By Leon on Wednesday, September 3, 2003 - 10:35 am:
Hi,
I am developing a method for Methotraxte (MTX) Analysis.
My method is based on USP method which use pre-mixed 100mM sodium phosphate buffer and 10% ACN. There is no gradient and the condition is: vydac C18 column on Waters Alliance system. My runs was Okay the MTX peak come out at ~~10min.
But if I don't premix the buffer, 90%A( Sodium phosphate) and 10% B (ACN), there is a extraneous peak at ~~8min and is very repeatable, even the blank got that peak. I ruled out the possibility that this extraneous peak is from the water or other chemical reaget because when I swich back to premix it's gone. Is it from the mixer? Did anyone else have similar problem with Waters mixer? Please help!!!
By Anonymous on Wednesday, September 3, 2003 - 03:05 pm:
Maybe there are bugs growing in the water when you don't premix. Also, what is the pH?
By Jan P on Wednesday, September 3, 2003 - 10:16 pm:
Hi Leon!
Avoid contacting 100% ACN with phosphates in mixer! At concentrations over 60 – 70% ACN in buffer, phosphates precipitate. As result You can to blockage the pump.
By Anonymous on Thursday, September 4, 2003 - 05:51 am:
I suspect there may be an impurity in the AcN which is removed-destroyed-modified by the addition of the aqueous phase. There isn't a mixer problem necessarily. If you run water/AcN gradients does the peak reappear? Also disappear with simple water-AcN premixed mobile phase?
These are simple experiments to give you a clue. It is possible that you may be rinsing some deposited material from the LC lines using the neat AcN. How reproducible in RT is this disappearing peak?
By R.C. on Thursday, September 4, 2003 - 06:03 am:
Another thing to check is differences between how you prepare the two eluents. Do you use the same grade of Na-phosphate and acetonitrile for both, do you filter the aqueous phosphate and not the mix, and do you use different types of plastic bottles for the aqueous and the mix. And so forth. To simplify the problem, you really should pick one method and stick to it.
By Anonymous on Thursday, September 4, 2003 - 10:49 am:
What's your blank?
By Tim on Friday, September 5, 2003 - 06:56 am:
Try running the mix without injecting anything - that should eliminate your diluent as the source and confirm it is the MP.
Your high conc of buffer in premix suggests it isn't a water soluble compound, so it is probably the MeCN that is causing it.
Do you see this on different systems or just one? if just one, suspect contamination being pulled out by the higher concentraion of MeCN through the lines and then the mixed MP eluting that.
Also try swapping the lines you use for the mix round, i.e. put buffer in line B, MeCN in line A, just to check it isn't peculiar to the MeCN in line B.