Aviv Amirav (1,2), Uri Keshet (1) and Albert Danon (3)
1. School of Chemistry, Tel Aviv University Tel Aviv 69978 Israel
2. Aviv Analytical LTD, 3 Haarad Street Tel Aviv 69107 Israel
3. Nuclear Research Center Negev P.O. Box 9001 Beer Sheva Israel
GC-MS with Cold EI (Electron Ionization of Cold Molecules in Supersonic Molecular Beams) provides ideal EI mass spectra which combine the usual library searchable EI fragments with enhanced molecular ions for improved library based identification probability, improved confidence in the sample identity and improved sensitivity and selectivity in the sample analysis. However, in some cases such as in the analysis of complex petrochemical matrices a soft ionization method that provides only molecular ions is desirable since it can generate carbon number distributions for petroleum homologues and since mass spectra with molecular ions only add a dimension of separation that can supplement and complement that of the GC. Field ionization is an example of a soft ionization method that can be combined with GC-MS in which the MS adds another independent dimension of information hence it was named GCxMS (F. C. Y Wang, K. Qian and L. A Green, Anal. Chem. 77, 2777-2785 (2005)). However, field ionization is three orders of magnitude less sensitive than EI, it is incompatible with library based identification, its response can be compound dependent and it may exhibit peak broadening/tailing. While 70 eV Cold EI provides the ideal mass spectra one can use the same fly-through Cold EI ion source at low electron energies in an attempt to observe molecular ion alone. Contrary to some perceptions, for most compounds lowering the electron energies does not affect much the observed EI mass spectra due to electron induced excitation of high lying vibrational states of the ions even at low electron energies. However, hydrocarbons are an example of compounds class that their EI mass spectra are relatively largely affected by the electron energy, and in Cold EI the elimination of internal heat amplifies the low electron energy effect.
We tested low electron energy Cold EI a few times in the past and although the molecular ion was the dominant MS peak we failed to eliminate the fragment ions and get molecular ion only. Thus, we decided to further investigate the reasons for our inability to get molecular ions only. We found that once the sample compound is cooled by the supersonic expansion it can be reheated via reflected scattered helium atoms near the skimmer. Furthermore, once a labile molecular ion is formed it can undergo undesirable collision induced dissociation (CID) the same as in MS-MS, and the magnitude of such CID can be significant for labile molecular ions such as of hydrocarbons. In order to reduce these adverse effects we evaluated the effect of reduction of helium pressure at the ion source and MS vacuum chamber via the increase of the nozzle-skimmer distance. We found out that this increased nozzle-skimmer distance resulted in noticeable increase of the ratio of molecular ions to low mass fragment ions.
Keep reading to find out how the Aviv Analytical 5975-SMB GC-MS with Cold EI uniquely improves low electron energy Cold EI and converts it into a Soft Cold EI while further approaching the ideal of molecular ion only ionization method.
Electron impact ionization is considered as a hard ionization method in which the molecular ion can often be fully dissociated hence the information content of EI is sometimes severely degraded. In GC-MS with electron ionization the molecular ion dissociates via four main reasons and we found that proper control of these reasons can largely improve the information content of EI and convert it all the way from being a hard ionization method into a most informative ionization method and even into a soft ionization method. The following are the main reasons for molecular ion dissociation in EI:
- Electron Induced Ion Excitation (and Electron Energy): The electron ionization process imparts up to several electron volts intra-molecular-ion internal vibrational energy that leads into molecular ion dissociation. This added vibrational energy can originate via direct excitation of high lying vibrational states of the lowest electronic state of the molecular ion or via the excitation of additional high lying electronic states of the molecular ion which undergo internal conversion into highly excited vibrational states of the ground electronic state of the molecular ion which leads into dissociation. The use of lower electron energy make these high lying electronic states of the molecular ion inaccessible hence the degree of molecular ion dissociation at low electron energies can be reduced. However, for certain compounds such as those with many heteroatoms high lying electronic states of the molecular ion are not accessible due to unfavorable Frank-Condon factors and for these molecules lowering the electron energies does not affect the mass spectra while it largely reduces the ionization yield. The simplest example is PFTBA that serves as the GC-MS calibration compound in which the molecular ion at m/z 671 cannot be observed at any electron energy.
