Optimal chromatographic separation is of utmost interest within the field of bioanalytical chemistry since the presence of even trace amounts of a given compound can mean the difference between success and failure. Supercritical fluid chromatography (SFC) can provide fast analysis times and efficient, baseline peak-to-peak resolutions. SFC also has the ability to be coupled to both gas chromatography (GC) detectors and high pressure liquid chromatography (HPLC) detectors, and thus SFC possesses a distinct advantage over the other separation techniques. Among the detectors available to SFC, mass spectroscopy (MS) provides the required selectivity and necessary sensitivity to detect very small to trace compound concentrations.

Aspects of Supercritical Fluid Chromatography. Lately SFC has found a niche in the field of pharmaceutical chemistry and has gained much support in the field of bioanalytical applications. In the overall ranking of chromatographic techniques, it has been judged that SFC falls somewhere between HPLC and GC as the chromatographic method of choice. Some researchers have used modified HPLC set-ups to gain SFC capabilities, reflecting the similarities between HPLC and SFC. Commercial instruments are also available and may offer better control over critical parameters through the use of system-controlling software.

The Role of SFC in Bioanalytical Chemistry. The success of SFC in the field of bioanalytical chemistry is well documented.^1, There are many examples within the literature testifying to the practicality of using SFC to separate specific compounds. The list of compounds separated by SFC includes steroids, bile acids, carbohydrates-oligosaccharides, barbiturates, opium alkaloids, antidepressants, stimulants, and diuretics just to name a few.²

Perhaps the most important contribution that SFC has made has been towards the separation of chiral compounds. To illustrate the importance of such separations in terms of pharmaceuticals, one enantiomer of a compound may have beneficial effects whereas the other may have deleterious effects. Separating the two could provide useful information about drug purity, efficacy and metabolism. SFC is enjoying great success in meeting the challenges of stereoisomer separation and may have already surpassed HPLC in the ability to provide appreciable selectivity of molecular stereoisomers.

Analyte Screening for SFC. Whether a given analyte will soluablize well in a supercritical fluid will largely depend on the compound’s size and the identity of its functional group(s). Non-polar to low-polarity analytes may be solublized in a pure supercritical fluid (SF), such as CO₂. Alternatively, non-polar analytes may be derivitized with a polar functional group and a polar-modified mobile phase (MP) may be used. Note that, in the case of SFC/MS, any added mass resulting from dirivitization would decrease the upper mass limit of the analyte. For high-polarity analytes, a polar solvent can be added to increase the overall polarity of the MP. Including chemical additives, like acids or bases, in the modifier can further enhance solubility. If the analyte is only soluble in an aqueous solution, it is probably a poor candidate for SFC.²

How SFC differs from HPLC and GC. SFC essentially uses compressed gases to solvate compounds, more or less as a liquid does. To date, the vast majority of compound separations have been performed through the use of GC or HPLC. SFC may be seen as overlapping the two, but there are distinct differences among them.

The biggest advantage that SFC holds over GC is the ability to separate thermally labile compounds. This is immediately appreciated in the pharmaceutical field since roughly 20% of all drug candidates fall into this category.² Capillary SFC compliments GC by allowing the separation of larger molecules which possess a low volatility and a low thermal stability. SFC also has the ability to interface with HPLC detectors. Because GC will accommodate easier selectivity adjustments than SFC could provide, GC should be considered first when analyzing compounds with high temperature tolerability.²

Several reviews are available in the literature for directly comparing SFC to HPLC.^2,, The biggest advantage that SFC has over HPLC lies within the differences in the MPs. Supercritical fluids are less viscous and possess a higher diffusivity than liquids under HPLC conditions. These properties cumulatively represent the ability to allow lower pressure drops along an analytical column. This provides not only the ability to increase column lengths, but also allows for faster flow rates. These factors in turn affect capacity ratios, selectivities and theoretical plate heights. It has been reported that 200,000 theoretical plates have been achieved by using eleven analytical (4.6 mm i.d.) columns in series. Additionally, SFC may be used with GC detectors. Finally, SFC can be set up for sub-ambient temperatures, which has been key in many chiral separations.

