Polar analyte analysis

[bisnettrj2]

I have an analyte that I have to analyze once in a blue moon, but I am attempting to optimize the analysis while I have a little down time. The analyte is nitroguanidine, an explosive propellant. I have tried cyano, C18, and PFP columns using 100% water mobile phase, but no phase will retain the analyte. For a 250x4.6 mm 5 micron PFP column, with a 1.0 mL/min flow rate of 100$ water, my RT is 5.2 minutes. I would really like to be able to retain this analyte for at least 10 minutes, in order to ensure I am correctly identifying this analyte in an unknown sample.

I thought, because this analyte is very hydrophilic, that HILIC might be useful for analysis, but I've never run anything with this method of analysis. If anyone can give me some good ideas on how to go about retaining this analyte and analyzing it via a UV detector, I would appreciate it.

[Uwe Neue]

I would say you nearly have no chance with a RP column, although I managed to get even sugars to retain on a suitable RP with a high salt concentration.

My opinion: a silica HILIC column (like Atlantis HILIC), probably with around 70% acetonitrile, 30% water. You should see the compound with little problem in low UV, so I would start with a gradient from 80% acetonitrile to 50% acetonitrile.

[bisnettrj2]

Thank you Dr. Neue. I normally use 270 nm (analyte is exceptionally responsive at 265-270 nm), and I have a Phenomenex Kinetex (their new 'partially porous') HILIC column on-order (it was too good a deal to pass up, even if it is relatively untested). Should I try to use a buffer:acetonitrile, or should I try to keep it simple and use just water:acetonitrile in order to try this analyte via HILIC?

Also, are there any particular ways to condition/prep a HILIC column for analysis?

[lmh]

use the buffer. Phenomenex have some references for their older HILIC chemistries that indicate that the buffer is absolutely vital to a decent reliable separation. Unless their new columns are radically different, the same rules are likely to apply.

If you haven't already asked them, they probably also have a recommended "first attempt" gradient for their column. It might be worth trying it.

[bisnettrj2]

I searched their site, and found a pdf titled "HILIC Method Development Guidelines" http://www.phenomenex.com/lib/gu71811109_l.pdf . Looks like a good place to start. Thanks for the tips.

[Uwe Neue]

I don't know about the Phenomenex column, but I do not see a reason for using a buffer on a decent silica column in HILIC for this analyte.

[bisnettrj2]

Well, when I get the column in-house, I'll start with just water:acetonitrile, and go from there. That would simplify the analysis, and since I only run this analysis every so often, ease-of-use is of high priority.

[bisnettrj2]

As luck would have it, I received the HILIC column today. I'm going to run 90:10 ACN:H2O at 1 mL/min for 2.5 min, then ramp to 50:50 over 7.5 minutes, and hold for 2.5 minutes, then re-equilibrate. The column is 50x4.6mm 2.7 um Kinetex column by Phenomenex.

Question - my injection solvent is 100% water - is that ok for HILIC, or am I going to have issues? I'm going to run 2 injections of my 0.2 ppm standard, one in 10:90 H2O:ACN, and the other in 100% water to check.

[Bryan Evans]

Your solute is ionizable. I would use pH modifier in sample diluent and mobile phase.

[bisnettrj2]

Bryan: I do understand that nitroguanidine is ionizable, but if I achieve decent retention without the use of a buffer, I'll be happy. I literally run this analysis two or three times a year for about 20 samples, so I'd rather make this as easy on myself as possible. I am, however, mentally prepared for the possibility that I may need to prep a buffer to use in conjunction with this analysis, in order to appease the chromatography gods.

[Uwe Neue]

It is good that you test the injection solvent. In HILIC, things are exactly the opposite as in RP, and an injection in 100% water can be problematic. If you see fronting, this is the problem.

I am puzzled... Can you help me understand the ionization pattern of nitroguanidine as a function of pH?

[bisnettrj2]

Dr. Neue, if I could answer your question, I would. However, I do like being agreeable. And, if I ever can help you understand something, then there has been a significant glitch in the Matrix. Or you've taken up water polo, in which case I would be Yoda to your Luke.

I know this much - my first attempt to run nitroguanidine (hereafter abbreviated NQ, per convention) at 0.2 ppm in 90:10 ACN:H2O (initial MP conditions) was unsuccessful. I saw no peak, but I did have a larger 'solvent front' (?) peak than in my blank at about 0.65 minutes (which, for a 4.6x50mm at 1 mL/min seems to me to be before the void volume of the column), and I saw no peaks at all running at the gradient I specified earlier. I had to shelve the analysis after this run, as time constraints presented themselves (there is always tomorrow, though ).

I did read somewhere that NQ has a pkA of 2.53, and from your HPLC Troubleshooting Guide, operating near the pkA of the analyte in question can be problematic. Since this is a diol-based HILIC column (rather than bare silica), and I have had no luck (in my one injection so far) running neutral HILIC on this analyte, should I try to run this analyte using a buffered mobile phase? If so, what pH/buffer should I try?

Also, I had to explain to my boss that it wasn't the size of the column in the separation, but the retention factor. I don't think she bought my explanation for why the column was so small....

[XL]

You will need buffer to separate ionized analytes.
The analyte needs to be dissolved in a diluent containing as much acetonitrile as possible.
The smaller the column volume, the more the retention is affected by the injection volume (especially when the diluent contains lower organic solvent than that in the mobile phase). Thus inject less and try different injection volumes to determine the optimal injection volume for your case.
Please keep us informed on the progress. In case the HILIC column doesn't give you satisfactory result. please let me know becuase I think one of our mixed-mode columns may be a good solution.
I was wondering how much the 4.6x50-mm 2.6-um Kinetex costs. Thanks!

[HW Mueller]

Since I had trouble guessing ionization characteristics of this molecule I tried google. Here is a relevant article:
http://pubs.acs.org/doi/abs/10.1021/ja01145a526

This would indicate that at room temp. one can not do much with pH variation, as Uwe seemed to indicate.

[Bryan Evans]

The resonance structure of the nitro functional group interacting with the
polar stationary phase is why I recommended the pH modifier to the mobile phase.
I thought it would help to sharpen the peak - if I'm wrong, I can admit it.

Unison UK-Amino may be another option if the diol doesn't retain this solute (and I would try 10-50mM ammonium formate).