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High-speed, low-pressure gas chromatography–mass spectrometry for essential oil analysis
Journal of Chromatography A, 1200 (2008) 28–33
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
High-speed, low-pressure gas chromatography–mass spectrometry for essential oil analysis Samuel D.H. Poynter, Robert A. Shellie ∗ Australian Centre for Research on Separation Science (ACROSS), School of Chemistry, University of Tasmania, Private Bag 75, Hobart 7001, Australia
a r t i c l e
i n f o
Article history: Available online 28 March 2008 Keywords: Essential oil Fast gas chromatography Mass spectrometry Low-pressure gas chromatography
a b s t r a c t Analysis of parsley and fennel essential oils was performed by using low-pressure gas chromatography– mass spectrometry (GC–MS). The low-pressure instrument configuration was achieved by fitting a GC–MS instrument with a 530 m I.D. capillary column and an appropriate capillary restrictor at the inlet of the column. Comparison of the performance of the low-pressure GC–MS setup was made with fast GC–MS using a narrow-bore capillary column. By comparing the two approaches side-by-side the benefits of low-pressure GC–MS for characterisation of moderately complex essential oils comprising less than 50 detectable components can be fully appreciated. Although efficiency is sacrificed, the improved sample capacity of the 530 m I.D. column leads to higher peak intensities and in-turn better mass spectral library matching thus providing highly satisfactory results. © 2008 Elsevier B.V. All rights reserved.
1. Introduction There are a variety of practical routes for achieving high-speed separations of complex samples [1] and many of them have been employed for essential oil analysis. Most commonly 10 m × 100 m I.D. columns (with appropriately reduced stationary phase film thickness) have been employed in place of 25 m × 250 m I.D. columns [2] and it has been shown many times this approach leads to a significant speed gain while preserving resolution [3]. In a similar way Mondello et al. [4] demonstrated fast analysis of lime essential oil using a 5 m length of 50 m I.D. capillary with a 0.05 m stationary phase coating. Lime oil analysis was achieved in around 90 s by employing the fastest possible temperature program rate of the GC oven (50–150 ◦ C in 75 s, 150–200 ◦ C in 43 s, 200–250 ◦ C in 55 s) as well as a higher than optimum average linear carrier gas velocity of 120 cm/s. An additional approach for achieving faster analysis includes direct resistively heated column GC [5] which, by marrying rapid temperature programming (up to 20 ◦ C/s) with fast data acquisition (using flame ionization detection) and high split ratio [6] can lead to essential oil analysis times between 40 and 100 s [5,6]. Fast analysis approaches often try to maintain efficiency, however having highlighted that the efficiency of a capillary column often exceeds analytical requirements, Bicchi and coworkers investigated the use of short 250–320 m I.D. columns with appropriate selectivity for the analysis of rosemary (Rosmarinus officinalis L.)
∗ Corresponding author. Tel.: +61 3 6226 7656; fax: +61 3 6226 2858. E-mail address: [email protected] (R.A. Shellie). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.03.069
and chamomile (Matricaria recutita L.) essential oils [7]. Both of these essential oils are considered as moderate complexity samples, and effective analysis was performed by substituting the standard (ca. 25 m) capillary column comprising around 150,000 theoretical plates with 5 m columns comprising 20,000–50,000 theoretical plates. A 5% phenyl- 5% vinyl-polydimethylsiloxane stationary phase was used for the separation of chamomile oil but lacked the selectivity to adequately resolve the key components of rosemary oil. Thus a polyethylene glycol stationary phase was used in its place. Analyses were performed 5–10 times faster than the corresponding analysis with conventional columns. Our primary interest in fast GC relates to its application in comprehensive multidimensional gas chromatography (GC × GC) where fast operation of the second dimension columns is particularly important [8]. Short (ca. 1.5 m) narrow-bore (100 m I.D.) columns have been utilised in more than 80% of published GC × GC applications [9] in order to produce chromatograms over the required retention window, which is typically 2–8 s. However, by considering the events taking place inside GC × GC columns, Beens and coworkers concluded that 100 m I.D. second dimension columns may not be the best choice for GC × GC [9]. The use of a narrow second dimension column leads to very high average linear velocity in the second dimension column; high resistance to flow in these narrow columns leads to a high midpoint pressure, which in-turn reduces the optimum average linear carrier gas velocity in the first dimension column [9]. The combined result of these phenomena is slow total analysis time coupled with reduced second column efficiency. Thus we are interested in fast GC approach that utilises columns with low flow resistance. In practical terms, this means the use of wide bore and/or shorter columns. This topic
S.D.H. Poynter, R.A. Shellie / J. Chromatogr. A 1200 (2008) 28–33
