Everything about Liquid Chromatography-mass Spectrometry totally explained
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).
Liquid chromatography
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 with respect to the internal diameter of the column and even more so with respect to flow rate since it scales as the square of the diameter. For a long time, 1 mm columns were typical for LC-MS work (as opposed to 4.6 mm for HPLC). More recently 300
m and even 75
m capillary 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.
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.
Mass spectrometry
Mass analyzer
There are a lot of mass analyzers that can be used in LC/MS. Single Quadrupole, Triple Quadrupole, Ion Trap, TOF (time of Flight) and Quadrupole-time of flight (Q-TOF).
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. Various deposition and drying techniques have also been used such as using moving belts; however the most common of these is off-line
MALDI deposition.
Applications
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 and exceptional specificity compared to UV (as long as the analyte can be suitably ionised), and quick analysis 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's 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.
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 proteomics 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.
Drug development
LC-MS is frequently used in drug development at many different stages including Peptide Mapping, Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity Screening, In Vivo Drug Screening, Metabolic Stability Screening, Metabolite Identification, Impurity Identification, Degradant Identification, Quantitative Bioanalysis, and Quality Control.
Further Information
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