1570 Reviews Supercritical Fluid Chromatography – Theoretical Background and Applications on Natural Products Authors Anja Hartmann, Markus Ganzera Affiliation Institute of Pharmacy, Pharmacognosy, University of Innsbruck, CCB, Innsbruck, Austria Key words Abstract DGC: dense gas chromatography l" SFC ! DPPH: 2,2-diphenyl-1-picrylhydrazyl l" supercritical fluid The use of supercritical fluid chromatography for ECD: electron capture detector chromatography natural product analysis as well as underlying ELSD: evaporative light scattering detector l" natural products analysis theoretical mechanisms and instrumental re- ESI: electrospray ionization l" preparative l" analytical quirements are summarized in this review. A FID: flame ionization detector l" applications short introduction focusing on the historical de- FPP: fully porous particles velopment of this interesting separation tech- FTIR: Fourier transform infrared nique is followed by remarks on the current in- spectroscopy strumental design, also describing possible detec- GC: gas chromatography tion modes and useable stationary phases. The GMP: good manufacturing practices overview on relevant applications is grouped HPGC: high-pressure gas chromatography based on their basic intention, may it be (semi) HPLC: high-performance liquid preparative or purely analytical. They indicate chromatography that supercritical fluid chromatography is still HPTLC: high-performance thinlayer primarily considered for the analysis of nonpolar chromatography analytes like carotenoids, fatty acids, or terpenes. IC: ion chromatography The low polarity of supercritical carbon dioxide, IMS: ion mobility spectrometry which is used with modifiers almost exclusively IPA: isopropylamine as a mobile phase today, combined with high effi- IR: infrared ciency and fast separations might explain the LOD: limit of detection popularity of supercritical fluid chromatography MALDI‑TOF‑MS: for the analysis of these compounds. Yet, it has matrix-assisted laser desorption received October 1, 2014 revised February 3, 2015 been shown that more polar natural products (e. ionization time-of-flight mass accepted March 4, 2015 g., xanthones, flavonoids, alkaloids) are separable spectrometry too, with the same (if not superior) selectivity and MS: mass spectometry Bibliography reproducibility than established approaches like MTBE: methyl tert-butyl ether This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. DOI http://dx.doi.org/ 10.1055/s-0035-1545911 HPLC or GC. NMR: nuclear magnetic resonance Published online April 23, 2015 ODS: octadecylsilyl Planta Med 2015; 81: OTC: open tubular column – 1570 1581 © Georg Thieme Abbreviations RP-HPLC: reversed-phase HPLC Verlag KG Stuttgart · New York · ! ISSN 0032‑0943 RSD: relative standard deviation ACN: acetonitrile SIC: silver ion chromatography Correspondence APCI: atmospheric pressure chemical SFC: supercritical fluid chromatography Assoc. Prof. Dr. Markus Ganzera ionization SFE: supercritical fluid extraction Institute of Pharmacy, CAD: charged aerosol detector TAGs: triacylglycerols Pharmacognosy CD: circular dichroism THF: tetrahydrofuran University of Innsbruck Innrain 80–82 DAD: diode array detector UHE/LP: ultrahigh efficiency/low pressure 6020 Innsbruck DCM: dichloromethane Austria Phone: + 4351250758406 Fax: + 435 1250758499 [email protected] Hartmann A, Ganzera M. Supercritical Fluid Chromatography… Planta Med 2015; 81: 1570–1581 Reviews 1571 Technical Aspects ! Historical development and current state of instrumentation It is maybe a surprising fact that the first report on SFC, at this time the technique was called HPGC or DGC, occurred in the year 1962 already, slightly before the evolution of HPLC started. Kles- per et al. used a mobile phase of supercritical fluoromethane de- rivatives to successfully separate a mixture of porphyrins on a polyethylene glycol stationary phase [8]. A few years later, Sie and coworkers described CO2 as a suitable eluent and UV as a possible detection mode [9]. In the 1970 s, SFC instruments with pressure programming were constructed [10], the first prepara- tive applications reported [11], and an SFC‑MS interface de- scribed [12]. The decade thereafter brought mainly improve- ments in column technology, leading to two different strategies. Fig. 1 Phase diagram of a compound indicating the supercritical fluid OTCs developed by Novotny and coworkers resembled the GC stage above critical pressure (Pc) and temperature (Tc). columns used today, i.e., the inner surface of a silica capillary with a small internal diameter (typically 50 µm) is coated with a thin film of stationary phase, e.g., a polysiloxane [13]. These ma- terials showed several disadvantages, such as