Liquid chromatography–mass spectrometry

Liquid chromatography–mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography - MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides structural identity of the individual components with high molecular specificity and detection sensitivity. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC-MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. Liquid chromatography–mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography - MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides structural identity of the individual components with high molecular specificity and detection sensitivity. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC-MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. In addition to the liquid chromatography and mass spectrometry devices, an LC-MS system contains an interface that efficiently transfers the separated components from the LC column into the MS ion source. The interface is necessary because the LC and MS devices are fundamentally incompatible. While the mobile phase in a LC system is a pressurized liquid, the MS analyzers commonly operate under high vacuum (around 10−6 torr / 10−7 'Hg). Thus, it is not possible to directly pump the eluate from the LC column into the MS source. Overall, the interface is a mechanically simple part of the LC-MS system that transfers the maximum amount of analyte, removes a significant portion of the mobile phase used in LC and preserves the chemical identity of the chromatography products (chemically inert). As a requirement, the interface should not interfere with the ionizing efficiency and vacuum conditions of the MS system. Nowadays, most extensively applied LC-MS interfaces are based on atmospheric pressure ionization (API) strategies like electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI).These interfaces became available in the 1990s after a two decade long research and development process. The coupling of chromatography with MS is a well developed chemical analysis strategy dating back from the 1950s. Gas chromatography (GC) - MS was originally introduced in 1952, when A.T. James and A.J.P Martin were trying to develop tandem separation - mass analysis techniques. In GC, the analytes are eluted from the separation column as a gas and the connection with electron ionization (EI) or chemical ionization (CI) ion sources in the MS system was a technically simpler challenge. Because of this, the development of GC-MS systems was faster than LC-MS and such systems were first commercialized in the 1970s. The development of LC-MS systems took longer than GC-MS and was directly related to the development of proper interfaces. V.L. Tal'roze and collaborators started the development of LC-MS in the early 1970s, when they first used capillaries to connect LC columns and MS ion sources. A similar strategy was investigated by McLafferty and collaborators in 1973. This was the first and most obvious way of coupling LC with MS, and was known as the capillary inlet interface. This pioneer interface for LC-MS had the same analysis capabilities of GC-MS and was limited to rather volatile analytes and non-polar compounds with low molecular mass (below 400 Da). In the capillary inlet interface, the evaporation of the mobile phase inside the capillary was one of the main issues. Within the first years of development of LC-MS, on-line and off-line alternatives were proposed as coupling alternatives. In general, off-line coupling involved fraction collection, evaporation of solvent, and transfer of analytes to the MS using probes. Off-line analyte treatment process was time consuming and there was an inherent risk of sample contamination. Rapidly, it was realized that the analysis of complex mixtures would require the development of a fully automated on-line coupling solution in LC-MS. The moving-belt interface (MBI) was developed in 1977. This interface consisted of an endless moving belt receiving the LC column effluent. On the belt, the solvent was evaporated by gently heating and efficiently exhausting the solvent vapors under reduced pressure in two vacuum chambers. After removing the liquid phase, the analytes would desorb from the belt and migrate to the MS ion source to be analysed. MBI was successfully used for LC-MS applications between 1978 and 1990 because it allowed coupling of LC to MS devices using EI, CI, and fast-atom bombardment (FAB) ion sources. The most common MS systems connected by MBI interfaces to LC columns were magnetic sector and quadropole instruments. MBI interfaces for LC-MS allowed MS to be widely applied in the analysis of drugs, pesticides, steroids, alkaloids, and polycyclic aromatic hydrocarbons. This interface is no longer used because of its mechanical complexity and the difficulties associated to belt renewal. Particle beam interfaces took over the wide applications of MBI for LC-MS in 1988. The direct liquid introduction (DLI) interface was developed in 1980. This interface was thought as a solution to the evaporation of liquid inside the capillary inlet interface. In DLI, a nebulizer was used to disintegrate part of the effluent coming from the column. A small diaphragm was used to form a liquid jet composed of small droplets that were subsequently dried in a desolvation chamber. A microbore capillary column was used to transfer the nebulized liquid product to the MS ion source. The analytes were ionized using a solvent assisted chemical ionization source, where the LC solvents acted as reagent gases. To use this interface, it was necessary to split the flow coming out of the LC column because only a small portion of the effluent (10 to 50 μl/min out of 1 ml/min) could be analyzed on-line without breaking the MS vacuum. One of the main