Anal, Chem. 1087, 59, 13-22
13
Mass Spectrometer Interface for Microbore and High Flow Rate Capillary Supercritical Fluid Chromatography with Splitless Injection Richard D. Smith* and Harold R. Udseth Chemical Methods and Separations Group, Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352
Improved methods for interfacing microbore and capillary supercritical fluid chromatography (SFC) with mass spectrometry, using a high flow rate interface with spiltiess injection, are described. The new Interface incorporated a mechanically pumped expansion region behind the chemical ionization repeller electrode, allowing higher gas flow rates (100-200 mg of COJmln) than previous interfaces and providing an improvement over direct pumping of the ion source In t m of background contrkulkns, ionlzatkn e f f k h c y , and sensitivity. The effects of sample flow rate, restrictor geom dry, and effluent heating were studied relevant to the transfer and detection of nonvolatile anaiytes. Separations are compared for both microbore and capillary columns for a polartty test mixture, pesticides, and the nonionic surfactant Triton X-100 polymer using supercritical carbon dioxide and carbon dioxide modified with methanol. Microbore columns were shown to have greater limitations for pressure programming, and superior resuits were obtained with capillary columns.
There is a growing interest in supercritical fluid chromatography (SFC), and its combination with mass spectrometry (SFC-MS), for more efficient separation and characterization of mixtures not amenable to gas chromatography (1-10). Potential applications would exploit the high efficiency (defined in terms of either the number of effective plates or separation speed) that can be obtained with capillary columns (1-8) or the high speed and altered mobile-phase properties of supercritical fluids with packed columns (9, 10). However, a major impetus for the interest in SFC also derives from the applicability of gas-phase detectors. In particular, the mass spectrometer is the only detector that has been demonstrated to provide direct interfacing with both high sensitivity and selectivity for detection regardless of the SFC mobile-phase composition (11-16). In previous work we have described our initial capillary SFC-MS interface designs for both chemical (11-13) and electron ionization (14), high-speed separations using rapid pressure programming (15,16), the introduction of solvent mixtures (17),a detailed evaluation of capillary restrictor performance (18),and potential analytical application to several problems where advantages exist compared to alternative GC-MS or HPLC-MS methods (19-23). Much of the promising initial work with capillary SFC has not yet addressed or resolved a number of current limitations that will play a key role in determining if SFC and SFC-MS will develop to be a viable, widely practiced analytical technology. Rather than replacing established chromatographic methods, most interest in SFC is for the characterization of materials where the more mature GC or HPLC methods are inappropriate. In particular, for the combination with mass spectrometry the advantages afforded by the greater number of effective plates or high separation speed (compared to HPLC) is often of secondary importance compared to more practical concerns (e.g., can the SFC-MS be readily converted 0003-2700/87/0359-00 1380 1.50/0
to GC-MS or LC-MS modes of operation?). Truly useful SFC or SFC-MS instrumentation must also address problems related to injection methods and quantitation. Nearly all capillary SFC work reported to date with small diameter (1100-pm-i.d.) capillaries has used split injection techniques, which can be unreliable and result in constraints on sample size due to the sample volume and solubility limitations. This is further complicated by the relatively small sample size typically required to avoid overloading such columns (typically ca. 1-10 ng for 50-pm columns with a 0.25-pm film thickness), explaining the continued interest in conventional (HPLC) packed and microbore columns for SFC due to their higher effective phase ratios (24, 25). Application of capillary SFC-MS using previous interface designs may also be limited by the performance of capillary restrictors (e.g., spiking with flame ionization detection), which we recently have shown can result in precipitation of nonvolatile analytes, particularly a t low flow rates (18). Thus, the development of practical SFC-MS applications would be greatly facilitated by improvementsin SFC injection methods, significantly increased sample capacity, greater detector sensitivity, and improved interface operation with less volatile analytes. In this work we describe the development and initial evaluation of new SFC-MS instrumentation that meets many of the criteria for improved performance. An important feature of the interface is a chemical ionization source which has an adjacent fluid expansion region that is directly pumped. The new interface provides the capability for operation with much higher gas flow rates, allowing operation with both microbore and capillary columns. Also incorporated is improved control of restrictor temperature, which has recently been demonstrated to be vital to the transfer of less volatile