By keith bayer on Tuesday, July 22, 2003 - 07:49 am:
I am running a gradient assay using ACN and 100 mM sodium Perchlorate buffer, pH= 2.7. The column is heated at 30 degrees C, run time =60 minutes. The last few times I have run this assay (on the same LC) I have noticed a decrease in RT from ru to run (seems fairly consistent during the run). I have checked the flow rate of both pumps, find no problems.
I tried a brand new column and experienced the same problem (spec is 24 to 30 minutes for RT of main peak), now I am seeing 22.9 minutes. I switched the MP to another system (same column, however) to no avail. I am trying one more test injection using a second column not new, but previouly used for the assay).
If this fails, I plan to remake the MP buffer and rerun system suitability. If the sodium perchlorate monohydate used in buffer has picked up water in storage, how might this affect what I am seeing? Thank you for your insight. Keith
By Anonymous on Tuesday, July 22, 2003 - 09:36 am:
Sodium perchlorate is an ion-pairing reagent. All the precautions to be followed regarding equilibration times with alkylsulfonic acids also apply to sodium perchlorate. Therefore I am not surprised about the shift in RT.
By Chris Pohl on Wednesday, July 23, 2003 - 10:00 am:
Keith,
One possible problem is reagent contamination. We've seen some customers experience similar problems on anion exchange columns with perchlorate eluents. We are not positive of the exact cause but suspect iron contamination of the perchlorate. You may want to try another reagent source (Fluka's highest purity grade is probably best). If it really is iron in your case, you may be able restore your column with overnight treatment using 0.1 M oxalic acid.
By Anonymous on Wednesday, July 23, 2003 - 05:47 pm:
How do you make a 100 mM sodium perchlorate buffer at pH 2.7?