Nick Bukowski

Despite the advantages of soft ionization ion-source technologies for improving confidence in the identification of a range of challenging analytes, soft ionization remains a niche technique for gas chromatography–mass spectrometry (GC–MS). This article discusses the reasons for this limitation as well as new developments that make lower-energy electron ionization possible without the disadvantages that have historically been associated with it. The specific benefits of this technique are discussed by reference to several examples across the GC–MS field.

The term "soft ionization" encapsulates a range of techniques that ultimately result in analyte molecules that become ionized without imparting excess energy to them. The result is a limited degree of analyte fragmentation, meaning that the molecular ion passes intact through the mass spectrometer and to the detector. This ability to provide information about the unfragmented molecule makes soft ionization of great value to analysts.  

For macromolecules, the ready fragmentation of the molecular ion means that soft ionization is the only suitable technique and, in this field, matrix-assisted laser desorption–ionization (MALDI) is a popular method for ionizing DNA, proteins, peptides, sugars, polymers, and dendrimers. 

However, for smaller analytes amenable to gas chromatography (GC), high-energy electron ionization (EI), typically at 70 eV, is by far the most popular technique. Large libraries of spectra such as those curated by the National Institute of Standards and Technology (NIST) and Wiley are available for matching and identification, and have long been used by analysts across a wide range of gas chromatography–mass spectrometry (GC–MS) applications. In this field, methods for soft ionization have historically been viewed as more specialized, and more often used in cases where EI at 70 eV does not provide adequate results. 

There are multiple reasons for this. The major factor is that EI, which although by its nature is applicable to almost every vaporized substance, cannot be used successfully at lower energies. This is because of inefficient channeling of electrons from the filament ("e-gun") into the ion chamber, giving an extremely low signal and an unacceptable loss of sensitivity. 

Therefore, to achieve soft ionization, analysts have turned to other techniques, of which chemical ionization (CI) is the most common. CI also results in a drop in sensitivity, but to a far lesser extent than EI. Unfortunately, however, CI requires a different ion-source configuration, with additional source pressurization and the use of reagent gases. If a single instrument is being used, this can be a time-consuming transition and a considerable drain on laboratory resources if required on a regular basis. 

The challenge, therefore, remained of developing a convenient soft ionization technique that retained the performance and wide applicability of standard 70-eV electron ionization. Hence, we were excited by the development of an e-gun design that removes the link between the energy of the electrons and the ionization efficiency, by introducing an additional electrostatic element between the e-gun and the ion chamber. This allows the ionization energy of the electrons to be varied on a sliding scale from conventional 70 eV to lower energies, without loss of sensitivity. 

The Advantages of Variable-Energy Electron Ionization

A key feature of this variable-energy electron ionization is the avoidance of sensitivity losses; absolute ion intensities at low energies have been found to be equal to or greater than at 70 eV. On its own, this would not be particularly useful, but in conjunction with the improved signal-to-noise ratios at low energies (see below), the result is lower detection limits for target compounds. Furthermore, greater sensitivity allows analysts to use the same sample loading and analytical conditions for both hard and soft ionization, meaning less time is spent on sample preparation and method development.  

Like other soft ionization techniques, using EI at low energies inherently results in reduced fragmentation, and (as a consequence) an enhanced molecular ion. Importantly, however, we have found that a degree of fragmentation is usually retained even at the lowest energies. The ions produced are typically those that arise from the lower-energy fragmentation pathways, that is, larger ions that are more significant in terms of understanding the structure of the molecule. These larger ions are more often those required for quantitation, and so their increased intensities also benefit calibration.  

In addition to reduced fragmentation of the analyte, fragmentation of chromatographic background and carrier gases is also reduced. This greatly improves the signal-to-noise ratios for analytes, reduces demand on the dynamic range, and improves limits of detection, particularly in complex or more "dirty" samples. 

Another important point to note is that switching from regular 70-eV ionization to lower energies is inherent to the design, and can be carried out by changing a method parameter in the MS software. As well as avoiding the inconvenience of changing from EI to an alternative soft ionization technique, this allows sequences of samples to be repeated automatically at different energies, with subsequent savings of time. 

The inherent variability of the technique is also helpful, by allowing the analyst to adjust the ionization energy to suit the situation, and discover optimal parameters during method development. Relatively small differences in ionization energies at the low end of the scale can have significant differences. For example, note the difference in relative intensity of the 

 105 and 182 signals at 16 eV and 14 eV for benzophenone (Figure 1). 

Figure 1: Comparison of mass spectra for benzophenone at 70, 16, and 14 eV.			

The variable-energy ionization technology described above was developed by modifying the ion source for a time-of-flight (TOF) mass spectrometer (BenchTOF, Markes International), which is able to produce reliable 70 eV spectra that are a close match to those in standard libraries. The following three examples illustrate the use of the new system.  

