Chromatography Forum

Hello

I am currently working with a HILIC column and have to say that I am pretty unsatisfied with the results. I am doing UPLC-MS metobolomic study. My aim is to see polar compounds in a leaf extract - we already try ACN/H2O, ACN/MeOH and ACN/(MeOH/H2O) as A1 and B1 solvent the HILIC column

any help or tricks are really welcome

kinds regards

Alain

Hi Alain,

Not sure which HILIC column you used and how you set the conditions. There are many types of HILIC columns. Our website has 3 HILIC columns from Venusil, PolyLC, and SIELC. They give very different results. Company SieLC also provide free method development if you send them the sample. You may find more information from www.gc-lc.com or www.sielc.com .

Best regards,

PromoChrom

It is not clear, what your dissatisfaction is. We commonly start (for a silica HILIC column) with 95% acetonitrile, 5% water, and run gradients to about 50% to see where the compounds are eluting. Then the method can be finetuned using mobile phase additives such as formic acid or ammonium formate. Methanol is a much stronger solvent than acetonitrile, and not that good a choice, unless you can get selectivities that are not possible otherwise.

May I ask you which company you purchased the HILIC column from? I also had a HILIC column, but we were not able even to repeat the test chromatography from the manufacturer.

thanks for these prompt replies guys!!

I am currently using an atlantis column form waters - made for th eUPLC system!

why i am not happy: 'coz the polar compounds are coming out far too fast and the detection with my MS is really poor in quality

A.

What is the max concentration of organic solvent (ACN) that you start your gradient?

Also, if you do not use any acidic modifiers, it is normal that you have sensitivity problems with your MS. As you can see from Petritis et al. J. Chromatogr. A 913, 2001, 331-340, you can have as much as 100 times less signal if you have high concentrations of organic solvents without any acid in them (of course it depends on your compounds and other conditions).

Are you using a mobile phase as suggested? Starting with 95% MeCN/5% aqueous?
MS sensitivity is commonly better with HILIC mobile phases. What is your mobile phase, and what is the nature of the analytes?

i have spiked A1 and B1 with 0.2% acetic acid - the analytes i am looking for are the one coming from green tomatoes leafs

is AA a bad mof^difier?! is it better to use other additives like ammonium formate or ammonium acetate?

i start with 95/5 ACN/H2O (both are spiekd with 0.2%AA)

is there any issue using acetic acid to as a modifier or is it much better to use formic acid

Acetic acid is fine. If you don't get good retention, I suggest to work at a roughly 3-fold lower concentration. Whether formic aicd or acetic acid work better, is mostly a question of the analytes and of selectivity. Either one can be used.

The same is true for the ammonium salts compared to the acids themselves. I would use a roughly 10 mM concentration.

For detection, I am puzzled that you get low MS sensitivity. Most people have reported higher sensitivity in HILIC mode, and we have seen the same. If your analytes are acidic, than adding acid is of cause not helpful, and a salt may be better.