Supercritical fluid mobile phases

D. E. Martire and R. E. Boehm, Unified molecular theory of clrromatography and its application to supercritical fluid mobile phases. 1. Eluid-liquid (absorption) clrromatography , J. Phys. Chem. 91 2433-2446 (1987). 

D. E. Martue, Unified theory of absorption clrromatography gas, liquid and supercritical fluid mobile phases , ]. Liq. Chmmatogr. 10 1569-1588 (1987). 

The coupling of supercritical fluid extraction (SEE) with gas chromatography (SEE-GC) provides an excellent example of the application of multidimensional chromatography principles to a sample preparation method. In SEE, the analytical matrix is packed into an extraction vessel and a supercritical fluid, usually carbon dioxide, is passed through it. The analyte matrix may be viewed as the stationary phase, while the supercritical fluid can be viewed as the mobile phase. In order to obtain an effective extraction, the solubility of the analyte in the supercritical fluid mobile phase must be considered, along with its affinity to the matrix stationary phase. The effluent from the extraction is then collected and transferred to a gas chromatograph. In his comprehensive text, Taylor provides an excellent description of the principles and applications of SEE (44), while Pawliszyn presents a description of the supercritical fluid as the mobile phase in his development of a kinetic model for the extraction process (45). 

The nature of a supercritical fluid enables both gas and liquid chromatographic detectors to be used in SFC. Flame ionization (FID), nitrogen phosphorus (NPD), flame photometric (FPD) GC detectors (p. 100 etseq.) and UV and fluorescence HPLC monitors are all compatible with a supercritical fluid mobile phase and can be adapted to operate at the required pressures (up to several hundred bar). A very wide range of solute types can therefore be detected in SFC. In addition the coupled or hyphenated techniques of SFC-MS and SFC-FT-IR are attractive possibilities (cf. GC-MS and GC-IR, p. 114 el seq.). 

Solute retention as a function of temperature at constant pressure is seen to be dependent on the partial molar enthalpy of solute transfer between the mobile and stationary phases, the neat capacity of the supercritical fluid mobile phase and the volume expansivity of the fluid. The model was compared to chromatographic retention data for solutes in n-pentane and CO2 as the fluid mobile phase and was seen to fit the data well. 

Equation 11 should be the relationship between retention-solubility and pressure at constant temperature for infinitely dilute solutions. The RHS of eq. 11 consists of three terms, the first term will be a constant whose value depends on the partial molar volume of the solute in the stationary phase. The second term is the solubility of the solute in the supercritical fluid mobile phase. 

The volume expansivity leads to the retention maxima seen in SFC near the critical point of the supercritical fluid mobile phase. The... 

Since SFC is in its infancy, the same is true of the hyphenated techniques that involve it. In general, it would be expected that SCF/MS should use interfaces like GC/MS since the supercritical fluid mobile phases become gases when reduced to atmospheric pressure, but the conditions are more severe because of the higher (critical) pressure. OT columns, because of their low pressure drops, are favored. The two interfaces that have been used are a direct fluid injection (DFI) and a molecular beam apparatus. DFI has been used with packed columns26 and with OT columns,27 using both chemical ionization and electron impact ionization. For a more complete discussion of both interfaces, see the chapter on SFC in the ACS Symposium Series edited by Ahuja28 the recent review on LC/MS25 also contains considerable information about SFC/MS. 

SFC is complementary to the other classic techniques of GC or normal phase HPLC. The migration of the solute results from a distribution mechanism between the apolar stationary phase and a slightly polar eluting mobile phase. The solvation capacity of the mobile phase is governed by both temperature and pressure of the supercritical fluid. Therefore as the density of the supercritical fluid mobile phase is increased, components retained in the column can be made to elute. The resistance to mass transfer between the stationary and the mobile phases is less than in HPLC because diffusion is about ten times greater than in liquids. The C factor in Van Deemter s equation being smaller, the velocity of the mobile phase can therefore be increased without an appreciable loss of efficiency (Figure 6.5). Moreover, as the viscosity of the mobile phase is close to that of a gas, GC... 

