Chromatography Forum

Hi,

I had been thinking about the gradient profile I am running on a LCMSMS.

This is what I have:

(A) 10 mM ammonium acetate in water
(B) ACN

0-1min (B) 95%
1-5min (B) 95%
5-7min (B) 50%
7-12min (B) 95%

I obtained this method from a FDA document.

My question is why can't I switch immediately to 95% (B) from 5.1 min. Why must I allow a 2 min gradient increase from 50% to 95% (B)?

A fellow scientist suggested immediately changing at 5.1min. But I wonder if there is a strong reason for that 2 min difference.

Thanks!

Regards,
Wan

Since I have no idea what kind or size of column you are using, what your flow rate is, what your instrument dwell volume is, or where your compounds are eluting, there is no way to give any meaningful advice beyond "try it and see what happens".

There is actually no problem at all in term of immediately switching your gradient back within 0.1min or within 2min.

In fact, for LCMS/MS or even HPLC analyses; it's recommended to switch the gradient back within 0.1min or less so as not to waste time for column re-equilibration.

What I am concern is that unless your 'melamine peak' - looking at your gradient, I am guessing that you're running melamine analysis ( also considering your other posts) eluted out within the stipulated gradient when you did the 2.0min switch, then you are not recommended to switch the gradient immediately else the solvent polarity in the column will affect the peak shape. If your peak eluted out before the time you switch to 50% or so, then it's recommended to switch immediately back to 95% within 0.1min or less.

Also, the 2 min 50% water is most probably needed to 'wash out' other polar compounds from your column; and not only the melamine analyte, which I think is very logical.

Hope this helps !!!

There is actually no problem at all in term of immediately switching your gradient back within 0.1min or within 2min.

Unless he's using a HILIC column (in which case ACN is the weak solvent and water is the strong solvent) and his analyte is eluting due to that 2 minute gradient (which we can't know without knowing the retention time, flow rate, and dwell volume).

Hi Tom and Ken,

Thank you for your comments and sorry for the missing info.

Tom: We're using an Atlantis HILIC column and FR is 0.35 ml/min. We're running melamine analysis and it eludes around 3 min.

Ken: Since the retention time of melamine is less than 5 min, I'll try switching at 5.1min.

Thank you all again! I appreciate all the help given.

Regards,
Wan

By the way, Wan, if I may, the word in chromatography is "elute", not "elude", e.g. peaks may elute, while answers to problems elude us.

Woops. Sorry. I'll make sure I use the right word from now on. Thanks!

Wan