Retention, Selectivity and Efficiency in SFC.² Retention is most affected by MP composition, especially by the percent of polar modifier added. Increasing modifier generally decreases retention. Selectivity is adjusted primarily by temperature modification. Reversals in chromatographic peak order may occur as a function of temperature modification. Efficiency (N) is most influenced by flow rate. The linear flow rate of the MP tends to drop with increasing pressure due to the increase in MP density. A 4.6-mm i.d. column exhibits its optimum linear flow rate with a pump flow rate of 2.5 – 3.0 ml/min in pSFC. Higher flow rates will moderately affect N, but will also increase the solvent load considerably.

Why SFC/MS? Bioanalytical chemistry has already benefited greatly from the use of MS as a detector. In the pharmaceutical industry, analyte concentrations in the picogram-per-milliliter range (and lower) are commonplace. Optimum chromatography is fairly useless if demands are not met concerning the detection of realistic concentration levels. The detector of choice would be universal, one that offers the highest sensitivity, the broadest selectivity and the best resolution. Currently, the detector that fits all of these criteria is the mass spectrometer.

MS vs. Other Available Detectors for SFC. The pressure requirements for pSFC significantly limit the type of detector that can be used successfully. LC detectors need to have pressure tolerances of up to 400 bar. This upper limit is possible with special pressure-resistant absorbance detector cells, but not with the orthogonal window cell designs found in fluorimeters.

GC detectors that have been used with both cSFC and (split) pSFC include mass spectrometers (below), flame ionization (FID)^1,2,, electron capture^,,, nitrogen-phosphorous, and phosphorous sensitive detectors, just to name a few. In all, these detectors give a wide variety of sensitive selectivity, but each in itself can be restricted in application. The only truly universal detectors available to SFC are FID and MS. FID will produce high background noise in the presence of SFC polar modifiers, concluding that the practical universal detector of choice should be MS. Figure 1 gives a comparison of SFC/FID to SFC/MS.⁹

Compatibility of SFC Hardware with MS Hardware. As stated previously, supercritical fluids have unique characteristics that require specialized modifications to SFC hardware. Many of these modifications are based on the need to keep analytes solvated in the SF. The components that require the most consideration are pressure regulation, injection, and temperature regulation.

Pressure Regulation. Of highest consideration is the need for pressure control. Should the SFC unit lose pressure, the supercritical fluid would quickly separate into its phase(s) inherent to the molecule at the decompressed temperature and pressure (CO₂ freezes upon rapid decompression). Pressure control is handled in one of two modes, "downstream" or "upstream".

"Downstream" mode is used primarily for pSFC. It requires a highly regulated pressurized environment. In most systems, the pressure is kept constant by a back-pressure regulator (BPR) which is coupled to a pressure sensor. BPR’s are attached after the analytical column, and usually after any LC detector that has been placed in-line, thus the regulation occurs "downstream" from the pump. The MP pump will maintain the pumping speed and inlet pressure accordingly in order to maintain a consistent, user-defined, outlet pressure. Figure 2 depicts a typical "downstream" set-up.

In "upstream" mode, pressure is controlled at the pump, making flow control poor and irreproducible.² Pressure regulation at the detector end is accomplished by using a small i.d. silica-based capillary restrictor. Thus, the pump maintains the system pressure "upstream" from the analytical column and detector. Figure 3 depicts a typical "upstream" set-up.

Injection: One of the biggest limitations to SFC is the small injection volume tolerated by the MP. Even if the injection solvent is the same as the modifier solvent, no more than 20 m L can usually be injected without chromatographic peak distortion.² With a 4.6 mm i.d. column, 5 m L of aqueous solvent can be tolerated, but only at a column temperature of > 60^oC.² SFs cannot solvate large aqueous injections, but on-line phase switching can address this problem. Temperature Regulation. As for GC, temperature manipulation is a practical way to modify chromatographic selectivity. Supercritical fluids have a distinct advantage over many LC liquids since SFs can be held below ambient temperatures, even to below zero. In fact, resolution between stereoisomers may be increased at sub-ambient temperatures.

