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mass spectrometry inlets
Journal of Chromatography A, 1356 (2014) 283–288
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Evaluation of hydrolysis and alcoholysis reactions in gas chromatography/mass spectrometry inlets Guomin Ai ∗ , Tong Sun, Xiuzhu Dong State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing 100101, China
a r t i c l e
i n f o
Article history: Received 5 March 2014 Received in revised form 9 May 2014 Accepted 11 June 2014 Available online 19 June 2014 Keywords: Hydrolysis Alcoholysis Septum particles GC inlet liner Methanol Ethanol
a b s t r a c t During gas chromatography/mass spectrometry (GC–MS) analyses using water and methanol as injection solvents, hydrolysis reactions after injecting water control and alcoholysis reactions after injecting methanol control or ethanol into a GC–MS system were observed and studied. Two dominant hydrolysis/alcoholysis product series were detected, and were identified as being HO (CH3 )2 Si OR and HO (CH3 )2 Si O (CH3 )2 Si OR, where R = H, methyl, or ethyl, when pure water, methanol and ethanol were injected. The chemical structures of the reaction products were cross-checked by injecting H2 O/D2 O and H2 O/MeOH/EtOH, and comparable EI mass fragmentation patterns were found. The water and alcohols injected reacted with silicones in septum particles which accumulated in the injection port liner after numerous injections, and both hydrolysis and alcoholysis reaction products gradually increased in concentration as the number of injections increased. Potential interferences from hydrolysis or alcoholysis reactions should be paid attention to, evaluated or eliminated when water or methanol was used as the GC or GC–MS solvent, and especially when underivatized methanol or ethanol was subject to GC and GC–MS analysis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Silicones have been used extensively as gas chromatographic (GC) stationary phases [1,2]. In addition, most septa used in GC instruments are made of silicone rubber materials, such as Agilent BTO® septa [3]. This type of silicone rubber-based septum may be prone to coring or shedding particles into the injection port liner after repeated injections, and this behavior is related to the inlet temperature and physical interactions with the syringe needle [3]. There has been much research into the thermal degradation of GC stationary phases, especially the degradation of silicones [1,4,5]. The formation of cyclic siloxanes from the thermal degradation of GC septum is also occasionally mentioned in such studies [4]. In addition to the thermal degradation of such GC stationary phases and GC septa, the re-activation of deactivated GC columns when water or alcohols such as methanol are introduced has been also observed based on the impairment of chromatographic performances that can be restored by resilylating the newly formed silanol groups on the column wall [6]. However, to the best of
our knowledge, almost no hydrolysis or alcoholysis products of GC stationary phases resulting from water or alcohols injection have been identified and reported in the literature, and no information is available on the occurrence of hydrolysis or alcoholysis reactions between the silicones that is used in the GC inlet and the polar solvents (e.g., water, methanol and ethanol) that are injected into GC or GC–MS systems. This short communication describes a study of the hydrolysis and alcoholysis reactions that occur in a GC inlet system when water and alcohols (methanol and ethanol) are injected, respectively. We started to acquire fundamental knowledge on the hydrolysis and alcohol hydrolysis products that may affect analytical performance and pose potential problem in identification of compounds in sample. The use of water and methanol as GC and GC–MS solvents, and the popular application of GC and GC–MS techniques for analyzing underivatized methanol and ethanol (such as distilled spirits), provided motivation for this study. 2. Experimental 2.1. Materials, reagents and chemicals
∗ Corresponding author. Tel.: +86 10 64807429; fax: +86 10 64807429. E-mail addresses: [email protected], [email protected] (G. Ai). http://dx.doi.org/10.1016/j.chroma.2014.06.036 0021-9673/© 2014 Elsevier B.V. All rights reserved.
Methanol (LC/MS grade) was purchased from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA). Water (LC/MS grade) and absolute
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Fig. 1. GC–MS chromatograms of the separate injection of pure water (a), ethanol (b) and methanol (c), 0.2 L each, in split (1/5) mode. Reaction products eluted at 9.263 and 11.002 min from 100% methanol injection, 9.542 and 11.195 min from 100% ethanol injection, and 13.792, 18.107, 22.018, and 23.765 min from 100% H2 O injection were detected.
ethanol (HPLC grade) were purchased from Duksan Pure Chemicals Co. Ltd. (Ansan, South Korea). D2 O (99.990% D) was purchased from Sigma-Aldrich (Steinheim, Germany).