is supported by outstanding theoretical and experimental works [10–12]. A straightforward means of operating 530 m I.D. columns at reduced pressure was realised by de Zeeuw et al. [11]. This method directly couples the outlet of the column to a mass spectrometer and the entire length of the column is operated at very low pressure. A restrictor has to be applied at the inlet side of the system to ensure that the column head pressure can be precisely maintained by electronic pressure control. Later van Deursen et al. [12] explored three options for producing the restriction at the inlet of the wide-bore column, including (1) the use of a micro-injection valve (2) the use of a supercritical fluid chromatography restrictor and (3) the use of a narrow capillary, as employed by de Zeeuw and coworkers. Comparable performance was reported for the latter two options, both being slightly better than the micro-injection valve, which was thought to contribute to band broadening by additional dead-volume effects. Optimum average carrier gas velocity u¯ is proportional to the average binary gas-phase diffusion coefficient and both of these vary inversely with pressure, so the low-pressure GC arrangement opens opportunity for fast separations. Low-pressure GC–MS has been used for analysis of lanolin steryl esters [13], pesticide analysis [14–17] and for environmental contaminants [18–21]. The authors know only of a single study of low-pressure GC–MS with 530 m I.D. columns for essential oil analysis [22] in which the analysis of Turnera diffusa (Ward.) Urb. essential oil was performed in 3 min and compared to conventional analysis using a 30 min temperature program with a 200 m I.D. column. It is noteworthy that van Deursen and coworkers specifically stated “A distinct disadvantage of wide-bore columns is that the plate-number is not very high. This system therefore is not very suitable for complex separations” [12]. While this point may have discouraged the application of low-pressure GC for essential oil separations, we have found that highly satisfactory results are achievable. Thus the present study investigates the benefits of low-pressure GC for the analysis of essential oils, in terms of peak capacity, separation speed, and sample capacity. The
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work described here is important because it compares wide-bore columns with the more accepted narrow-bore columns in fast GC–MS analysis. Only by directly comparing the performance of these two separation systems side by side, as we have done in the present study can these benefits be truly appreciated. Translation of these findings to GC × GC–MS with 530 m I.D. second dimension columns is currently underway and this will be reported in future correspondence. 2. Experimental All analyses were performed using a Shimadzu QP2010 Plus GC–MS equipped with split/splitless injector and AOC20 autoinjector (Shimadzu Scientific Instruments Oceania, Mt Waverley, Australia). A 12 m × 530 m I.D. capillary column coated with a thin layer (0.25 m film thickness) of 5% phenyl polysilphenylene–siloxane (BPX-5) stationary phase (SGE Analytical Science, Ringwood, Australia) was employed throughout for the low-pressure GC separations. A 0.60 m × 100 m I.D. length of deactivated fused silica tubing (SGE Analytical Science) was connected to the inlet of the analytical column using a stainless steel union and appropriately sized SilTite metal ferrules (SGE Analytical Science). The narrow-bore restrictor column was inserted a few mm into the wide bore analytical column to ensure that there were no dead-volume effects caused by the union. Narrow-bore GC–MS separations were performed using a 10 m × 100 m I.D. capillary column coated with a thin layer (0.10 m film thickness) of 5% phenyl polysilphenylene–siloxane (BPX-5) stationary phase (SGE Analytical Science). The chromatograms presented here were acquired using the following instrument settings. The injector temperature was 250 ◦ C in all cases. An injection volume of 1.0 L was delivered using the AOC20 autoinjector and a split ratio of 250:1 was employed for all injections. The carrier gas was helium and the average linear carrier gas velocity used was 89 cm/s for the 530 m I.D. column and 33 cm/s for the 100 m I.D. column. The MS transfer line was set at 250 ◦ C and the MS ion source was set at 200 ◦ C for all analyses. Full-scan mass spectra were acquired
Fig. 1. GC–MS (TIC) chromatogram of parsley essential oil acquired using a 10 m × 100 m I.D. BPX-5 capillary column (left) and using a 12 m × 530 m I.D. BPX-5 capillary column (right). Peak numbers refer to those in Table 1. Expanded chromatograms with complete annotation can be provided by the corresponding author upon request.
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S.D.H. Poynter, R.A. Shellie / J. Chromatogr. A 1200 (2008) 28–33
using a 70 ms scan period (14.29 Hz data acquisition) over the mass range of 40–455 u using a quadrupole scan rate of 10,000 u/s. The temperature programs employed for each column were 40 ◦ C (hold 0.21 min); 40–240 ◦ C in 4.28 min; 240 ◦ C (hold 0.21 min), giving a total time of 4.7 min for the 530 m I.D. column and 40 ◦ C (hold 0.5 min); 40–240 ◦ C in 10 min; 240 ◦ C (hold 0.5 min), giving a total time of 11 min for the 100 m I.D. column. A C8 –C20 n-alkane standard was purchased from Sigma–Aldrich (Castle Hill, Australia) and used as supplied. Fennel and parsley essential oils were provided by Essential Oils of Tasmania (Kingston, Australia). The samples were obtained by hydrodistillation. The oils were diluted to 1% (v/v) in dichloromethane (Sigma–Aldrich) prior to analysis. All data acquisition and analysis was performed using Shimadzu GCMS Solution software Version 2.51 and utilised commercial mass spectral libraries.
3. Results and discussion
Fig. 2. Comparison of resolution achieved between the limonene (9) and phellandrene (10) peak pair using a 10 m × 100 m I.D. capillary column (top) and a 12 m × 530 m I.D. capillary column (bottom).
Fig. 3. Comparison of sample capacity available by using a major component myristicin using a 10 m × 100 mm I.D. capillary column (top) and a 12 m × 530 mm I.D. capillary column (bottom).