analytes (18). Additional benefits of the interface include practical splitless injection for improved quantitation, increased sample capacity and dynamic range, and extended application to nonvolatile compounds. The operation of the new interface is demonstrated by using a variety of samples with C02or C02-modifierfluid systems, and the capillary and packed column SFC-MS approaches are compared. EXPERIMENTAL SECTION SFC Instrumentation. Most studies utilized a modified Varian 8500 syringe pump under microcomputer control to generate a high pressure and pulse-freeflow of mobile phase. The accuracy and stability of the pressure control was f O . l bar. In other experiments a Brownlee Labs MPLC dual syringe pump (software revision H)was used for mixed fluid generation. A modified Hewlett-Packard 5790 gas chromatograph oven provided constant temperature control to 1 0 . 1 "C. Sample introduction was accomplished without flow splitting using a Valco C14W injection valve with 0.06-pL and 0.20-pL rotor volumes at ambient temperature. A 1-m length of deactivated 100-pm4.d.fused silica capillary tubing was used to make a direct low dead volume connection between the injection valve and the chromatographic column. The mobile phases used in this work were 99.99% pure anaerobic carbon dioxide (Airco, Vancouver, WA), or carbon 0 1986 American Chemical Soclety
14
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987 Thermocouple and Heater Leads
-1
I
1 0 - 4 TORR
Insulation Probe Sheath
Capillary Column
CI Source Volume 10.2. 1 TORR) ,Ion Exit Aperture Volume Union Half Union
J
b Steel Sheath
10 - 50 TORR
Mechanical Pump
Flgure 1. Schematic illustration of the high flow rate (HFR) SFC-MS initerface.
dioxide modified with 1% (by volume) methanol. The carbon dioxide was dried and purified by passage through activated charcoal and alumina adsorbent traps before use. The binary fluid mixtures were prepared by charging the high-pressure syringe pump (cooled to 0 "C) with the appropriate volume of dry methanol and filling the remaining volume of the syringe with carbon dioxide. Mass spectrometry was used to confirm fluid mixture compositions. Columns. Two columns were used in this work. The first was a 30-m X 100-pm4.d. capillarycolumn coated with a 1-pm-thick film of 5% phenyl poly(methylphenylsi1oxane) stationary phase (SE-54) that was rendered nonextractable by extensive crosslinking with azo-tert-butane (26). The second was a Brownlee 25-cm X 1-mm4.d. (microbore) column packed with 5-pm silica particles coated with a CI8-modified phase. Separations were generally conducted at 50 "C or 100 "C for the capillary column and 50 "C for the microbore column (due to the greater thermal instability of the stationary phase). SFC-MS separations were obtained in isobaric, pressure-programmed,and fluid gradient modes (in the case of the microbore studies), but the best results were invariably obtained in the pressure-programmed mode for the capillary column and in the isobaric mode for the microbore column. Mass SpectrometricInterface. Two new SFC-MS interfaces were developed and evaluated in this work. Both interfacedesigns had the aims of (a) allowing more rapid restrictor replacement and operational convenience,(b) operation at high fluid flow rates, (c) improved control of restrictor temperature, and (d) enhanced sensitivity for less volatile compounds. The first design (not shown) was very similar to the second (Figure l), except mechanical pumping (760 L/min at the pump) was provided through a 0.6-cm opening in the chemical ionization (CI) source. In the second design, the fluid expands into an expansion region behind the CI volume, which is pumped directly through a 1.2-em-i.d. port. In both interface designs the SFC-MS probe was constructed from a 1.27-cm-0.d.stainless-steel tube which was temperature regulated using air flow heated by the chromatographic oven, as described previously (15). The probe could be easily withdrawn or introduced to the mass spectrometer vacuum chamber. The capillarycolumn, or deactivated fused silica transfer tubing from the microbore column exit, was connected to the capillary re-
-
First Ion Lens
strictor using a zero dead volume union. The capillary restrictors used for all reported results consisted of a 5-IO-cm length of 25-50-pm4.d. fused silica capillary tubing with a drawn region, typically 3-10 mm in length at its termination and an exit diameter of 8-10 pm. The restrictors were drawn in a reproducible fashion using a small electrically heated oven. A few experiments were also conducted using 4-6-cm lengths of 10-15-pm-id. capillary tubing for flow restrictors. A vacuum seal was made to the probe body via a "half-union" which was fabricated using a capillary stainless-steel sheath which extended approximately 0.7 cm beyond the end of the probe (see Figure l). The arrangement also incorporated a mechanism for easy probe disassembly and provided mechanical support of the "half-union" and thermal insulation (not shown). The restrictor sheath provided mechanical protection for the fragile drawn restrictors. The first interface (not shown) involved relatively minor modifications to our previous interface (11,13,15). The primary change was the addition of a 0.6-cm-i.d.direct pumping port to the CI