Chromatography Forum

Please just state exactly how you prepared your buffers, so that it is known whether you are lazy or not.
Bruce, you prepare the dil. acid/base everytime also, or are they stable in your hands? Maybe you are not so lazy after all?
On a more serious vein: Some of the more stable modern pH meters can be less accurate than the older ones, where one has to have a bit of patience.
Also, I just wonder how you people do this adjusting the pH with an acid/base solution. When I did that I almost always overshot, then needed more buffer sol. then overshot again . . . . Do you hang the electrode into your solution?
I guess I am not only lazy, but inept to boot.

Obviously, if required, prepare according to compendial instructions which often don't involve pH meters and take me about 30 - 60 minutes to set up, but otherwise for research work I perform the following.

I'm not suggesting that this is desirable or best parctice, but it's quick and expedient...

1. Transfer electrode from storage solution, check calibration, rinse with Milli-Q water, and store in 400 ml of Milli-Q water for at least 30 minutes before use.
2. Weigh salt into 30 ml plastic bottle on 4 figure balance.
3. Add to calibrated 500ml Duran bottle, rinse plastic bottle with Milli-Q
4. Make up to about 98% volume with Milli-Q.
5. Ultrasonicate to dissolve and degas.
6. Add magnetic follower and electrode ( on holder arm ) and start stirrer.
7. Using a 1 ml pipettor ( less preferred is pasteur pipette ), immediately add 10% acid or base solution to desired pH - takes about a minute. I can't see how you would overshoot, as most buffers require much more of the acid or base as they approach the desired pH.
8. Remove electrode ( in buffer for 1 - 2 minutes maximum ), rinse with Milli-Q and return to storage solution.
9. Make up solution to 500 mL mark - decant to container.

Typical time to prepare ( excluding a couple of minutes for initial electrode rinse at start of day ), from start to finish, is about 10 minutes.

Dilute acid ( H3PO4 or HOAc ) and base ( KOH, NH4OH ) solutions are stable for at least 3-6 months here.

The pH meter stays in calibration ( three buffer pH=4, 7, 10, with 2 used for low pH ) for months without adjustment. I've actually found modern electrodes to be faster responding, and more consistent, than older electrodes, but these days I'm seldom measuring biological samples.

Yes I'm very aware of the comments about not putting electrodes into buffers - hence I keep contact to minimum, but I found making up to a specific volume ( eg 480 ml ), then taking 10 mls aliquots for pH reading and dumping them, recording accurately the added volume of acid or base, and then correcting the final makeup volume rather tedious ( but easy to calculate ) and unnecessary.

Obviously adding the electrode to the buffer is undesirable, but makes for a quick preparation. I've not seen any difference when I've analysed samples using mobile phases that were prepared without contact with the electrode and as prepared above.

Please keep having fun,

Bruce Hamilton

I apologize in advance for dragging this conversation on, but this is important...

There are thousands of validated method with buffers and I don't get this mixing volumes approach. Never had a problem with traditional buffer preparation: dissolving salt in water, adjusting pH of aqueous solution and then mixing with organic. If person is not qualified no matter what approach he/she uses there is always a chance of screw up.

Vlad,

I do not understand your statement. For HPLC, and in particular for separations of polar ionic solutes,
consistent [ions] in the eluent is critical for reproducible retention. We can use the following aqueous eluents:

acid
salt
buffer

(pH metering or adjusting should not be used to prepare any of these aqueous eluents)

All the above eluents have known quantity of ions and can be easily reproduced.
The mixing method that Imtakt (and others) propose for buffer preparation is simple and
reproducible. It is a standard, textbook procedure often used in biochemistry:

http://www.sigmaaldrich.com/life-scienc ... enter.html

The method you propose does not agree with buffer theory. SIELC (and some of the other column manufacturers)
are encouraging chromatographers to develop bad habits.

Please inform your customers of the correct way to prepare buffers.

Thank you.

You guys use a pH meter to adjust the pH of your aqueous mobile phases? Ick.

Figure a recipie for your buffer at a given pH, then make it and use the pH meter on a small aliquot - 25 ml or so - that's been removed from the flask or bottle to check the pH. If the pH isn't within 0.1 units of where you want it, you've made the buffer wrong, your water is screwy, or your meter isn't caibrated. If you need the pH to be in a tighter range than target +/- 0.1 to get the chromatography you need, then your method isn't rugged enough and you need to rethink something.

If made (and used) anywhere near properly, buffers should be pretty rugged pH wise- that's their job.

Well Bruce, looks like I guessed correctly about hanging the electrode into the buffer. That´s an absolute " no no" for me (this has been chewed through so many times that I can´t do it again). I use ~1mL aliquots in polyethylene tubes when I follow the adjustment, of course, only when I mix equal conc. solutions of conj. base and acid.
Unless your buffer pH is right smack on the pKa the slope is quite steep.
It is all a matter of what you need to do. If ionic strength and a tight control of pH is paramount you need to prepare the buffer via weighing.
Also, as a teaching tool the quicky methods are not so pedagogically sound.

You asked, I provided. I'm not a teaching institution.

I've no wish to be seen as an advocate of a practice that will mislead naive child chemists and lead them down the path to rack and ruin.

I've used calculation methods, and will if the method requires it, but for most of my work they didn't justify the extra time. YMMV.

One of the reasons why I gave up on routine use of weighed salt buffers was an apparent increased tendency to unexpectedly precipitate at high solvent concentrations during gradients for method development. That problem greatly diminished for me when I went to acid/base/salt buffers.

I assume those that use the calculation method also save considerable time because they don't need to check the prepared buffers with pH meters, as the "right" way is so good.

Whereas I have so little confidence in the published weighed buffer formulations that I also like to independently check a 20 ml aliquot on my meter - naive I know, but great for my peace of mind..

Please keep having fun,

Bruce Hamilton

Brian,

I guess all these years at Pfizer we were preparing buffers wrong, as well as other companies are wrong too:
http://www.sigmaaldrich.com/analytical- ... thods.html
http://www.mac-mod.com/tr/pbprep-tr.html
Common, calibrate your equipment every time, measure acurately and write down how you do it that the guy after you prepares it the same way. This is not a rocket science...just Good Lab Practice
and another thing if you remove 4-5 times 25 ml aliquotes your concentration is not the same as at the beigining Unless you return these milliliters)

Vlad, if one mixes solutions of conjugade base and acid, both having equal concentration, one can remove any amount without changing anything. The final solution will ALLWAYS have the intended concentration and ionic strength. The only drawback is that the final pH depends on the pH meter.
Now all these uninformed chaps at Pfizer, etc., which you mention, were not wrong, just not so good.
(Apparently that is not so bad, if everybody was super at pharmaceutical companies a lot of physicians would be out of work.)