Chromatography Techniques and Spectrometry

Liquid Chromatography-Mass Spectrometry (LC-MS)

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Liquid chromatography (LC) is a widely used method which is frequently coupled with mass spectrometry for sample ionization prior to analysis. In LC-MS, solubilized compounds (the mobile phase) are passed through a column packed with a stationary (solid) phase. Based on their weight and affinity for the mobile and stationary phases of the column, this effectively separates the compounds. Its anionization through loss of H+ ions also leads to fragmentation of the sample. The sample passes into the vacuum chamber of the mass spectrometer by following this step.

For larger and non-volatile molecules such as proteins and complex peptides, LC is the separation technique of choice. LC-MS offers broad sample coverage due to different column chemistries, such as reversed phase liquid chromatography, can be used when combined with MS. The ideal method for separating isomers, which have the same mass and will otherwise not be differentiated by a mass spectrometer, is LC. As a result, LC has largely replaced gel electrophoresis for molecular separation due to its superior resolving power and broad mass range. Finally, LC helps in reduce ion suppression, which occurs when two different molecules interact with one another and impede the process of complete ionization.

HPLC, which is commonly known as high performance liquid chromatography, has improved and largely replaced LC. HPLC was initially defined as High Pressure Liquid Chromatography which operates at a higher ranging of pressure from 50-350 bar. In contrast, LC majorly depends on gravity for the passage of the mobile phase through the column.