Liquid chromatography-mass spectrometry
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Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (aka HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and specificity. Generally its application is oriented towards the specific detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture).
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[edit] Liquid Chromatography
[edit] Scale
A major difference between traditional HPLC and the chromatography used in LC-MS is that in the latter case the scale is usually much smaller, both regarding internal diameter of the column and even more so with respect to flow rate since it scales as the square of the diameter. 1 mm columns were standard for LC-MS (as opposed to 4.6 mm for HPLC) for a long time. More recently 300μm and even "capillary" 75μm columns have become more prevalent. At the low end of these column diameters the flow rates approach 100nL/min and are generally used with nanospray sources.
[edit] Flow Splitting
When standard bore (4.6 mm) columns are used the flow is often split ~10:1. This can be beneficial by allowing the use of other techniques in tandem such as MS and UV. However splitting the flow to UV will decrease the sensitivity of spectrophotometric detectors. The Mass Spec on the other hand will give improved sensitivity at flow rates of 200 μL/min or less. This is due to the fact that the analyte ions have to be vaporised (nebulized) in order to become charged. As the solvent flows into the MS the solvent evaporates and the gained charge remains, therefore the mass to charge ratio increases. This occurs until the charges repel each other in a process called Coulombic Explosion. If the above is not optimal for this process, the solvent does not evaporate and the ions do not become charged to the same extent. This results in a build up of solvent and uncharged ions in the source area of the MS, resulting in poor ionization and decreased sensitivity.
[edit] Mass Spectrometry
[edit] Mass Analyzer
Most mass spectrometers can be used in LC-MS however quadrupole and quadrupole ion traps are most common. Manufacturers of triple quadrupole mass spectrometers include Varian, Inc., MDS Sciex, Waters Corporation, Agilent, and Thermo Fisher Scientific.
[edit] Interface
Understandably the interface between a liquid phase technique which continuously flows liquid, and a gas phase technique carried out in a vacuum was difficult for a long time. The advent of electrospray ionization changed this. The interface is most often an electrospray ion source or variant such as a nanospray source; however fast atom bombardment, thermospray and atmospheric pressure chemical ionization interfaces are also used.
[edit] Applications
[edit] Pharmacokinetics
LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals. These studies tell us how quickly a drug will be cleared from the Hepatic Blood flow, and organs of the body. MS is used for this due to high sensitivity compared to UV (as long as the analyte can be suitably ionised), and quick analaysis time. The major advantage MS has is the use of Tandem MS-MS. You can program the detector to select out certain ions to fragment. The process is more complex than just a selection technique, but this is essentially what it does. This results in a response that is due to a chosen fragment of a molecule chosen by the operator and as long as there are no interferences or ion suppression the time taken during the LC separation can be quite quick. It is common now to have analysis times of 1 minute or less by MS-MS detection, compared to over 10 mins with UV detection.
[edit] Proteomics
LC-MS is also used in the study of proteomics where again components of a complex mixture must be detected and identified in some manner. The bottom-up LC-MS approach to proteomics generally involves protease digestion (usually Trypsin) followed by LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides.
