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MS atmospheric pressure ionization sources: their uses and applicability

Eleanor Riches, Steve Bajic, Efstathios Elia, G. John Langley, and Julie M. Herniman

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It can be said that a mass spectrometer of any geometric shape is nothing if there is no ion source-because if there is no ion generation, the mass spectrometer has nothing to separate and detect. In the past, the ion source was kept under vacuum to promote the ionization of the sample and allow the ions to be easily transferred to the high vacuum area of ​​the mass spectrometer. Ions are mainly formed by electron ionization (EI) or chemical ionization (CI). The analyte enters the ion source in the gas phase, or is formed as a gaseous substance in the ion source, for example, by thermal desorption. This low pressure/high vacuum requirement makes the coupling of LC and MS particularly challenging.

In 1982, Patrick J. Arpino described LC-MS as "difficult courtship" (modeling it as an attraction between fish and bird-a kind of water and a kind of air) [1]. The main difficulty is to contain a large amount of solvent into a very low pressure area and the consequent requirement for the instrument pump system.

Since the early days of API, the development of ion sources has continued unabated. There are more than 20 environmental (or close to environmental) ionization techniques [2,3] available for brave analysts. Although the ion source itself is essential, it is almost as important to correctly select the most suitable ionization source for the type of molecule being analyzed, as well as related optimizations and knowledge about the expected behavior of the ionization source. 

In this work [4], we outline different atmospheric pressure ionization techniques, including: electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), atmospheric pressure solids analysis probe (ASAP) and Waters' new UniSpray ion source. Include information about their ionization mechanism, optimization, and the types of small molecules they are most suitable for. UniSpray ionization produces ions compared with ions produced by ESI, APCI, APPI and ASAP ionization sources for a range of small molecules, including polycyclic aromatic hydrocarbons, pesticides, and polymer additives. However, due to time and availability constraints, many other ionization options have not been studied. One or more ion sources not covered here may also be suitable for the ionization of the compounds mentioned in this work.

Figure 1 shows a simple schematic diagram of the ionization process in electrospray ionization (ESI). There is still some debate about the precise mechanism of ion formation in ESI. Generally, analytes and solvent molecules are considered to undergo electrochemical reactions through redox reactions at the liquid/metal interface at the tip of the capillary or through acid/base reactions in solution [5]. These processes form ions in the solution; the diagram shows positive ions, but negative ions can also be generated in a similar way.

In order to transfer ions into the gas phase, two main general mechanisms have been proposed [6]: "Ion Evaporation Mechanism" (IEM), in which the electric field on the surface of highly charged small droplets is sufficient to directly desorb ions from the ion field. The surface, or "residual charge model," in which ions are finally desolventized when solvent molecules leave the surface of the droplet. There is evidence that smaller ions are more likely to enter the gas phase through the IEM, while larger multi-charged species are more likely to follow CRM [6,7]. Modifications to these two mechanisms or related processes are also proposed [8].

ESI is an efficient ionization process at low flow rates (<1 µL/min), and "soft ionization" occurs due to the small difference in proton affinity between analyte and reagent ions. However, due to the practical limit on the amount of charge that can be transferred to the droplet, ESI is known to be affected by the "ion suppression" effect, where the analyte competes with coeluting components and solvent contaminants for available charge. The latter effect is exacerbated at higher flow rates [9]. Extensions to the basic ESI theory, such as reducing liquid flow rates to extremely low flow rates-for example, to 30 nL/min in the case of nanoelectrospray-have proven effective, especially in limited studies of protein and amino acid samples [10 ].

Atmospheric pressure chemical ionization (APCI)

Horning first introduced APCI in 1973 to analyze volatile compounds using various introduction techniques, one of which is HPLC [11]. Figure 2 shows a simple schematic diagram of the ionization process in atmospheric pressure chemical ionization (APCI). In contrast to ESI, APCI does not apply a voltage to the tip of the capillary through which the analyte solution passes. Instead, it uses a corona discharge to initiate ionization in the gas phase. The high-energy electrons from the corona discharge will cause a series of ion/molecule reactions, which will eventually produce positive ions related to the analyte [12]. Figure 3 illustrates a series of possible reactions involving atmospheric substances [13]. Electrons initially ionize atmospheric substances (mainly nitrogen molecules) by electron bombardment. A series of aggregation and/or charge transfer reactions occur; finally, the protonated water clusters formed by these reactions can continue to produce positive analyte ions through the charge exchange or proton exchange mechanism. Alternatively, the electrons can interact with the gas phase molecules, and the gas phase molecules can then continue to react with the analyte, usually through proton extraction, leading to the formation of the negative ion species of interest.

