MISCELLANEOUS TECHNIQUES AND IMPURITIES

MT 158 DETERMINATION OF MERCURY ON TREATED SEEDS

WARNING.

Mercury(II) salts and organomercurial compounds are toxic and many are soluble
in water. Protect skin and respiratory system when handling the materials. Do not
pour solutions of mercurial compounds down the drain. Precipitate the mercury as
sulphide and dispose of it safely.

OUTLINE OF METHOD

The organic matter is destroyed by wet oxidation and the mercury is determined
colorimetrically with dithizone.

REAGENTS

Chloroform (CHCl3) see RE 64

Nitric acid (HNO3) concentrated, d20 1.42
Sulphuric acid (H2SO4) concentrated, d20 1.84
- 1% v/v solution
Hydrochloric acid (HCl) 0.1 mol/l (0.1N)
Hydrogen peroxide (H2O2) 100 volumes; 30% m/v (Note 1)
Hydroxylammonium chloride (HO ⋅ NH3Cl) 20% v/v aqueous solution
Dithizone (1,5-diphenyl-3-thiocarbazone); RE 59
- stock solution: dissolve 50 mg in chloroform (100 ml); store in a refrigerator
- working solution: dilute 2 ml of stock solution to 200 ml with chloroform.
Protect from sunlight.
Mercury(II) chloride (HgCl2); RE 135
- stock solution: dissolve mercury(II) chloride (67 mg) in 0.1 mol/l hydrochloric
acid (100 ml)
- working solution: dilute stock solution (1 ml to 100 ml) with water. Prepare
immediately before use; 1 ml contains 5 µg mercury.

APPARATUS

Round-bottom flasks 500 ml with two necks fitted with condenser and condensate
trap in the vertical neck and a tap funnel in the angled neck
Separating funnels 250 ml
Volumetric flasks 200 ml

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Spectrophotometer with 1 cm cells

PROCEDURE

Weigh 10 g of seeds, or sufficient to contain 100 to 150µg of mercury, (w g),

transfer to the flask, and add, through the tap funnel, water (2 ml), concentrated
sulphuric acid (3 ml) and nitric acid (5 ml). Warm very gently until the initial
reaction has subsided, then increase the rate of heating adding further amounts (2
ml) of nitric acid as necessary to prevent charring. When the solution is clear, allow
to cool, return the condensate from the trap to the flask and add hydrogen peroxide
(2 ml). Reflux for 1 h to remove excess nitric acid. The solution should be
colourless or a very pale yellow. Allow to cool, transfer to the volumetric flask,
make up to 200 ml with water, and mix thoroughly.
Transfer sufficient solution (v ml) to contain 10 to 15 µg of mercury (usually 20
ml) to a separating funnel, add sulphuric acid (100 ml of 1%) and
hydroxylammonium chloride (10 ml).
Add chloroform (1 ml) and shake to saturate the solution, releasing the pressure
carefully. Add dithizone working solution (25 ml) and shake for 1 min. Insert a
small roll of filter paper in the stem of the separating funnel, and run off the
chloroform layer into a 1 cm cell. Measure the absorbance at 490 nm against
chloroform as the reference. Determine the mercury content (c µg) from the
calibration graph.
Prepare a calibration graph to cover the range 0 to 25 µg of mercury by taking
suitable portions of the standard mercury working solution in separating funnels
and treating them as above, from: '.... add sulphuric acid (100 ml of 1%) and
hydroxylammonium chloride (10 ml) ....'
Run a blank determination omitting only the sample. Subtract the blank from the
values used for the calibration graph
c ⋅ 200
Mercury content = mg/g
w⋅v

Note 1 Hydrogen peroxide can cause severe burns. Wear gloves and protect eyes
when handling. Wash any spillages with copious amounts of water.

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Figure 49 Apparatus for the determination of mercury in treated seeds.

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MT 159 POUR AND TAP BULK DENSITY OF GRANULAR

MATERIALS

OUTLINE OF METHOD

A known weight of sample is put into a glass measuring cylinder and its volume
measured. The cylinder is then raised and allowed to fall vertically through a
distance of 25 mm on to a rubber pad. The volume is measured again after 100
'taps'.

