Anal. #em. 1092, 64, 1571-1577
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Supercritical Fluid Chromatography/Mass Spectrometry Using a Quadrupole Mass Filter/Quadrupole Ion Trap Hybrid Mass Spectrometer with External Ion Source J. David Pinkston' and Thomas E. Delaney The Procter & Gamble Company, P.O.Box 398707,Cincinnati, Ohio 45239-8707
Kenneth L. Morand and R. Graham Cooks Chemistry Department, Purdue University, West Lafayette, Indiana 47907
The comblnatkn of capillary supercritical fluid chromatog raphy (SFC) and Ion trap massspectrometrywlng an edernal, differentlaliy-pumped k n source Is described. Thb was accomplkhed uslng a capillary SFC Instrument with direct, on-column InJectlon,and a custom-bulit, dlrect-lntroductlon Interface to a quadrupole mass fliter/lon trap (Wtrap). The hybrid mass spectrometer was equipped with a standard EI/ CI Ion source and operatedwlth conventional Iontrap detector (ITD)electrodes and electronics. The hybrld mass analyzer system allows the lon trap to tolerate the hlgh mobile-phaw flow rates introduced to the lonlzatlon region of the mass spectrometer. The Instrunent was characterized and optimal operating conditions were determined with perfluorotrlbutylamlne, anthracene, and methyl docooanate. A sample of 80 fmol of anthracene provideda molecular Ion peak wlth a S/N ratio of 20. Once the operallng parameters were optlmlzed, two less volatile mixtures were characterized. The first was a poly(dlmethylslloxane) with an average molecular welght of 770. The second was a derlvatlzed surfactant for whlch the full structure was not known In advance but whlch was elucidated from the SFC/MS data.
INTRODUCTION Supercritical fluid chromatography (SFC), a technique which is complementary to gas chromatography (GC) and high-performance liquid chromatography (HPLC), has become an increasingly accepted method of analysis over the past few years. For example, SFC has been applied to mixtures of compounds which are too low in volatility1 or thermal stability2 for GC or which lack an appropriate functionalgroup for sensitiveHPLC detection? Compilations of SFC applications have been p u b l i ~ h e d . ~ , ~ Historically, most SFC has been performed with flame ionization detection. As the routine use of SFC has grown, so has ita combination with other more chemically informative detectors. Such is the case, for example, in supercritical fluid
* To whom correspondence should be addressed.
(1) Sheeley, D. M.; Reinhold, V. N. J . Chromatogr. 1989,474,83-96. (2) Pinkston, J. D.; Bowling, D. J.; Delaney, T. E. J. Chromatogr. 1989, 474, 97-111. (3) Pinkston, J. D.; Delaney, T. E.; Bowling, D. J. J.Microcolumn Sep. 1990,2,181-187. (4) Markides, K. E.; Lee, M. L. Compilation of SFC Applications, 1988 Workshop on SFC,Park City,UT,Jan 12-14, 1988; Brighman Young University Press: Provo, UT, 1988. (5) Markides, K. E.; Lee, M. L. Compilation of SFC Applications, 1989Symposiumf Workshop on SFC,Snowbird, UT, June 13-15,1989; Brigham Young University Press: Provo, UT, 1989.
0003-2700/92/0364-157 1$03.00/0
chromatography/mass spectrometry (SFC/MS).e As this field, in turn, has matured, many researchers have moved to mass spectrometers that are able to capitalize on some of the inherent advantages of SFC. In particular, a move to higher mass range instruments,' double-focusing sector1?*10 or Fourier-transformll-13mass spectrometers,has been apparent. Unfortunately,these instruments are generallylarge,complex, and expensive. A relatively recent entry into the field of commerciallyavailable mass spectrometers is the Paul ion trap.14Js The commercial ion trap detector is a low-cost, simple mass spectrometer for gas chromatography/mass spectrometry. Its mass-to-charge range is limited to a nominal 650 Ddcharge. However,the capabilitiesof ion trap mass spectrometers have steadily grown over the past few year~.'~J'Advances include chemical ionization,lsthe ability to study negative ions,lgthe ability to perform tandem mass spectrometry (MS/MS) experimente,2021and mlz-range extension to at least 70OO0.22-24 In fact, the ion trap mass spectrometer (ITMS) is itself becoming a versatile and powerful instrument. Thus, the ion (6)Smith, R. D.; Kalinoski, H. T.; Udseth, H. R. Mass Spectrom. Rev. 1987,6,445-496. (7) Pinkston, J. D.; Owens, G. D.; Burkes, L. J.; Delaney, T. E.; Millington, D. s.;Maltby, D. A. Anal. Chem. 1988,60, 962-966. (8)Kalinoski, H. T.; Udseth, H. R.; Chess, E. K.; Smith, R. D. J. Chromatogr. 