INTERFACING Ilona L. Davies, Mark W. Raynor, Jacob P. Kithinji, and Keith D. Bartle Department of Physical Chemistry The University of Leeds Leeds LS2 9JT United Kingdom
Paul T. Williams and Gordon E. Andrews Department of Fuel and Energy The University of Leeds Leeds LS2 9JT United Kingdom
Coupled-column chromatography is a technique whereby sample fractions are selectively transferred on line from a primary column to a secondary column for further solute separation. This approach increases the peak capacity of the system and significantly improves the resolution of individual components in the sample. Although capillary gas chromatography (GC) is a highly efficient separation technique, it is seldom powerful enough to fully 0003-2700/88/0360-683A/S01.50/0 © 1988 American Chemical Society
resolve every component in very complex mixtures. As a result, prefractionation of the sample is usually necessary, and this has traditionally been carried out by liquid chromatography (LC). Two sequential separations are particularly useful, for example, for specific-component isolation or chemical-class fractionation before chromatographic analysis. Recent trends in coupled-column chromatography have been toward totally automated analyses, incorporating sample preparation on line with the final analytical procedure. Significant
lows the isolation of an LC fraction of interest for direct introduction into a GC column. Thus detection limits are lowered, quantitation is improved, and the opportunity to automate interface valves is realized, with the result that routine analyses become less time consuming and more reproducible.
Limitations of LC/GC LC/GC is a potentially important area of chromatography, but a survey of the relevant literature indicates that this technique has been limited to a relatively narrow range of applications (see
INSTRUMENTATION advantages are gained by coupled LC/ GC, compared with an equivalent offline system. Indeed, off-line methods are inevitably labor-intensive and susceptible to solute loss and sample contamination, whereas on-line LC/GC al-
box on p. 684 A), which may be grouped as follows: petroleum fuels (18%), foodstuffs (26%), coal-derived fuels (31%), environmental samples (15%), and medical samples (10%). There are several reasons why LC/
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988 · 683 A
Figure 1. Illustration of a coupled-col umn system consisting of a primary col umn and two secondary columns. (Adapted with permission from Reference 2.)
GC has not been widely adopted as the technique of choice for all complex samples. First, GC is useful for only 1520% of all organic compounds, and sec ond, it is often physically difficult to interface two chromatographic systems that operate with different mobile phases. The physical nature of the LC mobile phase and the volume of LC eluent transferred into the gas chromatograph are key factors of successful LC/GC interfacing; the smallest possi ble LC peak volumes are desired in most cases. Obviously, some LC mobile phases are more compatible with GC than others. The low-boiling, nonpolar solvents used in normal-phase LC
Typical applications of LC/GC Sample analyzed Sorghum Butter Coal tars Petroleum fuels
Figure 2. Two-dimensional LC/GC retention index model for polycyclic aromatic compounds. (Adapted from Reference 3.)
(NP-LC) are favored, whereas the po lar, aqueous eluents used in reversedphase LC (RP-LC) are the most diffi cult to use. Because of these mobilephase difficulties, only 15% of previous LC/GC applications featured RP-LC/ GC, even though RP-LC accounts for 75% of all LC work. Two-dimensional chromatography
Analytes
Atrazine Pesticides PACs, PCBs PACs, chemical classes Diethylstilbestrol, Urine heroin metabolites Toothpaste Dyestuff Raspberry sauce Raspberry ketone Wax esters Olive oil Pesticides Hops Aqueous samples PCBs, pesticides Fish extracts PCBs Diesel particulate PACs extracts Plasma Broxaterol Plasticizers Triglycerides Chemical classes Shale oil, lignite tars Triglyceride Bacteria esterification products
Giddings defined two criteria for mul tidimensional separations: compo nents are subjected to two or more in dependent separate displacements and the separated components remain re solved throughout the process (1, 2). This situation is illustrated in Figure 1, where two secondary columns are cou pled to a primary column and all three columns operate in the elution mode. Each secondary column is fed a frac tion of duration Δί from the eluting stream of the primary column. This ap proach is analogous to transferring fractions of LC eluent on line into two GC columns. The peak capacity of an LC/GC sys tem depends on the efficiency of each individual separation and on the dura tion Δί of the primary column fraction fed into the secondary column. The primary LC column effectively elimi nates components present in the sam ple that would otherwise interfere with the resolution of the components of in terest in the secondary GC column. An
