2400
Anal. Chem. 19W359,2400-2404
is trapped inside NaCl crystals. However, when the analytes were adsorbed on 80% a-cyclodextrin-NaCl mixture, only 0.7-2% could be extracted. This indicated that the analytes were strongly bound to the a-cyclodextrin-NaC1 mixture by forming inclusion complexes with the a-cyclodextrin.
(12) Vo-Dinh, T. Environ. Sci. Technol. 1985, 19,997. (13) Vo-Dinh, T.; Bruewer, T. J.; Coiovos, G. C.; Wagner, T. J.; Jungers, R. H. Environ. Sci. Technol. 1984, 78, 477. (14) Schuirnan, E. M.: Parker, R. T. J . Phys. Chem. 1977, 81, 1932. (15) Von Wandruszka, R. M. A.; Hurtubise, R. J. Anal. Chem. 1977, 49, 2164. (16) Dalterio, R. A.; Hurtubise, R. J. Anal. Chem. 1984. 56, 336. (17) Niday, G. J.; Seybold, P. G. Anal. Chem. 1978, 50, 1577. (18) McAleese, D. L.; Duniap, R. B. Anal. Chem. 1984, 56,2244. (19) Bello, J. M.; Hurtubise, R. J. Anal. Lett. 1986, 79,775. (20) Bello, J. M.; Hurtubise. R. J. Appl. Spectrosc. 1986, 4 0 , 790. (21) Ford, C. D.; Hurtubise, R. J. Anal. Chem. 1979, 51, 659. (22) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 79,344. (23) Szejtii, J. Cyclodextrins and Their Inclusion Complexes ; Akadernini Kiado: Budapest, Hungary, 1982; p 109. (24) McMulian, R. K.; Saenger, W.; Fayos, J.; Mootz, D. Carbohyd. Res. 1973, 31 37. (25) Saenger, W. Isr. J . Chem. 1985, 25, 43. (26) Burreii, G. J.; Hurtubise, R. J. Anal. Chem. 1987, 59,965. (27) Lloyd, J. B. F. Analyst (London) 1975, 100,529. (28) Strarnbini, G. B.; Gabeliieri, E. Photohem. Photobiol. 1984. 39,725.
LITERATURE CITED (1) Hurtubise, R. J. Solid Surface Luminescence Analysis: Marcel Dekker: New York, 1981. (2) Vo-Dinh, T. Room Temperature Phosphorimetry for Chemical Analysis; Wiley: New York, 1984. (3) Von Wandruszka, R. M. A,; Hurtubise, R. J. Anal. Chem. 1976, 4 8 , 1784. (4) Bateh, R. P.; Winefordner, J. D. Anal. Left. 1982, 755,373. (5) Vo-Dinh, T.: Lue-Yen, E.; Winefordner, J. D. Anal. Chem. 1976, 4 8 , 1186. (6) Vo-Dinh, T.; Walden, G. L.: Winefordner. J. D. Anal. Chem. 1977, 49, 1126. (7) Vo-Dinh, T.; Garnrnage, R. B.; Martinez, P. R. Anal. Chim. Acta 1980, 718, 313. (8) Vo-Dinh, T.; Martinez, P. R. Anal. Chim. Acta 1981, 725,13. (9) Su, S. Y.; Asafu-Adjaye, E.: Ocak, S. Analyst (London) 1984, 709, 1019. (IO) Aaron, J. J.; Kaieei, E. M.; Winefordner, J. D. J . Agric. Food Chem. 1979, 27. 1233. (11) Vanelli, J. J.; Schulman, E. M. Anal. Chem. 1984, 56, 1030.
~
RECEIVED for review April 27, 1987. Accepted June 29, 1987. Financial support for this project was provided by the Department of Energy, Division of Basic Energy Sciences, Grant DE-FG02-86ER13547.
