Chromatography Forum

hi.. i'm new comer in HPLC..
i'm on the way to finish my thesys about determination heptaminol with HPLC. could you help me? i'm confuse about peak tailing in HPLC. Heptaminol is base, and the silanol residue group of C18 will interact with heptaminol and give tailing...
according to you, which one should i choose? let the heptaminol directly injected to column, or make the column more acidic and heptaminol will ionized?
thanks.. (i'm sorry, i can't tell exactly, cause i can't speak english well..)

To greatly oversimplify, basic analytes have a tendency to tail less when the mobile phase pH is low (e.g., 2-3) compared to when the pH is higher (e.g., 6-7). Suppression of silanol ionization has been suggested as the reason for this.

That said, tailing can result from a number of processes, so there is no simple universal answer. The only way to know for sure is to do the experiment.

By the way, there is nothing to be ashamed of in your English (it is much better than my Bahasa! ).

Alternatively, you will have much less problems with peak tailing by using a high pH, where the amine is not ionized. In order to do that, you need to get a column that is capable of being run at high pH, such as an XBridge C18 column from Waters.

can you give details of your application?
mobile phase composition
column used
temp
injection volume
signal response for conc.
tailing you get

You need to remember that being close to the Pka value of your compound(10.6 in your case) is not recommended either. I would probably not go over pH 9.0 here
so I would play around a standard C18 end capped column first since they generally have a pH range of 2-10 for most vendors

Why "is it not recommended to be close to the pKa value of your compound"?

The data below is for aliphatic amines on Unison UK-C18:
http://www.imtaktusa.com/site_media/fil ... TI458E.pdf

Similar conditions (HFBA additive in your mobile phase) may work. How are you detecting this compound?

Uwe, I'm scared to have a go at answering the question:

As I understand it, if pH of mobile phase is close to pKa, then minor changes in buffer will lead to large changes in the degree of ionisation of the analyte, and so to large changes in retention time, which means the method is less robust.

Worse, small temperature differences (e.g. between outside and inside of column) will lead to small differences in pH, which will be of relevance if pH is close to pKa, so analyte will travel at different speeds depending on where it is in the column, and so you will get peak broadening. I've heard this is particularly serious if the buffer capacity is weak.

But this is just what I've picked up as I've gone along, so perhaps a person with proper theoretical training can correct me.

The temperature effect is no different than any other temperature effect, so this argument is not valid.

It is true that around the pKa of the analyte small changes of the pH can make a large difference in retention. However, this is only a question on how well you control the pH of the buffer. If you rely on a pH meter, you get junk. If you prepare a buffer by weighing the components, you will get control over the retention of your analyte to better than 0.5% isocratically, even if your buffer is right at the pKa of your buffer.

Proper care in buffer preparation is all that is needed.

(1) Uwe, please could you clarify something for me in the first statement about temperature effects? The argument I heard was that for those temperature influences that are mediated by pH, the problem would be worse if the pH is close to pKa, on the grounds that if pH is nowhere near pKa, changes make little differences. It wasn't that pH/pKa considerations are the only thing that matters when worrying about temperature.

Are you saying that other temperature effects are so big that effects via pH are irrelevant? If so, I'm happy... (in any case, for most of us, temperature variations across a column are not something we can control; they're a fixed evil in the world).

(2) I have had very little success preparing ammonium acetate buffers reliably, but they really are jolly useful in LC-MS. Any thoughts on how to measure out ammonia from the NH4OH bottle gratefully received.

1. The temperature effects around the pKa are no worse than away from the pKa. I vaguely remember some publications by McCalley, but I need to find them and reread them to make sure.

2. You can buy ammonium acetate and mix it with acetic acid or with ammonia, depending on the pH that you need. With acetic acid, you can weigh out everything to be precise. With ammonia, I suggest to prepare a master buffer at high concentration, measure the pH, and dilute later for use at the concentration that you need.

I'am afraid that it will be difficult to have 100% non ionized specie in your case because at pH10,4 you'll have and equilibrium between the protonated amine group and the non protonated.
To have 100% non ionized species you will have to work at pH 12

(but Xbridge could work at pH 12)

Try to work with 0,1% HCOOH on a sunfire Column (end capped) hoping that you will have enough retention (your molecule will be more polar at this pH).

At pH between 2 and 7 you will have NH3+ only specie after as i said you will have 2 species (NH2 and NH3+)

What do guys you do when the fully ionized version doesn´t retain but a 50/50 (ionized/unionized) mixture at the pKa does retain sufficiently?
Also, when looking at buffer capacity curves (which can be considered slopes of a titration curve) one sees that at the pKa you are at an apex of the slope, and that for a narrow range around the apex the slope changes little.

If going to a high pH to avoid full ionisation doesn't work and using an endcapped column at a pH where there is ionisation doesn't work then I would try ion pairing chromatography.

The molecule lacks a chromophore and I'm not sure what detection is being used, but this way would definitely get retention on a c18 column. Low pH with some octane sulfonic acid.