Polymer based NH2 columns

[mashtikar]

Hey,

Has anyone worked with a polymer based NH2 columns? I am trying to separate a mixture of Phosphatidylcholine and Phosphatidylglycerol and not getting enough resolution with C18 column, so was wondering if I can shift to NH2 column. Polymer based as I am detecting them on CAD. On Silica column (normal phase) I get retention for PC but no retention for PG, NH2 column could help retain PG but how would a polymer based column retain PC?

Any suggestions/tips are welcome!

Thanks.

Mukul

[Gerhard Kratz]

Have a look on the Application notes of Ashahipak NHP column: These are polymer-based amino columns that incorporate an amino group in the polyvinyl alcohol based gel. They are suitable for analysis of saccharides in normal phase like silica-based amino columns. They are superior to silica-based amino columns in terms of reproducibility and durability because of their stability in a wide range of pH. And you can have a look on HILIC columns. Beside the Ashahipak NHP column most of them are based on Silica. Good luck

[mashtikar]

Thanks for the reply Gerhard,

Even I was thinking of either Asahipak or apHera from Sigma but both do you know anything about or have heard about their performance for resolving phospholipids?

I have never worked with saccharides before so its a little difficult to get a perspective of the column based on the analysis of sugars.

Also I dont want to buy a column worth €1200 and find out that it doesnt work.

[Andy Alpert]

Why does the column have to be polymer-based? Our silica-based anion-exchange material, PolyWAX LP, separates PC and PG nicely using HILIC conditions with volatile mobile phases. The columns cost a lot less than €1200 and we guarantee their performance.

[sepscientologist]

Andy- I haven't tried your column but I did evaluate polymer NH2 (Alltech, Aphera, Asahipak) vs silica based amino (Supelco, Phenomenex, Waters, Agilent)
and observed ~ 10x higher background noise by ELSD when using the silica based columns. People generally attribute this to silica coming off the column
but is that really what is going on?

[Andy Alpert]

Actually it's probably the aminopropylsilane that constitutes the coating. It's notorious for creation of a locally high microenvironment of pH that leads to the hydrolysis of the Si-O-Si bond through which the silane is anchored to the surface covalently. Consequently, standard "amino" columns steadily lose capacity. An exception may be the Shiseido Capcell Pak NH2, which is made of an oligomer that's vapor-deposited onto the silica. Also, Phenomenex claims that their Luna NH2 will tolerate a pH as high as 11. That's only possible if they specially treat the silica to make it resist high pH (possible but expensive), and I've seen no data that backs up that claim for high-pH stability. Our silica-based WAX material is based on linear polyethyleneimine (PEI) that's adsorbed to the silica and then crosslinked using a bifunctional amine-reactive reagent, rather like making an epoxy glue network on the surface. The resulting coating is not covalently attached to the surface but is actually more stable than the underlying silica.

What were your running conditions? Were you using an extreme of pH in either direction?

[sepscientologist]

I wasn't even buffering. Just 75% ACN and DI water. It was at 70C though. I also ran the Tosoh amide column for a long time and liked it
but did observe higher background by ELSD comared to polymeric amino. I also ended up not using the polymeric aminos because Tosoh's
silica based amide phase seemed to work better for sugars.

[Andy Alpert]

A sustained temperature of 70°C will strip a hydrophilic, silane-based coating off most silicas, and will accelerate the dissolution of the silica itself in the presence of any base. That probably accounts for the high background you observed with ELSD with the Tosoh column. I don't see why it's necessary to use high temperature for such an application; we got a nice separation of PC, PE and PS (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, resp.) at room temperature using a PolyWAX LP column and a gradient from 95-50% ACN with 5 mM ammonium formate, pH 6.5, and with ELSD detection.

The Tosoh Amide-80 column does work well for sugars in general, but reducing sugars will exhibit anomer separation unless the mobile phase is made somewhat basic by inclusion of something like 0.1% triethylamine. Using an amino column, polymeric or otherwise, renders that unnecessary because the stationary phase provides the locally high pH necessary to accelerate the mutarotation that causes the anomer peaks to collapse into a singlet peak.

[mashtikar]

Thanks all for the reply,

Andy - column doesnt have to be polymer based, I just need a column that I can use with Charged Aerosol Detector and standard silica based NH2 column can not be used.

I will look into PolyWAX LP as you have suggested. Could you give me some more details about your separation method for PC and PS? Did you guys try PG?

Thanks

Mukul

[Andy Alpert]

Mukul:

See for yourself:



PG (= phosphatidylglycerol): Yes. It coelutes with PS because they have the same net charge at the polar head and hence the same degree of interaction with the anion-exchange material.

I believe that there are a couple of recent papers in the literature on HILIC of phospholipids. You might try a systematic search for alternatives to our approach if you don't like this separation.

- Andy

[mashtikar]

Thanks for the chromatogram Andy... This column suits our needs...

Now just the matter of convincing the boss!

Thanks again!

Mukul

[XL]

Andy,

What detector did you use for the chrmatogram shown above? Also, did you try a lower buffer pH such as pH between 3 to 4.5? Phospholipids seem to be a popular topic and there are seldom too many methods for this application.

[Andy Alpert]

Hi, Xiaodong -

Actually, this chromatogram came from one of our customers. They were using an ELSD detector but I don't know the model. As for the pH: At pH 6.5 the phosphate groups are starting to acquire a second (-) charge, especially at the surface of an anion-exchanger with a locally high microenvironment of pH. I presume that would result in better retention than would be true at a lower pH.

Andy