3. Results and discussion
2.2. GC–MS analysis
When pure water, methanol or ethanol was injected into the GC–MS system, four product peaks (eluted at 13.792, 18.107, 22.018, and 23.765 min) were found from water, two peaks (eluted at 9.263 and 11.002 min) were found from methanol, and two peaks (eluted at 9.542 and 11.195 min) were found from ethanol, as shown in Fig. 1. The retention times of all the product peaks were constant during all of the experiments. However, none of the product peaks were detected when either air injections or empty injections (manually started GC–MS runs without an injection) were performed. Therefore, we concluded that the peaks were for products of reactions between water or alcohol and the organic matter in the GC–MS (specifically in the GC) system. These reaction products were identified (or speculatively identified), and the positions in the GC system where the reactions took place were identified, as described below.
GC–MS analyses were performed on an Agilent GC–MS system (7890A gas chromatograph and 5975C mass-selective detector; Agilent Technologies, Santa Clara, CA, USA) equipped with a DBWAX column (30 m long, 250 m i.d., 0.25 m film thickness; Agilent Technologies). The inlet liner was an Agilent MS-certified split/splitless liner (part no. 5188–6576; 4.0 mm i.d., 870 L volume, single taper, glass wool positioned in the middle of the liner). The septum was an Agilent BTO® septum (part no. 5183–4757; 11 mm diameter with “CenterGuide”). The GC conditions were as follows: split injection (injector temperature 230 ◦ C, split 1/5 was used to place as much sample onto the column as possible); oven temperature, programmed from 35 ◦ C (held for 3 min) to 47 ◦ C at 5 ◦ C/min, then to 100 ◦ C at 25 ◦ C/min, then to 145 ◦ C at 2.5 ◦ C/min, and then to 200 ◦ C (held for 5 min) at 25 ◦ C/min; the post-injection dwell time, 0.04 min; carrier gas and flow, He 1.0 mL/min; interface temperature, 160 ◦ C. The injection volume was 0.2 L. Under these conditions, the relative compounds to this work were (with elution times, in minutes, in brackets) methanol (3.8), ethanol (4.6), water (6.8), products 1 (9.3) and 2 (11.0) (from pure methanol injections), products 1 (9.5) and 2 (11.2) (from pure ethanol injections), products 1 (13.8), 2 (18.1), 3 (22.0), and 4 (23.8) (from pure water injections). The MS was used in electron impact (EI) ionization mode, with electron energy of 70 eV, an ion source temperature of 230 ◦ C, and a quadrupole temperature of 150 ◦ C. Data were acquired in full-scan (m/z 10–500) mode. Solvent delay of 12.0, 8.3, 9.0 and 8.55 min were used when pure water (and D2 O), methanol, ethanol, and a mixture of methanol and ethanol (1:1, v/v) were injected, respectively. Data were acquired and analyzed using Enhanced ChemStation (Version E.02.00.493, Agilent Technologies). In order to measure changes in the responses of the reaction products as the number of injections increased, water and the mixture of methanol and ethanol (1:1, v/v) were alternately injected. For reducing the sample run time, a shorter GC oven program was run from the 32nd to the 59th, the 64th to the 129th, and the 134th to the 179th injections. The shorter GC oven program was: 100 ◦ C, increased at 25 ◦ C/min to 200 ◦ C, which was held for 3 min. A solvent delay of 6.8 min (the total short run time was 7 min) was used, so that no analytes were detected during these sample runs.
3.1. Reaction product peaks caused by water/alcohol injection
3.2. Identification of the hydrolysis/alcoholysis products We acquired EI mass spectra of the water injection reaction products eluted at 18.11 and 22.02 min (a2 and a3 in Fig. 2), the methanol injection products eluted at 9.26 and 11.00 min (a1 and a2 in Fig. 3), and the ethanol injection products eluted at 9.54 and 11.20 min (b1 and b2 in Fig. 3). We found two reaction product series with m/z differences of 14 between the fragment ions, which were m/z 77 (a2 in Fig. 2), m/z 91 (a1 in Fig. 3) and m/z 105 (b1 in Fig. 3), and m/z 151 (a3 in Fig. 2), m/z 165 (a2 in Fig. 3) and m/z 179 (b2 in Fig. 3), from water, methanol and ethanol injection, respectively. The difference in the m/z ratios (14) is simply the difference in the molecular mass between water, methanol and ethanol, indicating the possibility that there were two kinds of hydrolysis and alcoholysis reactions. The fragment ion schemes for the six reaction products mentioned above were proposed and their chemical structures were identified and are shown in Fig. 4a. These results were confirmed using D2 O injections (b2 and b3 in Fig. 2). The chemical structures of the six hydrolysis/alcoholysis products were cross-checked using H2 O/D2 O injections and H2 O/MeOH/EtOH injections, which gave comparable mass fragmentation patterns. In the case of water injection, the two minor reaction products eluted at 13.8 and 23.8 min were speculatively identified, and we did not take any more measures to confirm their chemical structures. Their proposed chemical structures and EI fragmentation patterns are shown in Fig. 4b.
