Chromatography Forum

Hi guys

This is my first time posting in this forum and I would very much appreciate somebodys help on this matter.

I am developing an HPLC method for the analysis of phosphonates. This method involves a post column reaction where the phosphonates are converted into ortho-phosphate and detected by UV analysis at 800nm. I have been advised to use an internal standard in my method but I am finding it very difficult to find a compound which absorbs in this region. The internal standard chosen should not be involved in the reaction and be detected independently.

Would anybody have any ideas of what compounds I could maybe use or where I could look for information? Does anyone know if there is an online database or a book which would have the UV spectrum of compounds in it?

Thanks

UV analysis at 800nm

To be a bit nit-picky, 800 nm is waaay outside the UV range; in fact, it's just north of what is typically considered the visible range (400 - 700 nm ; http://en.wikipedia.org/wiki/Electromag ... Boundaries ). If you really want an IS that absorbs that far out, you need to look at published absorbance spectra of blue dyes.

That said, why do you think you need an internal standard in the first place? Internal standardization is typically used to improve precision where the dominant sources of error are things like sample workup or injection volume. In the case of post-column-reaction detection, the reaction is likely to make the biggest contribution to error, and by choosing an IS which sidesteps the reaction, you are quite possibly simply adding another source of error.

Internal standard was also used a lot back before there were accurate, repeating autosamplers (manual injections).

Most times when I see a method that states 254nm or internal standard, I run and hide, or at least modernize, can be holdover methods from 30 years ago.

Seems it would be better to use a non-target phosphonate as the internal standard that would go through the entire process. This would help compensate for any variation inherent in the whole analytical system and give much better calibrations.

Thank you all for your feedback - I apologise I havent been on here for a few months as the method was abondoned for a while but I am trying to get it up and running again. The points made have been very useful.

Thanks Again