APPARATUS

Measuring cylinder 250 ml to ISO 4788, BS 604: 1982

Rubber base pad this should have a BS hardness of 35-40 (BS 903 Part A: 1957).
Other materials of similar hardness have been found satisfactory, e.g. neoprene
sheet.
Glazed sampling paper

PROCEDURE

By a preliminary examination determine the weight of sample required to fill the

measuring cylinder to approximately 90% of its capacity. Weigh this amount of
sample (W g), to the nearest 0.1 g, on to the glazed paper. Form a chute with the
paper and pour the sample smoothly into the cylinder; gently level off the surface
of the granules and measure the volume to the nearest 1 ml (V1 ml).
Gently grasp the upper part of the cylinder and raise it 25 mm (Note 1), allow it to
drop on to the rubber base pad and repeat until a total of 100 taps has been made,
making 1 tap every 2 seconds (Note 2). Measure and record the volume of the
granules to the nearest 1 ml (V2 ml).

Calculate the densities (g/ml):

W
Pour density DP = g/ml
V1
W
Tap density DT = g/ml
V2

Note 1 A dropping box as described in BS 1460: 1967 (Jencons Scientific

Limited) may be found convenient for controlling the 25 mm lift.
Note 2 Rotation of the cylinder through about 10 degrees should be made during

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the lifting to help impart a level surface to the granules.

MT 160 SPONTANEITY OF DISPERSION OF SUSPENSION
CONCENTRATES

SCOPE AND LIMITATIONS

The method is suitable for aqueous suspension concentrates (SC) which are
substantially diluted with water before use, and which contain only one suspended
active ingredient. In some cases the method may be extended to concentrates
containing two or more suspended active ingredients (see below).
Although the method gives reasonably consistent results, great care must be
exercised in the interpretation of the results as the relationship between the
measured spontaneity and the suitability or otherwise in field use, depends on the
application equipment and its degree of agitation.

OUTLINE OF METHOD
The method is broadly similar to that used to determine the suspensibility of
concentrates (MT 161), except that it employs only one inversion and a 5 min
standing time. It involves preparing 250 ml of a mixture of formulation and water,
mixed with only one inversion of the measuring cylinder. After standing under
defined conditions the top nine-tenths is removed, and the remaining tenth assayed
chemically, gravimetrically or by solvent extraction. The spontaneity of dispersion
is readily calculated.
REAGENTS

Standard Water C (see MT 18) or such other standard water as may be specified
Flocculating agent, e.g. 0.1% solution of Magnafloc R140 (required only if
gravimetric or solvent extraction procedures are adopted (Note 1)
Solvent suitable for the active ingredient in question (required only if the solvent
extraction procedure is adopted.)
APPARATUS

Constant temperature bath large enough to take several 250 ml cylinders

immersed to the neck, and capable of maintaining the specified temperature
within ± 1°C. It is important that no vibration is transferred to the cylinders, as
this would alter the sedimentation rate.
Graduated cylinders conforming to ISO 4788 (identical to BS 604). Glass
stoppered, 250 ml capacity (See Fig. 50).

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Glass suction tubes about 40 cm long, 5 mm i.d. drawn out at one end to 2-3 mm
i.d.
Extraction apparatus (required only if gravimetric or solvent extraction procedures
are adopted). Either (a) or (b).
(a) Centrifuge capable of 3000 rpm, and suitable for at least 50 ml tubes
(b) Filtration assembly consisting of a suction filter flask, together with either a
Buchner funnel or a sintered glass crucible, porosity P16 (pore size 10-16 µm)

PROCEDURE

Normally the whole procedure is carried out in duplicate. Homogenize the sample
by gently stirring (Note 2).
The standard water, measuring cylinder and sample to be used in the determination
should be equilibrated to the required temperature before starting the test.
Determine the density of the formulation (MT 3.3) and calculate the mass of
formulation equivalent to 12.5 ml (w g). Pour standard water (237.5 ml) at the
required temperature into the graduated cylinder and stand this on a top-pan
balance; add the calculated mass of formulation from a small beaker held so that
the lip is 1 cm above the top of the cylinder. Complete the addition within a time
limit of 15 s (Note 3).
As soon as the formulation has been added, stopper the cylinder and invert once
(Note 4). Stand the cylinder in an upright position on a bench free from vibration or
direct sources of heat for 5 min ± 10 s. At the end of this time, carefully remove the
stopper and withdraw the top 225 ml of suspension by means of the suction tube
connected to a reservoir and suitable pump. Carry out the operation in 10-15 s by
maintaining the tip of the tube just below the falling level of the suspension, care
being taken to minimize any disturbance of the suspension. Ensure that the tip of
the tube is always only a few mm below the surface of the suspension (Note 5).
The 25 ± 1 ml of dilute suspension remaining in the cylinder must be assayed in
one of the following ways:

(a) Chemically, by a method approved for the active ingredient being examined.
This is the preferred method and, if more than one insoluble active ingredient is
present, then it is the only acceptable method.