In interfacing with MS, it should be noted that the nominal temperature at the tip of the interface, as well as within the ion source, are usually higher than in the chromatographic oven. The analytes coming out with the SFC effluent are subject to much lower temperatures due to Joule-Thompson cooling. Due to this cooling phenomenon, the amount of time the compound will experience the elevated temperatures within the MS interface will be brief. If degradation is observed, it is likely that the degradation occurs due to heat transfer from within the chromatography components.

Variations: Packed Column and Capillary SFC. SFs have physical and chemical characteristics simultaneously similar to both liquids and gases. Due to their low viscosity and high diffusivity, SFs function equally well with LC and GC columns. For pSFC, HPLC columns can be used. Meanwhile, cSFC generally use GC columns. Commercially manufactured SFC columns are now available. Together, it has been estimated that they can separate about 30% of all known compounds.² When comparing pSFC to cSFC, complimentary uses can be found.

Packed column SFC provides the means for higher flow rates and higher sample volumes. Higher efficiencies can be obtained in less time with pSFC as compared to cSFC. The number of theoretical plates (N) in pSFC is generally higher than N for the same packed column in HPLC. The flow maximum for pSFC reaches 5 ml/min on a typical 4.6 mm i.d. column (2-5 times that of HPLC) with only ~1/5 of the pressure drop along the analytical column.² In addition, pSFC allows for a wider range of selectivity (a ) adjustment which, along with it’s capacity factor (k’), can be modified with MP composition, stationary phase (SP) identity, temperature and pressure.²

Capillary SFC can easily generate a high N, which results in increased resolutions comparable to HPLC.² The biggest advantage cSFC has over pSFC may be in the ability to be coupled directly into GC detectors (i.e. FID) which pSFC cannot do, especially if a polar modifier is used. The advantage may arguably go to pSFC, especially when considering the use of MS as opposed to FID, but the useful practicality of cSFC remains due to the low flow rates and reduced solvent volumes inherent in capillary delivery systems. In cases where low flow volumes are necessary the SFC flow can be split, thus overcoming this advantage of cSFC.

Packed Column or Capillary SFC/MS? Since both capillaries and packed columns can be interfaced with MS, the choice of column type should be oriented towards the separation goal. Capillary columns may be introduced into the mass spectrometer ionization source directly, as in GC/MS. If a capillary is employed from the analytical column to the ionization source and a relatively low flow rate is used, then packed-columns may be coupled to MS in the same way.¹⁷

Packed columns may also be used with LC/MS interfaces, but the proper modifications must be made in order to meet the demands of retaining the integrity of the supercritical fluid (see below). The demands of pSFC are higher on the MS hardware than those of cSFC. Either more pumping will be needed to compensate for the increase in effluent volume, or a split must precede the ionization source. Prior to 1989 the bulk of the literature had typically supported cSFC/MS. Initial work done with pSFC was done using a moving belt interface; direct flow interfacing with pSFC was not introduced until around 1993.

SFC/MS Operating Requirements. The difficulty in interfacing SFC with MS lies within the differences among the individual separation and detection requirements. SFC operating parameters depend on the nature of the supercritical fluid used as the MP. The number of adjustable parameters usable in SFC is greater than either GC or HPLC. Separations can be modified by controlling percent modifier, modifier composition, MP density (by controlling pressure), temperature or flow rate. Density, temperature, or modifier gradients can be used.

The dynamic ranges of temperatures (31-300^oC for CO₂) and pressures (71-500 atm for CO₂) usable is of great value to pSFC. By adding modifiers and additives, the analyst changes the T_c and P_c of the MP.^, Much care must be taken in order to be certain that the supercritical MP remains as a single phase. Temperatures below T_c will still allow the SF to remain in a single phase, as long as the pressure is appropriate. This modified technique is referred to as subcritical fluid chromatography and allows MP performance at and below ambient temperatures (even sub-zero temperatures are usable). This aspect is of great benefit to chiral separations since the fine manipulation of MP-SP interactions are thermodynamically driven.

MP Considerations for SFC/MS. Carbon dioxide is the most popular supercritical fluid due to it’s low cost, high selectivity, inflammability, and low toxicity. It possesses a relatively low critical pressure (72.9 atm) and critical temperature (31.3^oC), which are easily achievable with the minimum of instrumental strain. Thus, in order to keep CO₂ as a single supercritical phase, all that is required is to keep the working pressure outside of the single phase boundaries given in the phase diagram for carbon dioxide (Fig. 4). Other SFs that have been considered for use in SFC include N₂O (highly flammable) and CCl₂F₂ (ozone damaging). Each SF will have unique critical parameters, which will dictate systematic conditions.

