Anal. Chem. 1988, 60,2715-2719
Registry No. PHPAA, 156-38-7; H20, 7732-18-5; H202, 7722-84-1; peroxidase, 9003-99-0. LITERATURE CITED (1) Van Beaten, C.; Marler, J. E. Nature (London) 1988, 277, 951. (2) Zlka, R. G.;Moffett, J. W.; Cooper, W. J.; Petasne, R. 0.; Saltzman, E. S . Qmchlm. C o s m h l m . Acta 1985, 4 9 , 1173-1184. (3) Zika, R. G.; Saltzman, E. S.; Cooper, W. J . Mar. Chem. 1985, 17, 285-275. (4) Petasne, R. G.; Zika, R. 0. 1987, EOS, Abstract 41F-12, AGU/ASLO Ocean Sciences Meetlng. New Orleans, 1988. (5) Cooper. W. J.; Saltzman, E. S.; Zika, R. G. J . Geophys. Res., C : Oceans 1987, 91 2970-2980. (6) Moffett, J. W.; Zika, R. G. Environ. Sci. Technol. 1987, 2 1 , 804-810. (7) Kok, 0. L.; Holler, T. P.; Lopez, M. B.; Natchtrleb, H. A.; Yuan, M. Environ. Sci. Technol. 1978, 72, 1077-1080. (8) Van Zoonan, P.; Kamminga, D. A.; Gooljer, C.; Velthorst, N. H.; Frel, R. W. Anal. Chim. Acta 1985, 167, 249-258. (9) Beltz, N.; Jaeske, W.; Kok, G. L.; Gitlln, S.N.; L a m s , A. L.; McLaren, S.;Shakespeare, D.; Mohnen, V. A. J. Atmos. Chem. 1987, 5 , 311-322. (10) Kleber, R. J.; Helz, G. R. Anal. Chem. 1986, 56, 2312-2315. (11) Tamaoku, K.; Murao, Y.; Akiura, K.; Ohkura, Y. Anal. Chim. Acta 1982, 136, 121-127, (12) Johnson, K. S.; Sakamoto-Arnold, C. M.; Willason, S. W.; Beehier, C. L. Anal. Chlm. Acta 1987, 207, 83-94. (13) Zika, R. G. Ph.D. Dlssertation, 1978. Dalhousle Univerisity, 346 pp. (14) Cooper, W. J.; Zika, R. 0.; Petasne, R. 0.; Plane, J. M. C. Environ. Scl. Technoi.. in press. (15) Cooper, W. J.; Zika, R. G. Sclence (Washington, D . C . ) 1983, 220, 711-712. (18) Holm, T. R.; George, G. K.; Barcelona, M. J. Anal. Chem. 1987, 5 9 , 582-588. (17) Lazrus, A. L.; Kok, G.L.; Gltlln, S. N.; Lind, J. A,; McLaren, S. E. Anal. Chem. 1985, 5 7 , 917-922.
2715
(18) Tanner, R. L.; Markovlts, G. Y.; Ferreri, E. M.; Kelly, T. J. Anal. Chem. 1986. 58, 1857-1865. (19) Kok, G. L.; Thompson, K.; Lazrus, A. L. Anal. Chem. 1986, 58, 1192-1 194. (20) Kelly, T. J.; Daum, P. H.; Schwartz, S. E. J. Geophys. Res., D : Atmos. 1985. 9 0 , 7861-7871. (21) Lee, Y.-N.; Shen, J.; Klotz, P. J.; Schwartz. S.E.; Newman, L. J. Geophys. Res. D : Atmos. 1986, 97, 13284-13274. (22) Zepp, R. G.; Skuiatov, Y. 1.; Rltmiller, L. F. Environ. Technoi. Left. 1988, 9 , 287-298. (23) Gullbault, 0.0.;Brignac. P. J.. Jr.; Juneau, M. Anal. Chem. 1968. 40. 1256-1283. (24) Gullbault, G. G.; Brignac, P., Jr.; Zimmer, M. Anal. Chem. 1988, 4 0 , 190-196 .- - .- - . (25) Bielski, B. J.; Gebicki, J. M. Biochim. Biophys. Acta 1974. 364, 233-235. (26) Dunford, H. B.; Stillman, J. S. Coord. Chem. Rev. 1978, 79, 187-251. (27) West, E. S.; Todd, W. R.; Mason, H. S.;Van Bruggen, J. T. Textbook of Biochemistry, Macmillan: London, 1966. (28) Job, D.; Dunford, H. B. Eur. J. Biochem. 1978, 66, 607-614. (29) Hwang, H.; Dasgupta, P. K. Anal. Chem. 1986, 5 8 , 1521-1524. (30) Dasgupta, P. K.; Hwang, H. Anal. Chem. 1985, 5 7 , 1009-1012. (31) Heikes, B. G.; Kok, 0. L.; Walega, J. G.; Lazrus, A. L. J. Geophys. Res., D : Atmos. 1987, 92, 915-931. (32) Hwang, H.; Dasgupta, P. K. Environ. Sci. Technoi. 1985, 19, 255-258. (33) Banoub, M. W. Arch. Hydfobiol. 1973, 77, 159-165.
