We report on the development of a new laser-ionization (LI) source operating at atmospheric pressure (AP) for liquid chromatography/mass spectrometry (LC/MS) applications. APLI is introduced as a powerful addition to existing AP ionization techniques, in particular atmospheric-pressure chemical ionization (APCI), electrospray ionization (ESI), and atmospheric pressure photoionization (APPI). Replacing the one-step VUV approach in APPI with step-wise two-photon ionization strongly enhances the selectivity of the ionization process. Furthermore, the photon flux during an ionization event is drastically increased over that of APPI, leading to very low detection limits. In addition, the APLI mechanism generally operates primarily directly on the analyte. This allows for very efficient ionization even of non-polar compounds such as polycyclic aromatic hydrocarbons (PAHs). The APLI source was characterized with a MicroMass Q-Tof Ultima II analyzer. Both the effluent of an HPLC column containing a number of PAHs (benzo[a]pyrene, fluoranthene, anthracene, fluorene) and samples from direct syringe injection were analyzed with respect to selectivity and sensitivity of the overall system. The liquid phase was vaporized by a conventional APCI inlet (AP probe) with the corona needle removed. Ionization was performed through selective resonance-enhanced multi-photon ionization schemes using a high-repetition-rate fixed-frequency excimer laser operating at 248 nm. Detection limits well within the low-fmol regime are readily obtained for various aromatic hydrocarbons that exhibit long-lived electronic states at the energy level of the first photon. Only molecular ions are generated at the low laser fluxes employed (∼1 MW/cm2). The design and performance of the laser-ionization source are presented along with results of the analysis of aromatic hydrocarbons. Copyright © 2005 John Wiley & Sons, Ltd.
An alternative approach to the ionization of non-polar analytes at atmospheric pressure was introduced recently by Syage et al.27, 28 as atmospheric pressure photoionization (APPI) and by Robb et al.29, 30 as dopant-assisted (DA) APPI. Both techniques are based on single-photon ionization (PI), which has long been known from applications in ion-mobility mass spectrometry31–34 and in photoionization detectors (PID).35–37 The ionization process in APPI is initiated by VUV radiation, which ionizes species with ionization potentials (IP) lower than the photon energy. Frequently, the VUV radiation used is obtained from rare-gas discharge lamps. The most widely applied photon source is the krypton lamp emitting at λ = 123.9 nm (10 eV) and 116.5 nm (10.6 eV). Other discharge lamps are commercially available, but are generally less stable than the Kr lamp in prolonged operation.
With APPI, as is generally true for all ionization techniques operating at atmospheric pressure, thermodynamically favored ion/molecule reactions control the distribution of ions entering the collision-free region of the mass analyzer. In some cases, particularly at lower collision rates such as in low-pressure photoionization (LPPI),38 kinetic control of the ion distribution is also observed.39 In positive ion mode, the ionization efficiency for a specific analyte is thus primarily dependent on the ionization potential (IP) and the proton affinity (PA) of all the compounds present in the high-pressure region. The initial step in the photoionization of a molecule M is photon absorption and subsequent electron ejection to form the molecular radical cation M.+. Provided that the IPs of all the other species present in the ionization region, in particular of the matrix molecules, are higher than the photon energy, then this step should be specific towards the analyte. However, in the presence of protic solvent molecules and/or other analytes present in large excess, [M]+. can undergo further transformations. This is of particular interest in dopant-assisted APPI (see below). Other ionization mechanisms for APPI have been invoked. As an example, APPI (10 eV photons) with acetonitrile (CH3CN) as solvent can lead to predominant formation of [M+H]+ ions, although the IP of CH3CN is more than 2.2 eV higher than the available photon energy.40 This result is rationalized by the following mechanism: First, matrix molecules present in large excess absorb a significant fraction of 10 eV photons to form electronically highly excited neutral species, which rapidly isomerize to products with IPs lower than the photon energy. Ionization of the isomers/clusters and subsequent reaction with further matrix molecules or cluster formation leads ultimately to relatively long-lived species that can transfer protons to the analytes present. Therefore, more polar drug compounds in CH3CN/H2O mixtures are usually observed as [M+H]+ in APPI, whereas non-polar compounds such as naphthalene usually form [M]+..41 A detailed mechanistic analysis of [M+H]+ formation is given by Syage.39