684 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
efficient primary separation may be wasted if Δί is greater than the average LC peak width, owing to the possible recombination of resolved peaks after transfer into the secondary (GC) col umn. As Δί increases, the system ap proaches that of a mere tandem ar rangement, in which the resolution gained in one column may be nullified by the elution order in a subsequent column. In two-dimensional (2D) planar sep arations, a sample is subjected to two displacement processes oriented at right angles to each other. If the peak capacity along the primary axis (z) is defined as nz, and the peak capacity along the secondary axis (y) is defined as ny, then the total theoretical peak capacity of the 2D system is given by njriy (2). This is possible in planar chro matography because both displace ments occur simultaneously, whereas coupled-column chromatography re lies on sequential separations. On-line LC/GC can only approach the efficien cies obtainable with a 2D system by having a large number of secondary columns numerically equal to nz. This approach is impractical; therefore, most LC/GC systems lie somewhere between 2D separations and tandemcolumn arrangements. The selectivities of the LC and GC columns have a substantial influence on the separating power of on-line LC/
GC. High peak resolution is achievable with 2D chromatography because of the increased probability that two indi vidual components will be separated after independent displacement along the two axes (1). These displacements may be classified as selective (S) or nonselective (N). Selective displace ments are usually associated with LC/ GC because solute separations in the primary column are largely indepen dent of those in the secondary column. Figure 2 is an on-line LC/GC retention index map that correlates the GC and LC retention indexes (IQ and 7L> re spectively) of standard polycyclic aro matic compounds (PACs) plotted along the two axes (3). In this case, die sel fuel was separated by LC into aro matic and aliphatic groups, with the aromatics eluting according to ring size. Specific LC fractions were trans ferred on line into the GC on a batch basis. This approach significantly im proves the accuracy with which compo nents can be identified in an unknown complex mixture. LC/GC interfacing concepts
In his far-sighted article on coupled methods, the late Tomas Hirschfeld defined a hyphenated instrument as "one in which both instruments are automated together as a single inte grated unit via a hardware interface . .. whose function is to reconcile the often extremely contradictory output limita tions of one instrument and the input limitations of the other" (4). On-line coupling of two established techniques limits both instruments to operating within conditions that are compatible with one another. The ideal interface is one that allows the optimum and inde pendent usage of each instrument while the couple still operate as an inte grated unit. Transfer of LC eluent into the gas chromatograph. It must be remem bered that the fundamental difference between LC and GC is the physical state of the mobile phase itself. Any attempt to couple LC to GC involves the following steps: isolation of the LC fraction of interest, transfer of the LC fraction into the GC column, and ther mal volatilization of the transferred LC solvent and solutes into the GC mobile phase. The introduction of relatively large volumes (10 μL-10 mL) of liquid LC eluent into a GC column has a great effect on the efficiency of solute resolu tion; at inlet temperatures below that of the LC eluent boiling point, gross overloading ("flooding") of the GC col umn occurs. Solutes are spread out along the GC column inlet, resulting in peaks that are distorted, split, or broadened ("band-broadening in space" [5]). However, this deleterious effect can be avoided if a 30-cm to 50-m
length of uncoated, deactivated capil lary is included as a GC precolumn or retention gap (RG). RGs are column inlets with reduced retention power (compared with the separating part of the column) that reconcentrate the sol utes by a combination of phase-ratio focusing and cold trapping (5, 6). Modes of solvent evaporation. In jection of large volumes of solvent into a GC column should be carried out at a GC column temperature near the sol vent boiling point, to accelerate volatil ization and to keep the flooded zone relatively short (5). The two modes of solvent evaporation that occur within the RG are known as concurrent sol vent evaporation (CSE) (7) and solvent flooding, respectively, according to whether the initial GC temperature is higher or lower than the boiling point of the LC eluent introduced. Figure 3 is a schematic of these two solvent evapo ration options (8). Under solvent-flooding conditions, the solvent evaporates from the rear to the front of the flooded zone at a rate that depends on the carrier-gas flow rate and the column temperature. At inlet temperatures just below the sol vent boiling point, solute material con centrates toward the solvent front dur-
