The analysis of amines by gas chromatography–mass spectrometry (GC–MS) using electron ionization (EI) has always been a challenge
for three primary reasons: These compounds are very polar and their chromatography is difficult; many of these compounds exist
as hydrochloride salts that produce an equilibrium between the free base (R3N) and the ionic form (R3N+H + –Cl) in polar solvents; and to avoid problems with the presence of peaks due to air (m/z 28 [N2] and m/z 32 [O2]), analyses are often started at m/z 35, which means that an important peak representing the diagnostic primary immonium ion (H2C=N+H2) at m/z 30 is not observed. In the course of studying the fragmentation patterns of various compounds with an amine group, a fourth
problem was discovered — the reaction of the analyte with common solvents. Methanol, ethanol, acetone, carbon disulfide, dichloromethane
(methylene chloride), and chloroform as purchased from various suppliers were found to pose potential problems of undergoing
reactions resulting in compounds other than the analyte being studied both in the solutions formed at room temperature and
in the injection port of the GC system. This presentation is Part I of a multipart series with the focus on aliphatic primary
amines in dichloromethane. This study also emphasizes the importance of finding a confirmation of a proposed structure after
a logical solution has been reached.
During the course of normal laboratory work involving electron ionization (EI) gas chromatography–mass spectrometry (GC–MS),
it was found that the primary straight-chain aliphatic amine stearylamine appeared to decompose/rearrange into other compounds,
or react with dichloromethane used as the diluent. A study was undertaken to see if other aliphatic amines behaved in the
same way as stearylamine. The other straight-chain aliphatic amines studied were C8, C10, C12, C14, and C16. These rearrangement/reaction phenomena were investigated to determine the identity of the reaction products and to determine
what might be a possible mechanism of the reactions by which they were formed. In most of the cases in this study, data representing
the target analyte were observed in the reconstructed total ion current (RTIC) chromatogram along with data representing the
reaction product.
The reactions of primary aliphatic amines with the dichloromethane occurred in solution before injection into the GC–MS system.
These reactions were not an artifact of the injection method. The discovery and identification of these products also led
to the realization that the mass spectra of several important compounds were missing from the NIST Mass Spectral Database.
This situation has now been rectified.
Experimental
Table I: Compounds used in study
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Table I lists all of the solvents and analytes and their purity, manufacturer, and purchased source. Although often not stated
on the container or in the catalog, it was found that the dichloromethane contained a trace amount of cyclohexene as a stabilizer.
More recent purchases did have this information printed on the bottle in some cases. All other chemicals were used as received
without any further purification.
Table II: Results of accurate mass assignments using MassWorks
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The GC–MS analyses were carried out using a model 7890A gas chromatograph fitted with a split–splitless injector (Agilent
Technologies, Santa Clara, California). The GC system was interfaced to a model 5975C Inert XL MSD EI mass spectrometer (Agilent
Technologies). An Agilent split–splitless injector insert was used. A 30 m × 250 μm column with a 0.25-μm film thickness of
XE-52 (SGE, Austin, Texas) was used for the chromatographic separations. The mobile phase was helium (99.995% purchased from
California Welding Supply Co., Stockton, California) and passed through VICI Getter gas purification system (Valco, Inc.,
Austin, Texas).
