Aviv Amirav, Tel Aviv University and Aviv Analytical Ltd.
Introduction - GC-MS Limits of Identification
Analytical conditions summary
Figure 1 clearly demonstrates several major observations:
- 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 ~6000 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 160 while in Cold EI the cholesterol TIC signal to noise ratio is ~2000.
- Response uniformity: The response uniformity of the HES is poor 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 and 300ºC is requires and even such high ion source temperature is not enough. On the other hand, Cold EI exhibits uniform compound independent response which is important for quantitation of unknowns.
- Mass Spectra: The mass spectrum obtained with the HES of n-C32H66 is of poor quality and has zero molecular ion abundance (undetected at any level) and it cannot be identified by the NIST library. Note that since it exhibits no molecular ion with the HES it can-not be identified not only at 1 ng but also at any higher level. In contrast, n-C32H66 exhibits dominant molecular ion in its Cold EI MS together with all the informative fragment ions and it is identified by the NIST library with 72% identification probability. In addition, the Cold EI mass spectrum of n-C32H66 enabled the use of the TAMI software that provided the n-C32H66 elemental formula, and improved the NIST library identification probability to 86% via using isotope abundance analysis with the limited 0.1 amu quadrupole MS mass accuracy. http://www.avivanalytical.com/Isotope-Abundance.aspx
As demonstrated in Figure 2, clearly the Cold EI mass chromatogram is far richer in information than the HES mass chromatogram. Several observations are made from Figure 2 including:
- Many more peaks in Cold EI. The TIC mass chromatogram of Cold EI exhibits many more peaks that are far more abundant than those in the HES standard EI mass chromatogram. In fact, all the 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. The tiny peaks that are indicated as hydrocarbons are indicated as such only via knowing about them from Cold EI and consequently searching peaks with RSIM on m/z=57. However, they remain as "not identified" since their TIC S/N is weak (about or below 1) and their mass spectra are dominated by vacuum background and column bleed and are without any molecular ions and or informative high mass fragments.
- Much better TIC sensitivity in Cold EI. Cold EI is much more sensitive in its TIC signal to noise ratio than the HES. For hydrocarbons Cold EI is about 10 times more sensitive while for the other groups of compounds (amides, cholestenone and Irganox 1076) they are detected in Cold EI with typical S/N of 40 for the amides while they are not detected at all in standard EI with the HES.
- Abundant molecular ions in Cold EI. As demonstrated in Figure 1 and Figure 4 below the Cold EI mass spectra are characterized by having enhanced molecular ions together with the standard EI fragment ions thus enable positive identification by the library which is supported by knowing the molecular ion identity. Thus, Cold EI provides far better identification than standard EI even with the HES.
|Figure 4. Cold EI mass spectra of Hexadecanamide, n-C29H60 and Irgaphos 1076 with their structures taking from the NIST library. These mass spectra were obtained from the indicated small TIC peaks shown in Figure 2.|
- Amides. We identified two amides, hexdecanamide and octadecanamide. We note that these compounds were not found at all in standard EI with the HES even at the 30 pg on-column amounts, possibly since they are reactive with the ion source metallic surfaces. We also note that we failed to find them even with ten times higher on-column amounts using splitless injection and thus we conclude that they are not amenable for GC-MS with standard EI analysis without derivatization. In contrast, identification was very good with Cold EI with a dominant molecular ion and good NIST library identification but a few isomers remained a possibility. The origin of these amides impurities is unknown and probably they emerge from the solvent that we used.
- Hydrocarbons. We found a range of linear chain hydrocarbons from C25H52 up to C34H70 plus the branched squalane C30H62. These saturated hydrocarbons were at the few pg levels (1 up to 6 pg) and were all identified via the availability of their molecular ions and typical fragmentation pattern of CnH2n+1 ions. Squalane has a very typical fragmentation pattern and thus was identified by the NIST library with impressive 69% identification probability. The TIC S/N of these hydrocarbons with the HES standard EI ion source was one or below one and only with 10 times higher on-column amounts using splitless injection they could be identified as hydrocarbons. However, since they do not have molecular ions, compound identification failed with standard EI (with the HES) even at this x10 higher level.
- Irganox 1076. Irganox 1076 exhibited dominant molecular ion at m/z=530 and a characteristic high mass fragment ion at m/z=515 and was easily identified with the NIST library with great identification probability of 97.1% and thus identification is assured. In contrast, we failed to find Irganox 1076 in the TIC of the standard EI with HES mass chromatogram even in RSIM on its fragment ions. Thus, 5977-HES completely failed to identify and even failed to detect Irganox 1076 at the 26 pg on-column level which with Cold EI was detected with high TIC S/N of 12 and its RSIM on the molecular ion was detected with S/N of infinite due to zero baseline noise. We note that we failed to find Irganox 1076 with the 5977B-HES even in the splitless mass chromatogram with x10 times higher on-column amount. Since we found (with the x10 times higher amount) that n-C34H70 eluted at 28.4 min at the isothermal plateau of the analysis near its 29 min end, we assume that Irganox 1076 simply did not elute in its standard EI analysis. In contrast, in Cold EI the use of shorter column and 8 ml/min high column flow rate reduced the elution temperatures and thus extended the range of compounds that eluted and could be detected and identified to include Irganox 1076.