Direct Coupling of Open-Tubular Liquid Chromatography with Mass Spectrometry Jos 5. M. de Wit,' Carol E. Parker, and Kenneth B. Tomer*
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 James W. Jorgenson*
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
The design and performance of a probe that allows direct llquld introduction of the total emuent of an opewtubular iiquld chromatography (OTLC) column into a mass spectrometer are described. No modlflcation of the mass spectrometer source is required, and the probe Is introduced through the direct insertion probe inlet. Because flow rates are less than 0.1 pUmin, this system can be used under both conventlonai electron Impact and chemical Ionization mass spectrometrlc conditions. The probe has been used with glass OTLC columns (0.5-mm o.d., 16-pm 1.d.) and fused sllica columns (0.35-mm o.d., and 5- or 10-pm 1.d.). Tapering and heatlng the end of the column allow for the detection of compounds of low vdatlky while malntalning good peak shape and mlnhnizlng band broadening. A separatlon of flve pesticides is shown, for whlch detection limits In the low picogram levels In the fuikcan mode were obtalned by ushg methane negative chemical knizatlon (MNCI). The analysts of two isomerlc triazine herbicides by using OTLC with electron impact ionlration mass spectrometry is also shown.
The development of open tubular liquid chromatography (OTLC) is dependent, in part, on detector technology. According to theoretical predictions by Jorgenson and Guthrie 'Also a t D e p a r t m e n t of Chemistry, University a t Chapel Hill, Chapel Hill, N C 27514.
of N o r t h Carolina
the optimum separation efficiency of OTLC columns will be achieved for columns with an inner diameter of approximately 2 pm ( I ) . Currently OTLC columns with inner diameters of 5 pm are easily produced. For these columns, peaks will elute in volumes of less than 1nL. This places great demands on the sensitivity of prospective detectors. Detection mechanisms suitable for OTLC include laser induced fluorescence ( 2 ) , electrochemical detection ( 3 , 4 ) ,and mass spectrometry (5-8). Liquid chromatography (LC) combined with mass spectrometry (MS) is one of the most powerful combinations of analytical techniques available today (9). An ideally coupled LC/MS system would be one in which optimum LC conditions can be employed. This would include the vast array of mass spectrometric techniques [e.g., electron impact (EI), positive and negative chemical ionization (PCI, NCI), and fast atom bombardment (FAB)] and a system where all the analyte is introduced into the MS. One type of interface currently receiving attention is the direct introduction interface (10, 11). Thermospray (TSP) (I2,13)and direct liquid introduction (DLI) are commercially available interfaces of this type. A serious problem with interfacing conventional LC with MS is the large amount of vapor generated by the mobile phase. Microbore DLI (14),which involves the use of microbore columns with flow rates down to 10-50 wL/min, allows introduction of the entire effluent and provides a more efficient use of sample than effluent splitting where only 1-5% of the effluent is introduced into the ion source (9). Alborn et al. coupled a packed fused-silica LC column with flow rates be-
0003-2700/87/0359-2400$01.50/0 @ 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
2401
ELECTRICAL CONNECTlONS
FOR HEATER 4NOCOWTROL
PROBE-TIP-, COPO-ER C A R T R I D G E HEATER
COLLiiC
Figure 2.
Yent
Figure 1.
Schematic of the OTLC system.
