Transfer the residue derived from 6.1.1 into a test tube, using a total of 10 ml cyclohexane-ethyl acetate mixture (VEx) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop of the gel permeation chromatograph (VR1) with 7 to 8 ml of the solution. Set the gel permeation chromatograph at the eluting conditions determined beforehand with a standard
284 Propiconazole
solution of propiconazole; cf. Cleanup Method 6, pp. 75 ff, Vol. 1 — Elution volumes ranging from 110 to 150 ml were determined for propiconazole on Bio-Beads S-X3 polystyrene gel using the cyclohexane-ethyl acetate mixture as eluant, pumped at a flow rate of 2.5 ml/min.
Collect the 110 to 150-ml fraction in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. Then proceed to step 6.2.2.
Check the elution range from time to time, and determine anew whenever a new gel column is used.
6.2.2 Column chromatography
Pour 15 ml hexane into the chromatographic tube. Slowly add 30 g aluminium oxide (free from air bubbles). Allow to settle, and then drain the hexane to the top of the column packing.
Transfer the residue derived from 6.1.1, 6.1.2, 6.1.3 or 6.2.1 to the column, using three 2-ml portions of toluene to complete the transfer. Drain the toluene to the top of the column pack- ing each time. Elute co-extractives with 50 ml of eluting mixture 1, and then elute pro- piconazole with 75 ml of eluting mixture 2, using a flow rate of 1 to 2 drops per s. Collect the eluate in a 100-ml round-bottomed flask, and rotary-evaporate to dryness.
6.3 Gas-chromatographic determination
Dissolve the residue derived from 6.2.2 in 2 ml ethanol-hexane mixture, and dilute to a suitable volume (VEnd). Inject an aliquot of this solution (V{) into the gas chromatograph.
Operating conditions
Gas chromatograph Hewlett-Packard 5710A/30A
Column Glass, 2 mm i.d., 1.5 m long; packed with 3% CP Wax 40 M on Gas Chrom Q, 80-100 mesh (Chrom- pack)
Column temperature 245 °C Injection port temperature 250 °C
Detector Thermionic nitrogen-specific detector Temperature 250 °C
Gas flow rates Nitrogen carrier, 36 ml/min Hydrogen, 3 ml/min Air, 50 ml/min Attenuation 16
Recorder 1 mV; chart speed 10 mm/min Injection volume 2 ul
Retention time for propiconazole 2 min 20 s
7 Evaluation
7.1 Method
Quantitation is performed by the calibration technique. Prepare a calibration curve as follows.
Inject 2 \x\ of each propiconazole standard solution (equivalent to 0.5 to 20 ng propiconazole) into the gas chromatograph. Plot the heights of the peaks obtained vs. ng propiconazole. Also
Propiconazole 285 inject 2-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of propiconazole from the calibration curve. Prepare a new calibration curve for each sample series.
7.2 Recoveries and lowest determined concentration
Recovery experiments were run on different untreated control samples of plant material, wine, soil and water, fortified with propiconazole at levels of 0.002 to 1.0 mg/kg. The recoveries are given in the Table; the values presented in this Table represent the means (± standard devia- tions) from 3 to 13 single experiments.
Table. Percent recoveries from plant material, wine, soil and water, fortified with propiconazole.
. . . , Added Recovery
Analytical material m g / k g n * s
Cereals Green matter Grains Straw Grapes Wine Soil Water
The routine limit of determination was 0.05 mg/kg for cereal green matter, straw and soil, 0.005 mg/kg for wine, 0.01 mg/kg for grapes and grains, and 1 jj.g/1 for water.
