Chromatography Forum

Hi,

Just wonder if this is an approach that is known to work...?

(using two pumps and a switch valve)

- Inject sample in a highly aqueous mobile phase on a C18 trap column
- Switch the valve and elute on a HILIC column with very high% acetonitrile

I assume that the trap column will contain some water that can destroy the HILIC separation? Or is it negligible amounts?
Thanks!

Wellcome to 2D HPLC.

Gerhard Kratz wrote:
Wellcome to 2D HPLC.

Not sure if this would qualify as 2D-HPLC... It is closer to on-line SPE

If it works it would certainly remove the problem of injecting aqueous samples in HILIC.

I guess the only way to find if the water in the trap column (left from the first "dimension") will disturb the HILIC separation is to try it.

"Why bother with theory when you can do the experiment"
(Some famous guy said this, cannot remember who)

It's indeed more of an online cleanup/trapping than 2D LC because you don't plan to separate anything on the first column, just get rid of stuff you don't need.

I haven't used this myself but I've read about it and it should work given there is time for development. Howerver, an important downside in my opinion is that it can lead to band broadening, and that it obviously increases the run time significantly.

Interestingly, you could do the opposite: use HILIC to trap and RPLC to separate. In this way you can inject samples in pure organic solvent.

Some article links:

http://onlinelibrary.wiley.com/doi/10.1 ... 4/abstract

http://www.sciencedirect.com/science/ar ... 7317302431

Rndirk wrote:
It's indeed more of an online cleanup/trapping than 2D LC because you don't plan to separate anything on the first column, just get rid of stuff you don't need.

I haven't used this myself but I've read about it and it should work given there is time for development. Howerver, an important downside in my opinion is that it can lead to band broadening, and that it obviously increases the run time significantly.

Interestingly, you could do the opposite: use HILIC to trap and RPLC to separate. In this way you can inject samples in pure organic solvent.

Some article links:

http://onlinelibrary.wiley.com/doi/10.1 ... 4/abstract

http://www.sciencedirect.com/science/ar ... 7317302431

Thanks!

This is also a very interesting approach! I have some very hydrophobic excipients that I want to get rid of, and this is actually an even better approach!

Are you talking about (phospho)lipids by any chance?

For this to work, the HILIC column has to trap your analytes of course.

Yes, they are lipids!

My analytes are peptides which are fairly hydrophilic. Could work!

Is a simple hexane extraction a possibility? Not as fancy as on-line cleanup, but should do the job.

Alternatively, you could use a (d)SPE cleanup.

Separating hydrophilic from hydrophobic compounds during sample prep is not rocket science

It took six months but now I have tried the approach of using a C18 trap column and an HILIC analytical column, and it works really well!

Now I can analyse all samples on HILIC, regardless of what they are dissolved in.