Using pure SF as the MP generally limits the range of compounds that can be solublized to non-polar compounds. Pure CO₂ is observed to have a polarity similar to pentane or hexane.² Polar solvents can be added on-line to increase the MP’s polarity. In this way, the range of compounds usable in SFC is dramatically increased to include polar compounds. In addition to these MP modifiers, additives, such as organic acids and bases, can be added to the modifier in low concentrations (i.e. 10^-4 M) in order to aid in chromatography as in HPLC. Inorganic salts and other ionic species will not likely be dissolved into the SF and will form deposits in the injector and within the narrow tubing. Additives will be necessary to promote chemical ionization in SFC/CI-MS.

Stationary Phase Considerations for SFC/MS. Stationary phases (SPs) used in HPLC can also be used in pSFC. Normal phase columns usually work best, but since MP components can cluster about the individual particles, they tend to perform more like reversed phase SPs.² In addition to the more common 4.6 mm i.d. columns, narrow-bore² (2.1 mm i.d.) and micro-bore (1.0 mm i.d.) columns have been used successfully, generally without a loss in flow rate.

SFC/MS Solvent Delivery Systems. There are several considerations to be addressed prior to constructing a SFC to MS interface. These special considerations revolve around the use of a supercritical fluid as a MP. The core of these requirements reflects the need for LC-like conditions needed for the high flow rate used in pSFC. The GC-like conditions used with cSFC fall within the acceptable values for popular GC/MS interfacing, barring excess pressure.

Flow. Capillary columns provide a gas flow output of 1 cm³/min; whereas packed columns tend to give 20 cm³/min of gas flow. A large volume of nonvolatile liquid effluent essentially accompanies this increase in gas flow. HPLC/MS interface systems have been modified in such a way as to remove the excess solvent from the ion source. These advances have made a way for SFC to directly deliver all of the effluent directly into an ionization source. The post-expansion effluent of SFC is less of a problem than that of HPLC, since it is composed of high-pressure gas (such as CO₂). This makes the effluent easier to pump away, but increases the source pressure considerably requiring a higher sweep gas flow.

MP Evaporation. Most SFC modifiers are adequately volatile and should not collect as a liquid under heated ion source temperatures. The boiling point of the modifier and of any additive should be considered for SFC/MS, especially when large volumes of modifier are essential to the separation. Even more importantly, the flammable nature of the MP components should be closely considered when an exposed electric charge is used to accomplish ionization.

MP Ionization Mechanisms. The solvating properties of supercritical fluids govern the success of chemical ionization in SFC/MS. Organic acids, such as trifluoroacetic acid (TFA), are only effective when they are stronger than acidic solutes.²¹ Minor differences in acid strengths between additives and analytes appear to have a significant effect on solute peak shapes and retention. This will affect the chromatography, but should not affect the mass spectrometry unless the sample is insufficiently ionized.

Restrictors. Since GC detectors require low flow rates and smooth, continuous nebulization, a short, straight, evenly heated flow restrictor is required.²² If there are any bends in the restrictor path, the low temperature of the SFC MP can promote poor performance from gradual sample build-up. The temperature of the restrictor should be maintained close to the temperature of the analytical column in order to avoid solute loss. The shorter the restrictor, the less the imparted temperature will affect the solute(s).¹⁰ Choosing the right restrictor is essential for the pursuit of SFC/MS, the consensus leaning towards the use of integral restrictors because of their turbulent flow characteristics prolongs their usefulness.^14,

Reasonable Expectations for Ionization Modes. Under vacuum, the supercritical temperatures that accompany SFs may cause the components of the interface to freeze. This may be overcome by heating the source. This is counter-intuitive to the reasoning for choosing SFC over GC, since the temperatures needed coincide with those required for GC-MS interfacing. However, the Joule-Thompson cooling effect, which occurs during nebulization, allows labile compounds to remain intact during heating in the probe.¹⁴ This advantage allows probe heating as per manufacturer’s recommendations promoting maximum detector sensitivity.

