Chromatography Forum

Hello everyone! I have developed a method to quantify Ambroxol HCl, dextromethorphan HBr and sodium benzoate in a syrup as following:
mobile phase: sodium 1- heptanesulfonate 0.005 M and buffered (phosphates) at pH= 2.8. (A)
acetonitrile (b)

There is agradient that goes from (80:20) (A:B) to 70:30 and later returns to 80:20.

column: 4.6 x 150 mm C18 Symmetry 3.5 micrometers

The standards and samples are disolved with mobile phase.

I obtain the three peaks correspondigs to my analytes, but later of around 70 injections the peaks don't present a gaussian form and look like rectangles. I have put through the column water:acetonitile after each batch sample and regenerated the column with dimethyl sulfoxide but i cant come back to my original chromatogram. The pressure in the column is similar to the beginning of its use.
The column has not been used with a different mobile phase.
The retention times are not changing.

What can I do to extend the life time of my next columns and prevent this situation?

please feel free to make any kind of suggestions to the entire method.

Isaac

Thanks

What else is in the syrup? What does your sample prep look like?

I prefer to avoid the "classical" ion-pairing reagents like the alkyl sulfonates with gradients because of equilibration time issues. Nevertheless, what you're describing sounds a lot more like contamination of the column. I would guess that something is sticking to the stationary phase.

The way to tell is to run a long series of injections of standards only. If you see the same problem, then there is something about the chromatography that needs to be addressed. A buildup of longer-chain alkyl sulfonates (present as contaminants) perhaps??

If you don't see the problem, then it is sample-related. A short-term solution is to use a guard cartridge and change *that* frequently. A better solution would be to address the sample cleanup to take out the junk (possibly SPE on a reverse-phase cartridge???).

Maybe a silly question but: Is the concentration of your samples more or less the same as at the beginning? Is the injection volume the same? I observe rectangular peaks usually with overloading a column.
Best,
Anna

Hi Isaac; I am Agree with Tom; even when your method seems very logical to me (low pH protonize amine groups and heptanesulfonate works as Ion pair) you will need longer time to stabilize your column after each gradient so I would prefer to work RP at bit higher pH.
Syrup can be a nasty sample and you would need precolumn and change it more often. I would try to use another column like sunfire, Atlantis, zorbax or other similar column instead of symmetry one of the reason can be higher end cap and more stability of silica.

Regards.

Oscar

There is a gradient that goes from (80:20) (A:B) to 70:30 and later returns to 80:20.
column: 4.6 x 150 mm C18 Symmetry 3.5 micrometers

If I read this correctly, I see that the highest percentage of ACN is 30%. At 30% ACN, lots of components could be potentially retained on your column; have you tried (after your three components of interest have eluted, so you wouldn't need to re-validate as your "assay" conditions are still the same) increasing the ACN percentage to 85% or 90% for a few minutes to clean off all the stuff off your column, before returning to 20% ACN?

so you wouldn't need to re-validate as your "assay" conditions are still the same) increasing the ACN percentage to 85% or 90% for a few minutes to clean off all the stuff off your column, before returning to 20% ACN?

I had missed the part about only going up to 30%

If you do run to a higher % ACN to clean junk off the column, be sure to study the effect of equilibration time (per my earlier comment about ion pair reagents with gradients).