tween 1 and 5 pL/min directly to a magnetic sector mass spectrometer and were able to obtain E1 mass spectra (15). The large amount of mobile-phase vapor present in TSP, microbore, and conventional DLI means that chemical ionization mass spectra are produced rather than electron impact spectra (7, 9). Chemical ionization usually gives molecular weight information, but little structural information. Open tubular LC, with flow rates lower than 100 nL/min, also allows direct introduction of the entire effluent into the ion source. The pressure generated by the mobile-phase vapor in OTLC is low enough that conventional E1 spectra, and thus the resulting structural information, can be obtained. In a recent paper by Niessen and Poppe (8)E1 spectra obtained by open tubular capillary introduction were reported. In this paper we report the development of an interface for combining an open tubular liquid chromatographic system and a quadrupole mass spectrometer, which offers significant improvements in performance over previous work (8). The OTLC system employs 10-pm-i.d. fused-silica columns coated with OV-17-V stationary phase, for which flow rates of less than 1 nL/s are normal. Actual separations of mixtures are demonstrated. Electron impact, chemical ionization, and negative ion chemical ionization of the effluent are routinely obtainable, depending on the operating conditions of the mass spectrometer. The E1 mass spectra reported here are comparable to those obtained by direct probe introduction. EXPERIMENTAL SECTION OTLC. The chromatographic system employed in this work has been described previously ( I ) . A brief description follows (Figure 1). The mobile phase is contained in two 75-mL stainless steel reservoirs under helium pressure, normally at approximately 400 psi. Flow rates can be varied by varying the helium pressure. As no pumps are employed, the flow rate is very stable. Two reservoirs are employed to allow easy switching between mobile phases to permit use of one-step gradient elutions. A Valco six-port injection valve (of which only four ports are used) is used to make injections. The injector valve is connected with 1/16-in. stainless steel tubing to the sample tee. The OTLC column is held in place in the sample tee with a Vespel ferrule. The other side of the sample tee is connected to a waste valve. An injection is made as follows: The injection valve is turned such that the syringe port is connected to the sample tee, and the waste valve is open. Approximately 0.2 mL is needed to fill the tubing and the sample tee with the analyte solution, the liquid displaced goes to waste. The waste valve is closed, and the injection valve is turned so that the mobile phase under pressure is connected back to the sample tee and the column. At this time the analyte solution enters the column. After the time of the injection, normally 1s, the waste valve is opened and the analyte solution is flushed out of the tubing and sample tee to waste. After a 5-10-s flush, the waste valve is closed again, and the chromatographic process begins. Two kinds of columns have been employed: 15-pm4.d. etched borosilicate glass capillaries with chemically
COUPLE-
Schematic of the OTLC/MS interface probe.
bonded octadecylsilane reversed phases (16, l a , and 10-pm-i.d. OV-17-V-coated fused-silica columns (18, 19). Both kinds of columns were approximately 1.5 m in length. For the interface development experiments columns of the same dimensions but without the stationary phase were used. MS. The mass spectrometers used included a Finnigan 3300 chemical ionization mass spectrometer, previously modified for negative ion detection (20),and a Finnigan 3300 electron impact mass spectrometer (Finnigan-MAT, Sunnyvale, CA). Both direct probe inlet systems had been previously modified for use with ‘I2-in. probes. A Finnigan/Incos 2300 data system is interfaced to both mass spectrometers. OTLC/MS Interface. The probe (Figure 2) is constructed from a 20-cm-long hollow stainless steel shaft (1.27-cm o.d., 1.00-cm i.d.), on which a copper tip is silver soldered. This copper tip, 3.70 cm long, contains a 40-W cartridge heater and a thermocouple. The temperature of this tip is monitored and controlled by an Omega 4001 temperature controller (Omega Engineering, Inc., Stamford, CT). A 29-cm-long (1.5-mm o.d., 0.75-mm i.d.) stainless steel tube is centered coaxially in the probe and protrudes 2.5 mm from the copper tip. This tube is silver soldered to the copper probe tip. Thus the probe shaft is isolated from the source vacuum and can be introduced and removed