Choosing a column for a polar analyte

A few years ago I was developing an LC-MS/MS method for psilocin in rat plasma at a preclinical CRO. I started where most people start, on a standard C18. The peak came off almost at the void volume. No retention, no separation from the matrix, and ion suppression sitting right on top of it.

I changed the gradient. Same result. I dropped the organic to 2 percent at the start, which is about as low as you can go before a standard C18 starts to dewet. Slightly better, still not a peak I could quantify.

The column was the problem. Psilocin is polar and basic, and a plain reversed-phase C18 has very little to hold it with.

Why polar compounds fail on a standard C18

Reversed phase retains by hydrophobic interaction. A polar analyte has almost no hydrophobic surface to offer, so there is nothing for the C18 chain to grip. The compound stays in the mobile phase and leaves with the void.

Dropping the organic content is the obvious fix and it only takes you so far. Below about 5 percent organic many standard C18 phases undergo phase collapse, or dewetting. The bonded chains fold in on themselves, retention drops instead of rising, and the effect is not always reversible. So the fix that seems free is the one that damages the column.

What to buy instead

Four options, in the order I would consider them.

An aqueous or polar-embedded C18. These are made to run in highly aqueous mobile phases without dewetting, and the embedded polar group adds a second interaction that holds polar compounds. This is the smallest change from what you already run, and for a moderately polar analyte it is often enough. Ask for the phase to be described as “AQ”, “aqua”, or polar-embedded, and check the stated minimum organic.

HILIC. Hydrophilic interaction chromatography retains polar compounds well, and it uses a high-organic mobile phase, which sprays better and usually gives you a real sensitivity gain on an MS. The cost is that HILIC is less forgiving. Equilibration is slow, and if you inject a highly aqueous sample onto a HILIC column you will distort the peak. Your extract has to end up in something close to the starting mobile phase.

Ion pairing. It works, and I would leave it last for an LC-MS method. Ion-pair reagents suppress ionisation and they contaminate a source in a way that takes a long time to wash out. If the instrument is shared, you will not be popular.

Derivatisation. Adds a hydrophobic group so a reversed-phase column can retain the compound. Real sample prep work, so only worth it when the other three fail.

What to check before you order

• The minimum organic percentage the phase tolerates. If you plan to start at 2 percent, a standard C18 is the wrong purchase.
• Whether the phase is described as aqueous compatible or polar embedded.
• The pKa of your analyte and where your mobile phase pH sits relative to it. A basic compound run near its pKa gives broad and irreproducible peaks, and no column fixes that.
• What solvent your extract will actually be in at injection. This decides whether HILIC is realistic.

The short version

If your compound is polar and your peak is at the void, another C18 will not save it. Work out how polar it is, check the pKa, then choose between an aqueous C18 and HILIC based on what your sample is dissolved in.

If you tell me the analyte and the matrix, I will tell you which of these applies before you order anything.