HPLC method of chromatography is in fact very simple to follow a well established procedure . Always keep the HPLC instrument clean including the chamber where the HPLC system is housed. Keep the room temperature to lower side and also fixed. Alteration in the room temperature during the HPLC method has effect on the detectors. All the solution, reagents, samples, standard solution, injections etc. should be arranged before hand.There should be no chances of power failures, it is better to have a substitute power back up in case of failure.
The HPLC method includes
♠ Fill the reservoir with Mobile phase which is throughly filtered, degassed. Clean the metallic filter at the end of pipe of mobile phase siphon with mobile phase and then dip into mobile phase in the reservoir. It is better to place reservoir at certain height to avoid unnecessary fall due to handling and other operator movements.
Check how to prepare HPLC mobile phase in detail.
♠ Switch on the HPLC system. In the following sequence. Pump→ Detector→HPLC software. First the pump, then the detector, then the computer with HPLC software. Then the software is set as per the pre-planned method. i.e the run time, the detector range of measurement, the wavelength of measurement etc.
♠ Then purge the system with pump for 5min. During this see if any air bubbles are passed from reservoir into the siphon pump and there by into the pump. Purging will remove any air bubbles if present in the entire HPLC system during hplc analysis.
♠ Then run the mobile phase by setting pump at fixed rate like 1ml/min or so as pre-planned and allow the detector to stabilise. This can be monitored on the computer software. The stabilization may take a few minutes and will give a constant and stable base line.
♠ Once the detector is stabilised, then set the method to run the samples in software and fix the flow rate and also run time like 10 min or 20 min as required by the sample. Run time varies due to sample, the flow rate, column length etc.
♠ Now once the run parameters are set, get the system ready for injection by commands on the software. Also set the rheodyne injector or loop injector to load mode. Inject the sample at fixed volume into the loop injector or rheodyne injector. Then give the command inject in the software. The sample in the loop injector is injected into the mobile phase passing into the HPLC column in the inject mode.
♠You can notice the reading in the monitor with peaks of sample detected by the detector. Once all peaks are formed, after the run time is over, stop the monitor and set for new injection. During this leave a gap of 5 to 10 min. for mobile phase to run through the column so as to wash the previous sample remnants if any.
♠ Then after confirmation of stable base line, go for next injection..
♠ To switch of the system, the sequence is detector→pump→HPLC software . First switch off the detector, then the pump and then the computer and software.
Tips for better resolution and performance in HPLC method.
1. Wash the column when you start and are also about to switch off the system for hplc analysis. This helps stabilize the detector, remove any sample remnants in the column. Any accumulation in the column can reduce the life of the column and also cause interference in the measurements.
2.Washing of column at the start of system should be with HPLC grade water and then the mobile phase. While at the system turn off, switch to mixture of methanol and water mixture and then to HPLC grade water for washing the column. Use a run time of at-least 30min. for better washing.
3. Always use a filter to inject sample or use a guard column to prevent any particle entry into the system. Filter the solvent or mobile phase with0.2 micron filter paper and also go for effective degassing. Presence of gas cause disturbance like alteration in pressure of flow etc.
4. Run the standard and note the readings at the starting of sample injections and also after the end of sample injections in every hplc analysis.
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