RECEIVED for review April 12,1988. Resubmitted September 6,1988. Accepted September 15, 1988. This work was supported by Sea Grant College Program Contract Number NA85AA-D-SG094.
Direct Heated Interface Probe for Capillary Supercritical Fluid Chromatography/Double Focusing Mass Spectrometry Eric C. Huang, Bruce J. Jackson, Karin E. Markides, and Milton L. Lee* Department of Chemistry, Brigham Young University, Provo, Utah 84602
A caplllary supercrltlcal fluld chromatograph was coupled to a double focuslng mass spectrometer wlth a direct heated probe Interface. The Interface contalned a fused silica transfer capillary contalnlng a frit restrlctor at the end whlch was heated to a temperature of 300-350 O C by reslstance heating. The total column effluent was transferred Into the mass spectrometer Ion source. This Interface not only preserved the chromatographlc efflclency but also provlded for ma88 spectral detection and ldentlflcatlon of various hlgh molecular welght, thermally labile, and polar compounds. Electron-Impact and chemlcal lonlzatlon mass spectra of 88lected natural products and pharmaceutlcals were obtalned at the low nanogram level.
Applications of supercritical fluid chromatography (SFC) for the separation of compounds that are nonvolatile or thermally labile have been rapidly increasing. Supercritical C02is the most frequently used SFC mobile phase, primarily because of its inert chemical properties, its ready availability in high purity, and its compatibility with the flame ionization detector. However, to fully take advantage of SFC, increased mobile phase polarity is essential. This can be achieved by doping C02with polar modifiers (I,2) or by simply using more 0003-2700/88/0380-2715$01.50/0
polar substances such as supercritical ammonia (3,4). However, problems with detector compatibility often arise when these mobile phases are used. Although various selective detectors such as UV absorbance (5),fluorescence (6),IR (7-9), and ion mobility spectrometry (10) have been demonstrated to be useful with SFC, the mass spectrometer is ideal, not only because of its mobile phase independence at low flow rates but also because of its inherent sensitivity and capability of providing structural information. In spite of the fact that SFC was first introduced in the 1960s, only recently has supercritical fluid chromatography/mass spectrometry (SFC/MS) been studied intensively. Capillary SFC/MS was f i t demonstrated by Smith et al. (11). They reported a direct fluid injection (DFI) probe, which utilized a laser-drilled orifice or a crimped metal capillary tube for flow restriction. On the basis of this study, several modified versions (12-15) have been designed and used for improvement of SFC/MS performance. Recently, high flow rate SFC/MS interface designs (16-18) and SFC/Fourier transform MS (19,20) have been successfully demonstrated. In addition to these efforts, a vacuum nebulizing interface originally developed for the combination of liquid chromatography (LC) with MS (21),a direct liquid introduction (DLI) LC/MS interface (22),and a moving belt interface (23)were modified to couple packed column SFC with MS. Each of 0 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
I-mrn o o stainierr s t e e l tube
Tobacco Co. (Winston-Salem, NC), valinomycin was purchased from Sigma Chemical Co. (St. Louis, MO), and benzylpenicillin 1'-[ (ethoxycarbonyl)oxy]ethylester and raclopride were obtained from Astra Llikemedel AB (Ssdertiilje, Sweden). Lindane (i.e., BHC), DDT, and chlordane were obtained from Pesticides Research Laboratory (Perrine, FL). Carbofuran was obtained from FMC (Middleport, NY). These samples were dissolved in methylene chloride, ethanol, or benzene and injected into the SFC at a split ratio of 201. Experimental conditions, both for the SFC and the MS, differed according to the requirements of the different samples. These are cited where appropriate in the text.
