- MTH 68
- Ver 1
SOP: HPLC - Pesticide Analysis
- SimuLab uses the HPLC for only one application in the Food and
- The application is to detect the presence or absence of the
pesticide, 'Kill-A-Tick', in cows' milk for human consumption.
The pesticide is used to kill ticks found on cows.
- The active component in 'Kill-A-Tick' is 'Sterilin' which this
method detects. The Health Department requirement is that there
be no detectable 'Sterilin' in milk.
Safety Checks - Daily:
- Check that electrical connections are firmly in place, that
cords are not frayed and that there is no liquid or encrusted
salts on or about the HPLC. Unidentified spilt chemicals should
be removed with extreme caution whilst wearing standard SimuLab
- Under NO circumstances is any staff member of SimuLab permitted
to open any sealed unit of the HPLC or attempt any procedures
not described in this SOP.
Set-up and Pre-use Checks - Daily:
- Ensure that the door to the instrument room is closed and that
the fans are turned on prior to any operations to ensure good
ventilation from acetonitrile (CH3CN or methyl cyanide).
Caution: acetonitrile is toxic in moderate amounts.
- Prepare new batches of each mobile phase solvent; HPLC-grade
acetonitrile and 0.05% w/v trifluoroacetic acid (TFA) in Milli-Q
water (caution: TFA is corrosive). Store at 4°C when
not in use.
- De-gas both liquids and carefully pour them into the 1 L flasks
on the top of the HPLC machine. Cover the flask openings with
clean aluminium foil.
- Press the ON button and allow five minutes for the machine to
- Turn on the UV-Vis detector and set the wavelength to 210 nm.
- Set the flow rate to 1.6 mL/minute.
- Set Pump A (0.05% TFA) to 100% and run the pump for ten minutes
(to flush column). This is achieved by setting the gradient controller
to run a 0% - 100% acetonitrile gradient over 15 minutes (to flush
- Allow 100% acetonitrile to flush the column for an additional
period of 15 minutes. This is achieved by setting the gradient
controller to reverse the gradient (ie 100% to 0% acetonitrile)
over 15 minutes.
- On the UV-Vis detector set the gain at 10%, and adjust the baseline
QC Check - Weekly:
- Run a 'Sterilin' Calibration Standard as a QC check sample after
set-up (as above) under the running conditions (as below). Check
that the standard elutes as follows:
| Elutes at:
9.5 - 10.5 minutes
|Base of Peak:
4 - 6 mm
| Height of Peak:
~ same as solvent
Running the Sample
- Inject 100 µL of sample into the injection port.
Select a linear gradient.
- Press the Run button - gradient elution will occur over 15 minutes
(0 - 100% acetonitrile).
- Check the trace (chart recorder) for the presence or absence
of the peak corresponding to 'Sterilin'.
- Clean column by holding at 100% acetonitrile for five minutes
then reverse gradient back to 0% acetonitrile over five minutes.