Why, oh why, are my peaks tailing? There are two culprits who are to blame:
1. 90% of the blame is on your column.
2. And the remaining 10% - is on you.
Method development is based on one fundamental principle - the more time your sample spends on the stationary phase, the better are your chances of getting a good chromatogram.
However, if your analytes spend too much time on the column, you get broad peaks and tailing.
Therefore, the objective of method development is to find a suitable middle ground between excessive retention and no retention.
So:
1) Choose the right column chemistry.
- Non-polar analytes = non-polar column. Like a C18 or C8.
- Strongly hydrophobic analytes (like carotenoids) = really non-polar column like a C30.
- Hydrophilic analytes (like carbohydrates) = think of HILIC.
- Proteins/peptides/macromolecules = consider a C4 column, 300A pore size.
- Ionised analytes (like pollutants in water) = ion exchange column.
- Ionisable analytes (like neurotransmitters, for example) = C18 column, with pH controlled mobile phase.
2) Choose the right column manufacturer and the right column brand.
The brand matters. The manufacturer matters.
There are dozens of brands. Each of them will have impressive specifications - on paper. The column's actual performance is an entirely different issue.
Several variables affect column performance. These are:
The purity of the base silica, uniformity of the particles, pore size distribution, bonding chemistry, and importantly, the column hardware and the column packing methodology.
Column performance really depends on how well the column has been packed. And it depends on the quality of the column hardware itself.
The column's inner wall must have a mirror-like finish, and must meet very stringent specifications. Cheap columns are made from inferior column hardware and are poorly packed.
The quality of the packing methodology in turn depends on the quality of the packing pump.
Across the world, there are very few manufacturers of top-quality column packing pumps. The best column manufacturers use only these pumps.
I should know. I've visited some of these column packing facilities myself.
Over the past thirty-five years, I've come to trust only a handful of really good column brands.
I'd rather pay more for a good brand that I trust, than experiment with a cheap brand.
But please note: Even the best columns may start tailing after a few weeks of usage. And that's because of you.
If you refuse to use a guard column, forget to rinse your column after using buffers, use extreme pH conditions, and store your column in the wrong solvents, you have only yourself to blame if your column retaliates with tailing peaks.
So. Choose your column brand wisely. Choose your vendor very wisely. Treat your column really wisely.
To take care of the tail, use your head!
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Resource Person: SK Srinivas
