[mandytan]

Hi,

My mobile phase for HPLC (high performance liquid chromatography) consists of Methanol (HPLC grade), Water and Glacial Acetic Acid. Is it necessary to filter (vacuum filtration) my mobile phase?

Thank you.

[Arkcon]

It really depends on conventions in your laboratory.  That is to say, you should do what everyone in the labs is doing. I'm betting you don't own the HPLC system and column, so doing things your own way would reflect badly on you if something goes wrong.

Generally, most vacuum filtration is done through a 0.2 u or 0.5 u filter.  A quick check on the bottles of your reagents will reveal that they are likely filtered at the factory to 0.1 u, or even less, so the knee-jerk response would be that filtering the mix yourself is stupid.  Now, I'm not trying to be a jerk here -- mathematically, you're wasting time, and filtering is just a procedure by rote, and people may act as though you're performing a ritual, and not thinking like a scientist.  You could look bad to others who want to be jerks.  That's where I'm going with this.

Some other points:  Is your water from a glass bottle, or dispensed from a distilled water tap?  Even the best maintained plumbing may release some rust, or maybe non-corroded metal particles.  I'm guessing you crack open (that is remove the security seal around the cap) of a fresh bottle of methanol (and maybe water too) or has it been opened before, by others and maybe left open to collect dust?  The acetic acid, you and everyone else, probably use the same bottle for months, how cleans is that.  Also, when you use a brand new bottle, look at it, is there dust around the top -- at least wipe that off with a damp lint free wipe before you mix.  If your sources are really dusty, you may want to filter anyway.

The vacuum filtering may be useful to insure that the eluent is degassed, but a modern system should do that for you.  Methanol-water mixes do release a lot of dissolved air when mixed.  Or filtering may be useful to insure thorough mixing, especially for a large volume.  It may also add extractable impurities to the mobile phase, which may interfere with some analysis, and not another one.   So you might want to be ready to change your method, if you encounter a problem you can't fix.

'Course, always filter when using a solid reagent in mobile phase.  No matter how pure and soluble a reagent is, there's always something in it.  Often lint from the cap liner, of some grit from who knows where.