Aviv Amirav, Tel Aviv University and Aviv Analytical Ltd.
Executive Summary
GC-MS with Cold EI includes, among its several benefits, a classical EI mode of operation. Mode changing is uniquely achieved in a few seconds via a click of the mouse method change that involves the reduction of the helium make-up cooling gas flow rate without touching any hardware. Classical EI with the Cold EI contact-free fly-through ion source (named Classical EI-SMB) provides classical EI mass spectra with high NIST library matching factors and identification probabilities. Furthermore, in comparison with the various other standard EI ion sources, Classical EI-SMB excels in having a contact-free fly-through ion source structure and as a result: a) It exhibits uniform compound independent response; b) It has no ion source peak tailing thus preserves the chromatographic separation; c) It is an inherently inert ion source thus it extends the range of compounds amenable for analysis via the analysis of polar and labile compounds such as free fatty acids and/or amides without derivatization; d) It has much lower noise and thus exhibits very high total ion count mass chromatograms signal to noise ratio. Classical EI-SMB is compared in this article with the Agilent 5977B with high efficiency ion source (HES) and all the above written benefits are demonstrated.
1. Introduction - GC-MS with Classical EI-SMB
GC-MS with Cold EI is known for its ability to enhance the molecular ions abundances in the Cold EI mass spectra via the cooling of sample compounds in supersonic molecular beams. However, some GC-MS users prefer to also have the ability to obtain classical EI mass spectra in the same GC-MS with Cold EI. It is our opinion (based on our long time experience) that Cold EI is the best ion source and with it there is no need for any other ion source. However, due to the psychological need of some users for classical EI, in this article we inform and demonstrate that the Cold EI ion source can be easily converted into a classical EI ion source (which we named as Classical EI-SMB) via a method change that requires only a few seconds and a few mouse clicks. We note that the cooling of sample compounds in supersonic molecular beams (SMB) requires the addition of about 60 ml/min helium as make-up gas, while the elimination or reduction of this cooling gas flow rate in order to obtain classical EI mass spectra is easy via a simple reduction of the helium make up gas flow rate, so that the sum of helium make-up and column flow rate will be 5 ml/min plus some minor changes in the ion optics lens system voltages. At such Classical EI-SMB operation conditions the GC-MS transfer line and nozzle temperature is 300-320ºC and the limited cooling reduce it to about 230ºC which gives very good matching to the NIST library that includes EI mass spectra that were obtained at about this ion source temperature. We note that there are several types of classical EI ion sources such as the Agilent Inert, Extractor, High efficiency source, EI ion sources of Time of flight mass spectrometers, EI ion sources of ion traps mass spectrometers etc. and each provide about the same 70 eV classical EI mass spectra with some small variations in the molecular ion abundances and fragmentation patterns. The reasons for these variations are that the ion optics, mass analyzer and/or ion detector could be different. For example, in Time of Flight MS the ion detector does not have a high voltage (10 KV) conversion dynode and thus the molecular ions and high mass fragment ions can have lower abundances, the Agilent HES ion source is a very closed ion source thus requires higher operational temperatures which result in reduced molecular ion abundances, while in ion traps MS the ion extraction process promotes undesirable collision induced dissociation MS-MS hence eliminates molecular ions in hydrocarbons.
Furthermore, all the standard EI ion sources are characterized by having metallic ion source surface that induce peak tailing, significantly reduce the response uniformity and degrade labile and certain polar compounds such as free fatty acids, amides and even steroids and alcohols at low levels that as a result require derivatization for their analysis. In contrast, the Classical EI-SMB ion source is based on a contact-free fly-through structure and thus it inherently eliminates all those downsides of standard EI ion sources as above.
In this article we demonstrate the Classical EI-SMB ion source in its provision of classical EI mass spectra and compare it with the Agilent high efficiency EI ion source (HES) in sample identification of several compounds, both at relatively high level (1 ng) and with impurities.
As demonstrated below, Classical EI-SMB uniquely excels via its contact-free fly-through ion source structure and: a) It exhibits uniform compound independent response; b) It has no ion source peak tailing thus preserves the chromatographic separation; c) It is an inherently inert ion source thus it extends the range of compounds amenable for analysis via the analysis of polar and labile compounds such as free fatty acids and/or amides without derivatization; d) It exhibits very high total ion count mass chromatograms signal to noise ratio.
