Strange behaviour (change in RT) of LC under same conditions

[saioa]

Hello,
These last days I have been trying to set up a LC-ESI(-)-MS method to determine different acidic pharmaceuticals(pKa from 3.2 to 4.4).
As reported in many articles I am trying to work in a negative mode so I tried different mobile phases and check which one gave me the best signal. The best one was a gradient with Acetonitrile-Ammonium acetate 50mM pH=5.47 adjusted with formic acid. My standards are prepared in 2%acetic acid (due to my pre-treatment step).
The column I use is a C18 (15cm, 2.1cm, 3um) and the flow 0.2 mL/min.
Last Friday, I developed a gradient where I could separate the 3 compounds. However on Monday when I tried to reproduced this, it did not work. 2 of the 3 compounds eluted with the mobile phase and the third one eluted much earlier (about 15 min before). I tried different things, like change the mobile phase to acidic one and finally I reduced the flow to 0.1 mL/min. That way I could see the 3 peaks (all confirmed by MS). This morning I have run again the same sample at 0.1 mL/min, twice. I could see the 3 peaks, however retention time was decresed a little. Then I changed back to 0.2 mL/min and I see the 3 analytes. I repeat the injection and then again the first 2 analytes elute with the mobile phase and the third one elutes as Monday.
A colleague said he has seen similar behaviour and the best is that I have an isocratic gradient.
I do not know how to explain this and how to solve it.
Hope someone can help me to figure out what it can be...as I have never seen this problem befer in HPLC.
Thanks a lot!

[rhaefe]

Hello Saioa,

What is your gradient?
What exactly do you mean when you say "2 of the 3 compounds eluted with the mobile phase"? I always assumed that all compounds that elute from a column, elute with the mobile phase.
Since you are separating acidic compounds and you are using a C18 column I would think the pH of the mobile phase should be low enough so that your analytes are 100% protonated. So pH is again one of the possible suspects. Is there a particular reason why you are using ammonium acetate and adjust the pH with formic acid?

best regards,

Robert Haefele
Hamilton Company

[saioa]

First of all, thanks for the answer.
The gradient I am using is the following:
t:0-t:0.5 40% ACN, t.1.5 60%ACN, t:8 min 65%ACN, t:20 min 70%ACN
Sorry for my bad explanation, I meant that the two more polar compound elute in the "solvent peak". I see this because of the MS.
I have used 0.1% formic acid ph=2.67 to have all the compounds neutral. But I got not improvement. I am using the ammonium acetate, so I do not have so low pH that all my analyte would be charged when they enter the MS.
The strange thing is that sometines it has worked with ammonium acetate and then suddenly it does not work. The retention time over all my experiments are not constant, they vary greatly.
I have been thinking that it could be the pumps, but the pressure pattern is the same with the different injections and I always leave quite much time (16 min) so the pressure becomes the same as at the begining of the run and that the baseline looks more or less stable.
So I do not understand why I can not reproduce the separation part.
Thanks again
saioa

[rhaefe]

saioa,

I still think you should lower the pH so that all your acids are protonated and neutral. Then lower the acetonitrile concentration at the begining of your gradient. If your compound is eluting in the void your mobile phase is too strong. For initial method development I recommend decreasing the steepness of your gradient (run time will be longer) and determine at what % acetonitrile your analytes elute.

best regards,

[saioa]

Hello Robert,
After some days of work and trying many things I think I have figure out the reson of my problems. As I said before I saw very big differences in retention time, I have changed mobile phase pH with no success. I also changed the strength of the mobile phase with no luck. Third I changed different solvent of my standard with same bad results.
Today I realized that I get nice peaks at the end of the day. So I think it might be something related with the equilibration of the column. I did not think it could be this, because when I worked the first days (it is a new column, about 1.5 week old) I did not remember to have this problem.
I will make short injections to equilibrate it at the begining of the day and hopefully I equilibrate the column quicker.
Thanks again for your answers and hopefully I will be right.
Regards,
Saioa