Even when using a TOF-MS that produces spectra free from mass discrimination, uncertainties can arise when compounds have weak molecular ions or extensive fragmentation at regular 70-eV ionization energy. As illustrated in Figure 2 for the common pesticide precursor hexachlorocyclopentadiene, reducing the ionization energy can greatly enhance the molecular ion, thereby providing an additional degree of confidence in identification.  

Figure 2: Comparison of 70-eV and 11-eV EI spectra for hexachloro-cyclopentadiene, showing the greatly enhanced molecular ion at the lower energy.			

Example 2: Differentiating Between Isomeric Compounds

The ability to provide enhanced molecular ions while retaining structurally significant fragment ions improves discrimination between compounds that have similar spectra at 70 eV. Figure 3 shows an example of this increased orthogonality for four hydrocarbons in a crude oil. 

Figure 3: Top: GCÃGCâTOF-MS contour plot of a crude oil, showing overlaid EICs (m/z 268 + 282 + 296 + 310 + 324 + 338 + 352), analyzed with an ionization energy of 14 eV. The panels below (aâd) show the 70-eV and 14-eV EI spectra for  each of the four labeled peaks AâD, respectively, illustrating that it is much easier to discriminate between these compounds using the low-energy spectra.			

Example 3: Enhancing Signal-to-Noise Ratios

At low ionization energies, the reduction in the degree of fragmentation of both analytes and chromatographic artifacts helps improve signal-to-noise ratios, with subsequent lowering of the limits of detection. This is illustrated in Figure 4 for the odorous compounds safrole and coumarin, in which the increase in molecular ion intensity and reduction in fragmentation on moving from 70 eV to 15 eV result in an approximately twofold increase in the signal-to-noise values for the molecular ions.  

Figure 4: Comparison of 70-eV and 15-eV EI spectra for the allergens safrole and coumarin, showing the enhanced signal-to-noise ratios at the lower energy.			

The variable-energy ionization approach described in this article is a rugged technique that has the potential to deliver significant benefits to GC–MS laboratories across a wide range of applications. 

The ability to easily switch between hard and soft EI allows both regular 70-eV spectra to be generated for matching against existing libraries, alongside lower-energy spectra that provide complementary information on the molecular ion and structurally significant fragment ions. This complementary information enables more confident identification and discrimination between isomers that previously would not have been resolved. 

Furthermore, the reduced fragmentation of analytes, matrix interferences, and carrier gases significantly improve signal-to-noise ratios for target substances, and allow lower limits of detection to be achieved.  

For more information about variable-energy electron ionization, and detailed experimental information on the examples covered in this article, please contact the authors. 

Laura McGregor, Nick Bukowski, and David Barden

 are with Markes International, at the Gwaun Elai Medi-Science Campus in Llantrisant, UK. Direct correspondence to: 

Articles

Despite the advantages of soft ionization ion-source technologies for improving confidence in the identification of a range of challenging analytes, soft ionization remains a niche technique for gas chromatography–mass spectrometry (GC–MS).

A New Outlook on Soft Ionization for GC–MS

Crude oils are some of the most complex mixtures routinely analyzed by gas chromatography (GC). They contain significant amounts of organic compounds, ranging from light hydrocarbons to complex biomolecules derived from the remains of ancient marine organisms and the bacteria that feed on them.  

The compounds of most interest to the petroleum industry are relatively volatile (with boiling points generally below 400 °C) and nonpolar, leading to separations predominantly being performed by GC with a nonpolar column. Unsurprisingly, the resulting chromatograms are complex and usually characterized by a matrix of unresolved material that appears as a significant background "hump" beneath the partially resolved nonpolar compound peaks (Figure 1). A suitable background subtraction and library search algorithm can identify the nonpolar compound peaks, but for those lost in the matrix, there is little hope for a positive identification. 

The inability of simple GC separations to cope with this level of complexity has led to a system of classification based upon the presence of discrete biomarker compounds. However, such an incomplete characterization limits understanding of the oil sample's geochemistry and provides no insight into issues that might arise during extraction, transport, or refining. This problem is becoming more acute as the more easily extracted "light" crude oils become depleted and oil companies move to reservoirs containing "heavier" crudes, bituminous shales, and tar sands, which contain higher proportions of involatile, polar compounds that can complicate the analysis (1). 

Two-dimensional chromatography (GC×GC) is an important technique for the petrochemical industry due to its ability to separate very complex mixtures (2,3). The usual configuration is a conventional nonpolar column connected to a short length of a polar column with a GC×GC modulator in between. The modulator collects time slices of effluent from the first column (typically 5-s wide) and reinjects them onto the short polar column. The result is a separation that combines both polar and nonpolar elements. In the case of crude oil analysis, GC×GC can start to tease apart the complexity present in the matrix and identify the major components present there. With two levels of separation, GC×GC has the resolution required to isolate many of the compounds of interest in complex samples.  

Figure 2 shows a relief representation of a GC×GC separation of the same lubricating oil sample shown in Figure 1. The silhouette outline on the top left is a representation of how the sample would look on a conventional, single-dimensional GC separation. The colored portion in the figure represents the GC×GC separation with the nonpolar aspect being from bottom to top and the polar aspect being from right to left. This separation allows us to visualize compound peaks invisible in a conventional GC separation. 

