Effect of affinity for droplet surfaces on the fraction of analyte molecules charged during electrospray droplet fission.

The effect of uneven fissioning of mass and charge from electrospray droplets on the amount of analyte charged during the electrospray process was explored. A surface selectivity factor (S) was developed to describe the affinity of an analyte for the droplet surface, and both theoretical and experimental response curves were compared for analytes with various S values. The theoretical response curves were generated by calculating the overlap between the charge and analyte spawned from parent droplets to determine the amount of analyte charged. This overlap was then graphed as a function of analyte concentration. Differences in the amount of analyte charged during droplet fission were predicted for analytes of varying surface affinities. The issue of analyte partitioning between the surface and interior phases of the ESI droplet was also included in the discussion. This was accomplished by applying the equilibrium partitioning model to a set of offspring droplets to determine the amount of analyte on their surfaces and then calculating the overlap between fissioning analyte and excess charge. Experimental response curves resembled theoretical ones, and S values predicted from theory were in excellent agreement with those predicted on the basis of the structural characteristics of the analytes.

Electrospray ionization detection of inherently nonresponsive epoxides by peptide binding.

A small organic molecule that is inherently nonresponsive to electrospray analysis, 1,3-butadiene diepoxide, was analyzed via electrospray ionization (ESI) by binding it to various peptides and observing the product at the characteristic mass shift. The epoxide reacted only with peptides with arginines in their sequence, most likely through a base-catalyzed ring opening to form a covalently bound product. A calibration curve linear over 3 orders of magnitude was generated for the butadiene diepoxide/peptide adduct. Several other epoxides were also reacted with the peptide of choice (angiotensin II), and adducts of these epoxides with the peptide were observed as well, demonstrating the versatility of this method for the analysis of small epoxides. This study demonstrates the possibility of assaying epoxides bound to peptides or proteins in biological samples. Furthermore, it demonstrates an important concept that could be applied to other analytical problems in electrospray: the ability to react an analyte that is nonresponsive to electrospray analysis with an analyte well suited for the technique, and accomplish quantitation based on the adduct formed between the two.

Predicting electrospray response from chromatographic retention time.

The relationship between electrospray ionization response and HPLC retention time was explored. For the series of small peptides studied, higher ESI response was observed for analytes with longer reversed-phase HPLC retention times. This correlation existed for both experimentally measured retention times and those calculated from amino acid retention coefficients. This study is useful t

Practical implications of some recent studies in electrospray ionization fundamentals.

In accomplishing successful electrospray ionization analyses, it is imperative to have an understanding of the effects of variables such as analyte structure, instrumental parameters, and solution composition. Here, we review some fundamental studies of the ESI process that are relevant to these issues. We discuss how analyte chargeability and surface activity are related to ESI response, and how accessible parameters such as nonpolar surface area and reversed phase HPLC retention time can be used to predict relative ESI response. Also presented is a description of how derivitizing agents can be used to maximize or enable ESI response by improving the chargeability or hydrophobicity of ESI analytes. Limiting factors in the ESI calibration curve are discussed. At high concentrations, these factors include droplet surface area and excess charge concentration, whereas at low concentrations ion transmission becomes an issue, and chemical interference can also be limiting. Stable and reproducible non-pneumatic ESI operation depends on the ability to balance a number of parameters, including applied voltage and solution surface tension, flow rate, and conductivity. We discuss how changing these parameters can shift the mode of ESI operation from stable to unstable, and how current-voltage curves can be used to characterize the mode of ESI operation. Finally, the characteristics of the ideal ESI solvent, including surface tension and conductivity requirements, are discussed. Analysis in the positive ion mode can be accomplished with acidified methanol/water solutions, but negative ion mode analysis necessitates special constituents that suppress corona discharge and facilitate the production of stable negative ions.

Simple cylindrical ion mirror with three elements.

A new cylindrical ion mirror has been designed to create an electric field that is non-linear or curved along the flight path axis for general-purpose time-of-flight mass spectrometers. The inclusion of one or two grids is found to improve the radial field homogeneity especially around the aperture. Only three cylindrical electrodes are used in the design. Changing the electrode dimensions and voltages affects the electric field distribution. Once the electrode dimensions are fixed, there are only two adjustable parameters for achieving optimum nonlinear electric field shape. Resolving powers of 7,000 and 16,100 have been achieved with kinetic energy variations of 34 and 10.5%, respectively. Simulations show that the electric field homogeneity in the radial direction enables the use of ion beam diameters up to 15 mm with only modest loss of resolving power. Increasing the mirror diameter could further increase the practical ion beam diameter. This article details the electric field distribution within the cylindrical mirror in both axial and radial directions. The voltages of the middle and rear electrodes affect the resolving power and the kinetic energy range over which focus can be achieved. The predicted arrival time spread for a single m/z value is narrower than that caused by the turn-around time of ions in a gas-phase ion source. In this case, the broad energy range over which good focus is achieved enables the use of higher extraction fields for turn-around time reduction.