1987,394, 3-14. (9) Reinhold, V. N.; Sheeley, D. M.; Kuei, J.; Her, G. Anal. Chem. 1988.60.2719-2722. (10) H G g , E. C.; Jackson, B. J.; Markides, K. E.; Lee, M. L. Anal. Chem. 1988,60, 2715-2719. (11) Laude, D. A,, Jr.; Pentoney, S. L.; Griffiths, P. R.; Wilkins, C. L. Anal. Chem. 1987,59,2283-2288. (12) Lee. E. D.: Henion.. J. D.:. Codv.R. _ . B.:. Kinsineer. - . J. A. Anal. Chem. 1987,59, 1309-1312. (13) Baumeister, E. R.; West, C. D.; Ijames, C. F.; Wilkins, C. L. Anal. Chem. 1991,63,251-255. (14) Todd, J. F. J. Presented at the 1989 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlanta, GA, March 610, 1989, Abstract No. 1001. (15) Stafford, G., Jr.; Kelley, P.; Syka, J.; Reynolds, W.; Todd, J. Znt. J. Mass. Spectrom. Zon Processes 1987, 75, 245. (16) Cooks, R. G.; Kaiser, R. E., Jr. Acc. Chem.Res. 1990,23,213-219. (17) Todd, J. F. J. Mass Spectrom. Rev. 1991, 10, 3-52. (18) Brodbelt, J. S.; Louris, J. N.; Cooks, R. G. Anal. Chem. 1987,59, 1278. (19) McLuckey, S.A.; Glish, G. L.; Kelley, P. E. Anal. Chem. 1987,59, 1670. (20) Kelley, P. E.; Stafford, G. C., Jr.; Syka, J. E. P.; Reynolds, W. E.; Louris, J. N.; Amy, J. W.; Todd, J, F. J. Presented at the 33rd ASMS Conference on Mass Spectrometry and Allied Topics, San Diego, CA, May 2631, 1985. (21) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, G. C., Jr.; Todd, J. F. J. Anal. Chem. 1987,59, 1677. (22) Kaiser, R. E., Jr.; Louris, J. N.; Amy, J. W.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1989, 3, 225. (23) Kaiser, R. E., Jr.; Cooks, R. G.; Moss, J.; Hemberger, P. H. Rapid Commun. Mass Spectrom. 1989, 3, 50. (24) Kaiser, R. E., Jr.; Cooks, R. G.; Stafford, G. C., Jr.; Syka, J. E. P.; Hemberger, P. H. Znt. J. Mass Spectrom. Ion Processes 1991, 106, 79. @ lag2 Amerlcan Chemical Soclety
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trap mass spectrometer potentially provides a worthwhile route to high-performance mass spectrometric detection for SFC. Todd and co-workers have interfaced an SFC instrument to a modified ion trap detector.14~25The Teflon spacer rings which separate the electrodes were removed to improve the rate of evacuation of the SFC mobile phase entering the ion trap. Recognizable SFCIMS and SFC/MS/MS spectra were obtained on low-molecular-weight aromatic and polycyclic aromatic hydrocarbons. However, the low pumping speed (nominally 40 LIS) of the ion trap detector was a major limitation at higher SFC flow rates. In addition, the performance of the ion trap was clearly affected by the introduction of relatively large quantities of the mobile phase, COz, into the trap volume and the authors indicated that further experiments were needed to optimize ~ e n s i t i v i t y . ~ ~ The present study is an attempt to follow up on this early and promising work. Carbon dioxide is the most common mobile phase used in SFC. Unfortunately, high levels of COz in the ion trap negatively influence the performance of the trap in at least two ways. First, a buffer gas, normally helium, is commonly used in ion traps to stabilize the trapped ions and increase mass resolving power. Carbon dioxide has been shown to be a poor buffer gas.14~25 Second, high levels of trapped ions derived from C02 may adversely affect performance of the trap.26 This phenomenon, due to coulombic repulsion or space-charge, results in a drop in mass resolving power and dynamic range. It thus appears that the ion trap must be operated in the absence of high levels of neutrals and ions derived from the mobile phase for adequate performance in SFCIMS. Morand, Horning, and Cooks27 have constructed and reported on the MSIMS performance of a tandem quadrupole mass filterlquadrupole ion trap (&/trap). This hybrid instrument may also present advantages for other types of studies. Ions are produced in a conventional ion source and are then transmitted through the quadrupole, with or without mass selection, to the ion trap. The ion source and analyzer portions of the instrument are located in separate, differentially pumped areas of the mass spectrometer. This tandem instrument is,therefore, well suited to combine the advantages of the ion trap as a mass analyzer with sample introduction1 ionization modes which require (or perform best) with ion production external to the ion trap. A similar instrument of Qlquistor (quadrupole ion storage device)/& configuration was constructed by Kofel, Reinhard, and Schlunegger for studying organic ion-molecule reactions.% Amagnetic-sector1 trap instrument has also been described by Schwartz et aLZ9 using a particle-bombardment desorptionlionization source. Note too, that methods in which external ionization and injection into an ion trap are achieved without use of a second mass analyzer are also well-known.3b32 However, the combination of capillary SFC and ion trap mass spectrometry using an external, differentially pumped ion source and a (25)Todd, J. F.J.; Mulchreest, I. C.; Berry, A. J.; Games, D. E.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988,2,55-58. (26)March, R. E.;Hughes, R. 3. Quadrupole Storage Mass Spectrometry; Wiley Interscience: New York, 1989;pp 344-347. (27)Morand, K. L.; Horning, S. R.; Cooks, R. G. Znt. J.Mass Spectrom. Ion Processes 1991,105,13-29. (28)Kofel, P.; Reinhard, H.; Schlunegger, _ - U. P. Org. Mass Spectrom. 1991,26,463-467. (29)Schwartz, J. C.; Kaiser, R. E., Jr.; Cooks, R. G.; Savickas, P. J. Int. J. Mass Spectrom. Ion Processes 1990,98, 209-224. (30)Louris, J. N.; Amy, J. W.; Ridley, T. Y.:Cooks. R. G. Int. J.Mass Spectrom. Ion Processes 1989,88,97-111. (31)Kaiser, R. E.,Jr.; Williams, J. D.; Lammert, S. A.; Cooks, R. G.; Zakett, D.J. Chromatogr. 1991,562,3-11. (32)VanBerkel, G. J.; Glish, G. L.;McLuckey, S.A. Anal. Chem. 1990, 62,1284-1295.
