about isomer separation,

[ahvivian]

I want to Development of a HPLC method for the determination of metal A using L as a precolumn derivatizing reagent
but I find the chelate of A-L is a mixture of two isomers.
so two peaks can be easlily seen in the chromatography.

u may advise me to begin quantitative determinations peak area or height of two isomers respectively, but....... the interesting thing was that I found there exsits a kinetic tansformation between two isomers, that is to say in the different reaction time,the peak area or height of each isomer is changing, one is increasing, the other is decreasing,

I developing a chromatography method to quantitative analysis, since the sum of the area or peak is invariable in a certain concertration, then the single peak is more fit for quantitative analysis?

could i try to find a suitable mobile phase composition to make the resolution of two isomers is nearly zero, or may be explained as the retention times are equalin, so a single peak will be seen in chromatography, then can i quantitative determine the concentration of metal A through measuring the peak area or height of chelate A-L?

it puzzled me for a long time, please give me some advise,ok?

[HW Mueller]

You have to analyze a compound which equilibrates between two isomers under your handling/chromatography conditions and you are looking for a standard of this material which does not do this? You can not have the same material with different characteristics.
Or are you looking for an internal standard which is close to your analyte? Then keep looking. To introduce lability into your analysis via an internal standard is the last thing you want to do. I see only one real other possibility: You find conditions under which there is no interconversion of isomers. (If you are only dealing with highly pure substances you may get away with allowing isomerization,..... but hard to accept for an old chemist)

[ahvivian]

to HW Mueller: thanks!


i hope to get more valuble advice from this forum!

[HW Mueller]

Hm...very confusing, this is editing? A completely new question? Also I don´t remember in detail what the other question was. Nobody will know why I gave the above answer. I have no inclination to play this any further.

[ahvivian]

hope to receive other valuble suggestion!

[Mark Tracy]

You cannot assume that the UV response is the same for the two isomers. You would need to make calibration lines for both, then add together the calibrated amounts, not the raw peak areas.

[ym3142]

my understanding:
1) try other L's to see if you can obtain just one isomer ?
2) if 1) failed, try to see if you can standardize the method so well that the ratio of the two isomers is contant and you can do quantification based on either isomer;
3) if 2) failed, try to prove the sum of the two peaks can be used for the quantification such as robustness test: test under variety of conditions;
4) if not 3), find a correction factor for one of the isomers and do the sum.
Good luck.

[ahvivian]

thanks a lot to all of u for giving good advice!

i hppe more,please,