Chromatography Drift :( Eluting off early.

[djaydearz]

Hi There.

I am having an issue with our dionex system, and I was wondering if I could get some advice?

I am seeing a fair drift in peaks, which are eluting off much earlier than usual, also peaks that usually come off near to each other, are now forming a split peak. The method has not been changed at all.

I have tested the following

• Re-made all mobile phases and injector solvents, to rule out any uncertantity.
• Back Flushed Column Procedure.
• Replaced Column With a brand new column (improved chromatography but the problem still remains)
• Checked pressure, baseline is smooth and pressure stable.
I run with 80& H20 and 20% ACN at 1.5 ml/min
Injector solvent is 90%H20 and 10% ACN.

I am now seeing my final peak has eluted at 7.075 mins, compared to how I usually observe of 13.92 mins.

I would really appreciate some advise to how to overcome the problem. Could it be the mixing chamber? Flow cell?



Kind Regards

James

[annaand]

I have several questions before suggesting anything:
1. Is sample composition exactly the same as previously (when you had 13.9min retention)? E.g. you do not have additional polymer or SiO2 nanoparticles as the excipient in drug which does not give you a signal.
2. Did the retention time decreased gradually or change suddenly?
3. Is the temperature the same?
4. When you used a new column what was the retention time at the beginning? 7min or 13.9?
5. Did you try to use the same method with a new column but make a series of blank injection? Let's say 10-20 injections. And then inject your sample - what is the ret. time? If is much smaller then usual (13min) then I would be looking closer to mobile phase. If it is normal then I would rather focus on the sample composition.
Best,
Anna

[djaydearz]

I have several questions before suggesting anything:
1. Is sample composition exactly the same as previously (when you had 13.9min retention)? E.g. you do not have additional polymer or SiO2 nanoparticles as the excipient in drug which does not give you a signal.
2. Did the retention time decreased gradually or change suddenly?
3. Is the temperature the same?
4. When you used a new column what was the retention time at the beginning? 7min or 13.9?
5. Did you try to use the same method with a new column but make a series of blank injection? Let's say 10-20 injections. And then inject your sample - what is the ret. time? If is much smaller then usual (13min) then I would be looking closer to mobile phase. If it is normal then I would rather focus on the sample composition.
Best,
Anna

Hi Anna thanks for your reply

1) Compostion is exactly the same.
2)it was a sudden change. right at the start of this month, looking in the audit files, nothing looks unsuhe al, ie no pressure change.
3)Temp is the same
4)The new column gave a rt of 7.9 mins also, so did not make any difference i.e not a column problem
5)With the new column, i just leave it pumping for an hour monitoring the baseline and then tried an injection, all using fresh primed solvents.

Im pretty sure its not the MP or sample compostion, as its exactly the same as i have always done.

James

[juddc]

Ok, I have a thought...

I'm assuming here that your method is isocratic, it's reverse phase, you can run a gradient, and that your aqueous phase won't precipitate anything when mixed at ANY ratio with ACN. If that's not the case, DON"T do what I'm about to suggest.

If so, try preparing fresh everything (mobile phase, etc), equilibrate at for ~10-15 minutes at 20% ACN, then ramp to 90-95% ACN over 10 min, hold for 5 min, then go back to 20% ACN. Repeat the process a second time, but equilibrate for 30-40 minutes before you run the same gradient. Don't inject any samples, just run the gradient. Look at the resulting chromatograms and tell us what you see.

If my hunch is correct, you'll see some junk toward the end of the first chromatogram and lots more junk toward the end of the second.

I think your MP may be contaminated, coating your column, and causing loss of retention. I could very well be wrong, but I've seen it happen.

Have you changed your ACN (lot, vendor, grade) and is your water OK? 10 times out of 9 when this happens, it's the water.

Do you premix your MP or use the pump to do it?

If the latter, rinse your water line with MeOH or ACN to rid it of any biological material that may be growing there.

Good luck and let us know what happens!!

Cheers!

CJ

[djaydearz]

Hi CJ thanks for your reply

i followed the steps you suggested, and also replaced the water line, which looked a bit dirty.
After all of your steps, i tried an injection, and it improved a little, with better seperation and the last peak eluting off at 8.30 mins.

However (under coinsedence?) when i was priming today the alarm kept going off. I looked at the purge valve and the end had snapped off and was stuck in the tranducer. However up until today i had still been able to prime (perhaps the little broken bit was just floating around?) So i had to blank off the ends, put the pressure up and force the lil bugger out.
After putting a new purge valve in, i ran an injection and result!! a perfect chromatogram back to normal.

So i pressume the purge valve was the cause, however i dont know why, as it is before the sample is introduced to the MP and the flow rate/pressure had not changed by much. Strange, but at least its fixed.

Thank you ever so much.

Kind regards
James

[juddc]

Glad you solved it!