(b) Gravimetrically, by separating the solids (either by centrifugation or by

filtration), drying and weighing (Note 6).

(c) Solvent extraction, by separating the solids as above (Note 6), extracting the
solvent-soluble portion into a suitable solvent, evaporating the resultant solution to

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dryness, and weighing the residue.

Assay the formulation by the same method. Do not mix methods.

CALCULATION

Each of the three assay methods yields a mass: chemical assay gives the mass of
active ingredient, gravimetric assay the mass of water-insoluble solids, and solvent-
extraction the mass of solvent-soluble residue.
Irrespective of the assay method used, and irrespective of which of the above
masses is therefore obtained, the masses required in the calibration are as follows.

Q = mass in the 25 ml sample at the bottom (g)

a = percentage by mass in the formulation
w = mass of formulation actually added to the cylinder (g)
c = wa/100 = mass in the whole cylinder (g)

111 (c - Q)
Spontaneity of dispersion = %
c
REPORTING

Report Spontaneity of Dispersion to the nearest 1% and specify the following

parameters:

(a) Water hardness (if other than Standard Water C)

(b) Water temperature

Note 1 Magnafloc R140 is supplied by Allied Colloids Co, PO Box 36, Low
Moor, Bradford BD120J2, U.K.
Note 2 A crust of dry material sometimes forms inside the neck of suspension
concentrate containers. Care must be taken to ensure that particles do
not break away from this and contaminate the sample used for the
determination.
Note 3 It may be helpful to weigh the beaker plus formulation before and after
the addition to the water to make a check on the amount of sample
added to the water.
Note 4 To invert the cylinder, hold it with one hand at each end, insulated with
a cloth, and rotate it through 180° and back again, through an imaginary
fixed point midway between the hands. Ensure no bouncing occurs.
Each inversion should take 2 sec.
Note 5 It may be helpful to fit a rubber bung, larger than the cylinder mouth, as

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an adjustable stop so that the final level of suspension left in the

cylinder is repeatable. The position of the bung can be determined by a
preliminary calibration.
Note 6 The general procedures of gravimetric analysis are outlined in books on
analytical chemistry. Suspension concentrates present special problems
because their particle size is very small, and these particles are
sometimes difficult to separate, either by centrifugation or by filtration.
Separation may sometimes be facilitated by adding 2.5 ml of 0.1%
flocculating agent solution (e.g. Magnafloc R.140, see Note 1) to the
25 ml of suspension in the cylinder. This should then be allowed to
stand for 5 min.

MT 161 SUSPENSIBILITY OF AQUEOUS SUSPENSION

CONCENTRATES

SCOPE AND LIMITATIONS

The method is suitable for aqueous suspension concentrates (SC) which are
substantially diluted with water before use, and which contain only one suspended
active ingredient. In some cases the method may be extended to concentrates
containing two or more suspended active ingredients (see below). If the diluted
suspension contains more than 1% insoluble solids, accuracy may be reduced
because of hindered settling.

OUTLINE OF METHOD

The method is broadly similar to previously published methods for determining the
suspensibility of wettable powders (e.g. MT 15.1). It involves preparing 250 ml of
diluted suspension, allowing it to stand in a measuring cylinder under defined
conditions, and removing the top nine-tenths. The remaining tenth is then assayed
essayed either chemically, gravimetrically or by solvent extraction, and the
suspensibility calculated.

APPARATUS

Constant temperature bath large enough to take several 250 ml cylinders

immersed to the neck, and capable of maintaining the specified temperature
within ± 1°C. It is important that no vibration is transferred to the cylinders, as
this would alter the sedimentation rate.