7.3 Calculation of residues
The residue R, expressed in mg/kg propiconazole, is calculated from the following equation:
£ _ WA • VEx ^VEnd
0.1 1.0 0.04 0.4 0.1 0.5 0.04 0.4 0.02 -0.4 0.04 -0.4 0.002 0.02
6 8 5 6 10 6 13 13 8 4 3 3
98 101 101 87 98 90 96 89 90 99 111 101
11 9 13 5 11 6 10 5 10 7 6 4
Vw-V,-G where
G = sample weight (in g) or volume (in ml)
VEx = volume of solution prepared for gel permeation chromatography in 6.2.1 (in ml)
286 Propiconazole
VR1 = portion of volume VEx injected for gel permeation chromatography (volume of sample loop) (in ml)
VEnd = terminal volume of sample solution from 6.3 (in ml)
Vj = portion of volume VEnd injected into gas chromatograph (in ul) WA = amount of propiconazole for Vj read from calibration curve (in ng)
8 Important points
For the analysis of extracts from water samples containing only small amounts of co- extractives, cleanup by column chromatography can be omitted.
Gel permeation chromatography can be performed also by the method described on pp. 65 ff, Vol. 1 (Cleanup Method 4).
9 Reference
B. Buttler, Gas chromatographic determination of propiconazole and etaconazole in plant material, soil, and water, J. Agric. Food Chem. 31, 762-765 (1983).
10 Authors
Ciba-Geigy AG, Agricultural Division, Basle, Switzerland, B. Buttler and W. D. Hormann
Sulphur
Apples, cucumbers, grapes, hops (foliage and cones), strawberries
Soil
184-B
High-performance liquid chromato- graphic determination (German version published 1985)
1 Introduction Chemical name Empirical formula Molar mass Melting point Boiling point Solubility
Other properties
Sulphur S8
256.51
112.8 °C (a-sulphur) 444.67 °C
Insoluble in water;
readily soluble in carbon disulphide (42.4 g/100 ml at 20 °C);
soluble in carbon tetrachloride;
sparingly soluble in commonly used organic solvents Solutions with low content of sulphur are sensitive to light
2 Outline of method
Sulphur residues are extracted from the sample material with a mixture of water, methanol and dichloromethane. An aliquot of the separated dichloromethane phase is rotary-evap- orated to dryness, and taken up in a mixture of isooctane and isopropanol. The extract is cleaned up by column chromatography on silica gel. Sulphur is determined by high-perfor- mance liquid chromatography using a UV detector.
3 Apparatus
Erlenmeyer flasks, 500-ml and 250-ml Laboratory mechanical shaker
Graduated cylinders, 250-ml, 100-ml and 25-ml Glass funnels, 10 cm and 4 cm dia.
Filter paper, 10 cm dia., Whatman 1 PS
Rotary vacuum evaporator, 40 °C bath temperature Chromatographic tube, 20 mm i. d., 20 cm long Round-bottomed flasks, 100-ml, with ground joints
288 Sulphur Ultrasonic bath Volumetric flasks, 5-ml
High-performance liquid chromatograph equipped with UV detector for measurement at 260 nm
Microsyringe, 100-ul
4 Reagents
Dichloromethane, dist.
Methanol, dist.
2-Propanol (isopropanol), dist.
2,2,4-Trimethyl pentane (isooctane), dist.
Solvent mixture 1: isooctane + isopropanol 8 :2 v/v Solvent mixture 2: dichloromethane + methanol 5 :95 v/v Eluting mixture: isooctane + isopropanol 97 : 3 v/v
Sulphur standard solutions: 1.0, 2.0, 5.0 and 10.0 ng/ml solvent mixture 2 Sulphur solutions for recovery experiments: 10 and 100 jig/ml dichloromethane Silica gel 60, 0.05-0.2 mm (Macherey-Nagel No. 81532)
Cottonwool
5 Sampling and sample preparation
The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1.