Simple geometry gridless ion mirror.

A gridless variation of the cylindrical ion mirror has been designed to create an electric field that is nonlinear in the axial direction and nearly homogeneous in the radial direction. The designs may include one or two chambers that consist of truncated cones. This new design concept yields ion mirrors with improved energy focusing over conventional single-field and multiple-field mirrors. Conventionally, ion mirrors with nonlinear field gradient use multiple diaphragm electrodes to which distinct voltages are applied. In this work, optimized nonlinear field distributions are achieved through shaping only two or three electrodes and applying only one or two voltages on the electrodes. The designs presented here offer high resolving power and low ion dispersion. SIMION simulations of performance from the ion source to the detector demonstrate resolving powers of 11,000 and 1,750 for ions with kinetic energy variations of 7.5% and 23.6%, respectively.

Importance of gas-phase proton affinities in determining the electrospray ionization response for analytes and solvents.

The effect of gas-phase proton transfer reactions on the mass spectral response of solvents and analytes with known gas-phase proton affinities was evaluated. Methanol, ethanol, propanol and water mixtures were employed to probe the effect of gas-phase proton transfer reactions on the abundance of protonated solvent ions. Ion-molecule reactions were carried out either in an atmospheric pressure electrospray ionization source or in the central quadrupole of a triple-quadrupole mass spectrometer. The introduction of solvent vapor with higher gas-phase proton affinity than the solvent being electrosprayed caused protons to transfer to the gas-phase solvent molecules. In mixed solvents, protonated solvent clusters of the solvent with higher gas-phase proton affinity dominated the resulting mass spectra. The effect of solvent gas-phase proton affinity on analyte response was also investigated, and the analyte response was suppressed or eliminated in solvents with gas-phase proton affinities higher than that of the analyte.

Relating electrospray ionization response to nonpolar character of small peptides.

Nonpolar regions in biological molecules are investigated as a determining factor governing their electrospray ionization (ESI) mass spectrometric response. Response is compared for a series of peptides whose C-terminal residue is varied among amino acids with increasingly nonpolar side chains. Increased ESI response is observed for peptides with more extensive nonpolar regions. The basis for this increase is examined by comparing values of nonpolar surface area and Gibbs free energy of transfer for the different amino acid residues. Comparisons of response with octadecylamine are also made, and this highly surface-active ion is observed to outcompete all other analytes in ESI response. These observations are rationalized on the basis of the equilibrium partitioning model, which is used successfully to fit experimental data throughout the concentration range for several two-analyte systems. This model suggests that because excess charge exists on ESI droplet surfaces, an analyte's relative affinity for the droplet surface determines its relative ESI response. Increased nonpolar character, which leads to enhanced affinity for the surface phase, results in more successful competition for excess charge and higher ESI response.

Electrical equivalence of electrospray ionization with conducting and nonconducting needles.

An electrical equivalent circuit is derived for the electrospray process. It is a series circuit which consists of the power supply, the electrochemical contact to the solution, the solution resistance (R(s)), a constant-current regulator which represents the processes of charge separation and charge transport in the gap between the spray needle aperture and the counter electrode, and charge neutralization at the counter electrode. A current i, established by the constant-current regulator flows throughout the entire circuit. Current-voltage curves are developed for each element in the circuit. From these it is shown that in the case where R(s) is negligible (the power supply is connected directly to a conducting needle) the shape of the current-voltage curve is dictated by the constant-current regulator established by the charge separation process, the gap, and the counter electrode. The solution resistance may be significant if a nonconducting needle is used so that the electrochemical contact to the solution is remote from the tip. Experiments with a nonconducting spray needle quantify the effect of the solution resistance on the current-voltage curve. Subtracting the iRs voltage from Vapp (power supply voltage) yields the current-voltage curve for the constant-current regulator. When iRs drop is a significant fraction of Vapp, the current-voltage curve of the constant-current regulator is changed substantially from the case when the solution resistance is negligible.

Effects of salt concentration on analyte response using electrospray ionization mass spectrometry.

The effect of salt concentration on analyte response using electrospray ionization mass spectrometry (ESI-MS) was measured and compared to that predicted by Enke's equilibrium partitioning model. The model predicts that analyte response will be proportional to concentration and that the response factor will decrease with increasing electrolyte concentration. The measured analyte response is proportional to concentration over four orders of magnitude when the electrolyte concentration is below 10(-3) M, as the model predicts. The concentration of excess charge ([Q]) generated by the ESI process increases significantly at 10(-3) M ionic concentration, but the response factor decreases at this concentration. Changes in shape of the spray that cause a loss of ion transmission efficiency may be the basis for the decrease in response. An increase in the analyte response factor with increasing electrolyte concentration is observed for electrolyte concentrations below 10(-3) M. An explanation for this based on the electrical double layer is proposed.