Characterisation of many essential oils requires very highresolution techniques and may not be amenable to high-speed analysis. However, those with fewer components are more suitable for fast GC using columns with fewer theoretical plates. Fennel and parsley essential oils are produced for the international flavour market and exhibit moderate complexity. Thus the present investigation examines at the feasibility of applying lower peak capacity separations to the analysis of fennel and parsley essential oils using a wide-bore column (530 m I.D.) that was operated at subambient column pressure. First, a 10 m × 100 m I.D. column was used to generate a set of benchmark results upon which the quality of the separations achieved using the 12 m × 530 m I.D. column could be measured. An initial carrier gas flow rate of 0.4 mL/min (helium) was used for all analyses using the 100 m I.D. column. This carrier gas flow rate was selected according to recognised criteria for opti-
S.D.H. Poynter, R.A. Shellie / J. Chromatogr. A 1200 (2008) 28–33
mising flow rates in GC [23]. The temperature program rate for essential oil analysis was empirically optimised by performing analysis of a C8 –C20 n-alkane mix using temperature program rates of 5, 10, 15, 20 and 28 ◦ C/tM . Predictably, the results of these analyses showed that a faster temperature program rate leads to shorter analysis time, however peak capacity, and therefore the suitability of the method, simultaneously decreases. A temperature program rate of 20 ◦ C/min (10 ◦ C/tM ) was selected as a good compromise between speed and efficiency by using the weighting function described by Blumberg and Klee [24]. There was no
31
observable difference in retention time repeatability between the separations performed using the 100 m or 530 m I.D. columns; neither approach exceeded 0.15% RSD for retention time repeatability of selected reference compounds (terpinen-4-ol, dodecane and decan-1-ol). Peak response precision did not exceed 10% RSD in the linear chromatography range in either approach. A typical chromatogram illustrating the separation of parsley essential oil using a 100 m I.D. capillary and employing the optimised conditions described above is shown in Fig. 1 (left). The separation is performed in around 600 s and provides satisfac-
Table 1 Peak assignments for parsley and fennel essential oil illustrating library match quality for identified components
The parsley essential oil was analysed using both column configurations. Peaks were identified using Shimadzu GCMS Solution library search similarity search results and linear retention index.
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S.D.H. Poynter, R.A. Shellie / J. Chromatogr. A 1200 (2008) 28–33
tory peak capacity to separate the moderately complex sample. For comparative purposes a typical chromatogram that illustrates the separation of parsley essential oil using a 530 m I.D. column operated at sub-ambient pressure is shown in Fig. 1 (right). Each separation was achieved using optimised flow rate and both use the same normalised heating rate of 10 ◦ C/tM . An approximate 2.5-fold speed gain is apparent for the 530 m I.D. column separation, but this is accompanied by a concurrent loss in efficiency. The loss of efficiency is highlighted (Fig. 2) by expanding the limonene/-phellandrene peak pair, which are baseline resolved using the narrow-bore column but these two components are almost completely overlapped in the chromatogram acquired using the wide-bore column. A reduction of the temperature program rate might partially alleviate the peak overlap observed on the widebore column, and it is expected that the use of a faster temperature program rate would speed up the analysis using the narrow-bore column, however neither of these steps were followed to maintain a valid comparison of the two approaches. Quantitative comparison of performance (using separation number SN) of the two separation systems was made with one of the fastest separations reported in the literature that employed a conventionally heated GC oven [4]. In this fast separation using a 50 m I.D. column SN = 28 for the C8 , C9 homologue pair was reported. In the present investigation the measured performance figures are SN = 27 and SN = 10 for the narrow-bore and wide-bore columns, respectively. For the C19 , C20 homologue pair SN = 20 was achieved with a 50 m I.D. column and under the analysis conditions used in the present investigation the measured values were SN = 13 and SN = 6 for the 100 m I.D. and 530 m I.D. columns, respectively. Clearly the performance of the wide-bore column set up falls far short of the narrower counterparts, however sample capacity is also an important consideration for GC–MS analysis of essential oils, particularly if the goal of the analysis is to completely characterise the sample. Lower total ion current (TIC) peak height often reduces the library match quality and leads to ambiguous peak assignments. While there are other important considerations that determine the suitability of a scanning mass spectrometer for fast GC peaks such as peak width, data acquisition rate and scan range [25], the effect of these variables was essentially cancelled out between the two systems, which used identical MS settings. Mass spectral skewing is an undesirable phenomenon that appears to be suppressed using new generation rapid scanning quadrupole instruments. Identification of all peaks in the current study was performed using a single scan around the peak apex (i.e. we did not average spectra across peaks). The occurrence of MS skewing seemed minimal in this work (data not shown), which is consistent with other studies using the same instrumentation employed in this work [26]. In general the 530 m I.D. column resulted in taller TIC peaks and better library match than the 100 m I.D. column. This trend was observed quantitatively by comparison of LOD values for selected standards; terpinen-4-ol, dodecane and decan-1-ol. LOD using the method described in Section 2 was determined to be 3.8 ppm versus 12 ppm (terpinen-4-ol m/z 71 extracted ion chromatogram, EIC), 1.1 ppm versus 2.5 ppm (dodecane m/z 57 EIC) and 11 ppm versus 15 ppm (decan-1-ol m/z 55 EIC) for the wide-bore versus narrow-bore columns, respectively. These values should be used for comparative purposes only because clearly lower levels could be reached by modifying the injection, but this effort is beyond the scope of the present investigation. For instance, the approximate percent abundance (measured as percentage of GC–MS total ion current) of ␣-phellandrene in parsley essential oil is <1% so it is considered to be a minor component. The peak shape of ␣-phellandrene in each of these chromatograms is satisfactory, with measured asymmetry being 1.2 and 1.1 for the 100 m I.D. and 530 m I.D. columns, respectively. However, by
Fig. 4. GC–MS (TIC) chromatogram of fennel essential oil acquired using a 12 m × 530 m I.D. BPX-5 capillary column. Peak numbers refer to those in Table 1. Expanded chromatograms with complete annotation can be provided by the corresponding author upon request.