Particle beam ionization interface. A modified particle beam interface has been successfully used to perform electron impact ionization (EI). Analytes were separated by pSFC and flowed through a heated restrictor to a two-stage momentum separator (Fig. 5), using helium as a coaxial (nebulizing) gas. The SFC effluent was formed into small particles and successfully eliminated. EI is performed by flash volatilizing the eluates. Low microgram levels of involatile, thermally labile analytes has achieved. Since this work was done (1989), efforts have been put forth into the optimization of particle forming mechanics. Factors influencing aerosol formation has been evaluated (Table 1). With these improvements came an increase in analyte sensitivity, and limits of detection (LOD) have been recorded in the 1 ng (EI, Fig. 6) to 10 ng (SIM, Fig. 7) range. Additionally, EI spectra are reportedly artifact-free comparable to on-line library spectra. Equal success was achieved for pure and modified CO₂. Figure 8 shows the nebulizer assembly and modified set-up. Another sample separation is shown in Figure 9.

Chemical Ionization (CI) interface. Ionization mechanisms in cSFC/CI-MS have been plagued with interferences caused by MP constituents. It has been discovered that adding a reagent gas to the ion source can reduce these interferences. A fused silica capillary column is modified by the addition of a heating mechanism. Reagent gas is flowed coaxially through an interface housing and separated from the capillary by a glass isolation tube (Fig. 10). The properties and pressure of the selected reagent gas control ionization. Protonization is increased with increasing reagent gas partial pressure (Fig. 11). The reagent gas also acts to break up solvent clusters. If an excess of reagent gas is used, the charge exchange processes are suppressed between MP constituents and solutes. Charge exchange then favors fragmentation over H⁺ transfer and the ionization spectra are comparable to EI spectra libraries (Figures 12-14).

Atmospheric pressure chemical ionization (APCI) interface. An APCI interface has been developed for the coupling of cSFC to MS. This was accomplished by inserting the cSFC restrictor directly into the APCI source. A flow rate of 2 ml/min is possible with a pressure range of 180 to 230 bar. No modifier was used which made the interfacing appreciably straightforward. Figures 15 and 16 shows a sample chromatograph and it’s mass spectra.

Electrospray ionization (ES) interface.¹⁸ ES can accommodate the higher flow rates (0.15-1.0 ml/min) used by pSFC, especially when modified CO₂ is necessary to achieve separation. Ionization efficiency is impacted by MP composition (Fig. 17) and temperature (Fig. 18). Mechanistically, flow is delivered through a restrictor, the end of which is inserted to about 5 cm and in direct line of sight with the ion-sampling aperture. Sufficient charge is imparted upon the effluent by covering the fused-silica polyimide coating with an electrically conducting layer of nickel-doped polyurethane paint over a 10-cm length. The restrictor end is kept at ground potential, being heated by a nitrogen curtain gas kept at 80^oC at the atmospheric pressure within the source. Figure 19 shows the schematic of the ES set-up. The SFC/ES-MS instrument is still limited to the determination of low mass samples owing to cold trapping on some critical surfaces. The SFC/ES-MS analysis of a pyridine derivative mixture is shown in Figure 20.

Ionspray ionization interface. Some modifications have been made to existing Ionspray technology in order to accommodate pSFC. A sheath-flow liquid is introduced coaxially to the fused-silica transfer line through a tee. The outlet does not need to be restricted. A second tee is used to deliver the nebulizing gas. The gas is delivered through a larger i.d. stainless steel (s.s.) tube such that the total effluent is nebulized. The back-pressure from the flow source is sufficient to control the outlet pressure in a way similar to a BPR. The liquid flows in the small area between the (slightly regressed) fused silica transfer line and an s.s. capillary, so as not to interact with the pSFC MP until they are united at the nebulizer tip. Figure 21 shows a diagram of the modified Ionspray interface. Both tees should be held at the nebulizer potential (4 kV) in order to maximize ionization efficiency. Figures 22 and 23 show the results of using the modified interface, as given in reference 27. There was no signal degradation when they accidentally lost their high voltage on the ES needle (Fig. 24), and the authors are actually a loss as to explain why. This interface seems to be the most promising for bioanalytical applications because of the ease in modifying existing technology to suit the needs of pSFC.