The total ion yield in APPI is rather low as a result of both the limited VUV photon flux and the above-mentioned series of reactions, which potentially occur in parallel. Consequently, Bruins and co-workers introduced dopant-assisted atmospheric-pressure photoionization (DA-APPI)29 as a new ionization method, in which the total number of ions produced by the discharge lamp is greatly increased by the addition of a large quantity of a directly ionizable compound (dopant) to the liquid stream. If the dopant is selected so that its photoions have a relatively high recombination energy, or a low proton affinity, then dopant ions may react with other compounds present in the ionization region by charge exchange or proton transfer. Besides acetone and toluene, anisole was recently introduced as an effective dopant for APPI.42 The dopant-induced subsequent ion/molecule reactions can improve the sensitivity, but may render the analysis of APPI mass spectra more difficult, e.g., because of adduct formation.29, 41, 42 Moriwaki et al.43 have demonstrated the ultra-sensitive detection of various aromatic hydrocarbons. The reported detection limits when using DA-APPI and single ion monitoring are well within the low-nmol/L range. The various AP ionization methods were described recently in reviews by Hayen and Karst44 and by Van Berkel.45
In this paper we present a novel ionization method, atmospheric-pressure laser ionization (APLI). APLI is based on resonant or near-resonant two-photon ionization of aromatic ring systems. To our knowledge this is the first report describing the analytical application of resonantly enhanced multi-photon ionization (REMPI) in conjunction with commercially available ion sources operating at atmospheric pressure. We note that the acronym APLI has been used in the literature before.46–48 In view of the presently described newly evolving laser ionization technique operating at ion source pressures around p = 1 atm, the acronym APLI becomes ambiguous. In order to describe the former technique more accurately, the term medium-pressure laser ionization (MPLI) has been introduced49 with the intention of better distinguishing the three pressure regimes in which currently applied laser ionization techniques operate. The commonly used inlet systems for laser ionization mass spectrometry are shown in Fig. 1. Figure 1(a) shows the traditional supersonic jet expansion method using a skimmed and collimated molecular beam. Ionization occurs far downstream of the high-pressure nozzle, usually at a distance of 5–20 cm, well behind the sudden-freeze surface50 of the gas jet. The ro-vibrational temperatures of the analytes present in the ionization volume are usually well below 10 K allowing for high-resolution spectroscopic investigations. However, since the molecular number density decreases roughly with 1/r2 from the nozzle,50 and the ionization volume is rather small, the method is not well suited for analytical applications. The jet-REMPI approach (Fig. 1(b)), as introduced by Oser et al.,51 with orthogonal beam geometry operates also in a low-pressure background but without using skimmers. The ionization region is located just downstream of the sudden-freeze surface of the gas jet. Ro-vibrational cooling is thus still efficient, but the ionization volume is greatly increased, leading to favorable detection limits. A comparable linear setup was introduced by Onoda et al.,52 as shown in Fig. 1(c). The ionization volume is further increased by application of a line focus setup. Using MPLI (Fig. 1(d)), the ionization region is located as close as 0.5 mm from the nozzle orifice and thus within the continuous region of the jet, approaching local pressures up to 10 mbar. Ro-vibrational cooling is limited, which favors ionization with broad-bandwidth laser sources. The high analyte density allows for selective ultra-sensitive measurements (see Refs. 46–49 for details). Employing APLI (Fig. 1(e)), as presented in this contribution, ionization occurs at atmospheric pressure, upstream of the mass spectrometer sampling orifice. Ro-vibrational cooling does not occur. The generated photoions are directed towards the sampling orifice of the mass spectrometer by an external electrical field and drawn into the differential pumping stage mainly through collisions with the bath gas.
Dichloromethane, acetonitrile, methanol, benzo[a]pyrene, fluoranthene, anthracene and fluorene were obtained from Merck (Darmstadt, Germany) and were of chromatographic or analytical grade.
