Liquid Chromatography - Mass Spectrometry
Liquid Chromatography Mass Spectrometry (LC/MS) is an analytical chemistry laboratory technique for identification, quantitation and mass analysis of materials. This technique allows for the structural elucidation of unknown molecules through fragmentation. Similar to HPLC, Liquid Chromatography Mass Spectrometry utilizes a compound’s intrinsic affinity for both a “mobile phase” (typically a buffered solvent) and a “stationary phase” (porous solid support with specialized coating). Essentially, a pump is used to provide a continuous flow of a solvent into which a dissolved sample is introduced. Once the sample is in the solvent flow, it travels through an analytical column. The compounds present in the sample mixture are then separated depending on their affinity to the coated particles in the column. After the components in the sample are separated, they pass through a mass detector. The mass detector response and the “retention time” (time it takes for a compound to pass from the injector to the detector) of the compound(s) of interest may then be compared to a reference material.
At EAG’s analytical labs, two ionization types are available for sample analysis, Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI). Both EI and APCI are similar in that the ionization of the analyte(s) of interest occurs at atmospheric pressure. The differences between the two techniques lie in the way the analyte(s) of interest are ionized. Using the ESI technique, the sample is effectively dispersed into a nebulized aerosol, removing the solvent and producing ions that are either positively or negatively charged. Typically, very little fragmentation or chemical reactions occur in ESI, and the molecule’s molecular weight is revealed. APCI, on the other hand, uses collisions with reactive gas ions to produce [M+H]+ ions and removes the solvent by heating. ESI is more ideal for polarized molecules, which may provide their own charge, while APCI is more effective with non-polar species, as the collisions with the reactive gas may provide the molecule with a charge.
Once the ions are produced, they may be trapped using an Ion Trap (IT) and subjected to further collisions from which the daughter ions or fragments may be observed. Any of the fragment ions may then be trapped and fragmented again producing a mass signature specific for a particular molecule. Numerous experiments may be conducted to fragment the molecule as long as there is ample concentration of the analyte in question. In this manner, a precise lineage of fragment ions can be obtained for any parent ion, which can ultimately provide valuable information about the compound of interest. Typical mass ranges that can be analyzed by LC/MS are between 50 – 4000 Daltons. ESI often provides multi-charged ions, thus the molecular weight range of analyst that can be identified using ESI-MS is relatively large.