1U l I Ilona L. D a w , Mark W. Raynor, Jacob P. KHhlnjl, and Keith D. Baltle 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 transferredon 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 0003-2700188/0360-683A/$01.5010
@ 1988 American Chemical Society
resolve every component in very complex mixtures. As a result, prefractionation of the sample is usually necessary, and thig 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
Iows the isolation of an LC fraction of interest for direct introduction into a (3 2 column. Thus detection limits are 1owered, quantitation is improved, and 1the opportunity to automate interface valves is realized, with the result that 1routine analyses become less time con!ruming and more reproducible.
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 LCIGC al-
box on p. 684 A), which may be grouped as follows: petroleum fuels (18%),foodstuffs (2673, coal-derived fuels (315% environmental samples (15%), and medical samples (10%). There are several reasons why LCI
1
ILirnitatlons of LClGC 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
ANALYTICAL CHEMISTRY, VOC. 60, NO. 11, JUNE l , 1988
e
683A
Figure 1. Illustration of a coupled-column system consisting of a primary col-
umn and two secondary columns. (Adept06 wllh permission horn Relerence 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 second, 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 possible 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 LClGC Sample analyzed wn
Analyte8
Atrazine Pesticides
r
iels
PAC% PCBs PACs, Chemical
classes Diethyistilbestroi, heroin metabolites Dyestu f I Toothpaste Raspberry sauce Raspberry ketone Olive oil Wax esters Hops Pesiicides Aqwus samples PCBs, pesticides Fish exlracts PCBs Diesel particulate PACs extracts Broxaterol Piasma Triglycerides Plasticizers Shale oil. lignite Chemical classes tars Bacteria Triglyceride esterification products
684A
Figurs 2. Twc-dir
compounds. (Adpled ham Reference 3.)
(NP-LC) are favored, whereas the POlar, aqueous eluents used in reversedphase LC (RP-LC) are the most difficult to use. Because of these mobilephase difficulties, only 15% of previous LC/GC applications featured RP-LCI GC, even though RP-LC accounts for 75% of all LC work.
Two-dimensionalchromatogaphy Giddings defined two criteria for multidimensional separations: components are subjected to two or more independent separate displacements and the separated components remain resolved throughout the process ( I , 2). This situation is illustrated in Figure 1, where two secondary columns are coupled to a primary column and all three columns operate in the elution mode. Each secondary column is fed a fraction of duration At from the eluting stream of the primary column. This approach is analogous to transferring fractions of LC eluent on line into two GC columns. The peak capacity of an LC/GC system depends on the efficiency of each individual separation and on the duration At of the primary column fraction fed into the secondary column. The primary LC column effectively eliminates components present in the sample that would otherwise interfere with the resolution of the components of interest in the secondary GC column. An
ANALYTiCAL CHEMISTRY, VOL. 60. NO. 11. JUNE 1, 1988
efficient primary separation may he wasted if At is greater than the average LC peak width, owing to the possible recombination of resolved peaks after transfer into the secondary (GC) column. As At increases, the system approaches that of a mere tandem arrangement, in which the resolution gained in one column may be nullified by the elution order in a subsequent column. In two-dimensional (2D) planar separations, a sample is subjected to two displacement processes oriented a t right angles to each other. If the peak capacity along the primary axis (2) is defined as n,, and the peak capacity along the secondary axis (r) is defined as ray, then the total theoretical peak capacity of the 2D system is given by nzny(2).This is possible in planar chromatography because both displacements occur simultaneously, whereas coupled-column chromatography relies on sequential separations. On-line LC/GC can only approach the efficiencies obtainable with a 2D system by having a large number of secondary columns numerically equal to n,. 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 LCI
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Rinsing function eliminates sampleto-sample carryover.
