Acetonitrile stability as a mobile phase

[quantonium]

Hi,

according to the "best practices" document by Agilent (https://www.agilent.com/cs/library/user ... ice_en.pdf), special care should be taken with acetonitrile as a mobile phase, but I can't seem to find any more information on this to confirm it. The document mentions how ACN should be kept in brown bottles to avoid photochemical reactions (I've also heard acids like formic acid degrades it?), as well as the need to monthly wash the LC system with 1 L of hot water to remove ACN reaction products. Is this common practice?

Also, as a bonus question: I'm very confused by the differing opinions on how long mobile phases last after mixing, with regards to microbial growth. It seems most agree that pure water with buffer at around pH 7 should ideally be changed daily. However, how long does water last with 0.1% formic acid? What about a 50H2O/50MeOH mix without acid?

In advance, thank you! (And thanks for all I've learned from browsing this forum for years!)

[Consumer Products Guy]

We used 100% acetonitrile as one of our mobile phase components, and never used any special precautions; we would simply refill the reservoir when it got low.

As to aqueous mobile phases: sometimes we just 100% water as the aqueous fraction, often we used water acidified with acetic acid or phosphoric acid. With 100% water we switched out more often.

When we used THF or a mobile phase containing THF, we used amber reservoirs and only made up as much as was needed that day. We used THF without preservative.

Disclaimer: I only used HP/Agilent 1084B, 1050, 1100, and 1200 units; I felt there was no reason to look into other vendors as our reliability with these units was so great.

Disclaimer 2: I'm not necessarily recommending any of the above practices, just detailing what I did for 35 years....

[per_oxid]

I have no clear answer but this are what I would guess, no actual data to prove it.

I think the question of 100% water as mobile phase and microorganisms was adressed well by the "trust you science" channel on youtube episode7. One day only. (0.1% FA i would assume not making much difference.)

I try to avoid 100% water when i can but then how long can i use a mobilephase with say 20% MeOH and 80% H2O? I dont know for sure but for mobilephase i made a guess of 2 weeks and for seal wash I accept 4 weeks, reasonable guess? If it is 50% MeOH i would guess it last a very long time probably 2 months. It is very practical to be able to use one batch of mobilephase for at least 1week of samples.

Volatile buffers can evaporate quite quickly so how good seal the bottle have makes big difference, investing in good better sealing lc caps is worth it. On my ms system the signal goes down over time as the buffer evaporates making ionisation less efficient. If bottle is in lc then i run no more than a week as caps are not sealed but if it is high in ACN % and sealed bottle cap and bottle is full then it can last many weeks, that is my guess and what I do.

ACN has been said to case sticky valves and a good practice is to switch a and b solvent lines to clean pumps from time to time as they are not cleaned by the gradient.

[quantonium]

Thanks Consumer Products Guy and per_oxid, good to hear what your practices are. I also watched the "trust your science" episode, and obviously got spooked

[DR]

ACN, when left in a system long enough, can cause "sticking" check valves.
I've been told that it tends to react on the surface of the ruby balls commonly used along with sapphire seats in check valves to form polymers and that this is what results in sticking, floating etc.

Switching to ceramic check valves has helped a lot with this (typically in Waters Alliance systems).

Aqueous solutions allow growth of things you do not want in your HPLC systems, solutions containing at least 5-10% organic (ACN/MeOH etc.) tend not to allow such growth, but also tend not to remain at 5-10% organic if left long enough too.

The above guidelines have been pretty common to the several places I've worked over the years and try to strike a balance between absolute prevention of system issues and inconvenience to the people operating them.

Also remember that your best, most reproducible gradient methods usually go from 100%A to 100%B, so do the math and premix those mobile phases to allow for this once you're beyond the development stage for your new methods. Premixing also provides the advantages of having that little bit of organic in most A phases and/or diluting your THF so that you need not worry about things like bug growth and peroxide formation for at least a few days .

[quantonium]

Sounds good. I'll likely add some methanol to the A solvent when I'm done with method development, then. I'm also considering doing the "hot water flush" as described by Agilent, as acetonitrile has been used a lot with this pump.

Thanks