Solvent Evacuation Through Vacuum Pumping.¹⁵ The high gas load inherent in SFC generally requires the employment of differential pumping. It is also necessary to continuously sweep the ion source with an inert carrier gas. Without such precautions, the increased flow and the gas expansion due to SF decompression may decrease performance of the MS.

Quadrupoles. In terms of SFC interfacing, quadrupoles might display the most advantages. They tend to be regarded as the mass analyzer of choice in SFC/MS. The direct fluid introduction (DFI) interface on a triple quadrupole mass spectrometer allows a m/z range of 4000 Da per unit charge.¹⁴ Features of merit include direct SFC interfacing as well as appreciable sensitivity, reasonable m/z range and moderate cost.

Magnetic Sectors. Carefully designed probes have been constructed in order to interface the SFC effluent to the high voltage source of mass sector analyzers.^, The gas effluent of cSFC should be handled fairly well by existing vacuum systems. The separation power of SFC would be a desirable compliment to the high sensitivity and wide m/z range of a magnetic sector.

Time of Flight (TOF). With the advance of electronic capability, TOF may soon be a contender for SFC interfacing. The limitations do not arise from SFC, but from the inability to deliver high flow rates into the TOF delivery system. When SFC/TOF-MS becomes available, it is conceivable that it should be very popular due to TOF’s cost effectiveness and it’s ability to analyze a broad range of m/z with high sensitivity and versatility.

Ion Cyclotron Resonance (ICR). The detection features of the SFC/ICR instrument would make ICR most likely the detector to beat in SFC/MS. However the high gas loads of SFC effluent puts the interface at a strong disadvantage. Limited success has been achieved by using a longer than usual interface line, but the most effective route might also include a method of differentially pumping the external ion source in order to cancel out these obstacles.

Quadrupole Ion Traps (QIT). As for ICR, QIT has difficulty with dealing with the high-flow gaseous effluent of SFC. Ion traps require high-pressure inert dampening gases, such as helium, to maximize ionization efficiency. Most SFC MPs are not good dampening gases.¹⁵ The system could benefit from a means to draw away undesirable SF MPs which fill the trap. A differential pumping, similar to the one suggested for use in ICR, should allow desirable results.

Biological Sample Considerations. The biggest obstacle to overcome introducing biological matrix to SFC arises out of the lack of solubility that water has with SFs. A few m l of water can be tolerated at 40^oC and up to 5 m l at T > 60^oC.² The issue of larger aqueous volume injection has been addressed by replacing aqueous matrix with supercritical MP via an on-line column-switching set-up. Success has been achieved for both pSFC and cSFC. This, however, adds considerable time to an analytical run and the pros and cons between SFC and HPLC must be weighed in this light. Fortunately, MS requires very little sample and the small amount of aqueous matrix soluble in SFs may be sufficient to generate reliable mass spectra.

Potential for Analysis of Chiral Compounds. As mentioned, the real breakthrough for SFC in the bioanalytical field has been its contribution to chiral separations. The nature of supercritical solvents has allowed unique and sufficient interactions with isomers that are useful in chiral recognition. There has been appreciable advancement in chiral separation within the field of subcritical fluid chromatography. There is an abundance of literature supporting chiral SFC, especially in selecting the proper column for chiral separation. There is little doubt that SFC/MS will impact how pharmaceutical companies screen drug candidates for chiral impurities and target analyte chiral inversions caused by metabolism.

Bioanalytical chemistry has much to gain from SFC/MS. The enforcement of strict quality standards has produced a need for fast, complete and sensitive analysis of drug candidates. SFC can provide the fast and complete analysis, and MS can provide the universal, sensitive detection. Much work has been accomplished by means of interfacing the two technologies. SFC/MS shows great potential in the field of bioanalytical chemistry, but especially in chiral compound separation and detection. Currently, it appears that the best SFC/MS combination available for bioanalytical applications might be a modified ionspray interface coupled to a quadrupole mass analyzer. As the science advances, it would be reasonable to foresee the practicality of this analytical application reach into the chemistry laboratory mainstream.