The success or failure of flow cell detection depends on the ability to cope with the absorption due to the supercritical fluid mobile phase. Because carbon dioxide is used as the supercritical fluid in the majority of applications, it is important to demonstrate its viability with FT-IR detection. 

Analytical systems do equip almost all laboratories. Many different kinds of particle sizes are used from 20 to 2 [tm (from HPLC to UPLC), using liquid or supercritical fluid mobile phases. An intermediate market is to be considered using 10 mm ID columns for small-scale purifications. The term preparative chromatography is dedicated to purification units using column diameters from 50 up to 1600 mm ID. 

Capillary Columns for SFC-MS. At present, the major limitation to broad application of capillary SFC technology is related to the availability of columns compatible with supercritical fluid mobile phases. The fused silica capillary columns used in this work were deactivated and coated with crosslinked and surface-bonded stationary phases using techniques similar to those reported by Lee and coworkers (40,41). Columns from less than 1 m to more than 20 m in length and with inner diameters of 10 to 200 ym have been examined. Colvimn deactivation was achieved by purging with a dry nitrogen flow at 350 C for several hours followed by silylation with a polymethylhydrosiloxane. Any unreacted groups on the hydro-siloxane were capped by treatment with chlorotrimethylsilane at 250 C. After deactivation, the columns were coated with approximately a 0.15-.25 ym film of SE-54 (5Z phenyl polymethylphenyl-siloxane) or other polysiloxane stationary phases. The coated stationary phases were crosslinked and bonded to the deactivation layer by extensive crosslinking with azo-t-butane (41). The importance of deactivation procedures for elution of more polar compounds, such as the trichothecenes, has been demonstrated elsewhere (42). 

Chromatographic separations using supercritical fluid mobile phases have been described for the first time in 1962 by Klesper, Corwin, and Turner, who are considered to be the discoverers of this technique. Since 1969, SFC is also one of the main activities of our own laboratory [7,11,13,40-43,54-62]. After slow progress, SFC has now found its place among the chromatographic techniques. For a compilation of books, publications, bibliographies, and commercial equipment see [11,13]. In the present book, SFC applications are also treated by King [89]. 

At the same time, analysis speed needs to be improved. The characterization techniques of biopolymers have made an cistonishingly fast progress, which enabled to complete the sequencing of human DNA. Employment of more variety of LC instrumentation, such as monolithic colunrn, capillary as well as open tubular column, supercritical fluid mobile phase, capillary electrophoresis, electrochro-... 

This means that a supercritical fluid mobile phase results in lower column backpressure than a liquid. Due to the higher diffusion rates in a supercritical fluid than in a liquid, the column effldency should, in principle, be higher in supercritical fluid chromatography (SFC) than in liquid chromatography (LC), but lower than that in gas chromatography (GC). However, for long columns with a density gradient over the column, improved efficiency compared to HPLC is not necessarily the case. 

As for preparative applications, the choice of technique is closely dependent on the amount of compound to be purified. The use of SFC will continue increasing, in combination with SMB mode or even in single-column mode, given the environmentally friendly character of the technique and the low cost in solvents. SMB, either with liquid or supercritical fluid mobile phases, allows for an improved use of the expensive stationary phase and it is the technique of choice if the purification of important amounts of enantiomers is required. However, one main disadvantage of SMB is the considerable inversion in equipment required, which is only recoverable in a reasonable period of time for companies dealing with a number of analytes or candidates in production. 

One area of preparative SFC that would benefit from further investigation is the sample injection technique. With the exception of on-line extraction/chromatogra-phy, the sample is usually introduced as a solution in an organic solvent aind injected onto the column by means of a loop rotary valve. The sample loop is then flushed with a high density liquid that eventually becomes the supercritical fluid mobile phase on entering the heated column. 

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