Coupling Stages

Introduction

Mass spectrometry is one of the most sensitive methods for molecular analysis - and provides at the same time insight into chemical structures. The AP ionization methods provide also some degree of selectivity – in case of APLI towards ionization of aromatic compounds. However, the analysis of complex real-world samples is still a tough challenge. Hyphenation of APLI-MS with LC as well as GC pre-separation unleashes the full power of the technique.

LC-APLI MS

To demonstrate coupling with a chromatographic pre-separation step, an HPLC stage was coupled to the heated probe.

Benzo[a]pyrene (trace a), fluoranthene (trace b), anthracene (trace c) and fluorene (trace d) were dissolved in CH3CN and then separated chromatographically for mass-selective detection (Figure 1). For comparison, the same mixture was analyzed by APLI (right panel) and APCI-MS (left panel) under otherwise identical chromatographic conditions. With APLI, the parent ion M+· was monitored, with APCI the [M+H]+ ion. The signals from the UV-diode-array detector of the HPLC were monitored simultaneously (trace e in both panels).

The results clearly show the advantages of APLI for PAH analysis: First, the spectra obtained with APLI are distinguished by reduced base-line noise. Second, the ionization efficiency for the various PAHs is generally orders of magnitude higher with APLI.

In APCI, only in the case of benzo[a]pyrene were comparable 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 greater ionization efficiency, lead to detection limits which are at least two orders of magnitude lower with APLI than with APCI.

Comparison of LC-Chromatograms obtained with APCI and APLI
Figure 1: Comparison of LC-Chromatograms obtained with APCI and APLI

GC-APLI MS

Since the development of the electron capture detector by Lovelock, 63Ni has been used as an electron emitter for ionization of analytes in ion-mobility spectrometry (IMS). To overcome certain problems with the 63Ni foil, one-photon ionization with a VUV lamp was developed in the late 1970s. Photoionization has thus long been recognized as a technique for ionization of organic compounds in GC.

Figure 2: APLI hyphenated with GC

We have hyphenated APLI as a selective ionization technique towards aromatic hydrocarbons with GC – enabling GC analysis with an LC-MS. The coupling stage consists of a temperature controlled heated transfer line form GC oven and the heated probe (Figure 2). The probe has the same dimensions as the original APCI probe and can be used without further modification of the source enclosure. Nebulizing gas is not required the heated desolvation gas flow is adjusted for optimum performance.

Figure 3: APLI-GC MS analysis of PAHs, alkyl-PAHs and heterocyclic PAHs
Figure 3: APLI-GC MS analysis of PAHs, alkyl-PAHs and heterocyclic PAHs

The potential of GC-APLI MS is demonstrated with an analysis of a mixture consisting of PAHs, alkyl-PAHs and heterocyclic PAHs (Figure 3). All components of the mixture are separated by GC, and each analyte is detected with outstanding sensitivity as radical cation. Minor fragmentation is observed in the case of alkyl PAHs (loss of a methyl group).

CEC-APLI MS

The heated AP probe used for direct injection, HPLC, and GC, respectively does not allow the coupling with CEC: the high temperature in the probe would lead to evaporation of the electrolyte in the capillary and thus to the formation of gas bubbles and interruption of the separation voltage.

Schematic of CEC-APLI hyphenation
Figure 4: Schematic of CEC-APLI hyphenation

With CEC coupling the laser radiation is directed into the spray emitted by a micro-ESI source (Figure 4). In the following example, a silica monolith CapROD-RP-18 from Merck (Darmstadt, Germany) was used as capillary. However, the construction of the interface and the external diameter of this monolithic silica capillary (360 µm) prevented extending the capillary all the way up to the tip of the ESI interface. The separation capillary thus had to be coupled with a transfer capillary (10 cm long, 192 µm external diameter, 75 µm internal diameter), which meant that the field strength for the CEC separation was less and consequently the analysis time was increased.

The analyte solution was vaporized by electrospray. While most polar compounds are already in the ionic form in the liquid phase, the non-polar aromatic analytes are ionized by the laser beam after spray evaporation (electrospray-atmospheric-pressure laser ionisation, ES/APLI). In contrast to the APLI probe, the laser beam in this set-up was gently focussed onto the spray; the total ion yield increased by a factor of ten as compared with an unfocused-beam delivery.

Figure 5: CEC-APLI analysis of a mixture of PAHs
Figure 5: CEC-APLI analysis of a mixture of PAHs

A mixture of PAHs consisting of fluorene (1), anthracene (2), fluoranthene (3), pyrene (4) and benzo[a]pyrene (5) was separated by CEC on the monolithic column, and the PAHs were detected by ES APLI-MS (Figure 5). Because fluoranthene and pyrene have identical molecular weights, the M+· signals typically obtained with APLI did not allow an unambiguous assignment; this was performed by the addition of fluoranthene and reanalysis. Of course, only a few practical analytical problems exist in which polar and non-polar substances need to be analysed in a single run by mass spectrometry. The strength of the system in comparison with the APCI coupling lies, however, in the generation of a “cold” spray. This allows the detection of thermally labile compounds or compounds with rather high vapor pressure.