Recently, there has been a growth in the application of SFC and SFC-MS to the analysis of compound libraries. The combination of mass spectrometry with
SFC offers the potential benefit of rapid, high-resolution analysis, separation, and purification with sensitive detection and identification. In addition, the SFC mobile phase is considerably more volatile than the aqueous-based mobile phases that are typically used with reverse-phase LC-MS. This condition allows the entire effluent to be directed into the MS interface and simplifies the coupling of the SFC with mass spectrometry with ESI and APCI.
One of the earliest reports of SFC interfaced with APCI was by Huang et al. . The authors used a pin-hole restrictor to maintain supercritical fluid conditions in a packed-column (pcSFC) system. Results for a mixture of five corticosteoids were described with an injection of 25 ng of each of the components. The system was also amenable for capillary SFC/MS applications with minimum modification. Sadoun and Virelizier  reported an SFC interface with ESI in which a two-pump SFC and a packed column were used with the outlet directly interfaced to an ESI source of a quadrupole mass spectrometer. Also, 1-30% (v/v) of polar organic modifier (MeOH-H2O 95:5) was added to CO2 mobile phase to help elute polar organic compounds. The setup was shown to allow analysis of polar organic compounds that were difficult to analyze with earlier implementations of SFC-MS with a chemical ionization interface. A recent review article is available on pcSFC-MS .
Baker and Pinkston  modified an LC/ESI-MS interface for use with pcSFC. The use of a concentric sheath-flow liquid provided ESI modifiers to assist the ionization of neutral, pcSFC-separated components. Postcolumn chromatographic fidelity was preserved with a pressure-regulating fluid, supplied under pressure control to the effluent and positioned just ahead of the sprayer. This modified interface has been used to characterize a variety of mixtures of compounds. Spectra produced with the pcSFC/MS interface are similar to LC/ESI-MS spectra. Hoke et al.  compared a pcSFC-MS/MS method to an LC-MS/MS method for quantitation of enantiomers in human plasma. Samples were prepared with automated solid-phase extraction in the 96-well format. Generally, most analytical attributes, such as specificity, linearity, sensitivity, accuracy, precision, and ruggedness, were comparable for both of these methods, with the exception that the pcSFC separation provided a roughly threefold reduction in analysis time. A 2.3-minute pcSFC separation and a 6.5-min LC separation provided equivalent, near-baseline-resolved peaks, and demonstrated significant time savings for the analysis of a large batch of samples with pcSFC. Hoke et al.  demonstrated the use of pcSFC for high throughput bioanalytical quantitation with dextromethor-phan as a model compound. Plasma samples were prepared by automated liquid-liquid extraction in the 96-well format prior to pcSFC-MS/MS analysis. A throughput of ~10 min/plate was achieved with acceptable relative standard deviation (RSD). Pinkston et al.  described a comparative study of 2266 diverse organic compounds with generic pcSFC (CO2 with 5-60%
MeOH gradient) and HPLC (3-95% ACN gradient) methods with ESI-MS detection, and concluded that the range of coverage is comparable for the two techniques.
The Markides group at Uppsala University in Sweden was also among the earliest in the development of SFC-MS, and demonstrated the application of the technique in the analysis of small organic molecules. Tyrefors et al.  described an APCI-MS interface for an open tubular SFC system. The interface was designed to permit transport of the supercritical mobile phase into the ionization region of the mass spectrometer while the temperature is maintained to within one degree of the chromatographic oven temperature. Temperature control of the interface-transfer line was achieved with preheated gas streams from the chromatographic oven and an active electrical insulation. An average retention-time reproducibility of 0.24% was demonstrated with an average 2.6% precision in relative peak height. Sjoberg and Markides  described an SFC interface probe for API-MS (including ESI and APCI). A sheath-liquid flow of 20 ^L/min in ESI provides optimal conditions for both separation and ionization. The new probe also allows for an easy ionization-mode conversion between ESI and APCI. An improvement to the previous setup was made  to obtain stable ion signals and better sensitivity. Factors that influence the ion signal intensity and stability have been studied, and include corona needle position; nebulizer gas flow; gas additives; spray capillary assembly dimension and position; liquid flow-rate; and composition. The achievable detection limits were in the 50-0.1-pg (i.e., 290 fmol-140 amol) range. The detection limit in APCI mode was improved by a factor of about 20-25 compared to an earlier design .
Ventura et al.  have interfaced an SFC system to a mass spectrometer and evaluated the system for applications requiring high sample throughput. The authors demonstrate the high-speed separation and accurate quantitation capability of SFC-MS. The LC-MS analysis cycle time was reduced threefold with a general SFC-MS high throughput method. Unknown mixture characterization was improved due to the increased selectivity of SFC-MS compared to LC-MS, and was demonstrated with the analysis of combinatorial library mixtures with negative-ion APCI-MS analysis. Rapid elution of SFC was also shown to reduce both sample carryover and cycle time. In an extension of their early work , positive-ion APCI-MS was used for the analysis of compounds with a wide range of polarities. The use of SFC-MS instead of LC-MS resulted in substantial time savings, increased chromatographic efficiency, and more precise quantitation of sample mixtures. The instrumental setup also allows for facile conversion between LC-MS and SFC-MS modes of operation.
Morgan et al.  described an optimized interface for coupling SFC with APCI-MS. Data presented demonstrate that the internal diameter and length of the transfer line between the SFC unit and the APCI source are not critical to maintain peak shape or retention time under the set of conditions tested. A comparison of responses from an in-line UV detector, two quadrupole mass spectrometers, and an ion trap was presented to demonstrate limits of detection and linear ranges for the SFC separation of a six-compound test mix. Villeneuve and Anderegg  developed an automated analytical SFC method to separate enantiomers based on a commercial instrument and column-selection valves. Similar racemic compounds, even those from the same molecular class, were separated with different column and modifier combinations. The optimal chiral separation of several compounds can be obtained within 24 hours with the fully automated system. Garzotti et al.  described a simple and economical method to couple a commercial SFC system with a high-resolution hybrid mass spectrometer (Q-TOF-MS). The setup provided on-line accurate mass SFC-MS measurements, and the fast spectral acquisition rate of TOF-MS facilitated data acquisition from rapid SFC separations.
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