through the direct probe vacuum locks, which reduces pumpdown time. The OTLC column enters the probe through a 1/16-in.stainless steel “tee”, which is connected to the 1/16-in.stainless steel tubing in the probe. The other port on the “tee” is used as the inlet for reagent gas when chemical ionization experiments are performed. The column is held in place by a Vespel ferrule in the “tee” which also serves as a vacuum seal. A Plexiglas handle houses the connections for the thermocouple and the cartridge heater. A positioning collar allows reproducible positioning of the probe in the ion source. Reagents. The mobile phases used were methanol, acetonitrile, water (HPLC grade, Fisher), and mixtures thereof. The pesticides were obtained from the U S . Environmental Protection Agency (EPA Pesticides & Industrial Chemicals Repository, Research Triangle Park, NC). RESULTS AND DISCUSSION Early experiments were performed by using only very volatile solutes such as toluene and hexane. These compounds were easily introduced and detected. It was noted, however, that slightly less volatile solutes gave very poor response (e.g., naphthalene) or no response at all (e.g., phenol and acridine). To improve the performance of the interface, several modifications were tested, such as tapering of the column end and use of interface heating. Effect of Tapering the Column End. Our initial approach to increase sensitivity for compounds with low volatility was to heat the interface, but this proved unsuccessful. Heating led to vaporization of mobile phase without vaporization of the solute, which caused solute deposition, plugging the column end. Tijssen et al. investigated the effect of tapering the end of OTLC columns to create a liquid jet (6). The combination of an increase in linear velocity of the mobile phase at the tip and a smaller surface available for vaporization might provide an answer for problems encountered with nonvolatile compounds. Tapering of the column end was achieved by quickly drawing the glass out in a flame, followed by cutting of the tapered end so that a 1-to 2-wm opening was created. The conical tip shape and orifice were verified by microscopic inspection. After some practice this procedure could be carried out with ease, but each time approximately 4 cm of the column
2402
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987 (00.0
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Figure 3. Effect of tapering on interface performance. Probe tip temperature is 200 "C. (A) Injection of phenol dissolved in hexane using a nontapered capillary. (B) Injection of phenol dissolved in hexane using a tapered capillary.
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Figure 6. Separation of the two isomeric pesticides propazine (scan 176)and trietazine (scan 183/4)(molecular weight 229).ion current traces for molecular ions (D) and several characteristic fragment ions (A, 0 , C).
20
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Figure 4. Effect of temperature on interface performance. (A) Injection of naphthalene and acridine (5 X lo-*M) at 100 OC by using a tapered column end. (B) Repeated injection, now with the interface temperature at 200 OC. (C) Same injection again at 300 "C. The arrows i n d i t e the peak width to compare with D. (D) Total ion current trace for the injection made at 300 OC. The arrow indicates the injection time.
was sacrificed. At ambient temperature no improvements in sensitivity were observed with the tapered column end; at higher interface temperatures, however, significant improvements were noted. Figure 3 illustrates the effect of tapering. Dummy columns consisting of 16-pm-i.d. glass tubing were used throughout these experiments. The solute used was phenol dissolved in hexane ( 5 mg/mL), and acetonitrile was used as the mobile phase. The probe tip temperature was maintained at 200 "C. The molecular ion traces for hexane (molecular weight 86) and phenol (molecular weight 94) obtained by electron impact ionization are shown. In order to study the vaporization process, long injection times were used resulting in broad, square-topped peaks. The peak for hexane had the expected shape, but phenol apparently collected around the column tip and came off as a small peak, several seconds after the hexane peak. The same glass tubing was then tapered (the change in elution time resulted from the use of the shortened piece of tubing, caused by the tapering process). The tapered column gave similar results for both compounds. I t appears that the degree of tapering had little effect on the peak shape.