A predictive model for matrix and analyte effects in electrospray ionization of singly-charged ionic analytes.

In electrospray ionization (ESI), droplets with a surface excess charge are created. The rate of production of surface excess charge is a constant and is equal to the rate of ion production. The ions appearing in the mass spectrum are postulated to be those that formed the surface excess charge at the time of droplet formation (or their collision products). An equilibrium model based on competition among the ions in the solution for the limited number of excess charge sites has been developed. This model accurately predicts the response curves of singly-charged ionic analytes as a function of the concentration of electrolyte and other analytes and provides an explanation for the selective effectiveness of ESI. At low concentrations of total analyte (micromolar and less), the response curves are linear, indifferent to the presence of other low concentration analytes, and suppressed by electrolyte concentrations in excess of the minimum required. At higher analyte concentrations, the response becomes independent of analyte concentration but highly affected by the presence of other analytes.

Composition-selective detection of polychlorinated biphenyls (PCBs) by oxygen-chlorine exchange reaction in a tandem mass spectrometer (MS/MS).

The exchange reaction of chlorine by oxygen with polychlorinated biphenyl (PCB) anions was explored as a method for composition-selective detection of PCB congeners in a gas chromatograph/tandem mass spectrometer (GC/MS/MS) experiment. The odd-electron molecular anions of PCBs react with molecular oxygen introduced into the collision cell of a triple-quadrupole mass spectrometer to form a product ion that has 19 mass units less (35Cl - 16O) than the precursor. The even-electron anions which also form in the source do not react when allowed to pass through the precursor analyzer. Multiple reaction monitoring allows the generation of separate chromatograms for each different composition from tetrachloro through nonochloro PCB. Most coelutants are resolved in this way. Response factors for the 12 coplanar congeners that exhibit dioxin-like toxicity relative to octachloronaphthalene vary from 1.6 to 580. Results of the analysis of an Aroclor 1242 sample for the coplanar congeners compare well with previously determined values.

Valved sampling cell for membrane introduction mass spectrometry.

Membrane extractors comprising a membrane house inside of a valve have been developed to separate compounds of interest from a sample matrix and introduce these compounds into a mass spectrometer. Experimental control over parameters that affect permeability or that may damage the membrane, such as the membrane temperature, is provided with the valve. The valve was tested for response and response times with the valve separated from the mass spectrometer by various interface tube lengths. Data for steady state response measurements showed no significant change with the valve at different distances from the ion source. Polar compounds show a strong response time dependency on the interface tube length. This adsorption phenomenon is minimized by simply heating the interface tube. Other factors affecting the performance of the device are discussed.

A mathematical study of sample modulation at a membrane inlet mass spectrometer-Potential application in analysis of mixtures.

An ANSI C program that simulates the diffusion profiles of sample modulation at a membrane inlet system has been developed to study the characteristics of modulated diffusion profiles. The program produces concentration profiles within the membrane and flux values at the exit side of the membrane as a function of time. Sample concentration on the inlet side can be switched between zero and an arbitrary value with a square or asymmetric cycle. Achievement of steady-state diffusion between alternations is not required. With this computer simulation, the flux profiles of analytes through a membrane inlet have been studied as a function of diffusion coefficient, modulation frequency, and concentration. The amplitude, shape, and time lag or phase angle of the flux profile are shown to be related directly to analyte concentration and diffusivity. A method that involves a set of linear equations is proposed to resolve mixtures of diffusing analytes based on differences in the time dependence of their flux profiles.

193-nm Photodissociation of ions from saturated and unsaturated aliphatic molecules.

To determine the analytical utility of photodissociation as a general fragmentation technique for tandem mass spectrometry of organic ions, the ability to fragment those ions considered least likely to absorb photons efficiently was investigated. To this end, the ability to photodissociate ions of aliphatic compounds by using 193-nm photons has been studied. Three fragment ions, the C4H 9 (+) ion from n-hexane, the C4H 7 (+) ion from 2-hexene, and C4H 5 (+) from 2-hexyne, have been photodissociated. The fragmentation efficiencies for all three ions studied were between 25 and 45%. The photofragment ion spectrum for each precursor ion studied is made up of characteristic fragments. These spectra demonstrate the ability to photodissociate aliphatic ions that originate from both saturated and unsaturated molecules. This provides substantial hope that virtually all organic ions will be able to be photodissociated by using 193-nm photons.

Method for the design of broad energy range focusing reflectrons.

