Archive for May 31st, 2012

Many HPLC methods can be used with an LC-MS with little or no modification. However, there are some things that don’t work, and some things where you may find that slight changes improve your signal wonderfully:

  • LC-MS relies on drying away all the solvent. It therefore follows that you cannot use solvents with non-volatile components. Phosphate buffers and salts are bad. They will crystalize out as the solvent is dried away, and fill the ion source with solid gunk. Ion exchange chromatography is therefore unlikely to work. But you can try changing non-volatile salts to volatile ones, such as ammonium acetate.
  • All the solvent must be dried away, so the less solvent there is, the better. Consider reducing your flow rate. Electrospray mass spec signals depend on concentration, not total amount of stuff squirted into source per minute, so reducing flow rate will also increase sensitivity!
  • You are no longer dependant on perfect separation of peaks, and good retention time, for the identification of your chemical of interest. But the machines are more expensive and in demand. Consider sacrificing a little separation for improved throughput. Most LC-MS people use very short columns, 100mm maximum, often less.

 

What matters to retention time is how fast the solvent flows linearly along the column. You can calculate a flow rate for a narrower column very easily.
The cross-sectional area of the column is πr2
The cross-section of a 4.6 mm column is about five times that of a 2.1 mm column.
Therefore you should use a flow of about one fifth, i.e. 200 µl.min-1 instead of 1000µl.min-1

Note that 200 µl.min-1 is absolutely ideal for most mass specs. Buy 2mm columns instead of 4.6mm where ever possible!

 

Written by : Dr. Lionel Hill (JIC, UK)