water and ACN can make higher system pressure?

[carls]

This particular thread was started by a user asking for assistance.

Anyone that would like to discuss tangentially related matters is encouraged to start another thread.

[carls]

is this HILIC?

Yes, this is HILIC.

[carls]

Any further discussion on the pro's/con's and good/bad of amino columns can be discussed in depth here:
viewtopic.php?t=13766

[Uwe Neue]

Interesting bonding strategy, but probably not entirely helpful. The issue is that a new amino column will automatically create a high pH (around 9+) when it is first exposed to the mobile phases used for sugar separations (acetonitrile/water 70/30 or similar). An at least mildly alkaline pH is needed for sugar separations, unless you want to see anomer peaks.
It is easy to test: take a new amino column, convert it to the mobile phase needed for sugar separations, and take a strip of pH paper to check the pH of the effluent as it comes out of the column. You can also watch the pH as the column equilibrates. If I am correct, the pH will be alkaline to start with.

[lmh]

sorry to have re-asked something that ought to have been obvious.

I use Luna Amino frequently for HILIC and have not encountered any problem in bleeding (using electrospray MS and UV detection). Nor have I encountered rising pressure. My buffer is generally mildly alkaline ammonium formate.

The real problem in HILIC is keeping the polar analytes and buffers in solution at the high concentrations of ACN needed at the start of the run. Carls is absolutely right: the tricky point is working out how much water you can get away with before the retention times decrease, and how much analyte/buffer you can get away with before things precipitate and the pressure goes up.

Incidentally, I am not affiliated to any manufacturer and have no axe to grind!

[carls]

sorry to have re-asked something that ought to have been obvious.

I also like to get a second opinion and this is what this forum is for.

Good luck!

Carl

[flora0975]

I missed initially flush column with IPA.

Standard amino columns bleed like crazy during the initial runs, and this can be the cause of clogged outlet frits.

Dr. Neue, that's an interesting point to consider. Luna Amino is not standard amino propyl chemistry and does not share their bleed charateristics.

That did remind me of another potential issue.

Flora,

Have you flushed this column with IPA? New amino columns are shipped with normal phase solvent (hexane/IPA) which must be purged with 100% IPA (10 column volumes,CV) prior to use in HILIC mode. Please perform this purge/flush prior to doing any further work. Be sure to reduce the flow rate so as to not over pressuize the column. Inadequate purging will cause a host of different problems including unpredictable and poor chromatographic performance as well as very slow equilibration. After purging with IPA equilibrate the column with 20 CV of your initial mobile phase.

[flora0975]

Now the pressure can be controled. I just inject water perior samples. so far the pressure is very consistent.

sometimes a shoulder peak can be observed, after I flush with water for 1hr, the column performance is getting better.

It's easy to create durability data for NH2 phase - just pump water through through the column.

Imtakt compared durability of Unison UK-Amino vs. standard NH2 phases:
http://www.imtaktusa.com/site_media/fil ... TI304E.pdf

[carls]

Hi Flora,

How is your analysis working out?

[flora0975]

Carls:

By your inspiration, I tried 5 injection of water, 5 injection of saline buffer (which I used for dilution) and then 5 injection of real sample. It turned out the pressure can be controled better for injection of water. But if I put 5 injection of water after samples, the pressure increased.

I think inject water first might work to wet in-line filter to avoid salt precipitation (since the previous run ends with 95% of ACN which easy to be dried)

I still have some puzzles, it is fun to have challenge. Thanks for all of your suggestions!

Hi Flora,

How is your analysis working out?

[carls]

Great to hear you're making progress.

I'm sure you'll let us know if there are any problems or revelations.