Chromatography Forum

Greetings

I think I used to prepare std in some pure organic, and then use different diluents (organic: H2O=X:Y or organic:buffer=X:Y etc.) for different sample matrix.

Currently people believe we have to use exactly the same diluent for std and sampe matrix. Is it necessary?

Thanks

It depends...

If you are doing quantitation by area, you will be able to inject in a different solvent. If you are quantitating by height only, I would be very concerned about your results.

Reason: the use of a stronger eluting solvent as the sample solvent can cause peak distortion. Such peak distortion will of course be very visible, if you inject a large sample volume, but they may not be visible for smaller volume injections.

Bottom line: if you are quantitating by area and if the possible peak distortion does not affect the separation, you will get away with injecting the standards dissolved in an organic solvent and your samepl dissolved in mobile phase.

Is it necessary?

Is it necessary from a technical perspective? I'd say "no", but we usually do. the injector delivers a volume, and your sample and standards are a certain concentration (such as nanograms per microliter) so an injection of (for example) 5 µl would still deliver the same number of molecules of analyte no matter what the solvent is. However, if the injection volume is large, and the solvent is a "strong" organic solvent, the solvent injected can affect the chromatography; so in that case the response would be "yes", and to match solvent type.

In the regulated world, follow whatever was validated and documented by your company. We use (at least) one validated assay where the calibration standard is made up in Organic Solvent 1, and the samples are extracted with Organic Solvent 2. The mobile phase is an aqueous-organic mixture containing neither Organic Solvent 1 or Organic Solvent 2. For this our injection volume is 5 µl.

Thanks a lot. very applausible.

"In the regulated world, follow whatever was validated and documented by your company. " - to add to this great comment, you should do things as is written in the method procedure, if the method was validated and shown to be fit for purpose then it is even if it calls for using different diluents for standards and samples

On the other hand, if we always did what we were "supposed" to do without a shadow of curiosity or compulsion to improvements, we would still be hiding in caves and using the majority of our conscious life to find (or kill) food.
Come on guys, haven’t you seen thousands, even millions of examples of poor chromatography or mindless procedures in general? And yet, most of them have been validated and even published various places.
So, never give up understanding, improving and innovating – even though the bureaucrats will do their best to suppress potential desires of the kind.

Best Regards

Come on guys, haven’t you seen thousands, even millions of examples of poor chromatography or mindless procedures in general?

See USP to share in the experience. Dust off those packed columns and wet chemistry glassware.

And: what's the highest amount of water permitted in 99.5% USP glycerin? 5.0% water. Figure that out.

How many different USP and NF procedures are there for trace DEG and EG, all calling for use of different USP RS reagents, sold only by (you guessed it).

"Come on guys, haven’t you seen thousands, even millions of examples of poor chromatography or mindless procedures in general? And yet, most of them have been validated and even published various places. " - we have, and yet if the method was validated it gives valid results, and these are not always best results

"On the other hand, if we always did what we were "supposed" to do without a shadow of curiosity or compulsion to improvements, we would still be hiding in caves and using the majority of our conscious life to find (or kill) food. " - that's were method development comes into play, you can always change the method and revalidate