Acidic analytes in caustic solution

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

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Hello All

I have an interesting task on hand. It will be solvable one way or another - I am not terribly worried. But I am thinking about elegant and simple approaches to sample prep for transfer to a production lab. This procedure should be fool-proof.

We have acidic analytes with very limited water solubility at low pH but excellent solubility at high pH. Samples will come in at pH 10-14, so up to 1M excess NaOH. I need to adjust pH before injection, especially since there will be a high throughput of samples. But, I do not want to lower pH so much that the analytes or ISTD might precipitate.

I like this approach because there is no danger of dropping the pH so much that the analyte will precipitate but at the same time the pH won’t be terribly high anymore.

I am thinking about this in case we use the internal standard approach:

Take 1g of sample solution and note weight.
Add 10 mL DI water
Add 1 mL of saturated sodium bicarbonate solution (1.2M at RT).
Add 2.00 mL of ISTD solution by dispensette (or weigh in)

1 uL will be injected into a column flow of 1 or 1.5 mL/Min of phosphate buffer at pH 2.1.

The mobile phase will be acidic and this is my worry with this approach. Will the bicarbonate in the injected sample cause trouble due to its transformation to H2CO3 / CO2? The pressure in the HPLC should keep it from bubbling out and so should the backpressure regulator at the UV detector.
An alternative approach would be to use a phosphate buffer around neutral pH.

Any thoughts?
I like such questions, making me think how I would solve the problem if it were mine.

But I miss some info here:
Is it a gradient method or could be turned into one (e.g. step gradient)?
How easy/difficult is the separation?
What kind of stationary phase (I guess C18) but what are the column dimension?
What LC system is it used on?

From the description it is likely that your analyte is an acid.
So trap and elute on an (an)ion-exchange column (online SPE? 2D-LC) could be one thing to establish, but may be only worth it, if a lot of samples need to be measured.

On the simple side, your approach may or may not work... maybe give it a try and keep a certain pressure on your detector cell.

The success depends on how fast your mobile phase can buffer the 1 uL (ca. 100 nmol) of bicarbonate injected, before the analyte reaches the column head. If the pH is still to high, you may get doubled peaks or other weird shapes.
So this time it may be favourable to include something like a mixer (e.g. just a piece of wide bore tubing) before the column. But this may/(will) distort the injection "plug" and may not be applicable for isocratic methods.
But if it's a gradient or you may lower the elution strength of your mobile phase and then do a step/steep gradient back for the (isocratic) elution, you may trap the inline neutralized but diluted sample again at the head of the column before the elution process starts and obtain a good peak shape. Of course, this may also affect other peaks present in your sample, so it may change your separation too much.
Somehow in the style of -> Waters "at-column-dilution" technique for semi-prep separations
I'm guessing this is reverse phase? If so, you presumably want to avoid running close to the pKa of the analyte. In any case, for both solubility and for good chromatography you only need to be a unit on the alkaline side of the analyte's pKa, whatever that is. I would be inclined to find a chromatography-compatible buffer that's good around that range, dilute the sample in that buffer one unit alkaline of the pKa, and run them in the same buffer/pH, selecting an alkaline-tolerant column, such as Phenomenex's EVO C18 (if you're using C18) or the equivalent from whatever column manufacturer you prefer to use (I'm sure Waters do alkaline-tolerant reverse phase columns too).
Thank you for the thoughtful answers, Hollow and lmh! I like the questions as well, because they induce us to think things through thoroughly.