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Graduated cylinders conforming to ISO 4788 (BS 604). Glass stoppered, 250 ml
capacity (see Fig. 50).
Glass suction tubes about 40 cm long, 5 mm internal diameter, drawn out at one
end to 2 - 3 mm internal diameter.
Extraction apparatus required only if gravimetric or solvent extraction procedures
are adopted. Either (a) or (b).
(a) Centrifuge, capable of 3000 rpm, and suitable for at least 50 ml tubes.
(b) Filtration assembly consisting of a suction filter flask, together with either a
Buchner funnel or a sintered glass crucible, porosity P 16 (pore size 10 -16 µm)

REAGENTS

Standard Water C (see MT 18) or such other standard water as may be specified
Flocculating agent, e.g. 0.1% solution of Magnafloc R140 (required only if
gravimetric or solvent extraction procedures are adopted; (Note 1)
Solvent suitable for the active ingredient in question. Technical grade is
satisfactory. (Required only if the solvent extraction procedure is adopted.)

PROCEDURE

Normally the whole procedure is carried out in duplicate. Homogenize the sample
by gently stirring (Note 2).
The mass of sample to be taken is that required to make 250 ml of diluted
suspension, with the concentration recommended on the label. If a range of
concentrations is recommended, the highest and lowest should be used (Note 3).
The standard water, measuring cylinder and sample to be used in the determination
should be equilibrated at the required temperature. Place 100 ml of equilibrated
standard water in a 250 ml measuring cylinder. Weigh the required amount of
sample into a 50 ml beaker and transfer quantitatively to the cylinder with more
standard water, finally making up to 250 ml. Stopper the cylinder and invert 30
times (Note 4). Place the cylinder submerged to the neck in the water bath in an
upright position, free from vibration, and allow it to stand undisturbed for the
specified time (Note 5).
At the end of the time, remove the cylinder from the constant temperature bath,
quickly insert the glass tube into the cylinder, and by means of a vacuum pump and
suitable reservoir withdraw nine-tenths (225 ml) of the suspension. Carry out the
operation in 10-15 sec by maintaining the tip of the glass tube just below the falling
level of the suspension, care being taken to minimize any disturbance of the
suspension.

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Figure 50 Sedimentation cylinder with critical dimensions.

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The 25 ± 1 ml of dilute suspension remaining in the cylinder must be assayed in

one of the following ways:

(a) Chemically, by a method approved for the active ingredient being examined.
This is the preferred method and, if more than one insoluble active ingredient is
present, then it is the only acceptable method.

(b) Gravimetrically, by separating the solids either by centrifugation or by

filtration, drying and weighing (Note 6).

(c) Solvent extraction, by separating the solids as above (Note 6), extracting the
solvent-soluble portion into a suitable solvent, evaporating the resultant solution to
dryness, and weighing the residue.
Assay the formulation by the same method. Do not mix methods.

CALCULATION

Each of the three assay methods yields a mass: chemical assay gives the mass of
active ingredient, gravimetric assay the mass of water-insoluble solids, and solvent-
extraction the mass of solvent-soluble residue.
Irrespective of the assay method used, and irrespective of which of the above
masses is therefore obtained, the masses required in the calculation are as follows.

Q = mass in the 25 ml sample at the bottom (g)

a = percentage by mass in the formulation
w = mass of formulation actually added to the cylinder (g)
c = wa/100 = mass in the whole cylinder (g)

111 (c - Q)
Then suspensibility = %
c
REPORTING

Report the suspensibility to the nearest 1% and specify the following parameters:

(a) Water hardness (if other than Standard Water C)

(b) Water temperature
(c) Duration of test (if other than 30 min)

Note 1 Magnafloc R140 is supplied by Allied Colloids Co, PO Box 36, Low
Moor, Bradford BD120J2, U.K.

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Note 2 A crust of dry material sometimes forms inside the neck of suspension
concentrate containers. Care must be taken to ensure that particles do not
break away from this and contaminate the sample used in the test.

Note 3 Recommended concentrations given on labels will refer to volumes of

product. The nature of suspension concentrates means that it is difficult to
avoid air bubbles when the product is poured out and, for accuracy, it is
necessary to determine the density of the product by MT 3.3 and use
masses of product in the method.

Note 4 To invert the cylinder, hold it with one hand at each end, insulated with a
cloth, and rotate it through 180° and back again, through an imaginary
fixed point midway between the hands. Ensure no bouncing occurs. Each
inversion should take 2 sec.

Note 5 A period of 30 min is appropriate for checking the suitability of the

suspension for spraying. For some special purposes, however, such as
detecting slight differences between formulations, or for detecting slight
differences in a formulation before and after a heat stability test, a time of
1 or even 2 h may be more appropriate.