- Thermal vibrational energy: In standard GC-MS the sample compounds can have up to 3NKT vibrational energy (heat capacity) in which N is the number of atoms, K the Boltzmann constant and T is the ion source temperature in Kelvins. Usually the high temperature heat capacity limit is not achieved and a closer estimate for the sample internal vibrational energy is NKT. For small molecules with up to 15-20 atoms this thermal energy contribution to the molecular ion dissociation is smaller than that of the electron energy. However, for large molecules this contribution is central and it serves as the dominant reasons for molecular ions dissociation. In Cold EI this thermal vibrational energy is practically eliminated and thus the molecular ions are enhanced and the larger the compound the greater is the effect of Cold EI on the enhancement of the molecular ions. You may read further material on this topic in another blog article. We note that while in Cold EI the intra molecular vibrational energy can be eliminated for small and medium molecules via the supersonic expansion, large molecules may require specially shaped nozzle and higher make up gas flow rate for effective full elimination of their internal vibrational energy and in general the skimmer used must be sharp and relatively large to reduce and/or eliminate sample compounds reheating via a reflected shock-wave that can be formed on blunt and/or small skimmers.
- Weaker Molecular Ion Chemical Bonds: Molecular ions by definition have one less electron than the neutral compound hence their weakest chemical bond is typically weaker than the weakest bond of the neutral molecule. Thus, molecular ions can be inherently unstable and sometimes dissociate even without any added intra-ion vibrational energy.
- Collision Induced Dissociation (CID): Once the molecular ion is formed, even if it is stable and not dissociated via the three reasons above it can dissociate in its flight path to the mass analyzer via collisions with the residual carrier gas such as helium that comes from the GC column. It is well known that in GC-MS with internal ionization ion traps such as the Varian Saturn 2000 or Varian 220 the molecular ions of hydrocarbons are often fully missing while they are small yet observed in Quadrupole based GC-MS. In these ion traps the molecular ions undergo CID inside the ion trap prior to its detection. We found in our 1200-SMB GC-MS with Cold EI which is based on a Varian 1200 triple quadrupole MS system, that when we select in the first quadrupole MS the molecular ion m/z=226 of hexadecane (n-C16H34) as a parent ion without adding any Q2 collision gas and CID voltage we observe in the scanning Q3 that about 30% of the hexadecane molecular ion is dissociated despite having double differential pumping in this system. As demonstrated below, CID could be an important residual fragmentation mechanism once the three above reasons are eliminated.
In this article we demonstrate recent improvement that we achieved with our Cold EI (EI of vibrationally cold molecules in supersonic molecular beams) and our further advance in having Soft Cold EI at low electron energies that further approaches the status of mass spectra with molecular ions alone.
Analytical Conditions Summary
GC-MS with Cold EI System: 5975-SMB GC-MS with Cold EI system of Aviv Analytical, based on the combination of an Agilent 5975 MSD (and 7890A GC) with the Aviv Analytical supersonic molecular beam interface and its unique fly-through ion source.
Samples: Squalane C30H62 and n-C24H50 as liquid and powder.
Sample Introduction: Aviv Analytical ChromatoProbe sample introduction device with 3 mm OD and 2.4 mm ID vials that were used for sample vaporization inside the GC injector.
Injector temperature: 250ºC for squalane and 220ºC for n-C24H50.
Column: 5m length, 0.25 mm ID with 0.25 µ DB-5MS UI film served as a transfer line between the ChromatoProbe and the supersonic nozzle.
He column flow rate: 4 ml/min.
GC Oven Temperature: 280ºC.
Cold EI Source: 8 mA emission current, 70 eV electron energy, 54 ml/min He make-up gas flow rate for standard Cold EI conditions. For soft Cold EI conditions we used 84 ml/min He make-up gas flow rate for extended cooling conditions and 18 eV electron energy. The nozzle backing pressures were 830 mBar for 54 ml/min helium make up gas flow rate and 1100 mBar for 84 ml/min.
