Trends in Analytical Chemistry
I.3 Coupling of UHPSFC to MS
Several detectors can be hyphenated to SFC, such as UV or ELSD. However, MS has recently captured the interest of the majority of research groups. Due to its unparalleled applicability range, as well as high selectivity and elevated sensitivity, MS represents the gold standard detector in numerous laboratories (28,29). Its coupling to chromatographic technique such as GC or, more importantly, LC has been the subject of different studies. Nowadays LC-MS represent the gold standard in metabolomics, anti-doping laboratories, food industry and for environmental analyses (28,30,31). Because of its usefulness, its hyphenation to SFC has been extensively tested. Due to the peculiar nature of the SFC mobile phase, specific interfaces have been developed throughout the years to increase the compatibility of SFC to MS, with several designs available on the market.
This section focuses on the current situation regarding the coupling of SFC to MS. Different MS ionization sources, as well as MS analyzers available on the market are described in the first half of this paragraph. A review article, providing a critical evaluation of SFC-MS and of its applications is presented at the end of this section.
I.3.1 MS ionization sources
Many typologies of MS ionization sources are available nowadays. Ionization chambers can work either under vacuum conditions, such as in the case of chemical ionization (CI) or electron impact (EI), or under atmospheric pressure (atmospheric pressure ionization, or API). The development and subsequent use of API sources, such as electrospray (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo-ionization (APPI), allowed the coupling of separation techniques such as LC and, lately, SFC to MS systems.
More specifically, ESI sources have proven to be decisive as they ensure the ionization and desolvation of a large spectrum of compounds, from small to large molecules, with intermediate to relatively low polarities.
ESI can be divided into three main phases (Figure 6). Initially, the mobile phase stream coming from the chromatographic system needs to be reduced into droplets released from the Taylor cone at the end of the capillary, with the aid of a nebulization gas as well as the use of high temperatures. These droplets will acquire a charge that can be either positive or negative, according to the applied capillary voltage. Subsequently, the charged droplets will undergo a reduction in their size, which would increase exponentially the ionic repulsion phenomenon as an excess of charges with the same polarity will accumulate on the surface of the droplets.
This leads to Coulomb fission of the larger droplets into smaller ones, igniting a chain of reactions which would lead, eventually, to the creation of gas-phase ions (32). Finally, the ions
in gas-phase will enter into the MS analyzer (under vacuum region), leading to their separation and detection.
Figure 6: Representation of the ESI process. Reprinted from (33)
While the working principle of ESI source does not change between LC and SFC, some considerations need to be made on the state of the mobile phase reaching the ionization chamber. In LC, the solvent is always in a liquid state, thus the ESI source needs to be highly efficient in generating the charged droplets and in reducing their size, to have consequently the production of gas-phase ions. Therefore, an important parameter in LC-MS analysis is the flow-rate employed during the analysis. Flow rates above 500-700 µL/min tend to be considered detrimental for LC-MS, as the ionization process would not be able to efficiently generate the charged droplets from the solvent entering into the ESI capillary. This leads to a general reduction in the MS sensitivity. Consequently, it is advised to work below such flow-rates (e.g. 250 µL/min) in LC-MS. With SFC, on the other hand, the situation is more complex.
As discussed in the previous section, this technique needs to work at high velocities (thus higher flow rates) to obtain the maximum possible chromatographic efficiency. Therefore, it seems at first that SFC does not fit well with ESI sources. However, the different SFC-MS interfaces currently available on the market tend to have a flow splitter before delivering the solvent in the ionization chamber (34,35). More details on the different SFC-MS interfaces available are given in the review article in section I.2.4. In particular, the “pre-BPR splitter with sheath pump” geometry used by Waters and Agilent on their SFC systems limit the flow-rates of modifier entering the ESI chamber to less than 250 µL/min, using various co-solvent percentages up to 40% in the mobile phase (Figure 7).
Figure 7: Simulated amount of MeOH entering into the MS as a function of the flow rates on the SFC pump and the sheath pump, with the Waters “pre-BPR splitter with sheath pump” interface (A–C) and Agilent “pre-BPR splitter with sheath pump” interface (D–F). Simulations have been performed with a mobile phase composed entirely of scCO2 (left column), 80% scCO2 and 20% MeOH (middle column), 60% scCO2 and 40% MeOH (right column).
Adapted, with permission, from (35).
A second factor also contributes to the lower amount of solvent reaching the ionization chamber compared to LC-MS. In the tubing employed to connect the SFC-MS interface to the ESI source, carbon dioxide experiences a state shift from a supercritical fluid to a gas, as it is not anymore under the influence of the BPR module. Therefore, when the mobile phase reaches the capillary, the CO2 is immediately removed and the actual amount of solvent entering into the ESI source is only composed of MeOH and eventual additives. Furthermore, the gaseous CO2 can improve the efficiency of the ionization chamber in the evaporation of the liquid solvent, with the potential to lower even further the limits of detection. More details on the sensibility achievable with SFC-MS compared to LC-MS are given in the review article of paragraph I.3.4.