Source
Quad
ImTrap
5 Flgurr 1. Schematlc diagram of the SFWltrap Instrument.
quadrupole mass filter as described above offers a number of positive features. These include good sensitivity, the capacity for performing MS/MS experiments, traditional electron and chemical ionization, the ability to reject ions such as those derived from the mobile phase or the chemical ionization reagent gas, and most especially the ability to tolerate relatively high flow rates of COz from the chromatograph. Here we describe the coupling of capillary SFC to the tandem quadrupolelion trap instrument described above.
EXPERIMENTAL SECTION The SFC-Q/trap instrument is shownin the schematicin Figure 1. The various portions of the instrument and the experimental parameters are described in the following sections. SFC. The SFC portion of the instrument has been described previ~usly.~J The SFC unit consita of a Model 8500syringepump (Varian Associates, Palo Alto, CA) modified for pressure control up to 560 atm,a an HP 5712Agas chromatographyoven (HewlettPackard, Avondale, PA, USA), and a custom-built interface probe.' Direct, on-column injection was performed with a Model EC14W.1 internal-loop injector (Valco, Houston, TX). The internal-loopvolume was 0.1 pL. The column was a 10-m-long, 50-pm-i.d. SB-methyl-100capillary column with a 0.25-pm film thickness (Dionex, Lee ScientificDivision, Salt Lake City, UT). It was linked directlyto the injection valve. Flow restrictionwas provided by a tapered, fused-silica c a p i l l e drawn from a 50pm i.d. toa 2.5-pm aperture over 2 cm. The restrictor was housed in the custon-built interface probe. The probe passed through the direct-insertion-probevacuum lock and mated directly with the rear of the ion-source ion volume. The stem of the interface probe and the SFC oven were held at a temperature of 100 "C, while the probe tip was heated to 300 "C. The SFC mobile phase was SFC-gradeCOz (ScottSpecialtyGases, Plumsteadville, PA). Other SFC conditions are listed in the Results and Discussion and in the appropriate figure caption. &/Trap. The tandem Q/trap mass spectrometer has been described in a previous p~blication.~'The ion source is a Model 4500 EIICI ion source (Finnigan MAT, San Jose, CA) held at 150 "C. Ionization is achieved by either 70-eV electron ionization (EI) with an emission current of 0.3 mA or chemical ionization (CI) with either methane (99.99% minimum, Matheson Gas Products, Dayton, OH) or a mixture of 1% ammonia in methane as reagent gas (Matheson). The quadrupole is a hyperbolic, mass-analyzing quadrupole (Model4500, Finnigan-MAT,San Jose, CA) with amass-to-charge (mlz)range of 1800Da/charge. The ions produced in the source are accelerated to the quadrupole, typically held at a dc-offset voltage of -5 V with respect to the grounded ion source. The quadrupole is normally operated in the radio-frequency-only(rfonly) mode at an operating voltage which eliminates a majority of the highly abundant carbon dioxide and chemical-ionizationreagent ions (ions of m/z < 63 Da/charge are not transmitted unless otherwise indicated). This is necessary to reduce spacecharging effects within the ion trap. Certain of the experimenta described here could have been performed without using the quadrupole to eliminate carbon ~
~
~
~~~~
(33)Chester, T. L.; Bowling, D. J.; Innis, D. P.; Pinkston, J. D. Anal. Chem. 1990,62,1299-1301. (34)Chester, T. L.; Innis, D. P.; Owens, G. D. Anal. Chem. 1985,57, 2243-2247.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1002
dioxide and chemical-ionization-reagent ions. The ion trap rf voltage could have been set at a level appropriate to eject these ions from the trap. However, this would not have provided the freedom to vary the injection rf level to optimize injection and dissociation efficiencies. Resonant ejection of carbon dioxide and chemical-ionization-reagent ions was not possible since the ion trap used for these experiments was not equipped with this capability. Ions exiting the quadrupole are focused on the entrance aperture of the ion trap using an einzel lens assembly. The middle element of this assembly is a gated-potential lens which also serves to define an ion-injection-time window. The potential placed on the element is typically set at -30 V (relative to the grounded ion source) for ion injection and at +330 V at all other times. The width of the ion-injection-time window is set via the software and may be as large as 650 ms and as small as 1ma. Varying the width of the window affects the number of ions admitted to the trap as well as the ultimate scan rate (scans/s) which can be achieved. The quadrupole ion trap has an internal radius of 1.0 cm and an actual m/z range of 667 Da/charge in the absence of auxiliary fields. Computer controlof the ion trap is effected by commercial, Model ITD 700 electronics (Finnigan)and a personal computer (ModelPC, InternationalBusiness Machines,Armonk, NY) with ITMS, version 2.00, software (Finnigan). Ions are injected into the trap at values of the Mathieu parameter, qe, appropriate for the ion of interest and ranging from 0.02 to 0.1. The value chosen for q1 (by selection of the amplitude of the rf voltage) determines the depth of the potential well which the ion experiences. Therefore, the