Chromatography Forum

Hello,

I am a GC neophyte in need of some help. I am trying to separate chiral isomers of a compound after derivatization to their respective diastereomers. I am trying to find a temperature gradient on the GC that could separate my compounds.

Does anyone know of any literature for a methodical approach to achieve my separation?

Any help would be greatly appreciated!

I would suggest you call Supelco technical service. They sell GC columns that separate chiral compounds and can assist you in making the best choice. There is no cost involved to ask for help.

The proper column choice depends upon the structure of the compounds involved. If you don't get help, you will end up buying column after column to try to separate the compounds, a waste of time and money.

Supelco will give you free research and technical help. All you have to do is to ask for it.

best wishes,

Rodney George
constultant

Hello,

I am a GC neophyte in need of some help. I am trying to separate chiral isomers of a compound after derivatization to their respective diastereomers. I am trying to find a temperature gradient on the GC that could separate my compounds.

Does anyone know of any literature for a methodical approach to achieve my separation?

Any help would be greatly appreciated!

Have you tried running one of the generic methods on the GC you have access to?

From what I've experienced, there is enough of a boiling point variance in diastereomers that nothing truly special needed to be done. For my thesis, I worked on organic synthesis. 3 of my intermediate steps generated chiral molecules. With one step's products being inseparable, at the third step I had 4 diastereomers. The regular temperature ramp used at the place I worked on a standard fused silica capillary column. I should mention that my molecules were strained ring systems, though, so your mileage may vary. Couldn't hurt to run it, though, right?

It's separating enanteomers that'll require you to have a column with some sort of chiral phase coating.

For developing the GC conditions, you need to consider the stationary phase, inlet temperature and the oven temperature profile.

Select a column appropriate to the type of compounds you are separating. You do not tell what kinds of molecules you have, so I'll give a couple of ideas for general guidance. Common stationary phases separate by boiling point, polarity, or interactions with pi orbitals. Classically these are methyl silicon, polyethylene glycol, and phenyl phases. look at your molecules and determine if there are differences in the molecules in which you can take advantage of these separation mechanisms. Go to the catalogs for the various column manufacturers and look to see if they have any separations demonstrated that involve similar molecules.

For the highest resolving power, your column should be small diameter, thin film and moderately long. Long is much over rated. The other side of this is the narrower the column and the thinner the film, the less material you can put on the column. I have come to like doing chromatography on a 10 m x 0.2 mm ID column with a 0.1 micron film. Going to 20 m may help – but doubling the length of the column does not quite double the resolving power. And if you get the column too long, you do not gain anything other than pressure problems trying to drive the thing. And you will need to inject from a sufficiently dilute solution and/or split the injection.

Sometimes you have a shared instrument and have to use the column that is there. I will confess that my favorite column for method development is usually whatever is in the instrument. If it works, I'm off to getting samples run.

Assuming a capillary column, set the linear velocity to 23 to 60 cm/second. On a really tough separation, sometimes you need to be very close to the theoretical optimum velocity. But if you are lucky, you can run the column a bit faster and get compounds off faster.

Normally samples are in a solvent. This should have a lower boiling point than the analytes of interest. Start your oven temperature below the boiling point of the solvent to allow the solvent to partition into the column. Your ramp rate will depend on the column dimensions and the characteristics of the compounds to be separated. With capillary columns, I will typically try 3 to 10 degrees per minute and see how things go.

Once you locate the peaks for your compounds, you can adjust conditions to improve the separation. A slower ramp rate or even an isothermal portion of the profile can help. Remember that it takes a time for a change in temperature ramp rate to affect the chromatography, so make your change in rate at a point where the temperature is about five to ten degrees cooler than where you expect to see the compounds elute. And then make further adjustments.

If there is a significant difference between the boiling point of the solvent and the temperature at which your compounds elute, you can either continue to inject with the oven at low temperature and then ramp the oven up quickly to a higher temperature or you can inject with the oven at the higher temperature. I tend to prefer the first technique to maintain any solvent focusing that I can get out of the injection. But some mixtures do just as well, if not better being injected with the oven warmer.

And, I mentioned inlet temperature - run it warm enough to volatalize the compoounds you need to see in the analysis, and not much higher. You avoid thermal degredation of your molecules. My habit is to use liners with glass wool (deactivated) to catch non-volitile stuff and septum crumbs. I change liners fairly often and keep from getting high molecular weight materials off the head of the column.

I hope this gives you some useful ideas. Post your results -- and with a bit more detail about what you are doing and how it is going, perhaps someone can give you more focused advice.