Halogenated aromatic oligomers and the aromatic iridium complexes were provided by the research group of Prof. U. Scherf, Center for Polymer Technology, University of Wuppertal, Germany.
All other chemicals were from Merck (Darmstadt, Germany) and of the highest purity available and were used without further treatment.
All experiments were performed with a MicroMass Q-Tof Ultima II orthogonal time-of-flight mass spectrometer (Eschborn, Germany) equipped with an APLI interface made by the machine shop of the Chemistry Department at the University of Wuppertal, Germany. The interface was based on the design of the original MicroMass Z-Spray™ interface. The heated AP probe was used without modification at T = 250–350°C. For APLI operation, the corona needle was removed, and an additional repeller electrode was installed. Typical voltages in the range +150 to +200 V were applied to the repeller when the instrument was operating in the positive ion mode. The ion-block temperature was generally maintained at T = 250°C. High-purity nitrogen was used as nebulizer, cone and desolvation gas with flow rates of approximately 0.3, 0.16, and 2.5 L min−1, respectively. The REMPI light source employed was a compact Lamdba Physik (Göttingen, Germany) OPTex excimer laser running at 248 nm (KrF*). Typical operating conditions were 100 Hz repetition rate, 15 mJ pulse energy, and 8 ns pulse duration. The laser beam was delivered unfocused to the APLI interface through fused-silica windows. No attempts were made to synchronize the orthogonal TOF pusher sequence with the laser pulse train (see discussion). The samples were injected into the probe with either a syringe pump (Harvard Apparatur, Hugstetten, Germany) at a flow rate of 500 μL/min or with a 30-μL loop in a Waters (Eschborn, Germany) 2690 HPLC system at a flow rate of 800 μL min−1. Figure 2 shows a schematic diagram of the setup.
RESULTS AND DISCUSSION
REMPI as analytical method
APLI utilizes resonantly enhanced multi-photon ionization (REMPI) as the primary ion production mechanism. The reactions listed below summarize well-described excitation pathways53–57 along with possible interfering processes, such as deactivation upon rapid intersystem crossing (ISC) and photodissociation at intermediate energy levels: Stepwise excitation
Precursor-ion fragmentation via ladder-switch mechanism
Photodissociation at intermediate levels
Deactivation via ISC
Reactions 1(a) and 1(b) represent a classical (m + n) REMPI process; for analytical applications in most cases m = 1, 2 and n = 1, 2. Although higher-order processes have been reported (e.g., (3 + 1) REMPI of CO58), they are very unlikely to occur with overall efficiencies sufficient for analytical applications. Even if m = 2, i.e., two photons are to be absorbed by the target molecule within a time frame of fs (or quasi simultaneously) for a resonant population of the intermediate level M*, the power density within the interaction volume has to be in the order of several GW/cm2. Under such conditions the ionization step 1(b) usually follows immediately, partly because the Franck-Condon (FC) overlap between a highly excited state (e.g., a Rydberg state) and low ionic states is often favorable.59 Although such power densities are easily achievable by tight focusing of a conventional ns laser beam with good beam quality, two effects have to be considered: First, the ionization volume (i.e., the region close to the laser beam focus) is significantly smaller compared to other ionization methods, severely limiting the detection limit. Second, when ns pulses are applied, further absorption of photons by the precursor ion is unavoidable, leading to fragmentation of the precursor ion via ladder-switch mechanisms53 (cf. reactions 2(a) and 2(b)).
Whereas rapid photodissociation (reaction 3) generally leads to unfavorable ion yields, deactivation of resonantly excited states through intersystem crossing processes (reaction 4) appears to be less critical for larger molecules. Upon the application of a second light source with higher energy photons, these states can be pumped above the ionization threshold provided that the FC factors are still favorable after ISC. One fundamental difference between APLI and APPI is the role of the mobile phase molecules (MP) of the HPLC stage or the solvent molecules (S) when using direct syringe injection. Since MP and S, e.g., H2O, CH3CN, CH3OH, are essentially transparent within the UV wavelength range used in APLI, significant absorption of the incident photons and thus any energy transfer steps from MP to the analyte M, isomerization of MP to yield reactive or ionizable species, as well as other processes involving highly excited MP or S molecules, as observed in APPI, can be excluded. It follows that the photon flux provided by the laser within the ionization volume is generally independent of the composition of the liquid phase entering the heated AP probe and fully available for analyte ionization.