ing solvent evaporation and is retained as a narrow band by the stationary phase at the inlet of the analytical col umn. Sharp GC peaks are then ob tained by temperature-programmed elution; these peaks have the same ap pearance as if they had been injected on column in a small volume. When the inlet temperature is above the boiling point of the LC mobile phase, solvent trapping fails to concen trate the solutes because the solvent evaporates at the front of the eluent plug; solutes are released simulta neously to migrate through the analyti cal column. Band-broadening then oc curs for solute peaks eluting less than 50 °C to 100 °C above the injection temperature because of the lack of con densed solvent within that tempera ture range. This unwanted effect may be reduced if conditions for phase soaking are favorable (i.e., if the sta tionary phase and solvent are of similar polarity and if the column temperature only slightly exceeds that of the solvent boiling point). The transfer of LC elu ent into a GC column under CSE con ditions is therefore often limited to the analysis of high-boiling solutes; peaks for low-boiling solutes can be signifi cantly broadened in this process. How-
Figure 3. Two options for carrying out solvent evaporation. (a) Concurrent solvent evaporation, (b) Negligibly or partially concurrent solvent evaporation (solvent flooding). (Adapted with permission from Reference 8.)
686 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE
1, 1988
WHEATON 0.1 ml PLASTIC AUTOSAMPLER VIALS W i t h Glass Inner Cone Precision molded plastic vials with borosilicate glass inner cones •
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ever, the advantage of CSE is that relatively large LC volumes (1-10 mL) can be directly transferred into GC columns equipped with only short RGs (1-5 m); no liquid actually enters the GC column. LC/GC interface designs
An interface can be defined as a surface forming a common boundary between two regions and a place, or piece of equipment, where interaction occurs between two systems (9). All the LC/ GC interfaces described in the literature can be classified into three main categories: the autoinjector, eluent-
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688 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
feed (or on-column), and gas-transfer (or loop-type) interfaces. Autoinjector interface. The first commercially available LC/GC interface was introduced at the 1979 Pittsburgh Conference as a GC autosampler injector that had been modified with a flow-through side-arm syringe (10,11). The interface was positioned between a conventional 4-mm i.d. LC column and a capillary-column (or packed-column) GC. Figure 4(a) shows how the interface was adapted to on-line LC/GC. With this arrangement, LC eluent flows through the autosampler syringe to waste until a selected volume (0.1-
3 μίι) is injected into the split/splitless flash-vaporization injector. RGs are not required with this interface be cause no liquid eluent enters the GC column. Unfortunately, the nature of this in terface limits the proportion of LC peak volume that can be transferred into the GC, making quantitation diffi cult {12). This problem has recently been circumvented by reducing LC peak volumes in three ways (13): re placing the conventional LC columns with microbore 1-mm i.d. packed col umns, positioning a bundled multicapillary stream splitter between the LC detector and the autoinjector interface, and operating the GC flash-vaporiza tion injector in the split mode. This system has been applied to the LC/GC of PACs in coal tar samples using mass spectrometric detection (MSD) (14). Eluent-feed interface. The first application of RGs to on-line LC/GC involved the introduction of several hundred microliters of LC eluent (cyclohexane) into a GC column via a 4port switching-valve interface (15). In this case, a 0.17-mm i.d. capillary was used to transfer eluent from the valve through the GC on-column injector into a 50-m RG precolumn, as shown in Figure 4(b). The GC column was main tained below the boiling point of the LC solvent during the transfer, and the solutes then eluted on temperature programming. The use of an RG en sured that there was no peak distor tion, even though 100-300-ML aliquots from the LC were switched into the GC. The rate and volume of LC eluent transferred into the RG was deter mined by the flow rate of the LC pump. LC eluent was exposed to GC carrier
Figure 4(c). Gas-transfer interface.
