isomers!

[amaryl]

do cis and trans isomers have same UV spectrum?

thanx for clearing my doubts in advance

regards

amaryl

[tom jupille]

Usually different. However, if you have an isolated double bond far removed from a stronger chromophore, the differences may be small.

[amaryl]

sir, thanx for the reply.

what kind of stationary phase is used to separate isomers n on what basis such separation is usually achieved apart from taking in to account the differences in the physicochemical properties of isomers.

thanking you

regards

amaryl

[HW Mueller]

What properties other than physicochemical are there? In my experience geometrical isomer separation has been among the simplest of exercises on ~all types of columns (no experience with ion exchange in this regard).

On UV: Tom´s reply got me to thinking about why one does not often see UV used to assign geometry, probably because the differences are usually too small to discern? Now, both the wave length and absorption coefficient can be highly different if steric hindrance prevents the cis isomer (usually) to mesomerize its double bonds.

[amaryl]

Is it possible to achieve cis trans isomer separation on C18 column.

Is their a particular approach followed in mobile phase apart from the physicochemical properties differences of isomers...to achieve such separation.

their uv spectra is different as stated by sir. do their pka even differ.

is this separation achieved by gradient mode or can even work with isocratic mode.

i juzt want to know what are things kept in to mind to achieve separation of isomers.


thanx

regards


amaryl

[HW Mueller]

Sort of partially answered this already, but: If you can choose a system where you get retention than chances are good that your geometric isomers are separating, especially on an adsorption column like a C-18 (not necessarily if the site of the geometric isomerization is buried in a huge molecule).
Remember: Isocratic elution is the one with higher resolution (small molecules).
What compounds are you thinking of in regard to pKa?
What is there apart from physicochemical properties?

[tom jupille]

Is it possible to achieve cis trans isomer separation on C18 column.

In principle, yes. In practice, maybe (depending on cis/trans with respect to what: a double bond? a ring?). If reversed-phase does not work, normal-phase often provides better selectivity for isomers.

Is their a particular approach followed in mobile phase apart from the physicochemical properties differences of isomers...to achieve such separation.

I'm not sure what you mean by "the physicochemical properties differences of isomers". The method development approach would be the same as for any other separation.

their uv spectra is different as stated by sir. do their pka even differ.

Again, maybe. Depends on where the ionizable group(s) are located with respect to the cis/trans center.

is this separation achieved by gradient mode or can even work with isocratic mode.

If your sample contains only the two isomers, then most likely isocratic will work. Gradient conditions are normally required where you have a wide range of retention (unlikely in the case of only two isomers).

[amaryl]

thanx for the reply

sir, if a drug is getting expired is it possible it may undergo cis-trans conversion even when stored at its required stated temperature conditions.

thanking you

regards


amaryl

[tom jupille]

Anything is possible. How probable it is depends on the drug.

[amaryl]

thanx for reply


sir, i had been working with glimepiride. well i was not getting answers to what ghost peak suddenly started appearing in my analysis. i looked in to every possible solution right from cleaning the flasks properly and using appropriate HPLC grade solvents.

the peak was close to my analyte peak. resolution was less. you can say it was tailing of one peak n emergence of another peak.

there r two peaks one big n other small when i injected 10 ug/ml and as i moved down the concentration the small one started disappearing and at some concentration it was not appearing. i was working with UV detector so i could not see spectral differences. my previous work with the same standard never showed two peaks n with passage of time started showing ghost peak.

i found a research paper where they had separated the cis-trans isomer using a C18 column though they used Cis and trans isomer reference standard and found the percentage of Cis isomer (which is reported to be inactive biologically) in refined in crude product of glimepiride.


well my analysis conditions had been 60:40 ACN: 20 mM phosphate buffer pH3.0. i used C18 column of particle size 5 micron 250*4.6 mmm.

Now when i performed the degradation analysis (this was HPLC with PDA detector) using the same standard which was approaching expiry showed this ghost peak along with other degraded products. the peak is very large compared to my analyte peak. even working with expired marketed tablets showed the same large peak.

what could be that large peak. i have injected my buffer n methanol (my analyte diluent) when i started getting this ghost peak n never found it in their injection.

do ghost peaks can be of so large intensity compared to my other interested peaks of my degradation analysis.

i still doubt its not ghost peak. i feel its cis trans isomer of glime.
thought their spectra as seen by PDA differs at 228 nm the large peak shows a little addition trail to the bottom.

i don't know how to paste the chromatogram on this site.

it may be difficult to answer without chromatogram

any suggestions will be appreciated a lot.

thanking you

regards

amaryl
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[HW Mueller]

You don´t have access to a MS?
Does your ghost exceed the original glimepiride (what is it?) in size, same vol and concentration injected?
What happens if you collect the two peaks, separatly, and reinject?

[tom jupille]

It is possible to display chromatograms. The instructions are here:
http://www.sepsci.com/chromforum/viewtopic.php?t=701

What you have to do is to upload the image of the chromatogram to an accessible web address similar to what you would use for posting photographs. Then you reference that address using the "Img" tag. The results should look like this:

[amaryl]

You don´t have access to a MS?
Does your ghost exceed the original glimepiride (what is it?) in size, same vol and concentration injected?
What happens if you collect the two peaks, separatly, and reinject?

well i don't have access to LCMS...this ghost peak is very large in comparison to my analyte peak...it started coming out in my reference standard which is now approaching expiry (though stored at stated conditions)... well i don't have fraction collector can't make out what that large ghost peak is!


when i didn't have access to PDA. Working with UV detector,
i prepared my standard solution juzt to one day discover it started showing two peaks. One was large and the other was small.

the 1st thing that came to my mind was small peak is ghost peak as i had injected (10 ug/ml) and as i lowered the concentration the small peak started decreasing in area and at some concentration (1ug/ml) it was not visible which should not occur since
a) working with PDA i found that the short peak is GLIME
b) and i got a LOQ of 0.207 ug/ml.

the spectra of large peak is bit similar to short peak the only difference is that large peak shows a slight bump before it reaches bottom.

my working wavelength is 228 nm

well i have no idea how isomers behave and how are they separated. the only thing that i wish to know is whats that large peak ...is it its degradant product or cis isomer.

thanking you

regards

amaryl

[HW Mueller]

Amaryl, you do not state ~how much larger the big peak is than your original standard (same volume and concentration injection, before 2nd peak appears).
What is this again, fraction collector needed for collecting peaks? One can do this better manually (less dead volume, etc., etc.).
One does not need a LCMS to get a mass spec. Nobody nearby has a MS?

[amaryl]

sir, i don't have access to MS.

thanx

regards

amaryl