In summary, for analytical applications of REMPI, overall resonant, low-order processes are the most favorable, in particular one-color (1 + 1) REMPI. Multi-color excitation significantly broadens the applicability of REMPI, but is not discussed here. In addition, ultra-fast excitation avoids a number of the interferences listed above, in particular photodissociation at intermediate levels,60–62 but the instrumentation currently available in the required energy range is not yet as user friendly as compact stand-alone ns laser sources.
REMPI of aromatic hydrocarbons
It is well known that numerous aromatic hydrocarbons are very efficiently ionized via (1 + 1) REMPI (see, e.g., Refs. 63 and 64, and references cited therein). The main reasons are high molecular absorption coefficients in the near UV, relatively large lifetimes of the intermediate S1 or S2 states, and favorable location of these states: Since the IP is often lower than the energy of the sum of two photons, ionization occurs readily from the resonantly pumped intermediate states. It follows that rather unfocused laser beams can be used, making this compound class ideally suited for REMPI analysis. A substantial body of reports on the analytical application of REMPI to the measurement of aromatics exists in the literature (see Ref. 64 for a recent review). The majority of these papers are concerned with the detection of toxic aromatic substances in exhaust gases such as from municipal waste incinerators,65–67 but also analysis of process gases,68–70 as well as vehicle exhaust measurements,71–73 besides many others, have been reported.
Generally REMPI is regarded as a highly selective ionization method: The target molecules are introduced into the ion source by means of a supersonic expansion stage, which drastically narrows the rotational population density in the ground state (cf. Fig. 1(a)). Tuneable narrow-bandwidth laser sources are required for efficient resonant excitation. At atmospheric pressure, however, the absorption features of aromatic hydrocarbons become broad and unresolved, and fixed-frequency lasers can be employed. Nevertheless, the ionization process remains selective towards the class of aromatic hydrocarbons with long-lived intermediate states: There are only a very limited number of non-aromatic analytes with molecular properties comparable to those described above. More importantly, the bulk gases N2 and O2, as well as virtually all traditional HPLC solvents (e.g., H2O, CH3OH, and CH3CN), exhibit only very small absorption cross sections in the near-UV region used for excitation. Furthermore, at least three photons would be required for their ionization; this is very unlikely to occur under the present experimental conditions, i.e., power densities in the order of 1 MW/cm2. As discussed above, this represents a major advantage of APLI over APPI, where the absorption cross sections of the bulk gases/solvents are comparably high in the energy range of the VUV lamp, leading to severe loss of photons available for direct ionization of analytes present.
APLI source operation
As mentioned above, for APLI operation the heated AP probe was used without modification. Since the corona needle is removed or disconnected from the high-voltage supply, an additional electrical-field gradient directing the laser-generated ions towards the sampling cone of the ion block is required. This gradient was introduced by installation of an additional repeller electrode, as shown in the inset in Fig. 2. Optimizing the repeller voltage and the distance to the sampling nozzle increased the total ion-signal intensity by a factor of 50–100 over operation of the source without this electrode present. A typical value of +180 V resulted in maximum sensitivity (the sampling cone was always held at a constant potential of +88 V). The positioning of the laser-beam axis relative to the sampling cone was less critical; however, the maximum sensitivity was achieved when the beam was positioned directly in front of the nozzle, as expected.
The TOF pusher pulse frequency, adjustable approximately from 4 to 30 kHz depending on the recorded mass range, was not synchronized with the laser-pulse train. The ion-signal intensity depended almost linearly on the laser-pulse repetition rate in the range 10–100 Hz and leveled off at higher frequencies. This observation is in accord with the assumption that below 100 Hz molecules in the ionization volume of approximately 1 cm3 are irradiated only once. This estimate is based on the assumption that the entire gas flow of approximately 3 L min−1 from the AP probe passes through the irradiated region. At higher laser-pulse frequencies, the gas in the ionization volume is only partly replaced.
