baseline problem

[annavera]

Hi everybody!
I am trying to separate by RP-HPLC using ion-pair reagents some compounds which are negatively charged. The column is an waters xterra MSC18 250x 3mm, 5 um, flow 0,5 ml/min; I used as solvents (A)water and (B) MeOH both with triethylamine 0.15% and formic acid 0.18%. the solvent program beginns with 99% A for 10 min., then goes to 100%B in 60 min. the wavelenght I am using are: 227 nm and 235nm. I did not have any problem with th ebaseline during the first 10 min. but during the gradient the baseline goes up to 300 mAu, it is characterized by small simmetric waves. I conditioned the column before each run for 20-25 min. at the strating conditions.
is it a conditioning problem? air bubbles in the system (I degas my solvents for 20 min with He before to start)? I also thought that the problem can be the UV absorbance of my solvents and additives but the wavelenghts I choose are the best for my compounds...

I hope to receive a lot of good hints from you as always!!!

thanks in advance
annavera

[SIELC_Tech]

Dear Annavera,

Please check the following link: no ion-pairing reagent, no base line problems, simple mobile phase and isocratic conditions.

http://allsep.com/brochures/June_2004.pdf (Retention of Polar Compounds without Ion-Pairing Reagents)


The articke has separate examples of acids and bases retained on Primesep mixed mode columns.

Here are few more links:

http://allsep.com/makeChr.php?chr=Chr_055 (chromate and bromate)

http://allsep.com/makeChr.php?chr=Chr_070 (lactic acid oligomers)

[annavera]

hi,
thanks for the suggestion but in this moment I cannot buy a new column since I bought the x-terra not long ago!!!:-)
I hope that someone can help me in using the system I already have.

yesterday I forgot to say that the HPLC is an Agilent HP 1050 with DAD.
I have a curiosity, maybe someone has experienced the same problem, during the gradient and in particular from the 40% MeOH to 100% I can see air bubbles go from the multichannel gradient quaternary pump to the active inlet valve in the head of the pump. Is in that case that my baseline beginns to go up and make waves....that happens at flow 0.3 and 0.5 mL/min.
why the air bubbles come from that percentage of MeOH? is it a coalescence problem?

thanks

[Newman768]

if you want to run gradient seperation on a Agilent HP 1050 you should buy a inline degasser. Degassing with He is may work with isocratic mode, but with gradient mode most of the time you have bubbles.
BTW: You are not restricted to Agilent degassers.

[WK]

Give both solvents a good 20-30ml purge when they are degassed - since their will be dissolved gas in the lines between degasser and valves.

[HW Mueller]

annavera,
take a look at the chain on He, not too long ago. From that one would deduce that you see He, not air bubbles. (That mkes it at least two persons that have "seen" He bubbles in this misnamed degassing technique). Andreas´suggestion to use an inline degasser would then be prudent (if you don´t have one, pull vacuum on your mobile phases). In isocratic mode it doesn´t matter so long as you don´t disturb equilibrium, since the raised baseline can be "zeroed" if you are not already near "blackout" (UV detector).
Purging? With a saturated He solution?

[Chris Pohl]

Annavera,

Although the symptom you describe could be due to outgassing during your gradient, it seems more likely that the underlying problem is mixing. One way to distinguish between the two is that mixing related "noise" is generally highly sinusoidal and should reproducibly appear in your chromatogram at a specific location. Although it can occasionally be fairly reproducible, gas bubble formation problems are rarely highly reproducible so if the problem is sporadic or only semi-reproducible the problem is more likely to be gas bubble formation related. Since you've been sparging with helium, I would be surprised if your problem was related to outgassing.

If your problem is mixing related, I would look into buying one of the various available mixers designed for HPLC. Keep in mind, though, that thorough mixing requires that the mixer volume must be comparable to the piston displacement volume (on some instruments it needs to be equal to the sum of the displacement of both pistons). So a simple mixing tee won't be adequate in most cases.

[Uwe Neue]

You are working at a wavelength range where the background absorbance of the formic acid is becoming significant. As you are changing the acetonitrile concentration, I would expect that the ratio of formic acid to formate might change, since you are dealing with a weak acid and a weak base. I don't have any books at home that give me the spectra of formic acid and formate, but it is there where I would look for a potential problem.

[HW Mueller]

If I read this correctly, then annavera saw "air" (= He here) bubbles. Vigorously gassing H2O with He gave me a raised baseline + spikes (typical for gas bubbles, UV detection).
Why doesn´t someone saturate some water with He, record a chromatogram without a column, then while keeping the chromatogram running, degas the H2O with vacuum (10 min, swirling once in a while to remove the He bubbles), then reconnect and watch the chromatogram. Then immediatly report on the forum....

[annavera]

Hi friends,
thanks for all replies and suggestions..
I have in part found a solution for my baseline drift problem switching to an isocratic elution. I prepared a mixture of 20% MeOH and 80% H2O (TEA O,1% +HCOOH 0,18%, pH 2.5) and run the 100% of this mix for 30 min. As I said I solved only in part the problem because I still have bubbles but much less than before...and the baseline is ok now.
now I would have another question for you: I am using an xterra MS C18 250x3mm, 5 um column, flow 0.3 mL/min (it is the first time I use one of this "non silica" column) I find impossible to use it with rapid gradients!!!
when I program gradients with short steps I have always very high pressure in between and the pressure stabilizes only after several minutes.....
must those columns to be used in particular way?

thanks in advance

[Uwe Neue]

The packing is behaving like silica, i.e. for your case, it is not compressible like poymeric packings.

I do not know as to the origin of your pressure spikes as you change the solvent composition. If you want to, you can give me the details of your solvent changes, and we can figure out together what the problem might be. We are running routinely gradients, even sharp gradients on XTerra columns, and the pressure behaviour is absolutely normal.

[Basil]

Hi all,

I am trying to separate by RP-HPLC some compounds which are basic The column is a Kromasil C-18 250x 4.6 mm, 5 um, flow 1,5 ml/min; I used as solvents (A) buffer at pH 5.3 that has been made with amonium acetate (B) ACN. The solvent program beginns with 60% A for 10 min, then goes to 10% A in 250 min.

I was first using the wavelenght at 254 nm and it no were any significant problem, but we have discovered a potential impurity taht eluates at the end of the chromatogram and has a very low response factor at 254. Then we have changed to 240 nm and the baseline has a big drift during the gradient change.

This methos is for an API and in the future it could be evaluated by the FDA. Can we change wavelenght during the HPLC method. Is there any problem if absorbance goes from 0 mAU to -60 mAu at the end of the chromatogram.

Thanks