The Huck Institutes of the Life Sciences

Sample preparation for MS analysis of small molecules

How to submit samples for small molecule analysis by LC-MS

Thanks in large part to the care with which our users prepare their samples, we are able to successfully analyze hundreds of samples each year by LC-MS. Each sample has its unique requirements in terms of preparation, ionization, detection and interpretation. We make every effort to analyze your sample successfully, but there are certain limitations. Analysis of pure, low-molecular weight organic compounds is easy; mixtures are harder; and comprehensive analysis of complex mixtures of high molecular weight compounds can be very time-consuming. We have to balance the routine demands of the many with the special needs of the few.

 

Less is better!

 

Unlike NMR, each sample introduced into the mass spectrometer never comes back out. While most of it ends up in the vacuum pump oil, a lot sticks to the interior of the instrument, so instrument cleaning is occasionally required. Fortunately, mass spectrometry only needs miniscule amounts of material to produce useable data. Our instruments can normally detect 1 to 10 ng injected or less. For a compound with molecular weight of 500 that is 2 to 20 pmol. (Compounds that are more difficult to ionize will require somewhat more sample.) We always try to introduce the minimum amount of material necessary to produce a useful spectrum because too much sample means higher background signals will build up requiring more downtime for cleaning.

 

Sample Preparation for LC-MS

  • A purified sample has the highest chance of a positive identification.
  • If possible, samples should be submitted as solutions dissolved in water, methanol, or acetonitrile since they will be injected into a stream of these liquids. Dichloromethane can be used.
  • Since it is virtually impossible to measure out ng amounts of sample, we assume that most samples contain roughly 1 to 10 mg per mL of solvent and therefore dilute the samples 50:1 with methanol or acetonitrile unless instructed otherwise.
  • Typical injection volumes are 50 uL, so if you submit a solution of a pure compound, it should contain a minimum of 2 to 20 ng sample in 100 uL of solvent.
  • Please make sure your samples do not contain any kind of additives such as:
    • Alkali salts and non-volatile buffers, because of signal suppression and ionization interference.
    • Polyethylene glycol (PEGS), plastics and plasticizers suppress the signal and clutter the spectrum
    • Detergents suppress ionization and compete for ionization, in particular, Tween (any), Triton (any) NP-40 and SDS.
    • Additives compete for ionization. Additives are carbohydrates, glycerol, EDTA, Urea, GnHCl.
  • Use:
    • Dilute volatile buffers (1-20mM), i.e., ammonium formate, acetate and carbonate
    • Dilute volatile acids for positive ion mode (formic, acetic acid, TFA)
    • Dilute volatile bases for negative ion mode (triethyamine, ammonium hydroxide)