Aviv Amirav (1,2), Uri Keshet (1) and Bogdan Belgorodsky (1)
1. Tel Aviv University
2. Aviv Analytical Ltd
Analytical conditions summary
- Standard EI ion sources, even of the most advanced GC-MS system such as the Agilent 5977, exhibit ion source related peak tailing.
- The ion source related peak tailing results in significant loss of peak height and peak area up to an order of magnitude loss for the compounds in Figure 1.
- The ion source related peak tailing strongly depends on the on-column sample amount and thus the sample signal dependence on its concentration is non-linear.
- The ion source peak tailing strongly depends on the sample features such as volatility, polarity and reactivity. As demonstrated the effect is small for the more volatile n-C16H34, intermediate for methylstearate and big for the low volatility cholesterol and n-C32H64.
- We found that cholesterol mostly degrades to cholestane at the ion source even at 250ºC, based on the ratio of m/z=386.4 (molecular ion) to m/z=368.4 of cholestane. Furthermore, this ratio was strongly increased with the ion source temperature and was more than doubled at 300ºC versus at 250ºC. In addition, the ratio of cholestane to cholesterol was increased at the tail of the GC peak. This finding is in contrast to the vendor claims that their ion source is inert. Perhaps it is more inert than other standard EI ion sources but it is still based on exposed reactive metal surfaces and thus highly reactive.
- Injector split: We found that the splitless injection of 40 times diluted sample resulted in about the same results as via the injection of undiluted sample with split 40.
- Increased injector temperature from 250ºC to 280ºC had no effect.
- The use of glass wool at the liner had no effect on the results.
- Increased standard EI ion source temperature from 250ºC to 300ºC had a significant effect on the peak tailing that was reduced but the noise was increased, the molecular ion was reduced and the degree of cholesterol degradation was more than doubled.
- This effect was observed with few column types.
Table 1 Explanations and Discussion
- TIC S/N Cold EI over Standard EI. This column shows the ratios of the total ion count (TIC) signal to noise ratio of Cold EI versus Standard EI. TIC signal to noise ratio (S/N) is probably the most universal sensitivity parameter that characterizes GC-MS. The reason for this is that it considers both the signal and noise level at their correct proportion of Signal/(Number of Noise Ions)0.5. The 5975-SMB GC-MS with Cold EI is better than the standard 5977 in the range of 1.1 up to 20 and the harder the compound for analysis the greater is the Cold EI sensitivity gain. We note that it is the lower noise in Cold EI due to elimination of vacuum background that contributed the most to this higher TIC S/N of Cold EI and not higher signal.
- Cold EI M+ increase. This column shows the enhancement in the molecular ion abundance alone. Cold EI always provide enhanced molecular ions that are dominant for all the test mixture compounds while they are weak or missing in standard EI.
- RSIM M+ S/N CEI/SEI. This column shows the magnitude of Cold EI signal to noise ratio improvement on the molecular ion. This is the parameter that currently serves as the common way of all vendors for the evaluation of GC-MS sensitivity (brochure specification and during installation) via the use of 1 pg OFN on-column and the measurement of its RSIM S/N (in RMS units) on its m/z=272 molecular ion. Cold EI is characterized by inherent vacuum background filtration and its noise is therefore very low and mostly made of low intensity column (and injector) bleed from its temperature programmable transfer line and ever-present trace PFTBA tune compound. As a result, when the Agilent data analysis is used with its internal (non-optional) software filters, one frequently observe that Cold EI RSIM peaks have clean baselines with no single ion noise around (prior to) the explored compound elution time as shown for Methylstearate in Figure 3 above. Thus, for four out of five compounds including OFN we wrote >100 which implies very high S/N gain over standard EI, in part due to zero noise in cold EI and in part due to more intense molecular ions.
- NIST identification probability Cold EI over Standard EI. All five compounds were identified by the NIST library from their Cold EI mass spectra (number 1 in the NIST hit list for four compounds and number 2 after an isomer for methylstearate) while only two were identified from their standard EI mass spectra (no background subtraction was attempted). Hexadecane Cold EI mass spectrum has lower matching factor than of standard EI but twice higher probability of identification. For Methylstearate, Cold EI mass spectrum library search resulted in an isomer of Methylstearate as number one and methylstearate as number 2 in the NIST search while in Standard EI it was number one thus better for Standard EI. However, isotope abundance analysis was much better with the Cold EI and provided excellent matching factor of 999 versus 955 in standard EI due to its higher molecular ion abundance and lack of vacuum background noise. Thus, the Methylstearate identification of its elemental formula was much better via the Cold EI mass spectrum. Cholesterol and n-C32H66 failed to be identified from their standard EI while they were easily identified from their Cold EI mass spectra. Thus, contrary to some perceptions Cold EI provides superior sample identification by the NIST library than standard EI and as further explained in our article discussing the subject.
- For n-C16H34 the TIC signal was increased by a factor of 1.5 but the molecular ion abundance was reduced by a factor of 2.3 thus the molecular ion signal was reduced by a factor of 1.5;
- For methylstearate the TIC signal was increased by a factor of 3.8 but the molecular ion abundance was reduced by a factor of 2.3 thus the molecular ion signal was increased by a modest factor of 1.6;
- For cholesterol the TIC signal was increased by a factor of 2.8 but the molecular ion abundance was reduced by a factor of 5.5 and thus the molecular ion signal was reduced by a factor of 2. In addition, the ratio of cholestane to cholesterol was increased by a factor of 2.5 due to increased intra-ion-source degradation at the higher temperature;
- For n-C32H66 no molecular ion was observed at any temperature for the 0.25 ng sample.
- We found that even for the more volatile OFN the increase of the ion source temperature to 300ºC resulted in ~50% TIC signal increase and 25% molecular ion signal increase but despite that signal increase the noise was also significantly increased hence the OFN S/N was reduced by a factor of 3.3 at the 300ºC ion source temperature. In fact, we found for all our sample compounds that the molecular ions RSIM signal to noise ratios were noticeably reduced with the increased ion source temperature.
- Peak Tailing: We demonstrated that standard EI ion sources exhibit significant ion source related peak tailing.
- Signal loss: The ion source related peak tailing results in both significant losses of peak height and peak area that can be over an order of magnitude.
- Non Linearity: The ion source related peak tailing strongly depends on the on-column sample amount and thus the sample signal dependence on its concentration is non-linear.
- Compound dependency of the peak tailing: The ion source peak tailing strongly depends and increases with the sample polarity, reactivity and low volatility.
- Ion Source Reactivity: We found that cholesterol mostly degrades to cholestane at the ion source and that the degree of degradation increases with the ion source temperature.
- Some Cold EI Advantages over Standard EI: In this article we demonstrated that Cold EI with its fly-through ion source has no peak tailing, no ion source reactivity and hence it exhibit linear signal increase with the sample amount for all compounds.
- Response Uniformity: Cold EI exhibits uniform compound independent response unlike standard EI in which the response uniformity is eroded for large polar and labile compounds due to ion source tailing and reactivity.
- Reduced Column Bleed with Cold EI: We found that surprisingly the level of column bleed is lower with Cold EI due to the elimination of ion source peak tailing.
- Reproducibility: Standard EI ion source peak tailing also adversely affects the reproducibility as the magnitude of peak tailing depends on the ion source temperature, cleanliness and history.