Polar analyte analysis

[bisnettrj2]

Vlad:

I'll talk to my manager about your proposal, but like I've stated before, this is not an important analysis to my lab, so my time is not well spent in developing novel solutions to this issue (as much as it pains me to state the situation, it is what it is). I really hope to use my columns at hand (this diol HILIC phase from Phenomenex, and my PFP(2) column from Phenomenex) to realize a good separation with this analyte. I do hope to use mbicking's tips for using my current PFP phase for analysis of this molecule, if acidifying my mobile phase and re-running on the diol HILIC phenomenex phase does not work.

[XL]

Since the column you have is quite small, I suggest 1 to 2 uL of injection volume instead of 10-uL. The concetration of the standard can be higher to compensate the decrease in injection volume.

I think you might want to work under isocratic conditions first such as 50%, 70% and 90% acetonitrile levels. It is more straightforward and in fact faster to get the result.

First inject the standard with no column connected and measure the peak area of the analyte. This way you will have a reference point when you looking for the peak of interest under different conditions. If you are using a PDA, use it and the life would be much easier.

Although Nitroguanidine is assumed to be neutral with varuous reasons, I still think it is better to use 10 mM ammonium acetate in the aqueous portion. The Diol column has quite a number of silanol groups. In case the nitroguanidine is charged somehow, non-buffer mobile phase will give poor peak shape.

[bisnettrj2]

XL - First, stop referencing my 'small' column. It's not the size of the column in the separation, it's the selectivity and mobile phase parameters... I'll give your suggestion a shot tomorrow, if I get a chance. I'll have to see if anyone in the lab has ammonium acetate. I do have sodium acetate - does anyone know if that will work in the short term?

[XL]

bisnettrj2,

you are right that selectivity and mobile phase condition are the keys. From my experience with the HILIC application, the peak shape and retention are often affected by the injection volume if the diluent is not the same as the mobile phase. Larger column volume is more tolerable to large injection volume. I learned this hard way. In your case, the sample diluent is the same as the initial mobile phase condition, thus should be ok. It safe to check it.
Sodium acetate and ammonium acetate work the same for UV detection. Ammonium acetate is more soluble in high organic solvent. Anywhere between pH4.2 and 5.2 is good.

[carls]

Sodium acetate is not soluble in 90% ACN. For solubility you’ll need to use an ammonium salt of formate or acetate.

Phosphoric acid is soluble in pure ACN and this may help. If the pKa of your analyte is ~2 then it will be lower (pKa~1) in 90% ACN. The pH of 0.1% phosphoric acid in 90% ACN will likely be >3 so the analyte will still be uncharged. If you use phosphoric be sure to flush the column with 20 CV of 90/10 ACN/H2O before using any buffers with it. The phosphate will precipitate in the presence of other cations and damage the column.

If the acid doesn’t work then try a salt such as ammonium acetate or formate. The concentration should be 10mM in the mobile phase (not in the aqueous only) and no pH adjustment is needed. Simply dissolve the salt in 90/10 ACN/H2O.

The injection solvent ideally should match the mobile phase in terms of ionic strength and organic composition. As others have stated, HILIC s very sensitive to the injection solvent and 100% H2O should be avoided. This is akin to injecting 100% ACN into an RP column.

[bisnettrj2]

Thanks carl. I'm bogged down with work today, but I'll probably get to work on this issue tomorrow. I think I'm just going to go with Uwe's idea of phosphoric acid in the mobile phase to try to get this analyte to retain on-column. I'll make sure I'm using the initial MP as my sample diluent as well, and I'll report my results when I get a chance.

[bisnettrj2]

Well, my second attempt at retaining nitroguanidine has failed. Acidifying the mobile phase did not affect retention. Right before I left tonight, I put 10 mM ammonium formate in 90:10 ACN:H2O and 10 mM ammonium formate in 50:50 ACN:H2O on the instrument, and re-ran the method I ran today with 0.1% phosphoric in the mobile phases. I re-made the standard in 90:10 ACN:H2O 10 mM ammonium formate. I'll report results again in the morning.

