Ultra Performance Liquid Chromatography coupled with Mass Spectrometry

Louvain-La-Neuve

Ultra Performance Liquid Chromatography is a technique similar to that of HPLC when it comes to separating different components of a sample, as well as identifying, quantifying and separating the components of a mixture. The major difference in UPLC is the particle size in the column which is less than 2µm providing better separation than in HPLC where the size of these is limited to 5µm. These smaller particles require higher pump pressures (100MPa vs 40 MPa) which makes this method very efficient with rapid analysis and higher resolution.

On leaving the UPLC, the separated compounds arrive in the detector, the Mass Spectrometer.

The choice of ionization of the sample depends on the purpose of the analysis and the nature of the compound analyzed. In the platform, we use the Electrospray Ionization Source (ESI) in positive or negative mode where relatively little energy is communicated to the analyte, which means that there is little fragmentation.

The liquid eluate leaving the chromatographic column is pumped through a metallic capillary maintained between 3 to 5 kV. It is nebulized at the end of this silica capillary where a fine spray of droplets is formed, either positively or negatively, depending on the mode chosen. To minimize contamination, this capillary is generally located perpendicular to the entrance to the Mass Spectrometer. The heat created by the electrical potential is used to quickly evaporate the droplets in an atmosphere of dry nitrogen.

Then, the ionized analytes are transferred to the high vacuum chamber of the Mass Spectrometer as the charged ions flow through a series of small openings using focusing voltages. Both positively and negatively charged ions can be detected and it is possible to switch between negative and positive operating modes.

The next step is the separation of the ions according to their mass/charge ratio (m/z) by a mass analyzer which is, in our case, a simple quadrupole.
The quadrupole analyzer consists of a set of four parallel metal tubes. A combination of constant and variable voltages allows the transmission of a narrow band of m/z values ​​along the axis of the tubes. By varying the voltages over time, it is possible to scan a range of m/z values ​​giving a mass spectrum. The separated ions are then directed to a photoelectric tube detector or electron multiplier, which identifies and quantifies each ion.

The main advantages of this technique are sensitivity, specificity and precision since the analysis is carried out at the molecular level. Ion analyzes contain structural information on the analyte which can be used to determine the mass of the analytes, their elemental and isotopic composition, to elucidate the chemical structure of the sample or to confirm identification.