that the mobile Introduction phase pressure could not be changed independently of the flow ! velocity, or the commonly used type of detector (FID) was incom- Whenever temperature and pressure exceed a substance-specific patible with organic modifiers. This explains why the interest in critical value (i.e., the critical point), a supercritical fluid of a com- this SFC variant faded rather quickly [14]. The second strategy, pound is obtained (l" Fig. 1). In this state, neither higher pressure which was developed at the same time and is standard nowa- can revert it to a liquid again nor elevated temperatures to a gas. days, is the use of packed columns as employed for HPLC [15]; In simplified terms, supercritical fluids can be seen as highly for further details on stationary phases and SFC, see the respec- compressed gases with liquid-like density [1]. They combine tive chapter in this review. The design of a packed column SFC characteristics of both physical states in a unique way, such as system is schematically shown in l" Fig. 2. As can be seen, it re- low viscosity and high diffusivity [2]. In terms of chromatography sembles the common HPLC setup to a large extent. In addition to (supercritical fluid chromatography), this results in a low pres- pumps for the mobile phase (CO2 and modifier), an injection and sure drop even at higher flow rates, faster separations, increased detection device, it requires two additional components: an oven solvating power, and efficiencies, so that fluids in supercritical to facilitate supercritical/subcritical conditions and a backpres- conditions often are considered as ideal mobile phases [2,3]. To- sure regulator, commonly positioned after the detector. Its func- day, carbon dioxide-based fluids are used almost exclusively be- tion is to adjust the fluid pressure above the critical pressure in- cause of several reasons [4]. Critical temperature (31°C) and dependent of the mobile phase flow rate or composition. Thus, pressure (74 bar) are easy to reach with the conventional type of this electronic device originally developed by Saito et al. is most HPLC instrumentation; CO2 is inert, nonflammable and cheap, it important to maintain SFC conditions throughout the entire sep- can be liquefied and stored in cylinders without problems, and it aration system [1]. is considered environmental friendly (“green”). The latter might not remain without opposition, but as carbon dioxide used for Stationary phases SFC is an industrial by-product, the overall environmental impact Pure, liquefied carbon dioxide is a solvent with nonpolar charac- of using it is zero (in contrast to the production/disposal of regu- ter, and SFC was often considered an alternative for normal- lar solvents) [5]. The polarity of supercritical CO2 is similar to phase chromatography only. In this context, polar stationary hexane, but by adding modifiers like methanol, ethanol, or aceto- phases like silica gel, cyano- or aminopropyl-bonded silica are This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. nitrile, solvent strength is adjustable, so that normal- or re- suitable options [16]. But as the eluotropic strength of CO2 can versed-phase stationary phases can be utilized [6,7]. Carbon di- be modified by polar additives, reversed-phase type materials oxide, due to its rather low critical temperature, is also ideal for (e.g., alkyl-bonded silica) are useable as well. Thus, in respect to the analysis of thermo-labile compounds and in case the isola- column technology, SFC, at least as it is most commonly used to- tion of constituents is attempted, no final evaporation of the sol- day, resembles “standard” liquid chromatography to a large ex- vent is required. At atmospheric pressure, CO2 converts to the tent. The same stationary phases (including aromatic, halogen- gaseous state and discharges, leaving the purified fractions/com- ated, or porous graphitic carbon and polymeric phases) and col- pounds behind. umn dimensions as in HPLC can be used regardless if the separa- tions are performed in the subcritical or supercritical stage [17, 18]. For a differentiation of the latter two, see the comments be- low (Theoretical Considerations). This indicates that SFC is rather versatile in respect to applicable stationary and mobile phases, and both need to be considered in order to improve chromato- graphic performance and selectivity during method develop- ment. Hartmann A, Ganzera M. Supercritical Fluid Chromatography… Planta Med 2015; 81: 1570–1581 1572 Reviews Theoretical Considerations ! By definition, SFC-based separations should be performed under supercritical conditions, which only occur above critical pressure and temperature. These values vary depending on the