Chromatography Forum

Dear All,

Recently I performed a routine analysis of chiral separation,which comprises of a chiral column and a mixture of isopropanol and hexane(40:60) for mobile phase. The results from differnent istrumentations and lots of solvent have no impact on quantitative analysis,however,the retention changes made me confused. The chromatograms as follows:

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First I think the changes may be resulted in dwell volume of instruments,but some literatures indicate this factor have no interferance on isocractic separation,then an idea came into my brain:whether mobile phase components interfere with the retention or not. OK, I tried this solution. In the latest analysis(3rd time, 20 Jul.), I used a new batch of IPA into the original pure IPA(not fresh IPA absolutely) and the retention close to the result from method validation,a slighly difference exist however. I sure that the mobile phase from different lots made the retention variability,because the IPA used in 2nd analysis(25 Jun.) are same as in method validation(02 Feb.),but the resevior isn't sealed and the solvent is exposure to air,so IPA absorbed the moisture in air and the composition changed, therefore the retention varied accordingly.I've little experience on chiral separation in NPLC, so I hope you give me some practical advices. Thanks

By the way, I read the FDA's reviewer guidance(1994)"validation of chromatograhpy method" and the sentence puzzled me:

" when used as an impurity test method, the sensitivity is enhanced if the enantiomer impurity elute before the enantiomeric drug."(Chiral chromatography)

So is it important that the impurity must be eluted before the main component for monitoring the impurity method?

" when used as an impurity test method, the sensitivity is enhanced if the enantiomer impurity elute before the enantiomeric drug."(Chiral chromatography)

because of tailing peaks, when impurity is on the tail there may be interference from the main peak, it is not obligatory that impurity is before main peak but it always look nice
baseline resolution is usually easily achieved when impurity elutes first

I would suggest to buy air conditioner and go down with the room temperature - it was common for us that in the summer we observed retention time fluctuations with normal chromatography

Hope this helps

I would guess that the water content of the IPA would be the biggest issue; normal-phase separations can be *very* touchy about water content. Temperature variations can also contribute.

Re "small peaks early", in addition to grzesiek's explanation, you can add the fact that in isocratic separations, earlier peaks are narrower (and hence more detectable).

Thanks Tom and Grezsiek!


"it was common for us that in the summer we observed retention time fluctuations with normal chromatography "-you mean that the compressibility of the two organic solvents vary greatly under different temperature, which may result in flow rate accuracy variation in on-line mixing mode? I also think this factor may interference the retention, but no ideas support this point. And I know the water content may lead the retention time in NPLC, but I'm not sure the impact on the chiral case. Would you give some advices? Thanks in advance

"you mean that the compressibility of the two organic solvents vary greatly under different temperature, which may result in flow rate accuracy variation in on-line mixing mode?"

i'm a bit pragmatic in such cases - room temperature control helped to solve the problem, it was the first thing that came to my mind

it was common for us that in the summer we observed retention time fluctuations with normal chromatography.
[...]
you mean that the compressibility of the two organic solvents vary greatly under different temperature, which may result in flow rate accuracy variation in on-line mixing mode?

Since chiral chromatography is based on really soft interactions between the phase and the analyte, I guess the room temperature is more relevant for your column equilibration than for solvent compressibility.
In any case if you think the IPA absorbed some water, you could check this by adding some drops of water in a freshly opened ipa and try to use it, if you obtain the same drift you found your problem.