Nozzle skimmer distance: 7 mm in standard Cold EI conditions and 16 mm in Soft Cold EI conditions resulting in 8x10-6 mBar and 1.5x10-6 mBar ionization gauge pressure reading for 7 mm and 16 mm nozzle-skimmer separation distances respectively while 54 ml/min helium make up gas flow rate was used. The pressure readings were increased by a factor of 1.5 from the above values when the helium make up flow rate was increased to 84 ml/min. The real helium pressures are higher by about x8 due to ionization gauge calibration with nitrogen.
Transfer-line temperature: 250ºC.
Mass spectral range: 50-500 amu at about 3 Hz scanning frequency.
Results – Soft Cold EI
In Figure 1 we show the Cold EI mass spectra of squalane which is a highly branched C30H62 isoprenoid hydrocarbon. Usually branched hydrocarbons do not exhibit any molecular ion in standard EI but as demonstrated in the bottom trace Cold EI provides about 60% relative abundance of the molecular ion combined with all the structurally important fragment ions that enable the elucidation of its structure. Furthermore, despite (due to is probably a better term) the enhanced molecular ion and high mass fragment ions the NIST identification actually improved and resulted in 79.7% identification probability which is very high for large hydrocarbons. The reason for this improved library identification is that while the matching factors are reduced due to the enhancement of the molecular ion the matching factors of other candidates are reduced even more since the molecular ion and high mass fragment ions are the most sample characteristic ions. This Cold EI mass spectrum was obtained with 84 ml/min helium make up gas flow rate plus 4 ml/min column flow rate for the total helium flow rate at the supersonic nozzle of 88 ml/min which is higher than our usual use of 60 ml/min combined make up and column flow rate. Since squalane is a highly branched hydrocarbon it requires improved cooling to optimize its molecular ion abundance. However, we found at 70 eV electron energy practically no effect of the flow rate increase on the obtained Cold EI mass spectrum. On the other hand, as demonstrated in the upper Cold EI mass spectrum the x2.3 increased nozzle-skimmer distance from 7 to 16 mm had a dramatic impact on the obtained Cold EI mass spectrum which now has a dominant molecular ion and about six times further enhanced ratio of the molecular ion to low mass fragment ions. Despite the six times further enhanced molecular ion squalane was still identified as #1 in the NIST library with 44% identification probability. As shown, under these conditions Cold EI provides dominant molecular ions to all hydrocarbons including highly branched large hydrocarbons. This demonstrated further enhanced molecular ion raises the question of what is the reason for this clear mass spectral effect. The main change in the experimental conditions is that the pressure at the ion source second vacuum chamber was decreased from 1.2x10-5 mBar to 2.3x10-6 mBar as measured with the ionization gauge. The actual pressures are about eight times higher than these values since ionization gauges are calibrated for nitrogen that has about x8 higher response than helium. Accordingly, reduction of both skimmer related neutral compounds reheating and collision induced dissociation of the generated molecular ions could be responsible for the observed effect of large enhancement of the molecular ion. As the nozzle-skimmer distance is increased, the gas density around the skimmer lips is quadratically reduced and as a result any reflected shock wave of scattered helium atoms at the skimmer lips that can reheat the sample compounds is substantially reduced. While at this time we cannot rule out skimmer induced reheating as the main cause of reduced molecular ion in the bottom trace of figure 1 we think that our Aviv Analytical skimmer is relatively large (0.8 mm) and sharp and of high quality and thus this effect is estimated as secondary. We believe that the main reason for extended molecular ion dissociation at 7 mm nozzle-skimmer distance is collision induced dissociation of the molecular ions. In Cold EI, the molecular ions of squalane are formed from neutral compounds with about 8-10 eV kinetic energy and after ionization they gain additional 2 eV (inner ion source cage potential) for total of 10 eV ion kinetic energy while traveling about 10 cm from the center of the ion source to the entrance of the quadrupole mass analyzer. During this path the ions are accelerated to 40 eV at the ion mirror, 20 eV at the ion mirror exit lens and to about 100 eV at the Agilent focus lens and the combination of the relatively high helium pressure and high ion kinetic energy is the main reason for the observed CID. Regardless of the exact reason, the effect is clear, large and unambiguous and we now know (as demonstrated for squalane) that for labile molecular ions increased nozzle-skimmer distance helps to further enhance molecular ions. We note that while the helium flux via the skimmer was reduced by a factor of 5.3 in these experiments, the total ion count was reduced by a little less than a factor of 2 while as shown the molecular ion of squalane was actually increased by a factor of 2.5. Thus, the trade-off of signal for enhanced molecular ion results only in relatively small loss in the total ion count signal. The Aviv Analytical 5975-SMB has a simple screw based control of the nozzle-skimmer distance that allows easy optimization of this distance which as we now know can be beneficial for certain hydrocarbons mixture analysis. We note that while the increase of helium make up gas flow rate from 54 ml/min to 84 ml/min did not make an effect on the Cold EI mass spectrum of squalane at 7 mm nozzle-skimmer distance, it made a large effect at 16 mm nozzle-skimmer distance. The simple explanation for this observation is that the improved supersonic expansion based cooling at the higher make up gas flow rate was offset by increased CID and/or skimmer reheating at the small nozzle-skimmer distance.