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Fig. 2. EI mass spectrum of water injection reaction products (a1–a4) and deuterated water injection reaction products (b1–b4). The products eluted at 13.78, 18.15 and 22.01 min were deuterated when 100% D2 O was injected. It is not certain whether the product eluted at 23.70 min was deuterated though m/z 207 was not 2 H-labeled, because this product could be formed from two pathways (Fig. 4b).
It is well known that hexamethylcyclotrisiloxane (D3) is the main thermal degradation product of polydimethylsiloxane [2,4], which gives a discernible peak at m/z 207 in EI mode. However, according to our results (described in Section 3.2), the formation of hexamethyl-1, 5-trisiloxanediol (trimerdiol) through the hydrolysis of PDMS is also reasonable. Trimerdiol and D3 give similar mass fragmentation patterns in EI mode [7], therefore, D3 and trimerdiol that was produced from H2 O and D2 O hydrolysis
cannot be distinguished from each other in EI mode (a4 and b4 in Fig. 2). For the other minor water injection reaction product that eluted at 13.8 min, we deduced that this product was created by a reaction between reactive hydrogen atoms (from water detained in the analytical systems) and PDMS, with one H atom being deuterated in the product molecule (a1 and b1 in Fig. 2). The proposed fragmentation schemes for the H2 O and D2 O injection reaction products are
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Fig. 3. EI mass spectrum of reaction products from the injection of methanol (a1–a2) and of ethanol (b1–b2).
shown in Fig. 4b. Repeatedly injecting samples onto a GC system can generate surfaces containing reactive hydrogen atoms, especially if moisture is present in the analytes [7], so the formation of H [(CH3 )2 SiO]n H is likely. 3.3. Influence of the number of injections on the hydrolysis and alcoholysis products Changes in the sizes of the hydrolysis and alcoholysis product signals as the number of injections increased were investigated. The injection order was water and then the methanol and ethanol mixture. Both the sizes of the hydrolysis and alcoholysis reaction products gradually increased with increasing number of injections. For water injection, the peak areas of extracted ion at m/z 77 for hydrolytic products (eluted at 18.11 min) of the 30th, the 62nd, the 132nd, the 182nd injection were 2.95, 5.12, 8.64 and 10.58 times that of the 4th injection, respectively. In the case of the injection of the methanol and ethanol mixture, the peak areas of extracted ion at m/z 91 for methanol hydrolysis products (eluted at 9.26 min) of the 31st, the 63rd, the 133rd, the 183rd injection were 1.58, 2.08, 2.93 and 3.35 times, and the peak areas of extracted ion at m/z 105 for ethanol hydrolysis products (eluted at 9.54 min) were 1.68, 2.22, 3.28 and 3.78 times that of the 3rd injection, respectively. These results showed that interferences from hydrolysis and alcoholysis reaction products should be monitored periodically throughout a series of runs, not only at the beginning or end. The increase in the hydrolysis and alcoholysis product signals when water and alcohols were used as the injection solvents might affect the necessary inlet maintenance time intervals (because of accumulation of septum particles in GC inlet liner and their reaction activity), in addition to the usual consideration of the ability of the septum to maintain the inlet pressure. 3.4. Interpretation of the reaction positions and mechanisms When both the GC septum and inlet liner were replaced with new ones the amounts of hydrolysis and alcoholysis products
dropped significantly. These significant decreases in the sizes of the reaction product peaks support the conclusion that the injection port is the main reaction site in the GC system rather than the main site being the GC column. When only the septum was replaced there were almost no changes in the hydrolysis or alcoholysis product signal sizes. Therefore, we concluded that the instant reactions between water or alcohol and the septum or septum particles instantly shed during that injection were not responsible for the hydrolysis and alcoholysis reactions, and that the reactions mainly occurred in the inlet liner. It is widely accepted that GC septa degrade relative to injector operating temperature and also based on the physical interactions with the syringe needle [3]. The interactions between the septum and needle may cause the shedding of particles that then accumulate in the inlet liner [3]. The reactions of water, methanol or ethanol with the septum particles accumulated in the injection port liner after repeated injections, therefore, appeared to contribute to the hydrolysis and alcoholysis reactions. The mechanism for the reactions studied appears to be that the silicones in the septum particles are first desilylated, and HO–PDMS is formed, then the HO–PDMS is subjected to further hydrolysis and alcoholysis reactions (Fig. 4). We deduced that the sizes of the hydrolysis and alcoholysis product signals was positively related