Chromatography Forum

I am new to this site and have a question concerning buffered mobile phases. I notice that most posts that identify mobile phases include buffered ones. I have routinely used HPLC for over ten years (as well as LC/MS/MS) and developed methods for numerous compounds. I never employ buffers unless it is absolutely necessary (like quaternary amines). It just seems to me that they are employed as routine elsewhere. Of course I use them more often in LC/MS/MS to enhance ionization but just kind of think that many of the compounds could be successfully chromatographed without buffered mobile phases. Anyone else feel this way?

If your compounds are ionizable compounds like amines or carboxylic acids, you need to control the pH of the mobile phase, if you want to get reproducible retention. In some cases, a careful control of the mobile phase pH is necessary. There are many discussions on this board that go into details.

Well, I guess I also made a mistake when saying I rarely use them because I do 90% of the time use either 0.1% formic for LC/MS analyses and 0.1% phosphoric for HPLC/UV (in conjuction with ACN or MEOH) but this is so routine that I don't think of them as buffered (my mistake). Its when you have to get out the KH2PO4, ammonium formate or acetate...etc ...etc and vacumm filter that buffered mobile phase begins to be a PITA and circumvented by other means.

Additionally (and diquat is good example) it takes much longer to equilibrate system with buffer. It takes as many as a 5-15 injections of a std before that peak settles in and is consistent. It sure is rock solid once there but I'm going to pull that colum (or use the same) the next day to look at another compound. Then I have to change reservior, rinse channel and column before use...another PITA. Maybe you are in a different environment where you are looking at the same thing for quite awhile in which case the buffer makes more sense. I am in a contract lab working with different compounds practically every day.

I think of the adjustment of pH as the most powerful tool in the control of retention of ionizable compounds. There is nothing that will scramble up your chromatogram more than going from formic acid to ammonia, for example, or from an ammonium formate buffer at pH 3.75 to an ammonium bicarbonate buffer at pH 10. If you need orthogonal methods (as sometimes required), this is the simplest way of doing this. I use this as a primary tool in method screening.

Of course, this does not do anything, if your analytes do not have ionizable functions. Then the acetonitrile/methanol route or the use of temperature or column chemistry are the tools that you are left with.

Exactly, get the right column and mobile phase composition and in most cases (I have seen) you can successfully chromatograph it. just finished a method in 3 diff matrices for a parent and 2 metabolites where sent first 2 min to waste then 3 min in positve mode for 1 metabolite, then switch to negative mode and pick up the other metabolite and parent all in one 12 min run. mobile phase 0.1% formic channel A, ACN channel B with gradient. I'm sure any one of the components might c-graph better with a buffered mobile phase but it was not neccessary. I also had LOQ of 50ppt on all 3 analytes. Matrices were soil, water and plant material.

Also used the same triple quad the next day for a different compound with a low std conc of 25.0 ppt. I don't want buffers in that system.

I have another post that showed pH as important factor in separation for ionized substances beside solvent strength.
Buffer is not needed if the substances are nonionic.

I'm not doubting the importance of buffered mobile phases for certain compounds and when necessary I employ them. Just saying that it appeared to me as though everyone on here seemed to be using buffered mobile phase and knew the pH of their mobile phase as though it was always critical. Out of the last 20 mobile phases I have prepared, I have measured the pH of maybe one.

There are very interesting issues already discussed in this forum concerning pH, ionic strenght, etc.

As a borderline, in my opinion, you should always buffer your solution, as it is a way to guarantee method reproducibility.

Actually, I'll (sort of) disagree with Rafael's approach. If I know there are no ionizable compounds in my sample, then a buffer has no advantage, and possible problems. If I don't know (or if I know there are ionizable species), then I will always buffer, for the reason cited by Rafael.

Like KC, I only use "true" buffers when absolutely necessary. For us, common aqueous phases used with ionizable substances like carboxylic acids, phenols, etc., include either acetic acid or phosphoric acid. Never trouble trouble until trouble troubles you.

I must admit we tend to follow what KC and CPG usually do i.e. very rarely use buffered mobile phases. We usually just use low pH mobile phases with either formic or TFA. Working in the pharma industry, a great many of our analytes have basic nitrogens, thus unless we are working at high pH (i.e. > 9 -10) an intermediate buffered pH mobile phase doesn't hold much value. Using a acid modified mobile phase as we do is also MS compatible.

Further we rarely see many problems with retention reproducibility. However perhaps the biggest problem we face is trying to achieve enough retention of our basic analytes in a low pH environment.

Oh dear, all this crazy stuff about "true" buffers and claims that things like TFA in the usual concentrations employed in HPLC (say 0.1%) are not buffers is coming out again.

There are some very nice posts by Bill Tindall on this subject. A buffer solution is one that resists pH change when small amounts of acid or alkali are added to it. The mechanism of buffer action is not relevant to this definition. Thus 0.1M HCl is a very good buffer, largely because it contains a large concentration of hydroxonium ions and the addition or neutralisation of a few of them does not make a lot of difference. The same applies in varying degrees to things like 0.1% TFA or even 0.1% formic acid. I suggest those of you who don't beieve this should obtain one of those computer programs (some are freely available on the web) whihc work out the buffer capacity of various solutions commonly used in HPLC. The reason why you are getting reasonable results is because you are using buffer solutions..... Of course, if like Tom says you have no ionizable compounds in your mixture then you can probably leave out any of these things (although you may show minor differences between buffered and non-buffered solutions due to some secondary effects).

hm... those people who use buffers without a need must have some good coolies.

Victor, acids at a ~ pH of 2 and lower are buffers (especially toward adding more acids), not because there are so many H+ present, but because the water removes some H+ at those low pH. (No matter what the pH, if you don´t have a substance which can remove or release H+ you get a change in pH that directly corresponds to the amount added. Thus if you add 10 mol of H+ to a 10-2M acid you would get a 10-1M, pH=1, acid if it was not for the water.)

And: Sometimes it is necessary to use buffers to manage ionizable matricies (for instance a protein matrix) in the sample, even though the analytes would not require that.