Thus, the goal of this article is not only to show that Cold EI has easy access to Classical EI ion source but also to describe and demonstrate that the Classical EI-SMB ion source outperforms standard EI ion sources in several important operational aspects.
2. Analytical conditions summary:
GC-MS Systems: A) The Agilent 7890B + 5977B-HES system that was used is a new 5977B with HES ion source that is working one month with its initially provided and assembled HP5MS column. The 5977B-HES passed all its installation OFN specifications and shortly after that we achieved with it impressive OFN LOD of 0.2 fg in SIM mode. B) 5977-SMB GC-MS with Cold EI system of Aviv Analytical, based on the combination of an Agilent 5977A MSD (and 7890B GC) from 2014 with the Aviv Analytical supersonic molecular beam interface and its fly-through ion source. The system used has a three years old fly-through ion source that was never serviced and was operated in the Classical EI-SMB mode via the reduction of the make-up gas flow rate to 2 ml/min .
Sample: Aviv Analytical standard test mixture sample with 10 ppm each n-C16H34, methylstearate, cholesterol and n-C32H66 in hexane. Most of the identified impurities were incurred in this test mixture. Split 10 was used thus 1 ng on-column amounts.
Injection: 1 µL at 260ºC injector temperature and split 10 using the Agilent auto-sampler. The liners were 4.0 mm I.D. Agilent ultimate inert with gooseneck and glass wool.
Columns: In the 5977B-HES it was 30 m long, 0.25 mm I.D. and 0.25µ HP5MS film while in the Classical EI-SMB system it was 15 m, 0.32 mm I.D. and 0.1µ DB1HT film. The selection of 15 m 0.32 mm I.D. column was made since it is the most flexible column for Cold EI operation and can be used with up to 32 ml/min column flow rate plus serve for faster analysis. Classical EI-SMB can be operated if desirable with any standard 30 m or 60 m 0.25 mm I.D. column.
He column flow rate: 1.2 min in the 5977B GC-MS with HES and 3 ml/min with the 5977-SMB GC-MS with Classical EI-SMB system.
GC Oven: For the 5977B-HES it started at 50ºC followed by 10ºC/min to 300ºC and wait 4 min for the total run time of 29 minutes. In the 5977-SMB GC-MS with Classical EI-SMB the temperature program start was 50ºC, its program rate was 40ºC/min and upper GC oven temperature was 300ºC for 1.75 min for the total of 8 min analysis time.
Classical EI-SMB Source: 7 mA emission, 70 eV electron energy, 2 ml/min He makeup flow.
5977B-HES EI ion source: The Agilent HES was auto-tuned and operated at 70 eV with 100 µA emission current. The HES ion source temperatures was 300ºC (Classical EI-SMB has no relevant source temperature).
Transfer-lines temperature: 300ºC for the 5977B-HES systems and 320ºC for the 5977-SMB GC-MS with Classical EI-SMB system.
Mass spectral range: 50-500 amu at about 3.2 Hz scanning frequency.
NIST library: The latest NIST 2017 library with its search algorithm was used.
Precautions: Exactly the same sample and vial were used in both systems, injections were automated in both cases with the same model of Agilent small autosampler, the washing solvents were fresh, each experiment was repeated a few times, we injected blank samples, we also used both higher split ratio of 40 and splitless injections and we also evaluated the data with other GC-MS systems such as an older 1200-SMB GC-MS with Classical EI-SMB.
3. Results
3.1 Response uniformity
In the data presented in Figures 1-4 we compare GC-MS operation and sample identification with Classical EI-SMB and the 5977-HES systems. Classical EI-SMB is demonstrated to be superior to the HES in sample identification.
In Figure 1 we show the total ion count (TIC) mass chromatograms of our test mixture compounds as obtained with the HES ion source at 300ºC (upper mass chromatogram) and Classical EI-SMB (bottom mass chromatogram). The analyzed test mixture included 1 ng on-column (10 ppm split 10) each hexadecane (n-C16H34), methylstearate, cholesterol and n-C32H66 in order of their elution times.