Note 6 The general procedures of gravimetric analysis are outlined in books on

analytical chemistry. Suspension concentrates present special problems
because their particle size is very small, and these particles are sometimes
difficult to separate, either by centrifugation or by filtration. Separation
may sometimes be facilitated by adding 2.5 ml of 0.1% flocculating agent
solution (e.g. Magnafloc R.140, see Note 1) to the 25 ml of suspension in
the cylinder. This should then be allowed to stand for 5 min.

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MT 162 DETERMINATION OF ETHYLENETHIOUREA (ETU)

(IMIDAZOLIDINE-2-THIONE)

NH
S
NH

SCOPE

The methods are suitable for determining ETU in technical and formulated
products containing ethylene-bis-(dithiocarbamate) (mancozeb, maneb, zineb and
metiram)

162.1 HPLC method (Referee method)

OUTLINE OF METHOD

The ETU is extracted from the sample with methanol. An aliquot of the methanolic
solution is evaporated to dryness and the residue is dissolved in water. The solution
is filtered and diluted, if necessary, and injected on to a column of Nucleosil C18 or
Spherisorb ODS. The ETU is eluted with water containing tetrahydrofuran and
detected at 233 nm (Note 1).

REAGENTS

Ethylenethiourea (ETU) standard sample at least 99% pure.

Calibration solutions Prepare a set of calibration solutions in water containing 1, 5,
10, 15, 20, 30, 40, and 50 µg of ETU per ml. Keep in a water bath at 20°C.
Methanol (CH3OH) HPLC grade. Keep in a water bath at 20°C
Tetrahydrofuran (C4H8O) Uvasol - Merck 8110 or equivalent
Distilled water RE 130

APPARATUS

Liquid chromatograph equipped with 10µl loop injector, variable UV detector, and
electronic integrator or recorder
Column stainless steel, 200 × 4.6 (i.d.) mm with Nucleosil 5 C18 or Spherisorb ODS

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(5 µm)

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Sintered glass funnel porosity P 16 (pore size 10-16 µm) approximate dimensions:
diameter at top about 45 mm, a sintered glass plate about 40 mm diameter, depth
from rim to plate about 55 mm. Attach the funnel with suitable adaptor to a
250 ml flask making sure that the end of the funnel tube is below the side arm of
the flask.
Millipore Filter, 0.45 µm, 2.5 cm (Ref HAWP 02500) or equivalent
Rotary vacuum evaporator
Water bath with accurate thermostatic control
Pipettes 10, 20 and 50 ml
Volumetric flask 100 ml
Conical flask 25 ml

PROCEDURE

(a) Operating conditions

Eluant distilled water containing tetrahydrofuran (0.05% v/v). Filter the
solution through a 0.45 µm filter
Flow rate 1.0 ml/min
Temperature ambient
Detector wavelength 233 nm
Injection volume 10 µl
Retention time about 4 min

(b) Preparation of calibration graph.

Inject 10 µl aliquots of one of the calibration solutions (e.g. the one containing 20
µg ETU per ml) to stabilize the instrument and to set the parameters. Inject
consecutively 10 µl of each of the calibration solutions to obtain their respective
HPLC responses. (Note 2).

(c) Extraction of ETU.

Weigh (to the nearest mg) sufficient sample (w g) to contain about 0.01 g ETU
(but not exceeding 20 g) on to the sintered glass funnel and add methanol (20 ml at
20°C). Stir the powder gently to disperse it and allow it to stand for 3 min, then
drain under gentle suction into the Buchner flask. Repeat with further methanol (3
× 20 ml). Transfer the filtrate into a 100 ml volumetric flask, wash the funnel and
Buchner flask with methanol transfer the washings to the volumetric flask,
equilibrate the extract at 20°C, and make up to 100 ml with methanol at 20°C.

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(d) Preparation of sample and determination.

Transfer by pipette 5 ml of the sample solution to a 25 ml conical flask. Evaporate
to dryness in the rotary evaporator at 35°C. Add water (20.0 ml) from a pipette to
the flask and shake for 3 min to dissolve the ETU. Filter the solution through the
Millipore filter. Inject 10 µl (Note 2) of the solution exactly 15 min after the
filtration. Allow the solution to remain undisturbed during this time. The
evaporation temperature, the time of shaking and of filtration, also the time
between filtration and injection must be rigidly observed. Allow the chromatogram
to run for 15 min and then inject 10 µl of the calibration solution with a
concentration of ETU near that of the sample.