It is pointed out that the performance of the arrangement described is far from optimum; however, in this contribution, we focus on the description of the efficiency of the APLI processes rather than the optimization of the APLI source geometry.
APLI of selected aromatic hydrocarbons
In the section below, we report APLI mass spectra of several aromatic hydrocarbons, which include polycyclic and polymeric structures, heterocycles, and coordination complexes. The polarity of the analytes was generally low. For selected PAHs, APCI measurements were also performed for comparison; in all other cases the quality of APCI mass spectra was not sufficient for a meaningful interpretation of the data.
Figure 3 shows a typical mass spectrum obtained for a representative non-polar PAH, benzo[a]pyrene. Only the precursor ion is produced with very high efficiency. The sample, dissolved in CH2Cl2, was directly injected with a syringe pump into the source via the heated AP probe. Current detection limits for benzo[a]pyrene are in the fmol range; however, as already discussed, the ion source is operating far from optimal performance. Similar results were obtained for anthracene, fluorene, and fluoranthene. APLI is thus extremely sensitive towards the determination of non-polar PAHs and could prove to be a powerful alternative to APCI and APPI. Recently reported detection limits for DA-APPI of the above PAHs among others are about an order of magnitude higher.43
More complex systems, with aromatic rings in oligo– or polymeric structures, were also investigated. In particular, the influence of heteroatoms, e.g., halogens, was of interest, since such molecules are often used as building blocks in advanced materials synthesis. An APLI mass spectrum of C50H45Cl is shown in Fig. 4 with the calculated isotopic pattern of the precursor ion in the inset for comparison. As in the case of the PAHs, the precursor ion signal is dominating the spectrum. Excellent agreement is found between the calculated and experimentally recorded spectrum. Minor signals are due to the presence of compounds which are either by-products of the synthesis or impurities resulting from thermal degradation within the AP probe. The synthetic procedure suggests that products with structures as shown in Fig. 4 may still be present after purification of the raw material. Since the AP probe temperature was relatively low (250°C) along with a rather high total gas flow present, we expect thermal degradation to be of minor importance. Another example is given in Fig. 5. The building block C36H22Br4 was introduced as 1 μM solution (in CH2Cl2) via the heated AP probe. Again, virtually no fragmentation occurred. The agreement between calculated isotopic pattern and experimental results is demonstrated in the inset.
The compound shown in the left panel of Fig. 6 was the target of a recent synthesis effort at the University of Wuppertal. We have employed APLI to investigate the synthesis products. The right panel in Fig. 6 shows an APLI spectrum obtained at an intermediate stage of the work. The signals in the two individual regions of the mass spectrum are fully compatible with the proposed structures shown in both lower panels of Fig. 6, as judged by the isotopic pattern of the signals recorded. The synthetic procedure has not yet been fully achieved, but, as shown, the approach is very promising. State-of-the-art analysis of such building blocks and reaction products includes field desorption (FD) MS. However, the sample preparation time for FD-MS is significantly longer than for APLI-MS. The latter method requires only a dilution step and loading of the syringe pump.
In conclusion, APLI represents a powerful new technique for mass spectrometric investigations of polymeric and polycyclic aromatic molecules. The quality of the spectra provides a basis for a critical evaluation of the progress of the synthesis and the purity of the products. The simplicity of the setup, which is comparable to APCI, allows for swift, and sensitive analysis of polymeric compounds with respect to their elemental composition and support of proposed structures, which are otherwise difficult to analyze.
