Injecting HCl solutions into HPLC

[zvi]

Hi All,
My samples dissolve very well in HCl(aq)/Methanol and I would like to inject these filtered solutions into a standard RP-HPLC (mobile phase: water, methanol, weak acid). Are there "safe" concentrations of HCl that can be injected?
Thanks.

[mbicking]

HCl is corrosive to stainless steel, so there is always a risk. However, if the concentration is relatively low, and the contact time is minimal (it is in it's most concentrated form only during the injection cycle), then you may not have any serious damage.

However, you your samples dissolve in HCl and methano, they will probably also dissolve in H3PO4 and methanol, or H2SO4 and methanol. Either option would be better than HCl.

Of course, you could also buy a metal free HPLC and continue to use HCl.

[peptidemetdev]

Just out of curiousity, at what sorts of concentrations will there start to be serious issues?

I know mobile phases that call for concentrations of 1-5mN HCl. I'm wondering if that's going to promote corrosion, or if that should be relatively benign.

I guess my question is: What are the limits at which an HCl containing mobile phase will start doing appreciable damage to the system, and what are the limits that an HCl containing analyte solution will start doing appreciable damage to the system?

[mbicking]

I don't know if there is a critical concentration or not; I'm thinking in very general terms, and considering that this reaction would be governed by the normal rules of kinetics (concentration dependence). Certainly, more dilute means slower reaction. I would think 1 mN would not be an immediate problem, but I would not let the system stand idle with that solution present.

There were some interesting articles a few years ago on the stability of stainless steel (Kenneth Collins, LCGC, 2000, but not on-line any more), including cleaning and passivation issues. If anyone has a link to that article it would be a good review.

[zvi]

Thanks Merlin for the tip about the other acids.
I was also worried about causing damage to the head of the column as the HCl concentration is of course highest at the point of injection, though the HCl is flushed out of the column fairly fast with the polar mobile phase. It all boils down to the low-pH tolerance of the column.
I'll also check on the stability of stainless steel.

Zvi

[mbicking]

Since this seems to be a solubility issue with acidic solution, I am assuming that you are also using an acidic mobile phase. Otherwise, you are creating a precipitation hazard that could be every bit as bad as any damaged caused by HCl.

[zvi]

Definitely using an acid in the mobile phase. In fact it's H3PO4.
I have also been experimenting with a DMSO+HCl system at 100 deg C for the dissolution of my samples. Is there any chemical or safety hazards that you know of using that system or DMSO+H3PO4 at 100 deg C?

Zvi

[mbicking]

DMSO/HCl at 100 C ???? What are you trying to dissolve (or shouldn't I ask)?

I see no obvious problems switching to phosphoric (not an expert on this combination), but it will be less acidic. And again, I assume that you are diluting your samples in mobile phase (or something very similar) and filtering before injection.

[zvi]

You can ask ...

I'm dissolving certain organo-metallic complexes - "lakes" - organic colorants that have been precipitated by reacting them with typically aluminum and iron salts. I'm interested in the organic part (the colorant) of the complex. So I need to use a generally strong acid to break up the metal-organo bond, and the organic solvent (DMSO or methanol) to dissolve the colorant. Do yiou have a better suggestion?

I'm also worried about injecting into an MS later. Can H3PO4 be injected?

[sfe-co2]

Hmm....this has got me thinking real weird. I see your point Merlin, but....

1) H3PO4 is not suitable for injection into MS, as I recall (not an expert here so please correct if I'm wrong). Formic acid may be a better bet.

2) I thought HCl is more active in diluted form because the proton is fully and quickly dissociated and thus become more active as a hydronium ion. This is why HCl (and other strong acids and bases) are transported in tankers in their most concentrated form....so the proton is more tightly bound and hence less active.

Anyway, in general, it is not a good idea to use HCl in a stainless steel system. I recall a stainless steel vessel pitted with holes within hours of HCl exposure......but then again, hey, I'm no expert of this. Just a curious and keen learner What do others think???

[mbicking]

The best acids for MS are going to be formic and TFA, but many MS people do not like TFA any more, because it tends to hang around even after you quit using it, and it complexes/forms ion pairs that can interfere with ionization (I think... it's that expert thing again!).

And acidity in an aqueous/organic environment takes on a whole new meaning.

It sounds like you need to do sample prep a little differently.

Could you dissolve in HCl to release the metals, then extract with an immiscible organic solvent? The metals should stay in the aqueous layer as the chlorides.

Or, could you pre-extract the metals with EDTA or some other hydrophilic chelator, then extract.

Just some random thoughts on only one cup of coffee.

[Kostas Petritis]

Using strong acids as injection solvents shouldn't pose any problem assuming that the injection volumes are relatively small when compared to the flow rate/column volume. Also assuming that your compounds are retained somewhat in the column you won't see any ionization suppression in mass spectrometry if you use TFA to dissolve your compounds... As for injecting non-volatile acids in MS, this is acceptable if you divert the flowthrough towards the waste and start the aquisition later (again your compounds need to be retained enough to do this).

[zvi]

Thanks again Merlin, and Kostas. You have good tips.

My "problem" is that I analyze archaeological colorants, dyes and pigment, from ancient objects, and as such I only have minute quantities to "sacrice" for the greater good of science. It's pretty much a one-shot game. Often, no second chance. Hence, I must minimize the treatments and extraction steps in getting the organic colorant dissolved, because each step retains part of the sample. Additionally, these colorants are slightly soluble in water, so in first using an aqueous solution, part of my sample will be lost.

In short, that's why I'm looking for that "pot of gold" where there is only one extraction step, which I found, but then again we're back to that HCl problem. Though I will try TFA. Any compatibility problems with it in DMSO?

I use a nylon membrane centrifuge tube filter for cleaning the sample prior to injection and I see that formic acid is not compatible with nylon. I haven't seen any compatibility charts regarding TFA with nylon. Any known info on that?

Maybe I can also try oxalic acid. Any problems with its injection into an MS system?

OK, thanks people.

Zvi

[Kostas Petritis]

When people talk about potential problem of HCl when being injected in LC they refer to long term column stability or HPLC stainless steel corrossion. Your column might not live for 5000 injections, you might only be able to make 4000 and so on. If you have a small number of injections I wouldn't even think about it. If you have established methods with HCl, use HCl.

You can test drive your methods by extracting colorants from something more conventional than archaelogical colorants or by just mixing together HCl and everything else you plan to use for extraction along with your standards... I would think that you are planning on doing somekind of quantitation anyways of your dyes/colorants?

Regarding your question about nylon and formic acid, I do not know why they are not compatible, but in general I would think that everything non compatible with formic acid won't be compatible with TFA either as it is much stronger acid, much more corrosive and slightly more hydrophobic...

[mbicking]

Strong acids are likely to hydrolyze the nylon molecule (an amide), although slowly. If your contact time is short, I wouldn't worry about it.

If you have enough organic solvent present, the Teflon filters should work fine. There are some hydrophilic polypropylene materials as well. Check the major suppliers like Whatman and Pall for other choices.