Why is methanol a preferred solvent for flushing machine?

[EmpowersBane]

I use UPLCs a lot in work and most of my methods use gradient methods with Acetonitrile and buffered mobile phases. I am always told by senior lab analysts to use methanol to store the lines in when you wont be using the system for a while plus to prime the lines and pump out with, as well as the detector for a few hours as this is supposed to be "easier" on the system that Acetonitrile.

Is there any truth in this?

[Multidimensional]

Methanol has superior solubility properties compared to other popular HPLC solvents (better than ACN).

Flushing a system down for several hours seems excessive. Depending on how it is used, perhaps 50 column volumes would be plenty for long term storage (BUT, you should flush it down until it is clean, however long that takes).

For most HPLC systems ("UPLC" is a trademark), consider flushing any column(s) used first with plenty of aqueous phase to rinse out any buffers or additives, then progressively rinse with less polar solvents to remove the water and leave it in a mostly organic phase (to prevent corrosion and growth of microbes or algae) a reversed phase system. It does not have to bee 100% pure organic, but we routinely flush RP systems down with Methanol over the weekend or when not in use. Note: For longer term storage (weeks), when the column is clean/flushed, remove it, place a restriction capillary or ZDV union in its place and cap the column for storage.

You should seriously think about reading one or more of the many great books on liquid chromatography ("Introduction to Modern Liquid Chromatography") as well as take some formal classes too (not the free ones at the shows!). These are basic questions and you will benifit greatly from putting the time in now to learn these routine maintenance tasks as well as chromatography fundamentals. They will help you a lot.

[EmpowersBane]

Thanks for the reply, I'm actually an experienced user of UPLC and HPLC instruments and the one thing I have learned the hard way over the last 15 years is that everyone has a different opinion on what is deemed correct and incorrect when it comes to best practice for sample prep, mobile phase, system prep, maintenance etc. The engineer you have hired to do PMs on your systems can give you completely different advice from the senior engineer on site- similarly, companies like Waters can provide training on a certain subject and then contradict themselves when you contact them on a later date.

I think people like John Dolan etc are the only absolute experts to trust when it comes to consistent and accurate advice.

[Multidimensional]

I agree that "everyone has a different opinion" on the matter, but what you left out is that many of those opinions are just plain wrong. Very little in chromatography is subjective, most is objective info. After all, everyone has an opinion, but very few of those people have learned or received quality training stressing the fundamentals of chromatography and put it to use. Few chromatographers seem to know and/or understand these fundamentals. This includes many of the service engineers at all of the major companies too. Many have never learned the basics, they just swap parts. Knowledge does not come from years on the job, it comes from both the practical understanding of the concepts and info plus use of the same in practice to solve problems.

Just like your question about methanol. The answer is not subjective. You can look up the properties of this solvent and compare them to other commonly used solvents. The physical data is well known and many studies have been done which demonstrate these properties and useful application.

[mattmullaney]

Hi Empowersbane,

Returning to the question posed...I have a question. Do you mean an HPLC flush with the column in place or without the column?

In the case without the column in place (of course, the column will be flushed and stored as per the column manufacturer's recommendations in the perfect lab), why is MeOH preferred for flushing the instrument? Here are my two cents worth:

1. Methanol is less expensive than acetonitrile, isopropanol, or THF.

1a. MeOH is chemically stable, unlike THF.

1b. MeOH is miscible with the solvents used in RP chromatography. Buffers are generally more soluble in MeOH than in either THF or ACN...that said, the buffers (if used in the prior work done on the instrument) would have been flushed out of the column/HPLC prior to flushing the instrument only with a union connector in place.

2. Neat (100%) methanol is more viscous than acetonitrile or THF. Sometimes more viscosity works better for cleaning...neat IPA is more viscous than neat methanol, generally, IPA is also regarded as being a good HPLC flush solvent (at least by Waters and Agilent--the latest I've read is that IPA is now the "Gold Standard" for instrument-only flushing prior to more extreme cleaning).

3. Here's a link worth skimming over:

http://www.chromatographyonline.com/che ... etonitrile

In the case of acetonitrile, CRUD builds upon the check valve seats causing them to stick over time (I don't think much has changed in ACN manufacturing since 2008). Why flush and leave the HPLC instrument in a solvent which is eventually inimical to the check valves? To my knowledge, no such complaints have been spoken of regarding methanol and check valve function.

Please see what you think, and thank you!

[EmpowersBane]

Thanks for the reply Matt.

I am referring to flushing down the HPLC or UPLC system with no column, just a union in place. At the end of my runs I always switch to a high aqueous wash for 2 hours to flush out any buffer. then a low flow of Acetonitrile for an hour then stop the flow.

For the washdown in Methanol, I put all the lines (except the seal wash line, I don't think that would be good!) into a bottle of Methanol, prime all lines and syringes for about 10 mins then flow A1:B1 50:50 at 0.5ml through the system for about 3 hours. Thanks for the info you provided, very interesting reading.

Its funny because I was having big problems with my check valves sticking/failing on my binary Waters UPLC for about a year- literally every 3rd run would go belly up with the check valve until a senior Waters engineer gave me a little tip that he said is not on any manual- if you put your mobile phase lines (for me A1 and B1) into a bottle of 90:10 Water:Organic after the run and washdown are complete and prime these lines for about 20 mins, apparently that leaves an aqeuos "seal" on the check valves increasing the lubrication and leaving it much less likely to stick when Acetonitroile is flowing again for the next run. I was sceptical because of all the different advice I get but ive been doing it now for 3 years on that machine and never once had I an issue with the check valves since!!

[mattmullaney]

@ EmpowersBane,

You're Welcome.

I am referring to flushing down the HPLC or UPLC system with no column, just a union in place. At the end of my runs I always switch to a high aqueous wash for 2 hours to flush out any buffer. then a low flow of Acetonitrile for an hour then stop the flow.

Just checking as to what was being flushed. Five- to ten-times the Acquity-H system volume (with or without the column in place, depending on what I'm doing) has worked okay for me. Probably no such thing as "over-flushing" regarding column/LC cleanliness, but I suppose it can be wasteful of solvent. The way you're flushing might be able to be reduced, but it's not a broken process, either, so why "fix" it?

Last thing I do (I'm running quaternary AcquityHs these days) after neat MeOH is to fill all of the solvent lines with 20:80 (v/v) MeOH/water and prime the Purge (usually something like 90:10 MeOH/water, depends on the method) and Wash lines (usually 10:90 MeOH/water).

Agreed again--neat MeOH in the Seal Wash (for me the Purge line) isn't a great idea.

[mattmullaney]

Hi Again,

You'll want to see this, too:

Controlling Contamination in LC/MS Systems - Best Practices 715001307, Rev. G

http://www.waters.com/waters/support.ht ... &type=SSPR