RESULTS AND DISCUSSION Copper g a s k e t ~ i g Uh D C U U ~ .hlgh u o l l a g e
i i e r i r o n ~connection
Figure 1. Schematic dlagram (not to scale) of the direct heated probe
SFClMS interface.
these interfaces represent quite different approaches. The majority of studies to date on capillary SFC/MS have involved quadrupole systems, mainly because of simplicity, low cost, and capacity for rapid scanning. However, a sector MS can provide high resolving power and expanded mass range, which are areas in which quadrupole systems are more limited. We recently reported the direct coupling of capillary SFC to double focusing mass spectrometry (24),in which the direct insertion probe, originally designed for the direct analysis of solid samples, was used to assist vaporization of the SFC effluent from the frit restrictor without modification of the MS source chamber or the pumping system. This paper describes the construction and operation of a direct heated probe for use with a frit restrictor for sample transfer and depressurization. Both electron impact (EI) and chemical ionization (CI) results are reported.
EXPERIMENTAL SECTION Details of the capillary SFC and MS equipment used in this study have been described previously (24). A Finnigan-MAT Model 8430 double focusing mass spectrometer (San Jose, CA) equipped with a dual EI/CI ion source was coupled to a Lee Scientific Model 501 supercritical fluid chromatograph (Salt Lake City, UT). SFC grade C02 (Scott Specialty Gases, Plumsteadville, PA) was delivered by the SFC pump. A schematic diagram of the new direct heated probe developed in this study is shown in Figure 1. The stainless steel probe body was designed and built in-house. The probe body consists of an access assembly welded to a 9 in. long stainless steel tube. The access assembly was fabricated from 23/4 in. 0.d. round stainless steel bar stock. This was hollowed out and machined to provide a knife-edge seal with a 23/4 in. 0.d. high-vacuum, high-voltage feed-through (Insulator Seal, Inc., Hayward, CA). Opposite from the feed-through, the access assembly was machined to accept a in. 0.d. stainless steel tube and a in. NPT fitting. At the l f g in. NPT fitting port, a Swagelock fitting was attached to provide Swagelock connection to the Finnigan-MAT flexible transfer line. A stock 7 / l s in. 0.d. x 9 in. long stainless steel tube was machined to an outer diameter of 11mm, and its surface was polished to provide a vacuum seal using a stock Finnigan-MAT gasket assembly. This tubing was then welded to the access assembly at the 7/le-in. port. The electrical wires for supplying current for heating at the tip were placed directly inside the stainless steel body and run through the high-vacuum, high-voltage feed-through. These wires were connected to the existing direct insertion probe interface heater outlet for accurate temperature control. The tip was made from brass and was wrapped with heating wire. A 2 mm i.d. hole was drilled through the center of the tip body in order to feed the frit restrictor. A well-deactivated 6 m X 50 pm i.d. fused silica capillary column (Polymicro Technologies, Phoenix, AZ) coated with a cross-linked 0.25-fim film of 25% biphenyl polysiloxane stationary phase was used throughout this study. Standard compounds were obtained from various sources: solanesol was obtained from R J Reynolds
It is well-known that the cooling effect (11,24),which results from the expansion of the supercritical fluid from the outlet of the SFC into the MS ion source chamber, is one of the most important factors affecting SFC/MS performance. Heat is required at the restrictor to prevent precipitation and to assist in the vaporization of the solute. Moderate heating of the fluid just prior to expansion and heating of the restrictor itself has been shown to be important in improving the detection limits for nonvolatile compounds (17). The direct heated probe interface developed in this study produced a heated region of 1 cm in length a t the probe tip, which is about the same length as the frit restrictor that is positioned in the probe tip. The preservation of the environment in which the ionization of gas-phase molecules takes place is the major requirement for good MS performance. The interface designed for coupling the chromatography to the MS should not only maintain the integrity of the chromatographic separation but also meet the unique requirements of the MS ion source and the proper transfer of the solute from the chromatograph to the MS. The influence of temperature in the fluid expansion area on ion source pressure has been discussed previously (24). By use of the direct heated probe interface with a tip temperature of 350 "C, a vacuum of 10-6-10-6 Torr was achieved during a typical density programmed SFC run with a 50 pm i.d. capillary column and a mobile phase linear velocity of approximately 1 cm/s. No additional ion source pumping was necessary. In this new interface design, a section of the column just prior to the restrictor was heated by conductance from the tip. This also assisted in the detection of solutes as has been demonstrated with a flame ionization detector (25). Since changes in fluid density result in changes in fluid solvating power, and fluid density is mainly controlled by pressure and temperature, the heat applied to the frit restridor should not exceed the necessary temperature required for compensating for the cooling effect. In this study, a tip temperature of 300-350 "C was found to be optimum. It is important to point out that when using supercritical C02 as mobile phase, a charge exchange ionization mechanism can produce an