HPLC with supercritical mobile phases

Supercritical mobile phases open up an area that is related to both gas and liquid chromatography but is also subject to its own set of laws. Some of the physical phenomena are both interesting and unusual  

The vapour pressure of volatile samples has a great influence on retention behaviour, this being one of the analogues with GC. Compounds that are potential mobile phases are listed in Table 23.1. The first two are obviously unsuitable for temperature-sensitive analytes. By far, carbon dioxide is used most frequently. Since it is very nonpolar a common B solvent is methanol. 

Accurate pressure- and ternperatiire-control facilities are required for supercritical chromatography. The mobile phase must be heated to the correct temperature in a spiral before the injection valve. The spiral, valve, column and detector should all be placed in an oven. A restrictor must be placed behind the detector so that the whole system can be maintained at a sufficiently high pressure. As columns it is possible to use either open capillaries, which allows us to obtain very high plate numbers, as well 

TABLE 23.1 Potential mobile phases for supercritical HPLC 

Polar analytes are less suited for SFC. The sample solvent can be methanol or everything which is less polar. 

A pure compound may be in the state of a gas, solid or liquid (or even multiphase) depending on the pressure and temperature, the interrelationship being shown in Fig. 22.5. Following the vapour pressure curve which separates the gas and liquid states in the direction of increased pressure and temperature leads to an area in which the densities of both phases are identical. A phase that is neither gas nor liquid follows on from the critical point P (shaded area) and this is known as the fluid or supercritical area. The fluid can be used as a mobile phase for chromatography which is then referred to as supercritical fluid chromatography (SFC). 

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With the demonstration of supercritical fluid extraction, an obvious extension would be to extract or dissolve the compounds of interest into the supercritical fluid before analysis with SFC.(6) This would be analogous to the case in HPLC, where the mobile phase solvent is commonly used for dissolving the sample. The work described here will employ a system capable of extracting materials with a supercritical fluid and introducing a known volume of this extract onto the column for analysis via SFC. Detection of the separated materials will be by on-line UV spectroscopy and infrared spectrometry. The optimized SFE/SFC system has been used to study selected nonvolatile coal-derived products. The work reported here involved the aliphatic and aromatic hydrocarbon fractions from this residuum material. Residua at several times were taken from the reactor and examined which provided some insight into the effects of catalyst decay on the products produced in a pilot plant operation. 

The first chiral separation using pSFC was published by Caude and co-workers in 1985 [3]. pSFC resembles HPLC. Selectivity in a chromatographic system stems from different interactions of the components of a mixture with the mobile phase and the stationary phase. Characteristics and choice of the stationary phase are described in the method development section. In pSFC, the composition of the mobile phase, especially for chiral separations, is almost always more important than its density for controlling retention and selectivity. Chiral separations are often carried out at T < T-using liquid-modified carbon dioxide. However, a high linear velocity and a low pressure drop typically associated with supercritical fluids are retained with near-critical liquids. Adjusting pressure and temperature can control the density of the subcritical/supercritical mobile phase. Binary or ternary mobile phases are commonly used. Modifiers, such as alcohols, and additives, such as adds and bases, extend the polarity range available to the practitioner. 

A large variety of normal phase HPLC columns (packed type) or GC columns (capillaries) are used. These two types of columns are complementary. For a packed column of HPLC type, the high flow rate of the supercritical mobile phase will render detection by FID or coupling with a mass spectrometer more difficult. Adding a modifier to the supercritical fluid would have the same effect. 

There is, however, one major problem with CO2 as a mobile phase and that is its low polarity. Thus only relatively non-polar analytes can be dissolved in CO2. Moreover, in columns packed with silica-based material there are always residual adsorptive sites. In reversed-phase HPLC the mobile phase deactivates these sites, but the CO2 is not polar enough to do that. As a consequence, the more polar analytes are adsorbed and these are then eluted as severely tailing peaks or are not eluted at all. It should be mentioned here that reports on more inert packings have been published (Li, Malik and Lee, 1994). There are some supercritical mobile phases other than CO2 that can be used, but those that are realistic to use are all non-polar. The only alternatives are the freons, of which chlorine-free freons are considered to be less harmful to the environment (Blackwell and Schallinger, 1994). 

Supercritical fluid chromatography (SFC) refers to the use of mobile phases at temperatures and pressures above the critical point (supercritical) or just below (sub-critical). SFC shows several features that can be advantageous for its application to large-scale separations [132-135]. One of the most interesting properties of this technique is the low viscosity of the solvents used that, combined with high diffusion coefficients for solutes, leads to a higher efficiency and a shorter analysis time than in HPLC. 

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.). 

In supercritical fluid chromatography (SFC) the mobile phase is a supercritical fluid, such as carbon dioxide [15]. A supercritical fluid can be created either by heating a gas above its critical temperature or compressing a liquid above its critical pressure. Generally, an SFC system typically has chromatographic equipment similar to a HPLC, but uses GC columns. Both GC and LC detectors are used, thus allowing analysis of samples that cannot be vaporized for analysis by GC, yet cannot be detected with the usual LC detectors, to be both separated and detected using SFC. SFC is also in other... 