visual comparison of a major component myristicin (ca. 20% abundance) within these chromatograms (Fig. 3), the reduced sample capacity of the 100 m I.D. column is clearly evident for peaks of quite similar peak area (2.6 × 106 in each chromatogram). Here the major component is quite severely overloaded in the narrowbore column (As = 0.34) while the peak is well within the bounds of linear chromatography in the 530 m I.D. column (As = 1.0). While the quantitative comparison of efficiency indicated that the narrow-bore column is far superior, the increased propensity for band broadening in the narrow-bore column indicates that maximum utilisation of the available peak capacity would be difficult to achieve while keeping the TIC peak height at a sufficiently high level to afford high quality spectra. This is further highlighted in Table 1, where the library match quality for the identified components of parsley essential oil is reported. On average the library match quality for separated components was 5% better in the separation performed with the wide-bore column. For some components, such as E--ocimene it was impossible to obtain unambiguous peak assignment using the narrow-bore column without increasing the mass of essential oil injected into the column. Identical conditions were employed for the analysis of fennel essential oil using the wide-bore column arrangement. The results indicated that the 530 m I.D. column configuration is also appropriate for the analysis of fennel oil which also exhibits low-moderate complexity. Qualitative results for this sample are presented in Table 1 and the chromatogram of the fennel essential oil is presented in Fig. 4. 4. Concluding remarks High throughput analysis is highly desirable in many situations and this can be performed by reduced performance employing fast GC approaches such as low-pressure GC–MS as described here. Parsley and fennel essential oil are among those produced in Tasmania for the international flavour market and while the Tasmanian essential oil industry has a reputation as a reliable supplier of consistent quality essential oil products, maintaining this reputation depends upon regular quality monitoring. The fast GC–MS approaches described here are highly satisfactory for this monitoring purpose. We are currently investigating the suitability of
S.D.H. Poynter, R.A. Shellie / J. Chromatogr. A 1200 (2008) 28–33
wide-bore second dimension columns in GC × GC–MS to determine how well the findings of this paper translate to GC × GC analysis, and have obtained satisfactory preliminary results [27]. These results will be reported elsewhere. Acknowledgements This research was supported under the Australian Research Council’s Discovery funding scheme (project number DP0771893). The generous support of Shimadzu Scientific Instruments is gratefully acknowledged. The authors also wish to thank Essential Oils of Tasmania for providing samples for analysis. References ´ H.-G. Janssen, E. Matisova, ´ U.A.Th. Brinkman, Trends Anal. Chem. 21 [1] P. Korytar, (2002) 558. [2] P. Donato, P.Q. Tranchida, P. Dugo, G. Dugo, L. Mondello, J. Sep. Sci. 30 (2007) 508. [3] F. David, D.R. Gere, F. Scanlan, P. Sandra, J. Chromatogr. A 842 (1999) 309. [4] L. Mondello, R. Shellie, A. Casilli, P.Q. Tranchida, P. Marriott, G. Dugo, J. Sep. Sci. 27 (2004) 699. [5] C. Bicchi, C. Brunelli, C. Cordero, P. Rubiolo, M. Galli, A. Sironi, J. Chromatogr. A 1024 (2004) 195. [6] P. Magni, R. Facchetti, A. Cadoppi, F. Pigozzo, C. Brunelli, LC–GC Eur. Appl. Book 59 (2004) 2. [7] C. Bicchi, C. Brunelli, M. Galli, A. Sironi, J. Chromatogr. A 931 (2001) 129. [8] P. Marriott, R. Shellie, Trends Anal. Chem. 21 (2002) 573.
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[9] J. Beens, H.-G. Janssen, M. Adahchour, U.A.Th. Brinkman, J. Chromatogr. A 1086 (2005) 141. [10] C.A. Cramers, G.J. Scherpenzeel, P.A. Leclercq, J. Chromatogr. 203 (1981) 207. [11] J. de Zeeuw, J. Peene, H.-G. Janssen, X. Lou, J. High Resolut. Chromatogr. 12 (2000) 677. [12] M. van Deursen, H.-G. Janssen, J. Beens, P. Lipman, R. Reinierkens, G. Rutten, C. Cramers, J. Microcol. Sep. 12 (2000) 613. [13] E. Jover, Z. Moldovan, J.M. Bayona, J. Chromatogr. A 970 (2002) 249. ´ S.J. Lehotay, J. Hajˇslova, ´ J. Chromatogr. A 926 (2001) 291. [14] K. Maˇstovska, ´ [15] F.J. Arrebola, J.L. Mart´ınez Vidal, M.J. Gonzalez-Rodr´ ıguez, A. Garrido-Frenich, N. Sanchez Morito, J. Chromatogr. A 1005 (2003) 131. ´ [16] A. Garrido-Frenich, F.J. Arrebola, M.J. Gonzalez-Rodr´ ıguez, J.L. Mart´ınez Vidal, N. Mora D´ıez, Anal. Bioanal. Chem. 377 (2003) 1038. ´ J. Hajˇslova, ´ S.J. Lehotay, J. Chromatogr. A 1054 (2004) 291. [17] K. Maˇstovska, [18] K. Ravindra, A.F.L. Godoi, L. Bencs, R. van Grieken, J. Chromatogr. A 1114 (2006) 278. [19] J.W. Cochran, J. Chromatogr. Sci. 40 (2002) 254. [20] P.E. Joos, A.F.L. Godoi, R. de Jong, J. de Zeeuw, R. van Grieken, J. Chromatogr. A 985 (2003) 191. [21] A.F.L. Godoi, K. Ravindra, R.H.M. Godoi, S.J. Andrade, M. Santiago-Silva, L. van Vaeck, R. van Grieken, J. Chromatogr. A 1027 (2004) 49. [22] A.F.L. Godoi, W. Vilegas, R.H.M. Godoi, L. van Vaeck, R. van Grieken, J. Chromatogr. A 1027 (2004) 127. [23] M.S. Klee, L.M. Blumberg, J. Chromatogr. Sci. 40 (2002) 234. [24] L.M. Blumberg, M.S. Klee, J. Microcol. Sep. 12 (2000) 508. [25] M. Adachour, M. Brandt, H.-U. Baier, R.J.J. Vreuls, A.M. Batenburg, U.A.Th. Brinkman, J. Chromatogr. A 1067 (2005) 245. [26] L. Mondello, D. Sciarrone, A. Casilli, P.Q. Tranchida, P. Dugo, G. Dugo, J. Sep. Sci. 30 (2007) 1905. [27] R.A. Shellie, S.D.H. Poynter, Alternative column dimensions for GC × GC–MS, in: 31st International Symposium on Capillary Chromatography and Electrophoresis, Albuquerque, NM, November 28–30, 2007.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
High-speed, low-pressure gas chromatography–mass spectrometry for essential oil analysis Samuel D.H. Poynter, Robert A. Shellie ∗ Australian Centre for Research on Separation Science (ACROSS), School of Chemistry, University of Tasmania, Private Bag 75, Hobart 7001, Australia
a r t i c l e
i n f o
Article history: Available online 28 March 2008 Keywords: Essential oil Fast gas chromatography Mass spectrometry Low-pressure gas chromatography
a b s t r a c t Analysis of parsley and fennel essential oils was performed by using low-pressure gas chromatography– mass spectrometry (GC–MS). The low-pressure instrument configuration was achieved by fitting a GC–MS instrument with a 530 m I.D. capillary column and an appropriate capillary restrictor at the inlet of the column. Comparison of the performance of the low-pressure GC–MS setup was made with fast GC–MS using a narrow-bore capillary column. By comparing the two approaches side-by-side the benefits of low-pressure GC–MS for characterisation of moderately complex essential oils comprising less than 50 detectable components can be fully appreciated. Although efficiency is sacrificed, the improved sample capacity of the 530 m I.D. column leads to higher peak intensities and in-turn better mass spectral library matching thus providing highly satisfactory results. © 2008 Elsevier B.V. All rights reserved.