The powerful desolvation capability of the heated nebulizer probe allows the use of APCI sources at very high flow rates (>2 mL/min). Compared with ESI, corona discharge ionization promotes the ionization of non-polar analytes and is compatible with normal-phase mobile phases [9].

Atmospheric Solids Analysis Probe (ASAP)

The Atmospheric Solids Analytical Probe (ASAP) [14] is an ionization technique that uses the APCI ionization mechanism to introduce samples as solid deposits, solutions or suspensions into the ion source. These samples are located at the tip of a small glass tube fixed by the probe. The heated atomizer gas desorbs molecules from the tip of the glass tube, as shown in Figure 4.

There is no chromatographic eluent, so this method is inherently dry compared to classic APCI. For ASAP, the ionization mechanism theory similar to APCI can be applied (Figure 3), but ASAP does seem to provide a way (or multiple ways) to ionize some substances that are not easily ionized by APCI, such as polyolefins [15]. This may be due to the absence of excessive solvent in the source atmosphere, resulting in fewer solvent-related clusters, which may enhance the charge exchange mechanism [16]. ASAP also provides the ability to perform a certain degree of thermal degradation or similar pyrolysis experiments, because the atomizer gas can be heated to more than 400ºC, which may be of interest to specific applications (such as polymer analysis). In addition, although there is no chromatographic separation, the heating ability applied in ASAP analysis can obtain boiling point curves and simplify highly complex samples by volatile components based on their respective boiling points [17].

Similar to APCI, APPI is a gas phase ionization technology in which a series of gas phase ion/molecule reactions trigger ion formation. Unlike APCI, APPI does not use corona discharge. Instead, a vacuum ultraviolet (VUV) lamp emits photons and photoionizes gaseous substances to form radical cations and electrons. Free radical cations and/or electrons can further react with other gas phase substances (such as solvent molecules) to produce analyte ions [18,19]. Figure 5 shows a simple schematic diagram of the ionization process in atmospheric pressure photoionization (APPI).

The most commonly used VUV lamp is a krypton lamp, which emits photons with an energy of about 10 eV. Any species in the source atmosphere can absorb photons. If the ionization energy (IE) (sometimes called ionization potential (IP)) of the species is less than 10 eV, it can be ionized and form radical cations and electrons. If the IE is lower than 10 eV, the analyte of interest may absorb photons and be directly photoionized; however, for many samples, this is statistically impossible because the analyte is The concentration is very low. To overcome the potential limitation of relying on direct photoionization, an additional solvent, called a dopant, is usually added, with an IE of less than 10 eV. Examples of solvents that can be used as dopants and their IE and proton affinity (PA) values ​​are shown in Table 1. The dopants are easily photoionized, and the resulting dopant radical cations initiate gas phase ion/molecule reactions, which then form analyte positive ions.

The dopant undergoes direct photoionization, as described in the following scheme:

    D + hν → D* → D+. + Electronics- 

(Where D = dopant molecule, hν is the energy of the photon).

Table 2 shows the key reactions believed to be related to the formation of positive ions in APPI. The IE and PA of all substances present in the atmosphere of the ion source will affect the ionization mechanism. In the positive ion mode, APPI can form a variety of different ions, including [MH]+ and [M-H2]+[21], as well as [M+H]+ and M+. The reactions shown in Table 2 depend on the relative gas phase acidity or alkalinity of the substances present in the ionization source.

A new UniSpray® (US) ionization source has been developed, which uses a unique method to generate ions for mass spectrometry (Figure 6) [22,23]. This atmospheric pressure ionization source includes a grounded capillary, from which the analyte solution flows out and is then atomized by high-speed nitrogen. The eluent spray acts on a cylindrical stainless steel target rod under high pressure, usually about 0.5-4.0 kV, providing the potential to ionize the analyte with higher efficiency. The point of impact is optimized to be offset from the center of the rod and upstream of the entrance of the mass spectrometer. Due to the Coandă effect, this causes the eluent spray stream to bend around the profile of the rod. The aerodynamic flow associated with the UniSpray cross-flow geometry can have many other important effects, such as droplet impact, surface microvortices, and scattered vortices, which are believed to affect source performance [22].