Some auto-injectorsoffer only basic injection functions. G i o n
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Between each injection, the 2311401 rinses the injection port, sample loading line and the outside ofthe needle. This
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Unlike any other auto-injector, the Ghon 231/401 monitors the, ietector signal. It automatidy stops the run if the baseline shifts or if an expected peak is not detected. It also dilutes concentrated samples if t height of a peak is off-sc
Stationary rack system ensures trouble-free operation. Carousel racks that rotat iinto position are subject 1to frequent mechanical failures. The Gilson 231/401 offers amore reliable stationary rack system with fewer moving p Plus, 21 different racks are available p handle up to 120 samples in popular via^ sizes.
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GC. High peak resolution is achievable with 2D chromatography because of the increased probability that two individual components will be separated after independent displacement along the two axes (I). These displacements may be classified as selective (S) or nonselective (N).Selective displacements are usually associated with LCI GC because solute separations in the primary column are largely independent of those in the secondary column. Figure 2 is an on-line LCIGC retention index map that correlates the GC and LC retention indexes (lo and IL, respectively) of standard polycyclic aromatic compounds (PACs) plotted along the two axes (3).In this case, diesel fuel was separated by LC into aromatic and aliphatic groups, with the aromatics eluting according to ring size. Specific LC fractions were transferred on line into the GC on a batch basis. This approach significantly improves the accuracy with which components can be identified in an unknown complex mixture.
LC/GC interfacingwncepts 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 integrated unit via a hardware interface. . . whose function is to reconcile the often extremely contradictory output limitations 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 independent usage of each instrument while the coude still onerate as an integrated unit. Transfer of LC eluent into the ea8 chromatograph. It must be remembered 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 thermal volatilization of the transferred LC solvent and solutes into the GC mobile phase. The introduction of relatively large volumes (10 rL-10 mL) of liquid LC eluent into a GC column has a great effect on the efficiency of solute resolution; a t inlet temperatures below that of the LC eluent boiling point, gross overloading (“flooding”) of the GC column 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 capillary 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 solutes by a combination of phase-ratio focusing and cold trapping (5,6). Modes of solvent evaporation. Injection of large volumes of solvent into a GC column should he carried out at a GC column temperature near the solvent boiling point, to accelerate volatilization and to keep the flooded zone relatively short (5).The two modes of solvent evaporation that occur within the RG are known as concurrent solvent evaporation (CSE) (7)and solvent flooding, respectively, according t o 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 evaporation options (8). Under solvent-flooding conditions, the solvent evaporates from the rear to the front of the flooded zone a t a rate that depends on the carrier-gas flow rate and the column temperature. At inlet temperatures just below the solvent boiling point, solute material concentrates 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 column. Sharp GC peaks are then obtained by temperature-programmed elution; these peaks have the same appearance 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 concentrate the solutes because the solvent evaporates at the front of the eluent plug; solutes are released simultaneously to migrate through the analytical column. Band-broadening then occurs for solute peaks eluting less than 50 “C to 100 “C above the injection temperature because of the lack of condensed solvent within that temperature range. This unwanted effect may be reduced if conditions for phase soaking are favorable (Le., if the stationary 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 eluent into a GC column under CSE conditions is therefore often limited to the analysis of high-boiling solutes; peaks for low-boiling solutes can be significantly broadened in this process. How-
I
686A
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sclvent evapa&n at rear ofsample layer
\
Carriergas flow
I
\
Fieure 3. Two oDtions for carrying out solvent evapor
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1. 1988
Samge liquid forming film on capillary wall
,w L
A
