlett livelli għandu jiġi annotat fuq ir-rapport finali.

Meta l-linji huma ikkunsidrati paralleli, ikkalkula il-logarithm ta’ l-attivita` 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

(c) log.A=

Għal tlett livelli

(d) log .A =

jew

(d’) log.A=

Attivita Reali = Attivita supposta × rattivita reali.

Meta l-linji huma ikkunsidrati mhux paralleli, irrepeti d-determinazzjoni. Jekk il-paralleliżmu ma

jirriżultax, id-determinazzjoni ikkalkula l-logarithm tar-relative activity (log A) permezz tal-formula

(c). Ir-riżultat għandu jiġi ikkunsidrat approssimat u dan għandu jitniżżel fir-rapport finali.

10. REPETIZZJONI

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 mal-ogħla valur fil-każ ta’ kontenut ta’ aktar minn 2 sa 10mg/kg;

-20% relatat mal-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 mal-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, l-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

Għal-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 bid-trattament

doppju fuq ion exchange resin.

Ir-reaġenti għandhom ikunu ta’ mill-inqas grad analitiku (a.p). Ghandek 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:1·19).

3.2. Hydrochloric acid a.p. (6 N).

3.3. Hydrochloric acid a.p. (0·5 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: 1·84).

3.6. Hydrogen peroxide a.p. (madwar 100 volumi ossiġnu (30 % meta jintizen) ).

3.7. Soluzzjoni b’ working standard tal-ħadid (1 000 µg Fe/ml) ippreparata kif ġej: ħoll 1 g of 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 tal-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 bl-ilma.

4. APPARAT

4.1. Muffle furnace b’ regolazzjoni tat-temperatura u recorder.

4.2. L-oġġetti tal-ħġieġ iridu jkunu tat-tip borosilicate reżistenti u huwa irrikkomndat li jintuża

apparat li hu apposta għad-determinazzjoni tat-trace elements.

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

4.4. Atomic absorption spectrophotometer li jissodisfa r-rekwiżiti tal-metodu li għandu x’jaqsam

mas-sensitivita’ u l-preċiżjoni fil-medda meħtieġa.

5. PROĊEDURA

5.1 Kampjuni b’kontenut ta’ materjal organiku

5.1.1 Ashing u preparazzjoni tas-soluzzjoni għall-analiżi *

*

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-għalf, għalhekk, din il-proċedura immodifikata taplika.

Imxi mal-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 sakeem il-materjal karboniku kollu jitlaq. Ħallih jiksah, 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 hames 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 (konċentrazzjoni ta’

HCL madwar 0.5N). Ipproċedi bid-determinazzjoni minn punt 5.1.3.

(i)

Poġġi 5 sa 10g mil-kampjun sa l-eqreb 0.2mg fi crucible tal-quartz jew tal-platinum (4.3) (ara nota

b), nixfu f’forn fuq temperatura ta’ 105 gradi u daħħal il-crucible fil-cold muffle furnace(4.1).

Agħlaq il-forn (are nota (ċ)) 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 jiksaħ (ara 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 axxjah f’450 sa 475 grad

Celsius. Dan l-ashing, li jeġtieġ 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 6

N hydrochloric acid (3.2). Saħħan u iffilta 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 biz-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 taz-żingu

hija partikolarment sensittiva għal ħafna tipi ta’ kontaminazzjoni eż mil-ħġieġ, reaġenti, trab,

eċċ.

(b) Il-piż tal-kampjun li se jiġi axxjat jiġi kalkulat mill-approximate trace element content fl-

għalf irrelat mas-sensitivita’ ta’ 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.

(c) L-ashing irid isir fi closed furnace fejn ma jkunx hemm arja jew ossiġnu.

(d) It-temperatura indikata mil-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 f’

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’ sensittivita’ tas-spectrophotometer użat. It-tabelli hawn taħt

juru, bħala eżempju, il-komposizzjoni tal-meddiet tipiċi ta’ calibration solutions; pero` skond it-tip u

s-sensittivita’ ta’ spectrophotometer użat jista’ jkun neċessarju li jintgħażlu konċentrazzjonijiet oħra.

Ħadid

µg Fe/ml

0.5

ml

soluzzjonijiet

standard li

jaħdmu

(3.7.1)

(1

ml=100 µg

0.5

Fe) +ml 6N

HCI (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

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

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

ml

soluzzjonijiet

standard li

jaħdmu

(3.10.1) (1

ml=100 µg

Zn) +ml 6N

0.5

HCI (3.2)

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

5.1.2.2 Preparazzjoni tas-soluzzjoni ta’l-analiżi

Għad-determinazzjoni tar-ram, is-soluzzjoni ipprepata minn punt 5.1.1 tista’ normalment tintuża

direttament. Jekk hu neċessarju biex tinġieb il-konċentrazzjoni fil-medda tal-calibration solutions,

tista’ tiġi ippipettjata 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 punt 8

‘osservazzjoni’)

5..1.2.3 Blank experiment

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

kampjun irid jitħalla barra.

Il-calibration solution “0” m’għandhiex tintuza bħala l-blank.

5.1.2.4 Tkejjil tal-atomic absorption

Kejjel l-atomic absorption tal-calibration solutions u tas-soluzzjoni li se 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 f’punt 5.1.1 (i) mil-bidu tat-tieni paragrafu . L-evaporazzjoni bil-hydrofluoric acid tista’

titħalla barra.

6. KALKULAZZJONI TAR-RIŻULTATI

Permezz ta’ calibration curve , ikkalkula l-kalkulazzjoni 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. REPETIZZJONI

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-itrace 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-itrace 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

Ca + Mg

2 , iż-żieda tas-soluzzjoni ta’lanthanum

chloride (3.11). Jekk, pero` fil-kampjun il-weight ratio

P

chloride (3.11) mas-soluzzjoni għall-analiżi u mal-calibration solutions tista’ titħalla barra.