gas only as it entered the on-column injector. This system has been used to determine azulene dyestuff in a tooth paste (15). Gas-transfer interface. This inter face (16) was designed to isolate an LC fraction in a switching-valve sample loop (load position); carrier gas was used to force the contents of the loop into the RG section of the GC column (inject position). Features of the gastransfer interface are illustrated in Fig ure 4(c). The interface valve is posi tioned close to the side of the GC oven so that the RG passes through a hole in the oven wall, and carrier gas enters the GC columns via the interface valve. Advantages of the gas-transfer inter face are that it is a type of direct oncolumn injector, making quantitation easy; it is easily automated; and it adapts well to CSE conditions. A fully automated system coupled by a twoloop, 10-port valve has been reported for the analysis of petroleum fuels and exhaust particulate extracts (16, 17). GC carrier-gas flow regulators may be incorporated for CSE operation to in crease the inlet pressure during eluent transfer, thereby decreasing the time required for solvent evaporation (18). This approach was used to determine a ketone in a raspberry sauce (18). LC/GC hardware optimization
LC pumps. Syringe pumps are proba bly most suited to LC/GC interfacing for three reasons. They are capable of stable flow rates in the I-IOO-ML min^ 1 range (which is particularly important for partially concurrent solvent evapo ration techniques and narrow-bore LC/ GC); syringe pumps are less prone to bubble formation associated with low-
boiling-point solvents such as pentane and diethyl ether; and the same pump may be used for supercritical fluid chromatography (SFQ/GC or for su percritical fluid extraction (SFE)/GC, if desired. LC columns. All types of analytical LC columns, from 4.6-mm i.d. conven tional columns (19) to 100-μπι i.d. packed fused-silica capillaries (20), have been used for on-line LC/GC. However, LC eluent splitting is usually required prior to GC introduction when 4.6-mm i.d. LC columns are em ployed, and quantitation therefore be comes difficult. LC/GC interfacing is always facilitated by small LC peak volumes; because the peak volume is proportional to the square of the LC column internal radius, the use of nar row-bore LC columns is favored. Mi crobore 1-mm i.d. columns are conve nient to use, have low solvent con sumption, and provide peak volumes that are approximately 20 times small er than those obtained when using 4.6-mm i.d. columns of equal length (for the same linear velocities). Packed-capillary columns are probably the most suitable LC columns for LC/ GC interfacing; however, relatively few laboratories have the specialized in strumentation required for successful capillary LC operation, because strin gent limits are placed on dead volumes within the system to avoid huge losses in separation efficiencies. GC columns. Effective solute recon centration by phase-ratio focusing in LC/GC requires a thicker stationary phase in the GC column (0.3-1.0 μτη) than would typically be used. The length of the RG primarily depends on whether or not CSE conditions are used, which also depends on many fac tors such as sample volume, GC inlet temperature, carrier-gas flow rate, and the solvent boiling point (21). Pub lished applications cite RGs varying in length from 2 m (under CSE conditions [18]) to 50 m (under solvent-flooding conditions [15]). The RG and analyti cal columns are usually joined by a Press-Fit connector or by a zero-deadvolume union with graphitized Vespel ferrules. The column should be devoid of active sites that would result in a chromatogram "hump" (20), broad ened solute peaks, or a badly tailing GC solvent peak. The RG must be deacti vated by a suitable silanizing agent. Even the use of pure graphite ferrules in the connectors could provide highly active sites if a good seal were not achieved. LC/GC detectors. Common LC de tector options include ultraviolet (UV) absorbance and refractive index (RI); only laser-based options of the latter can be used with narrow-bore LC col umns because of the large volumes of conventional RI cells (22). The LC de tector is often a nondestructive, flow-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988 · 689 A
cyclohexane/tetrahydrofuran (99/1 ). The binary azeotrope pentane/methanol (93/7) is useful for certain applica tions because of its low boiling point, intermediate polarity, and solvent strength. The use of solvent blends as LC eluents further complicates LC/GC op eration. The solvent blend should be chosen so that the nonpolar solvent is less volatile than the polar solvent; sol ute reconcentration is enhanced by fa vorable solvent effects of the increas ingly nonpolar nature of the liquid elu ent evaporating within the RG. Polar aqueous RP-LC solvents are particularly difficult to use with LC/ GC. Problems are encountered when several hundred microliters of aqueous eluent are transferred into a GC RG under solvent-flooding conditions; wa ter and alcohols do not wet the nonpo lar, deactivated surface of an RG, and these poor solvent effect properties re sult in severely distorted GC peaks. CSE conditions. Many of the prob lems experienced with RP-LC occur because aqueous liquid eluent enters the GC RG column. However, under CSE conditions, the GC temperature is adjusted so that LC solvent evaporates as it enters the RG, and the situation changes because the solvent-effect properties of the eluent are no longer crucial to solute reconcentration. In practice, high pressures are required to rapidly dispel the large volumes of LC eluent vapor formed. The volume of va por (per volume of liquid) of either wa ter or methanol is 3-5 times that of hexane (27); for water, this high-pres sure condition raises the minimum in let column temperature to 150 °C with the result that all peaks eluting before
