MS problems to detect sugars trough HILIC columns

[patomb]

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.

[tom jupille]

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.

[lmh]

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.

[carlo.annaratone]

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.

[carls]

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+).

[gxch43]

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,

[trackster]

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