GC×GC peak widths in this example analysis are typically 50–200 ms wide, which presents a problem for traditional GC–mass spectrometry (MS) systems. The maximum scan rate of a quadrupole mass detector will be in the order of 10,000 amu/s. A system set to scan from 50 to 550 

 would imply a maximum of 20 scans/s. When an interscan delay is taken into account, it is rarely possible to acquire more than 10 scans/s, implying a very poor definition of even the 200-ms wide peaks. To monitor such narrow peaks effectively, a much faster mass detector is required, and time-of-flight (TOF)-MS is currently the only viable technology. 

TOF-mass spectrometers differ from traditional scanning MS instruments in that TOF technology does not use a mass filter for the analysis. TOF technology does not scan and does not use a filter and therefore, no ions are discarded. Instead, the user defines the start- and end-mass range limits and there is no penalty for extending the mass range to 1000 amu or higher. As such, all the ions that are formed are also acquired, which is ideal when analyzing complex samples with tiny concentrations of compounds of interest. However, traditional TOF instrumentation has not been able to produce classic spectra that are identified easily in published spectral libraries. This shortcoming has forced analysts to create custom libraries, which can be a time-consuming and expensive process (4,5).  

A significant advantage of coupling TOF-MS with GC×GC is the extended mass range, as TOF enables the improved identification of compounds in complex samples when compared with traditional MS. Recent advances in TOF instrumentation have offered further benefits when combined with GC×GC, as newly developed instruments offer the ability to produce classical spectra, which can be identified in commercial or reference libraries.  

The study described in this article examines the use of a benchtop TOF mass spectrometer that offers full spectral information at levels previously only obtained with single ion monitoring (SIM) on quadrupole mass spectrometers.  

A crude oil sample was analyzed using GC×GC coupled with a TOF mass spectrometer to determine the level of classical spectral information generated by the instrumentation. 

 A model 7890A gas chromatograph (Agilent Technologies, Santa Clara, California) was configured with a thermal loop modulator (Zoex ZX1, Zoex Corp., Houston, Texas). This system was used for the comprehensive GC×GC separation. Conditions are listed in Table I. 

: A TOF mass spectrometer (BenchTOF-dx, ALMSCO, Llantrisant, UK) was used for data acquisition. Table II shows the conditions  used. 

Spectra obtained in this experiment included accurate representations of high boiling point hopanes (pentacyclic triterpanes) up to C

, which are highly diagnostic marker compounds in petrochemical applications. 

Confident identification was achieved using a commercial (NIST) mass spectral library. The high sampling frequency of the TOF enables the spectral quality and sensitivity to be realized in this GC×GC application and, equally, any high-speed GC analysis. This capability would benefit laboratories without the time, budget, or ability to develop custom spectral libraries or laboratories possessing proprietary spectral libraries previously developed on quadrupole systems. 

This new generation of TOF-MS instrumentation is capable of producing classical spectra that are comparable to commercial spectral libraries. This fundamental capability opens the door to harnessing the performance of a TOF for advanced chromatographic techniques such fast GC and comprehensive GC×GC techniques, while retaining the use of spectral libraries proprietary to petrochemical laboratories   

The power of such capability is illustrated here in the separation and identification of hopanes in a single analysis. Hopanes are used as markers for characterization of oil and the ability to analyze them in a single run with minimal sample preparation is an obvious advantage. Similarly, screening of other analytes in crude oil, particularly those not normally separated by conventional GC–MS, becomes possible. Such constituents are thought to contribute to a process of "solidification" of oil in transmission pipes, while others might have relevance to the environmental impact of oil, a factor that will gain further attention with the introduction of programs such as European REACH (Registration Evaluation Authorisation and Restriction of Chemicals), with its focus on the identification of chemicals in order to assess their toxicological impact.  

 is with ALMSCO International, Llantrisant, UK. 

 (CRC Press, Boca Raton, Florida, 2003). 

(2) C. von Mühlen, C.A. Zini, E.B. Caramão, and P.J. Marriott, 

(3) C.M. Reddya, R.K. Nelsona, S.P. Sylvaa, L. Xub, E.A. Peacocka, B. Raghuramanc, and O.C. Mullinsc,  

Crude oil is a generic term for the unrefined flammable liquid that is mined from the ground. It is an extremely varied and very complex medium that can contain many thousands of organic compounds, whose contents and concentrations vary enormously from one sample to another. This article discusses how recent advances in time-of-flight-mass spectrometry coupled with comprehensive two-dimensional gas chromatography is helping the petrochemical industry to characterize crude oils more fully and so provide solutions to common problems experienced during drilling, extraction, and refining.

Nick Bukowski

Articles

A New Outlook on Soft Ionization for GC–MS

TOF-MS: A Viable Solution for Crude Oil Extract Analysis