- Cholestenone. Cholestenone was detected and identified at the high time side of the cholesterol peak. It appeared as an impurity in the original Restek solution. We could easily obtain in RSIM on m/z=384.4 two separate peaks of the weak cholesterol M-2 fragment ion and the molecular ion of cholestenone. This compound MS also includes high mass fragment ions at m/z = 299 and 271. We can-not be sure that this compound is cholestenone but we are certain that its molecular ion is m/z=384 and that it is a steroid from the cholestenone and its isomers family. Cholestenone was fully missed by the 5977-HES that did not exhibit any such peak due to the big HES peak tailing even at 300ºC ion source temperature. In addition, RSIM on m/z=384.4 did not reveal any separate additional peak to the small M-2 cholesterol fragment ion and only at the splitless x10 times higher on-column amount mass chromatogram this cholestenone compound started to perhaps reveal itself with S/N ~1 in RSIM on its molecular and few fragment ions. Thus, this is another clear case of a complete miss by the HES compared with good detection and identification by Cold EI.
- OFN and easy to analyze compounds. The test mixture that was explored in this study also included octafluoronaphthalene (OFN) at 100 ppb thus 10 pg on-column amount with split 10 used, and it eluted before the elution times shown in figure 1. OFN is considered as a very easy compound to analyze, with relative very little mass spectral background and dominant molecular ion in standard EI. OFN 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. This is lower than the OFN specification but good considering almost doubled mass spectral range and different GC oven temperature program used for OFN specification. In Cold EI the S/N was infinite in both RMS and PTP using m/z = 272 +-0.3 since the noise was zero. NIST library identification of OFN with the 5977B-HES resulted in identification probability of 89.2% while in Cold EI it was much better with 97.9% identification probability. The reason for this superior Cold EI NIST identification probability is that the OFN TIC signal in standard EI was only 1.5 times higher than the vacuum background baseline level while in Cold EI it was ten times higher than the baseline.
- TIC detection. The response of standard EI is not uniform and in general declines with mass and polarity. In addition, the standard EI ion source response is highly non-linear and often vanishes at the low pg range as demonstrated and discussed in the Advanced GC-MS Blog Journal article titled "Linearity, Sensitivity and Response Uniformity Comparison of the Aviv Analytical 5975-SMB with Cold EI and the Agilent 5977A GC-MS with Standard EI". http://blog.avivanalytical.com/2014/05/linearity-sensitivity-and response.html Accordingly, thirteen impurities were detected in Cold EI with good sensitivity while in standard EI only a few of the hydrocarbons were detected with S/N of about 1 and none was identified.
- Separation. As demonstrated in Figure 3 standard EI with the HES is confronted with major peak tailing that eliminates the separation of cholestenone from cholesterol and similarly masks two other nearby eluting compounds. Cold EI was operated with 15 m 0.32 mm I.D. column and 8 ml/min column flow rate in order to speed up the analysis, lower the elution temperatures below the onset of column bleed and extend the range of compounds that can be analyzed (particularly with column flow program and higher GC oven temperatures that were not employed in this study). These operational conditions should result in reduced GC separation by a factor of 2.5-3 yet since Cold EI is used with a tailing-free fly-through ion source the GC separation with it is preserved and it is superior to that of the HES that is confronted with major ion source peak tailing.
- Ratio of the peaks to vacuum background. With Cold EI vacuum background is eliminated (zero field ion source with directional sample compounds kinetic energy) and sample compounds elute before the onset of column bleed. Thus, the ratio of the identified peaks to vacuum background is far better in Cold EI than in the HES standard EI. For hydrocarbons this ratio is over 300 times better in Cold EI than in standard EI while the other four impurities are undetected in standard EI.
- Having trustworthy molecular ions. This is a prerequisite for good identification in which Cold EI excels while standard EI often fails. In addition, the HES is a harsh EI ion source that exhibits lower molecular ions abundances than other ion sources or the NIST library and a partial reason for this is that it requires higher operational temperatures such as 300ºC to reduce peak tailing which serves to reduce the molecular ions abundances.
- Having informative fragment ions and NIST library identification. Cold EI excels in having a combination of enhanced molecular ions and the standard fragment ions and thus it is the only soft ionization method that is fully compatible with NIST library identification. We found that the enhancement of the molecular ion actually improves the NIST library identification probabilities as described in details in "Tal Alon and Aviv Amirav "How Enhanced Molecular Ions in Cold EI Improve Compound Identification by the NIST Library" Rapid. Commun. Mass. Spectrom. 29, 2287-2292 (2015)."
- Significantly higher range of compounds amenable for analysis. This is the most important Cold EI benefit (hexadecanamide, octadecanamide, cholestenone and Irganox 1076 in this study).
- Enhanced molecular ions with Cold EI versus reduced molecular ion in the HES.
- Improved NIST library identification probabilities.
- Much faster analysis. In this case the Cold EI analysis is four times faster than the HES (elution of the last to elute major peak at <6 min versus 27 min).
- Improved sensitivity particularly for difficult to analyze compounds, as demonstrates in this article.
- Lowest vacuum background noise.
- Inherently inert ion source even for low pg range polar compounds.
- Highest (by far) ratio of TIC peaks to column bleed (better S/N and identification). A factor of 300 better Cold EI versus HES TIC to vacuum background and column bleed ratio was measured for hydrocarbons.
- No ion source peak tailing for better chromatography and separation.
- Uniform response for better quantitation (as demonstrated in Figure 1).