If, however, the taper was too long, a long narrow channel was created, usually resulting in plugging of the column. Effect of Heating the Column End. The effect of temperature could only be investigated with a tapered column. The solutes in this experiment were naphthalene (bp 218 "C) and acridine (bp 350 "C), both a t 5 X M in acetonitrile, which also served as the mobile phase. Again, long injection times were used to allow study of the vaporization. Figure 4A shows the molecular ion traces for naphthalene (molecular weight 128) and acridine (molecular weight 179) at 100 "C. At this temperature the peak for naphthalene was very broad, and no signal a t all was obtained for acridine. At 200 "C, the peak shape for naphthalene was as expected, but the response for acridine was low and the peak was broad (Figure 4B). At 300 "C the responses for both solutes were similar, and good peak shapes were observed (Figure 4C). The combination of tapering the end of the column and heating the interface gives good results for less volatile compounds. The exact process of vaporization is not fully understood. The mobile-phase flow is too low to create a liquid jet, so processes similar to "filament-on" thermospray (also referred to as "hot DLI") may be the most accurate description. Effect of the Interface on Peak Broadening. The bottom trace of Figure 4D shows the total ion current (TIC) chromatogram for the injection made at 300 "C. The injection involves two interruptions of the mobile-phase flow: the first to fill the sample "tee", and the second to flush out the sample "tee". The time between those two interruptions is the actual injection time during which the sample enters the capillary LC tubing. Since the mass spectrometer was acquiring data over a mass range from 35 to 200 amu, most of the total ion
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
2403
I2?.8,
I
A
187
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248
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MI2 Figure 7. Electron impact mass spectra of propazine and trietazine by direct probe (DP) and OTLC: (C) trietazine, D P (D) trietazine, OTLCIMS.
current was contributed by the acetonitrile (molecular weight 41). The interruptions in the solvent flow caused the total ion current to decrease and the two valleys were separated by the time of injection. The width of the eluted peaks for naphthalene and acridine, indicated by the arrows, is the same as the width of the injection indicated in the total ion current chromatogram. This indicates that no significant extracolumn peak-broadening is generated by this interface. This is an especially important point since these results were obtained for compounds of lower volatility. Applications. For the combined technique of OTLC and MS to be useful, the separations achieved by OTLC must not be degraded by the interface. In addition, E1 or CI mass spectra obtained by using the interface should be comparable to those obtained from conventional sample introduction systems. The interface described in this paper allows acquisition in both electron impact and positive and negative chemical ionization modes. For chemical ionization, reagent gas is introduced into the source through the probe. This gas flows around the column into the ion source and may facilitate the vaporization process. Figure 5 shows the total ion current chromatogram of a synthetic mixture of five pesticides (Table I, compounds 1-5), obtained under methane negative ion chemical ionization conditions, and acquired in the full scan mode over the mass range from 60 to 350 amu. The concentration of each compounds was 500 ng/hL, dissolved in the mobile phase. The
(A) propazine, DP (6) propazine, OTLCIMS;
Table I. Compounds Studied RE1ENTION TIME
MW.
1 Metrlbuzln
430
214
2 Methyl parathion
4.55
263
3 Pmrathlon, ethyl
5:20
291
4 Morphos
6:15
290
s
7:10
135
6'10
229
6%
229
COMMON NAME
T)IfIwaIIn
FORMULA
STRUCTURE
2404
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
column used was a 1-m by 10-wm-i.d. fused-silica column coated with OV-17-V stationary phase. Since the injected volume was 2 nL (a 4-s injection with a mobile-phase flow of 0.5 nL/s, the actual amount of solute per peak was 1ng. The mobile phase used was 7525 methanol/water (v/v). The temperature of the probe tip was 320 "C. Excellent negative ion chemical ionization spectra were obtained. To establish the linear dynamic range, the original mixture was diluted 10-fold (to 100 pg) and a 10-fold decrease in ion intensities was observed. The linear dynamic range of the OTLC/MS system is small: the upper limit is determined by the sample capacity of the column (approximately 50 ng) and the lower limit by the sensitivity of the mass spectrometer. The limits of detection of these compounds under full scan mode conditions were determined to be approximately 100 pg. For these amounts of solute signal-to-noise ratios of 2:l to 8:l for characteristic ions were observed. Without introduction of a chemical ionization reagent gas, electron impact spectra are obtained. A mixture of propazine and trietazine (Table I, compounds 6 and 7),two isomeric herbicides (1.6 ng of each component), was separated on a 10-km-i.d. fused-silica column coated with OV-17-V and transferred to the mass spectrometer through the interface. The mobile phase used was 70:30 methanol/water (v/v), and the flow rate was 0.4 nL/s. Reconstructed ion chromatograms for some characteristic ions are shown in Figure 6. These isomers have distinguishable E1 mass spectra; spectra obtained with solid probe introduction are shown for propazine (Figure 7A) and trietazine (Figure 7C). Similar spectra were obtained from the OTLC separation on 1.6 ng per compound (Figure 7, parts B and D). Although the lower mass range is contaminated by incompletely subtracted background from the mobile phase and ion source, the high-mass region is virtually identical with that observed with the direct insertion solid probe. The I3C and 37Clisotope clusters observed for the major high-mass ions in the OTLC mass spectrum compare favorably with those observed in the solid probe mass spectra. No evidence of chemical ionization [ (M H)+ formation) is observed in the OTLC mass spectra.