EI-like mass spectrum. This is important simply because most of the mobile phases used in SFC are C02-based fluids. As a result, care must be taken to distinguish the E1 mechanism from the CE mechanism. The ionization of molecules via a charge exchange mechanism requires the use of a higher ion source pressure (0.5-1 Torr) and a higher electron energy (from 200 to 500 eV) (26).On the other hand, a conventional E1 mechanism is obtained a t high vacuum ion source conditions in conjunction with the use of an electron beam at an energy of 70 eV. For this reason, the instrument for E1 operation in this study was maintained at a source manifold pressure in the lo4 Torr range and with an electron beam of 70-eV energy. Therefore, it is reasonable to expect that the spectra obtained under these conditions were produced by an E1 mechanism rather than a CE mechanism. The performance of the SFC/MS interface was evaluated by using several compounds that possessed one or more of the
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 y 3
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2717
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Figure 3. E1 spectrum of solanesol (10 ng) obtained from SFC/MS. SFC conditions: 100 "C, linear density program from 0.25 to 0.7 g/mL at 0.009 (g/mL)/min after an initial 5-min isoconfertic perlod. MS conditions: 320 "C interface probe tip temperature, 250 "C source temperature, scan from 50 to 700 amu at 0.5 sldecade.
D
1
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Flgure 2. Chemical structures of (a) solanesol, (b) benzylpenicillin l'-[(ethoxycarbonyl)xy]ethyl ester, (c)raciopride, and (d) valinomycin. properties of low volatility, moderate to high polarity, and thermal lability. These samples are found to be difficult or impossible for analysis by gas chromatography/mass spectrometry (GC/MS). None of the compounds were derivatized before analysis. Solanesol (see Figure 2 for the structure) is a polyisoprenoid alcohol. It was first isolated from tobacco by Rowland et al. (27). Polynuclear aromatic hydrocarbons (PAH) might be generated from solanesol in the pyrolyzate of tobacco (28), and it has been suggested to be a precursor to plastoquinone9 (29). Recently, solanesol has been suggested as a stable tracer in environmental tobam smoke (30). Analytical methods that have been developed for the determination of solanesol include analysis by GC of its trimethylsilyl derivative (31),reversedphase LC (32),and magic angle spinning NMR with the nuclear Overhauser enhancement (NOE) technique (33). The E1 mass spectrum of 10 ng of underivatized standard solanesol obtained by using SFC/MS is shown in Figure 3. The mqlecular ion was detected (m/z 631) as well as the typical fragment of [M - 18]+ (m/z 613),which is normally seen in mass spectra of alcohols. The fragment ions at mlz 543,475, 407,339,271,203, and 135,clearly indicate the stepwise loss of a repeating unit of 68 amu in the solanesol structure. Another important application area for SFC/MS is in the analysis of pharmaceuticals. Most pharmaceuticals have low thermal stabilities and usually contain polar functional groups. In similarity to the analysis of other biologically important compounds, GC techniques are not readily applicable for the analysis of underivatized samples (34).LC has been the major technique used in this application area for the last 10 years, mainly due to the remarkable developments in reversed-phase column technology (35)and to the developments in coupling
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Flgure 4. E1 spectrum of raclopride (10 ng) obtained from SFC/MS. SFC conditions: 80 "C, linear density program from 0.2 to 0.7 g/mL at 0.015 (g/mL)/min after an initial 10-mln isoconfertlc period. MS conditions: Same as described in Figure 3, except scan from 40 to 350 amu. LC to MS (36). With all of the advantages inherent in capillary SFC, including the ease of interfacing with MS, attention is being turned to SFC for the analysis of many such compounds. Raclopride is known to have strong antidopaminergic properties. It was reported (37) that raclopride had a stereotype-hyperactivity separation more than twice that of sulphide, while being 100 times more potent in blocking the apomorphine effect in antidopamine activity tests. Therefore, it has been chosen for clinical trials for schizophrenia. Figure 4 shows the E1 spectrum (10ng) of raclopride (see Figure 2 for structure) obtained from a density programmed SFC/MS run. The molecular weight (m/z 346)as well as rich structural information was obtained from this analysis. The most intense peak is a t mlz 98, and with the appearance of the m / z 248 peak, it is suggested that cleavage of the C-C bond, which generates the ethylpyrrolidinyl ion, is dominant. Penicillin is one of the most frequently used antibacterial drugs. The pharmaceutical behavior of penicillin can be c M g e d by varying its lipophilicity (38),which can be achieved by slightly modifying its chemical structure. Investigations have been conducted (39) to determine whether penicillins with small molecular differences vary with regard to their capacity to evoke allergic reactions after application to the
2718
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 E
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Flgure 5. Total ion chromatogram (€1) of benzylpenicillin l'-[(ethoxycarbonyl)oxy]ethyi ester (10 ng) obtained from SFC/MS. SFC conditions: 80 OC, linear density program from 0.15 to 0.7 g/mL at 0.015 (g/mL)/min after an initial 5-min isoconfertic period. MS conditions: same as described in Figure 3, except scan from 30 to 500 amu.