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ver, the advantage of CSE is that rela.velylarge LC volumes (1-10 mL) can e directly transferred into GC colmns equipped with only short RGs 1-5 m);no liquid actually enters the iC column. C/GC Intetface designs ,n interface can be defined as a surface )ruing a common boundary between wo regions and a place, or piece of quipment, where interaction occurs etween two systems (9). All the LC/ :C interfaces described in the literaure can be classified into three main ategories: the autoinjector, eluent-
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688A * 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 a t the 1979 Pittsburgh Conference as a GC autosampler injector that had been modified with a flow-through side-arm syringe (IO, 2 2 ) . The interface was positioned between a conventional 4-ma 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 pL) is injected into the split/splitless flash-vaporization injector. RGs are not required with this interface hecause no liquid eluent enters the GC column. Unfortunately, the nature of this interface limits the proportion of LC peak volume that can he transferred into the GC, making quantitation difficult (12). This problem has recently been circumvented by reducing LC peak volumes in three ways (13): replacing the conventional LC columns with microhore 1-mm i.d. packed columns, positioning a bundled multicapillary stream splitter between the LC detector and the autoinjector interface, and operating the GC flash-vaporization injector in the split mode. This system has been applied to the LCIGC of PACs in coal tar samples using mass spectrometric detection (MSD) (14). Eluent-feed interface. The first application of RGs to on-line LCIGC 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 maintained below the boiling point of the LC solvent during the transfer, and the solutes then eluted on temperature programming. The use of an RG ensured that there was no peak distortion, even though 1W300-pL aliquots from the LC were switched into the GC. The rate and volume of LC eluent transferred into the RG was determined by the flow rate of the LC pump. LC eluent was exposed to GC carrier
gas only as it entered the on-column injector. This system has been used to determine azulene dyestuff in a toothpaste (15). Gas-transfer interface. This interface (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 Figure 4(c). The interface valve is positioned 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 interface 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 (IS,17). GC carrier-gas flow regulators may be incorporated for CSE operation to increase 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). LClGC harbware optimization LC pumps. Syringe pumps are prohably most suited to LC/GC interfacing for three reasons. They are capable of stable flow rates in the 1-100-rL min-1 range (which is particularly important for partially concurrent solvent evaporation techniques and narrow-bore LCf GC); syringe pumps are less prone to bubble formation associated with low-
1 LC
boiling-point solvents such as pentane and diethyl ether; and the same pump may be used for supercritical fluid chromatography (SFC)/GC or for supercritical fluid extraction (SFE)/GC, if desired. LC columns. All types of analytical LC columns, from 4.6-mm i.d. conventional columns (19) to 100-pm i.d. packed fused-silica capillaries (ZO), 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 employed, and quantitation therefore hecomes difficult. LC/GC interfacing is always facilitated hy small LC peak volumes; because the peak volume is proportional to the square of the LC column internal radius, the use of narrow-bore LC columns is favored. Microhore l-mm i.d. columns are convenient to use, have low solvent consumption, and provide peak volumes that are approximately 20 times smaller 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 instrumentation required for successful capillary LC operation, because stringent limits are placed on dead volumes within the system to avoid huge losses in separation efficiencies. GC columns. Effective solute reconcentration hy phase-ratio focusing in LCIGC requires a thicker stationary phase in the GC column (0.3-1.0 pm) 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 factors such as sample volume, GC inlet temperature, carrier-gas flow rate, and the solvent boiling point (21). Published applications cite RGs varying in length from 2 m (under CSE conditions [IS]) to 50 m (under solvent-flooding conditions [15]). The RG and analytical 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” (ZO), broadened solute peaks, or a badly tailing GC solvent peak. The RG must be deactivated 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 detector options include ultraviolet (UV) absorbance and refractive index (RI); only laser-based options of the latter can be used with narrow-bore LC columns because of the large volumes of conventional RI cells (22).The LC detector is often a nondestructive, flow-
ANALYTICAL CHEMISTRY. VOL. 60. NO. 11. JUNE 1. 1988
*
689,.