value of qehas a direct effect on injection efficiency of the selected ion by influencingallowablekineticenergies which may be successfully trapped.3S@ Mass analysis using the ion trap is achieved with the usual mass-selective-instabilityscanmode.16J7 In the ion trap analogue of selected ion monitoring (hereafterreferred to as the "narrowscan-range mode"), the scan is performed over a 5 Da/charge range centered on the ion of interest. Only the data acquired over a 1 Da/charge envelope centered on the ion of interest is actually stored. Ion detection is performed with an off-axis multiplier and conversion dynode, the operating voltages being -1400 and -5OOO V, respectively. The off-axis design maintains a high signal-to-noise level by eliminating line-of-sight effects. This minimizes problems which can arise from ions which are not trapped during the injection process or fast neutral species which pass directly through the ion trap. As is usual,helium is added to the ion trap to provide a means by which injected ions may be collisionally stabilized and overall resolution increased during mass analysis. The ion trap is pressurized with helium to a readingof 1.7 X lo-'Torr as measured by an Bayard-Alpert ionization gauge. This value is uncorrected but corresponds to approximately 1mTorr of helium within the ion trap. The mass spectrometer was tuned at an SFC pressure of 200 atm with perfluorotributylamine since tuning the mass spectrometer at a relatively high SFC pressure was found to be beneficial in earlier SFC/MS A typical scan sequence is as follows: the ion gate is opened for 650 ms and a mass analysis scan is performed over an m/z range of 70-650 Da/charge at a scan rate of 180 ps/Da. Only two scans are averaged to produce a fiial, recorded mass spectrum in order to preserve the chromatographic-peak profiles. This results in a total acquisition time of approximately 1.51 s for a full mass spectrum. Chromatographic peaks are approximately 20 s in width (fullwidth at half-maximum) allowing a total of 10 or more mass spectra to be acquired per peak. All analyzer components are attached to an opticalrail, allowing elements to be modified and quicklyand accurately repositioned. The ion-optical components are contained within a glass-top vacuum chamber. The ion source and analyzer regions are differentially pumped to minimize the passage of SFC-carrier gas (36) Dawson, P. H. Quadrupole Mass Spectrometry and i t s Applications: Elsevier Scientific Publishing Co.: New York, 1976. (36) Major, F. G.; Dehmelt, H. G. Phys. Reu. 1968,170,91-107. (37) Bowling, D. J.; Pinkston, J. D. Presented at the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson,AZ, June 3-8,1990; p 1232.
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and chemical-ionization-reagent gas to the analyzer and so maintain optimum performance. Otherwise, resolution and sensitivityare degraded due to collisional scattering, while new ions appear by such routes as charge exchange and collisioninduced dissociation. The ion source region is pumped by a 270 L/s turbomolecular pump (TPH 270, Balzers/Pfeiffer, Liechtenstein), while the analyzer region is pumped by a 170 L/s turbomolecular pump (TPH 170, Balzers). The base pressure in the analyzer region is 3 X lO-' Torr. Without CI reagent gas or He buffer gas flowing into the system, the analyzer pressure was 2.2 X 1Oa Torr (uncorrected)at an SFC-mobile-phasepressure of 100 atm. The differential-pumpingcapability of the custombuild Q/trap was significantly poorer than that of a commercial instrument such as the Finnigan-MATTSQ-70systema2JUnder similar conditions, the TSQ-70analyzer pressure would lie in the (5-7) X 10-8Torr range. It was difficultto routinely monitor the rise in ion-trapanalyzer pressure during the pressure-programmed SFC runs. With He buffer gas admitted tothe trap, the ionizationgauge signal was in the (1.5-3.0) X lo-' Torr range. The rise in the signal level was less than 1 X lo-' Torr over the course of an SFC run. Sample Preparation. Anthracene (Fluka AG, Buchs, Switzerland),methyl docosanate (Alltech Associates, Deerfield, IL), and 770-average-molecular-weight,methyl-terminatedpoly(dimethylsiloxane) (Polyscience,Inc., Warrington, PA) were used as received from the supplier. The first two samples were dissolved in toluene (American Burdick &Jackson, Muskegon, MI) to the appropriate concentration,while the last was dissolved in d i d o romethane (American Burdick & Jackson). Perfluorotributylamine (PFTBA) (Scientific Instrument Services, Ringoes, NJ) was also used as received from the supplier. The ethoxylated alcohol standard (produced in-house) was dissolved in a 3 2 mixture of N-methy-N-(tert-butyldimethylsily1)trifluoroacetamide (MTBSTFA, Pierce, Rockford, IL) and pyridine (Pierce) and heated at 80 "C for 1h to form the tertbutyldimethylsilyl(TBDMS)derivative. The alkylethoxysulfate surfactantmixture (produced in-house)was dissolved in methanol (American Burdick & Jackson) and reacted with diazomethane in ether at 0 "C until the reaction was complete in a manner similar to that previously described.= The reaction mixture was evaporated to dryness with dry nitrogen and then dissolved in methanol to a concentration of approximately 4% (w/v).
