Chromatography Forum

Can anyone suggest the best column and conditions for highly polar compounds, Thanks in advance

Which technique would you like to utilize? HILIC?
Are your compounds ionizible? Are they large?

Many questions, but these are some of the first questions you should ask yourself before throwing yourself into method development.

Best Regards

yes, it is ionizable, and also not a big molecule

By any chance, can you tell us what the analyte is?

In general, RP columns have limited use for highly polar compounds (such as organic acids, and nucleic acid bases, etc). On the other hand, HILIC columns seem suited for many highly polar compounds (such as carbohydrates, drug metabolites, etc). Sometimes, solubility of the analytes in the diluent may impose problem. However, you don't have too much control or flexibility in selectivity when using above two approaches.
When dealing with ionizable highly polar compounds and RP columns fail to provide satisfactory results, the alternative you might consider is either reversed-phase/anion-exchange or reversed-phase/cation-exchange mixed-mode columns, depending on the charge of the analyte. Acclaim Mixed-Mode WAX-1 (for anionic compounds) and Acclaim-Mode WCX-1 (for cationic compounds) could be the solution to your challenge. The common limitation of above two approaches. The main feature of these columns is adjustables selectivity which is explained in details in the attched links. The WAX-1 can retain anions as polar as chloride and lactic acid and the WCX-1 can retain cations as polar as lithium, sodium, and catecholamine. Of course, the success of the analysis depends heavily on other compounds in the sample and the sample matrix, too.
Please feel free to contact me if I can be of further help.
http://www1.dionex.com/en-us/webdocs/65 ... PN2024.pdf
http://www1.dionex.com/en-us/webdocs/48 ... 021407.pdf

There are several chromatographic modes/conditions that would satisfly your (very general) requirements. Ion pairing chromatography, ion-exchange, mixed mode columns, HILIC, PGC (unders certain circumstances) etc... If you want more specific conditions you need to be more specific on the charge/pka of your analyte and it's polarity...

What a beauty, a compound almost as polar as the conditions up here in the far north of Sweden from where SeQuant originates . See http://webcam.educ.umu.se/

It seems that you can control the dissociation of this compound by pH of the mobile phase. Then it make sense to use the ZIC®-HILIC column that maintains its character regardless of pH, but offer robust separation of both large and small polar compounds.

HILIC is a very straightforward technique that is easy to use and enable robust conditions for separation of complicated hydrophilic compounds. I am convinced that the "LC community" very soon will forget old ion-pairing methods or RP mixed mode separations.

Please search for more information at www.sequant.com

Wow, Einar you are funny!

This is a very bold prediction and I hope that it is not all that money from Merck got into your head (joking, congrats on the sale). I am sure that you did not mean to state that HILIC will take over the world of chromatography. My guess is that HILIC is accounted for less than 10% of the column market right now (estimated to be 500M??). It is true a lot of companies introduced new HILIC columns recently (Pittcon), as well as…mixed-mode, but this is purely extension of product lines. Reverse phase chromatography is pretty much done developing (as a C18/C8, end-capped, etc.) and people are looking for the next “scienceâ€

Are these (HILIC or mixed moxe like WAX-1) available for flash LC as I am using RP for highly polar and complexing compounds and facing many problems

Or simple normal Phase is sufficient?

'Hypercarb columns based on porous graphitic carbon have routinely been used for analysis of compounds with negative log P.


Details of this column can be found on www.thermo.com/hypercarb

Einar,
I sort of loath this, but you are practically soliciting this repetition: If one uses your primarily recommended mobile phase (fair concentration of ammonium salt, ACN), even Waters´s silica HILIC does not show any SiO-interactions. On the other hand, under conditions where the Waters silica HILIC shows these IX interactions, your ZIC HILIC does likewise.

Hans:

I do not know what you are referring to.

1. Ion-exchange on silica works wonderfully - no peak tailing, good peak shapes.

2. Why do you think that ZIC-HILIC would show ion-exchange, and what kind of ion exchange, if any?

OK, it seems that repetition is in order.
I was referring to Einar´s statement
"Then it make sense to use the ZIC®-HILIC column that maintains its character regardless of pH, but offer robust separation of both large and small polar compounds."
From this I conclude that it is claimed that ZIC does not give rise to any interactions with SiO-. I repeatedly stated or gave evidence that it does at low ionic strength and higher pH. As a matter of fact it behaves parallel to the Waters Silica HILIC (Atlantis): At pH and ion conc. where the Waters sequesters Na+ (22Na+ is adsorbed at the start of the column) or causes X- exclusion (negative ions, like Cl- TcO4-, elute ahead of to) the ZIC does it also. At high Ammonium salt concentrations (as recommendet by Sequant) or at low pH both columns do not show evidence of ion interaction with SiO- .

So: My statement has no qualitative comment on ion exchange on silicas.

Well, I have never seen a silica-based packings that is free of silanols. However, contrary to common perception, silanols are nothing bad. They are only bad if buried under a C18...

Dear All,

In principle HILIC stationary phases can be divided into three different groups:

Neutral - no electrostatic interactions
Diol phases, amide phases

Charged - strong electrostatic interactions
Plain silica phases, aminopropyl phases

Zwitterionic - weak electrostatic interactions
Merck SeQuant ZIC®-HILIC and ZIC®-pHILIC phases

Good and Bad Secondary Interactions
With stationary phases that change their properties as a function of pH (such as plain silica), there is a complex relationship between the retention and the pH, making it harder to predict and optimize the separation.

The separation selectivity between different analytes can be favored by the electrostatic (ionic) interactions that are contributing to the retention with charged HILIC stationary phases. With too strong electrostatic interactions, however, the presence of high concentrations of salt ions in the eluent is required to disrupt these interactions during elution, but this is disadvantageous since such eluent conditions reduce the sensitivity for several detection techniques (including MS). Balancing the charge with a zwitterionic stationary phase is the design strategy SeQuant have used to get the best of both worlds.

One also should bear in mind that many compounds are pH dependent, and will change its behaviour depending on the pH. With a zwitterionic phase having a balanced charge, you may set the experimental conditions best suited for the analytes and not to the column. This may be confusing in the beginning but when one learns to utilise this, it gives a new dimension to explore in method development.