A practical approach to metabolomics. Rob Linforth Food Sciences – Biosciences University of Nottingham. Metabolomics. Goal – The analysis of everything in anything biological Reality – The analysis of anything in everything
Food Sciences – Biosciences
University of Nottingham
Effectively targeted analysis, or, broad analyses where many compounds are present, but, many at levels too low for detection in the sample matrix.
Sampling, injection, separation
Where the sample gets in
Hot to ensure compounds
volatilise and enter column
Where the compounds
leaving the column
Enters injector and
Gas used typically Helium
Where the compounds in the
sample are separated
As temperature increases, compounds move….
Dependent on partition with gum (polarity) and volatilityGas Chromatography: Column
Compounds enter a high vacuum region where they are bombarded by high energy electrons that cause compounds to fragment. Fragmentation patterns are dependent on the structure of the compound. Ions are guided to the analyser where an electric field separates them on the basis of their mass and they are detected.
Later peaks are
Higher boiling point
With GC this is the mass spectrum
Library spectra: C11 acid ester
Library spectra: C19 acid ester
Me-SalicylateExample of Tea analysis
Boiling Point of compounds increases
C12Fatty acid profiling
Fatty acid profile of sample compared with that of standard (mix of 36 saturated and unsaturated FA).
What fatty acids are there and in what proportions.
Lipid can be fractionated (polar vs. non-polar) and “sub-profiles” determined.
Used in product authentication or diet impact studies.
Fatty acid methyl esters produced by derivatization of lipid: transesterification with trimethyl sulfonium hydroxide in methanol
1 – 5,000psi
Tubing, fittings etc have to be
designed to cope with high
Compounds are retained on the column to different extents. This depends on the affinity of the compound for the column packing
(stationary phase) relative to its affinity for the solvent. Plus the competition of the solvent molecules for the sites where the analyte is absorbed.
Essentially dependent on the polarity of the compound and the stationary and mobile (solvent) phases
The solvent font is the time at which
un-retained molecules arrive at the
end of the column/detector
% MeOH in
Water increased from
10% to 60% over 2 ramps
separated by an isocratic phase
Lots of charge per molecule mass spec is a mass/charge analyser. Work out original mass by reversing maths
Objective: purification of an unknown for identification. But, still a significant number of peaks – and hence compounds in sample (40L of bacterial broth now in a volume of 1mL).
Active compound detected by separate bioassay.