Chromatography Forum

A debate started yesterday at work - does the buffer mass/composition need to be exact or within a certain percentage (we argued 5-10%).

IMO, the molar concentration wouldn't change that much (0.01% for 1.2 g of NaH2PO4 AND 0.0092 for 1.1g) and the adjusted pH would be the same in both cases.

What are your experiences when preparing buffers? Do you weight the exact amount, water volume and pH? Or do you have +/- tolerance for buffer mass and pH and/or use measuring cylinders instead of volumetric flasks ...

Or does it depends on the robustness of a particular method?

Depends on the robustness of the method. Usually the USP monographs say 'about', which is interpreted as 10%. I would recommend the same language in your SOP's and not think too much.

A debate started yesterday at work .

What are your experiences when preparing buffers? Do you weight the exact amount, water volume and pH? Or do you have +/- tolerance for buffer mass and pH and/or use measuring cylinders instead of volumetric flasks ...

I'd like to comment, my 2 cents worth.

Even when we followed a procedure from elsewhere (compendial, supplier, etc.), I often found instructions for preparation of buffers to be vague. Some did not even specify if a certain pH was measured for just the aqueous portion, or for the mix including the organic.

First thing I'd do would be to follow instructions as written to confirm that the procedure actually worked. But I also measured how much phosphoric acid (for example) was added to reach the specified pH in the procedure. Then from then on, I'd make the buffer adding components by weight or volume, and did not use a pH meter, much faster, and in my mind more accurate and reproducible. Of course my pointy-haired boss

figured EVERYTHING I did was wrong, so he didn't like that, wanted us to use pH meter. I tried telling him that other chromatographers also liked the non-pH meter way after working things out, and that I did use pH meter initially, but he'd periodically return to ME making buffers wrong.

Anyway, buffers exist so that small variations do not affect pH significantly. Yes, I wrote stuff so it would state like" "add 3.20 - 3.40 grams of Na2HPO4" so that anything within that range was acceptable, and anything out of that range would be NOT acceptable, and non-cGMP, and would need to be re-made.

Love the cartoon!

Yeah, your pointy-haired boss was wrong, wrong, wrong.

As far as pH is concerned, buffer preparation by weight is *much* more accurate and precise than using a pH meter. Quite aside from issues about meter / electrode calibration and the ambiguity in the definition of pH when the system is not 100% water, pH is a logarithmic function. To have the pH off by 0.1 unit, you would have to make a 20-30% error in weighing -- and if you can't weigh to closer that 20% of a target value, then pH is the *least* of your problems!

As far as ionic strength is concerned, most reversed-phase methods are relatively insensitive to the salt (buffer) concentration, so long as it is high enough to control ionization. The USP and EP default method adjustment limits allow buffer concentration to vary by +/- 10%. That said, if your separation involves a significant contribution from ion exchange / ion exclusion / ion-pair mechanisms, then it may be more sensitive to ionic strength effects.

Now, if only I could get my labmates out of the habit of pouring their organic into a cylinder and then topping to mark w/ water...

I keep explaining that "conservation of mass is a thing, conservation of volume is not a thing (when mixing organic solvents w/ water)."

Love the cartoon!

Yeah, your pointy-haired boss was wrong, wrong, wrong.

Boss really was like that.

Even though my computer skills are very poor and elementary, anything that went wrong with the Agilent Open Lab and his separate server was "my fault", as if I press the ENTER key differently than others.

By the way, Tom - it may have been from one of your writings in LCGC Magazine or somewhere which I read about preparing buffers by weight. And when I started doing this the pointy-haired boss misunderstood at first that I did re-confirm pH before I wrote up such procedures, so he kept throwing that up in my face !!!

Follow-up (I'm retired now) is that pointy-haired boss is no longer in charge of the Analytical Chemistry department. So I lasted longer in that position than he did.