Chromatography Forum

I understand that columns with embedded polar groups are not particularly useful for HPLC-MS because they give rise to a lot of background.

I also understand that it is not that these columns lose stationary phase more readily but that the stuff that bleeds off ionises readily in the MS (at least in ES mode).

Does the same apply to columns which have bonded ionic groups like Primesep ? A simplistic view would be that if the column contained negatively charged groups for the analysis of positively charged solutes, then bleed of this group from the column might be a problem in negative ion MS but not positive ion MS, and the reverse for columns with groups of oppoisite charge. I would welcome any comments from SIELC or anyone who has experience of this issue. Comments on the EPG bleed issue would also be appreciated-did I get it right above?

Let’s do some math. A typical 150 mm column with 2.0 mm id holds about 0.3 g silica with about 12% carbon load on Primesep type material. This translates to about 36 mg of stationary phase.
With molecular weight about 200 Da total amount of ligand is 36/200=180 mcM
Since columns typically withstand without significant lost of ligand 1000 injections with average run time 10 min and flow rate of 0.25 ml/min the column will be in contact with about 2.5 L of a mobile phase. Assuming 10% ligand is washed (actual lost of retention in 1000 injection within recommended pH range is fraction of per cent) we would have ligand concentration about 180/ (10x2.5) = 7 mcMol in the total MP volume.
Typical buffer that we recommend and people use has 5-25 mM concentration which is 1000 times higher than potential ligand concentration.
You can probably argue that ligand is not volatile while TFA, HCOOH, AcOH are volatile. But if amine is not volatile which usually is the case in HPLC then volatility of the acid is not so much important. pKa of the acid may be more important. Our ligand has relatively low pKa value from 1.5 to 3.5, which is less than TFA but more than AcOH. This calculation is probably explains why Primesep phases used widely as MS phase for polar amines, AA, and peptides. I would not be surprised that in gas phase with molecules in high energy state all equilibriums that we apply for water based solutions are not very useful.

I've been using a polar embedded columns, Polaris 50 x 4.6, for LCMS for several years. We use it to survey samples for all the chemists in the company.

Works fine, run the sample in 4 modes (low pos for MW, high pos for substructure, low neg for MW, high negative for substructure) and diode array (multifunction experiment all in one run.

Normally use water containing 2.5 mmolar ammonium acetate and 30 ml/acetonitrile (retard microbial growth) per liter water. The organic solvent is either acetonitrile, methanol, or mix. Add 0.1 ml post column of 25 mmolar ammonium acetate in methanol to enhance ionization at 100% organic.

Ours is a polaris column..

http://www.metachem.com/metasil/Index.htm