cyclohexane/tetrahydrofuran (99/1). The binary azeotrope pentanelmethanol(93/7) is useful for certain applications because of its low boiling point, intermediate nolaritv. _ . and solvent strength. The use of solvent blends as LC eluents further complicates LC/GC operation. The solvent blend should he chosen so that the nonpolar solvent is less volatile than the polar solvent; sol'ute reconcentration is enhanced by favorable solvent effects of the increasingly nonpolar nature of the liquid eluent 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; water and alcohols do not wet the nonpolar, deactivated surface of an RG, and these poor solvent effect properties result in severely distorted GC peaks. CSE conditions. Many of the problems 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 vapor (per volume of liquid) of either wa.ter or methanol is 3-5 times that of hexane (27): for water, this high-pressure condition raises the minimum inlet column temperature to 150 "C with the result that all peaks eluting before
LC eluent properties required for suceesstul on-line LClGC hterfacing To facilitate LC/GC interfacing,the LC eluent should
* solubilize the solutes and elute them in a narrow band;
not b e 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 vess~ls;and be free from wntaminants such as buffer salts and particulate matter
ed hydrocarbons form hydrochloric acid and soot in the FID and are often associated with GC solvent peak tailing. In addition, ketones irreversibly adsorb on amino (-NHJ LC stationary phases. Solvent-flooding conditions. Although the good solvent effect properties 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 polar 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 literature; some examples include pentane/ diethyl ether (80/20);hexane/dichloromethane (90/10); diethyl ethedmethanoVdiethylamine (91.5/8.0/0.5): cyclohexane/dichloromethane (50/50):and
through type. Alternatively, microcolumn LC eluents may he split to a GCtype detector such as flame-ionization (FID) or electron-capture (23). Although these two detectors are also the most commonly used GC detectors in LC/GC, MS is worthy of further investigation: as yet, Fourier transform infrared (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 detector 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-') without substantial loss of separation efficiency. High carrier-gas velocity is desirable because of the increased solvent evaporation rate and accelerated movement of the solute hand through the RG. This effect produces a narrower bandwidth of solutes at the beginning of the analytical column. Even so, many laboratories are prepared to sacrifice 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 he 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 solvents 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 altogether. Aromatic solvents are not usually used with UV detection because of strong absorption, and they also deposit soot on the FID collector. Chlorinat6 9 0 1 * ANALWiCAL
CHEMISTRY, VOL.
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Table 1. Properiies d solvents for LC. S0)rmt
Perfluoroaikane Pentane Hexane Cyclohexane Toluene Diethyl ether Dichlofomethane Tetrahydrofuran Acetone Acetonitrile Ethanol Methanol Water
E
db
-0.25
6.0
0.00
0.01 0.04 0.29 0.38 0.42 0.45 0.56 0.65
0.88 0.95
large
7.1 7.3 8.2 8.9 7.4 9.7 9.1 9.9 11.7 12.7 14.4 23.4
RI 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
BP d
Y'
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
-
Reference 25 and Brislow. P. A,: Liquid WvMvltOgrephy in Fmcrice; HETP: Wilmslow. Cheshire. U.K.. 1976. * t0 = values fw alumina according Io Snyder (26). d = solubility parameter (mi C ~ - ~ ) O ~ . RI = refractive index at 20 'C. BP = boiling point in OC at 1 am pressure. a y = UV CUTOR;wavelength in nanometers at lYhiCh transmission Bllr lo 10% in a 10-mm cell. Source:
60.
NO.
11. JUNE
1. 1988
1
Silica is good. But for tough HPLC methods development, Hamilton’s PRP-t reversed phase column is better. First, the exceptionally stable, polymer-based PRP-1 accepts virtually m y mob& p h e with a pH between 1 and W, giving you greater flexibility over your chromatographic conditions, Second, the PRP-1 lets you separate most compounds with no need for ion-pairing reagents. But even if ion-
pairing reagents are required, column degradation is minimized. Third, the fact that PRP-1 columns are polymer-based means the purified samples do not contain silica impurities which must subsequently be removed. Lastly, composition of the polymer-based particles is consistent from surface to center. No “new” type of surface becomes exposed to cause the selectivity variations common to silica columns.
The PRP-1 is available in Spin and 10pm analytical, and lOpm semi-preparative and preparative columns. So whether you need high speed and resolution, or run large-scale separations, there is a Hamilton PRP-1 column that can significantly improve performance of your HPLC. For complete information, call us toll-free at: (800) 648-5950, or (702) 786-7077 Hamilton Company, EO. Box 10030, Reno, Nevada 89520. . . .
