The compound is isolated from a plant extract and there are two possible positional isomers. Their peaks are very close that sometimes it looks like a single peak with a small shoulder. I have tried separating using binary solvent systems ( Acetonitrile in water, acetonitrile in methanol). The elution happens at higher percentage of acetonitrile.
Will three solvent systems of the above mentioned solvents work? Or could someone suggest me a better alternative?
Are you running a gradient? If so, run the mixture isocratically for increased resolution. I didn't see mention of a solvent modifier, so try adding one (TFA, formic, or acetic acid). Some phenolic compounds have a pKa close to neutral.
Another possibility is to try normal phase- I saw the reverse phase elution uses a "higher percentage of acetonitrile".- what concentration? The separation can be evaluated on TLC before commitment to a column, using a different column media and solvents may allow a different selectivity.
Positional isomers should be easy to separate on a C-18 column. You didn't mention the solubility of your compounds in acetonitrile or methanol. Shoulder can mean anything (impurity, sample solvent effect, actual positional isomer, decomposition product ...plenty of possibilities). Normal phase is also an option , the key question is solubility in hexane/EtOH system. NP is very good for shape selective separations.
By chance, do you have a bare silica column? You can try a technique called HILIC in which you use a high percentage of acetonitrile (80 to 95%) and (20 to 5 % water containing a buffer which is usually ammonium acetate. Again it will all depend on the solubility of your compound in this HILIC system.
Thank you everyone for the suggestion.
1. I had tried with solvent system 0.1% TFA added to acetonitrile and water. The TFA didn't have any effect on the retention time. When the gradient was run with increasing acetonitrile in water, the elution happened after the run time i.e., 100% or higher concentrations of acetonitrile is required when water is used as the other solvent. But when methanol-acetonitrile gradient was used, elution happened at 32% of acetonitrile in methanol (retention time 6.25 min with 1ml/min flow rate).
2. This compound is soluble in methanol, acetonitrile and almost all the semipolar organic solvents. Compound also doesn't have a chiral center.
3. In TLC the compound gives only single spot. I had purified the compound in batches. In most of the cases, the compound shows proper single Gaussian peak. But in some batches, the shouldering of the peak occurs. LC-MS of that particular batch revealed same mass for both the peak and the shoulder, which is why I had suspected positional isomer forms.
4. There is a limitation of column types. I have access to only c18 right now for both analytical and preparative mode. So I will have to play around with the mobile phases.
Are there any other possibilities.
Going forward, please tell us the details of your experiment and also what have you have already tried. You would have received better answers.
Running a methanol/acetonitrile reverse phase gradient is non-aqueous reverse phase, it is a useful technique, but not commonly used. It is very useful for non-polar compounds, such as yours.
Try running an NMR to verify you have positional isomers, and are not dealing with a chromatography issue that causes minor peak splitting. Run NMR on both the major peak and the shoulder. A mass specc on both may also be useful since you may see different fragmentation patterns.
You can try other solvents to affect the selectivity such as methanol/dichloromethane, methanol/tetrahydrofuran, and water/tetrahydrofuran; the first solvent in the pair is the "weak" solvent, the second is the "strong" solvent. These are all reverse phase methods.
Try a column such as diol, which can run as normal phase, or cyano, which can also run as normal phase with organic solvents.
Hello Mr. Silver,
Thank you for the wide range of suggestions. They are very helpful to look at the problem from various perspectives.
Those were the techniques and methods that I had tried previously.
Now recently, I had tried the three solvent system wherein the percentage of acetonitrile was kept constant (50%) and the methanol was increased in gradient in water (0-50% gradient). The resolution of the peaks are a little better. Since I was unable to purify the isomers I only did the NMR of the mixture. They were matching with the expected structure (isomer form of the compound was non-distinguishable with NMR) and as mentioned the mass was also same.
I will try the solvent systems mentioned by you and see if I could resolve the peaks even further.
It wasn't obvious you were using a 3 solvent system. Most chromatography is done with a binary solvent system. If the solvent system contains water, non-aqueous reverse phase isn't an option, since the solvent system will be too strong. You can try tetrahydrofuran, and the other normal phase options.
However, I'm not convinced you actually have an isomeric impurity. The NMR should look different, and the mass spectrum should have a different fragmentation pattern. Chiral isomers may act that way, but they aren't resolvable on C18. The first eluting peak in the attached image is not an impurity, but is almost certainly some sort of injection artifact because this mixture is well known to me. Your peak shoulder may be a different compound from your main peak, but the other instruments haven't confirmed it. Perhaps show the chromatogram?
I had done MS in ESI mode, so the fragmentation wasn't there.
PFA. 1. acetonitrile gradient in water with elution after the run time (peak expanded) 2. three solvent system with little impurities. In the second image, I am not sure the other small peak next to the large one is an impurity or isomer or is the shoulder that was seen in the previous image as its intensity is very low.
I guess a way to confirm this is to purify both the peaks using preparative HPLC and then perform the analytical techniques.
Since I do not have tetrahydrofuran in my lab, I will try it as soon as I get.
Hello,
As you don't have a chiral center at your compound, you don't need a chiral column to resolve them. If you can find the correct solvent system, you will certainly be able to resolve them using a c-18 in reverse mode.
Regarding the possibility of you are dealing with positional isomers, nmr techniques should be able to confirm it. Try to analise the 1H and 13C chemical shift of each atom individually, so that it will be possible to look for small variations that may help you to discriminate them.
Dear Sanjana, you can use phenyl and biphenyl column for the separation of aromatic positional isomers. It is not that chiral column separate only chiral impurties we have separated some positional isomeric impurities on normal phase chiral column after failing it to separated on reverse phase chromatography C18, C8 and phenyl columns. Some times its work dude.
You can use a chiral column. For this, β-cyclodextrin adobebent will be used.
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