I'm currently having to problems with our Agilent LC/MS, my first problem is that the pressure becomes a bit high when I switch from 100% ACN to 50% water and 50% ACN. The max bar I usually see under this condition is 140 bar for the Sunfire Prep C18 column, but now the pressure reaches 200 bar and my max is set to 250. My second problem is that as soon as the needle injects my sample, the upper pressure limit error message pops up.
A 50/50 mixture of water and ACN is more viscous than a solution of pure ACN. So the pressure is supposed to be much higher in this case. You may not be observing a problem, you may be observing normal operation. Now, to investigate and stop the over pressure alarm from triggering and terminating the analysis run, simply go to the Instrument, PUMP, screen and set the upper pressure limit to 350 bars. Save the method with this change. Assuming that your column or HPLC system is not clogging, that should allow you enough dynamic headroom to continue the gradient (or iso) run to completion. One concern I have is that the injection pressure may be rising too much because: (1) you are introducing a sample which is not soluble in the mobile phase; (injecting sample of too large a volume for the column; or (3) introducing particulate matter or unfiltered material into the injector flow path. All of these things would be abnormal and you should seek the assistance of an experienced chromatographer before you proceed. **The pressure over protection alarm is something that is set by the operator. It will be different for different methods. You are responsible for choosing a value which is appropriate. The fact that it triggers during your injection is proof that it is doing its job as programmed. IOW: It works.
In the future, please be sure and review "HPLC Solvent Properties" (please see link for a table of common properties) in advance so you can predict what pressure effects may occur due to changing viscosity values. Higher flow rates will also increase the back pressure too. Make sure all samples fully dissolve in the mobile phase and all mobile phase components are fully soluble and filtered, before use. Optionally, you may be able to reduce the overall flow rate of the method to reduce the back pressure observed.
http://hplctips.blogspot.com/2011/11/hplc-solvent-properties-table.html
A 50/50 mixture of water and ACN is more viscous than a solution of pure ACN. So the pressure is supposed to be much higher in this case. You may not be observing a problem, you may be observing normal operation. Now, to investigate and stop the over pressure alarm from triggering and terminating the analysis run, simply go to the Instrument, PUMP, screen and set the upper pressure limit to 350 bars. Save the method with this change. Assuming that your column or HPLC system is not clogging, that should allow you enough dynamic headroom to continue the gradient (or iso) run to completion. One concern I have is that the injection pressure may be rising too much because: (1) you are introducing a sample which is not soluble in the mobile phase; (injecting sample of too large a volume for the column; or (3) introducing particulate matter or unfiltered material into the injector flow path. All of these things would be abnormal and you should seek the assistance of an experienced chromatographer before you proceed. **The pressure over protection alarm is something that is set by the operator. It will be different for different methods. You are responsible for choosing a value which is appropriate. The fact that it triggers during your injection is proof that it is doing its job as programmed. IOW: It works.
In the future, please be sure and review "HPLC Solvent Properties" (please see link for a table of common properties) in advance so you can predict what pressure effects may occur due to changing viscosity values. Higher flow rates will also increase the back pressure too. Make sure all samples fully dissolve in the mobile phase and all mobile phase components are fully soluble and filtered, before use. Optionally, you may be able to reduce the overall flow rate of the method to reduce the back pressure observed.
http://hplctips.blogspot.com/2011/11/hplc-solvent-properties-table.html
I agree with Bill Letter. But, I think 200 bar is quite high for 50% ACN, but not too much. You can remove the colum and use a connector and check where exactly th pressure is developing. If the pressure is due to column, you must clean it following recommended procedures.
Other comments as my first response did not address all of your concerns (sorry about that):
It is impossible to say what is "too much pressure" without knowing details of the method used. If you would be so kind as to provide the exact column dimensions (ID x Length x particle size) and the flow rate to us. This is very important info and may allow us to provide you with better suggestions.
Make sure you are not overloading the column or fouling the column with sample. The pressure will rise if you contaminate and clog the column (or frit). You must have a proper cleaning method in place which dissolves EVERYTHING and elutes it off the column. The wash method should be run after the analysis method and include solvent(s) which are stronger than the ones you use in your method. If you have clogged the column, then you will see higher than normal pressures. In some cases you can reverse the column and wash it. In other cases a few hours with stronger solvents washing through it will remove the debris. If you do a search, you will find many suggestions on how to wash the column, but start with the ones suggested by the column manufacturer and my earlier advice about using a stronger mobile phase (please wash the column with it disconnected from the MS source).
Your HPLC system also has an inlet frit (PTFE) installed on the pump module (inside the Prime Purge Valve). It is there the protect the rest of the HPLC system from particulate matter missed by the solvent inlet pickup filters and/or normal pump seal wear debris. This 10u filter should be replaced every month. It is a maintenance item and very inexpensive. When maintenance is ignored, this filter clogs and then contaminates the rest of the flow path. It also raised the instrument back pressure too. If you have not done so already, check that a fresh clean frit is installed (should be snow white in color).
If your injector is always triggering the issue, then you need to insure that: your samples are filtered; the volume of the injection is appropriate for the column and method (not too large); That the sample is dissolved in the mobile phase; The A/I rotor seal or needle seat have not been contaminated or clogged [ More info on this ttopic can be found here: http://hplctips.blogspot.com/2013/06/troubleshooting-hplc-injectors-manual.html].
That is good news. Always try and filter those samples before placing in injector vials. Also, try and make sure the samples are dissolved in mobile phase. Both of those things will help reduce the chance of any clogs. If a clog or partial blockage is going to happen, then it will generally be right where the needle inserts itself into the HPLC flow path (i.e. the needle seat). Always a good idea to have one spare needle seat on hand.
Good luck to you!
Before going to analysis the samples, proper washing to be given to HPLC column. Increase the flow rate of pump to column is also very important (0.1 mL, 0.2 mL, step by step etc). Flow rate sudden changes is not good for HPLC column. Proper filtration techniques should be followed for buffer, solvents and as well as samples also.
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