APLI vs. APCI
A comparison of the performance of APLI and APCI is presented in Figs. 7 and 8. First, a solution containing benzo[a]pyrene in CH3CN was injected directly through the heated AP probe by syringe. For APCI operation, the corona needle was installed, and the source parameters were then optimized. For APLI, the source geometry was identical, but the needle was removed and the repeller electrode (see above) installed. In Figs. 7(a) and 7(b) the mass spectra obtained with APLI and APCI, respectively, are shown. The former method generates only parent radical ions [M]+. (along with background noise), whereas the latter generates [M+H]+ ions, as expected. However, the signal intensity [M]+. vs. [M+H]+ is at least a factor of 100 higher in APLI with simultaneously greatly reduced noise. The two effects result in a limit of detection (LOD) for benzo[a]pyrene that is about three orders of magnitude lower than with APLI (see above). It is pointed out that photoions generated in only 100 laser shots were co-added within the accumulation time of 1 s, since the laser pulse frequency was set to 100 Hz. In contrast hereto, using APCI with continuous ion generation, signals from more than 10 000 spectra are co-added in the same time period, because the TOF pusher frequency in both cases was about 30 kHz. These results suggest the possible further improvement of APLI upon phase locking the laser and TOF pusher pulse frequencies.
In a second set of experiments, an HPLC stage was coupled to the AP probe. Four PAHs (benzo[a]pyrene, fluoranthene, anthracene and fluorene) were dissolved in CH3CN and then separated chromatographically (cf. Table 1 for details) for mass-selective detection. For comparison, the same mixture was analyzed using APLI and APCI-MS, respectively, under otherwise identical chromatographic conditions. For APLI, the precursor ion [M]+ was monitored, for APCI [M+H]+. The signals from the HPLC UV-diode-array detector were monitored simultaneously. The results are summarized in Figs. 8(a) (APLI) and 8(b) (APCI).Gradient program for the HPLC analysis. Column used: RP18e (LiChrosphere 100; 250 × 4 mm; 5 μm) from Merck (Darmstadt, Germany). The PAH mixture: benzo[a]pyrene, fluoranthene, anthracene, and fluorene (20 mg/L of each PAH)
|Time [min]||Eluent A methanol/water (4:1) [%]||Eluent B methanol [%]|
The results clearly show the advantages of APLI for PAH analysis. First, the spectra obtained with APLI are distinguished by a reduced base-line noise. Second, the ionization efficiency for the various PAHs is generally orders of magnitude higher for APLI. Using APCI, only in the case of benzo[a]pyrene were relatively high signal intensities obtained, while, for anthracene, significantly lower signal intensities were recorded and fluoranthene und fluorene were even below the APCI detection limit in this concentration range. Both effects, base-line noise reduction and much larger ionization efficiency, lead to detection limits which are at least two orders of magnitude lower for APLI as compared to APCI.
APLI is a very promising addition to the existing ionization sources operating at atmospheric pressure, in particular APCI, ESI, and APPI. Currently, the method is selective towards the analysis of simple, polymeric or polycyclic aromatic hydrocarbons, which may also contain heteroatoms. For these compound classes, APLI is extremely sensitive, with current detection limits in the fmol range. It is expected that this limit will significantly improve upon optimization of the source geometry and synchronization of the laser and mass spectrometer. In contrast to past applications of REMPI as an analytical technique, APLI is simple to operate. Most AP mass spectrometers can easily be retro-fitted with an APLI stage.
Future directions of APLI include: (i) Multicolor excitation. This approach allows for selective, efficient excitation of analytes exhibiting more complex absorption features or strong ISC processes. (ii) Dopant-assisted APLI. Since aromatic hydrocarbons are efficiently ionized, as described, addition of an aromatic dopant to the HPLC effluent widens the applicability of the technique significantly. In particular, non-aromatic compounds will be ionizable. All mechanisms and processes as described in the literature for DA-APPI also hold for DA-APLI. (iii) Electron attachment ionization mass spectrometry at atmospheric pressure. Thermal electrons are generated by the interaction of the laser beam with metal surfaces. Resonance and/or dissociative electron capture then leads to ionization of the analyte. The work described above is in progress, and results will be described in upcoming contributions.
The authors thank Prof. Dr. U. Scherf, Center of Polymer Technology, University of Wuppertal, Germany, for providing samples. This work was funded in part by the State of NRW, Germany, through the ‘Innovations Fond’ program.