Chromatography Forum

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

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.

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

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!)

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.

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.

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.

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