(e) Calculation.
Determine the ETU content in the sample solution by comparing its peak height
with that from the calibration solution (Note 2).
ETU content = Hw⋅c g/kg
H s ⋅ w ⋅ 2.5
where:
Hw = peak height of the sample (mm)
Hs = peak height of the calibration solution (mm)
w = mass of sample (g)
c = concentration of ETU in the calibration solution (µg/ml)

162.2 Paper chromatographic method

OUTLINE OF METHOD

The ETU is extracted from the sample with methanol. Portions of the sample and
standard solutions are spotted on to filter paper and developed with butan-1-ol +
ethanol + water. The chromatogram is sprayed with pentacyanoaminoferroate
[PCAF] reagent which produces a blue spot with ETU. The ETU content of the
sample is determined by comparing the spot with those from the standards (Note 3).

REAGENTS

Butan-1-ol {CH3[CH2]3OH}
Ethanol (CH3CH2OH) or Industrial Methylated Spirit
Running solvent butan-1-ol(4 vols) + ethanol (1 vol) + water(1 vol) Mix well and
store in a well-stoppered glass bottle.
Visualising agent pentacyanoaminoferroate (PCAF reagent); RE 146 (Note 4)

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Methanol (CH3OH); RE 47
Ethylenethiourea (ETU) standard sample at least 99% pure
- strong standard solution. Dissolve ETU (0.1 g) in methanol (100 ml). 1 µl
contains 1 µg ETU
- weak standard solution. Dilute strong standard solution (10 ml) with
methanol to 100 ml. 1 µl contains 0.1 µg of ETU

APPARATUS

Whatman slotted chromatographic paper No 1

Beaker 600 ml squat form without spout
Chromatography spray
Sintered glass funnel Size 11, porosity P16, see MT 162.1
Syringe 10 µl
Volumetric flask 100 ml

PROCEDURE

(a) Extraction of ETU As for MT 162.1, (c) Extraction of ETU

(b) Determination.

Using the 10 µl syringe, add the following amounts to the paper channels:
Strong standard solution: 0.5, 1.0, 1.5 and 2.0 µl
Sample solution: 0.5, 1.0, 1.5 and 2.0 µl
Clip the edges of the paper together to form a cylinder and put vertically in a 600
ml beaker containing an approximate 1 cm depth of the running solvent. Cover with
a clock-glass and allow to run at room temperature until the solvent front coincides
with the top edge of the slots. Dry in warm air, and then develop the chromatogram
by spraying with PCAF reagent (Note 4). Allow to develop fully for 10 min and
then note in which channel spots are just visible.
From this observation, the highest possible limits of concentration of ETU in the
original sample are determined.

(c) Calculation
Volume of standard spot just visible
⋅ 1µg = y µg/µl ETU
Volume of sample spot just visible

100 ml methanol extract contains 100 y mg ETU or 0.1 y g ETU

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0.1 y ⋅ 1000 100 y

ETU content = = g/kg
w w

Volume of standard spot just visible ⋅ 100

or ETU content = g/kg
Volume of sample spot just visible ⋅ w

where:
w = mass of sample (g)

For a more precise measurement, transfer the following portions of the test
solution and the weak standard solution to the paper:

1.0, 2.0, 3.0, 4.0, 5.0 µl of the test solution

2.0, 4.0, 6.0, 8.0, 10.0 µl of the weak standard solution

Run the chromatogram and visualize as before and calculate a more precise limit
from correlation with the standard.

Volume of standard spot just visible ⋅ 10

ETU content = g/kg
Volume of sample spot just visible ⋅ w

Note 1 Based on the method of J C Van Damme, M Galoux and J Verdier, J

Chromat, 1981, 206, 125-31.

Note 2 Because of the influence of temperature on the HPLC response, a

calibration graph can only be prepared if the column and injector are kept
at a constant temperature. This graph can be used for calculating the ETU
content but should be checked at intervals to ensure that the response of
the HPLC has not changed. If not so controlled, it will be necessary to
prepare the standard solutions and to inject them each time an analysis is
carried out.

Note 3 Method supplied by Robinson Brothers Limited, based on that of E I

Johnson & J F C Tyler, Chem & Ind, 1962, 305-6.

Note 4 PCAF is not specific for ETU, and reacts to differing degrees with many
sulphur compounds.

404