6 Procedure
6.1 Extraction
Weigh 50 g of the analytical sample (5 g for hops) (G) into a 500-ml Erlenmeyer flask, add 25 ml water, 25 ml methanol and 150 ml dichloromethane, stopper the flask tightly, and shake for 1 h on a mechanical shaker. Filter the flask contents through a filter paper in a funnel, and collect in a 250-ml Erlenmeyer flask. The separated dichloromethane phase contains the whole of the sulphur residue (VEx = 150 ml). Take a 75-ml aliquot (VR1) of the dichloro- methane phase, and rotary-evaporate to dryness. Dissolve the residue in 2.0 ml of solvent mix- ture 1.
6.2 Column chromatography
Pour 50 ml isooctane into the chromatographic tube, and slurry in 15 g silica gel. Drain the solvent to the top of the silica gel layer. Then add the solution derived from 6.1 to the column.
Wash with 3 ml of eluting mixture, and allow the solution to trickle into the column. Next elute the sulphur with the same mixture. From the time of addition, collect a forecut of 25 ml
Sulphur 289 (0-25 ml), and discard. Collect the main eluate of 30 ml (25-55 ml) which contains the sul- phur, in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. Dissolve the residue in 4 ml of solvent mixture 2; if necessary, use an ultrasonic bath. Filter the solution through a cottonwool pad placed in a small funnel, collect in a 5-ml volumetric flask, rinse with 1 ml of solvent mixture 2, and fill up to the mark with the same solvent mixture (VEnd).
6.3 High-performance liquid chromatographic determination
Inject 100 ul (Vj) of the solution derived from 6.2 into the high-performance liquid chromatograph.
Operating conditions
Pump Orlita No. AE-10-4-4 fitted with pulse dampener Injector Valco injection valve fitted with 100-ul sample loop Column Stainless steel, 6 mm i.d., 25 cm long; packed with
Polygosil 60-5 C18 (Macherey-Nagel) Mobile phase Solvent mixture 2
Flow rate 1.3 ml/min Temperature 20 °C
Detector Uvikon 722 LC UV detector (Kontron) Wavelength 260 nm
Recorder 10 mV; chart speed 5 mm/min Injection volume 100 ul
Retention time for sulphur 4 min 12 s
7 Evaluation
7.1 Method
Quantitation is performed by the calibration technique. Prepare a calibration curve as follows.
Inject 100 ul of each sulphur standard solution (equivalent to 0.1 to 1.0 ug sulphur) into the high-performance liquid chromatograph. Plot the heights of the peaks obtained vs. ng sul- phur. Also inject 100-ul aliquots of the sample solutions. For the heights of the peaks obtained for these solutions, read the appropriate amounts of sulphur from the calibration curve.
7.2 Recoveries and lowest determined concentration
The recoveries from untreated control samples of hops, fortified with sulphur at levels of 5 to 400 mg/kg, ranged from 85 to 100% with a relative standard deviation of ± 5%. The recov- eries from untreated control samples of the other materials, fortified with sulphur at levels of 1 to 10 mg/kg, averaged 100% for cucumbers, 90% for strawberries and apples, 85% for grapes, and 75% for soil. The routine limit of determination was 5 mg/kg for hops, and 1 mg/kg for all other substrates. Relatively high blanks may usually be expected, e.g.
0.2 mg/kg for cucumbers.
290 Sulphur
7.3 Calculation of residues
The residue R, expressed in mg/kg sulphur, is calculated from the following equation:
R_ wA-vE x-vE n d
V R I - V S - G
where
G = sample weight (in g)
VEx = volume of dichloromethane used for extraction of analytical sample (in ml) VR1 = portion of volume VEx used for cleanup in step 6.2 (in ml)
VEnd = terminal volume of sample solution from 6.2 (in ml)
Vj = portion of volume VEnd injected into high-performance liquid chromatograph (in Hi)
WA = amount of sulphur for Vj read from calibration curve (in ng)
8 Important points
During the analysis, care should be taken not to expose the solutions to sunlight.
Extracts with a high content of sulphur and a very low content of co-extractives need not be subjected to the cleanup described in step 6.2
Experience so far gained indicates that sulphur residues can be determined also in crops other than those listed in the heading of the method.