I.3.2 MS analyzers
Once ions are passed in their gaseous state and have acquired a charge, they will be transferred in the mass analyzer of the MS instrument. Mass analyzers are fundamental, since their role is to guarantee the discrimination between the different ions that have been previously generated in the MS ionization source. Mass analyzers are classified according to their resolution power, following equation 3:
𝑟𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑝𝑜𝑤𝑒𝑟 = ,
∆, (3)
where m is the ion’s mass and Dm is the resolution or full width at half maximum (FWHM).
Using equation 2, it has been possible to classify mass analyzers in two main groups: those with a resolution power < 10 000, called low-resolution mass analyzers, and those with a resolution power > 10 000 (high-resolution mass analyzers). In the first group quadrupole (Q), and ion trap (IT) analyzers are present. These are not capable to discriminate ions with the same nominal mass, due to the unit resolution they offer. In compensation, they are able to provide excellent performance in the context of targeted analyses, especially when quantification is needed. High resolution mass analyzers, such as time-of-flight (TOF) or Orbitrap©, are capable in separating ions down to 0.001 atomic mass unit of difference and are, therefore, mainly used in untargeted analyses to obtain information regarding the structure of unknown analytes. In this thesis work, single quadrupole, triple quadrupole and time-of-flight analyzers have been employed. More details regarding how they function and their application with chromatographic systems such as LC and SFC can be found in the previous thesis works of Dr. Alexandre Grand-Guillaume Perrenoud (University of Geneva, Thesis n°4717) and Dr.
Vincent Desfontaine (University of Geneva, Thesis n°5181), as well as in the review article present in paragraph I.3.4.
I.3.3 Matrix effect
Application involving SFC-MS have started to greatly increase with the development of the latest generation of SFC-MS interfaces. Among those, the analysis of biological matrices has attracted the attention of researchers. Indeed, the possibility to benefit from the complementarity given by SFC in the chromatographic separation, in combination with the typical detection range of MS was seen by many as an interesting alternative to LC-MS.
However, when compounds present in complex matrices are analyzed with MS, a phenomenon called matrix effect (ME) starts to arise. Matrix effect has been defined as the alteration of the ionization process due to the presence of co-eluting substances generally invisible in chromatograms (36). ME has been known for several years in LC-MS, with the first example described by Tang and Kebarle in 1993 (37). Two ME types have been identified so far: ion suppression, in which the signal intensity of the desired ion present in the matrix is lower than the signal generated by the same analyte as a standard, both at identical concentration levels. The second ME phenomenon is ion enhancement, in which an increase of the signal intensities can be observed, in opposition to ion suppression. Although almost 30 years have passed, there is no clear theory that can fully explain how ME actually occurs. The main hypothesis states that ME is due to a competition phenomenon between non-volatile matrix components and the ions of interest at the droplet surface for their subsequent transfer in the gaseous state and, thus, entrance in the MS analyzer. However, while this hypothesis
can be useful in understanding ion suppression, it does not correlate with the ion enhancement phenomenon.
Several strategies have been developed to provide an estimation of the ME generated during the analysis. One of the most common approach was introduced by Matuszewski et al. (38), in which the ratio between the peak area of one analyte spiked after the extraction procedure applied on the biological matrix over the peak area of the same analyte, at the same concentration, in neat solution is calculated. ME is, then, obtained using the following equation 4:
𝑀𝐸 (%) = .*&/ &+*& #0 1#2% *3%+&$%4#' 214/*5 2&,1(*
.*&/ &+*& #0 2%&'5&+5 4' '*&% 2#(6%4#' ∗ 100 (4) Another methodology was introduced by Bonfiglio et al (39), in which a post-column infusion of the analyte is made during injections of the biological matrix and neat solvent (Figure 8).
Figure 8: Post-column infusion system. Reprinted from (39).
As previously mentioned, ME is due to the coelution phenomenon of the desired analyte with substances present in the tested biological matrix. As the latter can interact with the stationary phase, it is expected that the choice of the chromatographic technique can impact the level of ME generated within the analysis, thus reducing its extent. SFC, therefore, can provide a quite different ME profile compared to that of RPLC and HILIC, due to its orthogonality and separation complementarity. Additional information are present in the review article of paragraph I.3.4.
I.3.4 Review article: Recent trends for SFC-MS
In the following review article, published in 2019, different aspects related to SFC-MS analyses, such as the type of SFC-MS interface, matrix effect as well as achievable sensitivity compared to LC-MS? are discussed. Examples of the latest applications using SFC-MS are also given.