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Article by: Leonard O. Hargiss*, Mark J. Hayward*, Richard Klein**, John J. Manura*** , *Novartis Pharmaceuticals, 556 Morris Ave., Summit, NJ 07901 **Thermo Finnigan, ***Scientific Instrument Services
Open access molecular weight confirmation, by means of mass spectrometry using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), is common in many laboratories. However, some classes of compounds do not ionize readily with these techniques, and results may be misleading due to wide disparities in sensitivity.
A novel automated probe inlet device is described, which interfaces with a mass spectrometer employing electron and chemical ionization (EI and CI). This combination provides sensitivity and ease of use, even for most highly nonpolar molecules. Principles, instrument setup and applications are described.
The use of atmospheric pressure ionization (API) mass spectrometry as a routine self-service molecular weight confirmation tool by synthetic chemists has long been established. However, API techniques do not efficiently ionize all organic compounds. Both false negatives and positives may arise due to the discriminatory nature of API, leading the chemist to incorrect conclusions about the state of the reaction. False negatives, where the desired product fails to sufficiently ionize, may lead the chemist to think that the reaction yield is unacceptably low; false positives, where the target compound ionizes more efficiently than the starting material, may erroneously be interpreted as evidence that the reaction has reached completion.
Chemical ionization (CI) and other gas-phase techniques offer more energetic ionization, an advantage for less polar compounds with a lower probability of ionization in solution. However, sample introduction for CI has commonly relied upon manual direct exposure probes, which require operation by a specialist at a high cost in time to both the chemist and the analyst.
Automated direct probes have been developed [3-5], but thus far have not included the software component essential for true self-service operation.
A novel automated probe inlet device has been developed, which is controlled by open access software (Thermo Finnigan Xcalibur), and interfaces with a commercially available mass spectrometer (Thermo Finnigan Trace-MS). The system comprises a CTC LC/PAL autosampler and automated desorption chemical ionization (DCI) probe with pneumatically actuated vacuum lock.
Relative to open-access GC approaches, the automated DCI probe enables analysis of compounds in a broader volatility range and with less probability of thermal decomposition. This device enables relatively universal molecular weight confirmation for small molecules by the non-specialist.
In the Desorption Chemical Ionization inlet technique, a drop of solution containing analyte is deposited on a metal filament and evaporated. The filament is then inserted into the ion source and heated to evaporate the sample. Reagent ions present in the ion source (e.g. NH4+, C4H9+ or free electrons) impart a charge to the analyte molecules; the mass spectrometer then measures the mass-to-charge ratio (m/z) of the resulting ions.
Instrument Setup I. Independent control of two autosamplers and two inlet devices requires either two COM ports and two external 9-pin switching devices, or a total of four COM ports. We added an ISA card containing two additional COM ports (MultiTech #ISI552PC) to the TraceMS Control PC (IBM NetFinity 3000). A separate PC may be used for sample login. LC/PAL currently uses the A200S driver in Xcalibur.
Instrument Setup II. ThermoFinnigan provides separate ion sources for probe and GC inlets. A hole for the GC transfer line may be drilled in the Probe EI/CI Ion Source Block (part #C587A) in order to permit use of both GC and probe inlets without breaking vacuum. Compromise in CI performance appears to be minimal.
Instrument Setup III. It is convenient to route CI gases through a central switching valve. Reagent gas may be changed in less than 5 minutes with vacuum purge.
The customer dissolves the sample in a volatile solvent, and enters his name and sample description at the login screen. A menu of experiments may be presented (EI, positive-ion or negative-ion CI). Solvent evaporation parameters, DCI current ramp and processing parameters may be independently set for each method. Sample history is documented in an Access database.
Robustness of Autoprobe / Trace-MS EI/CI System
1. Runs ~250 samples/month, capacity much higher
2. Source cleanings about every 1000 samples
3. Burned out 1 DCI filament in five months
4. Fully automated--runs nights, weekends
Advantages of electron / chemical ionization with Autoprobe
• Fast analysis time (~ 2 min)
• More energetic ionization than API (useful for low polarity compounds)
• Compatible with aprotic solvents, chemically reactive compounds
• May use either positive- or negative-ion polarity
• Unattended by specialist
• Choice of reagent gases provides selectivity
• EI spectrum provides "fingerprint", library-searchable
• Instrument stops when error condition is encountered (filament burnout, sample not present)
• Results delivered in either Word 97 or hard-copy format
• With minor source modification, Xcalibur open access MS may be configured for either GC or Autoprobe inlet on TraceMS without breaking vacuum
• Robust operation (>250 samples / month)
Limitations of Autoprobe
• Extremely volatile compounds may be "lost" (but often suitable for GC)
• Thermally labile compounds may decompose
• CI gas not user-selectable (requires operator intervention)
• M/z limit of Trace-MS is 1000
• Full autosampler control lacking due to lack of fully functional software driver for LC/PAL
• Open-access software not tolerant of misplaced vials
The SIS Autoprobe / ThermoFinnigan Trace-MS combination has proven to be a reliable, robust and convenient tool for molecular weight confirmation by EI or CI in the open access mode of operation. The ability to control the analysis with open-access ("no-brain") software makes this combination unique and significantly more productive than previous EI/CI instruments. The instrument has been made available to a community of > 100 untrained chemists for reliable walk-up use. Current use is > 250 samples per month, and growing as chemists learn what OA-DCI/MS can do for them. A growing number of chemists are now finding that EI/CI analysis can now be performed in an open access environment as easily as the well established flow injection API-MS.
1. Pullen, F.S. et al, J Am Soc Mass Spectrom 1995, 6, 394-399
2. Czarnik, Anthony W., Anal Chem 1998, 70(11), 378A-386A
3. Heinrich, H. et al, Anal Chim Acta 1979, 112, 123-132
4. Martin, D.J. et al, Biomed Environ Mass Spectrom 1989, 18, 733-737
5. Huang, N. et al, J Am Soc Mass Spectrom 1999, 10, 1212-16
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Netfinity is a trademark of IBM Corporation.
Trace-MS, Trace-GC and XCalibur are trademarks of ThermoFinnigan Corporation.
AutoProbe is a trademark of Scientific Instrument Services.
Multitech is a trademark of Multi-tech Systems, Inc.
LC/PAL is a trademark of CTC Analytics, AG.
Whitey is a trademark of the Swagelok Company.
Sigma is a trademark of Sigma-Aldrich Corporation
Word 97 is a trademark of Microsoft Corporation
Access 97 is a trademark of Microsoft Corporation
Yttria coated filament at start
Yttria coated filament after 16,000 cycles