wavelength

[kahlon]

1.My previous mail was validation--peak distorted. I am writing in response to that.

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. Working at wavelength 210 nm. how can we avoid extra peaks?

Please help
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kahlon123

[DR]

If your method requires a given pH ±0.1 or even less, put it in the method. Sometimes, you can't get around that. You may even want to specify the cal. range of pH meter (2, 4) and specify that the buffer be pH adjusted before addition or organic (almost always a good idea).

The fewer opportunities you provide for Q-analysts in a hurry to screw up, the fewer phonecalls you'll get later on.

[kahlon]

You are right DR,

pH range will be specified in the method. Can you suggest better solvent/filter or may be wave length for excipients peaks/humps. we are at 210 nm. trying to work on extra peaks

[Uwe Neue]

In your previous message, you suggested that some of the extra peaks may be related to the polymer in the matrix. This may be resolvable with SPE, or with a precipitation technique.

[kahlon]

Yes Uwe, It could be polymer. We still are working on that. hopefully will have more answers. it appears as a hump in the chromatogram , doesnot interfere with impurities.

SPE could help. Precipitation--Could you please send some detailed information?

Thanks for your reply.

[Uwe Neue]

Hump in the chromatogram... sounds like a polymer.

How to get rid of it depends on the type of polymer and the molecular weight. If the MW is large enough, SPE with Oasis HLB will work.

Procedure:
1. Activate the packing with methanol, then with water.
2. Load your sample in exactly the same solution as you use right now.
3. Wash with water.
4. Elute your sample with methanol.
5. evaporate and reconstitute.

For good quantitation, you will need to add an internal standard to you sample up front.

Alternatively, you may be able to precipitate all or most of the polymer by adding another solvent to your sample. What to use will depend on the solubility characteristics of your polymer.