Beside the use of MIPs in conventional HPLC, Mi-polymers may also be established in supercritical fluid chromatography, which is characterized by faster equilibration times combined with the use of the environmental friendly C02 as mobile phase. Although preliminary results show relatively broad peaks, chiral separation could be performed based on polymers imprinted with an enantiomer. However, the long-term stability of the photochemically generated polymers seems to be a problem [89]. 

Fig. 10 HPLC chromatograms of supercritical fluid extracts of (A) an unfortified wheat sample and (B) a vitamin A-fortified bran-based ready-to-eat breakfast cereal. Column, 5-/rm Altex C8 (octyl) (150 X 4.6-mm ID) mobile phase, acetonitrile/2-propanol/aqueous 25 mM sodium perchlorate (45 45 10), 2.0 ml/min amperometric detection (oxidative mode), glassy carbon electrode, +1.2 V, vs saturated calomel electrode. Peak (1) retinyl palmitate. (Reprinted from Ref. 90, Copyright 1997, with the kind permission of Elsevier Science-NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)...

CF SFC directly coupled with supercritical fluid extraction and HPLC-UV (272 nm). SIL C18 column. Mobile phase H20-MeOH. Roasted coffee beans Grounding, H20 addition, extraction on cartridge. SFC (T — 48°C) extract with C02 extract trapped on activated charcoal cartridge. Elution with MeOH. 314... 

Pure fluids. Carbon dioxide is often the mobile phase of choice for SFC, since it has relatively mild critical parameters, is nontoxic and inexpensive, chemically inert, and is compatible with a wide variety of detectors including the flame ionization detector (FID) used widely in GC and the UV absorbance detector employed frequently in HPLC (7). The usefulness of carbon dioxide as a mobile phase in many instances is somewhat limited, however, because of its nonpolarity (8), and many polar compounds appear to be insoluble in it. For a sample containing polar compounds, pure carbon dioxide may not be the proper mobile phase. The elution of polar compounds is often difficult and the peak shapes for these polar compounds are sometimes poor. This latter difficulty is commonly observed with nonpolar supercritical fluids and may be due to active sites on the stationary phase rather than any inherent deficiency in the fluid itself. 

Janicot et al. presented the separation of opium alkaloids using sub-critical and supercritical fluid chromatography [20]. Carbon dioxide-meth-anol-triethylamine-water mixtures were used as the mobile phase with packed aminopropyl or bare silica columns. The influence of aminated polar modifiers such as methylamine, ethylamine, and triethylamine was studied. Figure 7.15 shows the separation of six opium alkaloids narcotine, papaverine, thebaine, codeine, cryptopine, and morphine on a Lichrosorb Si-60 column. The method gave comparable results with HPLC. 

Sample introduction is a major hardware problem for SFC. The sample solvent composition and the injection pressure and temperature can all affect sample introduction. The high solute diffusion and lower viscosity which favor supercritical fluids over liquid mobile phases can cause problems in injection. Back-diffusion can occur, causing broad solvent peaks and poor solute peak shape. There can also be a complex phase behavior as well as a solubility phenomenon taking place due to the fact that one may have combinations of supercritical fluid (neat or mixed with sample solvent), a subcritical liquified gas, sample solvents, and solute present simultaneously in the injector and column head [2]. All of these can contribute individually to reproducibility problems in SFC. Both dynamic and timed split modes are used for sample introduction in capillary SFC. Dynamic split injectors have a microvalve and splitter assembly. The amount of injection is based on the size of a fused silica restrictor. In the timed split mode, the SFC column is directly connected to the injection valve. Highspeed pneumatics and electronics are used along with a standard injection valve and actuator. Rapid actuation of the valve from the load to the inject position and back occurs in milliseconds. In this mode, one can program the time of injection on a computer and thus control the amount of injection. In packed-column SFC, an injector similar to HPLC is used and whole loop is injected on the column. The valve is switched either manually or automatically through a remote injector port. The injection is done under pressure. 

In normal high pressure liquid chromatography, typical sample volumes are 20-200 p.L this can become as little as 1 nL in capillary HPLC. Pretreatment of the sample may be necessary in order to protect the stationary phase in the column from deactivation. By employing supercritical fluids such as carbon dioxide, pretreatment can be bypassed in many instances so that whole samples from industrial and environmental matrices can be introduced directly into the column. This is due to the fact that the fluid acts as both extraction solvent and mobile phase. Post-column electrochemistry has been demonstrated. For example, fast-scan cyclic voltammo-grams have been recorded as a function of time after injection of microgram samples of ferrocene and other compounds in dichloromethane solvent and which are eluted with carbon dioxide at pressures of the order of 100 atm and temperatures of 50°C the chromatogram is constructed as a plot of peak current vs. time [18]. 

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