I somehow cannot receive notifications on topics. Emails from chromforum never reach me, so sorry for the delay.
Regarding the info you think is missing (I am not sure I agree that it is pertinent here, but that doesn't matter really):
1. It is isocratic. This is strongly preferred.
2. The separation is trivial.
3. column: Reversed phase on an AQ-C18 type column. 150x3mm, 1 mL/min, at the moment just 0.1% H3PO4 with 10% ACN.
4. Matrix: Just water, up to 1 M NaOH, maybe some aliphatic amines, which will not be retained in acidic RPh and won’t absorb.
5. LC System: Dionex 3000, Agilent 1100, Agilent 1050, Thermo Accela. It has to run on a variety of systems in yet unknown labs.
6. I agree that the trap and elute should work. Online SPE or 2D-LC is out of the question due to technological limitations. Why would you recommend this sample prep, rather than just dilute and shoot?
We have a PEEK capillary post UV cell anyway for the pressure. I will give it a shot for sure. I want to keep everything as easy as possible and hard to screw up for future users who will NOT be analytical chemists.
We may go with a phosphate 2 buffer (1 M) as well.
Your column-head thoughts make sense! I also understand why a gradient / step method would be nice. It would be possible to do a 0% water to 10% water step gradient, but then the method run time would be longer than isocratic, especially on old systems like HP 1100… I need a high pH to dissolve the acid analyte but a low pH to get decent retention. Samples are expected to have between 0.5 and 25 wt% of the analyte, so solubility needs to be very high for sample prep.
There are no other peaks of interest, except maybe a very similar ISTD, so that is nice.
I was not familiar with “at-column-dilution”

Regarding lmh’s thoughts on mobile phase pH: I would prefer RPh, but you made me consider doing high pH isocratic HILIC. High pH reverse phase is not suitable. I like the YMC columns for such applications.
5. LC System: Dionex 3000, Agilent 1100, Agilent 1050, Thermo Accela. It has to run on a variety of systems in yet unknown labs.

if you rely on "neutralization" on the way to the column, be careful on different systems. We once had issues when injecting standards in high organic solvent but low organic initial. On the original system (Waters 2695) we never had any issues but going to an Agilent 1100/1200, the peak had severe fronting. That's because the 2695 injects the sample into a tiny stream of mobile phase, and therefore it is mixed more on its way to the column, while the 1100/1200 keeps the sample as a plug with only few mix up at the borders of the injection volume.
So, this won't be that fool proof unless some checkpoints are implemented during the method transfer.

6. I agree that the trap and elute should work. Online SPE or 2D-LC is out of the question due to technological limitations. Why would you recommend this sample prep, rather than just dilute and shoot?

was just an idea, I don't have such a system neither.
Same idea as with the step gradient. If dilute and shoot is not working well, then you should have something to convert and/or trap the neutralized form before it starts moving through the column. That's when the ion-exchange SPE came into mind. But of course, it would be dedicated to one system and out of scope here (>> missing information ;) )

Now some new thoughts:
Why would you like to introduce another, alkaline buffer system and not only stay with plain NaOH? I'm just speculating but as a real buffer, it may take longer to bring the pH down? Of course, your idea was to prevent going to low when using only water.
But maybe just dilute the sample with NaOH of a certain concentration (0.1M) will work too?

As written before, from my point of view, the success depends on how good the injection volume will mix with the mobile phase (and neutralize) before reaching the column head. If you still have some ionized molecules, they will move further into the column head until they stop. But then with isocratic mode, all molecules will move through the columne at the same speed, resulting in weird peak shapes. (that was the idea to start with a lower organic initial and then to re-focus the molecules with the ste(e)p gradient back to normal MP composition for the separation).

Maybe it already helps, if you install some pre-column inline filter as a static mixer just before the column head. This would also be easy to transfer to other systems (and even protects the column).

If you're thinking about alkaline HILIC.
Why not go with a dedicated IEX column and eluents?
Hi folks

thanks for your advice again. An update:

The method has been successfully completed with 98-102% recovery, a sufficient LOQ, and good linearity. Everyone is happy.

I did go the gradient route eventually, as per your advice. This "magically" solved high/low recovery problems and reproducibility issues. It's still a method with just under 10 min turnaround time - that is acceptable for us.

For sample prep we are doing a 1g in 25mL dilution of the caustic sample (up to 1N NaOH) in 100mM NaH2PO4 @ pH 2.15. This ensures acidic pH while also ensuring that we have a sufficiently low concentration of analyte to avoid precipitation. It is also a sample prep procedure that is easy to carry out in almost any lab with a halfway decent scale and a volumetric flask. The LOQ is more than sufficient, so we can get away with this large dilution and a 1 uL injection.

We ended up using an old-school 150x4.6x5u column and 1.5 mL/min flow-rate. That is ok for us since we use very little expensive organic solvent. Most of our mobile phase is water with 0.1% H3PO4, which is all but free for us.
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