RESULTS AND DISCUSSION A standard compound with little propensity for fragmentation, anthracene, was chosen for a series of experiments performed to optimize the ion-injection time. This interval was varied from 10 to 650 ms, and the signal-to-noise ratio (S/N) was found to be greatest a t the maximum ion-injection time of 650 ma. Thus each scan averaged by the ion trap's data acquisition system to produce a stored spectrum was preceded by a 650-ms ion-injection time. Since the chromatographic peaks were generally 20 s or less in width, it was necessary to keep the number of scans averaged to a minimum to preserve the chromatographic peak shape. Figure 2 shows the mass chromatogram a t 178Ddcharge and the E1 spectrum recorded for 780 fmol of anthracene injected on column. The scan range was 60-200 Ddcharge, and the mass chromatogram exhibits a S/N ratio of approximately 10. The degree of fragmentation in the anthracene E1 spectrum is much greater than that typically observed. This is the result of gaseous collisions of the anthracene molecular ions during transmission through the quadrupole and upon injection into the ion trap. The fact that COz as well as helium is present at relatively high pressures accounts for this propensity toward collision-induced dissociation. This effect has been discussed p r e v i ~ u s l ybut ~ ~is~enhanced ~ here due to collisionswith the heavier target COZ. As with the present case and all other experiments, the fact that an injected ion (38) Knapp, D. R. Handbook of Analytical Derivatization Reactions; Wiley Interscience: New York, 1979 p 402.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
100
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EO
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Figure 2. (Top) mass chromatogram for the molecular ion of anthracene, 178 Dalcharge, for a 780-fmoi sample examined using EI. (Bottom) E1 SFWltrap spectrum of 780 fmol of anthracene. The SFC pressure was ramped from 70 to 80 atm at 2 atmlmin upon injection. I t was then ramped at 10 atmlmin to 300 atm. The ion injection energy was 20 eV, the Injection time was 650 ms, the scan range was 10-200 Dalcharge, and the value of q2during injection for the molecular ion was 0.081.
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Scan Number Figure 4. Narrow-scan-range-modechromatogramof 179 Dalcharge, the protonatedmolecule of anthracene, acquiredfor a 78-fmol Injection under methane C I conditions. The SFC pressure conditions are listed in Figure 2, while the mass spectral parameters are listed In Figure 3.
i
355
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I 17a
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Flguro 3. (Top) mass chromatogram for the protonated molecule of anthracene, 179 Dalcharge, for a 780-fmol sample examined using methane C I conditions. (Bottom) methane-CI, SFC-Qltrap spectrum of anthracene. The SFC conditions are identlcal to those listed in Figure 2. The scan range was 60-200 Dalcharge and the value of q2 upon injection was 0.202 for the protonated molecule. The other mass spectral parameters are identical to those listed in Figure 2.
may collide with one of the ion trap electrode surfaces during the injection process cannot be overlooked. Such a process, termed surface-induced dissociation (SID), has been shown to lead to substantial fragmentation27929 of the injected ion. To what extent either process (CID and/or SID) determines the fragmentation observed is only an estimation, and both mechanisms should be considered for each experiment. All subsequent work was performed using chemical ionization with either methane or ammonia as a reagent gas. Perfluorotributylamine (methane reagent) was used to optimize the ion-injection energy (trap offset voltage) and the He pressure. The injection energy was varied from 0 to 40 eV, and the optimum injection energy waa found to be 20 eV. The helium pressure was found to be optimized at 2.0 X Torr after examining the range 4.0 X 10-4to 6.5 X 10-5Torr (all values uncorrected). Figure 3 shows the methane CI spectrum and maw chromatogram at 179 Da/charge recorded for 780 fmol of anthracene on column. The mass chromatogram exhibits a S/N ratio of approximately 15. Figure 4 shows results from an experiment performed with just 78 fmol of anthracene injected on column, in which the protonated molecule at 179 Da/charge was monitored in the narrow-scan-rangemode (as described in the Experimental Section). The S/N ratio is approximately 15. These data are indicative of the detection limit of the technique even at this relatively early stage of method development.
Flgure 5. Methane CI, SFWltrap spectrum of 28 pmol of methyl docosanate. The SFC pressure was ramped from 100 to 300 atm at 10 atmlmln upon injection. The ion injection energy was 20 eV, the injection time was 650 ms, the scan range was 150-360 Da/charge, and the value of q2 during injection for the protonated molecule was 0.102.