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230 "C are band-broadened because of poor phase soaking (28). The initial GC oven temperature required for different eluent combinations has been experimentally determined for CSE conditions (28).The addition of a high-boiling, nonpolar cosolvent can provide some solvent effect reconcentration at high GC inlet temperatures (8). Normal-phase LC/GC. NP-LCI GC is the most common form of LCIGC coupling because of the general suitability of NP-LC (with nonpolar mobile phases) as a cleanup technique prior to GC analysis. NP-LC is particularly useful for grouptype separations of hydrocarbon mixtures, such as gasoline (29); ring-size fractionation of PAC8 (3);and the isolation and on-line analysis of specific trace components in a complex mixture (30). Figure 5 is a g o d example of the power of on-line NP-LC/GC (31). Turkish shale oil, which had been pyrolyzed at 520 OC, was injected directly into the GC on-column injector without prior LC cleanup, as shown in Figure 5(a). Figure 5(b) was obtained after coupling a liquid chromatograph to the same GC system by a gas-transfer interface valve; only the total aromatic fractionwas switched into the gas chromatograph. In Figure 5(c), the two-ring subfraction of the pyrolyzed shale oil
has been isolated for hieb-resolution " GC analysis. Reversed-phase LCIGC. RP-LC/ GC is useful for the analysis of toxic, organic compounds such as polychlorinated biphenyls (PCBs) and PACs in aqueous environmental samples. However, 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. Instead these applications use either the autoinjection interface (32) or pure acetonitrile eluent with an RG and gastransfer interface (20)or a solvent-exchange interface (33,34). Most of the solvent-exchange interfaces involve solute transfer into a nonpolar solvent prior to GC introduction. These techniques include liquid-liquid extraction, extraction into GC column stationaryphases, and adsorption onto solid LC phases. The latter RP-LC/GC interfacemethod is similar to that originally developed for on-line NP-LCI 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 eluent, and then redissolving the solutes in a passing stream of nonpolar solvent before transfer into the GC RG column.
An extension of the solvent-exchange 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 important feature of LC for controlling solute elution, yet there have not been any reports of applying this concept to LCI 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 little has been published on gradient elution with microcolumns because of the difficulties of accurately mixing such small eluent volumes (37). However, gradient elution LC/GC is quite feasible if all the solvent compositions are suitable for transfer to the GC or if a solvent-exchange interface is used.
LClGC 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 temperature to the volume and physical
~
Figure 5. PVrolyzed shale oil analysis by on-line LCIGC.
692).
ANALYTICAL CHEMISTRY, VOL.
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,'
It works likea dream Exchanging one LC column for anotherinstantly and effortlessly
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tion valve-and it eliminatesthe need to physically disconnect a column and reconnectanotherinits
handle to rediict flow from line (see right diagram). one column to another. For technicalnotes and product Our Model literature, phone your Rheodyne 7OOOvalve selects dealer. Or If more convenient, one of two coladdress Rheodyne, Inc.,EO. Box t urrms (see photo 996, Gtati,California 94928,USA. and left dkgmm). phone (707) 664-9050, Two of our Model 7060 valves select any of five columns or a bypass flushing
SR RHGODYNG THE LC CONNECTION COMPANY
CIRCLE 140 ON READER SERVICE CARD
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properties of the LC eluent transferred (5); begin GC temperature programming only after the solvent peak has passed the GC detector: use CSE conditions 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 hexandtetrahydrofuran); for aqueous RP-LC/GC, use an in-line microcolumn solvent-exchange method, replace LC/GC with SFC/GC or SFE/GC when possible.
Supercrltkalfluid chromatography Chromatographic properties of SFs. Supercritical fluids (SFs) exist a t temperatures and pressures above the supercritical point of a compound. The fundamental differences between LC, SFC, and GC lie in the physical properties of the liquids, SFs, and gases, the most important of which are the "density" of the mobile phase, diffusivity of solutes in the mobile phase, and viscosity of the mobile phase (38).The ranges of these parameters are shown in Table I1 (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; conversely, 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 disadvantages 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 separations). A useful feature of the supercritical state is that the solvent strength of the eluent is closely related to ita density. This property adds another operating parameter to SFC-pressure/density (184,.
