Past, Current, and Future Directions in Supercritical Fluid Chromatography


Special Issues

LCGC SupplementsSpecial Issues-10-02-2013
Volume 31
Issue 10

Supercritical fluid chromatography has become a viable option for the separation scientist in diverse areas, and the field now seems more adequately described as an extension of HPLC, or perhaps as "carbon dioxide–based HPLC".

At the turn of the 21st century, supercritical fluid chromatography (SFC) experienced a re-birth prompted by a demand to separate polar analytes, initially in the pharmaceutical industry. Instrument design and performance improved in tandem with this demand. Preparative SFC has also improved significantly over the last decade and chiral stationary phases have been introduced for the SFC of racemic drug-related mixtures. The introduction of more polar stationary phases and unique mobile phase additives has enabled separation of water-soluble, ionic, and high molecular mass analytes. This article looks at the past, present, and future of SFC.

Early Days: 1962–1981

Supercritical fluid chromatography (SFC) was first proposed in 1958 at an international gas chromatography (GC) meeting by James Lovelock, one of the research pioneers in GC, while at Yale. Lovelock conceived the idea of using supercritical fluids as chromatographic mobile phases to increase solvating power, and to facilitate the elution of nonvolatile substances (1). In 1962, Ernst Klesper was the first person to use a supercritical fluid as a chromatographic mobile phase, utilizing it for the analysis of nonvolatile materials. Klesper used a 30-in. long column packed with 33% Carbowax 20M on Chromosorb W to separate a mixture of nickel porphyrins with supercritical dichlorodifluoromethane and monochlorodifluoromethane mobile phases. The technique was called "high pressure gas chromatography" (2). Work in the area of supercritical fluid technology continued with a number of applications, including the analysis of petroleum mixtures and silicone polymers (3). Between 1962 and 1981 nearly all of the 91 SFC-related papers published involved the use of packed columns (4), but SFC remained relatively underdeveloped as a viable separation alternative. In 1983, Hewlett Packard (HP) modified a limited number of their Model 1084 liquid chromatography (LC) instruments for packed column SFC (pcSFC). They were flow-controlled and able to accommodate the use of various modifiers. In the mid-1980s, a second commercial instrument was introduced that incorporated open tubular, wall coated fused silica column technology (5). This action afforded unprecedented litigation that prevented further packed column SFC development for several years. During this time, capillary SFC experienced explosive growth because it was seen as an extension of GC where the conventional sample base could be extended to less volatile, more reactive analytes. Nevertheless, many experimental issues associated with capillary SFC were not readily addressed, despite its wide acceptance at the time.

Open Tubular Column SFC: 1981–1995

Despite worldwide acceptance, in the late 1980s SFC using capillary columns was in bad shape. There were some prominent, unsolved experimental issues:

  • No mobile phase flow control

  • Addition of modifier was not possible

  • Operating temperatures were high

  • Retention times were long

  • Helium-padded CO2 was necessary

  • UV detection was feasible but not practical

  • Fragile fused silica restrictors were employed

  • Injection was through a retention gap

Extension of the technology to small bore packed columns by Lee Scientific and Brownlee, Inc. afforded minimal advantage. Regardless, the technique was viewed as an extension of GC that was amenable to primarily high molecular mass, non-polar analytes. Separation scientists generally agreed that SFC seemed to be finding its niche as a technique that could handle larger molecules than GC, at higher efficiencies than high performance liquid chromatography (HPLC). However, although numerous SFC vendors offered GC instruments with basic modifications, there was no long-term vendor commitment to the technology. Columns tended to be operated well above the critical temperature of the supercritical fluid, while pressure (or density) programming replaced conventional temperature programming. Capillary SFC was most often practiced using (a) columns with inner diameters considerably smaller than GC columns (50 μm i.d.); and (b) highly cross-linked non-polar stationary phases to avoid significant CO2 promoted column bleeding. Capillary columns dominated the development of both SFC theory and instrumentation during this period (5). A syringe pump was used as a pressure source, with a fixed restrictor at the column outlet limiting flow through the small inner diameter open tubular column. Primary vendors in the development of capillary SFC during the 1980s were Dionex and Isco Inc.