source volume. In this design the capillary restrictor sheath terminated at the plane of the CI source repeller electrode, opposite the ion exit aperture. The capillary sheath fit snugly into a larger stainless-steel capillary which could be heated directly using a dc power supply, and the temperature of the sheath was monitored by using a thermocouple welded to the middle of the sheathed region. The maximum flow rate to the ion source was limited by the conductance of the direct pumping port and the ion source pressure necessary to obtain a reasonable compromise between the factors relevant to sensitivity (electronentrance and ion exit aperture sizes and ion source pressure) and the maximum desirable pressure in the ion source vacuum chamber (2-4 X lo4 torr). The second interface (Figure 1) was developed as a result of drawbacks in the arrangement described above and was used in all reported work. Details of the restrictor heating and expansion region are shown (not to scale) in Figure 2 for this arrangement. The 0.5-mm-0.d. capillary sheath protected the restrictor which fit inside a larger 0.7-mm4.d. (0.8-mm-0.d.) stainless-steel capillary and was aligned with an aperture in the CI source repeller electrode. The repeller also served to isolate the two regions. This larger 0.6-cm-longcapillary was directly attached to the expansion volume so as to mate with the probe (Figure 2). Thermocouple wires and an electrical lead of high-resistance wire were welded
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
15
DFI
Flguro 2. Detail of probe tip heater and expansion region for the high flow rate SFC-MS interface.
at the free end of the capillary heater. The high-resistance electrical lead prevented a cold spot near the end of the capillary and produced a temperature gradient along the sheath giving the highest temperature at the capillary restrictor exit. This design provided the direct resistive heating of the 0.7-cm capillary, which in turn heated the capillary protection sheath and the capillary restrictor. The diameter of the CI source entrance aperture (in the repeller) was selected to give a desired “split” between the source and the direct pumping port, which was approximately 1:25 in this work. The CI source was a 0.6-cm-diameter X l-cmlong cylindrical volume, which was otherwise similar to that reported earlier (13,15). CI reagent gases (methane,isobutane, or ammonia) were introduced directly to the CI volume by an additional inlet line (not shown in Figure 1). The mass spectrometer and methods for control and data acquisition were conventional and have been described previously (11, 13, 15).
RESULTS AND DISCUSSION Expansion through Capillary Restrictom for SFC-MS. A major aim of this work was the development of an enhanced SFC-MS interface which would allow operation at higher fluid flow rates and provide for detection of nonvolatile analytes. The improved performance for less volatile analytes was anticipated on the basis of an extensive study of restrictor performance in SFC (18). The previous work derived simple approximations for fluid expansion through straight-walled capillary restrictors based upon the limitation of fluid velocity in the capillary to the speed of sound, which is typically attained within a few orifice diameters of the capillary exit. By use of reasonable values for the Fanning friction factor, an ideal gas assumption, and operation at a temperature sufficient to avoid a two-phase isenthalpic expansion (generally reduced temperatures greater than 1.3), it was shown that fluid flow rates could generally be predicted within 30% using the following relationship:
-
Q = 31.3FQld2(M/7“)1/2 (1) where Q is the mass flow in g/min, PI is the fluid pressure in bar, d is capillary diameter in mm, M is molecular weight, T is the fluid temperature in K, and F, is a flow reduction coefficient based upon fluid viscosity and capillary dimensions (18). It was also shown that fluid density, within a few orifice diameters of the capillary exit (where ita velocity becomes
sonic), could be estimated based upon similar assumptions. Previous work with flame ionization detection (FID) for capillary SFC has shown that the pressure and temperature of the fluid prior to the restrictor, restrictor dimensions, restrictor temperature, and possibly the temperature of the expansion region can all impact performance (18). Heating of the fluid just prior to expansion and heating of the restrictor itself have been shown to improve and extend detection for compounds having low voltilities. It has been shown that volatility plays a major role in detection of such analytes, both directly and indirectly, since fluid-phasesolubilities (at a given density) generally mirror analyte vapor pressures (18, 23). However, for truly nonvolatile compounds heating of the fluid before or during expansion can be counterproductive, due to lower fluid densities and the resulting lower fluid-phase solubilities (18). For nonvolatile analytes improved FID results are obtained by using shorter restrictors with higher flow rates (18). It was also shown that excessively long restrictors and low flow rates resulted in a precipitation mode, where the analyte collected on restrictor surfaces, preventing detection and ultimately plugging the