Polar Compouds Separation_Column Recommendation

[Molly]

All:

I am trying to develop a method for a pharmaceutical mixture that contains highly polar compounds. The polar compounds (300-500MW) have multiple COOH and NH2 groups. Due to sample stability issue, the diluent has to be neutral pH between 6-7, which means COOH will be deprotonated (negatively charged) and NH2 will be protonated (positively charged). I am currently using YMC ODS-AQ column (15X0.46, 3um) and ACN/pH 6.8 phosphate buffer as mobile phase. The gradient was 0%ACN hold for 5 min and 0%ACN to 30%ACN from 5 to 20min. The method did a good job of separating most of the compounds, except that there are three early eluters have retention time of 2.11, 2.27 and 2.43min. The peaks are not baseline separated, and all elute during the first 5min 0% ACN hold. Temperature did not help the separation.

What should I do next? Some other RPLC column that can be good at 100% aqueous (any recommendations?). Will HILIC column work? The sample is in pure Aqueous, will that be a problem for HILIC? Some literature work suggests ion pair with sulfonic acid, but I would rather not go there unless I have to.

Any suggestions will be appreciated.

Thanks, Molly

[Uwe Neue]

My recommendation is to use the Atlantis T3 column from Waters. It generates in many cases a roughly 2-fold higher retention than other reversed-phase packings that are compatible with 100% water.

[Molly]

IS that a HILIC column? What mobile phase would you recommend?

Thanks, Molly

[Uwe Neue]

No it is a C18 compatible with water and with maximized retention.

[SIELC_Tech]

Molly,

Try mixed mode columns they will retain your compounds by combination of ion-exchange and reverse phase mechanisms. You don't even need to go to lower organic. You can even develop an isocratic method. There is no need to use ion-pairing reagent either. Here are few examples of hydrophilic compounds retained on Primesep columns:

http://www.sielc.com/compound_108.html
http://www.sielc.com/compound_002.html
http://www.sielc.com/compound_106.html
http://www.sielc.com/compound_005.html


or all applications sorted by compound:
http://www.sielc.com/Applications_By_Compound.html

You can use the following step by step method development guide. It will ask you bunch of questions and then show recommended column and mobile phase:

http://www.sielc.com/MethodDevelopment_ ... dType.html

If you send us sample we will screen it for you on of our columns (free of charge)

Regards,

Vlad

[Yury Zelechonok]

Most zwitterions can be retained by combination of ion-exchange and RP mechanism on Primesep column without any buffer in the mobile phase.
See this example:

http://www.sielc.com/application_080.html

k’ over 5 can be obtained with only water and acetonitrile (MeCN can be of any amount from 0 to 80% to adjust selectivity). In this system acids will be deprotonated and bases protonated as you need for stability. With concentration of MeCN over 80% retention can be too long.

[Bryan Evans]

Hi Molly!

I would suggest our Unison UK-C18 column. It's great at retaining
polar analytes and can be used in 100% aqueous mobile phase.

http://www.imtakt.com/TecInfo/TI269E.pdf

[Patrik Appelblad]

Dear Molly,

With such post title, plenty of alternatives will be given…and I’ll try to give my five cents of worth as well!
Your compound might very well be retained by these previous alternatives, but as you specifically asked about HILIC, I’ll try to explain why that option would be a superior alternative.

Your existing method is running on an AQ-phase, but still you have little retention for the three early eluters. That would indicate that an opposite retention mode like HILIC would be superior rather than testing another brand of AQ columns. Secondly, your polar compounds require neutral pH between 6-7, which rules out the use of TFA or any other acidic ion-pair reagent as mobile phase modifier. Instead, I’d use a HILIC column, and in particular one of the SeQuant columns that have permanent zwitterionic stationary phases. With such stationary phase, you do not have to tweak the experimental conditions to best suite the column, rather set the pH interval most suited for the analytes. At neutral pH, your analytes will be zwitterions, and for such compounds we have several application examples, like aminolevulinic acid, betaine, etc but unfortunately they have not been posted on the web yet.
However, Polymer Labs have produced an application note using ELSD detection after ZIC®-HILIC separation for Quantification of Betaine in Chinese Lycium using Hydrophilic Interaction Chromatography (HILIC) and ELS Detection.

Aqueous samples are not a general problem in HILIC it all depends on the system set-up. If you inject a large sample volume, there will definitely be a risk that the sample is self-eluting or affecting the retention, as water is the strong solvent in HILIC. However, the same is true with high portion of organic solvent in sample solutions used under RPLC…and that I guess is done everyday around the globe!

[Uwe Neue]

Well, you got the list....

I think that it is easiest to stick with a well designed more retentive C18 as I had proposed above.

If you try more than one thing, it will be interesting to see what your results were...