LC eluent properties required for successful on-line LC/GC interfacing To facilitate LC/GC interfacing, the LC eluent should • solubilize the solutes and elute them in a narrow band; • not be irreversibly retained by the LC stationary phase; • be compatible with both LC and GC detectors; • have a lower boiling point than that of the solutes of interest; • have a high vapor pressure to promote rapid solvent evaporation; • have a low polarity to efficiently wet the silanized RG surface under solvent-flooding conditions; • have a polarity similar to that of the GC stationary phase to improve phase-soaking characteristics; • be distilled, LC grade, degassed, filtered, and stored in glass vessels; and • be free from contaminants such as buffer salts and particulate matter.
through type. Alternatively, microcol umn LC eluents may be split to a GCtype detector such as flame-ionization (FID) or electron-capture (23). Al though these two detectors are also the most commonly used GC detectors in LC/GC, MS is worthy of further inves tigation; as yet, Fourier transform in frared (FT-IR) spectroscopic detection has not been applied to LC/GC. In some cases, to avoid contamination it may be necessary to bypass the GC de tector during the solvent evaporation step (24).
LC/GC mobile phases Hydrogen is the preferred GC carrier gas for LC/GC because it can be used at high flow rates (4 mL min - 1 ) without substantial loss of separation efficien cy. High carrier-gas velocity is desir able because of the increased solvent evaporation rate and accelerated movement of the solute band through the RG. This effect produces a narrow er bandwidth of solutes at the begin ning of the analytical column. Even so, many laboratories are prepared to sac rifice the advantages of hydrogen for the safety of helium. Compromises have to be made in the choice of LC mobile phase for on-line LC/GC because of the many variables involved; there is no guarantee that the solvent employed as an LC eluent can suitably be introduced in relatively large volumes into the GC. Several properties are required of an LC eluent to ensure successful LC/GC coupling (see box). For example, some of the sol vents commonly used for LC (Table I) are difficult to use with on-line LC/GC because of the following problems. Compatibility with both LC and GC detectors is required. Fluorocarbons are decomposed to hydrogen fluoride in the FID, so that the detector either has to be gold coated or bypassed alto gether. Aromatic solvents are not usu ally used with UV detection because of strong absorption, and they also depos it soot on the FID collector. Chlorinat
ed hydrocarbons form hydrochloric acid and soot in the FID and are often associated with GC solvent peak tail ing. In addition, ketones irreversibly adsorb on amino (—NH2) LC station ary phases. Solvent-flooding conditions. Al though the good solvent effect proper ties of low-boiling, NP-LC solvents such as pentane, hexane, and cyclohexane make them ideal eluents for LC/ GC interfacing, they often lack the solvating ability of the stronger, more po lar solvents. Nonpolar solvents may be modified by the addition of miscible, polar solvents such as diethyl ether, methanol, dichloromethane, or tetrahydrofuran. Many solvent mixtures have been reported in the LC/GC liter ature; some examples include pentane/ diethyl ether (80/20); hexane/dichloromethane (90/10); diethyl ether/methanol/diethylamine (91.5/8.0/0.5); cyclohexane/dichloromethane (50/50); and
Table 1. Properties of solvents for LC. Solvent Perfluoroalkane Pentane Hexane Cyclohexane Toluene Diethyl ether Dichloromethane Tetrahydrofuran Acetone Acetonitrile Ethanol Methanol Water
£0a
d"
Rlc
BP"
Ve
-0.25 0.00 0.01 0.04 0.29 0.38 0.42 0.45 0.56 0.65 0.88 0.95 large
6.0 7.1 7.3 8.2 8.9 7.4 9.7 9.1 9.9 11.7 12.7 14.4 23.4
1.267 1.358 1.375 1.427 1.496 1.353 1.424 1.408 1.359 1.344 1.361 1.329 1.330
57.0 36.2 86.2 81.4 110.6 34.6 40.1 66.0 56.5 82.0 78.5 64.7 100.0
210 210 200 210 285 220 235 215 330 190 210 205
—
Source: Reference 25 and Bristow, P. Α.; Liquid Chromatography in Practice; HETP: Wllmslow, Cheshire, U.K., 1976. a £° = values for alumina according to Snyder (26). b d = solubility parameter (cal cm- 3 ) 0 5 · 0 RI • refractive index at 20 ° C. " BP = boiling point in °C at 1 atm pressure . • y = UV cutoff; wavelength in nanometers at which transmission falls to 10% In a 10-mm cell.