9 References
K. Wenzel, Die HPLC-Bestimmung von elementarem Schwefel in Lebensmitteln, Z. Lebensm.
Unters. Forsch. 170, 5-6 (1980).
R.M. Cassidy, A selective method for elemental sulfur analysis by high-speed liquid chroma- tography, J. Chromatogr. 117, 71-79 (1976).
10 Author
BASF, Agricultural Research Station, Limburgerhof, J. Elzner
Thiabendazole 256-A
Apples (peel), bananas (peel), grapefruit (peel), oranges Fluorimetric (peel), potatoes (peel) determinination on
TLC chromatograms (German version published 1979)
1 Introduction
Chemical name Structural formula
Empirical formula Molar mass Melting point Vapour pressure Boiling point
Solubility (in 100 ml at room temperature)
2-(Thiazol-4-yl)benzimidazole (IUPAC)
Other properties
C10H7N3S 201.25
304-305 °C, sublimes from 250 °C No data
No data
Slightly soluble in water (approx. 1 g) at pH 2, very sparingly soluble at pH 3-12;
soluble in dimethylacetamide (6.47 g), dimethylform- amide and dimethylsulphoxide (8 g);
slightly to sparingly soluble in acetone, benzene, tert.butanol, chloroform, dichloromethane, diethyl ether, ethyl acetate and methanol (0.93 g) Stable in solution, fluoresces in UV light
2 Outline of method
Thiabendazole residues are extracted from plant material with dichloromethane. The extract is concentrated and the concentrate made up to a definite volume with ethanol. In order to separate thiabendazole from plant co-extractives, an aliquot portion of this solution is chromatographed on a silica gel thin-layer plate, together with a series of standards for com- parison. Thiabendazole is determined directly on the TLC plate by fluorimetric measurement.
3 Apparatus
Soxhlet extractor, capacity of extraction tube approx. 500 ml, fitted with 500-ml round-bot- tomed flask and reflux condenser
Rotary vacuum evaporator
292 Thiabendazole Volumetric flask, 50-ml
Equipment for thin-layer chromatography Micro-capillaries, 2-^1
Spectrophotometric scanner, suitable for fluorimetric measurements on TLC plates
4 Reagents
Dichloromethane, p.a.
Ethanol, p.a., 96% vol.
Ethyl acetate, p.a.
Methyl ethyl ketone, p.a.
Mobile phase for TLC: ethyl acetate + methyl ethyl ketone + formic acid + water 5 : 3 : 1 : 1 v/v/v/v
Thiabendazole standard solutions: 0.1, 0.2, 0.3 and 0.4 mg/10 ml in ethanol Formic acid, p.a., 98-100%
Pre-coated TLC silica gel 60 glass plates, 20 cm x 20 cm, without fluorescent indicator (e.g.
Merck No. 5721), or equivalent aluminium sheets Cottonwool
5 Sampling and sample preparation
For preparing the analytical sample, weigh 2 to 2.5 kg of the laboratory sample (see pp. 17 ff, Vol. 1), peel the fruits or potatoes, and weigh the peel. Finely chop the peel and mix thoroughly.
6 Procedure
6.1 Extraction
Insert a cottonwool plug into the extraction tube of the Soxhlet extractor. Transfer the peel, equivalent to 400 g (G) of fruit or potatoes, into the tube and top with another cottonwool plug. Next, add dichloromethane to the tube until it begins to siphon, allow to drain into the round-bottomed flask, and half-fill the tube with dichloromethane anew. Mount the reflux condenser, heat to reflux, and extract for 3 h. Allow the extract to cool, and rotary-evaporate it to a volume of a few ml. Quantitatively transfer the concentrate to a 50-ml volumetric flask, using ethanol to complete the transfer, and make up to the mark with ethanol (VEnd).