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m/z
Figure 6. E1 spectrum of benzylpenicillin 1'- [(ethoxycarbonyl)oxy]ethyl ester. Conditions: Same as described in Figure 5.
skin. The total ion chromatogram and E1 spectrum of benzylpenicillin 1'-[(ethoxycarbonyl)oxy]ethyl ester (10 ng) from a typical SFC/MS run are given in Figures 5 and 6, respectively. The total ion chromatogram indicates the excellent sensitivity (signal to noise) obtained in the scanning mode for only 10 ng injected. In the mass spectrum, structural information as well as the molecular weight (m/z 450)are obtained. Previously, LC/MS using thermospray (40) or direct liquid introduction (41) was the only technique for definitive analysis of such compounds. However, it is difficult to obtain EI-type spectra from LC/MS. This limits the structural information available in the spectra. In contrast, E1 spectra at low nanogram levels using SFC/MS is obtainable. In comparing E1 operation with CI operation, in many m e s , higher sensitivity or more simplified spectra in which molecular weight information is provided can be obtained by carefully selecting a reagent gas for chemical ionization. The performance of the interface under CI conditions is illustrated in Figures 7 and 8. Figure 7 shows the Spectrum resulting from negative ion chemical ionization (NICI) SFC/MS of valinomycin (10 ng) using methane as the reagent gas. Valinomycin is an antibiotic ionophore. It is distinguished by the unique ability to transport potassium cations across natural and artificial membranes selectively (42). The conformation of valinomycin has been verified by several spectroscopic studies (43-45). Positive identification of this molecule can be obtained quite easily by SFC/MS. The quasi-molecular ion [M + 11- at m / z 1112 is the most abundant ion in the spectrum. Figure 8 demonstrates the separation of a mixture of pesticides and herbicides under NICI conditions. The amount of each individual component is at the subnanogram level. The direct heated probe SFC/MS interface adequately handles the typical flow rates generated from capillary SFC
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Flgure 8. Total ion current chromatogram of a mixture of pesticides and h e r b i i s obtained under methane NICI SFC/MS conditions. SFC conditions: C02, 120 OC, linear density program from 0.2 to 0.5 g/mL at 0.0075 (g/mL)/min; 5 m X 50 bm i.d. column coated with 25% biphenyl-substituted polydbxane statkmty phase. MS condttions: 200 OC interface probe tip temperature, 210 OC source temperature, scan from 100 to 500 amu. Peak identifications: (1) carbofuran, (2) a-BHC, (3) y-BHC, (4) 6-BHC, (5) impurity from peak no. 6, (6) chlordane, and (7) DOT.
systems, and in most cases, the chromatographic integrity is preserved. However, in the analysis of valinomycin, a relatively low ionization efficiency and poor total ion current peak shape were observed. It is believed that this resulted from insufficient heat transfer to the restrictor, which resulted in poor vaporization of this large molecule. Reduction of the tip internal diameter would provide more intimate contact, which could improve heat transfer and eliminate this problem.
ACKNOWLEDGMENT The authors thank Lon Que Adams for his help in construction of the interface probe.
