Chromatography Forum

i just want to know how do you ensure peak purity? i have isolated few impurities of an api by TLC, i dont have the stanards for these impurities, how can i ensure the purity of isoalted impurities? how do you asess purity of a substance isoalted from a mixture by chromatography? i am very confused by this, my friend told me that you can not be sure of absolute purity of an analyate? then how do i go about it, my softwere has a peak purity function, but gives some number like 998, how do i ensure that the peak is pure, i dont have a MS? please help on this

Aniket

your software should also give a treshold value? the peak is pure if the purity factor is within the calculated treshold limit.

998 is a pretty good number. However remember that if your impurity and the compound of interest has the same UV profile, you will not be able to see any difference by peak purity.

The best way to approach to run LCMS, assuming your method is LCMS friendly. You can try to go for a contract lab.

Thanks.

I agree with Roy. You need to use the proper tools for the job.

If you are not having 'peak purity value' more than threshold value (i.e. peak purity value is negative) it confirms that peak is impure and is result of more than one co-eluted components.

In case you are having positive peak purity values (i.e. values greater than threshold) it however does not absolutly confirms lack of co-elution. Simply because if UV spectra of co-eluting compound may be same (as Roy pointed).

Therefore, 'Homogenous peak' rather 'pure peak' terminolgy is used sometime.

We have found UV spectra of simvastatin and its degrdation product 'beta hydroxy acid' is identical qualitatively and most probably quantitatively as well. If in any HPLC method both peaks co-elutes, Peak purity value fails as it will suggest single component elution.

LC-MS should be used for absolute confirmation. 'Peak purity' shall provide confirmation of 'impure peaks' rather 'pure peaks'.

i jsut want to know if you have a mixture of streoisomers, then how do you ensure that they do not co elute? even LC-MS will not detect tose? then how do you go about that? this was the question asked to me for an interview? can any one explain?

One needs two strongly differing chromatographic methods, or entirely different analytical methods, to even approach certainty.
Another factor seems to have been forgotten in these discussions on purity of peaks via UV spectra: The light absorption intensity of substances differ enormously. It is no problem to get an indication of very high purity of a peak, via inspection of its spectrum, even though it consists mostly of an impurity.

No single technique can give absolute peak purity but they just provide you confidence. To prove your peak is pure one has to combine data from different technique.

If one is sure of existance of stereoisomers , that can be proved by different means i.e. polarographic detectors, optical rotation , DSC, NMR etc.

JM