In Figure 2 we show a comparison of Soft Cold EI mass spectrum (upper trace), Cold EI mass spectrum (middle trace) and the NIST library mass spectrum of squalane C30H62 (bottom trace). The middle trace shows the obtained Cold EI mass spectrum at our standard Cold EI conditions of 70 eV electron energy, 7 mm nozzle-skimmer distance and 54 ml/min helium make up gas flow rate while the upper trace shows the obtained Cold EI mass spectrum at 18 eV reduced electron energy, 16 mm nozzle-skimmer distance and 84 ml/min helium make up gas flow. We named Cold EI mass spectra that were obtained using the latter conditions as "Soft Cold EI". As shown in the Soft Cold EI mass spectrum low mass ions are highly suppressed and even for a labile molecular ion of a compound such as squalane the highly dominant molecular ion brings Soft Cold EI a step closer to the ideal of molecular ion only. GC-MS with Cold EI and its fly-through ion source can uniquely serve via a simple few minutes method change to provide mass spectra in three modes of operation of Cold EI, Soft Cold EI and Classical EI which we named as Classical EI SMB (A. Gordin, A. Amirav and A. B. Fialkov "Classical Electron Ionization Mass Spectra with GC-MS with Supersonic Molecular Beams" Rapid. Commun. Mass Spectrom. 22, 2660-2666 (2008)). Classical EI SMB is achieved via the simple reduction of the sum of column flow rate and make-up gas flow rate to 5 ml/min hence suppresses the SMB cooling. While the classical EI mass spectrum as shown is the NIST library MS (figure 2 bottom trace) is with little structural information the Cold EI mass spectrum provides the highest level of structural information and best NIST identification probability and the Soft Cold EI mass spectrum provides the most selective EI mode of operation that is approaching to molecular ion only.
In Figure 3 we show a comparison of Soft Cold EI mass spectrum (upper trace), Cold EI mass spectrum (bottom trace) and NIST library mass spectrum (bottom trace insert) of n-C24H50. As demonstrated, Soft Cold EI with a linear chain hydrocarbon such as n-C24H50 provides a mass spectrum in which the molecular ion is over 50 times higher than any other fragment ion and its three isotopomers amount to 67% of the total ion count. This largely enhanced molecular ion is not as high as obtained with field ionization in which K. Qian and G. J. Dechert, Anal. Chem. 74, 3977-3983 (2002) reported over 94% molecular ions from the total ion count for C10-C16 hydrocarbons. However, we believe that it serves as a major advance in approaching molecular ion only and in improved selectivity in the analysis of complex hydrocarbons mixtures. The bottom trace Cold EI mass spectrum exhibits the usual hydrocarbons fragments plus a dominant molecular ion. NIST search of this Cold EI mass spectrum resulted in n-C24H50 as number one in the hit list with 53.4% identification probability while when we analyzed n-C24H50 with Agilent 5975 GC-MS with standard EI n-C24H50 was only number 4 in the NIST hit list with 10.2% identification probability. Thus, Cold EI via its enhanced molecular ions improves the NIST identification probabilities.
