High Pressure Liquid Chromatography
Instructions for use
1. Ensure the pump reservoir is filled with the appropriate filtered mobile phase.
2. Turn on the power to the HPLC pump.
3. If the pump has not been used for some time it may be necessary to prime the pump with fresh
solvent by connecting a syringe to the drain port ( make sure pump is not running ), opening the
drain and collecting about 30 mls solvent in the syringe, then closing the drain.
4. Select an appropriate flow rate ( usually 1 ml/min ) and start the pump by pressing the [run/stop]
5. Inspect the column connections for leaks and ensure the waste runs to the waste collection
vessel ( check not overfull ).
6. Turn on the power to the UV detector and set the appropriate wavelength ( typically 254 nm ).
7. Turn on the power to the chart recorder and ensure there is enough paper.
8. Allow about 20 mins for the chromatographic system to equilibrate.
9. Set the UV detector range ( typically 0.2 ) then press the autozero button on the UV detector and
check the output is stable.
10. Once the baseline is stable the system is equilibrated and ready for analysis.
11. Inject a sample with the 100 ul syringe ( this feeds the 20 ul internal loop ) using about 60-100
ul with the injector valve in the load position.
12. Then turn the injector valve to the inject position and simultaneously press the [run/start] button
on the recorder.
13. After about a minute remove the syringe, clean with methanol and turn the valve back to the
14. When the peaks have appeared on the recorder press the [stop] button on the recorder to get a
printout analysis and tear off the paper.
15. If the peaks are off-scale set the range to a higher value and repeat the injection.
1. Always flush the system at shutdown .Switch the pump off, change the reservoir to pure solvent
( e.g. methanol ), then pump through for 10 mins.
2. Switch off the recorder and remove any charts required.
3. Switch off the UV detector.
4. Ensure syringe is cleaned with solvent and stored safely.