Once the ion-injection time, ion-injection energy, and He pressure had been optimized as described above, a highermolecular-weightstandard, methyl docmanate,was examined. Though still relatively low in molecular weight (3541,methyl docoeanate more closely approximates the types of samples that might typically be run by SFC/MS than does anthracene. Figure 5 shows the methane CI spectrum of 28 pmol of methyl docosanate. The scan range was 150-360 Ddcharge with five scans averaged per spectrum. The base peak of the spectrum is the protonated molecule at 355 Ddcharge. Injection of 280 fmol of methyl docosanate while the signal at 355 Da/charge is monitored in the narrow-scan mode provided a S/N ratio of 2. The mass spectral peaks in some of the following spectra are broadened due to space-charge broadening in the ion trap or due to relatively high levels of COZpresent in the trap. Space-charge broadening is due to the high concentration of similarly charged species confined within the trap.26 Compared to helium, carbon dioxide is a poor buffer gas for the ion trap, aa discussed in the Introduction. Both of these phenomena result in a loss of mass spectral resolution and degradation of the spectral quality. Space-chargebroadening is especially apparent in selected cases where high concentrations of samples were analyzed. Ethoxylated alcohols (AEs) are widely used nonionic eurfactants. A pure AE standard with a carbon-chain length of 12 and an ethoxylate-chain length of 3 (CIZE~) was derivatized to form the tert-butyldimethylsilyl(TBDMS)derivative
ANALYTICAL CHEMISTRY, VOL. 84, NO. 14, JULY 15, 1992 loo
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Flgurr 6. Ammonla CI, SFWltrap spectrum of 170 pmoi of the TBDMS derivative of a pure ethoxylated alcohol standard with an alkyl chaln length of 12 and an ethoxylate chaln length of 3. The experiment was performed at an isobaric SFC pressure of 210 atm. The ion Injection energy was 15 eV, the Injection time was 850 ms, the scan range was 100-500 Dalcharge, and the value of qr upon Injection for the ammonium adduct ion was 0.0987.
with a nominal molecular weight of 432. Ammonia CI was used to effect ionization. The influence of the ion kinetic energy on the spectrum of this sample was first investigated. As expected, the ion-injection voltage had a strong effect on the relative abundances of the protonated molecule (433Da/ charge) and the ammonium adduct ion (450 Da/charge). At 20 eV, the abundance of the protonated molecule was approximately twice that of the ammonium adduct ion. At 10-eV ion kinetic energy, the ratio was reversed, with the abundance of the protonated molecule being approximately 70% that of the ammonium adduct ion. At 15 eV, the two ions are approximately equal in abundance. These results clearly demonstrate that collision-induceddissociation in the course of injection of ions into the trap has an effect on the appearance of the spectra, as found in earlier studies.27 Figure 6 shows the NH3 CI spectrum of 170 pmol of C~EB-TBDMS at an ion-injection energy of 15 eV. The fragment ions at 131 and 257 Da/charge correspond in mass to [TBDMS - Ol+ and [C12H26 - (OCHZCHZ)~I+, respectively. They are reasonably represented as structures l and 2, respectively, the enhanced C-O cleavage between ethoxylate units 2 and 3 being accounted for by the rearrangement which yields ion 2.
c4y9 +
CH3-Si /
CH3 1
=O
CHz
CH,
CH, ‘ 0 ’
CH, 2
The ion-injection energy was held at 15 eV while the amplitude of the rf voltage (which sets the value of the Mathieu stability parameter qz)was varied during repeated injections of the C12E3-TBDMS standard. The amplitude of the rf voltage affects the low-mass cutoff (Le., the ion of lowest m/z which can be trapped within the ion trap). It also affects the efficiency of trapping of higher-m/z ions, as well as the appearance of the spectrum itself. When the amplitude was varied from 178 V(rf, zero-to-peak), corresponding to a lowmass cutoff of 15.9 Da/charge, to 625 V(rf, zero-to-peak), corresponding to a low-mass cutoff of 55.5 Da/charge, the abundance of the protonated molecule and the ammonium adduct ion were found to be maximized at a rf voltage of 535 V(rf, zero-to-peak), corresponding to a low-mass cutoff of
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500
Flgurr 7. Ammonia CI, SFWltrap run of a methykapped poly(dimethylsiloxane)with an average molecular weight of 770. The peak labels refer to values of n in structure 3. The SFC pressure was ramped at 2 atmlmln from 70 to 80 atm upon injectlon. It was then ramped at 10 atmlmln to 400 atm. The trap was scanned from 140 to 850 Dalcharge. (h during Injection for the ammonlum adduct ions of the oligomers within the mass-to-charge range examined varied from 0.0987 to 0.0538. The ion injection energy was 15 eV, and the injectlon time was 850 ms.
47.6 Da/charge. At this rf voltage, the protonated molecule and the ammoniumadduct ion of the ClZEa-TBDMS standard were also approximately equal in abundance. At higher rf voltages, the relative abundance of the ammonium adduct ion dropped below that of the protonated molecule. The relative abundance of the fragment ion at 257 Da/charge (structure2: loss of TBDMS+(CH2CH20)-H from the protonated molecule) also increased in relative abundance at the higher rf voltages. These effects are probably due both to changes in the trapping efficiency at differing m/z values as the rf voltage is increased and to increased dissociation of the ammonium adduct ion and of the protonated molecule at the higher rf voltages where more energetic collisions of trapped ions occur with the helium buffer gas. Poly(dimethylsi1oxane)s (PDMSs) are widely used in industrial processes as lubricants, coolants, antifoaming agents, and pharmaceutical actives. They have been used to evaluate the performanceof SFC/MS instruments7since they provide a series of chromatographic peaks separated by 74 Da. The NH3 CI spectra of the PDMS oligomers are dominated by the ammonium adduct ions, with small (generally less than 10% relative abundance) signals due to the protonated molecules. We chose a relatively low-molecular-weightPDMS (average MW 770) to further evaluate the performance of the SFC-Q/trap instrument. The structure of this “methyl-capped” PDMS is shown in structure 3.