LITERATURE CITED (1) Levy, J. M.; Ritchey, W. M. HRC CC, J . H@'h Resolut. Chometogr. Chromatogr. Commun. 1987, 10, 493-496. (2) Fields. S. M.; Markides, K. E.;Lee, M. L. J . Chromatogr. 1987, 406, 223-235. (3) Grolimund, K.: Jackson, W. P.; Joppich, M.; Nussbaum, W.; Anton, K.; Wldmer, H. M. Proc. Int. Symp. Capillary Chromatogr.. 7th 1988, 625-636.
Anal. Chem. 1988, 60, 2719-2722 Kuel, J. C.; Markbs, K. E.; Lee, M. L. H1C CC,J . Hygh Resolut. chrometw. chrometw. Camwr. 1987, 70, 257-262. Fields. S. M.; Markides, K. E.; Lee, M. L. Anal. Chem. 1988, 60, 802-808. FJek)sted, J. C.; Richter. 8. E.; Jackson, W. P.; Lee, M. L. J . Chromatw. 1983, 279, 423-430. Jlnno, K. chrometopaphk 1987. 2 3 , 55-82. CLIfflths. P. R.; P e n t m y , S. L., Jr.; Gbrgenl. A.; Shafer, K. H. Ana/. Chem. 1986, 5 8 , 1349A-1388A. W s i k , S. V.; French, S. B.; Novotny, M. Anal. Chem. 1988, 5 8 , 2258-2258. Monissey, M. A.; HIII, H. H., Jr. Northwest Regional ACS Conference, June 1985. Smith, R. D.; Fellx, W. D.; Fjeldsted, J. C.; Lee. M. L. Anal. Chem. 1882, 5 4 , 1883-1885. Smith, R. D.; Kallnoskl, H. 1.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1884, 5 6 , 2476-2480. Wright, B. W.; Kallnoskl, H. 1.; Udseth, H. R.; Smkh, R. D. !+?C CC.J . High Resolut Chromatogr Chromatogr. Commun 1986, 9 , 145- 153. Kallnoskl, H. T.; Wseth, H. R.; Wrlght, B. W.; Smith, R. D. A M I . Chem. 1986, 58, 2421-2425. Lee, E. D,; Henion, J. D. M C CC, J . Hlgh Resolut. Chromatogr. chromatogr. Commun. 1988, 9 , 172-174. Cousin, J.; Arplno, P. J. J . chrometoy. 1987. 398. 115-141. Kallnoskl, H. 1.; Wseth, H. R.; Chess, E. K.; Smith, R. D. J . chromet q . 1987, 394, 3-14. Smlth, R. D.; Wseth, H. R. Anal. Chem. 1987. 5 9 , 13-22. Lee, E. D.; Henlon, J. D. Anal. chem.1987, 5 9 , 1309-1312. La&. P. A., Jr.; Pentoney, S. L.; oriffiths, P. R.; Wlklns, C. L. Anal. Chem. 1987, 5 9 , 2283-2288. Matsumoto, K.; Tsuge, S.; Hkata, Y. Anal. Chem. 1988, 2 , 3-7. Crowther, J. 8.; Henion, J. D. Anal. Chem. 1985, 5 7 , 2711-2718. Berry, A. J.; Games, D. E.; Perkins. J. R. J . C k o m a t w . 1986, 363, 147-158. Huang, E. C.: Jackson, B. J.; Markides. K. E.; Lee, M. L. chrometographle 1988, 2 5 , 51. Chester, T. L. J . U w o m a t w . 1984, 299, 424-431. Elndf. N.: Munaon. B. Int. J . Smctrom. Ionphvs. 1872. 9 . 141-160.
