By Anonymous on Saturday, February 21, 2004 - 07:50 am:
As a beginner,i hav some questions to ask:
1.Is it ok when the pressure is above 5000psi?
2.Do I need to degas the solutions,whenever I use them?
3.if I analyze the same sample in different days,do i need to flush the column in between?
Thank you!
By Anonymous on Saturday, February 21, 2004 - 10:46 am:
no, yes, ??
By Chris Pohl on Saturday, February 21, 2004 - 02:02 pm:
Anon,
1). It depends on your system, but in general you'll have problems above 5000 psi. Generally, this is in excess of the pressure limit for the injection valve, although there are some valves which will work at higher pressures.
2). This depends on whether or not you're instrument has a built-in degassing module (or an add-on degassing accessory). If you don't have such a device, you should definitely degas your mobile phase. If you do have such a device, someone still argue that you're instrument works better if you degas your eluent but in my experience it's not necessary in this case.
3). I'm not sure exactly what you mean by this. If you mean, do you have keep the instrument operating with the pump on in the time between analyses spending several days, I would say this is an unnecessary requirement. If you mean, should you reequilibrate the system before performing the assay on a subsequent day, I would say this was a standard requirement. Perhaps you can clarify this question in case I haven't guessed right on the intent of your question.
By Dipendra on Saturday, February 21, 2004 - 09:09 pm:
In response to beginner Anonymous question three:
"if I analyze the same sample in different days,do i need to flush the column in between?"
Chris Pohl responded well. I would like to further add, if the question really mean analysing the same drug after some time (say after weekend), it should be flushed with the mobile phase to get equilibrium. If you are using after long time, it is better to flush with ACN first and then with your mobile phase.
By Anonymous on Saturday, February 21, 2004 - 09:40 pm:
Thank you very much for your answers. Maybe I need to explain my last two questions. For question 2,what i meant by that is: I degassed a bottle of solution but and used half of it. Do I need to degas it again when I use it in the future? In the process of moving the tube, some air did enter the bottle. For question 3,I need to analyze a sample several times,all the conditions are the same. But i can't finish all them in one day. If I continue my analysis tomorrow, should I flush the column tonight?
Again, Thank you!
By Anonymous on Saturday, February 21, 2004 - 09:41 pm:
Thank you very much for your answers. Maybe I need to explain my last two questions. For question 2,what i meant by that is: I degassed a bottle of solution and used half of it. Do I need to degas it again when I use it tomorrow? In the process of moving the tube, some air did enter the bottle. For question 3,I need to analyze a sample several times,all the conditions are the same. But i can't finish all them in one day. If I continue my analysis tomorrow, should I flush the column tonight?
Again, Thank you!
By A.Mouse on Sunday, February 22, 2004 - 10:31 am:
When we run the same mobile phase on subsequent days, we do not prepare the column for storage. If the column is not used for several days, we put it into the storage solvent recommended by the manufacturer. Look for instructions by the manufacturer in the column box.
By HW Mueller on Sunday, February 22, 2004 - 11:43 pm:
Another thought on 3.:
If you use a mobile phase with characteristics near the limit of stationary phase stability criteria it would be advantageous to flush. Also, if your samples are very "dirty". One needs to be careful here, I have seen matrix proteins "cake" up after putting the column on MeOH over night (backpressure was higher the next day). This did not happen if the column was kept on water instead of MeOH over night.