A novel method for the design of reflections capable of focusing large kinetic energy ranges is presented. The design method itself is a numerical approach that provides a geometrically flexible alternative to traditional analytical design solutions. This design method has been used to produce a reflectron that provides unit mass resolution for product spectra in a tandem reflectron time-of-flight (TOF) mass spectrometer despite a kinetic energy range of 1950-2700 eV. In this application, the systematic progression of reflectron design results in a practical, nonlinear field reflectron with the use of only two grids. Design improvements are proposed for more flexible systems, although geometric constraints in the current instrument limit their experimental evaluation.

A segmented ring, cylindrical ion trap source for time-of-flight mass spectrometry.

An ion trap source has been designed for use with time-of-flight (TOF) mass analysis. Two thin diaphragms make up a segmented ring electrode; the end cap electrodes are planar wire mesh. The potential field produced by the rf voltage applied between the ring and end cap electrodes resembles that of the cylindrical ion trap. The trapped ion population for ions created by electron impact exhibits linear growth against a first-order loss that has a time constant of about 50 µs; no ion loss occurs when the electron beam is off. The observed value of q z at low-mass cutoff for rf ion storage is -0.84. Pulsed extraction of all ions is accomplished by switching the trap electrodes from rf to voltages required to provide a linear dc extraction field. The TOF flight path includes a wide energy range reflectron. Better than unit mass resolution is achieved through m/z 500 without collisional ion cooling. With an extraction rate of 1 kHz and a recording rate of 4 spectra per second, a linear working curve is obtained between 36 pg and 18 ng of chlorobenzene delivered chromatographically. The system has demonstrated the potential to achieve a very high sample utilization efficiency at high spectral generation rates.

Tandem reflectron time-of-flight mass spectrometer utilizing photodissociation.

A tandem time-of-flight (TOF) mass spectrometer has been designed to obtain complete MS/MS spectra from compounds eluting from a gas chromatograph. This application requires high spectral generation rate, unit mass resolution for both precursor selection and product spectra, and efficient ion utilization. These objectives are achieved by reflectron TOF mass separation in both stages and laser photoinduced dissociation as the ion fragmentation method. Careful timing of the laser pulse relative to ion extraction allows ions of a single m/z value up to m/z 1000 to be photodissociated while ions with adjacent m/z values are essentially unaffected. The convergent foci of the ion packet and laser pulse results in ion fragmentation efficiencies as high as 79%. An ion gate prevents the nonselected precursor ions from convoluting the product spectra. Product spectra can be generated at the maximum laser repetition rate (currently 200 Hz). To achieve unit mass resolution for all product m/z values simultaneously, a novel reflectron was designed for the second TOF stage.

Mechanistic study of hydrogen/deuterium exchange between [M - 1](-) ions of chlorinated benzenes and D 2O or ND 3.

Low-energy collisions between [M - 1](-) ions derived from the three isomers of dichlorobenzene and deuterated water and ammonia are found to produce distinctive hydrogen/ deuterium (H/D) exchange reaction product patterns. The predominant products observed for p-, m-, and o-dichlorobenzene are 1, 2, and 3 sequential deuterium exchanges, respectively. The reactivity is substantially higher for D2O than ND3 We postulate a mechanism that involves the formation of a five-membered-ring intermediate. The intermediate is thought to be initiated by the attack of ND3 or D2O at the localized negative charge site on the aromatic ring. A successful exchange is followed by the relocation of the charge site to the adjacent carbon. Ion products with higher degrees of deuterium substitution than the expected predominant products of their corresponding isomers are believed to be the results of isomerization of the reactant ions occurring in the ion source. The proposed mechanism fuily explains the observed product spectra derived from al1 the isomers of chlorinated benzenes. The trends for the formation of various H/D exchange products represented by the sweated product-time plots based on the proposed mechanism compare well with the similar trends obtained from the experimental product-pressure plots. The reaction is useful for the mass spectrometric differentiation of chlorobenzene isomers.

High Efficiency Photo-Induced Dissociation of Precursor Ions in a Tandem Time-of-Flight Mass Spectrometer.

High efficiency photo-induced dissociation (PID) has been demonstrated in a tandem time-of-flight mass spectrometer. This instrument focuses isomass ion packets to temporal and spatial dimensions similar to those of the focused laser pulses from a high power excimer laser. This high density overlap of photons and ions yields highly efficient fragmentation and also provides high resolution selection of specific precursor ion mass-to-charge ratio values. Using 193 nm photon excitation of the molecular ion of bromobenzene (m/z = 1561, fragmentation, collection, and PID efficiencies af 79%, 132%, and 104%, respectively, were obtained. Characteristic fragmentations of toluene, nitrobenzene, acetophenone, triethylamine, N,N-diethylformamide, N-methylacetamide, and cyclohexene have also been demonstrated.