Challenging separation of moenomycins by ion-pair HPLC

[cyanogen78]

I'm afraid only that the molecules will not have adequate retention, they will elute too early ... But I'll try. About acidity/basicity I have to admit I'm wrong.

[MestizoJoe]

[these moenomycins appear to be negatively charged detergents. So it should be advantageous to go to a acidic mobile phase, not only for the reasons just given, but also to prevent charge exclusion. I suspect that addition of the sulfonate increased the rt only by obliterating this exclusion.
]

Question for HW. What is charge exclusion? [/quote]

[HW Mueller]

MestizoJoe, that´s actually a very good question and for a certain reason I don´t want to discuss the mechanism here. It usually manifests itself in that negative ions which are not retained are coming out of the column ahead of tm (tO). Some people do their ion chrom. ahead of tm, akin to molecular size separation via SEC. I think Bio-Rad sells some of their IC materials with a pamphlet that describes ion exclusion techniques for carboxylic acids.
The effect is easily obliterated by salt concentrations of about 0.1M and, of course, is removed by eliminating SiO- via pH.

[cyanogen78]

Hi guys,

Tomorrow I'll try your proposals and I'll post the results here. But I want to comment the buffer concentration in ion-pair LC ... In case of poor retention of the analytes even at pH 2.5 I want to try ion-pairing with tetrabutylammonium sulphate at pH=7.0. I have read that it is desirable for buffer concentration to be as low as possible in IP-LC because of possibility for competition between beffer ions and those of IP-reagent, regarding IEX interactions. If this is true - isn't 50 mM phosphate too high for IPC ? Thanks to all

[carls]

I have read that it is desirable for buffer concentration to be as low as possible in IP-LC because of possibility for competition between beffer ions and those of IP-reagent, regarding IEX interactions.
Thanks to all

I'd try 0.1% phophoric acid in 10% ACN first which will give pH ~2.1 and has reasonable buffer capacity (~15mM)

If you NEED an IP reagent/buffer to increase retention you can use 0.1% phosphoric acid with 5mM TBA.H2PO4 (or TBA.HSO4)

[danko]

Salts are not just salts for the sake of buffering alone. They also change the structure of the water hence the mobile phase. In addition to that and especially in protein context, salts influence the analytes’ conformation.
I personally never saw a “beautifulâ€

[carls]

[quote="danko"]I personally never saw a “beautifulâ€

[danko]

Since this compound has a PO4 in its structure its a good idea to have PO4 in the mobile phase. A salt is not necessarily needed especially since there are no basic groups in this compound.

Maybe I didn’t pay too much attention in school, so please correct me if I’m wrong, but I thought there was some PO4 in phosphate buffers at pH 2.5
As for the need for salts in situations where there is or isn’t basic groups in the compound, I don’t remember mentioning basic groups as an argument for salts in the mobile phase

Best Regards

[carls]

I have had good exerience with 0.1% H3PO4 as a buffer in HPLC:

H3PO4 pKa1 = 2.1

pH 0.1% H3PO4 (~15mM) in H2O = ~2.1

pH = pKa+/-1 = good buffer (pH = pKa = max buffer capacity)

I'm referring to the PO4 in both the analyte of interest and the mobile phase (H2PO4-).

[HW Mueller]

The "PO4" in this molecule and PO4--- or H2PO4-, are about as different as Cl2 and Cl-, still I stand by my statement from above to try acidic conditions without IP.

[carls]

The "PO4" in this molecule and PO4--- or H2PO4-, are about as different as Cl2 and Cl-, still I stand by my statement from above to try acidic conditions without IP.

I believe we have come back to my original recommendation (0.1% H3PO4, no IP) which seemed to intiate this discussion.

Not sure what point you are making regarding PO4 in the molecule vs. H3PO4 in the mobile phase. They have similar physical properties (pKa, ion pairing, geometry, etc). My recommendation for H3PO4 was based on previous observations where phosphate containing compounds showed poor chromatographic performance in RPLC.

[danko]

I understand HW Mueller’s point. Strictly speaking, at pH 2.1 there is no PO4--- species. Neither at pH 2.5 for that matter. We just made it easy on our selves.
And now to the real point: You might achieve pH using solely phosphoric acid, but the concentration would be low and you know that of course.
So, here, it might be a good idea to remember that buffer capacity isn’t only dependant on the pKa, but on the concentration too – even in greater degree I should say.
Taking your suggestion as an example (addition of pure phosphoric acid) the concentration is ultimately limited by the desired pH, which wouldn’t be the case if a phosphate salts were utilized.

Best Regards

[carls]

Not sure where PO4-- came from (should be HPO4--). I've been talking about PO4- (analyte) and H2PO4- (buffer).

Prepare a solution of 0.1% H3PO4 in water and measure pH.

Typically I see pH ~2.1 for this solution.

pka1 H3PO4 = 2.1

From the knowledge that concentrated H3PO4 is ~15M,

0.1% H3PO4 in solution is ~15mM which is typically sufficient buffer capacity, esp. since pH = pKa1

[HW Mueller]

Ah, that´s where the 15mM comes from, I estimate via head calc ~10mM for 0.1% H3PO4 via:
0.1% is 1g H3PO4
1g/98g/M ~ 0.010M
In my opinion ~0.01 is not ~0.015.

For the few physical and chemical similarities there are many grave differences in the P species, I wouldn´t dream of using H3PO4 for the similarities. Phosphate buffers have some advantages for many substances, most of which don´t have any P in them.

[carls]

Ah, that´s where the 15mM comes from, I estimate via head calc ~10mM for 0.1% H3PO4 via:
0.1% is 1g H3PO4
1g/98g/M ~ 0.010M
In my opinion ~0.01 is not ~0.015.

I suppose I shouldn't assume chromatographers think in terms of v/v%: 1mL conc H3PO4 dissolved in 999mL H2O ~15mM

or 1.69g conc H3PO4 (1mL) dissolved in 999g H2O ~15mM

For the few physical and chemical similarities there are many grave differences in the P species,

Could you elaborate on this and how these differences affect chromatographic behavior?