Preparation of TEAP

[miro2009]

I have little experience on dealing with triethanol amine and preparing buffers that contain it, and I'm confused between triethylamine, triethanol amine, and an abbreviation I often see in literature TEAP.
Basically, I've found a certain RP-HPLC method where the following is described : "linear gradient of acetonitrile in triethylammonium phosphate pH 6". Regardeless of the acetonitrile, how is the aqueous phase here prepared? What I have available in my lab is a bottle of triethanol amine, do I use it and adjust pH to 6 with phosphoric acid, or is TEAP purchased already prepared? and how many ml is used? do I have to know the molarity for eg? or is it a default of 1 ml in 1 liter water?

I have googling this topic and found a lot of different methods and left out confused with the above questions, hoping someone bears with me and help me understand this in more detail.

[tom jupille]

Good gravy! where to even begin??

The procedure you cited ("linear gradient of acetonitrile in triethylammonium phosphate pH 6") does not contain sufficient information to run the method. Missing information:
- initial and final ACN concentrations
- rate of change of ACN
- buffer concentration

If you read the Forum regularly, you'll see that the most common response from "veterans" to questions is "You didn't provide enough detail".

In the case of buffer concentration, the ambiguity can get even worse than suggested above. Take, for example, this thread:
viewtopic.php?t=12482

In the specific case of buffers, look at the three articles I cited in the above thread for more details.

[miro2009]

Thanx Tom for the links, they gave me more insight about buffer preparation concept, generally. As I said I have no problem with the organic phase here and its % and gradient, I will deal with that as I'll need to try several gradients myself.

I need to specifically ask about the above mentioned aqueous part, ie. preparing an 100% aqueous phase of this buffer.
The salt here is not common to me as HCl, acetate and phosphate buffers.. I just need to understand what's the difference between triethyl amine and triethanolamine phosphate as both have the common abbreviation TEAP? and which is commonly used with RPC and in what molarity range (0.1, 0.5M...)?
Probably someone around here uses such buffer commonly, apparently it is used in a universal setup as the case for eg. with TFA aqueous phases (0.1% usually?)
Awaiting further assistance for a newbie in need!

[cody84]

TEA is triethylamine, we use this in most of our buffers, I think it helps with peak shape and retention of the sample on the column. I have heard once you use TEA in a column it is really hard to remove.

TEAP I have not heard of.
I know what Triethanolamine is, but we do not use it in HPLC here sorry.

[tom jupille]

triethanolamine:


triethylamine:



Both are commonly abbreviated as "TEA", which is why you always have to be careful about TLA's (three-letter acronyms) and FLAs (four-letter acronyms), and why I cringe when someone assumes that the whole world uses the same acronmym in the same way. In an HPLC contect, TEAP is most likely to be "triethylammonium phosphate".

Buffer concentrations in HPLC with UV detection are typically in the 10 - 50 mM range, with 25 mM being the most common. The molarity refers to that of the species doing the buffering; in your case, pH 6, that would be phosphate (H2PO4- / HPO4=), since the pKa of triethylamine is 10.75. The second pKa for phosphate is 7.1, so you're on the ragged edge of its buffering range in any case.

Buffer preparation depends on what salts are available. In the simplest scenario, Aldrich (and others) sell both 1M and 2M solutions of 1:1 triethlyamine / phosphoric acid. Dilute that to 25 mM (or whatever!) and adjust pH with concentrated triethylamine (to take the pH up) or with 25 mM phosphoric acid (if you ever wanted to take the pH down). Doing it that way, you stay at 25 mM in phosphate.

[miro2009]

Dear Tom, thanx for ur reply it was really very helpful and covers all my questions, but I hope you bear with a little further as a few questions came up to me after reading your explanations, this specific details are usually difficult to find in literature or text books:

1- In my case the main buffering agent is the phosphate so it's molarity should be precisely adjusted, ie. adjust pH also with 25mM phosphoric acid, but to bring pH up I could use concentrated triethylamine, so final molarity of triethyl amine now would change, this is not important?

2-I already only have a bottle of conc. triethyl amine (will plan to get the 1:1 triethlyamine / phosphoric acid from sigma, apparently it's easier to use), but is it possible to use just the conc. triethyl amine and conc. phosphoric to prepare the buffer? in that case I would need to calculate volume of each needed to prepare a 25mM solution for eg. then adjust the pH with 25 mM phosph. acid?

3-as cody mentioned, it's hard to remove, I've also read that you even should dedicate a column for it, but if I use it once or twice and decide I won't use it anymore, is there a special way or solvent to remove it?

Thanks all for your patience!!

[tom jupille]

To take the questions one at a time:

1. The Hendserson-Hasselbaclh equation http://en.wikipedia.org/wiki/Henderson- ... h_Equation has three parameters: concentration of the acid, concentration of the base, and pH. Once you specify two of them, the third is a dependent variable. So, for pH 6 and some concentration of phosphate, the triethylamine concentration is fixed.

By the way, most of the time, buffer concentration is not terribly important in reversed-phase HPLC (but then, most of the time you can drive right through stop signs without getting hit). In ion-pair or ion-exchange methods, that's not true, so it's better to be careful! I like to keep as many parameters as possible under control.

2. You could simply take 25 mM phosphoric acid and add concentrated triethylamine until the pH gets up to 6. All that matters is the relative concentration of phosphate and triethlyamine. The assumption behind using concentrated TEA is that it won't add much to the volume, so you stay at approximately 25 mM in phosphate.

3. Triethylamine is a lot harder to get rid of than is phosphate. Flushing the column with something like 50/50 25 mM phosphoric acid / methanol is as good as anything, but there is no guarantee that you will not have altered the column surface chemistry. My general recommendation is to dedicate columns ("Columns are cheap, your time is valuable").

[miro2009]

Thanx again Tom for taking the time to answer all my questions, everything now is clarified, will try it out... and will sure let you you know if I need further assisitance
Thanx again

[HW Mueller]

One can add here that the preparation of buffers has been discussed at length here many times.
Calculators, especially

http://www.liv.ac.uk/buffers/buffercalc.html

help in understanding what is involved.

Be careful with TEA, it has a strong tendency to oxidize. It should be safer to buy TEAP, as acidified TEA does not oxidize easily.

[rhaefe]

I have proabably prepared a few hundred liters of triethylamine (TEA) based buffers in my former life using all sorts of counter ions: acetate, oxalate, phosphate (hydrogen and dihydrogenphosphate), hydrogencarbonate, nitrate, perchlorate and of course chloride and bromide.

Here a few more things:
If you are using TEAP in a RP-Ion Pairing method make sure that the triethylamine you are using is of good quality and not too old. I used double distilled TEA from Aldrich or Fluka (now the same company) and stored the TEA under Argon in a freezer. You can get good results with lower quality TEA but I wouldn't count on it. The variation I experienced with "regular" 99%TEA was huge and annoying.

TEA has a hight UV cut-off. If your TEA concentration varies from batch to batch so will your base line shift (if you are running a gradient). Higher pH means more free TEA and a more pronounced base line shift. It is usually not a problem.

When preparing the buffer try to use weight measurements whenever possible. It is more accurate and more reproducible.

[rachely476]

One can add here that the preparation of buffers has been discussed at length here many times.
Calculators, especially

http://www.liv.ac.uk/buffers/buffercalc.html

help in understanding what is involved.

Be careful with TEA, it has a strong tendency to oxidize. It should be safer to buy TEAP, as acidified TEA does not oxidize easily.

Thanks for sharing the link!