Packed column SFC was not viewed with much favour because it almost always used: (a) Silica-bonded stationary phases such as cyano– and amino–propyl designed specifically for HPLC; (b) binary or ternary CO2-based mobile phases; and (c) ultraviolet (UV) detection via a high pressure flow cell. Once modifiers were added to the CO2, mobile phase composition became more important than CO2 pressure or density in determining retention. By necessity, capillary SFC exclusively focused on non-polar analytes; whereas packed column SFC found limited application in the separation of polar analytes (6). Packed columns were usually operated near the critical temperature of the fluid, with less than precise flow control pumps and electronically controlled back pressure regulators. The combination of upstream flow control and downstream pressure control allowed, to a limited extent, both volumetric mixing of the main fluid and modifier. With these developments, packed column SFC was increasingly viewed as an extension or subset of HPLC. The most important idea that supercritical fluids brought to separation science during this period was probably the recognition of the unity in the separation methods, and that a continuum existed between gases and liquids.

In 1992, Gilson, Inc. and Hewlett Packard, Inc. both introduced commercial analytical scale SFC systems that allowed computer control of all variables. The HP system was capable of both packed and capillary column operation; while the Gilson instrument delivered a higher flow rate. This prompted several unsuccessful attempts to create a small-scale semi-preparative instrument using 1-cm inner diameter columns. The HP product line was sold in 1995 to Berger Instrument where semi-preparative SFC was found to be successful for both achiral library purification and chiral-like stacked injections.

Packed Column SFC Rediscovered: 1990–2012

SFC with packed columns underwent a renaissance at the end of the 1990s when (a) the limitations of capillary SFC became obvious to separation scientists; and (b) important progress in pressure gradient techniques of mixed mobile phases was achieved. Advancements in this area were halted by misconceptions about the role of modifiers in the SFC chromatographic system. For example, it was widely thought that modifiers only covered active sites on the support and did not dramatically increase solvent strength. Currently, the overall effect of adding a modifier to the mobile phase is considered to be a combination of mobile phase modification effects and stationary phase effects. Despite the early success enjoyed by SFC with modifiers and additives, at the time there was considerable resistance to the use of more complex and expensive binary pumping systems, and UV detectors with packed columns (7).

Today, pcSFC is generally agreed to be an ideal replacement for normal phase LC. SFC (a) exhibits faster retention; (b) re-equilibrates extremely fast; (c) allows steep gradients; (d) tolerates significant amounts of water; (e) allows very long columns; (f) can incorporate very small particles with modest column pressure drops; and (g) is "green" technology. SFC outperforms normal-phase HPLC and even reversed-phase HPLC for some applications. Interest in packed column SFC is higher than ever, as it is capable of generating peak efficiencies similar to those observed in GC, and separations can be run at much higher flow rates than in HPLC. Furthermore, pcSFC is scaleable, detector-friendly, and relatively economical. Unlike reversed-phase HPLC, in which almost all work is performed on a variant of a single stationary phase (C18), SFC employs a wide range of polar stationary phases with significantly different selectivities (8).

During this period, pcSFC was observed to offer many advantages for the rapid separation of enantiomeric products over HPLC because of its greater separation efficiency per unit time (9). Lower temperature was beneficial as chiral selectivity usually increased with decreasing temperature. Low temperature also reduced the risk of analyte racemization and thermal decomposition. To successfully perform chiral pcSFC, a polar modifier was added to the CO2 mobile phase and, in many cases, operation across a gradient became desirable during the separation. pcSFC was ideal for chiral screening that provided rapid identification of the most appropriate column and modifier. Finding a highly selective chiral stationary phase was a key step in method development and the column screening strategy proved to be highly successful.

The use of SFC for preparative separations (PrepSFC) has received considerable attention in the past three years (10). The volume of solvent and waste generated is reduced, offering an economic bonus, and making PrepSFC especially attractive for the provision of purified materials on a kilogram scale. The product of SFC is recovered in a more concentrated form relative to HPLC, reducing the amount of solvent that must be evaporated. The higher pcSFC flow rates also contribute to higher productivity relative to HPLC methods. The faster SFC process makes the separation cycle time significantly shorter, so that it is practical to make purification runs by "stacking" small injections in short time windows, without compromising throughput. In this way, the utilization rate of expensive column material is much higher.