[Einar Ponten]

In objection to Uwe I think it is questionable if the column he suggest is "well designed", although it could retain "polar compounds". Everybody may have noticed marketing activities where Waters recently introduced the reversed phase T3 columns, claimed to be suitable for polar compounds, and suggested by Uwe in the previous post.

A RP phase will require a relatively high ratio of water in the mobile phase and that results in a limited possibility to control and modify the selectivity, and also often is very negative for sensitivity in many detectors.

Todays need for sensitivtity often require electrospray MS, or in terms of general detectors evaporative light scattering detectors (ELSD or "Corona" from ESA). Then the water content of the mobile phase will be of crucial importance for the analytical performance, the less water the better performance

Mollys concern about stability for her compounds at low pH rules out most "HILIC phases", simply since they are basically only applicable at low pH in order to avoid very strong ion exchange interactions (and high salt concentrations to break these).

This is where the zwitterionic stationary phase (ZIC®-HILIC) stands out since its behavior is more or less independant of mobile phase pH. This means that the conditions can be adjusted and optimised to fit the analytes and the application for best selectivity and sensitivity, at a relatively high ratio of volatile solvent (typially acetonitrile).

The best choice in this case is quite obvious and I agree with Molly that ion-pairing really is no alternative regardless of detector.

[HW Mueller]

Einar, your Zic-HILIC column behaves like any other silica based column which I have tried in this way: They were all extremely pH sensitive toward retention of 22-Na+. As one gets closer to basic the Na+ gets stuck on the column, at the more acidic pH it comes at or near the to (tm). Thus, I was quite negatively surprised at the neglect of taking silanols into account in a recent article of LCGC by Risley and Pack.
About a year with the Waters Atlantis Silica and the SeQuant Zic-HILIC my conclusion is: Use HILIC only when all types of polar RP columns completely fail.
And to the manufacturers of LC-MS: Work full blast on apparati which are compatible with H2O and nonvolatile buffers/salts.

Uwe, in a 2005 chain on HILIC you said something like that a well designed zwitter ionic column should not show any silanol activity toward small organic(?) cations. Can you expound on this?

[Uwe Neue]

I need to disagree with Einar. There is nothing wrong with a well designed reversed-phase packing for the retention of polar compounds, such as the family of Atlantis products, the Atlantis dC18 and the Atlantis T3. These C18 columns can be used without difficulty in 100% water. The T3 bonding is an improvement over the dC18 bonding, with better peak shape and still some gain in retention compared to the dC18. But both columns beat anything else in retentivity in fully aqueous mobile phases.

Considering the problem that Molly needs to resolve, I continue to think that the most universal approach is to use a well designed C18 that is compatible with a fully aqueous mobile phase. I am a fan of HILIC, but one needs to understand that HILIC is a completely different tool, and it may not work as well for a broad range of compound hydrophobicities. HILIC will definitely require a complete redevelopment of the method, while this may not be as bad with the use of the C18s optimized for this application.

[SIELC_Tech]

IBut both columns beat anything else in retentivity in fully aqueous mobile phases.

Now I would like to disagree with Uwe (i guess we agree to disagree on this forum). I think that ion exchange and mixed mode columns will provide you with a longer retention in aqueous MP. With mixed mode you can achieve K'=5-10, how long you can retain amino acids (or other zwitter ions) on Atlantis T3? (I don't have this column but will take a word from anybody).

http://www.sielc.com/pdf/SIELC_January_2005.pdf

You can achive even greater retention if you can play with pH and have carboxylic acid fragment in non-ionized state. In this case you can analyze amino acids as basic compounds (amines):
http://www.sielc.com/pdf/SIELC_June_2005.pdf


P.S. Dear Uwe, we are willing to do comparison study of Atlantis T3 (performed in Water's labs) and Primesep (at SIELC)-we just need to agree on a set of compounds. Einar can joint us in this "friendly" competition with his HILIC columns.

[Einar Ponten]

I was about to object to Hans, but apparently I’m caught by the cross-fire...

Mollys original post was a question of general interest. The key facts we got for a discussion was:
A. develop a method
B. highly polar compounds (charged, zwitterionic?)
C. due to sample stability a mobile phase pH 6-7 is required
D. Three peaks fairly retained/resolved despite 100% aqueous mobile phase on a “polar RP columnâ€

[Uwe Neue]

Vlad,

Please read the constraints that were posted by Molly. The compounds have a dual charge, cationic and anionic, and one is not free to manipulate the pH at will due to the instability of the compound(s) outside neutral pH. Thus one has severe limitations to change the charge of the compounds and thus their interaction with ion exchangers. Zwitterions do not interact with ion exchangers, unless you can manipulate the charge ratio.

This is exactly the situation where a highly retentive reversed-phase packing with full compatibility with a fully aqueous mobile phase is the most promising approach.