1. Introduction There are a variety of practical routes for achieving high-speed separations of complex samples [1] and many of them have been employed for essential oil analysis. Most commonly 10 m × 100 m I.D. columns (with appropriately reduced stationary phase film thickness) have been employed in place of 25 m × 250 m I.D. columns [2] and it has been shown many times this approach leads to a significant speed gain while preserving resolution [3]. In a similar way Mondello et al. [4] demonstrated fast analysis of lime essential oil using a 5 m length of 50 m I.D. capillary with a 0.05 m stationary phase coating. Lime oil analysis was achieved in around 90 s by employing the fastest possible temperature program rate of the GC oven (50–150 ◦ C in 75 s, 150–200 ◦ C in 43 s, 200–250 ◦ C in 55 s) as well as a higher than optimum average linear carrier gas velocity of 120 cm/s. An additional approach for achieving faster analysis includes direct resistively heated column GC [5] which, by marrying rapid temperature programming (up to 20 ◦ C/s) with fast data acquisition (using flame ionization detection) and high split ratio [6] can lead to essential oil analysis times between 40 and 100 s [5,6]. Fast analysis approaches often try to maintain efficiency, however having highlighted that the efficiency of a capillary column often exceeds analytical requirements, Bicchi and coworkers investigated the use of short 250–320 m I.D. columns with appropriate selectivity for the analysis of rosemary (Rosmarinus officinalis L.)
∗ Corresponding author. Tel.: +61 3 6226 7656; fax: +61 3 6226 2858. E-mail address: [email protected] (R.A. Shellie). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.03.069
and chamomile (Matricaria recutita L.) essential oils [7]. Both of these essential oils are considered as moderate complexity samples, and effective analysis was performed by substituting the standard (ca. 25 m) capillary column comprising around 150,000 theoretical plates with 5 m columns comprising 20,000–50,000 theoretical plates. A 5% phenyl- 5% vinyl-polydimethylsiloxane stationary phase was used for the separation of chamomile oil but lacked the selectivity to adequately resolve the key components of rosemary oil. Thus a polyethylene glycol stationary phase was used in its place. Analyses were performed 5–10 times faster than the corresponding analysis with conventional columns. Our primary interest in fast GC relates to its application in comprehensive multidimensional gas chromatography (GC × GC) where fast operation of the second dimension columns is particularly important [8]. Short (ca. 1.5 m) narrow-bore (100 m I.D.) columns have been utilised in more than 80% of published GC × GC applications [9] in order to produce chromatograms over the required retention window, which is typically 2–8 s. However, by considering the events taking place inside GC × GC columns, Beens and coworkers concluded that 100 m I.D. second dimension columns may not be the best choice for GC × GC [9]. The use of a narrow second dimension column leads to very high average linear velocity in the second dimension column; high resistance to flow in these narrow columns leads to a high midpoint pressure, which in-turn reduces the optimum average linear carrier gas velocity in the first dimension column [9]. The combined result of these phenomena is slow total analysis time coupled with reduced second column efficiency. Thus we are interested in fast GC approach that utilises columns with low flow resistance. In practical terms, this means the use of wide bore and/or shorter columns. This topic
S.D.H. Poynter, R.A. Shellie / J. Chromatogr. A 1200 (2008) 28–33
is supported by outstanding theoretical and experimental works [10–12]. A straightforward means of operating 530 m I.D. columns at reduced pressure was realised by de Zeeuw et al. [11]. This method directly couples the outlet of the column to a mass spectrometer and the entire length of the column is operated at very low pressure. A restrictor has to be applied at the inlet side of the system to ensure that the column head pressure can be precisely maintained by electronic pressure control. Later van Deursen et al. [12] explored three options for producing the restriction at the inlet of the wide-bore column, including (1) the use of a micro-injection valve (2) the use of a supercritical fluid chromatography restrictor and (3) the use of a narrow capillary, as employed by de Zeeuw and coworkers. Comparable performance was reported for the latter two options, both being slightly better than the micro-injection valve, which was thought to contribute to band broadening by additional dead-volume effects. Optimum average carrier gas velocity u¯ is proportional to the average binary gas-phase diffusion coefficient and both of these vary inversely with pressure, so the low-pressure GC arrangement opens opportunity for fast separations. Low-pressure GC–MS has been used for analysis of lanolin steryl esters [13], pesticide analysis [14–17] and for environmental contaminants [18–21]. The authors know only of a single study of low-pressure GC–MS with 530 m I.D. columns for essential oil analysis [22] in which the analysis of Turnera diffusa (Ward.) Urb. essential oil was performed in 3 min and compared to conventional analysis using a 30 min temperature program with a 200 m I.D. column. It is noteworthy that van Deursen and coworkers specifically stated “A distinct disadvantage of wide-bore columns is that the plate-number is not very high. This system therefore is not very suitable for complex separations” [12]. While this point may have discouraged the application of low-pressure GC for essential oil separations, we have found that highly satisfactory results are achievable. Thus the present study investigates the benefits of low-pressure GC for the analysis of essential oils, in terms of peak capacity, separation speed, and sample capacity. The