+
CONCLUSION The OTLC/MS interface presented here offers several distinct advantages over other LC/MS interfaces: the in-
terface is simple in construction and operation; the interface does not require dedication of a mass spectrometer; E1 mass spectra are routinely obtainable; and CI mass spectra can be readily obtained by use of a reagent gas as in conventional CI. Limitations in the combined technique are the mass spectral sensitivities needed for the detection of the very low absolute amounts of sample eluting and the vaporization of compounds of low volatility.
ACKNOWLEDGMENT The authors thank P. R. Dluzneski, of the University of North Carolina a t Chapel Hill, for fabricating fused-silica capillary columns and Bob Hall, of the NIEHS, for all of the machining involved in this research. LITERATURE CITED Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56, 483-486. Guthrie, E. J.: Jorgenson, J. W.; Dluzneski, P. R. J . Chromatogr. Sci. 1984, 22, 171-176. White, J. G.; St. Claire, R. L., 111; Jorgenson, J. W. Anal. Chem. 1988, 58, 293-298. Whlte, J. G.; Jorgenson, J. W. Anal. Chem. 1988, 58, 2992-2995. Ishii, D.; Takeuchi, T. J . Chromatogr. Sci. 1980, 18, 462. Tijssen. R.; Bleumer, J. P. A.; Smit. A. L. C.; van Kreveld, M. E. J . Chromatogr. 1981, 218 137-165. Niessen, W. M. A.: Poppe, H. J . Chromatogr. 1985, 323, 37-46. Niessen, W. M. A.; Poppe, H. J . Chromatogr. 1987, 385, 1-15. Covey, T. R.; Lee, E. D.; Bruins, A. P.; Henion, J. D. Anal. Chem. 1986, 5 8 , 1451A-1461A. Niessen, W. M. A. Chromatographia 1986, 2 1 , 277-286. Niessen, W. M. A. Chromatographia 1986, 21, 342-354. Blakley, C. R.; Carmody, J. J.; Vestal, M. L. J . Am. Chem. SOC. 1980, 102, 5931-5933. Blakely, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750-754. Lee, E. D.; Henlon, J. D. J . Chromatogr. Sci. 1985, 23, 253-264. Alborn, H.; Stenhagen, G. J . Chromatogr. 1985, 323, 47-66. St. Claire, R. L., 111; Jorgenson, J. W., submitted for publication in J . Chromatogr . St. Claire, R. L., 111 Ph.D. Thesis, University of North Carolina at Chapel Hill, 1986. Dluzneski, P. R.; Jorgenson, J. W. submitted for publication in HRC CC , J . High Resolut . Chromatogr . Chromatogr Commun , Dluzneski, P. R. Ph.D. Thesis, University of North Carolina at Chapel Hill, 1987. Friesen, M. Ph.D. Thesis, Kansas State University, 1977.
.
RECEIVED for review February 24, 1987. Accepted June 29, 1987.