operational problems of the DLI interface was the frequent clogging of the diaphragm orifices. The DLI interface was used between 1982 and 1985 for the analysis of pesticides, corticosteroids, metabolites in equine urine, erythromycin, and vitamin B12. However, this interface was replaced by the thermospray interface, which removed the flow rate limitations and the issues with the clogging diaphragms. The thermospray (TSP) interface was developed in 1983 by Vestal laboratories at the University of Houston. The interface resulted from a long term research project intended to find a LC-MS interface capable of handling high flow rates (1 ml/min) and avoiding the flow split in DLI interfaces. The TSP interface was composed by a heated probe, a desolvation chamber, and an ion exchange skimmer. The LC effluent passed through the heated probe and emerged as a jet of vapor and small droplets flowing into the desolvation chamber at low pressure. The ionization of solutes occurred by direct evaporation or ion-molecule reactions induced by the solvent. This interface was able to handle up to 2 ml/min of eluate from the LC column and would efficiently introduce it into the MS vacuum system. TSP was also more suitable for LC-MS applications involving reversed phase liquid chromatography (RT-LC). The TSP system had a dual function acting as an interface and a solvent-mediated chemical ionization source. With time, the mechanical complexity of TSP was simplified, and this interface became popular as the first ideal LC-MS interface for pharmaceutical applications comprising the analysis of drugs, metabolites, conjugates, nucleosides, peptides, natural products, and pesticides. The introduction of TSP marked a significant improvement for LC-MS systems and was the most widely applied interface until the beginning of the 1990s, when it began to be replaced by interfaces involving atmospheric pressure ionization (API). The frit FAB and continuous flow-FAB (CF-FAB) interfaces were developed in 1985 and 1986 respectively. Both interfaces were similar, but they differed in that the first used a porous frit probe as connecting channel, while CF-FAB used a probe tip. From these, the CF-FAB was more successful as a LC-MS interface and was useful to analyze non-volatile and thermally labile compounds. In these interfaces, the LC effluent passed through the frit or CF-FAB channels to form a uniform liquid film at the tip. There, the liquid was bombarded with ion beams or high energy atoms (fast atom). For stable operation, the FAB based interfaces were able to handle liquid flow rates of only 1-15 μl and were also restricted to microbore and capillary columns. In order to be used in FAB MS ionization sources, the analytes of interest should be mixed with a matrix (e.g., glycerol) that could be added before or after the separation in the LC column. FAB based interfaces were extensively used to characterize peptides, but lost applicability with the advent of electrospray based interfaces in 1988. Liquid chromatography is a method of physical separation in which the components of a liquid mixture are distributed between two immiscible phases, i.e., stationary and mobile. The practice of LC can be divided into five categories, i.e., adsorption chromatography, partition chromatography, ion-exchange chromatography, size-exclusion chromatography, and affinity chromatography. Among these, the most widely used variant is the reverse-phase (RP) mode of the partition chromatography technique, which makes use of a non-polar (hydrophobic) stationary phase and a polar mobile phase. In common applications, the mobile phase is a mixture of water and other polar solvents (e.g., methanol, isopropanol, and acetonitrile), and the stationary matrix is prepared by attaching long-chain alkyl groups (e.g., n-octadecyl or C18) to the surface of irregularly or spherically shaped 5 μm diameter silica particles. In HPLC, typically 20 μl of the sample of interest are injected into the mobile phase stream delivered by a high pressure pump. The mobile phase containing the analytes permeates through the stationary phase bed in a definite direction. The components of the mixture are separated depending on their chemical affinity with the mobile and stationary phases. The separation occurs after repeated sorption and desorption steps occurring when the liquid interacts with the stationary bed. The liquid solvent (mobile phase) is delivered under high pressure (up to 400 bar or 300.000 torr) into a packed column containing the stationary phase. The high pressure is necessary to achieve a constant flow rate for reproducible chromatography experiments. Depending on the partitioning between the mobile and stationary phases, the components of the sample will flow out of the column at different times. The column is the most important component of the LC system and is designed to withstand the high pressure of the liquid. Conventional LC columns are 100–300 mm long with outer diameter of 6.4 mm (1/4 inch) and internal diameter of 3.0-4.6 mm. For applications involving LC-MS, the length of chromatography columns can be shorter (30–50 mm) with 3-5 μm diameter packing particles. In addition to the conventional model, other LC columns are the narrow bore, microbore, microcapillary, and nano-LC models. These columns have smaller internal diameters, allow for a more efficient separation, and handle liquid flows under 1 ml/ min (the conventional flow-rate). In order to improve separation efficiency and peak resolution, ultra performance liquid chromatography (UPLC) can be used instead of HPLC. This LC variant uses columns packed with smaller silica particles (~1.7 μm diameter) and requires higher operating pressures in the range of 310.000 to 775.000 torr (6000 to 15000 psi).