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work described here is important because it compares wide-bore columns with the more accepted narrow-bore columns in fast GC–MS analysis. Only by directly comparing the performance of these two separation systems side by side, as we have done in the present study can these benefits be truly appreciated. Translation of these findings to GC × GC–MS with 530 m I.D. second dimension columns is currently underway and this will be reported in future correspondence. 2. Experimental All analyses were performed using a Shimadzu QP2010 Plus GC–MS equipped with split/splitless injector and AOC20 autoinjector (Shimadzu Scientific Instruments Oceania, Mt Waverley, Australia). A 12 m × 530 m I.D. capillary column coated with a thin layer (0.25 m film thickness) of 5% phenyl polysilphenylene–siloxane (BPX-5) stationary phase (SGE Analytical Science, Ringwood, Australia) was employed throughout for the low-pressure GC separations. A 0.60 m × 100 m I.D. length of deactivated fused silica tubing (SGE Analytical Science) was connected to the inlet of the analytical column using a stainless steel union and appropriately sized SilTite metal ferrules (SGE Analytical Science). The narrow-bore restrictor column was inserted a few mm into the wide bore analytical column to ensure that there were no dead-volume effects caused by the union. Narrow-bore GC–MS separations were performed using a 10 m × 100 m I.D. capillary column coated with a thin layer (0.10 m film thickness) of 5% phenyl polysilphenylene–siloxane (BPX-5) stationary phase (SGE Analytical Science). The chromatograms presented here were acquired using the following instrument settings. The injector temperature was 250 ◦ C in all cases. An injection volume of 1.0 L was delivered using the AOC20 autoinjector and a split ratio of 250:1 was employed for all injections. The carrier gas was helium and the average linear carrier gas velocity used was 89 cm/s for the 530 m I.D. column and 33 cm/s for the 100 m I.D. column. The MS transfer line was set at 250 ◦ C and the MS ion source was set at 200 ◦ C for all analyses. Full-scan mass spectra were acquired
Fig. 1. GC–MS (TIC) chromatogram of parsley essential oil acquired using a 10 m × 100 m I.D. BPX-5 capillary column (left) and using a 12 m × 530 m I.D. BPX-5 capillary column (right). Peak numbers refer to those in Table 1. Expanded chromatograms with complete annotation can be provided by the corresponding author upon request.
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using a 70 ms scan period (14.29 Hz data acquisition) over the mass range of 40–455 u using a quadrupole scan rate of 10,000 u/s. The temperature programs employed for each column were 40 ◦ C (hold 0.21 min); 40–240 ◦ C in 4.28 min; 240 ◦ C (hold 0.21 min), giving a total time of 4.7 min for the 530 m I.D. column and 40 ◦ C (hold 0.5 min); 40–240 ◦ C in 10 min; 240 ◦ C (hold 0.5 min), giving a total time of 11 min for the 100 m I.D. column. A C8 –C20 n-alkane standard was purchased from Sigma–Aldrich (Castle Hill, Australia) and used as supplied. Fennel and parsley essential oils were provided by Essential Oils of Tasmania (Kingston, Australia). The samples were obtained by hydrodistillation. The oils were diluted to 1% (v/v) in dichloromethane (Sigma–Aldrich) prior to analysis. All data acquisition and analysis was performed using Shimadzu GCMS Solution software Version 2.51 and utilised commercial mass spectral libraries.
3. Results and discussion
Fig. 2. Comparison of resolution achieved between the limonene (9) and phellandrene (10) peak pair using a 10 m × 100 m I.D. capillary column (top) and a 12 m × 530 m I.D. capillary column (bottom).
Fig. 3. Comparison of sample capacity available by using a major component myristicin using a 10 m × 100 mm I.D. capillary column (top) and a 12 m × 530 mm I.D. capillary column (bottom).
Characterisation of many essential oils requires very highresolution techniques and may not be amenable to high-speed analysis. However, those with fewer components are more suitable for fast GC using columns with fewer theoretical plates. Fennel and parsley essential oils are produced for the international flavour market and exhibit moderate complexity. Thus the present investigation examines at the feasibility of applying lower peak capacity separations to the analysis of fennel and parsley essential oils using a wide-bore column (530 m I.D.) that was operated at subambient column pressure. First, a 10 m × 100 m I.D. column was used to generate a set of benchmark results upon which the quality of the separations achieved using the 12 m × 530 m I.D. column could be measured. An initial carrier gas flow rate of 0.4 mL/min (helium) was used for all analyses using the 100 m I.D. column. This carrier gas flow rate was selected according to recognised criteria for opti-
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mising flow rates in GC [23]. The temperature program rate for essential oil analysis was empirically optimised by performing analysis of a C8 –C20 n-alkane mix using temperature program rates of 5, 10, 15, 20 and 28 ◦ C/tM . Predictably, the results of these analyses showed that a faster temperature program rate leads to shorter analysis time, however peak capacity, and therefore the suitability of the method, simultaneously decreases. A temperature program rate of 20 ◦ C/min (10 ◦ C/tM ) was selected as a good compromise between speed and efficiency by using the weighting function described by Blumberg and Klee [24]. There was no
31
observable difference in retention time repeatability between the separations performed using the 100 m or 530 m I.D. columns; neither approach exceeded 0.15% RSD for retention time repeatability of selected reference compounds (terpinen-4-ol, dodecane and decan-1-ol). Peak response precision did not exceed 10% RSD in the linear chromatography range in either approach. A typical chromatogram illustrating the separation of parsley essential oil using a 100 m I.D. capillary and employing the optimised conditions described above is shown in Fig. 1 (left). The separation is performed in around 600 s and provides satisfac-
Table 1 Peak assignments for parsley and fennel essential oil illustrating library match quality for identified components
The parsley essential oil was analysed using both column configurations. Peaks were identified using Shimadzu GCMS Solution library search similarity search results and linear retention index.
