Need help please! polysulfated disaccharide analysis

[dkollmorgen]

I have been struggling for some time now with this analysis. I am attempting to use a published method that calls for an amino column (silica based) and a high molar concentration (1M) of ammonium sulfate as the mobile phase and refractive index detection. I am having trouble understanding the exact mechanism of the separation, which is making it difficult for me to troubleshoot it. I think it is ion exchange, but why put this on an amino column? I am currently using a Spherisorb amino. Are there others on the market that would provide more retention? Tailing is also a big problem. I ordered a polymer amino thinking it would eliminate silanol interaction, but then saw no peak, which made me think the amino group wasn't part of the retention mechanism at all. Can someone please attempt to explain to me what my mechanism of retention is (perhaps it's multiple mechansims?) Does anyone have any suggestions on what to try? Any information would be greatly appreciated!

[Uwe Neue]

Looks like the procedure is an ion-exchange separation: anionic analytes, a weak anion exchanger, and a high salt concentration for elution.

You probably should control the pH to get a consistent ionization of the amino group, and you can reduce the total ionic concentration to increase retention.

[dkollmorgen]

The only problem is when I increase retention, the tailing also gets bad (~4 ! ). Is there anything else I can try to get a more efficient chromatogram?

[Bryan Evans]

Below are LC-UV and LC-ELSD applications for Unsaturated Chondro-Disaccharides on Unison UK-Amino:

http://www.imtaktusa.com/site_media/fil ... TI462E.pdf

These methods use gradient elution. Do you have access to UV or ELSD?

[Uwe Neue]

As mentioned above: you need to control the pH using a buffer of your choice. With RI you can use anything. Phosphate buffer pH 7 is probably OK. Use a high buffer concentration initially to equilibrate the packing, then use a low buffer concentration later for your separations with a salt gradient.

[dkollmorgen]

Uwe, the pH is being controlled at 3.5 (pKa of the compound of interest is around 1). The problem I'm having is getting enough sensitivity and maintaining decent peak shape. I don't quite understand your last post. When you say "equilibrate" the packing with a higher concentration of buffer, then use a low conc for separation with a gradient . . . how can I "equilibrate" the column with something different than what I will be using for the mobile phase? If the mobile phase is ionically weaker, won't it eventually just wash off and "de-equilibrate"?

Bryan, thanks for the reference, I will check it out. My problem with using ELSD (we have one) is my sample matrix - it's full of "junk" - and most of it is water-insoluble (like my compound of interest). I tried UV at 234 nm, but it did not give me the sensitivity I need (5 µg/mL).

[Uwe Neue]

Until now, you have not yet given us information what you use to control the pH. Please be specific - buffer, pH, concentration, at both the beginnign and the end of the gradient etc.

[dkollmorgen]

Uwe, I am not using a gradient. As stated previously, I am using a 1 molar ammonium sulfate solution at pH 3.5. I am using refractive index detection currently, and it is not possible to use a gradient with RI. This is the ONLY mobile phase I am using currently.
Thanks.

[Uwe Neue]

The question about a gradient was stupid on my side. I had forgotten that you use RI. Sorry. But ammonium sulfate is not a buffer. Use a phosphate buffer pH 7, as advised before.

[dkollmorgen]

Well, I looked at some of the references above and started to do some ELSD work. I landed on a method using 40 mM ammonium bicarbonate (pH 7.0) at a flow rate of 0.8 mL/min. ELSD: 60C, and 2.5 mL/min N2 flow. The problem I have now is that my main peak and its degradant are too closely eluting. I have tried adjusting the pH, but there is little effect. Would adding in a small amount of methanol do anything to affect the specificity? What other things can I do to this system to change the specificity? Oh, the column is an Aphera amino column (polymer based) 4.6 x 250 mm, 5 micron. The peak shape is WAAAYYY better than on the previous column/system.
Thanks for any additional advice you can offer.

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[Uwe Neue]

It is good that you have made progress. I am not to keen on an ammonium bicarbonate buffer at pH 7, because there could be some issues due to volatility, but you will see if it is gives you reproducible results. In order to potentially change selectivity drastically, you could go to pH ~ 9 and a lower buffer concentration. It is possible that this does not do a lot. You could add an organic modifier. Before doing this, read the C/U manual of your column to see if it will tolerate the addition of the organic solvent.

[dkollmorgen]

I am currently using ammonium bicarbonate as my buffer, but I want to take the pH to 10.5 or 11.0 (I'm using a polymer column, so it's OK). Since I am using ELSD detection, the mobile phase has to contain only volatile components. Sooo, how can I get the pH all the way up there? I have tried using concentrated ammonium hydroxide, but it seems to be taking ALOT to get there. Is there anything else you would recommend? When I use too much NH4OH, it seems I cannot get my baseline to settle on the ELSD, so I am assuming there is too much in there to volatilize. Any suggestions? Thank you!

[Uwe Neue]

Piperidine? BP 106, pKa 11.2

[dkollmorgen]

I assume you mean using the piperidine as the buffer in lieu of the ammonium bicarb?

[dkollmorgen]

Piperidine and pyrolidine are not listed as volatile buffers. Remember, I am using ELSD which requires a volatile buffer. I am new to this type of detection, so I'm not sure if this is a safe bet or not.