$4 $4 CH,-(Si-O),-Si-CH, CH, CH, 3
Figure 7 shows the reconstructed-total-ion-current (RTIC) chromatogram of the NH3 CI, SFC/MS run of this PDMS sample. The peak designations in Figure 7 correspond to values of n in structure 3. A significant amount of the ion current produced for peaks of n value greater than 7 (MW 606) was beyond the mass range of the ion trap in these experiments. The RTIC therefore does not represent the distribution expected for a sample of average MW 770. The later-eluting peaks in this and other chromatograms of the PDMS sample recorded using the SFC-Q/trap instrument are doublets. This observation is not understood since previous and later runs of this sample using SFC-FID and other SFC/MS instrumentation show singlets rather than
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100
z
1
-
loo
3
1
i l
A
,238
loo
0 800
238
B 223,
11
'j c
loo
260
460
'
1200
1400
Scan Number Flgure 9. Ammonia CI, SFW/trap run of an alkylethoxy sulfate surfactant mlxtwe subbcted to derlvatkatlon with diazomethane.The SFC pressure conditions and the mass spectral parameters are listed In Figure 7. qz upon In)ectlon for the ammonlum adduct ions ranged from 0.218 to 0.0690, depending upon their masses.
549
'
loo0
I
1
512
500
m/Z
Flgure 8. (A) Ammonia CI, SFcQ/trap spectrum of the n = 5 ollgomer of the methyl-capped poly(dlmethyisiioxane)(see structure 1) from the run shown in Figure7. (B) Ammonia CI, SFW/trap spectrum of the n = 6 digomer. The mass spectral scan range and trapping parameters are listed in Figure 7.
doublets and since the mass spectra of the two peaks in each doublet appear identical. Each peak in each doublet has the width of a typical singlet in previous and later runs of this sample. Moreover, other samples run on the SFC-&/trap instrumentation do not exhibit this unusual chromatographic behavior. We speculate that the doublets are due to poor focusing of the higher-molecular-weight PDMS oligomers upon injection. Evidence does exist that different injection solvent/solute combinations may exhibit dramatically different chromatographic behavior.39 Despite the presence of the doublets, the mass spectra are instructive. Figure 8 shows the NH3 CI spectra of the n = 5 and n = 6 oligomers. Both spectra show the protonated molecule and an ammonium adduct ion, yet both spectra also exhibit more fragmentation than was observed in NH3 CI SFC/MS runs of similar PDMS samples using other SFC/ MS in~trumentation.~This is expected, given the kinetic energy necessary for optimum performance and the energetic collisions which occur upon injection. This result is to be compared with that discussed above for anthracene. Supercritical fluid chromatography is a useful tool for characterizing surfactant mixt~res,4~5~*~ and the final test of the present SFC-&/trap system was an attempt to elucidate the structure of a surfactant. The surfactant chosen was an alkylethoxysulfate (AES) with the reported structure 4, where the value of m is equal to 11,13,or 15 and the average value of n is 3. CH,
- (CH,),-
(OCH,CHJ,
0
- 0 - f - OH 0
4
Such anionic surfactants may be separated by SFC, using a relatively nonpolar mobile phase such as COz, only after ~~~~
~
(39)Chester, T. L.; Innis, D. P. Unpublished results, Cincinnati, OH, 1991.
(40)Kalinoski, H. T.; Jensen, A. JAOCS, J . Am. Oil Chem. SOC.1989, 66,1171-1175.
495
I
0 200
300
400
500
600
m/Z
Flguro 10. Ammonia CI, SFC-Q/trap spectrum of the n = 7, hydroxyltennlnated ethoxylated alcohol produced during the attempted derivatizatlon of the AES surfactant(see structure 4). The mass spectral parametersare listedin Figure 7. qzduring injection for the ammonium adduct ion was 0.0867.