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tory peak capacity to separate the moderately complex sample. For comparative purposes a typical chromatogram that illustrates the separation of parsley essential oil using a 530 m I.D. column operated at sub-ambient pressure is shown in Fig. 1 (right). Each separation was achieved using optimised flow rate and both use the same normalised heating rate of 10 ◦ C/tM . An approximate 2.5-fold speed gain is apparent for the 530 m I.D. column separation, but this is accompanied by a concurrent loss in efficiency. The loss of efficiency is highlighted (Fig. 2) by expanding the limonene/-phellandrene peak pair, which are baseline resolved using the narrow-bore column but these two components are almost completely overlapped in the chromatogram acquired using the wide-bore column. A reduction of the temperature program rate might partially alleviate the peak overlap observed on the widebore column, and it is expected that the use of a faster temperature program rate would speed up the analysis using the narrow-bore column, however neither of these steps were followed to maintain a valid comparison of the two approaches. Quantitative comparison of performance (using separation number SN) of the two separation systems was made with one of the fastest separations reported in the literature that employed a conventionally heated GC oven [4]. In this fast separation using a 50 m I.D. column SN = 28 for the C8 , C9 homologue pair was reported. In the present investigation the measured performance figures are SN = 27 and SN = 10 for the narrow-bore and wide-bore columns, respectively. For the C19 , C20 homologue pair SN = 20 was achieved with a 50 m I.D. column and under the analysis conditions used in the present investigation the measured values were SN = 13 and SN = 6 for the 100 m I.D. and 530 m I.D. columns, respectively. Clearly the performance of the wide-bore column set up falls far short of the narrower counterparts, however sample capacity is also an important consideration for GC–MS analysis of essential oils, particularly if the goal of the analysis is to completely characterise the sample. Lower total ion current (TIC) peak height often reduces the library match quality and leads to ambiguous peak assignments. While there are other important considerations that determine the suitability of a scanning mass spectrometer for fast GC peaks such as peak width, data acquisition rate and scan range [25], the effect of these variables was essentially cancelled out between the two systems, which used identical MS settings. Mass spectral skewing is an undesirable phenomenon that appears to be suppressed using new generation rapid scanning quadrupole instruments. Identification of all peaks in the current study was performed using a single scan around the peak apex (i.e. we did not average spectra across peaks). The occurrence of MS skewing seemed minimal in this work (data not shown), which is consistent with other studies using the same instrumentation employed in this work [26]. In general the 530 m I.D. column resulted in taller TIC peaks and better library match than the 100 m I.D. column. This trend was observed quantitatively by comparison of LOD values for selected standards; terpinen-4-ol, dodecane and decan-1-ol. LOD using the method described in Section 2 was determined to be 3.8 ppm versus 12 ppm (terpinen-4-ol m/z 71 extracted ion chromatogram, EIC), 1.1 ppm versus 2.5 ppm (dodecane m/z 57 EIC) and 11 ppm versus 15 ppm (decan-1-ol m/z 55 EIC) for the wide-bore versus narrow-bore columns, respectively. These values should be used for comparative purposes only because clearly lower levels could be reached by modifying the injection, but this effort is beyond the scope of the present investigation. For instance, the approximate percent abundance (measured as percentage of GC–MS total ion current) of ␣-phellandrene in parsley essential oil is <1% so it is considered to be a minor component. The peak shape of ␣-phellandrene in each of these chromatograms is satisfactory, with measured asymmetry being 1.2 and 1.1 for the 100 m I.D. and 530 m I.D. columns, respectively. However, by
Fig. 4. GC–MS (TIC) chromatogram of fennel essential oil acquired using a 12 m × 530 m I.D. BPX-5 capillary column. Peak numbers refer to those in Table 1. Expanded chromatograms with complete annotation can be provided by the corresponding author upon request.
