By Anonymous on Thursday, January 9, 2003 - 12:37 pm:
i have a polyLC PolyHYDROXYETHYL Aspartamide column, i would like to try HILIC mode.
i have a polar small molecule that retains well on this column with 70%ACN/30%water. but there is a little tailing, so i wonder if 0.05%TFA will hurt the column?
all instructions i got for the column is to run it with buffer salts, but my molecule is not electrolyte, nor peptide or protein, so i figure just water/ACN should work, right?
but they never mention if it's ok to run with TFA, just as in RP HPLC.
anybody has the experience?
BTW, the water/ACN solvent pH with 0.05%TFA is about 2.2, so i am not sure if it's ok.
thanks.
By Einar Pontén - SeQuant AB on Thursday, January 9, 2003 - 02:22 pm:
HILIC seems to be a good choice for your application.
It is a silica based column and should be stable at pH 2.2. Andrew Alpert (PolyLC) has used 0.1 %TFA at least in one paper (prion protein). Unfourtunately, the Journal information is not readable in my copy, but it is a paper together with D. Lin and J.R. Yates.
Else, we recommend formic acid or ammonium acetate when buffering is required. A phosphate buffer may also do. However, one should keep in mind that the solubility decreases with a high content of acetonitrile.
One reason for using a buffer in the mobile phase is to reduce the effect of different matrices in the samples (ion strenght, pH).
By Uwe Neue on Thursday, January 9, 2003 - 04:34 pm:
There is nothing in the chemistry of this column that would prevent you from using TFA. At the same time, I do not see hwat the addition of TFA should do for you, since your molecule is not an electrolyte. The PolyLC columns have been designed for large molecules. If your sample is a small molecule, this tailing could be caused by small pores in the matrix. If this is the case, you may be better off with a column designed for HILIC, such as SeQuant's zwitterionic column, or just plain silica.
By Anonymous on Friday, January 10, 2003 - 06:57 am:
to Einar and Uwe:
thank you for the help. i tried plain silica(phenomenex silica) first with normal phase mode, the baseline is too messy to get any result.
it is true that the pore size on HEA is 20nm, but i also try another polar compound which is similar on structure size, but with perfect peak shape. so i wonder what is the factor to affect the tailing here.
anyway, since 0.05%TFA does no harm, i am going to try it to see how it goes.
By Uwe Neue on Friday, January 10, 2003 - 03:20 pm:
I was not talking about the macropores, but the micropores. The Alpert packings are unlike standard HPLC packings. Standard packings contain a monomolecular layer attached to the silica surface. The Alpert packings contain a rather thick layer of polymer attached to the silica surface. This is good for macromolecules, but may show up as tailing for small maolecules that can penetrate this layer.
By Anonymous on Monday, January 13, 2003 - 07:38 am:
well i tried with TFA. the result is the peak shape improved but elutes earlier.
my guess is the ion-pairing makes it less polar?
By Uwe Neue on Monday, January 13, 2003 - 03:09 pm:
You said that your analyte is not ionic. Then the reason for the lower retention of your compound is that you made the mobile phase more polar, which results in less retention in HILIC.
Think of true HILIC as a partitioning between your mobile phase and the polar water-saturated surface! if you make the mobile phase more polar, you get less retention.
By Einar Pontén - SeQuant AB on Monday, January 13, 2003 - 03:33 pm:
I agree.
When using plain silica for HILIC I suggest that you initially use a mobile phase with no or very low ion strength and run a gradient by increasing the content of water/decreasing acetonitrile.
I suspect that plain silica columns intended for HILIC mode separations require some "conditioning" procedure. Often these columns previously have been used in various other applications and that may affect the reproducibility. Dilute sodium hydroxide?
By Uwe Neue on Monday, January 13, 2003 - 03:46 pm:
There is no conditioning required. Silica loves water, and equilibration is as fast as reversed-phase equilibration. Water on silica is only a problem if you want to go back to normal-phase chromatography.
By Einar Pontén - SeQuant AB on Monday, January 13, 2003 - 03:53 pm:
Yes, I was concerned about "fouling" and the variability of silanols due to previous use (acids, alkaline, nucleophiles).
By Uwe Neue on Tuesday, January 14, 2003 - 04:17 pm:
Under conditions of HILIC, the state in which the silanols are in is completely reversible. You need to use the precautions that are commonly used in aqueous mobile phases. If your analyte (or any part of your sample) is ionic, you need to control the pH - just like in RP. There is no magic nor any special difficulty with silica in HILIC.