restrictor, or causing relatively large analyte particles (on the order of 1pm diameter) to be propelled from the restrictor resulting in a distinctive “spiking“ in the FID signal. Larger diameter or shorter restrictors were found to transmit nonvolatile analytes more efficiently to the gas phase, where larger concentrationslead to rapid nucleation and particle growth. These small particles, typically well under 0.1 pm diameter for solute concentrations under 100 ppm, apparently form rapidly ( 10000) and porous (multipath) frit restrictors (27) (L/d >> lo5)for transport of nonvolatile analytes (18). (Fluid velocity in the porous frit restrictor is expected to be subsonic (18), and the tortuous flow path combined with the relatively long residence time should provide for more efficient heat transfer to the expanding gas. We believe this results in improved transport due to enhanced volatility, but prevents detection of compounds with insufficient vapor pressures at the restrictor temperature.) Calculated volumetric flow rates and exit densities for various restrictor diameters and lengths are given in Table I. The calculations are based upon adiabatic expansion of carbon dioxide at 100 "C and 300 bar ( p = 0.66 g/cm3), and the volumetric flow rates are given for liquid carbon dioxide at 300 bar and 25 OC ( p = 0.95 g/cm3). The calculated fluid density at the capillary exit is directly proportionalto the mass flow rate for a given capillary diameter. This is physically reasonable since the fluid velocity will be sonic near the capillary exit and the variation in the speed of sound over the range of relevant fluid conditions is less than 10%. The calculated flow rates given in Table I are less reliable for the smallest diameter and longer restrictors, since the adiabatic assumption breaks down and heating through the restrictor walls becomes effective (18), particularly for the porous frit restrictor (27). Figure 3 gives calculated mass flow rates and exit densities for carbon dioxide at 100 "C and 300 bar expanding through a 10-pm-i.d. capillary as a function of restrictor length. Previous SFC-MS instrumentation was limited to a flow rate of about 20 pL/min (liquid) for optimum
Restrictor Length (mmi
Flgure 3. Calculated mass flow rate and density at restrictor exit as a function of restrictor length for C02 adiabatic expansion from 300 bar and 100 O C through a 10-pm-i.d. capillary.
performance. Since typical capillary SFC flow rates for 50pm-i.d. columns are in the range of 0.5-5 pL/min, capillary restrictors with a diameter of about 5 pm have been most widely used. The exit diameters of drawn restrictors for these flow rates are in the range of 2-3 pm (30) and are somewhat dependent upon the temperature of the fluid just prior to expansion, since a higher temperature will result in a lower density at the exit and a lower mass flow rate (18). The higher flow rates feasible with the new SFC-MS interface and practical limitations on restrictor dimensions indicate restrictor diameters of 10-12 pm, or slighly smaller tapered restrictors (5,18),should be optimum. This diameter allows flow rates of 100-200 pL/min for easily fabricated tapered restrictors, while providing relatively high fluid densities at the capillary exit. The high exit fluid density is advantageous since it will delay solute nucleation to the later stages of the expansion and make transfer to the mass spectrometer ionization region more efficient. SFC-MS Interface Operation. Two interfaces were constructed and evaluated in this work, and the second was found to give superior performance. The aim of the first interface was to allow higher SFC flow rates by direct pumping of the CI source volume. A maximum COz flow rate of -30 mg/min was obtained for a pressure in the ion source vacuum chamber of 3 x lo4 torr. The limitation upon pumping speed was imposed by the conductance of the connecting tubing. Investigation of a number of configurations failed to produce more than a factor of 3-5 increase in the maximum SFC flow
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
rate with this arrangement. Additionally, operation resulted in a significant decrease in total ion current and sensitivity. The reason for this is that source performance drops at higher pressures due to changea in the rates of important CI reactions, decreased penetration of the electron beam into the source, and the necessarily smaller diameters of both the electron entrance and ion exit apertures. Introduction of a CI reagent gas other than the chromatographic mobile phase would require an even greater increase in total gas flow to the source. Clearly, most of the ions produced in the CI source were removed through the auxiliary pumping port. The second high flow rate (HFR) interface was designed as a result of our dissatisfaction with the direct ion source pumping approach. The design shown in Figures 1and 2 met ow requirements of allowing SFC operation at high flow rates, providing efficient analyte transfer to the CI region, unrestricted CI operation at optimum pressures, maintenance of normal CI reagent gas flow rates, and a relatively minor increase in complexity. The additional pumping is provided by an inexpensive two-stage mechanical pump, similar to that required for