The spectrum generated when UniSpray is used is very similar to the spectrum analyzed by ESI. Therefore, although no voltage is applied to the tip of the capillary, the eluent may contain ions formed by solution-phase redox reactions and other physical processes. The surface effects of the impinging needle and additional gas phase phenomena may also further promote ion formation. Compared with other atmospheric pressure ionization technologies, UniSpray's response has increased [23]. It is well known that droplet size plays an important role in ion generation rate [24,25]. Therefore, a large part of this observed increase seems to be attributable to the formation of smaller droplets when the eluent spray interacts with the impinging needle, and the subsequent rapid ion desolvation of these droplets.

The performance of each source was studied using simple techniques that did not involve any chromatography. For ESI, APCI, APPI and UniSpray, standard solutions covering a wide range of small molecules are combined with a suitable representative LC flow through the flow path of the on-board instrument. In the case of ASAP, the glass capillary is directly immersed in the solution. Examples of representative compounds from each standard mixture can be seen in Table 4.

• Use a suitable solvent to prepare a solvent standard solution at a suitable analytical concentration: ~0.1-1.0 µg/mL for small molecule mixtures, ~0.1% for engine oil, and ~1 mg/mL for crude oil samples.

• UniSpray response was evaluated at three different impact pin voltages: 0.5 kV, 1.0 kV, and 3.0 kV.

• The APCI response was evaluated at four different corona currents: 1 µA, 5 µA, 10 µA, and 12 µA.

• The ASAP response was evaluated at two different corona currents: 1 µA and 12 µA.

• Use the SYNAPT G2-Si HDMS instrument to obtain high-resolution mass spectrometry data with ion mobility and review in MassLynx v.4.1 MS software.

• Source temperature: 120°C

• IMS wave speed: 1000 m/s (fixed)

• IMS wave height: 40 V

• IMS unit pressure: 3.3 mbar

• All data are obtained by combining the sample solution with a representative mobile phase (1:1 MeOH:H2O, 100% MeOH or 1:1 MeOH:Toluene), depending on the ionization technique considered or the analysis The compound category.

• A separate evaluation was conducted specifically on the response of oilfield additives analyzed by ESI and UniSpray.

• Use an Ultra High Performance Supercritical Fluid Chromatography (UHPSFC) system coupled with a tandem quadrupole mass spectrometer to separate C12 quaternary ammonium salts and 12OH amine compounds.

• Solvent A: Supercritical CO2

• Solvent B: MeOH + 2% H2O + 50 mM ammonium acetate

• Column: ACQUITY HSS C18 SB, 1.8 µm, 3.0 x 100 mm

• Temperature: 40°C

• Injection volume: 2 µL

• Gradient table (table 3):

Table 5 summarizes the response of each ion source for the representative compounds shown in Table 4. The values ​​highlighted in yellow indicate the maximum response of each compound and are therefore the best ion source for these types of compounds. X means that there is no reliable detection response for a given compound using this ionization technique. All representative compounds form protonated substances, but PAH compounds also form free radical cations (M+.), and sulfamethazine, which is selected as the representative of the cosmetic and allergen mixture 1, also forms sodiumized molecules.

The data shown in Table 6 focuses on a small compound mixture of polymer additives. For the four liquid flow ion sources studied, the responses of all components of the mixture are shown. In each case, the strongest ion observed is given. The color of the text indicates the type of ion: black = protonated molecule, blue = sodiumized molecule, red = hydride ion abstraction, brown = radical cation . The highlighted yellow value represents the maximum response for each compound and is therefore the best ion source for that particular compound.

It is also noted that the optimal striker voltage depends on the type of adduct formed. Protonated substances give a better response at higher applied voltages (for example, 3.0 kV), while sodiumized substances give better response at lower voltages (for example, 0.5 kV). Figure 7 further illustrates this phenomenon. The figure shows the axis-linked spectra of two polymer additives Uvitex OB and Irganox 245. Uvitex OB facilitates the formation of ions through protonation, while Irganox 245 facilitates the formation of ions through sodiumization. The different responses to different applied striker voltages can be clearly seen.

To illustrate the performance of different ionization sources with different classes of compounds, axis-linked spectra were generated. Figure 8 shows a magnified area of ​​the mass spectrum obtained from the analysis of organic light-emitting diode (OLED) compound mixtures. The exemplary target compound forms ion isotope clusters near m/z 762. UniSpray showed the strongest absolute response, and APCI and ASAP produced almost similar strong responses.

