I AM DEVELOPING METHOD OF ANLYSIS FOR ONE OF DRUG WHICH IS HYDROPHOBIC IN NATURE AND HAVING CIS AND TRNS FROM IN THAT FORMS ITS H--c--C00H group which is changing its position .
i tried with the tetrabutyl ammonium hydrogen sulphate ion pair for this to seperate on rp hplc with C18 column and flow rate of 2.0ml/min and setting a rt at around 25-30 minutes , but still not able to resolve it .

anybody has idea how to deal with thiS WITH c18 column and revesephase seperation . wheather NORMAL phase can work for it how , or RP is only the good idea but has to change mobile phase.

You have not clearly mentioned about your method-is it a gradient / isocratic. Try with C8 250 x 4.6 mm column. Try to optimise the gradient conditions. clearly specify your mobile phase conditions....

Generally speaking, reversed phase separation of cis and trans isomers of a carboxylic acid are fairly straightforward by reversed phase. But, since you are obviously having difficulty with this separation, let me ask a few questions and make a few suggestions.

First, what is your operating pH? Do you have any added electrolytes or is your eluent system simply ion pair reagent, buffer and solvent?

In general, in order to maximize the selectivity for this separation you should try to increase the concentration of ion pair reagent adsorbed on the stationary phase. So, you are actually probably better off using less solvent and increasing elution strength through the addition of an added electrolyte. For example, you might want to cut back your solvent composition 10% and add 50 mM sodium sulfate to your mobile phase. Also, since you are using an ion pair reagent you need to make sure that you've chosen a pH sufficient to fully ionize your analytes. Otherwise, I would consider operating in the ion suppression mode if you want to operate at low pH. Finally, because of its high steric selectivity, anion exchange can usually accomplish such separations with ease. You might want to consider anion exchange as an alternative if you are unable to get the reversed phase separation to work.

How fast is the equilibration between the forms?

Generally the equilibrium between the cis and the trans forms is extremely slow but you need to avoid exposure of your sample to light since this induces the transformation fairly efficiently.

I am not sure that ion-pair chromatography is the best solution to the problem. Ionic interactions work in the bulk, while reversed-phase interaction work on the surface. My recommendation would be to get a fresh reversed-phase column that has not seen an ion-pair reagent, and work at acidic pH to protonate the carboxylic acid group, and then to use simple reversed-phase chromatography.

Normally, the cis and trans forms have completely different surfaces, therefore I agree with Uwe. So unless you have a huge molecule they should be easy to separate on RP.
If your acid itself, or the matrix, is not colored there will be no input of energy from light (also, isolated double bonds do not absorb light in the visible) it can not isomerize due to light. Strangely, there are all kinds of myths surounding the isomerization of unsaturated fatty acids, if they did that so easily we would not exist.

actually i am trying to seperate nateglide new type two antidiabatic drug .
i am using isocratic separation .
witho ion pair , i tried at both upper 7.5 pH AND ALSO AT LOWER 3.0 PH STILL DIFFICLUTIES TO ACHIEVE SEPERATION .

Hans

The acid does not need to be colored to undergo cis-trans transformations upon exposure to light. Aconitic acid is colorless and is stable at room temp for weeks but if left out in room light, there will be significant conversion of the cis to the trans form in a few hours.

How does this happen? Some UV component in the light and a container which does not shield it? Or was there some colored "dirt" in the solution? Or is the coloration so weak that one couldn´t see it, maybe you had autoxidation products in it and are getting free radical chains?
You have to put energy into the molecule to isomerize it.

Is it "nateglide" that you are working with, or nateglinide? If it's nateglinide, you might want to look at the Journal of Liquid Chromatography & Related Technologies® , Volume 26 , Issue 11.

Since this is a hydrophobic molecule and a C/T
separation I wonder why no one has suggested
normal phase HPLC -silica gel col w a hex/ipa
mobile phase for a start. Ive had considerable
success using this type of system w C/T
separations.

paper which is given in journal of liquid chromagoraphy and related technologies is i think related to chiral isomer seperation , i am dealing with the positional isomer that it cis and trans one paper related to this is published in www.chromatographia.de but its not working giving working with 40buffer ammonium phosphate 0.01m ph 3.0 : 50acn:20 methanol on C18 column
this is giving very sharp peak for nateglinide but this method is not working for cis and trans seperation ? how he has published the paper i do not know?

