Separation of 2 peaks elutaing at approimately the same RT

[grzesiek]

link will do

"isomer number 1,2 and 3 penicilloic acid" - I'm confused, I just don,t see where these isomers could be

Injecting imp E you get two peaks right? is this imp E pharmacopoeial standard?

[grzesiek]

"About the difference of pHs of moblie phase and diluent...well thats the way they have it in the EP!" - it happens all the time with these methods, that's why I personally hate them

[seamoro]

yes...EP standard...when you inject you get a small broad peak then a peak with good shape about 1-1.5mins away. the intial broad peak could consist of a number of peaks!...the longer the penicilloic is in solution the larger the intial broad peak becomes...untill eventually it becomes a good shape peak as well.

[grzesiek]

ok, so this seems to be problem with the stability (of one form?) of imp E

if imp E would be one peak, this late one, would it be then easier/easy for you to develop method?

if so, than maybe you should focus on changing parameters (diluent) so that this problem does not occur

how is this impurity quantified according to EP? should be one or two peaks?

[seamoro]

its not a stability issue...the penicilloic acid comes at a particular purity....the impurtity or isomer will always be present underneath that sodium benzoate peak giveing an over estimation of the sodium benzoate....and this over estimation will only increase during a stability study thus giving incorrect sodium benzoate quantation.

[grzesiek]

"the impurtity or isomer will always be present underneath that sodium benzoate peak" - true, but I don't know stable is original peak (acid), and if this impurity of acid will come in stability studies

anyway, improving this impurity peak shape could lead to better resolution of the compounds

are you able to show any chromatograms of how the situatin looks like?

[juddc]

Interesting...

A couple of thoughts:

1. Your MP pH is a bit close to the pKa of benzoic acid and being that you're outside the effective buffer range of phosphate, I'd say that you might have some cause for concern there. If I recall correctly, the pKa for benzoate is ~4.2.

2. I just had a look at the impurities and if I was faced with developing that separation, I'd probably use a perfluorinated carboxylic acid ion-pairing agent (TFA, HFBA) to alter selectivity if I couldn't get anywhere otherwise.... I'd also look into some of the multi-mode columns currently available.

I understand that you might be bound to the EP, but I would think that all that would save you would be a full validation. I don't think there is any rule against using an alternate method as long as it can be validated, is there? As far as I'm concerned, a demonstrably superior separation is a sufficient argument...

[grzesiek]

juddc, I think we all agree on that - "I don't think there is any rule against using an alternate method as long as it can be validated"

unfortunatelly I met some people who don't think this way, these are called bosses

[juddc]

Yup...and that's why I'm in R&D. I'm the PITA employee that keeps asking "Why!?"

[grzesiek]

I was working in R&D, the question came from my mouth few times a day, I never got THE answer

What I got though was "cos we always do so"

If I wasn't forced to do "as they always did" I think I would be still working there

[seamoro]

ok..good news i got some separation with the t3 water column (moblie pH3.7, flow rate 1.0) but its not perfect yet!.i got the sodium benzoate coimng off just before the smaller broad peak of the penicilloic acid but there is some overlapping of the tails of the peak. i know that a resolution off 1.5 is usually sought but only if the peaks are comparible in size! so i may be able to justify the poor resolution due to the fact that the sodium benzoate peak is alot larger..........am i right?

[grzesiek]

as arnold would have said "you're wrong"

treat it as a joke, I couldn't help it

you don't have to justify resolution, you have to show accuracy and precision, on the levels you expect these analytes to be

[danko]

I don’t understand people investing so much effort in justifying unsuccessful chromatography (or anything else for that matter) instead of fixing the problem.

I saw (some of) you guys mentioned the boss tyranny as a serious obstacle to achieving high quality methods (for instance). And I agree; it is so – often anyway. I, for instance, was told (by my previous boss) that I ruined the cooperation with other people and departments, every time I concluded that the problem which caused a certain invalid result, or something like that, was the quality of the method. So, trouble-shooting always had to end with some minor error in the lab which should justify a retest. Had I accepted all this crap I would sill be working for that company and secretly hated my self (probably similar story to Grzesiek’s).

The point I want to make is: If /when we have the opportunity for doing something right, we should seize that opportunity and do so.

Seamoro, I’m wondering what made you raise the mobile phase pH to 3.7. Didn’t you start off with 3.5? What’s more important; did you try pH around 2?

Best Regards

[grzesiek]

"What’s more important; did you try pH around 2?" - danko, if I understand his problem, he probably wants to be in line with acceptable changes of EP method which is 3.3 - 3.7, and I think it is his boss dream, not his

[krickos]

[quote="danko"]Now, why would the pH of the sample solvent be 6.5 whilst the mobile phase’ pH is intended to be 3.5?
I write “intendedâ€