HILIC Gradient troubles

[agrubert]

Hello All,

I am working on developing a HILIC method for detection of organic acids (oxalic acid, etc.) and am having trouble setting up a gradient that, when run with no injection doesn't look like a pyramid set up in the middle of the run. I've tried several different timelines, %A vs. B mobile phase transitions, and curve adjustments, but nothing is working. I'm not sure if my mobile phase is majorly contaminated or if it's my gradient but I would like to get it figured out asap. My mobile phases are 95/5 ACN/Water with 10mm Amm. Acetate pH 9 and 50/50 ACN/Water with 10mm Amm. Acetate pH 9, running at 1ml/min, with an Amide column at 35 deg. C, 210nm detection.

Any advice is appreciated!

Thanks.

[Andy Alpert]

210 nm is appropriate for detection of the acids; their carboxyl- groups absorb there. So do the carboxyl- groups of the acetate in your mobile phase. 10 mM acetate will give you a baseline about 0.3 AU high at 210 nm. It doesn't take much of a disturbance to a baseline that high to give you an interesting profile, as you've seen. Now, people usually use acetate when they want a mobile phase that's volatile. In your case, though, you're monitoring absorbance. Why not use a buffering salt that's transparent at 210 nm? A lot of buffers will meet that criterion. Also, why do you need to buffer at so high a pH? Acetate ion has no buffering capacity above pH 5.7 anyway. If the objective is to make sure that the carboxyl- groups of your acidic analytes are completely ionized, then any pH above 6 should suffice, which opens the door for a number of buffers (example: triethylammonium phosphate).

[zokitano]

By a coincidence i have tried several dicarboxylic acids including oxalic on XBridge amide column under HILIC conditions. Have tried different buffers to optimize their separation in LC-DAD. As Andy said, in order to avoid background absorption at 210nm it is adviceable to use more "transparent" buffer. I have tried ammonium hydrogencarbonate to make a buffer at pH 9, and the background absorbance is very much diminished compared to ammonium acetate/formate. For some dicarboxylic acids i observed higher retention at pH9 than at pH 5-6.8.
Particulary for oxalic acid, its peak shape became more symmetrical only with higher end concentrations of the buffer salt (> 40 mM) in the mobile phase. With lower salt (buffer) concentrations i could not get symmetrical peak, but ugly "forever" tailing peak.
Also used an ammonia solution with acetonitrile, but the peak shapes were awful and the retention unsufficient. Bufer is always the right choice for HILIC separations.

These acids are also well detected by MS (if you have access) and ammonium hydrogencarbonate is MS friendly salt

Best regrads

[agrubert]

Thanks to both Andy & Zokitano! I will get going with a new buffer shortly and see what happens.

[carls]

Carefully check inorganic buffer solubility in your initial mobile phase to avoid preciptitaion. The solubility of inorganic buffer salts is GREATLY reduced in the high concentrations of ACN commonly used in HILIC. As suggested, ammonmium bicarbonate is a good choice for high pH. Unfortunately I do not know the solubility limit at different %ACN for this buffer. A good reference for the solubility limit of phopshate in ACN, MeOH and THF at different pH's is given here:
http://chromatographyonline.findanalyti ... rticle.pdf

[zokitano]

Carefully check inorganic buffer solubility in your initial mobile phase to avoid preciptitaion. The solubility of inorganic buffer salts is GREATLY reduced in the high concentrations of ACN commonly used in HILIC. As suggested, ammonmium bicarbonate is a good choice for high pH. Unfortunately I do not know the solubility limit at different %ACN for this buffer. A good reference for the solubility limit of phopshate in ACN, MeOH and THF at different pH's is given here:
http://chromatographyonline.findanalyti ... rticle.pdf

Agree with carls.
I also tested the solubility of ammonium hydrogencarbonate in the initial ACN/water mixture i needed for my method. In 95% ACN and 5% H2O mixture you cannot have 10mM of the amm. hydrogencarbonate as end (bulk) concentration, due to precipitation of the salt (at room temperature). But, 10mM final salt concentration in this ACN/water mixture could be maintained without risk of precipitation when ammonium formate or ammonium acetate is used instead.

As a source for ammonium hydrogencarbonate i used the highest purity available for chromatography (from Sigma, i think it was LC-MS grade, but i am not sure).

Best regards

[Andy Alpert]

Instead of using ammonium salts, try the corresponding triethylammonium salts. They are more soluble in predominantly organic solvents.

[agrubert]

Well, I tried Triethyammonium Bicarbonate at 10mM concentration in ACN/H20 and now I've got a beautiful baseline at 210nm.... but that's it. No matter what I inject (mobile phase, ACN, standard), at 210 I see nothing. At 220, nothing.. at 240 I at least see my void volume dip/peak. So now my problem is the opposite of what it was before. Any thoughts on what may have gone wrong?

Any advice would be greatly appreciated!