Hans,

I'm not sure why you think this is so implausible! This phenomenon has been observed many times in our laboratory. Fluorescent lights definitely emit in the ultraviolet and unless you're using amber bottles, the glass is sufficiently transparent to transmit ultraviolet at the right wavelengths. I've done photo polymerization in glass bottles so I know they are sufficiently transparent in the 300 nm region. This is all that's required to cause interconversion between the two forms. You don't need 200 nm wavelength to accomplish this.

As noted above, that is what I suspected: The miniscule UV component and low filtering of the container. I have gotten used to ignoring these UV components as just about everything stored in the lab is in amber bottles. But: one ought to be able to do photochemistry even in amber bottles if one "blasts" them with light.
The only cis-trans isomerization I ever saw at room temp., etc., was that of an osazone (without light, of course, this is typical).
Also, strong enough "H+" can isomerize....

i THINK subject is littile changed , i want to discuss on nateglinide cis and trans isomer seperation by RP HPLC , nateglinide spectrum start s from 200 and ends at 258 and shows very narrow absorabance after 220 , so selection of wavelength is offcourse 210 for the analysis of method .

After looking at the structure of this compound, I am not sure that I understand what you are trying to do. Are you talking about the cis and trans forms based on the substitutions on either side of the cyclohexane ring? Is the material that you are working with racemic at the chiral center with the COOH group attached?

In the interest of making progress with this
inquiry please note that Nateglinide (a
phenylalanine derivative) has 2 stereoisomeric
components which may require separations:
An asymmetric carbon alpha to the amine i.e.
enantiomers. The carboxyl group is attached to
this carbon and is not involved in cis/trans
isomerism
A cyclobutane substituted in the 1,3 positions
- these are involved in cis/trans isomerism

These 2 sets of isomers obviously wont be
separated by the same system.

Also, since this molecule has "low aqueous
solubility" the classification of "hydrophobic" is
quetionable.Semantics notwithstanding a RP
approach is reasonable and the RP chromatography
of the July 12 correspondence may work for a c/t
separation.

For starters what is the capacity factor of the
peakfound using that rp system?

You are absolutely coreect what i was trying to convince my self , but with presnt system i got the peak at 12 minutes retention time and capacity factor you are talking about it good enough , i injected a cis isomer standard and got the retention at same retention time .so what could be next.. done

First a correction - its a cyclohexane ring not a
cyclobutane ring thats involved.
Has the cis standard been confirmed by NMR?

First a correction - its a cyclohexane ring not a
cyclobutane ring thats involved.
Has the cis standard been confirmed by NMR?

STILL MY SEPRATION ISSUE IS NOT BEING DISCUSSED AS IT NEEDS WHICH BUFFER AND COLUMN TO SEPERATE ON REVERSE PHASE COLUMN

Strange that no one have talk about silver ion mode.
It's a very useful technique in rotine analysis of fatty acids, work very well resolving positional and geometical isomers of C18:2 or C18:3.

This post is going nowhere.

I too, have looked at the structure of this compound.

Can the original poster please answer the question of anonymous dated 16th July 11:15am- 'What are you trying to do?'

The Chromatographia On Line, 57,Feb3/4,2003
reference reports the separation of the c/t
isomers of Nateglinide on a C18 column using a
mobile phase of ACN/MeOH/0.1M NH4H2PO4 (the
dihydrogen phosphate at 0.1M):5:2:4. This mobile
phase differs from your July 12th communication.
Are you indeed using the published conditions?

This paper is absolutely rubbish he has not mentioned the brand of the column , this method is not at all working we have tried same mobile phase with the different brands of columns , if any body is wokring on same method and had workout this method.

Dear,
pl you try with followng combiniation you may get separatin between cis and trans isomer
use always for isomer sepration ACE brand column with particle size 3 µ, use modifie as THF and try with different buffer like phosphate buffer, p
H range between 3 to 8

I have read some times ago an article about cis trans separation with new type of HPLC separation columns at journal of chromatography. The columns called Caltrex. May be it can help you!