Figure 9 shows a similar magnified area of ​​the mass spectrum obtained by analyzing a mixture of polycyclic aromatic hydrocarbons (PAH) compounds. Exemplary target compounds form clusters of ionic isotopes near m/z 252 because these compounds generally form free radical cations. Interestingly, ESI can ionize compounds, while UniSpray has almost no reaction. APPI produced the strongest response, APCI showed a similar ion pattern but with lower intensity, and ASAP showed little or no response.

Safaniya vacuum residue petroleum samples were analyzed using direct injection. Figure 10 shows the full spectrum obtained with each ionization source. Here, we can see the value of using different ionization techniques to ensure full coverage of such a complex sample.

The key comparison of UniSpray and ESI's analysis of oilfield additives shows that, compared with ESI, the response is greatly improved when UniSpray is used. Figure 11 shows the calibration curve for the 12OH amine additive, and Figure 12 shows the calibration curve for the C12 quaternary ammonium salt.

Both compounds were analyzed in a concentration range of 10 ppt to 2 ppm. For 12OH amine, UniSpray's response increased by 17 times, while the response of C12 quaternary ammonium salt increased by 6 times.

The key structural features of any analyte can indicate which ionization technique may be suitable for the analyte. Some of the structural features are summarized in Table 7.

Source code optimization and usage guide [4] 

    • ASAP

    o Use corona current instead of corona voltage for acquisition.

    o Evaluate several different corona currents, including higher values, such as 10 µA.

    o For fast, similarly classified sample analysis, a 30-second ballistic temperature ramp can be used to volatilize the sample and evaluate the visible ions.

    o In order to separate according to the boiling point curve of the sample, a slower temperature ramp can be used.

    o In most cases, dopants will enhance the ionization process.

    o Try toluene as a dopant first, which is usually very effective. If needed, try other dopants based on their IE and the IE of your analyte or multiple analytes.

    o For accurate mass data acquisition, the dopant can be prepared as 1:1 dopant: MeOH, in which leucine enkephalin is dissolved in MeOH to obtain locked mass ions in function 1. Leucine enkephalin ions can be used for internal mass correction.

        o Use a low to medium rejection voltage, such as 0.5 kV.

    o Make sure to push the lamp fully into the source housing (position 2 on the source housing).

    o APPI shows better response at lower flow rates.

    o Ideally, the dopant flow rate should be in the range of 10% to 50% of the eluent flow rate.

     o Use corona current instead of corona voltage for acquisition.

    o Evaluate several different corona currents, including higher values, such as 10 µA.

    o Generally speaking, values ​​as high as 5 µA are sufficient for less complex samples.

    o The amount of water in the source may affect the ionization efficiency, because water clusters play a role in the ionization mechanism of APCI.

    o Try several different impactor voltages to optimize for the compound of interest.

    o Always check sodium adducts because they are easy to form for many of the compounds studied in this work.

    o It is very important to optimize the position of the jet to the surface of the impact pin. Make sure it is slightly offset from the center of the MS entrance to take advantage of the Coandă effect. 

ESI may be the first choice for most day-to-day analysis, and UniSpray should also be evaluated as an early option when available. If chromatographic separation is not required, then ASAP technology is recommended because it provides a very wide range of compound categories and can be evaluated within a few minutes to determine its suitability for analysis. In general, for problem-solving laboratories, having a wide range of available ion sources will facilitate the ionization of the widest range of different molecules. Once the appropriate ion source for a particular analysis is determined, the selected technique can be routinely implemented; however, if new ionization techniques are developed, such as UniSpray, they may provide a better response to the established analysis.

• UniSpray has been proven to have broad applicability to several types of compounds, but it is not necessarily the best ionization source for all molecules.

• UniSpray is a valuable add-on in the "toolbox" of mass spectrometers that can be used to solve the problem of sample diversity.

• Other complementary ionization techniques, such as APCI and APPI, are also needed to ensure maximum coverage of the most challenging samples.

• According to the adduct formed by the target analyte (sodiumization and protonation), UniSpray has been observed to have different impinging needle optimized voltages.

• Compared to ESI, UniSpray showed a significant improvement in response when analyzing selected oilfield chemicals.

• The structure and functional properties of the molecule influence the choice of the most appropriate ionization technique.

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