thermospray LC-MS (28), and introduces no significant problems due to the volatility of most supercritical mobile phases. The HFR interface does impose some operational restrictions to avoid contamination of the CI source with oils from the mechanical pump and vacuum lines. Care must be taken to minimize back-streamingof oils, particularly when the SFC is not in operation. In the absence of a significant gas flow, pump oils may migrate to the expansion region and be subsequently introduced to the CI region during normal operation. It was found that this problem could be largely eliminated by installing a valve on the direct pumping line which was always closed immediately after fluid flow into the expansion region was stopped. When not in operation (with interface probe in place or withdrawn from the ion source region), a small flow of CI reagent to the source also appeared to reduce the background. While these procedures largely eliminated the contamination problem, the background below m/z 150 was always somewhat greater than in our previous interface designs. However, in the mass range of most interest to SFC-MS ( m / z >150), the background was not significantly larger than observed in previous interfaces. It should also be noted that while substantial nonvolatile deposits were always observed when cleaning the expansion region, they were not observed in the CI volume, which could typically be operated for several weeks between cleanings. Interface Performance. The HFR SFC-MS provided greater sensitivity and dynamic range than previous interfaces. A greatly reduced plugging frequency was obtained with operation typically extending for several weeks. While the actual sample size injected on-column was much greater due to the splitless operation, sensitivity in terms of sample concentration was significantly enhanced. The enhancement was most obvious for higher molecular weight components,suggesting that the enrichment process may be due in part to alignment of the capillary restrictor with the CI sample entrance orifice (see Figure 2). Such a process would be directly dependent upon molecular weight and should not impact the potential for improved quantitation. Other factors likely contributing to the improved performance include the more ideal expansion process and delayed solute nucleation, optimized CI source operation, and analyte injection into a volume of the source from which ions are more efficiently created and sampled. A t this point the relative importance of these contributions to improved performance remains uncertain. The improved performance for higher molecular weight components is demonstrated in Figure 4, which shows a capillary SFC-MS total ion chromatogram (TIC) for injection
17
Triton X-100
co2. 100'C 100 gm ID i 30 m Splltlsrr
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,
185
186
, 210
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236
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Figure 4. Capillary SFC-MS TIC of the nonionic surfactant Triton X-100 obtained using the HFR interface with splitless injection (0.2 pL) and a 2.5 barlmin pressure increase. Triton X-100, CO,, 100°C, 100 pm x 30 m. Splitless mlz = 400 n - 4
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m/z
-
-488
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r mlr
-
576
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185
,
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Figure 5. Selected ion chromatograms for the selected oligomers from the Triton X-100 separation shown in Figure 4.
of a 0.2-pL solution of the nonionic surfactant Triton X-100. The separation was obtained by using supercritical carbon dioxide a t 100 "C as the mobile phase with a 100-pm-i.d. X 30-m fused silica capillary column (see Experimental Section). The separation utilized a pressure program of 2.5 bar/min from 185 bar starting 2 min after injection. Mass spectrometric detection utilized ammonia chemical ionization, but good results were also obtained using methane CI. Capillary SFC with FID has shown the Triton X-100 n = 9 or n = 10 oligomer to have the greatest abundance, consistent with the SFC-MS results. Capillary SFC-MS with the previous interface designs (13,15)was limited to n 16. The selected ion chromatograms shown in Figure 5 show that the n = 16 oligomer can be easily detected. The n = 19 oligomer was also readily observed, although it has a concentration at least an order of magnitude smaller. (It should be noted that even for ideal interface operation, the TIC will show discrimination against the heavier components compared with FID. This is due to the decreased transmission efficiency of quadrupole mass filters at high m / z and the fact the FID response is
-
18
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987 100 A
1 m/2 559
Methane Chemical Ionization Triton X 100 CH3 CH3
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n,C-t-CH~-C--Q-iOCHIcn~,*
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Ammonia Chemical Ionization n m 8 Oligomer Triton X.100
'tI $
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260
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Flgure 7. Capillary SFC-MS TIC of Triton X-100 at a pressure increase of 12.5 bar/min; see Figure 4 for comparison.
