Chromatographic Separation Techniques coupled to Mass Spectrometry for the Analysis of Complex Samples

The use of supercritical fluids (SFs) for chromatography was first reported in 1962 by Klesper (Klesper, et al., 1962). In that study supercritical dichlorodifluoromethane and monochlorodifluoromethane were used as mobile phases to separate nickel etiporphyrin II from nickel mesoporphorin IX dimethyl ester. Since its introduction, SFC had a rapid rise as a new separation topic in the late 1980s and 1990s, before starting a slow decrease and almost waning for over a twenty-year span, in the shadow of other separation techniques. The last decade has been characterized by a renewed interest in this technique, as the introduction of a new generation of commercial instruments has given new stimulus to the development and application of SFC-based methodologies. SFC may be considered as a valuable alternative to conventional chromatographic techniques, and as such is being exploited by separation scientists and employed for a wide range of sample matrices. In the 1980s, SFC was mainly used with capillary columns and FID, while nowadays packed columns and LC type detectors are preferred, as this configuration is more robust and can be easily adapted to a broad range of analytes. Lee et al. (Lee & Markides, 1987) and Novotny (Novotny, 1989) were the first to introduce long capillaries or open tubular stationary phases for SFC in 1981, contributing in this way to the propagation of SFC among GC chromatographers. The latter were strongly attracted by the possibility to widen the power of GC techniques in terms of range of compounds that could be analyzed, while making only slight modifications to GC hardware to properly function with a supercritical fluid. Capillary SFC (cSFC) required pure CO2 as mobile phase and was coupled with flame ionization detector (FID), while temperature and pressure ramps were programmed to modify the elution strength in separations. Various passive devices were adopted for maintaining the critical pressure within the chromatographic instrument, from capillary tubing of small I.D. to integral frits; however, such homemade systems were often little rugged and suffered from poor reproducibility. Moreover, the properties of pure SF CO2 limited its use to only lipophilic compounds, and the strong restrictions in terms of application caused the decline of cSFC in the 1990s. At the same time, a radical change of philosophy occurred based on works of Gere et al. (Gere, et al., 1982) and Berger (Berger & Deye, 1990a; Deye, et al., 1990; Berger & Deye 1990b). Enormous efforts were done for the development of SFC instruments dedicated to the use of LC-like packed columns (pSFC). The first commercial SFC system, based on LC equipment, was commercialized in 1983 by Hewlett Packard. The configuration included an innovative binary pump, offering the capability to modulate the properties of pure SF CO2 by directly modifying the mobile phase composition with the addition of a modifier. Moreover, the backpressure could be maintained constant within the system, thanks to the presence of an active backpressure regulator (BPR), even under gradient elution. The technique was reported to provide better selectivity, shorter analysis times, and broader applicability also to polar compounds (Saito, 2013) than cSFC. However, the poor compatibility of pSFC with FID and the lower efficiency afforded by the use of short columns and 5 or 10 μm particles, beside the commercial success of LC characterized by superior repeatability and more rugged design of instrumentation, have contributed to the almost disappearance of SFC for nearly 20 years from analytical laboratories. Over the last decade, a renewed interest arouse within the chromatographic community, following the introduction of a new generation of instruments by a number of manufacturers (Waters, Agilent, Shimadzu). These pioneering systems are well suited to different analytical purposes, and are capable to deliver efficiency and sensitivity rivaling with those offered by UHPLC. The novel BPR design, the higher upper pressure limits, and the reduced void volumes in fact make these instruments fully compatible with low diameter particles (<2 μm) and core-shell columns (Nováková, et al., 2014; Lesellier, 2012). On the other hand, preparative SFC (prep-SFC) has also proved to be a valuable tool for the isolation of sample constituents, or the purification of extracts (Pettinello, et al., 2000; Alkio, et al., 2000), strongly supported from the industry, to replace toxic and expensive normal phase solvents commonly employed at a preparative scale in HPLC.