Procedural question for the addition of TEA

[jmac]

Hi everyone-

So this why engineers don't need to be performing analytical chemistry work. If I read a procedure that call for a 35mM NaPO4 pH=7.5 w/ 1% TEA buffer w/ ACN gradient and I want to run salt on channel A and ACN on channel B, how do you make the salt solution?

In the past I've I've made an the pH=7.5 Na-PO4 up and then added the TEA at 1% and never thought about it. But I realized today that after adding TEA the pH shoots up to 11.xx. Am supposed to titrate after the TEA addition or am I doing it correctly by titrating the salt first then adding the TEA after............I think I've been doing this wrong because wouldn't you lose buffering capacity after adding TEA and pH shooting up?

[tom jupille]

That's a textbook example of ambiguous (i.e., bad) documentation; it could be interpreted either way.

If what you are doing now works OK, then document the *exact* procedure you are using and follow that in the future.

My strong recommendation is that buffers in mobile phases should be specified by weight (e.g., "weigh out x.xx grams of NaH2PO4, y.yy grams of Na2HPO4, and add z grams of TEA into a 1 liter volumetric flask and make up to the mark with deionized water"). Weighing (and volumetric glassware) is *much* more accurate and precise than pH measurement.

[BHolmes]

I agree wtih tom jupille, if your separation is sufficient and the standard peaks match those in quality control samples, then keep making the salt solution as you have been. If you change how you make salt solution now, your separation will change as a result of changing the elution strength.

Ideally you would have added 4.13g of NaPO4 and 10mL of TEA to a 1L volumetric flask and made it up to the mark with deionized water, but with the way your method is written it leads to several possible interpretations of a denoted buffer solution (make a 1% TEA and adjust pH to 7.5 using 35mM NaPO4....or make 35mM NaPO4 and adjust pH to 7.5 using 1% TEA....the list goes on).

A great HPLC method will not only have the concentration of final mobile phase, but it will also have step by step procedure on how to make it so that there is only ONE way to make it.

Here is a link I found that explains the imortance of a properly made buffer solution for HPLC: http://www.shimadzu.com/an/hplc/support ... 40lab.html

[jmac]

Yeah, I agree that it could mean all kinds of things but didn't know if there was a standard practice for when you add TEA. Maybe one of the pharma guys will chime in. My experience with TEA is that it's most commonly used in sharpening up broad (amine-containing) drug peaks.

[KM-USA]

My strong recommendation is that buffers in mobile phases should be specified by weight (e.g., "weigh out x.xx grams of NaH2PO4, y.yy grams of Na2HPO4, and add z grams of TEA into a 1 liter volumetric flask and make up to the mark with deionized water"). Weighing (and volumetric glassware) is *much* more accurate and precise than pH measurement.

My thoughts exactly, and I've written procedures which utilize added amounts of modifiers to minimize errors (after the investigations to confirm that doing that delivers the same pH and results).

Our robustness studies intentionally vary the amounts of such modifiers, instead of trying to vary the pH using a pH meter.