PH adjustment of mobile phase.

[zlb215]

Hi,guys;
I found there have been two kinds of way to adjust pH of mobile phase,even in USP: before or after addition of organic modifiers.So is it necessary that two different ways exist in one pharmacopoeia? What's the advantages and disadvantages for each one?
Personally, I always adjust pH of buffer and mix it with organic phase.So , I was confused when I met a different method. On the other hand, I don't think a normal pH meter has ability of determination pH in organic solvent.
I'd like to give these word as a begining of discussion.Any correlative idea will be welcome and appreciated.
Best regards!

[tom jupille]

First of all, consistency is more important than correctness, so it doesn't matter which way the method calls for, so long as it is always run the *same* way.

Second, the whole topic of pH was discussed very thoroughly here in the early days of the Forum (around 2000). If you search the archives for "pH" and "Tindall" you will find a number of very informative posts.

Third, shortly thereafter, Bill Tindall wrote a series of three articles for John Dolan's "LC Troubleshooting Column" in LC/GC. They cover the topic very thoroughly:

http://chromatographyonline.findanalyti ... rticle.pdf

http://chromatographyonline.findanalyti ... rticle.pdf

http://chromatographyonline.findanalyti ... rticle.pdf

[freemab]

I agree with you. pH is undefined in non-aqueous media. Furthermore, the reading you get may depend upon the pH meter. Glass electrodes will not read the same before and after organic solvents are added to the buffer. I hear that FET pH meters DO read the same. So, at very least, you must specify what kind of meter you're using. Furthermore, the presence of organics can affect the behavior of the salt bridge in a glass electrode, so you have to be careful.

I have found it possible to correlate very closely the apparent pH in the mobile phase with the actual pH of the buffer. So, as Tom says, it really doesn't matter as long as you're consistent.

[Consumer Products Guy]

Agree, better to adjust the pH of the aqueous portion only, but what's really important is to write instructions clearly without any ambiguous parts. And USP is chock full of tough to understand, archaic, and unclear stuff.

What are the thoughts about adding buffering agents by weight or gravimetrically, like if one adds 0.005 M of a phosphate salt, shouldn't that always take the same amount of acid (such as phosphoric acid) to reach stated pH?

[freemab]

Re: adding buffer salts gravimetrically.

I've had good luck doing this. It seems to me I wrote such a procedure into methods using acetate buffers, but it would probably work for other buffers as well. Just make sure you use sufficient quantities that the accuracy of weighing is not an issue. Knowing your chemical supplier helps as well.

The trick is that pH generally is not all that critical a parameter. If a shift of 0.1 pH unit greatly affects your chromatography, you may want to reexamine the pH region you're working in and find a more robust region. Then, too, the nature of a buffer is to have a stable pH - providing you're working within, say, 1 pH unit of the pKa. Hence, small errors in weighing should have negligible effects on buffer pH, which, in turn, should have negligible effects on the chromatography.

One thing sometimes missed: Use the least buffer concentration that provides adequate buffering capacity. And remember that the further from the pKa, the greater the concentration will have to be.

[Bryan Evans]

Here is a quote from our instruction manual:

"Buffers prepared with pH meters (e.g. titrate to desired pH) are not reproducible. Precision for [salt] and pH is
crucial for buffers used with HPLC. It is strongly recommended to prepare buffers by measuring appropriate
volumes of (equal concentration) acid and base solutions"

Example: Make 2 solutions - a). 20mM CH3COOH and b). 20mM CH3COONH4
Whatever ratio you mix the 2 solutions in is the final pH. Add (volumetrically) appropriate amount of organic as needed.
Throw the pH meter in the trash.

If you think of retention as a combination of RP + IEX, this makes a lot more sense.

I understand having to follow outdated USP methods that say "25mM phsophate buffer, titrate with acid till pH 3.0..."
But newer methods should not be developed this way - it's bad practice for HPLC.

[Bryan Evans]

Also you can read this quick section:

L.Snyder, J.Kirkland, J. Glajch. Practical HPLC Method Development. 2nd Ed.
1997. Appendix IV.

[shahis77]

ADJUST THE PH OF BUFFER (AQUEOUS PHASE)
AND ADD ORGANIC PHASE

[HW Mueller]

Shahi, if you were to repeat a method from the literature in which the pH was given for an aqu./org. mixture, would you rework the method, using pH determination only in the aqueous phase? If so why, especially in view of the links given by Tom?

(Incidentally, some have pointed out that using capitol letters, bold print, etc., can be considered as shouting).

[Uwe Neue]

It is nice to be aware about correct ways of preparation of the mobile phase pH. However, if you want results that agree with the method by a pharmacopoeia, you need to prepare your mobile phase exactly as they prescibe, and in no other way.

We need to realize that some of us have learned a thing or two since the time (often some 30 years ago) that many of the pharmacopeial methods have been created. Today, many of the methods could be described in a more meaningful way, or even with better methods of preparation. But you should NEVER substitute your own method for a method prescribed in the pharmacopoeia. If it says "mix base with organic solvent, and then adjust the pH to pH..." that is exactly what you need to do to copy the method.

[JGK]

If given a method involving a mobile phase at a given pH, I would normally adjust the pH of the aqueous portion and add the organic component.

However, I was once supplied with a method where even the wording of the method indicated the mobile phase should be prepared in this manner. however when this was done I had an analyte retention time of 25 minutes rather than 7 indicated by the method. When I contacted the method author, I found that the author was adjusting the mixed mobile phase to the required pH. Once i did this the problem was sorted.

As long as you are clear in the method how you prepare reagents there shouldn't be a problem reproducing methods.

[danko]

Sometimes (read often) the pharmacopoeia people like to describe the methodology (i.e. mobile phase preparation etc. – even calibration) as they’ve always done - despite the fact that we (as Uwe correctly mentioned) have learnt a thing or two over the time. I’ve experienced that several times in the context of publishing my methods. They always seem to know better – even if it means the method wouldn’t work if they get to write what they feel like. And maybe that is the main reason for the existence all those pharmacopoeia methods that are impossible to reproduce. Who hasn’t experienced that kind of methods? Only those who never tried to set up a pharmacopoeia method!

Best Regards

[lmh]

[rant]
Scientific literature is riddled with methods that are either so ambiguous that they are useless, or that don't work when you run them. We've had methods where incredients aren't even soluble at the concentrations at which they were supposed to have been prepared.

I blame journal editors, and to some extent reviewers.

When the methods section is relegated to the end of the paper, in small print, and subject to tight word-limit, you know it's likely not to be worth the paper it is no longer written on. In an electronic age where one picture takes the memory of 10000 words, there's no excuse for a telegraphically-abbreviated, meaningless text. We were taught at the age of 13 that a measurement that could not be repeated was rubbish; this is still true today.

[Consumer Products Guy]

The USP is what it is. I even contacted the editor of an in-process draft revision about a few items, one an outright copy/paste mistake which detailed use of the WRONG solution, and the other that many GCs cannot track 50C/min. His response was only that <621> allows one to modify the chromatography, and was unchanged in final monograph. How many locations have qualified packed-column GCs these days? Qualified temperature-programmable inlets on QC GCs? Also, USP doesn't seem to set any limits on how long a solution is good for - I assume they feel that all solutions should be made fresh every day - any thoughts here? They may say "as long as you validate stability"....

I've also received test procedures from suppliers which stated to disssolve the standard in a certain solvent, and no way would it dissolve, even with agitation, heat, sonication, etc. So that threw their believability down the toilet...