visual comparison of a major component myristicin (ca. 20% abundance) within these chromatograms (Fig. 3), the reduced sample capacity of the 100 m I.D. column is clearly evident for peaks of quite similar peak area (2.6 × 106 in each chromatogram). Here the major component is quite severely overloaded in the narrowbore column (As = 0.34) while the peak is well within the bounds of linear chromatography in the 530 m I.D. column (As = 1.0). While the quantitative comparison of efficiency indicated that the narrow-bore column is far superior, the increased propensity for band broadening in the narrow-bore column indicates that maximum utilisation of the available peak capacity would be difficult to achieve while keeping the TIC peak height at a sufficiently high level to afford high quality spectra. This is further highlighted in Table 1, where the library match quality for the identified components of parsley essential oil is reported. On average the library match quality for separated components was 5% better in the separation performed with the wide-bore column. For some components, such as E--ocimene it was impossible to obtain unambiguous peak assignment using the narrow-bore column without increasing the mass of essential oil injected into the column. Identical conditions were employed for the analysis of fennel essential oil using the wide-bore column arrangement. The results indicated that the 530 m I.D. column configuration is also appropriate for the analysis of fennel oil which also exhibits low-moderate complexity. Qualitative results for this sample are presented in Table 1 and the chromatogram of the fennel essential oil is presented in Fig. 4. 4. Concluding remarks High throughput analysis is highly desirable in many situations and this can be performed by reduced performance employing fast GC approaches such as low-pressure GC–MS as described here. Parsley and fennel essential oil are among those produced in Tasmania for the international flavour market and while the Tasmanian essential oil industry has a reputation as a reliable supplier of consistent quality essential oil products, maintaining this reputation depends upon regular quality monitoring. The fast GC–MS approaches described here are highly satisfactory for this monitoring purpose. We are currently investigating the suitability of
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wide-bore second dimension columns in GC × GC–MS to determine how well the findings of this paper translate to GC × GC analysis, and have obtained satisfactory preliminary results [27]. These results will be reported elsewhere. Acknowledgements This research was supported under the Australian Research Council’s Discovery funding scheme (project number DP0771893). The generous support of Shimadzu Scientific Instruments is gratefully acknowledged. The authors also wish to thank Essential Oils of Tasmania for providing samples for analysis. References ´ H.-G. Janssen, E. Matisova, ´ U.A.Th. Brinkman, Trends Anal. Chem. 21 [1] P. Korytar, (2002) 558. [2] P. Donato, P.Q. Tranchida, P. Dugo, G. Dugo, L. Mondello, J. Sep. Sci. 30 (2007) 508. [3] F. David, D.R. Gere, F. Scanlan, P. Sandra, J. Chromatogr. A 842 (1999) 309. [4] L. Mondello, R. Shellie, A. Casilli, P.Q. Tranchida, P. Marriott, G. Dugo, J. Sep. Sci. 27 (2004) 699. [5] C. Bicchi, C. Brunelli, C. Cordero, P. Rubiolo, M. Galli, A. Sironi, J. Chromatogr. A 1024 (2004) 195. [6] P. Magni, R. Facchetti, A. Cadoppi, F. Pigozzo, C. Brunelli, LC–GC Eur. Appl. Book 59 (2004) 2. [7] C. Bicchi, C. Brunelli, M. Galli, A. Sironi, J. Chromatogr. A 931 (2001) 129. [8] P. Marriott, R. Shellie, Trends Anal. Chem. 21 (2002) 573.
33
[9] J. Beens, H.-G. Janssen, M. Adahchour, U.A.Th. Brinkman, J. Chromatogr. A 1086 (2005) 141. [10] C.A. Cramers, G.J. Scherpenzeel, P.A. Leclercq, J. Chromatogr. 203 (1981) 207. [11] J. de Zeeuw, J. Peene, H.-G. Janssen, X. Lou, J. High Resolut. Chromatogr. 12 (2000) 677. [12] M. van Deursen, H.-G. Janssen, J. Beens, P. Lipman, R. Reinierkens, G. Rutten, C. Cramers, J. Microcol. Sep. 12 (2000) 613. [13] E. Jover, Z. Moldovan, J.M. Bayona, J. Chromatogr. A 970 (2002) 249. ´ S.J. Lehotay, J. Hajˇslova, ´ J. Chromatogr. A 926 (2001) 291. [14] K. Maˇstovska, ´ [15] F.J. Arrebola, J.L. Mart´ınez Vidal, M.J. Gonzalez-Rodr´ ıguez, A. Garrido-Frenich, N. Sanchez Morito, J. Chromatogr. A 1005 (2003) 131. ´ [16] A. Garrido-Frenich, F.J. Arrebola, M.J. Gonzalez-Rodr´ ıguez, J.L. Mart´ınez Vidal, N. Mora D´ıez, Anal. Bioanal. Chem. 377 (2003) 1038. ´ J. Hajˇslova, ´ S.J. Lehotay, J. Chromatogr. A 1054 (2004) 291. [17] K. Maˇstovska, [18] K. Ravindra, A.F.L. Godoi, L. Bencs, R. van Grieken, J. Chromatogr. A 1114 (2006) 278. [19] J.W. Cochran, J. Chromatogr. Sci. 40 (2002) 254. [20] P.E. Joos, A.F.L. Godoi, R. de Jong, J. de Zeeuw, R. van Grieken, J. Chromatogr. A 985 (2003) 191. [21] A.F.L. Godoi, K. Ravindra, R.H.M. Godoi, S.J. Andrade, M. Santiago-Silva, L. van Vaeck, R. van Grieken, J. Chromatogr. A 1027 (2004) 49. [22] A.F.L. Godoi, W. Vilegas, R.H.M. Godoi, L. van Vaeck, R. van Grieken, J. Chromatogr. A 1027 (2004) 127. [23] M.S. Klee, L.M. Blumberg, J. Chromatogr. Sci. 40 (2002) 234. [24] L.M. Blumberg, M.S. Klee, J. Microcol. Sep. 12 (2000) 508. [25] M. Adachour, M. Brandt, H.-U. Baier, R.J.J. Vreuls, A.M. Batenburg, U.A.Th. Brinkman, J. Chromatogr. A 1067 (2005) 245. [26] L. Mondello, D. Sciarrone, A. Casilli, P.Q. Tranchida, P. Dugo, G. Dugo, J. Sep. Sci. 30 (2007) 1905. [27] R.A. Shellie, S.D.H. Poynter, Alternative column dimensions for GC × GC–MS, in: 31st International Symposium on Capillary Chromatography and Electrophoresis, Albuquerque, NM, November 28–30, 2007.