In order to differentiate the relative roles of CID from skimmer induced reheating we explored Soft Cold EI and Cold EI of PFTBA. This compound that serves for MS tune in GC-MS is unique in two aspects: a) Its cooling effect in Cold EI is small and; b) Its m/z=264 fragment ion is relatively stable in MS-MS based CID while its abundant m/z=219 fragment ion is weak and easily dissociates into m/z=131 and 69 daughter ions. In the NIST library the ratio of m/z=264/219 ratio is 17% in the main library MS while in the replica it can be up to 26%. In Cold EI with nozzle-skimmer distance of 16 mm and 84 ml/min helium make up gas flow rate we found that the ratio of m/z=264/219 is 25% while at 7 mm nozzle-skimmer distance it was 28%. Under these conditions the m/z=131/219 ratio increased from 31% to 45%. While such increase in the m/z=131/219 can be explained by both increased CID and skimmer induced heating, the increase in the m/z=264/219 must originate only from CID as skimmer induced heating should slightly reduce this ratio.
While Soft Cold EI exhibits highly dominant molecular ions as shown in the figures above it should be emphasized that hydrocarbons are uniquely suitable for Soft Cold EI while with other compounds it may not be as successful. One example is 1-octanol which has no molecular ion at all in standard EI since water elimination is very easy and requires very little energy due to the formation of a double bond in the remaining octene ion. In Cold EI this compound exhibits 1% relative abundance molecular ion at 7 mm nozzle-skimmer distance (84 ml/min helium make up gas flow rate) and 2.5% relative abundance of the molecular ion at 16 mm nozzle-skimmer distance. For this compound the reduction of the electron energy made no effect on the relative abundance of the molecular ion while the signal was markedly reduced. Thus, while having 2.5% molecular ion is far more than having zero molecular ion, it is far from being a dominant molecular ion.
Conclusions and Discussion
GC-MS with Cold EI uniquely provides full control over the electron ionization mass spectrum. With the Aviv Analytical 5975-SMB GC-MS with Cold EI one can easily change (few minutes method change) the operation of the ion source and SMB interface and obtain the following three main modes of operation and their resulting types of mass spectra:
- Classical EI mass spectra can be obtained via the simple reduction of the helium make-up gas flow rate to reduce the sample vibrational cooling.
- Cold EI mass spectra are provided as the main and most useful mode of operation, typically with 60 ml/min combined column and helium make up gas flow rate, 70 eV electron energy and 7 mm nozzle-skimmer distance. Cold EI provides the most sensitive, selective and informative EI mass spectra combined with the best NIST library identification probabilities.
- Soft Cold EI mass spectra are provided as a complementary mode of operation, typically with 90 ml/min combined column and helium make up gas flow rate, 18 eV electron energy and 16 mm nozzle-skimmer distance. Soft Cold EI provides the most selective mode of EI mass spectra which is approaching to molecular ion only MS.
In 1995 Shai Dagan and Aviv Amirav published their paper "Electron Impact Mass Spectrometry of Alkanes in Supersonic Molecular Beams", J. Am. Soc. Mass Spectrom. 6, 120-131 (1995). In this article GC-MS with Cold EI analysis of hydrocarbons was demonstrated and discussed for the first time. Since that time we improved the vibrational cooling with a shaped nozzle and combined the Cold EI technology with advanced commercial GC-MS systems such as the Agilent 5975 or 5977 MSD. In this article we explored our new discovery that the softness of Cold EI mass spectra can be further improved via the reduction of both skimmer re-heating effects and collision induced dissociation of the molecular ion after its formation. We demonstrated our improved Cold EI via the Soft Cold EI mass spectra of squalane and n-C24H50 which demonstrated highly dominant molecular ions at 84 ml/min helium make up gas flow rate, 16 mm nozzle-skimmer distance and 18 eV electron energy. As demonstrated above, we further approached the ideal of molecular ion only and for n-C24H50 we obtained a molecular ion that was over 50 times higher than any fragment ion and which amounted to 67% of the total ion count. We tested also lower than 18 eV electron energies but found only little further increase in the ratio of molecular ion to residual fragment ions while the ionization yields were markedly reduced. It should be emphasized that in standard EI the reduction of the electron energy has only limited effect on the enhancement of the molecular ion, and the bigger the compound the lower is the low electron energy effect due to increased internal heat capacity. In contrast, in Cold EI once the intra-ion thermal energy is removed, the reduction of electron energy induces much greater effect and the bigger the compound the greater is the effect of vibrational cooling in Soft Cold EI on the enhancement of the molecular ions.
