My labmates tried a number of different columns in a number of different conditions to settle on HILIC with amino column at pH of 9 for best results. They did this testing a number of spiked in compounds and using a derived scoring method to analyze peak ‘goodness.’ After settling on this, they discovered retention times to improve the scan method and were able to quantitate over 100 metabolites. They also ran an experiment in E. coli with 13C-labelled glucose and carbon starvation to compare metabolomes, by running samples from labeled/starved with unlabelled/unstarved and labeled/unstarved with labeled/starved, they could determine intensity differences.
Some side notes:
- Cellular metabolites make up less than 3% of dry cell weight in E. coli
- Gas chromatography is good for low-weight, but not for low volatility and thermal stability (such as phosphates)
- Metabolites sensitive to environmental change, 39 significantly changed between conditions, notably FBP and IMP
- Reverse Phase Chromatography: non-polar nonmobile/polar mobile phase, compounds that have more surface area to interact with nonpolar elute later (nonbranched, saturated)
- HILIC: Hydrophilic Interaction Chromatography: polar nonmobile/nonpolar mobile, aqueous layer around nonmobile which grabs hold of polar analytes
This paper is relatively straightforward, a good layout of another column type to use to capture more metabolites accurately and expand the abilities of mass spectrometry to further metabolomics. A good, brief experiment comparing compound levels between two growth-types of E. coli.
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