Throughput in preparative chromatography is dependent on separation factors and the solubility of compounds in mobile phases. While chiral discrimination is required for purification, good racemate solubility (ideally greater than 20 mg/mL) is also necessary for a successful preparative resolution. A compound with poor solubility is more problematic in preparative purification than analytical chromatography because the typical ratio of sample to both mobile and stationary phases is much higher in preparative chromatography (11). Traditionally, mobile phase solubility measurement is overlooked when performing preparative SFC, because it is difficult and time-consuming.

The use of PrepSFC for high-throughput purifications initially focused on UV-based fraction triggering systems. Productivity enhancements quickly higlighted the need to have mass-directed PrepSFC capabilities. The principal advantage of mass guided preparative chromatography is that the collection of fractions can be limited to only those compounds eluting within the target mass range. Mass-directed systems accomplish this by monitoring the extracted ion signal from the mass spectrometer to trigger fraction collection (12). The triggering mechanism is compound specific, in contrast to previous unselective UV triggering mode.

In summary, pcSFC (as have most analytical techniques) has had a tortuous development history. However, it now appears that analytical- and preparative-scale SFC methods for purification are currently on the strongest foundation ever with vendors that are strongly committed to advancing the technology. New developments regarding achiral pcSFC and a broader spectrum of applications in the field are anticipated in the future.

Future SFC

(a) Method Development for Analytical SFC: Even though academic interest in SFC has basically died and numerous publications by private industry have not been forthcoming, the future looks exceedingly bright. From an internet and scientific meeting perspective, pcSFC applications in the food, environmental, biomedical, natural products, and energy areas are expanding and complementing many of the well-publicized pharmaceutical areas. The most widely used approach for resolving complex mixtures has been to change selectivity of the chromatographic system (not increase plates or increase retention time). One can, therefore, expect the introduction of a new range of polar stationary phases for pcSFC in the future, such as the popular ethyl pyridine phase which was introduced several years ago by Princeton Chromatography, Inc. specifically for pcSFC. While these developments will focus on nitrogenous bases such as amide and imidazole functionalities, the future continues to look promising for highly pure, bare silica, and related hydrophillic interaction liquid chromatography (HILIC) phases for separation of highly polar analytes. Phases that reduce the need for mobile phase additives in the separation of ionizable analytes will also be of interest for PrepSFC. Porous silica particles that are smaller than 5 μm will prove beneficial along with porous surface layer particles of similar size as faster, more efficient separations are desired. Additional polar stationary phases and the introduction of mobile phase additives that are either acidic, basic, or neutral have led to promising separations of water-soluble, ionic, and high molecular mass analytes. Ion-pairing–SFC and HILIC–SFC are expected to be fertile areas for expansion into applications of achiral separation of biomarkers and metabolites for example (13). The maturity of SFC–UV technology has truly created a seamless interface with both gas chromatography–flame ionization detection (GC–FID) and HPLC–UV. The overall acceptance of pcSFC will depend on its future robustness and analytical sensitivity in comparison with conventional and equally popular analytical techniques (14).

Figure 1: Suggested separation strategies for achiral SFC illustrating that SFC spans a wide range of polarity. Adapted with permission from Aurora SFC Systems.

While method development in chiral pcSFC has evolved into an economically expensive, multi-column, trial and error approach, achiral pcSFC development will not be receptive to this type of strategy as analytes have greater variation. Forty years ago, pcSFC was thought to be most applicable for non-polar analytes, but today (and into the future) pcSFC will "fit-in" anywhere between nonpolars and highly charged analytes (see Figure 1). Four protocols can be envisioned with packed column technology based upon preliminary reported studies (13):

(i) Pure CO2 for elution of hydrocarbons, ethers, and esters.

(ii) CO2 + methanol (or acetonitrile) for elution of alcohols, amides, and aniline.

(iii) CO2 + methanol + trifluoroacetic acid (or isopropyl amine) for elution of ionizable acids and bases.

(iv) CO2 + methanol + ammonium acetate + water for elution of amphoterics, peptides, carbohydrates, water soluble vitamins, and inorganic ions.