[Andy Alpert]

Seconding Carls advice: Unbuffered acids are chaotropes. Chaotropes promote binding in RPC but have the opposite effect in HILIC. Use a buffered salt instead. If you want one that's transparent at 215 nm, use triethylamine phosphate (try pH 3). If you want one that's volatile, try ammonium formate (again, pH 3). The sample and the starting mobile phase should contain 15-20 mM of the salt in question. The sample should also contain as much ACN as the starting mobile phase. If your sample is still not retained when starting with 90% ACN, then use a column with a thick, well-hydrated coating such as PolyLC's PolyHYDROXYETHYL A or Tosoh's TSK Amide-80.

[Vlad Orlovsky]

When we were developing method for cyanoguanidine we tried HILIC on several columns (SeQuant, bare silica, Obelisc N) at differnet pH, we had no luck retaining cyanoguanidine at any pH. Only switching to normal phase (heptane/etahnol) produce retention with K'>4.

[bisnettrj2]

Running 10mM ammonium formate last night didn't help. I prepped a 50mM ammonium formate in water solution this AM and pumped a couple hundred mL's to make sure I had sufficiently loaded the column with ammonium formate. I then switched to 10mM ammonium formate in 50:50 H2O:ACN, pumped 50 mLs, then switched to 10mM ammonium formate in 90:10 ACN:H2O and pumped 50 mLs again. I then restarted the sequence I had yesterday, and also programmed in a method where I would run exactly the same parameters, except a 1 uL injection instead of 10 uL.

Since I think I'm exhausting my options with this column, I might try mbicking's recommendations on using a PFP column.

[Vlad Orlovsky]

PFP Approach: You will need to go to very low organic (small amount of MeOH, THF or 100%aqeous). Another approach might be phenyl column in pure water or in the absence of ACN (THF or MeOH) in order to explore weak pi-pi interaction. You will need to find phenyl column which is stable in 100% water and is not collapsing. Adding 2% THF can address this issue.

HILIC/Ion-exchange: According to literature, nitroguanidine pKa is 3.5, so with ammonium formate or acetate at pH 5-7 you can assume that nitroguanidine is ionized and polar, so if there is no retention at 90% ACN, then HILIC is not going to work, and you need to make step towards normal phase.

[bisnettrj2]

So, I gather per Vlad's suggestion, I could use my diol HILIC column in NP mode... however, are NP solvents compatible with a diol HILIC column? And what are the guidelines in normal-phase chromatography? Miscible yet different-polarity solvents? Like MeCl2 (dichloromethane, MC) and IPA?

Also, I already attempted using a PFP column in 100% H2O, had a k-prime under 0.5. So much for that idea. Unless I can use the PFP with NP solvents...

The only problem is that Agilent recommends different seals in their pumps for NP mobile phases. Also, my current extraction method is a DI extraction of soil on a shaker table or simply a DAI injection for waters, so switching to NP (I assume) would require some major changes to injection solvent? NQ is thermally stabile, so I may be able to evaporate the water and transfer into MC or IPA, but this will be adding a lot of work to an analysis I do about three times a year....

[HW Mueller]

Vlad, do you have a reference for this pKa = 3.5?

[carls]

Nitroguanidine appears to be one of the very few compounds that seemingly do not behave in accordance with their (published) physical properties. This is rare. If you have a strong cation exchange silica-based column you try that in HILIC mode.

The diol column is compatible with normal phase but be sure to rinse all buffer (25 CV H2O) from the column before transitioning thru IPA.

[bisnettrj2]

I am hesitant to try Normal Phase due to the recommendation by Agilent that the pumps have different seals installed when using normal phase solvents. I do have access to a Restek Ultra Cyano column - is there any chance that I could get HILIC to work on cyano versus the diol HILIC phase?