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(29) Soli, J.; Kemmerllng, M.; Schub. 0. Arch. B h h e m . BIophys. 1980, 204. 544-550. (30) W e n , M. W., R J Reynolds Tobacco Co., Winston-Salem. NC, personal communlcatbn. 1987. (31) sheen, S. J.; b v i S , D. L.; DeJone, D. W.; Chaph, J. F. J . A@. Focd Chem. 1978, 26. 259-282. (32) Prenzel, U.; Llchtenthaler, H. K. J . chromatcgr. 1982, 242. 9-19. (33) Wooten, J. B. J . A@. FocdChem. 1985. 3 3 , 419-425. (34) Novotny, M.; Wlesler, D. Seperetlon Methods: New comprehensive Blochemlsby; Deyt, E. Ed.; Elsevler: New York. 1984; Vol. 8, p 41. (35) Kirkland, J. J.; De Stefano, J. J. J . C h o m a t w . Scl. 1970, 8 , 309-314. (38) Walner, I.W. Liq& Ctvomtogephy In phemceut!.cal Development; Aster Publ. Co.,OR 1985; p 239. (37) d e Paults. 1.; K m r , Y.; Johansson, L.; Mmsby, S.; Hall, H.; allemark. M.; Angeby-Mik, K.; Ogren, S . 4 . J . Med. Chem. 1986, 2 9 , 81-89. (38) Claman, H. N.; Miller, S. D.; Conlon, P. J.; Moorhead. J. W. A d . . Immunol. 1980, 3 0 , 121-157. (39) Kristofferson, A.; Ahlstedt, S.; Enander, I. Int. Arch. Allergy Appl. Immund. 1982, 6 9 , 318-321. (40) Blakley, C. R.; Vestal. M. L. Anal. Chem. 1983, 5 5 , 750-754. (41) Apffel. J. A.; Brinkman. U. A. lh.; Frel, R. W.; Evers, E. A. I.M. Anal. Chem. 1983. 5 5 , 2280-2284. (42) Ovchinnkov, Yu. A.; Ivenov, V. 1. Tebehecton 1875. 37,2177-2209. (43) Tabeta. R.; Saito, H. Blcchmkby 1985. 2 4 , 7898-7702. (44) Liddle, W. K.; Wlllis, T. W.; Tu, A. 1.; Sankaram, M. B.; Devarajan, S.; Easwaran, K. R. K. C b m . phys. Wids 1985, 36, 303-308. (45) ByStrOV, V. F. J . Mol. Stnrct. 1985. 726, 529-548.
RECEIVED for review December 15,1987. Resubmitted May 17, 1988. Accepted September 6, 1988. Major financial support for this work was from the U S . Department of Energy, Contract No. DEFG02-86ER604.45. This work was also supported in part by the Gas Research Institute, Contract No. 5084-260-1129, and by Finnigan-MAT. Any opinions, fin-,
Analysis of High Molecular Weight Samples on a Double-Focusing Magnetic Sector Instrument by Supercritical Fluid Chromatography/Mass Spectrometry Vernon N. Reinhold,* Douglas M. Sheeley, Jacob Kuei, and Guor-Rong Her Department of Nutrition, Harvard School of h b l i c Health, Boston, Massachusetts 021 15
Capillary column supercritical fluid chromatography/mass spectrometry (SFCIMS) has been demonstrated for derivatized oilgosaccharldes in excess of 5 kDa. Columns were prepared with integral pressure restrlctors and directly coupied to a chemical ionization chamber without sklmmers or other Intervening devices. The Interface was maintained at the column temperature with slightly hlgher temperatures at the column tip (ca. 200 "C). The SFC moMie phase was carbon dioxide with ammonia Introduced separately In the chemical ionization chamber to generate (M NH,)' ions. Samples were dedvatited to maintain moblie phase miscC bility, and sample elutlon was provided by a programmed Increase In denslty. Under these conditions the total Ion current could be attributed primarily to molecular-weight-related Ions, and these plots showed no losses in chromatographic fidelity when compared to those for SFC with flame Ionization detection. The current interface provided a slgnal to noise ratio of 1O:l for a 3-ng sample injection.
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The introduction of fast atom bombardment mass spec0003-2700/88/036O-2719$01.50/0
trometry (FAB MS) (1) and its application to the structural characterization of biopolymers has advanced our understanding of these materials considerably. For peptides and small proteins the impact has been most significant on those samples that have undergone posttranslational modifications and are therefore not amenable to standard analytical procedures (2, 3). The application of FAB MS (4-6), and of comparable soft ionization techniques (e.g., direct chemical ionization (DCI) (7)and laser desorption (LD) (8-10)), has introduced the first hope of a sensitive method for studying carbohydrate structures. Unfortunately, for those techniques requiring a desorption matrix (FAB),a problem arises in the biased presentation of sample components, and it is not uncommon for selected samples to exhibit very poor sensitivity or not work a t all, (e.g., sample suppression). Suppression appears to be less of a problem with the use of a continuous flow FAB probe where the dynamic motion of the surface minimizes this phenomenon (11). For DCI MS, samples are always detected, but heat-initiated desorption leads to pyrrolysis of high molecular weight materials and seriously limits high-mass analysis. An alternative technique not requiring an applied matrix or exhibiting sample suppression has been 0 1988 American Chemical Society
