Need help, splited peak and SDS

[Uwe Neue]

Chemgeek: one does not get split peaks by having a buffer at the pKa of the analyte. This is a completely unfounded but hard to kill rumor. You will get a perfectly good peak if you use a proper buffer - no problems....

If you care, I will send you an article that proves the point....

[chemgeek]

Chemgeek: one does not get split peaks by having a buffer at the pKa of the analyte. This is a completely unfounded but hard to kill rumor. You will get a perfectly good peak if you use a proper buffer - no problems....

If you care, I will send you an article that proves the point....

From his descrption he has 2 pka's, I have seen in my work, a peak split resulting from a molecule that is incompletely protonated, particularly with multiple pka's. (FWIW, I have also seen a peak split just due to the instantaneous rotational confirmation that induces a local temporary dipole split at about a 95-5%, even rarer and only with a charged stationary phase...)
It does happen. I have seen the MS data to support it. I agree it may not be as prevolent as once thought but why even tempt fate when it is so easy to avoid the issue all together?

[HW Mueller]

This "instant rotational confirmation" of a molecule which changes rotational isomers in solution faster than the chromatography also seems from the realm of mystique. I wonder whether the concept of idiot relationships is still in use.

[chemgeek]

Apologies for the thread drift...

This "instant rotational confirmation" of a molecule which changes rotational isomers in solution faster than the chromatography also seems from the realm of mystique. I wonder whether the concept of idiot relationships is still in use.

I agree, I'm still not quite sure about the mechanism, this was the best we could come up with...

The Details: we saw a peak that was splitting that looked like, for lack a of abetter description, an extremely lopsided silloute of batman's head with one point (left one, later eluting) about 5% the area of the other with an elevated baseline connecting the 2, which were about 1 minute apart. The mass spec data of this anomoly displayed the same mass distribution across the entire period, but qualitatively proportional to the respective response at the elution time. The molecule in question would have a momentary dipole that may have interacted with the stationary phase causing this(the stationary phase was propriatary and we couldn't get much out of the manufacturer...)

Anyway, back to the question at hand, peak splitting, SDS, and buffer choice.

[HW Mueller]

chemgeek, that´s typical for a number of causes like mobile phase, or pH mismatch, or some of the reasons given in this chain.

[chemgeek]

Yes, I know and these were all evaluated by someone who was much wiser than I was then (and most likely now as well) and considered to be a local expert by many more than I (both inside and out of my former employer)...

[mohan_2008]

SDS is used as a part of an aqueous formulation to provide a uniform suspension of a hydrophobic analyte.

It is nothing more than that. I don't think it is going to impart/ or provide solubility of a very hydrophobic substance, such as forming a solution.

If you want to improve solubility, it can be done only by choosing a proper organic modifier/aqueous combination.

[HW Mueller]

Surfactants cause severe changes of the properties of water even if no micelles are present. The most evident change leads to reduction of the surface tension (thus the synonym used here). A change of solubility in water modified in this way should be obvious. However, most people would use detergents as a last resort, even when proteins are involved, to influence solubility.

[zeq]

What he means is that your buffer does not have any buffer capacity at pH of 6.8...

I know that, i was trying to ask why does he suggest a ph=4.75, that is between the two pka´s of the molecule, it didn´t seem correct to me at first sight.

[Uwe Neue]

I suggested acetate at pH 4.75 because it is a buffer. I don't care where it is with respect to the pKa of your analytes. This does not make a difference, as long as you have a reproducible way of preparing the buffer.

[Alex Buske]

SDS does change the solubility of hydrophobic compounds in water. It is sometimes used to provide sink conditions in dissolution testing.
Alex

[Fabiano]

Apologies for the thread drift...

This "instant rotational confirmation" of a molecule which changes rotational isomers in solution faster than the chromatography also seems from the realm of mystique. I wonder whether the concept of idiot relationships is still in use.

I agree, I'm still not quite sure about the mechanism, this was the best we could come up with...

The Details: we saw a peak that was splitting that looked like, for lack a of abetter description, an extremely lopsided silloute of batman's head with one point (left one, later eluting) about 5% the area of the other with an elevated baseline connecting the 2, which were about 1 minute apart. The mass spec data of this anomoly displayed the same mass distribution across the entire period, but qualitatively proportional to the respective response at the elution time. The molecule in question would have a momentary dipole that may have interacted with the stationary phase causing this(the stationary phase was propriatary and we couldn't get much out of the manufacturer...)

Anyway, back to the question at hand, peak splitting, SDS, and buffer choice.

Sorry to join the group of the "concept of idiot relationships " but I also saw what chemgeek described.

We were running sugars on NH2 column with (75:25 - ACN:H2O - Only!) then decided to inject ascorbic acid. The peak splited with almost 1 minute of RT difference. After Mobile phase acidification it eluted as normal single peak. At time, boss said the column was resolving the dissociated-undissociated forms as 2 peaks. This was hard to get in my mind, the column resolving faster that chemical equilibrium...

[Uwe Neue]

What makes you think that ascorbic acid would elute at all from a NH2 column in unbuffered water/MeCN?

[Fabiano]

What makes you think that ascorbic acid would elute at all from a NH2 column in unbuffered water/MeCN?

What about a large peak with same spectrum as Ascorbic Acid? If it's not AA what it would be?

[Uwe Neue]

OK you got me there. My comment was a bit to premature. If you have a well worn amino column with plenty of silanols, it is possible that ascorbic acid elutes. Since you got spectra, was there a difference in the spectrum for both peaks?