Chromatography Forum

Hi

Were were trying to develop reverse phase-HPLC method for a compound and discovered that it precipitates at lower pH (was using ACN+0.1% TFA and Water+0.1% TFA)…
Do anyone know any mobile phase composition which allows me to work at high pH range?? Is there any ion pairing agent that could let me achieve this??

(we are trying to avoid using any buffer such as phosphate buffer??)

Thanks a lot

Were were trying to develop reverse phase-HPLC method for a compound and discovered that it precipitates at lower pH (was using ACN+0.1% TFA and Water+0.1% TFA)…

Do you sure that only pH is responsible for your compound's insolubility. You said that it precipitates at lower pH (lower than what? pH value)

At which pH do you want to run your analyses? What's the pKa of the compound?

Regards

You can work with a phosphate buffer at pH 7, or you can work with ammonia or ammoniumbicarbonate at pH 10. I recommend Symmetry C18 for pH 7 and XBridge C18 for pH 10. With many packings, you do not get a good peak shape at neutral pH, and most packings are not stable at pH 10

Hi rick1112,

i am guessing by looking at the mobile phases that you are doing a separation of peptides or proteins.
am i correct?
this is generally the basic setting for such separations.
any particular reasons that you favor not using buffers like phosphates?

Hi

Thank you all for your replies

Well yes I am dealing with a protein whose pKa is 3.0

Well I don’t have an problem for using phosphate buffer, but it just that we are habitually using ACN + 0.1% TFA and Water +0.1% TFA, so wanna know is just using a high pH buffer the only option or can I use some other buffer composition for this exercises ??

I have not yet worked with a protein at high pH, but I do not see why this should be impossible. We have done reversed-phase separations of peptides (protein digests) at pH 10 with ammonia or ammonium bicarbonate. Works as well as the TFA or formic acid (on special columns) at acidic pH. Since one can do protein ion-exchange separations at high pH, high pH reversed-phase could be a perfect alternative to what you are doing now.

Depending on the protein you might have a lot of losses and your peak shapes might suffer in basic pH.

hi

Uwe Neue wrote

With many packings, you do not get a good peak shape at neutral pH

may i know why is this???

It is a question of silanol acitivity. Silanols are weak acids and ionize around pH 7. This creates difficulties with the peak shapes on reversed-phase packings for analytes with basic functional groups that interact with the silanols. One can reduce this problem by improving the preparation procedure of the packing: high-purity silica, very good bonding and endcapping procedures, ligands with embedded polar groups, etc.

Silanols are weak acids and ionize around pH 7

Actually it is worse than that - pKa (silanol) is approx. 3.5. And this would mean that the ionization starts already at pH > 2.5.
Another good reason for choosing low pH (e.g. under 2.5) in RP separation of proteins is, the fact that proteins them selves are typically ionized (i.e. carboxylic acid functional groups) at higher pH values, which significantly increases their polarity, which in turn shortens the retention factor dramatically.

Best Regards

On a high-purity silica, the average pKa of silanols is around 7. There are indeed some more acidic silanols around, however, a titration curve will give you a value around 7. Only on low-purity silicas or on silicas with silly pretreatments will you get silanols with a "pka of 3.5". If you have such a packing, throw it away and join the modern world.

One can run really nice chromatography for peptides at alkaline pH with equivalent retention as at acidic pH As I mentioned above, I have not tested this for proteins, but I do not see a reason why it should not work as well as under acidic conditions with TFA.

One can run really nice chromatography for peptides at alkaline pH with equivalent retention as at acidic pH As I mentioned above, I have not tested this for proteins, but I do not see a reason why it should not work as well as under acidic conditions with TFA.

Hi Uwe,

The reason is as described under my second point (previous post); the ionized carboxylic acid functional groups to be found in any protein.
If everything’s equal, the low pH conditions will result in longer protein retention compared to the high pH ditto. I’ve worked with proteins for years and I’ve tested many cases. F. ex. you can try phosphate buffer at pH 2.3 and 7.2 – same concentration e.g. 0.05 M. Please choose a protein with pI away from 7, for this means trouble. A good test candidate would be human growth hormone (pI = 5.2). You’ll need at least twice as much ACN to elute it at pH 2.3 compared to pH 7.2. The same goes for all other proteins I’ve tested and they are many.
Regarding the modern world: I’ve joined it already – I use ACE, Xbridge, poroshell, biosuite, Jupiter and many, many other modern columns. Actually sometimes, the so called older materials can prove to be very valuable indeed with respect to selectivity. As I use to say: The right column is the one that works for the compound of interest. Different silicas provide different selectivity and this is especially true in the case of protein separation. I’m sure we can agree on the importance of selectivity

Best Regards

Danko,

I was not talking about running proteins at pH 7. I was talking about high pH, and by that I mean ammonia at pH 10. Under these circumstances, you should not loose retention compared to pH 2, since now a lot of the amine functions on a protein are not ionized any more. Therefore the retention should be - on average - equivalent to the retention at pH 2.

As I said several times before, I have not yet done this, but I see nothing that speaks against it, at least not from the standpoint of chromatography. Don't you agree?

As I said several times before, I have not yet done this, but I see nothing that speaks against it, at least not from the standpoint of chromatography. Don't you agree?

Very much so! I’ve done pretty successful protein separations at higher pH myself - forced to do so due to selectivity advantages specific to these cases. But then again silanols get something to say which typically manifests itself through tailing and general peak broadening. The latter could be counteracted, playing with salt concentration and temperature, but it needs more attention and experience. One should also expect the opposite effect of the ionized solanols, namely some repulsion of the ionized carboxylics. So it gets somewhat more complicated, unpredictable and specific. Less experienced chromatographers would have less trouble with low pH as a starting point. And that is the point I tried to make.
Otherwise I admire your “non conformityâ€

One can easily obliterate any repulsion due to SiO- on negatively charged analytes by adding some ions (salt) to the mobile phase (or using a highly concentrated buffer).