Trouble resolving isomers

[PercyG]

Hi Chromatography Forum,

I have four isomers m/z 247.88 [M+H]+ that I am trying to purify on a Waters e2695 separations module with a Phenomenex Luna C18 column. My issue is that while the two outer isomers separate easily, the two in the middle are very difficult to separate (presumably because they are very similar in structure - http://imgur.com/aqJ26sU). My method is as follows:

A = Water
B = Acetonitrile

0 min: A=95%, B=5%
0.5 min: A=95%, B=5%
16 min: A=40%, B=60%
18 min: A=5%, B=95%
19 min: A=5%, B=95%
20 min: A=95%, B=5%

at 35°C.

What are some other parameters that I can modify? Are they any other solutions besides getting a chiral column that would be able to resolve those peaks? Is this pattern common, possibly due to peptides that epimerize?

Any advice would be appreciated.

Thanks,

[tom jupille]

You haven't given a whole lot of information (what kind of isomers? What size column? What flow rate? Detector? Wavength? Loading?) So all I can do is make some general comments.

1. Why a gradient? Your 4 peaks are close enough that an isocratic separation would be feasible.
2. In general, you will get the best throughput (mass recovery perunit time) with a fairly low k' (e.g., somewhere around 1 - 2) an multiple injections. Running at higher k' gives you better recovery per injection, but the runs are disproportionately longer.
3. In general, a chiral column should not be necessary unless your "isomers" are actually enantiomers. Diastereomers (again, in general) should be separable with an achiral system. That said, cyclodextrin bonded phase columns are reputed to have good selectivity for stereoisomers.
4. C18 columns, as a class, retain on the basis of hydrophobicity and therefore tend to mre selective for hologs and less selective for isomers. Columns with low surface coverage (and so high levels of exposed silanols) tend to be somewhat better that fully-covered columns (gotta love those secondary interactions).
5. When all is said and done, there are only six ways to change selectivity in reversed-phase:
- change the solvent strength (aqueous/organic ratio)
- change the temperature
- change the solvent type (ACN / MeOH / THF)
- change the pH
- add or change buffers, additives, etc.
- change the column

[mattmullaney]

Hi Percy,

Along the lines of what Tom suggests, I've had good luck with diastereomer separations using polar-embedded phases and lower temperature (say 20 -25 degrees Celsius).

Most of the diastereomers I've had to work with were Schiff bases.

Please see what you think, and thank you.

[lmh]

well, you're not doing too badly! The two difficult peaks are actually very nearly base-line separated, which means that if you're purifying and finding that the fractions are contaminated, then the mixing-up is happening post-detector (possibly because the time-delay between the detector and the fraction collector isn't quite right, or possibly because the fraction collector is equipped with about 5m of wide-bore tubing, something manufacturers seem to do!).

Because the separation is almost good enough, you might get it to be really good enough by using a longer column, or if pressure is an issue, considering moving to Kinetex rather than Luna; I find that Kinetex is a bit less retentive, but Phenomenex can advise on the most appropriate improved column.

In terms of other changes, temperature is easy to play around with and might just make a difference; pH is useful, but only if your isomers are likely to behave differently in response to a pH change; sometimes changing solvent completely can help, and is easy to do, but it's a bit like pressing a "randomize" button: it may just move the peaks around, and they may end up in worse places than they are now.

Changing column is expensive, but if you want to avoid a difficult and really expensive thing like a chiral column, the more "rigid" reverse phase columns are reckoned to be better at separating isomers - i.e. short-chain things, which have less flexibility to wrap themselves round the analyte, and whose interactions with the analyte are therefore more dependent on the exact shape of the analyte.

By the way, does anyone know why, given modern fiber-optic technology and miniature detectors, why manufacturers don't supply a fraction collector with a miniaturised PDA-cell built into the very end of the dispensing tip? It might not give the best signal ever, but sometimes it would be really nice to know (roughly, at lower sensitivity) what's happening where it matters, rather than (with high quantitative precision) 200uL upstream.