HILIC (Carbohydrate NH2 column) Injection solvent effect

[Karen01]

While I have a LOT of years of HPLC experience, I am new to HILIC as well as Agilent HPLCs.

For the previous 15 years I had been mostly dealing with very hydrophobic compounds (saturation at 3-50ug/mL in water) doing RP work on Water's equipment. There I had a good feel for how much pure organic sample solvent I could inject without hurting the chromatography.

Now I'm developing a crabohydrate analysis method on an Agilent 4.6X150mm 5um carbohydrate column using an Agilent 1260 Infinity HPLC system with an ACN/Water 80:20 mobile phase.

These samples are all in aqueous media. Can anybody give me a ballpark idea of the largest injection volume I can safely use without the water messing up the chromatography?

I can always dilute the samples to say 50% ACN if I have to, but if I can get good enough sensitivity with a smaller injection volume without having to dilute with ACN, life will be much easier.

Thanks,
- Karen

[tom jupille]

The general rule of thumb when the mobile phase is used as the diluent is to limit the injection volume to less than 15% of the baseline volume (baseline width x flow rate) of the narrowest peak of interest. If the diluent is stronger than the mobile phase (for HILIC, more water), then you have to decrease; if the diluent is weaker you can increase, but there is no good rule of thumb (that I know of, anyway) for how much but it's easy enough to do a series of quick experiments to see what you can get away with.

[Karen01]

The general rule of thumb when the mobile phase is used as the diluent is to limit the injection volume to less than 15% of the baseline volume (baseline width x flow rate) of the narrowest peak of interest.

And I usually inject much less than that anyway because i don't need more sensitivity (at least with HPLC - in UPLC you have to be more careful). In any case I prefer injecting in a weaker solvent than the mobile phase, but I can't here.

If the diluent is stronger than the mobile phase (for HILIC, more water), then you have to decrease;

And that depends on the plumbing before the column (hence why I mentioned the specific HPLC) as well as the column and mobile phase.
For RP on an Alliance with a 4.6X250mm C18 I know I can almost always get away with between a 5-10 uL injection of MeOH... For this Carbohydrate column I had no clue how much water I could get away with.

if the diluent is weaker you can increase, but there is no good rule of thumb (that I know of, anyway)

Now THAT, at least on RP, I have lot of experience with because i had to deal with very hydrophobic compounds in aqueous solutions and needed sensitivity!

for how much but it's easy enough to do a series of quick experiments to see what you can get away with.

I know...

When i was an analytical R&D manger at my last job I used to have my direct reports do injection volume studies routinely as part of method development...

Because I have no HILIC experience I was trying to narrow the range to look at so minimize the # of injections to save time as in this job i have to do everything myself and not just method development! (as well as learn the Agilent hardware and software)

Thanks,
- Karen

[lmh]

In case it's useful to you: I do small oligosaccharides from time to time on a 100*2mm Luna NH2 column in Hilic mode, and get away with injecting 10uL of 50% ACN. I haven't tested exhaustively how much further I could go, but I believe 10uL of 20% MeOH, 80% water was disastrous (but this was during a setting-up phase when I was, stupidly, varying too many things at once, and I have no great drive to go back and make a method that's working stop working!)

[Karen01]

In case it's useful to you: I do small oligosaccharides from time to time on a 100*2mm Luna NH2 column in Hilic mode, and get away with injecting 10uL of 50% ACN.

I haven't tested exhaustively how much further I could go, but I believe 10uL of 20% MeOH, 80% water was disastrous

Thanks... That give me a ballpark idea. I did no get to the injection volume study today because other things came up...

Tomorrow I'll try 100% water in the range of 3-10 uL and 50% ACN in the 5- 20 uL range. I REALLY wish I had a more sensitive detector...

BTW does anybody make a good Carbohydrate column with packing the 2-3 particle size range? I also need speed and my boss keeps coming up with more carbohydrates he wants me to measure.

I can't use the Water's UPLC Amide column because the HPLC I have only can handle up to 8700 psi and i was told by Waters that column, at reasonable flow rates, runs at higher pressures.

[Bintang]

If you can stack eluents you could stack your sample between plugs of pure acetonitrile to increase the injectable volume.
If you are looking for sensitivity I think it is better to inject larger volumes in 50/50 than going with 100% water.

[Karen01]

It turns out I can inject almost 10 uL of my analytes in water without a negative effect on the chromatography (start seeing a minor issue at 10 uL)... 8 uL should give me the sensitivity I need and be safe.

[tom jupille]

Thanks for the update (it's always frustrating when we *don't* find out what worked!).