Hi Crong,
You said,
Hi Matt, sorry for the confusion. That 5.5 min at the end of the gradient program is re-equilibration to mobile phase A which is the buffer. The 90% water is only used at the end of the whole sequence before shut down. What I did was after all the samples in the sequence I add one injection of H2O without derivatization and set the mobile phase to 90% H2O 10% B for 30 min at the same analysis flow rate, after which it is changed to 70% H2O 30% B, reduced flow rate, and the instrument shut down initiates. Shut down method turns off the oven and waits until oven temperature is below 35 C before turning off the pump (maybe another 5-10 min). All in al0l, the column is flushed with mobile phase that doesn't contain buffer for at least 35 min. I will definitely check the eluent pH next time to see if it's still basic, thanks for that suggestion! And flush with higher % ACN prior to storing the column. Hopefully that will have some improvement.
Thanks again.
Actually--I wasn't confused on either count (seems to me to be some terminology inconsistency on my part, I'll try to be clearer). Just suggesting that to remove all of the basic eluent may take longer than that re-equilibration step in-between separation runs (and thanks, now I see you've been routinely using 30 minutes of 0.7 mL/min for the post-sequence run step of 90:10 water/ACN) within a sample sequence and at the same flow rate of 0.7 mL/min. I also understood from the first that the 90:10 water/ACN wash you perform occurs at the end of the ENTIRE sample sequence.
If you have pH paper handy, it still may make sense to check the effluent pH after the 30 minutes of pumping the 90:10 mix just as a check. Please let me know what you find with the pH check of the pH of the post-sequence run effluent--just seems that based on what you're observing, this could be why the stationary phase may be being adversely affected.
I agree with Imh too, to cause further confusion...I've had the easiest time with AccQ.Tag derivatization myself (life is full of these oxymorons, seemingly). I used to prepare the eluents for AccQ.Tag on my own using Steven Cohen's reference paper...these days that AccQ.Tag separation method has changed a bit (I think a BEH stationary phase is used these days rather than NovaPak). Oh...I don't work for Waters (or Agilent) either...just hard to change something that is working (Thermo's OPA/MPA and separation) for something new--time and money.
Is there a point of return on costs for injections on the Hypersil Gold columns/guards that could be satisfactory to your lab's needs?
Anyhow--I continue to wish you well, Crong!
Hi Rndirk: As to these guard columns, if they helped with something I was trying to develop, or were required to execute something I needed to run...I used them. If not, then not--guess it's safe to say that I liked them when they were specified for use or made a difference in what I was doing.
For example, the older AccQ.Tag method specified a guard for the NovaPak analytical column...there is no such specification for the modified AccQ.Tag separation with the BEH phase.