690 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
230 °C are band-broadened because of poor phase soaking (28). The initial GC oven temperature re quired for different eluent combina tions has been experimentally deter mined for CSE conditions (28). The ad dition of a high-boiling, nonpolar cosolvent can provide some solvent ef fect reconcentration at high GC inlet temperatures (8). Normal-phase LC/GC. NP-LC/ GC is the most common form of LC/GC coupling because of the general suit ability of NP-LC (with nonpolar mo bile phases) as a cleanup technique pri or to GC analysis. NP-LC is particular ly useful for group-type separations of hydrocarbon mixtures, such as gasoline (29); ring-size fractionation of PACs (3); and the isolation and on-line analy sis of specific trace components in a complex mixture (30). Figure 5 is a good example of the power of on-line NP-LC/GC (31). Turkish shale oil, which had been pyro lyzed at 520 °C, was injected directly into the GC on-column injector with out prior LC cleanup, as shown in Fig ure 5(a). Figure 5(b) was obtained after coupling a liquid chromatograph to the same GC system by a gas-transfer in terface valve; only the total aromatic fraction was switched into the gas chro matograph. In Figure 5(c), the two-ring subfraction of the pyrolyzed shale oil
has been isolated for high-resolution GC analysis. Reversed-phase LC/GC. RP-LC/ GC is useful for the analysis of toxic, organic compounds such as polychlorinated biphenyls (PCBs) and PACs in aqueous environmental samples. How ever, the technical difficulty of RP-LC/ GC interfacing remains a problem. Successful applications have avoided transferring large volumes of aqueous eluent into the gas chromatograph. In stead these applications use either the autoinjection interface (32) or pure acetonitrile eluent with an RG and gastransfer interface (20) or a solvent-ex change interface (33, 34). Most of the solvent-exchange inter faces involve solute transfer into a nonpolar solvent prior to GC introduction. These techniques include liquid-liquid extraction, extraction into GC column stationary phases, and adsorption onto solid LC phases. The latter RP-LC/GC interface method is similar to that orig inally developed for on-line NP-LC/ RP-LC (35). The approach involves passing the aqueous LC eluent through a packed concentrator microcolumn, purging the concentrator microcolumn with an inert gas to evaporate the elu ent, and then redissolving the solutes in a passing stream of nonpolar solvent before transfer into the GC RG col-
An extension of the solvent-ex change technique incorporates a derivatization column on line between an LC microbore 1-mm i.d. column and a capillary column GC, using an RG and an autoinjector interface. This system was applied to the on-line separation, esterification, and analysis of lipids in bacteria (36). Gradient-elution LC/GC. Varying the solvent composition is an impor tant feature of LC for controlling solute elution, yet there have not been any reports of applying this concept to LC/ GC. Perhaps the major reason for not using gradient elution is that gradient elution is usually carried out in the RP mode with conventional LC columns, whereas LC/GC favors NP-LC with small-bore columns. Comparatively lit tle has been published on gradient elu tion with microcolumns because of the difficulties of accurately mixing such small eluent volumes (37). However, gradient elution LC/GC is quite feasi ble if all the solvent compositions are suitable for transfer to the GC or if a solvent-exchange interface is used. LC/GC guidelines The following guidelines are suggested for facilitating LC/GC interfacing: use narrow-bore LC columns; adjust the length of the RG and the GC inlet tem perature to the volume and physical
Figure 5. Pyrolyzed shale oil analysis by on-line LC/GC. (a) Total sample solution. 1-μί aliquot injected on column without prior LC cleanup, (b) On-line GC of 150-fd. LC (total aromatic) fraction, (c) On-line GC of 150-μί LC (two-ring aromatic) fraction. 692 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE
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Table II. Properties of mobile phases used in chromatography. Mobile phase
Density (g rtiL-1)
Viscosity (poise X 10-")
Diffusivity (cm 2 s~1)
Gas Supercritical fluid Liquid
(0.6-2.0) Χ ΙΟ -3 0.2-0.9
0.5-3.5 2.0-9.9
0.01-1.0 (0.5-3.3) X 10"
30-240
(0.5-2.0) X 10-
0.8-1.0
Source: Reference 39. Copyright 1987 by the AAAS.
