Chromatography Forum

Apologies for bringing up yet another buffer prep issue. I was reading over some archived threads about weights-and-volumes practice, and the use of online buffer prep calculators has thrown up a question for me.

If I wanted to mildly amuse myself by solving the Henderson-Hasselbach equation for [A-] and [HA], how do I take account of the affect of ionic strength on apparent pKa?

For example, preparing a 100 mM acetate buffer at pH 5.7 from sodium acetate (Mr 82.03) and acetic acid (Mr 60.05) by solving the HH equation gives:

[A-] = 0.0897 (equiv. 7.358 g sodium acetate)
[HA] = 0.0103 (equiv. 0.619 g acetic acid)

However, the online calculator at Rob Beynon's site adjusts the apparent pKa in the HH equation by ionic strength which when solved gives:

[A-] = 0.0918 (equiv. 7.530 g sodium acetate)
[HA] = 0.0082 (equiv. 0.492 g acetic acid)

I'm interested to know the significance of the ionic strength correction and thus should I just carry on using that particular website for my weights-and-volumes?..

Regards
JA

I'm sorry but I don't understand your question.... "the significance of the ionic strength correction ". Doesn't the difference between what you calculated and the value calculated considering activity coefficients provide a measure of the significance? The example you chose is one where the difference will be relatively small because the activity correction for a neutral like acetic acid will be tiny. Try again for a doubly or triply charged buffer species at 0.1M concentration in which case the activity coefficient will be quite significant, eg. a high pH phosphate buffer.

sorry for being a bit vague my question was a bit further up the post.. "how do I take account of the affect of ionic strength on apparent pKa?"

It's not that hard to solve for [A-] and [HA] so I was tending towards wanting to prepare more of our buffers by weight/volume and do away with titrating to desired pH with a meter. The problem for me is that ionic strength seems to have a complicated affect on the commonly accepted pKa values you can get from the text, and this would put me off.

I was also trying to gauge whether those people already using weight/volume approach just do away with the mathematics and throw their numbers into a "predictor" like the one at the site I listed.

I know that for consistent chromatography we should have a consistent method of buffer preparation. Looking at some of my colleague's practices I have never been a fan of titrating using a meter. Contract method development and validation has really picked up at my workplace and I would like to be able to bring a better working standard to our lab.

In my (humble) opinion, the only thing that counts for a chromatographer is to have a method description that allows him/her to do reproducible chromatography. If you specify a real buffer in a reproducible way, it makes no difference whatsoever, if the pH is measured or not. Of course, those who want to measure the pH with the pH meter, can still measure it. I, for example, would do so, but I will not get excited over the measurement, unless it indicates that the buffer prepared by weight is not in the expected buffering region.

sorry for delay in response but I have been unable to get on this site for a few days...
The answer to your question is both simple (what Uwe posted) and more involved. I can think of a number of cases.

1. First time method development One can develop methods with a completely empirical approach to pH. Presumably one knows from the appearance of the separation whether the pH is too high or too low, or acceptable, and one knows about what pH is required. Let's guess we need about pH 5. I would make up an equal molar mix of acetate and acetic acid and test the separation. I know that I will have excellent buffering with a ratio of acetate to acetic acid in the range of 1/10 to 10/1 (which will move me +/- about 1 pH units. I will get good buffering for rations between 1/100 and 100/1. ( in a pinch you may be able to get by with even lager ratios. A ratios of 1/1000 is still orders of magnitude better buffering than water) So, I can empirically change ratios of acetate and acid in these ranges until separation is optimum. If the separation does not optimise in this range then I will pick a weaker or stroger acid and diddle with ratios for the new acid/anion pair. Once optimized, I know the weights and volumes that got me the desired separation so that I can repeat reliably using these values.

2. By some means you "know" you need pH 5.1 to do the separation. I could get out my pH meter and empirically adjust ratios of acetate and aceitc acid until I hit target. I would keep track of what weights and volumes I used and then make repeat buffer preparations using these weights and volumes. Or, I could use the buffer calculator that used activity coefficient corrections and prepare the buffer according to the recipe provided. (this is what I would do in practice) Or, I could get out my solution thermodynamics book, refresh my mind on the details of calculating activity coefficients, and slog through the tedious calculations. A final option is to realize that the target pH provided, 5.1, probably was not done rigorously and that it has an uncetainty of at least +/- .2 pH units. In this case, use Case 1 approach and establish you own optimim buffer recipe.

While fretting over pH it is well to keep in mind what Uwe said..... once a separation is established, by any means empirical or fundamental, repeatability of preparation is what is most important. In my experience, and what was standard practice up until the invention of biochemistry and LC =:),
"weights and volumes are most repeatable and there is less ambiguity reporting a procedure with weights and volumes".

The buffers you use to standardize your meter are prepared by weights and volumes according to NIST recipes.

thank you both Uwe and Bill for your replies.

I think the approach I'm going to try is to stick with the pH meter where method dev is concerned and a few buffers of largely differing pH can be made up reasonably accurately and in short time. Once the ideal pH for the separation has been found one could switch to the weight/volume approach and ensure the same separation is achieved.

If the method needs validating, transferring or will find long term use in QC or stability trial situations perhaps this allows us to eliminate or reduce some of the sources of error.