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Figure 6. Chemical ionization (CI) spectra of the n = 8 oligomer of Triton X-100 obtained during SFCMS separatbns using methane (top) and ammonia (bottom) GI reagents.
approximately proportional to analyk mass flux, whereas MS ion current is proportional to molar flux. Similarly, the nonuniform ion transmission efficiencies with m / z will often cause the TIC to appear qualitatively different from FID chromatograms; this is particularly evident for Figure 4 due to the limited fragmentation and large m / z range.) An advantage associated with SFC-MS is the flexibility in selection of CI reagent (13,15,21). For a COz mobile phase even large partial pressures (up to 80%) in the ion source do not substantially affect the sensitivity or the positive ion mass spectra due to the low proton affinity of COz compared to normal CI reagents (methane, isobutane, and ammonia). Figure 6 gives the methane and ammonia CI mass spectra obtained for the n = 8 oligomer of Triton X-100 (at a MS resolution of 400). The ammonia CI mass spectrum (bottom) shows a dominant ammonium ion adduct, (M 18)+,and a much smaller protonated molecular ion (M H)+. The more energetic methane CI reagent yields a protonated molecular ion as the base peak, but also structurdy significant fragment ions at mlz 447 and m / z 487, due to cleavages of the hydrocarbon terminal group, and at m / z at 321, due to loss of (OCH2CHJ60H. It should also be noted that although little fragmentation is evident in the ammonia CI spectrum, the single-ion chromatograms (as shown in Figure 5, for example) clearly reveal fragmentation pathways and demonstrate the large dynamic range and sensitivity available. A major advantage associated with capillary columns is the ease of pressure (or density) programming (16).The speed and resolution of separation can be easily manipulated by changing the programming rate (16). This is illustrated in Figure 7, which shows a separation of Triton X-100 obtained for a pressure increase of 12.5 bar/min under similar conditions as in Figure 4. Although a much faster separation is obtained, a significant loss of resolution is evident. Depending
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Figure 8. Capillary SFC-MS TIC and selected Ion chromatograms for m l z 268 and m l z 020. The restrictor heater temperature was -650 O C , resulting in substantial pyrolysis. Note the time delay between the n = 9 digimer molecular ion (m/z 620) and the pyrolysis products (e.g., m l z 268).
upon the analytical requirements this trade-off of speed vs. resolution may be advantageous, particulary given the selectivity of the mass spectrometric detector. Methods for predicting the effect of changes in pressure or density programming rates have been described previously (16). Recent work with capillary restrictors for both FID and MS detection has shown that heating of the fluid just prior to (or during) the expansion through the restrictor can facilitate transfer of less volatile compounds (14,15,21,29,30). The improved transport to the detector is primarily associated with the enhanced vapor pressure or delayed precipitation of less volatile analytes (18). It was anticipated that the HFR interface would provide for improved analyte transport to the detector and remove the need for excessive heating of the capillary restrictor, which may result in pyrolysis of labile compounds. The HFR interface allowed heating of only a small segment (-0.6 cm) of the restrictor, with the maximum heating obtained at the restrictor exit. Consistent with expectations, no significant changes in interface performance were noted when the maximum restrictor heater temperature was varied between 150 O C and 400 "C. It was observed, however, that high restrictor heater temperatures (>450 "C) resulted in decomposition of sufficiently labile analytes. This is illustrated in Figure 8 for a Triton X-100 separation identical to that shown in Figure 7, but with a restrictor heater temperature of 650 "C. Figure 8 gives the TIC and selected ion chromatograms for m / z 268 (a pyrolysis product corre-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987 Coal Tar
Pesticides & Herblcides
COP 50% 320 bar 1 mm x 250 mm, 5 pm
/,A, 1
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Flgure 0. TIC and selected Ion chromatograms for the microbore SFC-MS separation of a coal tar extract. Addltlon of 1% methanol did not result in a Significant change In the TIC.