properties of the LC eluent transferred (5); begin GC temperature program ming only after the solvent peak has passed the GC detector; use CSE con ditions if the analytes have boiling points 100 °C higher than that of the LC eluent and if short analysis times are required; for NP-LC/GC, use lowto medium-boiling alkane LC solvents and modify with lower boiling ethers (pentane/diethyl ether or hexane/tetrahydrofuran); for aqueous RP-LC/GC, use an in-line microcolumn solvent-ex change method; replace LC/GC with SFC/GC or SFE/GC when possible. Supercritical fluid chromatography
Chromatographic properties of SFs. Supercritical fluids (SFs) exist at temperatures and pressures above the supercritical point of a compound. The fundamental differences between LC, SFC, and GC lie in the physical proper ties of the liquids, SFs, and gases, the most important of which are the "den sity" of the mobile phase, diffusivity of solutes in the mobile phase, and viscos ity of the mobile phase (38). The ranges of these parameters are shown in Table II (39). SFs possess physical properties that are in between those of liquids and gases. As chromatographic mobile phases, SFs confer chromatographic properties intermediate to LC and GC; the low densities and high diffusivities of gases give GC superior resolution per unit of time than either LC or SFC; converse ly, high liquid densities are responsible for the good solvating power of LC. GC is limited to the analysis of volatile compounds of relatively low molecular mass and good thermal stability; LC is limited by long analysis times and a lack of sensitive, universal detectors; capillary SFC overcomes the disadvan tages of capillary GC (limited volatility and thermal stability of many organic compounds) and capillary LC (long analysis times and impractical narrowbore columns required for efficient sep arations). A useful feature of the supercritical state is that the solvent strength of the eluent is closely related to its density. This property adds another operating parameter to SFC—pressure/density
programming. The ability to solvate compounds at lower temperatures by controlling the mobile-phase density makes SFC particularly suitable for the analysis of nonvolatile and ther mally labile compounds. In general, the choice of chromatographic method should be GC, then SFC, then LC. SFC hardware. SFC instrumenta tion can be adapted from conventional LC and GC systems, but many com mercial systems are now available. The mobile phase is pumped as a liquid and heated to above the critical tempera ture before passing through the column as an SF via an injection loop valve. A pressure restrictor is incorporated af ter the column to ensure that condi tions throughout the column remain supercritical. SFC uses either packed LC-type columns or 25-100-μπι i.d. open-tubular fused-silica columns, with a variety of nonpolar, polar, or novel chiral-bonded stationary phases. The most commonly used mobile phase in SFC is carbon dioxide (CO2) because it has a critical temperature (Tc) of 31.05 °C and a critical pressure (Pc) of 72.9 atm, which are easy to work with; it is nontoxic and nonflammable; and it is readily available at low cost. Modifiers such as methanol are some times added to change solute elution characteristics. Important features of
SFC are the ability to interface SFC with both LC (UV, RI) and GC (FID, FT-IR, MS) detectors and the ease with which SFC can be coupled to GC. On-line SFC/GC. Interfacing packed-column SFC to capillary GC is essentially analogous to LC/GC, except that SFC is inherently more compati ble with GC because many SFs decom press into gases under GC conditions and are not detected by the FID. One SFC/GC application passed a heated capillary restrictor from a 4.6-mm i.d. packed column directly into a hot GC split vaporization injector to isolate PACs from a complex liquid hydrocar bon sample (40). The result is shown in Figure 6. The future holds great prom ise for the development of automated SFC/GC systems. Other combinations. The interfac ing of packed-capillary SFC to highresolution, open-tubular capillary SFC is eagerly awaited; this coupled tech nique would avoid LC/GC mobilephase interfacing problems and be ap plicable to a wider range of samples than SFC/GC. Why not replace RPLC/GC with RP-LC/SFC? Supercritical fluid extraction