Figure 1 clearly demonstrates two major observations:
- Response uniformity: The response uniformity of the HES is not good even at 300ºC and for example its cholesterol peak height is only ~6% of that of the n-C16H34. Clearly the HES response is reduced with the sample compounds size (as with other EI ion sources) and 300ºC is required and even such high ion source temperature is not enough. On the other hand, Classical EI-SMB exhibits uniform compound independent response which is important for quantitation of unknowns and for the evaluation of chemical reaction yields.
- Signal strength: The HES exhibits very high signal for 1 ng relatively volatile compounds such as n-C16H34 (the first to elute compound among the four). Furthermore, the total ion count (TIC) signal to noise for the n-C16H34 is approaching 10,000 which is impressive. However, while the HES TIC sensitivity is very high for the volatile n-C16H34 its TIC signal to noise ratio for cholesterol (and n-C32H66) is only ~200. On the other hand, in Classical EI-SMB the cholesterol TIC signal to noise ratio is ~1600 and it is similarly high for all compounds including for the sample impurities. A one way to compare EI-HES and Classical EI-SMB is the case of OFN which serves for GC-MS sensitivity specifications. The test mixture included 10 pg OFN (on-column amount) that was detected with the 5977B-HES with S/N (RMS) = 4800 using RSIM on m/z = 272 +- 0.3 and S/N (RMS) = 12600 while using m/z=272+-0.05. In Classical EI-SMB the S/N was infinite in both RMS and PTP using m/z = 272 +-0.3 since the noise was zero (no single ions noise).
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| Figure 1. The analysis of a test mixture with 1 ng on-column (10 ppm split 10) each hexadecane (n-C16H34), methylstearate, cholesterol and n-C32H66 (in order of their elution times) by the Agilent 7890B GC and 5977B MS with HES ion source (upper mass chromatogram) and by the Aviv Analytical 5977-SMB GC-MS with Classical EI-SMB (bottom mass chromatogram). |
3.2 Classical EI-SMB Mass Spectra
In Cold EI the molecular ions are enhanced but the degree of enhancement depends on the intra-molecular heat capacity which is increased linearly with the number of atoms in the sample compounds. Thus, for small molecules with up to about 20 atoms such as volatile organic compounds, benzene and/or OFN, the Cold EI mass spectra are similar to classical EI MS and naturally so are the Classical EI-SMB mass spectra. On the other hand, large compounds with over 40 atoms and particularly hydrocarbons are characterized by largely enhanced molecular ions in Cold EI which for hydrocarbons are dominant while in classical EI they are small or even absent. Thus, to demonstrate the Classical EI-SMB mass spectra similarity to the NIST library we selected n-C16H34 which is the first to elute compound in Figure 1 (about 2.72 min in the bottom mass chromatogram). In Figure 2 we show the Classical EI-SMB mass spectrum of hexadecane (n-C16H34) and its direct comparison with the NIST 2017 classical EI mass spectrum and identification parameters.
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| Figure 2. Classical EI-SMB mass spectrum of hexadecane (n-C16H34) and its direct comparison with the NIST 2017 classical EI mass spectrum. The classical EI-SMB mass spectrum is the red upper MS trace while the library mass spectrum is the bottom blue MS. The left side of the figure shows that hexadecane is rated as #1 in the NIST library hit list with matching and reversed matching factors of 915 and identification probability of 40.7% |
The classical EI-SMB mass spectrum is the red upper mass spectrum in Figure 2 while the library mass spectrum is the bottom blue one. The left side of the figure shows that hexadecane is rated as #1 in the NIST library hit list with matching and reversed matching factors of 915 and identification probability of 40.7% which is considered as high for hydrocarbons since they all share similar fragmentation patterns. Clearly the Classical EI-SMB mass spectrum is visibly similar to that of the NIST library EI mass spectrum and thus the Classical EI-SMB ion source provides classical EI mass spectra and as a result with it there is no need for any additional EI ion source.
Table 1. A comparison of NIST library based sample identification parameters by EI with the HES ion source and by Classical EI-SMB.
