Chromatography Forum

Hi

I am having great difficulties trying to quantitate the free form of the surfacatant. I am using a Biosep2000 column with 50mM P04 buffer, 0.5MNaCl at pH7 usng a PDA and a RI detector.

The MW of the surfactant micelle is about 60000Da while the free form should be below 1000Da. The CMC is about 300-500ppm. I have tried making sufactant solutions below its CMC (ie. 100, 300, 500ppm) and above its CMC (ie 6000ppm, 10000ppm) and inject in low and high column (10 - 1000ul). But I don't seem to be able to determine where the free form is coming out or the micelle for that matter.

Any suggestions would be greatly appreciated.

Is a SEC column a requirement for your separation or can you use a column (RP type) as in the link below?

http://www1.dionex.com/en-us/webdocs/25 ... et_V24.pdf


If you have to stick to the SEC type maybe you can increase the organic content of your mobile phase (if compatible with the column…) to disrupt the micelles.

vliu, what are the kinetics of your micelle formation/dissociation? (Especially: How fast do the micelles dissociate in relation to the time involved in the chromatography?)

bhuvfe:
SEC is not a requirement. I am trying to develop a method for quantifying the free form of the sufractant at this stage - so any method that works. I thought SEC would be suitable and simplest for this application therefore puchased the Biosep2000 column. I have read about the Acclaim column just recently (after I bought the Biosep column). Have you tried this cloumn before?

My purpose is to quanitfy the micelles as well as the free form as the free form is toxic (maybe there isn't any free surfactant at all in the formulation!), therefore I don't think I would want to disrupt the micelles by increasing the organic content of my mobile phase.

HW Mueller:
I cannot answer your question - I can only find very limited info on my surfactant. Is there a site specifically contains info on different surfactants??

Maybe vendors/manufacturers, like Calbiochem have sufficient info, certainly there is lots of info around. I was only interested in surfactants for washing columns and for modifying proteins. The impression I got is that micelles are in rapid equilibrium with the "free" surfactant, too fast for chromatography. Are there exceptions??

I've tried the column only with anionic surfactants (sulfonates) they are well retained and for those samples it worked fine. The product manual below was quite informative to me.

www1.dionex.com/en-us/webdocs/41787_65087-01_Acclaim_Surfactant_V25.pdf

But I doubt that you can separate micelles by RP-HPLC...

Hi

definetively rusty on this subject, but pretty sure (PW Atkins elements of physical chemistry) that micelles only form above the CMC and the Krafft temeperature.

Did not dig into that (Krafft temperature) so much when I used to analyse particles and similar as we used liquid nonionic surfactants as they can form larger micelles than inonic surfactants.
But temperature could be a factor depending on surfactant.

I doubt that you can do what you want to do. The amount of micelles will of course depend on the surfactant concentration. Since there is no surfactant in the mobile phase, the micelles will not be stable, and they will become dissolved surfactant molecules. The only question is how fast this happens. Considering that surfactants are not giant molecules, I would think that the dissociation of the micelle will happen in a time frame that is MUCH faster than chromatography, such as fractions of a second.

As far as I remember a formation of micelles is a rapid process. Also a dissociation will be rapid too. After crossing cmc you always have micelles and monomers in equilibrium. If concentration of your surfactant still increases you will have more micelles formed of the same shape and size, but the monomer concentration will stay the same as in cmc. At some point if your surfactant conc. still increases micelles are growing in size and they change shape, and then I would assume that a thermodymanic equilibrium changes as well, so your monomer conc. is different too.
I think that you are not able to separate micelles and monomers. Micelles will be destroyed if there is no monomers around. Because they exist only in equilibrium with monomers.
Anna

Thank you all for the replies.

Getting very disappointed that I might not be able to quantify the micelle and it's free form. From the trial injections, I think I managed to determine the elution of the micelle but not the free form. Maybe it's not an assay that can be done by HPLC.

If all of us, who think that the equilibrium between surfactant and micelle is too fast for the chromatography time scale, are correct then you always saw both coming out together.

I still believe ( want to believe - I can be quite stubbon sometimes) that the quantification of the monomers can be done - as suggested by annand that "After crossing cmc you always have micelles and monomers in equilibrium." which means that in any given time there is micelles and monomers in the column and due to their huge size difference, SEC would be able to sepearte the 2.

Not if the equilibrium is faster than the chromatography.