14
STANDARD METHOD FOR DETERMINATION OF PROPICONAZOLE AND TEBUCONAZOLE
IN WATERBORNE FORMULATIONS AND IN TREATING SOLUTIONS BY HPLC
Page 2 of 2
© 2015
The chromatographic system must be re-equilibrated with
the start conditions before performing another analysis
(typical equilibration time is 5 minutes). These
chromatographic conditions are typical but not essential. As
already mentioned, adaptation of some parameters (flow
rate, injection volume, elution solvent, column temperature,
etc.) may be necessary to avoid interference. Available
equipment may also require adaptation of the prescribed
method.
10. Standardization: Inject the standard solutions and
calculate the peak areas. Propiconazole may give a peak
splitting. In this case, the integrator peak-grouping mode
should be used (adding the peak areas is also possible). Plot
the peak areas of the standards (x-axis) vs. concentrations of
active ingredients in mg/l (y-axis). Use linear regression
analysis to determine the slope , the intercept and also the
correlation coefficient. This latter parameter should be
higher than 0.99 to justify the linear approximation. If not,
linear regression should be applied on a smaller range of
concentrations. In this case, the nominal concentration of
each active ingredient in the solution to analyze should be
situated within this linear calibration range. Another
possibility is to apply a polynomial regression of order 2 or
higher. If the calibration has a correlation coefficient greater
than 0.99 and the intercept slope ratio is less than 2, a single
point calibration may be used based on the highest
concentration standard used. Single point calibration can be
used for any sample solution with a concentration within the
calibration range.
11. Calculations:
11.1 Concentration of active ingredients. Calculate the
concentration of active ingredients C i, in mg/l from the peak
area P i:
C i P
where:
is the slope of the linear regression
is the intercept (see § 10).
i
11.2 Treating solutions. The concentration (retention) of
active ingredient in the treating solution is easily calculated
via the equation:
mg a.i. / l treating solution
C i V d
V s
where:
Ci
is the concentration obtained with the calibration
solutions (as explained in 11.1)
V d is the volume of solvent (expressed in milliliters) used to
dilute the treating solution sample
V s is the amount (expressed in milliliters) of treating solution
used.
12. Precision Statement:
12.1 The following statements and table should be used to
judge the acceptability of an analysis using the method under
the conditions below. The precision of the data were
developed following the guidelines in ASTM Method E691.
Repeatability. Triplicate single determinations on the same
sample by the same operator using the same equipment
should not be suspect at the 95% confidence level if they do
not differ from one another by equal or less than the limiting
percentages shown in the following table.
Reproducibility. Duplicate single sample determinations on
the same sample by different operators in different
laboratories should not be suspect at the 95% confidence
level if they do not differ from one another by equal to or
less than the limiting percentages shown in the following
table.
Range of
Propiconazole (ppm)
500
1000
2500
5000
Confidence Limits %
Repeatability
0.0016
0.0052
0.0106
0.0226
Reproducibility
0.0059
0.0111
0.0288
0.0612
- 219 -
A30-14
AMERICAN WOOD PROTECTION ASSOCIATION STANDARD
© 2015 All Rights Reserved
STANDARD METHOD FOR THE DETERMINATION OF
4,5 DICHLORO-2-N-OCTYL-4-ISOTHIAZOLIN-3-ONE (DCOI)
IN WOOD AND SOLUTIONS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Jurisdiction: AWPA Technical Committee P-5
Initially adopted in 2000; amended in 2006 and 2008. Revised and reaffirmed in 2014.
This AWPA Standard is promulgated according to an open, consensus procedure. Using this standard in no way signifies standardization of a
chemical or wood protection system in AWPA Standard U1.
1. Scope:
1.1 This method provides for the quantitative determination
of DCOI preservative in treated wood and treating solutions.
The methodology utilizes High Performance Liquid
Chromatography (HPLC) with ultraviolet (UV) detection.
1.2 The method is applicable to the determination of DCOI
in wood samples in the form of sawdust or wood meal and
aqueous solutions samples.
2. Summary:
2.1 The chromatographic method permits determination of
DCOI.
2.2 Wood meal samples are extracted in methanol using an
ultrasonic bath. Solution samples are diluted with methanol.
The filtered extracts and prepared solutions are analyzed by
HPLC with UV detection at 275 nm.
2.3 The concentration of DCOI in the sample is determined
using an external standard single point calibration method.
3. Interference:
3.1 Inert ingredients in the DCOI calibration standard do not
cause interference.
