Chromatography Forum

Another thing to consider when mixing volumetrically is the accuracy of measuring cylinders, in other words what are the differences among cylinders - which is poorer than the precision (repeatability) of repeated measurements with the same cylinder.

The problem here is not volumetrics vs gravimetrics, but an instruction that was not clearly expressed in terms of take this and do that with it.

Happy New Year

Peter

What about method Robustness, folks? If it matters whether it is 505 mL or 495 mL, then the method is not Robust. This MUST be built in for Quality methods due to training issues and time constraints. I can't imagine our Quality folks taking time to weigh solvents. (We do have an SOP on what kind of glassware to buy for both R&D and Quality.) I should say that we have about over 100 products that can go through our release labs, so time is precious.

Although I doubt that a 1% change in mobile phase major components is unlikely to have profound effect on most methods, robustness should not be an issue.

My recollection is that if measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled. The danger is that over-specification of required performance could make some instruments unsuitable for the method.

One advantage of weighing is that samples can be weighed into, and mixed, in the same container. For binary isocratic solvents, weighing can be very quick - especially if solvents are stored in cold DG stores, and there's usually less glassware to clean.

If I have to repeatably make up the same simple binary mobile phase, weighing is preferred, especially if a calibrated 3 kg balance is handy.

Measuring cylinders are available in two formats, " To Contain = In" and " To Deliver = Ex". I often find people using "In" format cylinders to prepare mobile phases, when they should be using "Ex" - unless the cylinder type is specified in the method. Usually people just look for the Grade ( "A" or "B" ), rather than the calibration format.

I doubt it affects chromatography much, but the Ex grades are calibrated with water, so will give incorrect values for solvents with different viscosities and surface tensions anyway.

The simplest solution is to follow the precribed mobile phase preparation method, as that will cause least grief with auditors.

Bruce Hamilton

FWIW, I never adjust the pH of a buffered MP (bulk). I figure out how to make it such that it ends up at the desired pH, then check a small aliquot of the aq phase (only) to be sure that I did what I thought I did. I'm rarely more than 0.1 pH units off, which won't impact anything in a properly designed separation anyway. As long as one is working within the proper buffering ranges of the stuff they're using, I find this technique to be quite rugged. I don't want a pH electrode near my (bulk) MP anyway...ick.

How does one do this? I'm used to the standard method of weighing out a salt, dissolving, checking the bulk with a pH meter and then adjusting with acid.

For instance, I recently did an analysis with 15mM potassium phosphate at pH 2.45. The salt alone resulted in a solution with pH around 4.5, so I had to add a couple mLs of conc. phosphoric acid to adjust down to pH 2.45. If there's a quicker and more repeatable way of making acidic buffers, then I'm all for it. I just don't know how to do it.

According to page 736 in my trusty dusty copy of Practical HPLC Method Development, 2nd Ed, I would combine roughly 331 ml of 0.1M phosphoric acid with 669 ml of 0.1M sodium (or potassium) phosphate monobasic to yield a solution of about pH 2.45, double check that against a pH meter, modify as needed, then calculate how much monobasic phosphate and phosphoric acid need to be diluted in 1L to make my MP.

Please note that I plotted the data given in the chart in the book and interpolated between the 2.4 and 2.6 pH points to obtain rough quantities for your 2.45 pH point:

mL of 0.1M H3PO4 = -1098.6 ln (pH) + 1315.8

331 = -1098.6 ln [2.45] + 1315.8

Phosphoric acid qty/L:

Conc (85%) phosphoric acid is 14.7M/L, so you should have (0.0331M*1000ml/L / 14.7M/L) = 2.25mL phosphoric acid.

NaH2PO4 monohydrate Qty/L:
=13.8 gm * 0.669 = 9.23 gm

Please remember that this will get you 100 mM/L, so you'd obviously need to scale down to 15 mM.

What I might do there is prepare a 15mM solution from the 0.1M solution prepared above (dilute 150 ml to 1L), check the pH and if it is where you want it (at 2.45), I'd simply scale the recipie to 0.338 ml 85% phosphoric acid and 1.38 gm NaH2PO4 monohydrate and QS to 1L.

If that all falls into place, then in the future, all you'd need to do is prepare that recipie accurately and you should geet a liter of MP at the same pH, which you could check by simply taking a few mL to check on your pH meter. You then discard the aliquot leaving your MP bulk untouched.

Of course, your way certainly works well if you have a dedicated pH probe, but I work around cosmetic formulators (great chemists all) and therefore the possibility of goop getting onto a pH probe here is pretty high, which is why I do things this way.

Hope this helps...

Chris

Yes, thanks, that does help. I have the first edition of Practical HPLC Method Development, but it looks like I need to get the second edition as well!