Damon B. Robb

Atmospheric pressure photoionization (APPI): a new ionization method for liquid chromatography-mass spectrometry

Atmospheric pressure photoionization (APPI) has been successfully demonstrated to provide high sensitivity to LC-MS analysis. A vacuum-ultraviolet lamp designed for photoionization detection in gas chromatography is used as a source of 10-eV photons. The mixture of samples and solvent eluting from an HPLC is fully evaporated prior to introduction into the photoionization region. In the new method, large quantities of an ionizable dopant are added to the vapor generated from the LC eluant, allowing for a great abundance of dopant photoions to be produced. Because the ion source is at atmospheric pressure, and the collision rate is high, the dopant photoions react to completion with solvent and analyte molecules present in the ion source. Using APPI, at an LC flow rate of 200 microL/min, it is possible to obtain analyte signal intensities 8 times as high as those obtainable with a commercially available corona discharge-atmospheric pressure chemical ionization source.

Excess partial molar enthalpies, entropies, Gibbs energies, and volumes in aqueous dimethylsulfoxide

The excess partial molar enthalpies, the vapor pressures, and the densities of dimethylsulfoxide (DMSO)−H2O mixtures were measured and the excess partial molar Gibbs energies and the partial molar volumes were calculated for DMSO and for H2O. The values of the excess partial molar Gibbs energies for both DMSO and H2O are negative over the entire composition range. The results for the water-rich region indicated that the presence of DMSO enhances the hydrogen bond network of H2O. Unlike monohydric alcohols, however, the solute-solute interaction is repulsive in terms of the Gibbs energy. This was a result of the fact that the repulsion among solutes in terms of enthalpy surpassed the attraction in terms of entropy. The data in the DMSO-rich region suggest that DMSO molecules form clusters which protect H2O molecules from exposure to the nonpolar alkyl groups of DMSO.

Comparison of dopants for charge exchange ionization of nonpolar polycyclic aromatic hydrocarbons with reversed-phase LC-APPI-MS.

Atmospheric pressure photoionization (APPI) is capable of ionizing nonpolar compounds in LC/MS, through charge exchange reactions following photoionization of a dopant. Recently, several novel dopants—chlorobenzene, bromobenzene, 2,4-difluoroanisole, and 3-(trifluoromethyl)anisole—have been identified as having properties making them well-suited to serve as dopants for charge exchange ionization under reversed-phase LC conditions. Here, we report the results of experiments comparing their effectiveness to that of established dopants—toluene, anisole, and a toluene/anisole mixture, for the charge exchange ionization of model nonpolar compounds—the 16 polycyclic aromatic hydrocarbons (PAHs) identified by the US EPA as priority pollutants—when using a conventional reversed-phase LC method. Chloro- and bromobenzene were found to be much more effective than toluene for all the PAHs, due to the relatively low reactivity of their photoions with the solvent. Their overall performance was also better than that of anisole, due to anisole’s ineffectiveness toward higher-IE compounds. Further, the experiments revealed that anisole’s performance for higher-IE compounds can be dramatically improved by introducing it as a dilute solution in toluene, rather than neat. The two fluoroanisoles provided the highest overall sensitivity, by a slim margin, when introduced as dilute solutions in either chloro- or bromobenzene.

Investigation of substituted-benzene dopants for charge exchange ionization of nonpolar compounds by atmospheric pressure photoionization

Atmospheric pressure photoionization (APPI) using a dopant enables both polar and nonpolar compounds to be analyzed by LC/MS. To date, the charge exchange ionization pathway utilized for nonpolar compounds has only been efficient under restrictive conditions, mainly because the usual charge exchange reagent ions—the dopant photoions themselves—tend to be consumed in proton transfer reactions with solvent and/or dopant neutrals. This research aims to elucidate the factors affecting the reactivities of substituted-benzene dopant ions; another, overriding, objective is to discover new dopants for better implementing charge exchange ionization in reversed-phase LC/MS applications. The desirable properties for a charge exchange dopant include low reactivity of its photoions with solvent and dopant neutrals and high ionization energy (IE). Reactivity tests were performed for diverse substituted-benzene compounds, with substituents ranging from strongly electron withdrawing (EW) to strongly electron donating (ED). The results indicate that both the tendency of a dopant’s photoions to be lost through proton transfer reactions and its IE depend on the electron donating/withdrawing properties of its substituent(s): ED groups decrease reactivity and IE, while EW groups increase reactivity and IE. Exceptions to the reactivity trend for dopants with ED groups occur when the substituent is itself acidic. All told, the desirable properties for a charge exchange dopant tend towards mutual exclusivity. Of the singly-substituted benzenes tested, chloro- and bromobenzene provide the best compromise between low reactivity and high IE. Several fluoroanisoles, with counteracting EW and ED groups, may also provide improved performance relative to the established dopants.