The mass spectrometer was tuned using the Agilent Standard Spectrum Tune file provided with GC, GC–MS ChemStation software
(version E.02.00.493), and perfluorotributylamine (PFTBA, also known as FC-43). The following GC–MS settings were used:
Column Oven Temperature Program:
Equilibration time : 0 min
Oven program: On 40 °C for 3 min; 15 °C/min to 270 °C, hold for 2 min
Pressure: 11.182 psi
Flow: 1 mL/min
Average velocity: 25.398 cm/s
Holdup time: 1.9686 min
Flow program : On; 1 mL/min for 0 min
Injection:
Run time: 20.333 min
Syringe size: 10 μL
Injection volume: 1μL
Injection repetitions: 1
Solvent A washes (PreInj): 3
Solvent A washes (PostInj): 3
Solvent A volume: 8 μL
Solvent B washes (PreInj): 3
Solvent B washes (PostInj): 3
Solvent B volume: 8 μL
Sample washes: 0
Sample wash volume: 8 μL
Sample pumps: 3
Dwell time (PreInj): 0 min
Dwell time (PostInj): 0 min
Solvent wash draw speed: 400 μL/min
Solvent wash dispense speed: 2500 μL/min
Sample wash draw speed: 400 μL/min
Sample wash dispense speed: 2500 μL/min
Injection dispense speed: 5000 μL/min
Viscosity delay: 1 s
Sample depth disabled
Injector:
Mode: Splitless
Heater: On; 290 °C
Pressure: On; 11.182 psi
Total flow: On; 18 mL/min
Septum purge flow: On; 2 mL/min
Gas saver: Off
Purge flow to split vent: 15 mL/min at 2 min
The following MS settings were used:
Solvent delay: 6.00 min
EMV mode: Absolute
Resulting EM voltage: 1729
Scan Parameters:
Low m/z: 20.0
High m/z: 300.0
Threshold: 150
Sample number: 3; A/D samples 8; Translates to 2.6 spec/s
MS Temperature Zones:
MS Source: 250 °C; max. 250 °C
MS Quad: 150 °C; max. 200 °C
TUNE PARAMETERS for SN: US73337238
Trace Ion Detection: OFF
EMISSION: 34.610
ENERGY: 69.922
REPELLER: 19.904
IONFOCUS: 74.973
ENTRANCE_LE: 0.000
EMVOLTS: 1776.471
Actual EMV: 1729.41
GAIN FACTOR: 1.21
AMUGAIN: 1595.000
AMUOFFSET: 127.500
FILAMENT: 1.000
DCPOLARITY: 0.000
ENTLENSOFFS: 14.306@3; 14.306@ 50; 11.294@ 69; 13.302@131; 12.549@219; 16.063@414; 16.565@502; 16.565@1049
MASSGAIN: –678.000
MASSOFFSET: –38.000
The atmospheric pressure chemical ionization (APCI) data were acquired with a JMS-T100LC AccuTOF mass spectrometer (JEOL,
Peabody, Massachusetts). Orifice 1 was set to 80 °C and 20 V with the desolvation chamber set to 280 °C. The needle voltage
was 3000 V; ring lens and orifice 2 were set to 10 V and 5 V, respectively. The solution was introduced to the APCI system
by direct infusion at a rate of 20 μL/min. The nitrogen nebulizing gas and drying gas were set to 1.5 mL/min and 1 mL/min,
respectively.
Accurate mass calculations were made with the Bioscience MassWorks (Cerno Bioscience, Danbury, Connecticut) software version
2,0,6,0. Within the data file of the stearylamine analysis, a 1-min acquisition of PFTBA was used to calibrate the file using
a five-spectra average after the partial pressure of the PFTBA equilibrated. The data file was acquired with the acquisition
molded (Acq. Mode) in the MS SIM/Scan Parameters dialog box set to Raw Scan, and the Threshold (counts) in the Threshold and
Sampling Rates tab of the Edit Scan Parameters dialog box was set to 0. All other instrument settings were the same as reported
earlier. Nine ions from the PFTBA acquisition were used for the calibration of the data file, m/z 69, 100, 114, 119, 131, 169, 218, 264, and 414. Five spectra from the chromatographic peak of the analyte were averaged to
produce the spectrum for the elemental composition calculations in the MassWorks software. The calculations were done by clicking
on the mass spectral peak and selecting the CLIPS Search function. Depending upon the mass spectral peak of interest, the
elemental limits of carbon, hydrogen, and nitrogen were set with a mass tolerance of 20 mmu. The profile mass range was set
to -1.00 for start and 1.50 for end.
Stock solutions were prepared at a concentration of ~1 μg/μL. The stock solutions analyzed were then diluted to ~20 ng/μL.
Not only were data obtained on individual analytes in solution but also on a mixture of the six n-alkylamines. The spectra of the unknown (identified) compounds submitted to NIST for inclusion in the NIST/EPA/NIH Mass Spectral
Database were from data files obtained on individual compounds. The submitted spectra were background subtracted using AMDIS
(NIST's Automated Mass spectral Deconvolution and Identification System) to eliminate mass spectral peaks due to the n-alkylamine that was eluted just before each unknown.
All solutions were spotted on thin-layer chromatography (TLC) plates, which were developed using iodine.