As shown in Table 1, Classical EI-SMB is fully compatible with NIST library identification and compared with the Agilent EI with the HES ion source the Classical EI-SMB provides somewhat lower matching factors but consistently higher identification probabilities and thus it is superior in sample identification to the HES. The main reason for this observation is that the HES requires higher operation temperatures such as 300ºC or risk greater response non-uniformity and signal loss for relatively low volatility compounds. Additional important observation is that octadecanamide was not detected in EI-HES while at the level of about 20 pg on-column it was easily identified by the Classical EI-SMB with impressive identification probability of 81%. We note also that dotriacontane was not identified with the HES by the NIST library in view of having no molecular ion and the similarity of fragmentation patterns to all other hydrocarbons while having 1% molecular ion abundance in Classical EI-SMB was enough to bring it to #3 in the NIST hit list. Thus, we conclude that Classical EI-SMB serves well in sample identification via the NIST library and even better than the Agilent EI-HES.
3.3 Classical EI-SMB sensitivity and peak tailing elimination
In Figure 3 we show the mass chromatograms of Figure 1 zoomed about 40 times in the intensity scale at around the elution times of methylstearate, cholesterol and n-C32H66. The upper mass chromatogram was obtained by the Agilent 5977B MS with HES ion source while the bottom mass chromatogram was obtained with the Aviv Analytical 5977-SMB GC-MS with Cold EI operated in its Classical EI-SMB mode. As demonstrated in Figure 3, the Classical EI-SMB mass chromatogram is far richer in information than the EI-HES mass chromatogram and it exhibits many more peaks which are far more abundant than those in the EI-HES standard EI mass chromatogram. In fact, all the EI-HES mass chromatogram peaks (aside the four main compounds) are relatively small, on top of major mass spectral background and none of them can be identified, aside a few siloxanes that emerge from pieces of septa at the liner and not from the sample. Other tiny EI-HES TIC peaks are of hydrocarbons as assumed from searching peaks with RSIM on m/z=57. On the other hand, the Classical EI-SMB mass chromatogram is much more sensitive in its TIC signal to noise ratio than the EI-HES. For hydrocarbons the Classical EI-SMB is over 10 times more sensitive while the indicated hexadecanamide and octadecanamide are detected in Classical EI-SMB with typical TIC S/N of 40 (PTP) while they are not detected at all in standard EI with the HES.
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| Figure 3. Zoom on the mass chromatograms of Figure 1 around the elution times of methylstearate, cholesterol and n-C32H66. The upper mass chromatogram was obtained by the Agilent 5977B MS with HES ion source while the bottom mass chromatogram was obtained with the Aviv Analytical 5977-SMB GC-MS with Cold EI in its Classical EI-SMB mode of operation. Note the superior sensitivity of Classical EI-SMB to the several sample impurities |
We note that the identification of the two amides in the Classical EI-SMB mass chromatogram by the NIST library as hexdecanamide and octadecanamide is of importance. These compounds were not found at all in standard EI with the HES even at the estimated ~20 pg on-column amounts, probably since they are reactive with the ion source metallic surfaces. The origin of these amides impurities is unknown and probably they emerge from the solvent that we used. The importance of this observation is that it demonstrates another benefit of Classical EI-SMB that uses contact-free fly-through ion source and thus any compound degradation on the standard EI in sources such as HES is inherently eliminated in it. Thus, Classical EI-SMB actually extends the range of compounds that are amenable for GC-MS analysis to include underivatized fatty acids, amides and low levels of alcohols and steroids.
In Figure 4 it is demonstrated that EI with the HES ion source is confronted with a major peak tailing problem that eliminates or suppresses the separation and detection of impurity peaks around the elution times of cholesterol and n-C32H66. On the other hand, the Classical EI-SMB mass chromatogram is characterized by exhibiting narrow symmetric and tailing-free peaks of both cholesterol and n-C32H66. As stated before, Classical EI-SMB is operated with a tailing-free fly-through ion source and thus with it the GC separation is preserved and it is superior to that of the EI-HES that is confronted with major ion source peak tailing for semi-volatile large compounds.
