validation-peak distorted

[kahlon]

Column Hypersil thermo C18 BDS 5 um 4.6X250 MP: phosphate buffer 3.8 pH and ACN gradient 10% ACN to %70 ACN in 50 minutes. Compound is nonpolar. Degs assay. compd conc. is .48 mg/ml.

The peak is tailing. with different column lots it shows double peak, peak tailing. The other problem is chromatogram shows lots of small peaks around that compd. ret time is around 46 mins. How can this be resolved.
Validation did not pass. We have other five degradants in the same method eluting at 15 minutes to 25 minutes well resolved peaks. Polarity is ranging from somewhat polar to non polar.

Any guidance will help.

[tom jupille]

First of all, phosphate is not an effective buffer at pH 3.8

Second, tailing of the parent is fairly common when looking at degradants, because you have to overload the parent compound in order to detect the degradants.

Beyond that, you really haven't provided enough information:

what is the buffer concentration?
What is your sample dissolved in?
what concentration is your sample?
what volume are you injecting?
are any of your analytes (the parent or degradants) ionizable? if yes, acid or base? can you estimate the pKa?

Do all the peaks tail, or only the parent compound? And when you change columns, do you see the tailing/splitting immediately or only after some time?

[kahlon]

Tom,

buffer concentration is 0.01 M phosphate buffer pH 3.85
samples are dissolved in 0.05M phosphate buffer:ACN (90%:10%)
concentration of the samples is .0048mg/ml
inj voume 50uL
only one degradant peak is tailing
There is a parent compd and 5 degrardants.
the degradants are

cyclohexane diacetic acid monoamide
Carboxymethyl-cyclohexanecarboxylic acid
3-Azaspiro{5,5}undecan-2,4-dione
2-Azaspiro(4,5)decan-3-one
1,1 cyclohexanediacetic acid
{1-(3-oxo-2-aza-spiro[4,5]dec-2-ylmethyl)-cyclohexyl]-acetic acid.

polarities are range, the last one being the least polar.

Please respond.

Thanks in advance.

First of all, phosphate is not an effective buffer at pH 3.8

Second, tailing of the parent is fairly common when looking at degradants, because you have to overload the parent compound in order to detect the degradants.

Beyond that, you really haven't provided enough information:

what is the buffer concentration?
What is your sample dissolved in?
what concentration is your sample?
what volume are you injecting?
are any of your analytes (the parent or degradants) ionizable? if yes, acid or base? can you estimate the pKa?

Do all the peaks tail, or only the parent compound? And when you change columns, do you see the tailing/splitting immediately or only after some time?

[kahlon]

Tom,

Validation was performed on the method. It failed in robustness. Different column lots shows different reults for the last imp. some had peak tailing tailing factor more than 2.0. some had splitting.

thanks.

First of all, phosphate is not an effective buffer at pH 3.8

Second, tailing of the parent is fairly common when looking at degradants, because you have to overload the parent compound in order to detect the degradants.

Beyond that, you really haven't provided enough information:

what is the buffer concentration?
What is your sample dissolved in?
what concentration is your sample?
what volume are you injecting?
are any of your analytes (the parent or degradants) ionizable? if yes, acid or base? can you estimate the pKa?

Do all the peaks tail, or only the parent compound? And when you change columns, do you see the tailing/splitting immediately or only after some time?

[Uwe Neue]

I agre with Tom. A significant increase in the buffer capacity by going to pH 2.5 to maximum pH 3 could solve the problem

[kahlon]

Thanks Uwe,

I should try that tommorow. I will post the details.

[tom jupille]

That eliminates a lot of possibilities:

The fact that only one peak tails means we can eliminate column headspace problems.

The fact that it's a degradant that tails rather than the parent means we can eliminate overload

Your diluent is comparable in strength to your mobile phase, so that peak "smearing" from a too-strong diluent is not likely.

Your injection volume is reasonable.

The most likely problem is pH control. If you want to use phosphate, follow Uwe's suggestion to drop the pH to something closer to phosphate's effective buffering pH (phosphoric acid has a pKa of about 2.1, so up to about 3.1 is the upper end). If you need to work at 3.8, then formate or acetate would be better buffer choices.

[kahlon]

I have some other problems on the same method that needs to be resolved. i will post the details. Thanks for your reply. I should try lower pH first aND WILL KEEP YOU INFORMED. THANKS AGAIN.

[kahlon]

1. I tried lowering pH. peaks are resolved(Rs slightly more than 2.0) at pH3.1. If I go lower at around 2.9, I see other degs peaks merging.My question is regarding robustness, if this will create problem in robustness later during validation? Run time is around 50 mins. 5 imp. peaks eluting form 16 mins to 25 mins. the nonpolar imp at 46mins. How can I reduce the run time. I have gradient 10% organic to 70% org in 50mins.


2. Samples ( polymer formulation) shows lots of exc peaks and humps in the chromatograms. Is there any better way of preparing spls? we are dissolving in 90% buffer :10% ACN--no filters using guard columns.