Injecting HCl solutions into HPLC

[Uwe Neue]

I have to disagree with Kostas. Packings, including siilica-based packings, can be exposed to low pH, of course this depends on the details of the surface chemistry used. Some surface modifications are better than others, i.e. a trifunctional surface modification or one with sterically hindered silanes is better than a standard monofunctional silane. For the packing, HCl has no different properties than HClO4 or H2SO4. However, for the stainless steel, HCl is problematic. This can be solved by using special hardware, from titanium columns or frits to PEEK columns and frits. If one wants to use HCl, a special instrument is required.

[Kostas Petritis]

Uwe,

We agree that we speak about the same thing which is: using HCl as part of the injection solvent, not in the mobile phase. Assuming that you use a 10 uL of injection loop and 200 uL/min of flow rate HCl will be very quickly be diluted. With the exception maybe of the valve, other parts of the system will come in contact with HCl a very small amount of time (before either HCl is washed off or diluted to harmless amounts)...

[Uwe Neue]

HCl as part of the injection is rarely a problem.

[zvi]

Thank you all.

You've started to alleviate my fears about injecting samples dissolved in dilute aqueous HCl-organic solutions. (I would never God-forbid use HCl in my mobile phase). In fact, I was just told today that in certain pharmaceutical analyses, some samples are dissolved in, and subsequently injected, 0.1% HCl solutions. I'd love to get a refeerence for that if any of you know about this. I'll also check up on it.


Zvi

[Kostas Petritis]

Per your request, a couple of papers where the analyte was dissolved in 0.1% HCl before analysis by LC...

Chromatographia 2006, 63, May (No. 9/10) 431-436

Journal of AOAC INTERNATIONAL Volume: 90 | Issue: 5 Page(s): 1266-1271

[peptidemetdev]

Also, I've heard of a small number of people suggesting the use of 5mM HCl mobile phases to separate proteins/peptides (as an alternative to TFA). These suggestions have never come with a "Do not use this mobile phase on stainless steel systems" warning before, though, so I'm wondering if they were mistaken.

As an example, here is a Vydac article I've seen posted in a few different places: Why TFA as an ion pairing agent.

To the best of my knowledge, Vydac doesn't even make PEEK columns, so they must be advising people to try 5mM HCl on stainless steel. Is this just really bad advice, or is it one of those things that's just considered to be acceptable damage should it result in better chromatography (like using a buffer a bit outside the stable range of the column, or heating a column to higher than what's recommended)?

[Kostas Petritis]

I wanted to say bad advice but in their application note HCl gives the worst resolution and peak shape when compared to phosphate and TFA mobile phases, so I wouldn't say that they recommend or advice the use of HCl.

I think that everybody agrees that HCl should not be used in the mobile phase unless you have specialized equipment.

[peptidemetdev]

I don't know if I agree with that Kostas. Of the three they show, HCl is definitely the worst, but they still recommend that it is considered as an option (along with a few formate and acetate mobile phases that they do not give an example for). But nowhere in the article do they explain that HCl should not be used with stainless steel instruments.

[zvi]

Thanks Kostas. In the Chromatographia article, though, the HCl solution was neutralized prior to injection.
I got an email today from a technical instrument rep who said that 0.1% HCl solutions can be injected.

[zvi]

Forgot to ask if the 0.1% HCl solution can also be injected into an LC-MS and not just into HPLC?

[ljc]

Just use a PEEK valve and a guard column so you can finish your work ! Your project re: the analysis of the "lakes" is far more intriguing to me than the HCl question ! I would like to read about your work and approach if it is published, please send me a reprint: ljc300@charter.net. I am particularly interested in hearing about the nature of your analytical approach and how you characterize the complexes after breaking them up and chromatographing them, if I understood it correctly.

I did have some experience with chloride containing samples (reaction mixtures of chlorosulfonation reactions) eating up injection valves over time, this even though the samples were diluted about 1000x before injecting. Fortunately I had the benefit of very highly absorbing analytes. Have you considered trying to increase your sensitivity somehow so you could use a more dilute sample or smaller injection volume, so that the chloride issue is negligable ?

Best of luck to you.

[zvi]

Hi ljc,
My samples are "one-of-a-kind" and miniscule (see above), and can't dilute them further.
Just sent off an article to you.
Zvi

[JGK]

Thank you all.

You've started to alleviate my fears about injecting samples dissolved in dilute aqueous HCl-organic solutions. (I would never God-forbid use HCl in my mobile phase). In fact, I was just told today that in certain pharmaceutical analyses, some samples are dissolved in, and subsequently injected, 0.1% HCl solutions. I'd love to get a refeerence for that if any of you know about this. I'll also check up on it.


Zvi

I've prepared Insulin standards in 0.1N HCL solution (but 0.1% seems a bit too concentrated)

[Rob Burgess]

Analysts all over the world regularly inject 0.1M HCl samples as this is the most common dissolution media for pharma products.

Not a problem from my experience.

[zvi]

Thanks, can you give me a published reference for injecting HCl?