(b) Validation of SFC: There are a number of manufacturers, past and present, producing equipment for pcSFC. Most users appear to assume that all instruments produce the same, or similar, flow and composition performance with minor variations even though there are at least three different reported kinds of pumps used to perform pcSFC. There also appears to be no recognition that there are significant pumping differences in various designs. In other words, no consensus exists among the separation community as to what constitutes a standard approach (15). Furthermore, there appears to be no analysis of how design differences might affect validation of methods when transferring a method from one laboratory to another (that is, robustness). For the record, there is only one reference in the literature that proposes specifications for the validation of equipment for pcSFC in the pharmaceutical industry (16). Although more than 10 years old, this proposal has not been adopted. Adding to the difficulty, many suggested measurement techniques are not very accurate or reproducible. Specifications of control variables, such as pressure or flow rate, must allow a wider range of variability in the measurement result than the variability in the sensors used to monitor the process (15). Furthermore, flow values should be corrected to conditions at the pump rather than to condition at the meter. Steps toward validation and robustness in pcSFC must be considered by vendors in the future.

(c) Sample Throughput: High sample throughput is often required in applications with large numbers of samples such as library screening. It is often true that chromatograms can be generated in less than a minute, but the system cycle time is extended by an additional 1–2 min to prepare to inject, perform detector balance, store the method with the previous runs raw data, external needle wash, needle and loop wash steps, and so on. Throughput can be significantly increased by reducing the delay time between the end of one run and the injection of the next sample. Efficient user-friendly protocols should be perfected by all vendors in the future.

(d) Trace Analysis: SFC is almost never used for trace analysis. Poor sensitivity and inadequate dynamic range are two prevalent issues. These limitations have, in the past, discouraged the use of pcSFC in environments such as quality assurance (QA) and quality control (QC) and relegated it to major–minor component analysis. A significant improvement in sensitivity could expand the role of pcSFC out of pharmaceutical analysis into a much wider range of applications. A review of the literature only produces a limited number of papers attempting to quantify impurities or metabolites at the 0.1% level and below. In traditional pcSFC systems, the compression of carbon dioxide is thought to be the dominant cause of UV detector noise. By isolating compression and metering, the detection limits and dynamic range have been shown by Berger and colleagues (17) to be improved well over an order of magnitude. In pcSFC, the compressibility is as much as 20 times higher than in aqueous-based HPLC, thus greatly exaggerating compression problems. The investigators in this work believe the low noise is the result of separating compression from the metering of the fluid. In most pumping systems, the same pump both compresses and meters the flow. Direct quantitation, however, means both the main peak (100%) and the trace contaminants (>0.1% of the main peak) are in a linear region of the detector with adequate signal to noise. If the main peak is 2 AU tall and S/N>10:1 is required to quantitate the trace components, the noise (peak to peak) must be <0.2 mAU and preferably lower to provide margin. The improvements noted here appear to be related to a new approach to pumping carbon dioxide which should open the door for more widespread use of pcSFC in regulated environments that require an extended dynamic range and better sensitivity. The future for pcSFC will hopefully see more vendors embrace technology that enhances pcSFC–UV sensitivity.


In summary, with faster separation, higher sample throughput, less organic solvent usage, and normal-phase mode purification, packed column-SFC has become a viable option for the separation scientist in diverse areas. Its applicability for achiral separations, its ability to separate water soluble analytes, and its feasibility for coupling columns of varied chemistries is only beginning to be appreciated. New areas of application can be envisioned such as flash SFC and its correlation with thin layer chromatography. The role of mobile phase components in changing both the polarity of the stationary phase and the mechanisms of retention are worthy of study (18). While the SFC area was given its first "push" as an extension of GC over 30 years ago, the field now seems more adequately described as an extension of HPLC. As a matter of fact, the area might be more readily advanced if it were thought to be "carbon dioxide-based HPLC".


Informed discussions with Mehdi Ashraf-Khorassani (Virginia Tech, Blacksburg, Virginia, USA) regarding the preparation of the manuscript have been very helpful.

Larry T. Taylor is emeritus professor of chemistry at Virginia Tech in Blacksburg, Virginia, USA. Please direct correspondence to:


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