Chemistry Reference
In-Depth Information
allowed to stand for 30 min before extraction to allow the spiked solutions to penetrate the
samples and attain the fungicide distribution in the samples.
2.3.3. Calibration
Quantification of triazoles were performed and compared by using calibration standards
involving both matrix-matching by adding standards to blank extracts and non-matrix
matching (standards in solutions) based on a calibration curve. For matrix matching, blank
extracts were fortified with the pesticide working standard after dispersive clean-up. The
calibration solutions were prepared daily at 7 levels of concentrations ranging from 0.05 to
2.0 µg/ml. The LOD's and LOQ's were calculated by multiplying the standard deviation of
the calculated amount for each triazole by 3 and 10 respectively.
3. Results and discussion
3.1. High Performance liquid chromatography-mass spectrometry
3.1.1. HPLC
A C18 reversed phase column (4.6mm x 75mm, 3.5 um particle size) was used in this study
to generate less back pressure as it allows more flexibility to adjust the flow-rate. A short
column was also used to obtain shorter separation times that produce narrower peaks
because there is less time for diffusive broadening. The small particle size used helps to
generate more pressure and generally give higher separation efficiencies. Smaller particle
size column is necessary to maintain resolution in the short column used. The HPLC column
had been run at different flow rates; 0.8 mL/min, 1 mL/min, 1.2 mL/min and 1.4 mL/min
during optimization and it was found that it gives better resolution at a flow rate of 1.2
mL/min A common operating temperature is 40
o
C as higher temperature is better in
producing sharp peaks and earlier elution [19]. For this study, the effects of column
temperature were also evaluated at various temperatures; 20
o
C, 25
o
C, 30
o
C, 35
o
C and 40
o
C.
Figure 1 showed that 25
o
C column temperature found to give better separation after
running triazole standard mixture.
Figure 1.
Acetonitrile/ H
2
O mobile phase, column temperature 25
o
C
