Liquid Chromatograph - Mass Spectrometry
LC-MS

The system we use is composed of two instruments. High Pressure Liquid Chromatography (HPLC) separates the compounds by passing them through a silica-based column that contains a bonded stationary phase selected to optimize separation of various compound classes. Compounds are eluted by a liquid mobile phase - usually an organic solvent, water, or a mixture. The HPLC can also be used preparatively - an aliquot of the separated compounds can be split to a UV detector, which measures the absorbance spectrum from 190 – 800 nm and collects the eluent in a programmable fractions collector. In some cases we can separate pure compounds and subject them to further analysis such as NMR or radiocarbon analysis.

The ionization source is the interface between the HPLC and the mass spectrometer. We use both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) for lipid analysis. Both types of ionization occur under atmospheric pressure in a nitrogen atmosphere. ESI operates by applying a strong electric field (~10⁶ V/m) to a capillary containing the liquid mobile phase and analytes from the HPLC. The field causes charge to accumulate on the surface of the liquid, which breaks into small droplets (the “spray”) . As the droplets break apart the charge is desorbed from the droplet to the analyte. APCI is a softer ionization technique that results in more molecular ions and fewer fragmentations. As solvent moves into the APCI source it is heated to a high temperature, such that both the mobile phase and analytes are gaseous by the time they encounter the corona discharge electrode. The corona discharge forms primary ions of N 2 ^.+, which reacts with the evaporated mobile phase to form secondary ions which then react with the analyte. ESI and APCI can be selected for positive ion or negative ion modes, which give complementary structural information.

The mass spectrometer that we use to detect is a Q-Tof micro. This instrument is a two stage mass spectrometer consisting of both a quadrupole and a time of flight (i.e. “Q-Tof”) with a collision chamber between. This means that we can do true MS-MS experiments, using the quadropole to select a parent mass of interest (for example the molecular ion of an IPL) then applying a voltage to the collision cell causing fragmentation, then using the ToF to detect the daughter mass produced. Additionally, the ToF has a mass resolution of 5 mDa, allowing us to determine unique elemental composition by the exact mass of a compound.

some details

• HPLC is a Waters 2795 Separations Module
• MS is a Waters QTof Micro
  

references

• Talbot H.M., Summons R.E., Jahnke L.L. and Farrimond P., 2003. Characteristic fragmentation of bacteriohopanepolyols during atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 17, 1-9.
• Sturt H.F.,  Summons R.E., Smith K., Elvert M. and Hinrichs K.-U., 2004. Intact polar membrane lipids in prokaryotes and sediments deciphered by ESI-HPLC-MS n  New biomarkers for biogeochemistry and microbial ecology. Rapid Communications in Mass Spectrometry 18, 617-628.
• Jahn U., Summons R., Sturt H., Grosjean E. and Huber H., 2004, Composition and source of the lipids of Nanoarchaeum equitans and their origin in the cytoplasmic membrane of its host Ignicoccus sp. KIN4/I. Archives of Microbiology 182, 404-413.