Key concept: - Orthogonal separation. Any separation technique that can be used to sort a population of molecules by some characteristic other than what is being measured can be called an orthogonal separation. The most common liquid chromatography approach in mass spectrometry involves sorting molecules by hyrdophobicity, which is a characteristic that is not directly correlated - and hence orthogonal - to the mass of a molecule. Some LC installations now employ multidimensional LC, where separations are used that are orthogonal to each other, for increased resolution of molecular populations.

Liquid Chromatography

Because of the complexity of many biological samples, the addition of various liquid chromatography (often abbreviated LC) techniques is of immense value to mass spectrometry. An approach that combines LC with sinlge mass spectrometry is often abbreviated LC-MS (designating that LC comes before mass spec), whereas an appliction using tandem mass spec will often be abbreviated LC-MS/MS. For the purpose of this section, the abbreviation LC-MS will be used as a short, blanket abbreviation for LC-MS or LC-MS/MS, unless otherwise noted.

The general objective behind using LC prior to MS is to acheive another dimension of separation for the sample. Many papers published that involve LC-MS have additional graphs that show this, often by relating separation to time in a graph that has intensity (or ion current) on the Y axis and time on the X axis.

LC chemistry is generally the deciding factor of how molecules are separated in LC. In an LC-MS/MS application examining a trypsin digest, many of the molecules in the mixture will start out with similar masses, so another separation needs to be aplpied; frequently these peptides will be separated by hydrophobicity.