Based on our experiments with PFTBA (and further additional indirect evidence) we conclude that CID is an active mechanism that explains the further enhancement of molecular ions at increased nozzle-skimmer distance in both Cold EI and Soft Cold EI. On the other hand, skimmer induced heating effect may or may not be active and it cannot be ruled out.
As described above, the conversion of Cold EI into Soft Cold EI requires three changes of:
- Increased helium make up gas flow rate. This increased cooling gas flow rate is not required for small molecules with molecular weight of 300-400 and even for n-C25H50 it made no observable effect. However, for large molecules and those with highly labile molecular ions such as of squalane it is important and helpful although it may reduce the TIC by a factor of less than 2.
- Reduced electron energy. With hydrocarbons the reduction of the Cold EI electron energy can significantly increase the relative abundance of the molecular ion and suppress the fragments ions while losing about x10 in TIC signal at 18 eV. The effect of reduced electron energy is not easy to predict and in some cases it has no effect. Electron energy reduction below 18 eV has limited further gain in the ratio of molecular ion to fragment ions while the TIC signal can be largely reduced.
- Increased nozzle-skimmer distance. This newly discovered effect of increased nozzle-skimmer distance on further enhanced molecular ion in Cold EI mass spectra seems essential for having Soft Cold EI. It can generate significant gain in the molecular ion while losing less than a factor of 2 in the TIC signal. Since this increased nozzle-skimmer distance requires a mechanical change in the system this change cannot be automated and thus should be implemented mostly for those experiments in which top sensitivity is not needed while enhanced molecular ion is highly desirable such as in complex hydrocarbon mixtures analysis and/or service GC-MS for synthetic organic chemistry. Once the nozzle-skimmer distance is set, the fly-through ion source can serve with automated method based change in all the three modes of Soft Cold EI, Cold EI and classical EI-SMB.
We note that Soft Cold EI is now closer to field ionization in the production of molecular ions alone since even field ionization can exhibit fragment ions (E. M. Chait and F. G. Kitson, Org. Mass. Spectrom., 3, 533 – 547 (1970)). However, in comparison with field ionization Soft Cold EI is two orders of magnitude more sensitive, Soft Cold EI can be changed in few minutes via a method change into Cold EI (or Classical EI-SMB) for NIST library compatibility, the response of Soft Cold EI is uniforms and compound independent, it does not exhibit any peak broadening/tailing and its fly-through ion source is highly robust and rarely require any service. Another soft ionization mode is chemical ionization (CI). However, CI is not as soft as Soft Cold EI and Cold EI shows molecular ions to broader range of compounds unlike CI which is incompatible with certain classes of compounds such as hydrocarbons. In addition, Cold EI is far more sensitive than CI and unlike CI is compatible with NIST library based sample identification. CI requires system venting to replace the ion source hence it is rarely used while the Cold EI ion source has three modes of operation that can be changed via a few minutes method change. Furthermore, CI requires more frequent cleaning and service and its closed ion source induces earlier onset of ion source peak tailing and degradation while Cold EI and Soft Cold EI significantly extend the range of compounds amenable for GC-MS analysis to include much bigger and more thermally labile compounds. Thus, Cold EI provides the most sensitive most selective and most informative mass spectra and contrary to some perception it improves the NIST library identification probabilities.
We believe that with such great flexibility of the Cold EI fly-through ion source it should bring the next GC-MS revolution as Cold EI has only advantages over standard EI and with it there is no reason to have a standard EI ion source.
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