sponding to the ammonium ion adduct from cleavage of the 0-CH2 bond of the second -OCH2CH2- unit) and m / z 620 (the ammonium ion adduct for the intact n = 9 oligomer). The pyrolysis products were observed to peak 3-5 s after the intact oligomer, as evident in Figure 8. The time delay associated with observation of the pyrolysis producta clearly suggests an origin not associated with the gas-phase expansion since any time lag should be too small to observe. The pyrolysis products are almost certainly due to early precipitation of the analyte (due to warming of the fluid and loss of solvating power) and flow to the restrictor exit where it could undergo decomposition on hot surfaces of the capillary heater. SFC-MS with Packed "Microbore"Columns. The HFR interface allows operation with packed columns, and the effective split ratio used in this work is well suited for 1-mm4.d. "microbore" columns. Simple replacement of the CI source repeller disk, which separates the expansion region and the CI source volume, with a disk having a different sample entrance orifice diameter would d o w operation in different flow rate ranges. Operation with a conventional 4.4pm-i.d. HPLC column a t similar linear velocities should be feasible by reduction of the sample entrance orifice diameter by a factor of -5. Figure 9 shows the TIC and several selected ion chromatograms for a coal tar extract separated using the 1-mm4.d. X 250-mm "microbore" column with a CI8 stationary phase (see Experimental Section). Supercritical carbon dioxide a t 50 OC and 320 bar inlet pressure (280 bar outlet pressure) at an average linear velocity of 0.62 cm/s was used as the mobile phase. It should be noted that a pressure drop along the column typically results in increasing linear velocity and greater retention (k? as the fluid density drops. (However, for larger column diameters the cooling effect from expansion may cause the fluid temperature to be significantly lower than the column walls. This results in higher fluid densities and, combined with the temperature change, can result in a complex dependence of retention upon linear velocity.) The selected ion chromatograms given in Figure 9 begin to suggest the sample complexity, shown previously by higher resolution capillary SFC-MS analyses (14). Greater resolution of the earlier eluting peaks was obtained by separation at lower pressur s, but the value of this approach is largely negated by the fact that retention for moderate molecular weight components (e.g., m/z 226 at 7.5 min in Figure 9) became even greater. The benzopyrene isomers, readily eluted by capillary SFC (14), had retention times in excess of 25 min. Higher preasures at 50 "C resulted in only minor decreases in retention within the limitations of our instrumentation (500 bar) since COz density only increases from 0.85 g/cm3 at 320 bar to 0.96 g/cm3 at 500 bar. Clearly, stationary phases with lower phase
e
Pesticides & Herbicider CO IMsOH 11% vlvl 504.460 bar 1 mm x 260 mm. 5 rm. RP.18
I
Time Imlnutasl
Flgure 10. Microbore SFC-MS TIC of a pesticide and herbicide mixture obtained using carbon dloxide (A) and carbon dioxide modified with 1% (by volume) methanol (B) at 50 OC.
ratios and giving less retention than the CI8 phase (for the dominant polycyclic aromatic hydrocarbons), or a mobile phase with greater solvating power than C02, would be desirable. The analysis of labile pesticides and herbicides constitutes a potentially important application for SFC-MS (20, 31). Figure 10A shows the TIC for an eight-component mixture separated using the microbore column with carbon dioxide at 50 "C and 400 bar inlet pressure (350 bar outlet). Although all eight components were easily identified from the mass spectra, the separation was relatively poor and significant tailing was observed for the most polar compounds. The addition of solvent modifiers in relatively low concentrations is often utilized in SFC with packed columns to improve peak shape and reduce retention (9,10). The mass spectrometric detector for SFC has the advantage of applicability to essentially any fluid or fluid mixture. The addition of 1% of methanol to carbon dioxide results in a dramatic improvement in the separation of the polar pesticide and herbicides, as shown in Figure 10B. In contrast to other components diuron and carbaryl showed substantial improvement in peak shape and reduced tailing ( N improved from 30 to 400 and from 80 to 500, respectively), but no significant reduction in k ' was observed upon addition of methanol. Previously we have demonstrated that such small amounts (
