By Anonymous on Thursday, May 15, 2003 - 03:17 am:
What parameters should I change in the following method in order to test its robustness (except for wavelength, flow rate, amount of acetic acid and columns)
A: Acetonirile + 1% acetic acid
B: Water + 1% acetic acid
0 min – 20 % A
20 min – 45 % A
30 min – 90 % A
Thanks in advance for your help.
By Anonymous on Thursday, May 15, 2003 - 03:31 pm:
You should change the flow rate, the composition at the beginning of the gradient, at 20 minutes, and at 30 minutes, and you should run the method on another instrument. If you are in a method development department and are developing a method for QC, you should run it on the type of instrument available in QC.
I also do not see why you do not want to change the acetic acid concentration or the column. This is all part of robustness testing.
By Anonymous on Thursday, May 15, 2003 - 11:11 pm:
Thanks for your answer
A small misunderstanding. The parameters in brackets are the ones I know that I will be changing. The question was about what changes to the mobile phase should I apply.
Will 21%/46%/91% (ACN) and 19%/44%/89% be enough, or should I check all the possible combinations.
I hope I made it clear this time (English is not my native language)
Best regards.
By ananda on Friday, May 16, 2003 - 10:37 am:
I think what you are planning to do is OK. One step lower and one step higher should be good enough but if you dont see any changes in resolution, sensitivity and separation, you should try another combination to find out the righ limit for the gradient. Again how about HAC%? So I think there is no limit. You can change the gradient, and HAC% . Since there is no standadrd protocol or guidlines to check the robustness of this particular gradient system, I think one can check a few things like above to check the robustness.
By Tom Mizukami on Friday, May 16, 2003 - 12:59 pm:
Anon,
Is your method run at ambient temperature or is it thermostatted. Temperature is a normal robustness parameter.
I also like to check gradients on the highest and lowest dwell volume systems.
By Anonymous on Friday, May 16, 2003 - 03:43 pm:
In the gradient, I would do one more thing, since if there is a change it will be the largest: 21 - 44 - 91. The reasoning is that in a gradient the slope is most important.
By Anonymous on Monday, May 19, 2003 - 05:01 am:
Thank you for your posts.
Ananda, I am not sure what you mean by “Again how about HAC%?”. If you mean change in conc. of acetic acid, then, of course I will be checking this parameter as well.
Tom, unfortunately I don’t have thermostat in my unit so I won’t be able to test this one.
Anonymous (may 16), thanks for suggestion. I will try 21-44-91 and, following your thought, I will also do 19-46-89.
Thank you all for help
Best regards