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| Figure 4. Comparison of EI-HES mass chromatogram (upper trace) and Classical EI-SMB mass chromatogram around the elution times of cholesterol and n-C32H66. Note the visible peak tailing in the EI-HES mass chromatogram (at 300ºC ion source temperature) that is eliminated in Classical EI-SMB due to its contact-free fly-through structure. |
4. Classical EI-SMB and Cold EI
It is our opinion that the most important and unique advantage of Classical EI-SMB over any other standard EI ion source is that Classical EI-SMB can be converted in two clicks of the mouse via a simple method change into Cold EI which offers the best GC-MS technology. While this article is not about Cold EI we note that it improves all the central performance aspects of GC-MS including improved identification capability, significantly extended range of compounds amenable for analysis to include much bigger and lower volatility compounds, enabling much faster analysis, exhibiting uniform response and improving the sensitivity particularly for difficult to analyze compounds.
In Figure 5 we show and compare the Classical EI-SMB and Cold EI mass spectra of Octadecanamide. As shown, the molecular ion abundance is increased from ~5% in Classical EI-SMB to become dominant in Cold EI.
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| Figure 5. Classical EI-SMB mass spectrum of Octadecanamide (upper MS) and Cold EI mass spectrum of Octadecanamide (bottom MS). Note the enhancement of the molecular ion in Cold EI combined with the availability of all the fragment ions for its NIST library identification. |
While both Classical EI-SMB and Cold EI mass spectra exhibit some molecular ion the much more abundant Cold EI molecular ion facilitates better provision of its elemental formula with the TAMI software that is based on the conversion of experimental isotope abundance and the measured mass with the limited quadrupole mass accuracy (http://www.avivanalytical.com/Isotope-Abundance.aspx). We note that about 33% of the NIST library compounds do not show molecular ions in classical EI and for those with molecular weight greater than 400 amu over 50% do not exhibit molecular ions and thus for these compounds the Cold EI provision of trustworthy molecular ions is critical for their proper identification. In addition, Cold EI often improves the NIST library identification probabilities as described in details in T. Alon and A. Amirav "How Enhanced Molecular Ions in Cold EI Improve Compound Identification by the NIST Library" Rapid. Commun. Mass. Spectrom. 29, 2287-2292 (2015). One example is dotriacontane (n-C32H66). As shown in table 1 this compound failed to be identified by the NIST library with its EI-HES mass spectrum while in Classical EI-SMB it was #3 in the NIST library hit list via its 1% molecular ion abundance. In contrast, in Cold EI due to its combination of dominant molecular ion and availability of all the fragment ions dotriacontane was identified by the NIST library as #1 in the hit list with impressive 73% identification probability.
5. Conclusions and discussion
In this paper we showed that GC-MS with Cold EI also includes a Classical EI mode of operation with easy and fast mode changing without touching any hardware. Classical EI-SMB is operated the same as Cold EI with a contact-free fly-through ion source and it provides classical EI mass spectra with high NIST library matching factors and identification probabilities. In comparison with various other standard EI ion sources, and as demonstrated in the comparison of Classical EI-SMB with EI with the Agilent high efficiency ion source (HES), Classical EI-SMB uniquely excels in: a) It exhibits uniform compound independent response; b) It has no ion source peak tailing thus preserves the chromatographic separation; c) It is an inherently inert ion source thus it extends the range of compounds amenable for analysis via the analysis of polar and labile compounds such as free fatty acids and/or amides without derivatization; d) It exhibits high total ion count mass chromatograms signal to noise ratio.
Over ten years ago we explored Classical EI-SMB with our older 1200-SMB GC-MS with Cold EI system and wrote a full length paper about it, A. Gordin, A. Amirav and A. B. Fialkov, "Classical Electron Ionization Mass Spectra in Gas Chromatography/Mass Spectrometry with Supersonic Molecular Beams" Rapid. Commun. Mass Spectrom. 22, 2660-2666 (2008). In this article we explore this topic again but now the comparison is with Agilent's most recent high efficiency ion source.
We hope that this paper is convincing in showing that with Cold EI there is no need for any other ion source since Cold EI also includes easy and fast access to Classical EI in the form of Classical EI-SMB.
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