Liquid chromatography mass spectrometry, what is it, how does it work and why is it useful?

So in the past, we’ve talked quite a lot about different chromatographies, playlist linked by the end of the video, in addition to a few techniques relating to mass spectrometry such as MALDI-TOF. Now, liquid chromatography (LC) is a technique used for separating compounds in a sample based on their affinity to some other molecule and can be paired with mass spectrometry (MS) for further analysis. In other words, it is the merging of these two different techniques! After chromatographic separation, the compounds are desolvated into the gas phase and ionized at an ionization source, and are then introduced into the mass spectrometer for mass analysis.

Liquid chromatography is a good separation technique, especially for larger and non-volatile molecules such as proteins and complex peptides. Combining this with mass spectrometry is especially useful since we can use different types of column chemistries for broad sample coverage. Liquid chromatography is in fact so efficient that it has largely replaced gel electrophoresis for molecular separation. LC is also especially well suited for pairing with MS since it reduces ion suppression which in turn would impede complete ionization needed for MS. An improved version of LC called high performance liquid chromatography also exists and has largely replaced regular liquid chromatography. The four main types of LC are partition chromatography, ion-exchange chromatography, size-exclusion chromatography and affinity chromatography and you can find all four of these chromatographies in my protein purification playlist, linked by the end of this video.

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In a nutshell, LC workflow is carried out in the following manner: A column, contains a mobile phase as well as a stationary phase. The interaction between our sample and the stationary phase, causes the different compounds in our sample to separate based on some unique difference in quality of these compounds such as size, charge or level of hydrophobicity. When the mobile phase flows out the column, it passes through a detector which “registers” a certain physical or chemical property such as refractive index or light absorption. This is displayed as a signal or “peak”, which corresponds to the amount of the component in the sample. The time at which the analyte is detected is called its RT and compounds of the sample can be confirmed through comparison of their RT to known RTs. However, this is not very reliable and requires secondary confirmation.

Now, the sample can move on to the mass spectrometer. The LC and the MS are coupled through an interface. The solvent is evaporated through the application of heat and analyte molecules are vaporized and ionized. This is crucial because the MS can only measure gas phase ions. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the two most commonly used ionization methods in LC-MS analysis. Then the analyte ions are drawn into the mass spectrometer, where they are subjected to electric fields and/or magnetic fields. The flight paths of the ions are altered by varying the applied fields which ensures their separation from another on the basis of their mass-to-charge (m/z) values. Post-separation, the ions can be collected and detected by a variety of mass detectors, the most common one being the electron-multiplier. The abundances of the ions measured during the analysis of a sample by LC-MS are plotted as a total ion chromatogram (TIC). The plot displays the peak intensities of the analyte ions versus their RT. Further, each point in the chromatogram is associated with the mass spectrum which depicts the ion abundances versus the measured m/z values.