3.2 A blank wood sample (material which has not been
treated with preservative) of Southern pine (SP) was
analyzed the same manner as a sample. It did not show any
interfering components. However, it is recommended that an
analysis of a blank sample be done to check for presence of
interfering components before conducting analysis of other
wood species. Modification of the HPLC conditions may be
necessary if interfering components are present.
4. Safety:
4.1 Personnel should be trained in the laboratory and safety
procedures.
4.2 Methanol is a volatile, flammable and toxic organic
solvent. Work only in a well ventilated environment and
wear proper safety attire when working with methanol.
4.3 Consult the MSDS of the chemicals involved for the
appropriate handling instructions and PPE.
5. Apparatus:
5.1 Sample and Standard Preparation Equipment
5.1.1 Analytical balance with 0.1 mg sensitivity
5.1.2 Top loading balance with 1 mg sensitivity
5.1.3 Ultrasonic bath with sufficient power and capacity to
extract the wood samples in the indicated time
5.1.4 Disposable nylon or Teflon 0.45 m filters
5.1.5 28x73 mm screw cap glass vials with Teflon lined
caps, 30 ml capacity
5.2 Liquid Chromatographic Equipment
5.2.1 High Performance Liquid Chromatograph with a
binary gradient elution capability
5.2.2 Inertsil ODS-3, 150 x 4.6 mm, 3 µm column. A
guard column with the same material is recommended to
extend column life. Substitution with a similar C18 column
can be made, provided the desired separation of the active
ingredient with sample components is achieved.
5.2.3 Variable wavelength ultraviolet detector or diode array
detector
5.2.4 An appropriate data system for data collection and
quantification
6. Reagents and Standard:
6.1 Methanol, HPLC grade or better
6.2 Water, HPLC grade or better
6.3 DCOI Analytical standard solution, 25% DCOI
(available from Rohm and Haas Company)
7. Preparation of Calibration Standards:
7.1 400 ppm Standard - Accurately weigh 160 mg (to
nearest 0.1mg) of solid 25% DCOI analytical standard into a
100 ml volumetric flask and dilute to mark with methanol.
Correct the standard weight for purity and calculate the
concentration (µg/ml) of active ingredient (AI) for this
standard.
7.2 The 400 ppm standard is stable for four weeks at 7°C.
Note: Refrigerate all dilute DCOI standards when not in use.
7.3 Working Standard - 20 ppm. Transfer 5 ml of the 400
ppm DCOI standard with a volumetric pipette to a 100 ml
volumetric flask and dilute to volume with methanol.
Calculate the concentration (µg/ml) of DCOI in the working
standard. This standards is stable for at least one week
stored in a refrigerator.
8. Preparation of Samples:
8.1 Wood samples.
8.1.1 Wood samples taken for analysis are ground in a
Wiley mill to pass a 30 mesh sieve. The samples are dried in
a convection oven at 80°C to a constant weight.
- 220 -
A30-14
STANDARD METHOD FOR DETERMINATION OF 4,5 DICHLORO-2-N-OCTYL-4-ISOTHIAZOLIN-3-ONE
(DCOI) IN WOOD AND SOLUTIONS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Page 2 of 3
© 2015
8.1.2 Weigh 500-600 mgs of wood meal into a glass screw
cap vial and record the sample weight to the nearest 1 mg.
8.1.3 Add 20 ml of methanol by volumetric pipette and
screw the caps on tightly. Place the vial in an ultrasonic
bath and sonicate 3 hours mixing the sample by swirling
every 30 min. Allow the mixture to cool and settle after
extraction.
8.1.4 Filter the extract through a 0.45 m filter prior to
HPLC analysis. Note: Each lab should determine if
additional extraction time is required with their specific
ultrasonic bath and sample load.
8.2 Solution samples.
8.2.1 Weigh a well mixed sample (to nearest 1 mg) into a
100 ml volumetric flask, dilute to volume with methanol,
cap and invert several times to mix. The weight of sample
taken is such that the DCOI concentration is 15-20 ppm in
the prepared solution.
8.2.2 Filter the prepared solution sample through a 0.45
µm filter prior to HPLC analysis
9. Chromatographic Parameters:
9.1 The following chromatography conditions are
recommended:
Column temperature: Ambient
Flow Rate: 1.0 ml/min
Reservoir A - Water
Reservoir B - Methanol
Solvent program:
Time (min)
0
10
10.5
12.5
13
17
%A
17
17
0
0
17
17
%B
8370
83
100
100
83
83
using equation 1.
RF = C std/ As