Separation of zwitter ionic drug molecule

[kvgpal]

Dear Experts,
Thanks to all of them who r contributing their intelectual help for current problem.
SYX, Drug i am using has two pKa 's: pKa1 = 4.8 and pKa2 = 10.1. Then what should be the pH of the media?

Best regards

[syx]

SYX, Drug i am using has two pKa 's: pKa1 = 4.8 and pKa2 = 10.1. Then what should be the pH of the media?

Just try pH 2 points above or below the pKas. You can try to use pH 2.8 or 6.8. Good luck.

[HW Mueller]

Kostas, generally speaking I completely agree with you, but, regarding ion pairing agents I have had such bad experience that it seems like a very good answer if somebody points out that they should be avoided if possible (also, remember that recommendations are to limit a column to ion pair use....). Now, unfortunatly, your hypothetical example could suggest that it´s allright to tell someone to go ahead with an analysis even if his apparatus is not capable. I am sure you didn´t intend that. (I knew what you tried to say, already, without this example)


syx, did you try any other pH besides those "theoretical" ones? For instance, I have had good results at the isoelectric point, even though one often sees recommendations to stay away from that. Thus, my suggestion to kvgpal is to try a wide range of pH.

[Kostas Petritis]

HWM,

Personally I had a pretty good experience with ion-pairing chromatography and I find that it is pretty powerfull approach especially if you want to separate several ionic and hydrophilic compounds. Furthermore, I guess that the problems that you are refering to, had to do with gradient elution ion-pairing methods that are more challenging.

I would agree that if you only have one compound to analyse you can always find a suitable alternative method, but again analysis of only one compound can be done with ion-pairing chromatography under isocratic conditions...

Anyway... moving on...

[Einar Ponten]

Still, I am confused on some conclusions in this discussion.

The fact we have been given are:
"zwitter ionic drug molecule"
"pKa1 = 4.8 and pKa2 = 10.1"

As I read this information the molecule is completely zwitterionic only between pH 5.8 and 9.1 ("isoelectric range"?). Below pH 3.8 it is positively charged and above pH 11 negatively.

I agree that a large zwitterionic molecule may be "soft" and "diffuse" that it also shows hydrophobic properties, like e.g., proteins. However, this "drug molecule" is likely to be small and then the pH range 5.8-9.1 seems to very unsuitable for reversed phase separations regardless if ion-pairing agents are used. Consequently, (my assumptions) below pH 3.8 the molecule will be positively charged and then an ion-pair with e.g., heptansulfonic acid may give retention, or at a pH around 11 the molecule will only be negative and form ion-pair with a base (like TBA+).

Neither of these alternatives looks attractive and are similar to the situation for retaining most amino acids (without pre-column derivatization).

If, we still are hooked on ion-pairing then a low pH and sulfonic acid or even trifluoroacetic acid can be used.

[HW Mueller]

Gee, I thought there would be more coming here...
kvgpal, you should have had enough time to get something useful by now, would be interesting to find out. Or, at least tell us what your substance is, that may help to predict a method.

[kvgpal]

Dear HW Muller,
As i am in acadamic institute please give me some more time to come up with a result.
Regards

[Uwe Neue]

With the pKa values that you mentioned above, you will get the most stable retention in the doubly charged range, i.e. around pH 7. If you do not use a MS detector, the best will be to use a pH 7 phosphate buffer with a modern C18. You did not mention the % organic that you have used until now to get retention. The only disadvantage of the doubly charged state of the molecule is that it gives you less retention than the singly charged state. If you can't get retention even with 10% water in the mobile phase, you may need to go to one of the singly charged states, probably best at pH 2.

[kvgpal]

Dear Experts,

Finally i got some retention in c18 column, 125 mm column C18 endcapped - 5 µm.
Mobile phase: ACN and aqueous phase (KH2PO4 + TBA+ + SDS - pH adjusted to 3.5 with KOH or H3PO4)

Retention time improved from 0.9 to 1.98 min and assymmetric factor is below 1.5.

pH 6 is showing very very less sensitivity at the same time peak shape is completely distorted. So i used pH 3.5 - at which i got best results.

Still i have few questions:
1. Still how can i improve retention?????????
2. Can i go pH below 3 for LiChrospher 100 - LiChroCart - C18 - Endcapped?
3. If i take longer column, will i have any advantage?

Best regards

[Kostas Petritis]

kvgpal,

At this point the best way to increase your retention is to decrease the % of ACN in your mobile phase. Pay attention though as this will increase the equilibration time of your column.

Another way would be to increase the concentration of SDS.

If you drop the pH at 2.8 (which you can do quite safely with these type of columns) could further improve things a little bit. A longer column won't be that much of advantage.

[kvgpal]

Dear experts
Please check the question with the title "Carbon content of the column". This question is for analysis of the same zwitter ionic drug i am woking on and i asked few of my doughts.
Regards

[Chris Pohl]

kvgpal,

Since you have noticed improved retention of your zwitterionic analyte by adding SDS to a tetrabutylammonium based ion pair reagent system, this suggests two options:

1). Determine the effect of SDS without tetrabutylammonium ion. Generally, a combination of both anionic and cationic ion pair reagents is inadvisable due to the relatively low solubility of the combination and the propensity of the combination to preclude retention of other analytes. Since you have observed additional retention by adding a reagent which one would expect should reduce retention, one possible explanation is that you have supplied SDS in a molar excess relative to the tetrabutylammonium ion concentration (you haven't mentioned any concentrations so I'm just guessing) and that your enhanced retention is due to the excess of SDS.