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 thermally labile compounds. In general, the choice of chromatographic method should be GC, then SFC, then LC. SFC hardware. SFC instrumentation can be adapted from conventional LC and GC systems, but many commercial systems are now available. The mobile phase is pumped as a liquid and heated to above the critical temperature before passing through the column as an SF via an injection loop valve. A pressure restrictor is incorporated after the column to ensure that conditions throughout the column remain supercritical. SFC uses either packed LC-type columns or 26100-pm 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 (Cod because it has a critical temperature (T,) of 31.05 "C and a critical pressure (P,)of 72.9 atm, which are easy to work with; it is nontoxic and nonflammable; and it is readily available a t low cost. Modifiers such as methanol are sometimes added to change solute elution characteristics. Important features of
Flgure 8. SFC/GC heartcut of chrysene rfm (Adapted wlbl pBrmllUion lrom Reference 40.)
ANALYTICAL CHEMISTRY, VOL. 60. NO. 11, JUNE 1, I988
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. O n - l i n e SFC/GC. Interfacing packed-column SFC to capillary GC is essentially analogous to LC/GC, except that SFC is inherently more compatible with GC because many SFs decompress into gases under GC conditions and are not detected hy 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 PAC8 from a complex liquid hydrocarbon sample (40).The result is shown in Figure 6. The future holds great promise for the development of automated SFC/GC systems. Other combinations. The interfacing of packed-capillary SFC to highresolution, open-tubular capillary SFC is eagerly awaited; this coupled technique would avoid LC/GC mobilephase interfacing problems and be applicable to a wider range of samples than SFC/GC. Why not replace RPLC/GC with RP-LC/SFC?
SupemHical fluid exbactlon Extractive properties of SFs. Many of the properties of SFs that make them useful chromatographic phases also make SFE an attractive possibility. The low viscosities and high solute diffusivities of SFs allow efficient mass transfer during extraction, and relatively low extraction temperatures (4050 "C) reduce the risk of analyte degradation. 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 solvating power of the SF can be varied by controlling the extraction pressure or
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A
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tose, hctose.
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Figure 7. SFElGC interface.
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by the addition of modifiers such as methanol; analytes are isolated from the extraction solvent simply by decreasing 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 involve passing the SF and dissolved material from the extraction vessel into a heated zone, whereas isothermal methods 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 precipitate out. Selectivity of SFE. One potentiallyimportant facet of SFE is the ability to selectively extract compounds by varying the extraction pressure. It has been demonstrated (41) that SFE can selectively extract alkanes and PACs from diesel exhaust particulates using supercritical CO? a t 45 "C; 85% of the alkanes were removed by extracting the particulates for 5 min a t 75 atm, and 90%of the PACs were retained until extracted a t 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 COz at 50 OC, progressively larger PACs were selectively extracted from a solid matrix by increasing the SFE pressure (42).
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ANALYTICAL CHEMISTRY, VOL. BO. NO, 11. JUNE 1. I988
S F E hardware. Analytical SFE cells have volumes ranging from microliter to milliliter proportions. Supercritical pressures are maintained within the heated extraction cell by using a fused-silica capillary restridor as the outlet. The internal diameter of the outlet restrictor controls the rate a t which SF eluent flows out of the cell during extraction and therefore controls the volume of SF used per extraction. For example, using supercritical COz, flow rates of approximately 150 mL min-' are typically obtained with 20-pm i.d. linear restrictors a t 300 atm; 30-pm restrictors operating under the same conditions give flow rates of approximately 300 mL min-I (43). In general, large internal diameter restrictors yield higher extraction efficiencies per unit of time than narrow restrictors, because a larger volume of S F passes through the extraction cell. On-line SFE/GC. Possible applications 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 procedures that are often far more time consuming than the actual chromatographic analysis. Whereas these traditional 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 whereby solutes are lost or contaminants introduced. In addition, the on-line technique is readily automated by interface valves. SFEIGC need not require any modifications to a commercially available GC. The simplest interfacing method is the equivalent of the eluent-feed LC/ GC interface; the extraction-cell restrictor is inserted directly into the GC capillary column through a standard on-column injector (Figure 7). Extracted components are deposited inside the column, which is in a cooled (