Extractive properties of SFs. Many of the properties of SFs that make them useful chromatographic phases also make SFE an attractive possibili ty. The low viscosities and high solute diffusivities of SFs allow efficient mass transfer during extraction, and rela tively low extraction temperatures (4050 °C) reduce the risk of analyte degra dation. Extraction of organic compounds from solid samples by SFs has many advantages over conventional liquid solvent (Soxhlet) extraction. Higher recoveries are achieved with SFE in shorter times (10-30 min); the solvat ing power of the SF can be varied by controlling the extraction pressure or
Figure 6. SFC/GC heartcut of chrysene from a complex hydrocarbon stream. (Adapted with permission from Reference 40.)
694 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
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Figure 7. SFE/GC interface.
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by the addition of modifiers such as methanol; analytes are isolated from the extraction solvent simply by de creasing the density of the SF; and SFE is easily coupled to chromatography for on-line extraction and analysis. Components dissolved in SFs can be isolated by any method that reduces the density and hence the solvating ability of the SF. Isobaric methods in volve passing the SF and dissolved ma terial from the extraction vessel into a heated zone, whereas isothermal meth ods involve expansion of the SF as it leaves the extraction cell by dropping the pressure below the critical point; in both cases, the SF density decreases and the extracted components precipi tate out. Selectivity of SFE. One potentially' important facet of SFE is the ability to selectively extract compounds by vary ing the extraction pressure. It has been demonstrated (41) that SFE can selec tively extract alkanes and PACs from diesel exhaust particulates using su percritical C 0 2 at 45 °C; 85% of the alkanes were removed by extracting the particulates for 5 min at 75 atm, and 90% of the PACs were retained un til extracted at 300 atm for 90 min. Similarly, ring-type fractionation of PACs such as that obtained by NP-LC (3) is also possible with SFE. Using C0 2 at 50 °C, progressively larger PACs were selectively extracted from a solid matrix by increasing the SFE pressure (42).
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696 A • ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
SFE hardware. Analytical SFE cells have volumes ranging from micro liter to milliliter proportions. Supercri tical pressures are maintained within the heated extraction cell by using a fused-silica capillary restrictor as the outlet. The internal diameter of the outlet restrictor controls the rate at which SF eluent flows out of the cell during extraction and therefore con trols the volume of SF used per extrac tion. For example, using supercritical CO2, flow rates of approximately 150 mL min - 1 are typically obtained with 20-μπι i.d. linear restrictors at 300 atm; 30-Mm restrictors operating under the same conditions give flow rates of ap proximately 300 mL min - 1 (43). In gen eral, large internal diameter restrictors yield higher extraction efficiencies per unit of time than narrow restrictors, because a larger volume of SF passes through the extraction cell. On-line SFE/GC. Possible applica tions of coupled SFE/GC are listed in the box. This technique is the method of choice for the analysis of organic compounds adsorbed on a solid matrix because it eliminates the traditional extraction and fractionation proce dures that are often far more time con suming than the actual chromato graphic analysis. Whereas these tradi tional methods can take days to complete, coupled SFE/GC is often carried out within one hour with higher extraction efficiencies; the extraction itself is faster, and there are no inter-
mediate reconcentration steps where by solutes are lost or contaminants in troduced. In addition, the on-line tech nique is readily automated by interface valves. SFE/GC need not require any modi fications to a commercially available GC. The simplest interfacing method is the equivalent of the eluent-feed LC7 GC interface; the extraction-cell restrictor is inserted directly into the GC capillary column through a standard on-column injector (Figure 7). Extract ed components are deposited inside the column, which is in a cooled (