2). There is a small body of literature demonstrating enhanced retention of electrolytes in the presence of zwitterionic media (a commercial source of such media frequently posts recommendations to use their zwitterionic material for HILIC applications in this forum). However, never or at least rarely mentioned is the use of zwitterionic "ion pair" reagents. There are a number of references which demonstrate the fact that surfactants containing both a quaternary functional group and a sulfonate functional group can be used to retain salts. A number of articles have been published which refer to "Electrostatic Ion Chromatography" which is based on this principle (the name is a misnomer, however, since all ion exchange has an electrostatic basis whereas the name would seem to suggest that only zwitterionic retention is based on electrostatics). Anyway, it's possible that your retention mechanism is parallel to this process. If so, I would suggest that you try one of the zwitterionic surfactants commonly employed in this work such as Zwittergen 3-12 from Calbiochem. This might possibly get you even better retention than the combination of ion pair reagents you are currently using. One point to keep in mind is that in this retention mode, increasing ionic strength increases retention (at least up to 50mM) so you might want to investigate the effects of increasing the ionic strength of your eluent system.

[Yury Zelechonok]

To kvgpal

I will try to summarize what we already know and what was said within two separate threads (this one and about carbon load).
1. You have a zwitterionic compounds which has no retention on C18 column without ion-pairing reagent (I assume you tried to use zero organic mobile phase already).
2. At pH 3 you start to see, small retention with strange combination of ion-pairing reagents SDS and TBA.
3. Your compound has limited and pH dependant solubility.
4. And peak symmetry is rather poor.

Your situation is not unique and there are some good solutions out there.

In order to get good chromatography you have five main approaches that often are used for amino acids:

1 Analyte’s derivatization the amino group or carboxylic group or both. (drawback - additional step is involved)
2. Use HILIC interaction with any bare silica column or special HILIC column (some times it works, but hard to resolve close structural analogs with small difference in carbon skeleton, unpredictable).
3. Use ion-pairing reagent but only one, SDS at pH below 3 and TBA above 9. Don’t use both ion-pairing reagents at the same time. You basically neutralize positive effect of one IP reagent by another one. One should do the job. You would be able to observe any retention you want with these systems.
4. Use a column with embedded ion-pairing reagent but still pH below 3 to create a positive charge on your molecule (Primesep A is typical examples of this type of the column). Simple and proved approach for amino acids.
5. Ion-exchange chromatography. Again pH <3 or >9 no organic in the mobile phase. (Ask Alltech or Dionex people for best column; may not work for the other parts of your formulation).

Now, your peak shape can improve significantly when you start to retain your compound. If not, then problem can be in solubility. Ideally your sample should be dissolved in the mobile phase. Sometimes poor peak shape is the result of slow conversion of the molecule from one form to another. Imidine group such as in arginine are known for that. In this case use temperature and pH study to find condition when two forms interconvert rapidly or coeluted.

I don’t think carbon load has anything to do in your situation. From what we know it is not RP interaction so you need RP stationary phase to retain SDS which will retain well almost with any RP column. I don’t think you need a column with polar embedded groups. But if you have one it’s nothing wrong with those. IP reagent can not destroy a column, but hard to get rid of them. So designate this column for IP methods. End-capped column I would avoid. Some of them looses end-capping in time. You don’t need end-capped column if you use IP-reagent.

[Einar Ponten]

I think the latter contributions in this matter make sense so in most parts I agree with Chris and Yury. It is required that one deal with the problem in a systematic way based on the fundamental facts of the target, namely its zwitterionic properties.

However, it is very surprising that Chris today suggests a zwitterionic agent to the mobile phase. That will ruin most future possibilities to use the separation method in combination with ESI-MS, which soon or later is likely to be a required technique for a drug molecule/candidate. Moreover, the examples of success using zwitterionic detergents are rare. In the past, zwitterions used to attract interest, due to their zero net charge (low conductivity), as mobile phase ions in Ion Chromatography with conductivity detection (in a way to escape the need for an [expensive] suppressor).

I regret that Yury has the experience that it is [quote] "hard to resolve close structural analogs with small difference in carbon skeleton" [end quote] using HILIC, but I assume that Yury's access to a zwitterionic HILIC column has been just as low as Chris.

We are happy to send you both a free column so you don't need to tumble around in groundless speculations. Some information may also be retrieved at; http://www.lcgcmag.com/lcgc/article/art ... ?id=120236

[Yury Zelechonok]

To Einar Ponten
You are absolutely right, my knowledge of HILIC mostly based on normal phase chromatography experience. I would be glad to try one of your columns with some of our difficult separations. My conclusion came from the typical usage of normal phase or HILIC at this point, they require high amount of organic modifier. At such condition small difference in hydrophobicity would be not sufficient to resolve molecules of similar nature and different in, for example of length of alkyl substitute. It is not an accident that RP chromatography is a main separation tool in HPLC. Your pdf link is not convincing either.