Chromatography Forum

Hi!

Well, I tried different HILIC columns (Agilent Carbohydrate / Luna NH2 with ACN 80% isocratic flow) to separate and detect hexoses by MS (ESI pos mode) without good results, sugars are not detectable, however I can detect different sugars with infusion injections or using C18 columns but without reaching a good separation (Detection: 203,05 m/z ion, the hexose+H+Na adduct).

Sugars seems to still retained in HILIC columns after 30 min running (0,5 ml/min).

Could some body help me with suggestions about what is happening or proposing additional protocols to test?

I´m feel so frustrated with my procedures.

Thanks in advance.
Regards.

One possibility is that it may require more than 20% water to elute your compounds from those columns.

If this were my problem I would get hold of an RI detector (which is universal) and establish the separation conditions before moving on to the MS.

retention on the Luna NH2 is also very dependent on pH. I'd recommend you use a buffer rather than straight water. Phenomenex will happily advise on suitable buffers and gradients for their column and will be more up-to-date on the best method than I am.
Do remember that sugars don't ionise particularly well in electrospray compared to most other compounds, so you may need to be injecting 10-100 times more on a molar basis than you would of a "typical" organic molecule.

Using a ZIC-HILIC, you need to add some buffer. I used the protocol adapted from DOI: 10.1002/rcm.3519

solvent A: CH3CN with 0.1% formic acid
solvent B: 20 mM ammonium acetate pH4

i start at around 10% solvent B but i guess you can tweak the ratios to achieve the retention you prefer. Without buffers (only acid) the carbohydrates were not eluted from the column.

There's an App for that

http://www.phenomenex.com/Application/D ... ion/Search

http://www.phenomenex.com/Application/D ... ion/Search

This is a classic separation for an amino column where only ACN/water are used for the mobile phase. For MS detection addition of some salt (0.1 mM ammonium acetate, no pH adjustment) may help ensure you see an adduct (NH4+).

just for your inforamtion

1. 20%Water (80%ACN) is not enough to elute the sugar from the HILIC column. please increase the percent% of water, you can make a gradient elution with 20%water to 90%water, I think you can find the sugar peak.
2. add some buffer to your mobile phase maybe help,

Carl's suggestion of adding ammonium source to eluant may help with ESI detection. The other thing I would try is APCI as a detection mode instead, although when doing that beware that you may have some water losses.

Randy