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Background:
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| 1. |
SimuLab uses the HPLC for only one application in the Food and Beverage area.
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| 2. |
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.
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| 3. |
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.
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Safety Checks - Daily:
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| 1. |
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 PPE.
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| 2. |
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.
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Set-up and Pre-use Checks - Daily:
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| 1. |
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.
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| 2. |
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.
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| 3.
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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.
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| 4.
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Press the ON button and allow five minutes for the machine to warm up.
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| 5. |
Turn on the UV-Vis detector and set the wavelength to 210 nm.
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| 6.
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Set the flow rate on each pump to 1.6 mL/minute.
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| 7. |
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 column).
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| 8. |
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.
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| 9.
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On the UV-Vis detector set the gain at 10%, and adjust the baseline to zero using the baseline knob.
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Calibration - Weekly:
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| 1.
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Run the 'Sterilin' standard solution 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 peak |
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Running the Sample
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| 1.
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Inject 100 mL of sample into the injection port.
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| 2. |
Press the Run button - gradient elution will occur over 15 minutes (0 - 100% acetonitrile).
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| 3.
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Check the trace (chart recorder) for the presence or absence of the peak corresponding to 'Sterilin'.
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Clean Column
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| 1.
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Clean column by holding at 100% acetonitrile for five minutes then reverse gradient back to 0% acetonitrile over five minutes.
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