their anionic head group is derivatized. An attempt was made to derivatizethis AES sample with diazomethane,as described in the Experimental Section, to form the methyl ester. Figure 9 shows the SFC-&/trap reconstructed-total-ioncurrent chromatogram of the derivatized AES. As expected, the chromatogram resembles that of an oligomeric series. However, the correspondingmass spectral data indicate that the sulfate group was cleaved from the ethoxylated alcohol during the derivatization procedure, producing the ethoxylated alcohol series. Figure 10 shows the NH3 CI spectrum of the m = 11,n = 7 oligomer. As discussed earlier, the mass spectral peaks are broadened due to space-chargebroadening in the ion trap, especially for larger sample sizes (the largest peak in the chromatogram, with n = 6, represents approximately 300 ng injected on column). The spectrum shown in Figure 10is dominated by the ammonium adduct of the ethoxylated alcohol at 512 Da/charge. There is also evidence in the SFC/MS data for the presence of a group of compounds in which the ethoxylated alcohol is terminated by a methoxyl group rather than a hydroxyl group. Figure 11 shows the spectrum of the methoxyl-terminated oligomer with values of n and m corresponding to 7 and 11, respectively. The molecular weight of this species is 508, and the spectrum in Figure 11 is dominated by the ammonium adduct ion at 526 Da/charge. Other ions in the spectrum shown in Figure 11 correspond in mass to the protonated molecule at 509 Da/charge, to the fragment ion at 493 Da/ charge formed by the loss of methane from the protonated molecule, and to the fragment ion at 477 Da/charge resulting from loss of methanol from the protonated molecule. Con-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992 526
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However, a smaller amount of the 14-carbon alkyl moiety (m = 13) is also present in the sample. On the 100% methyl stationary phase, the species with n and m values equal to x and 13,respectively,elutesjust before the oligomer withvalues of n and m equal to x 1and 11. Evidence for the presence of the C14-component is shown in Figure 12. The mass chromatograms for the ions at 496 and 512 Da/charge, the ammonium adduct ions for the n = 6, m = 13, and the n = 7, m = 11 hydroxyl-terminated oligomers, respectively, are shown in Figure 12. The n = 6, m = 13 species elutes before the n = 7, m = 11 species. The SFC-Q/trap data were thus valuable in the resolution of a practical problem, the derivatization of the AES surfactant. The data revealed that treatment of the AES surfactant with diazomethane cleaved the terminal sulfate, yielding ethoxylated alcohols. The results described in this work demonstrate that the combination of SFC and ion trap mass spectrometry using an external ion source has the potential to be a powerful analytical tool. Low-molecular-weightstandards were used to study the influence of various instrumental parameters on performance. Once these parameters were optimized, it was possible to use the SFC-Q/trap to study mixtures containing higher-molecular-weight species, namely a poly(dimethylsiloxane) and a derivatized alkylethoxy sulfate. The SFC-Q/ trap data were sufficient to establish the structures of the species present in the reaction mixture resulting from the attempted derivatization of the AES. Despite these successes,significantprogress must be made before the SFC/ion trap combination will reach its full potential. The transmission of ions from the external ion source to the ion trap and the trapping of ions in the trap are areas which need further optimization. Greater pumping capacity, as well as better isolation between the ion source and analyzer portions of the vacuum chamber should also improve the performance of the SFC-Q/trap instrument. Interfacing SFC with an ion trap mass spectrometer capable of MS/MS experiments and resonant ion ejection will provide an even more powerful combination. The high mass range, increased mass resolving power, and MS" capabilities achievable with the use of auxiliary fields in the ion trap mass spectrometer will help fulfill the promise of the SFC-Q/trap as a high-performance analytical tool.
+
2
0 200
300
400
600
500
nvz Flgunll. AmmoniaCI,SFCQ/traps~ofthen=7,methoxyltminated ethoxylated alcohol present Inthe reactkinmixture following the attempted derivatlzatkm of the AES surfactant (seestructure 4). The mass spectral parameters are listed in Figure 7. 9zupon injectbn for the ammonium adduct ion was 0.0844.
@-
$ # a -,,,, 1200
,',"'' 1210
1220
1230
1240
1250
Scan Number Flguro 12. Mass chromatograms from the run shown in Figure 9 for the ions at 496 Dalcharge (a:0.0895), 512 Da/charge (qz: 0.0867), and 526 Da/charge (a:0.0844), the ammonium adduct Ions for the m = 13, n = 6 (see structure 4) hydroxyl-terminatedoligomer and for the m = 11, n = 7, hydroxyl- and methoxyl-terminated oligomers, respecthrely.
f m a t i o n of this structure is provided by the chromatographic separation. Were the species of molecular weight 508 simply the hydroxyl-terminatedoligomerincorporating an additional methylene group in the alkyl chain (n = 7, m = 12), this specie8 of molecular weight 508 would elute after the molecular weight 494 species (n= 7, m = l l)on the 10076 methylpolysiloxane stationary phase. Figure 12 shows the mass chromatograms of the ions at 512 and 526 Ddcharge, the ammonium adduct ions of the molecular weight 494 and 508 species. The species of molecular weight 508 elutes before the molecular weight 494 species. This must be due to lower polarity of the molecular weight 508 species and provides confiiation that the molecular weight 508 species carries a methoxyl rather than a hydroxyl group. Previous SFC-FID and SFC/MS results41using other derivatization methods have shown that the most abundant alkyl chain length of the AES (m 1 in structure 4) is 12.
+
ACKNOWLEDGMENT We thank Donald J. Bowling and Thomas L. Chester for useful discussions and for help in reviewing the manuscript. Donald J. Bowling and Rosemary T. Hentschel are acknowledged for their help in preparing these experiments. The work at Purdue was supported by the National Science Foundation (Grant CHE 8721768).
RECEIVED for review December 26, 1991. Accepted April 20, 1992. (41)Pinkaton,J. D.; Bowling,D. J.;Delaney,T. E. Unpubliehedtesulta, Cincinnati, OH,1990.