Are Clusters Important in Understanding the Mechanisms in Atmospheric Pressure Ionization? Part 1: Reagent Ion Generation and Chemical Control of Ion Populations

AbstractIt is well documented since the early days of the development of atmospheric pressure ionization methods, which operate in the gas phase, that cluster ions are ubiquitous. This holds true for atmospheric pressure chemical ionization, as well as for more recent techniques, such as atmospheric pressure photoionization, direct analysis in real time, and many more. In fact, it is well established that cluster ions are the primary carriers of the net charge generated. Nevertheless, cluster ion chemistry has only been sporadically included in the numerous proposed ionization mechanisms leading to charged target analytes, which are often protonated molecules. This paper series, consisting of two parts, attempts to highlight the role of cluster ion chemistry with regard to the generation of analyte ions. In addition, the impact of the changing reaction matrix and the non-thermal collisions of ions en route from the atmospheric pressure ion source to the high vacuum analyzer region are discussed. This work addresses such issues as extent of protonation versus deuteration, the extent of analyte fragmentation, as well as highly variable ionization efficiencies, among others. In Part 1, the nature of the reagent ion generation is examined, as well as the extent of thermodynamic versus kinetic control of the resulting ion population entering the analyzer region. Figureᅟ

Method of atmospheric pressure charge stripping for electrospray ionization mass spectrometry and its application for the analysis of large poly(ethylene glycol)s.

We introduce a new atmospheric pressure charge stripping (AP-CS) method for the electrospray ionization mass spectrometry (ESI-MS) analysis of heterogeneous mixtures, utilizing ion/ion proton transfer reactions within an experimental ion source to remove excess charge from sample ions and thereby reduce spectral congestion. The new method enables the extent of charge stripping to be easily controlled, independent of primary ionization, and there are no complications due to adduct formation. Here, we demonstrate AP-CS with a Xevo G2-S Q-TOF from Waters-Micromass using an ion source originally designed for atmospheric pressure-electron capture dissociation (AP-ECD) experiments; repurposing the AP-ECD ion source for AP-CS requires only adding a supplemental reagent (e.g., a perfluorocompound) to scavenge the electrons and generate anions for the charge-stripping reactions. Results from model peptides are first presented to demonstrate the basic method, including differences between the AP-CS and AP-ECD operating modes, and how the extent of charge stripping may be controlled. This is followed by a demonstration of AP-CS for the ESI-MS analysis of several large poly(ethylene glycol)s (PEGs), up to 40 kDa, typical of those used in biopharmaceutical development.

A New Ion Source and Procedures for Atmospheric Pressure-Electron Capture Dissociation of Peptides

We introduce a new atmospheric pressure-electron capture dissociation (AP-ECD) source in which conventional nanospray emitters are coupled with the source block and photoionization lamp of a PhotoSpray APPI source. We also introduce procedures for data collection and processing, aimed at maximizing the signal-to-background ratio of ECD products. Representative data from Substance P are presented to demonstrate the performance of the technique. Further, we demonstrate the effects of two important experimental variables, source temperature and vacuum-interface declustering potential (DP), on the method. Last, we show that even when a high source temperature is used to maximize efficiency, AP-ECD fragments of a model phosphorylated peptide retain the modification.

Atmospheric pressure-electron capture dissociation of peptides using a modified PhotoSpray ion source.

An improved in-source atmospheric pressure-electron capture dissociation (AP-ECD) method is described. Building upon the early example of Laprévotes group, photoelectrons generated within a commercial PhotoSpray atmospheric pressure photoionization source are used to induce ECD of multiply charged peptide ions originating from an upstream heated nebulizer device. To attain high sensitivity, the method makes use of a novel electropneumatic-heated nebulizer to assist in the creation and transmission of multiply charged ions from sample solutions. Here, we demonstrate that readily interpretable AP-ECD spectra of infused peptides can be acquired from 100 fmol sample consumed, on a chromatographic time scale, using a conventional quadrupole time-of-flight (Q-ToF) mass spectrometer otherwise incapable of ECD/ETD experiments. Though much work remains to be done to develop and characterize the method, the results indicate that AP-ECD has the potential to be a practical new tool for the mass spectrometric analysis of peptides and proteins.

Liquid chromatography-atmospheric pressure electron capture dissociation mass spectrometry for the structural analysis of peptides and proteins.

Atmospheric pressure electron capture dissociation (AP-ECD) is an emerging technique with the potential to be a more accessible alternative to conventional ECD/electron transfer dissociation (ETD) methods because it can be implemented using a stand-alone ion source device suitable for use with any existing or future electrospray ionization mass spectrometer. With AP-ECD, no modification of the main instrument is required, so it may easily be retrofitted to instruments not originally equipped with ECD/ETD capabilities. Here, we present our first purpose-built AP-ECD source and demonstrate its use in conjunction with capillary LC for the analysis of substance P, a tryptic digest of bovine serum albumin, and a phosphopeptide mixture. Quality ECD spectra were obtained for all the samples at the low femtomole level, proving that LC-AP-ECD-MS is suitable for the structural analysis of peptides and protein digests, in this case using an unmodified quadrupole time-of-flight mass spectrometer built ca. 2002.

A critical investigation of the effects of the radio frequency potential on the trapping of externally injected ions in ion trap mass spectrometry.

The sensitivity of all ion trap mass spectrometry (ITMS) methods is dependent on the trapping efficiency of the instrument. For ITMS instruments utilizing external ion sources, such as laser desorption, trapping efficiency is known to depend on the phase and amplitude of the radio frequency (RF) potential applied to the ring electrode at the time of ion introduction. It is remarkable that, in a considerable body of literature, no consensus exists regarding the effects of these parameters on the efficacy of trapping externally generated ions. In this paper, a summary of the literature is presented in order to highlight significant discrepancies. New laser desorption ion trap mass spectrometry (LD-ITMS) data are also presented, from which conclusions are drawn in our effort to clarify some of the confusion. Copyright 1999 John Wiley & Sons, Ltd.