to both the amount of water/alcohol injected onto the GC column and of the HO–PDMS accumulated in the GC inlet liner. In this work, a low split ratio (1/5) was chosen and relatively strong reaction product signals were detected. Therefore, GC/GC–MS run with splitless injection would produce more effects, and with split injection using normal split ratios (1/20–100) would produce less effects than this (using split ratio of 1/5), respectively. In fact, When 0.2 L of the mixture of 1% methanol and 1% ethanol (v/v) in acetone or acetonitrile with a split ratio of 1/20 (relatively high concentrations of methanol and ethanol for the routine GC–MS analyses in scan mode) was injected, no alcohol hydrolysis products were detected in scan mode (data not shown). When 0.2 L of 2% (v/v) Chinese liquor samples (containing about 45% ethanol in water, v/v) in acetonitrile, acetone or water, with a split ratio of 1/20, was injected, the amounts of
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Fig. 4. The proposed reaction pathways of injected water, methanol and ethanol with PDMS in GC inlet. (a) The main products from water hydrolysis and alcohol hydrolysis of PDMS were identified based on mass spectra (Figs. 2 and 3), D2 O injection, and the m/z difference of 14 among the fragment ions of H2 O, methanol and ethanol injection. The main (alcohol) hydrolysis products were HO (CH3 )2 Si OR and HO (CH3 )2 Si O (CH3 )2 Si OR, where R = H, methyl, or ethyl, when pure water, methanol and ethanol were injected and subjected to GC–MS analysis, respectively. (b) Products eluted at 13.8 min and 23.8 min were speculatively identified.
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hydrolysis products decreased markedly when using acetonitrile or acetone as a solvent for diluting liquor sample replacing water (data not shown). PDMS swells more when it is in contact with the nonpolar solvents, and swells less when it is exposed to the polar solvents [2,8]. Attention should be paid to the potential uncertainty and unpredictability of hydrolysis and/or alcoholysis reactions in a GC inlet liner because they could be affected by different solvents or by substances that act as catalysts. 4. Conclusions The work presented here clearly demonstrates that the hydrolysis or alcoholysis of silicones can occur in the GC injection port when water or alcohol (methanol or ethanol) is repeatedly injected into a GC system, and that these reactions form reactive silanol groups (R Si OH, where the H atom comes from water or the alcohol hydroxyl group). The reactive silanol groups formed can then be subject to further hydrolysis or alcoholysis reactions to mainly form two series of products, which have been identified as HO (CH3 )2 Si OR and HO (CH3 )2 Si O (CH3 )2 Si OR, where R = H, methyl, or ethyl, when pure water, methanol, or ethanol is injected. Although the observed hydrolysis and alcohol hydrolysis of silicones came as no surprise, no direct (alcohol) hydrolysis products of the silicones used in GC systems have previously been identified. To our knowledge, this is the first report of hydrolysis and alcoholysis reactions in GC inlets. These reaction products can potentially interfere with compounds eluting from the GC column and cause problems in identifying compounds of interest. Such a case is commonly encountered when water and methanol are used as solvents in GC and GC–MS analyses. The potential reaction product peaks from injected water/alcohol should be separated from the analyte peaks. It would, therefore, be better if such solvents were avoided or used minimally in GC and GC–MS analyses, if possible. Analysts should be aware of the potential for the yield of hydrolysis or alcoholysis products increasing with the number of injections. In addition, the potential for alcoholysis reactions occurring in routine
methanol or ethanol determinations using GC, GC–MS, or GC–IRMS methods should be paid attention to, monitored and evaluated. Presently, the magnitude of alcohol hydrolysis effects on quantitative GC, GC–MS analysis of and GC–IRMS isotope ratio analysis of the alcohols themselves has been unknown yet. Acknowledgements This research was supported by the Technical Innovation Program on Development of Instrumental Function of the Chinese Academy of Sciences (No. YG2011024, entitled “Establishment and application of analytical platform for microbial metabolic fluxes based on GC–IRMS”), and by the National Natural Science Foundation of China (No. 30621005). References [1] S. Schmidt, S. Hoffmann, L.G. Blomberg, Gas—chromatographic massspectrometric analysis of compounds generated upon thermal degradation of some stationary phases in gas-chromatography-Part II, HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 8 (1985) 734–740. [2] S. Seethapathy, T. Górecki, Applications of polydimethylsiloxane in analytical chemistry: a review, Anal. Chim. Acta 750 (2012) 48–62. [3] J. Westland, K. Organtini, F.L. Dorman, Evaluation of lifetime and analytical performance of gas chromatographic inlet septa for analysis of reactive semivolatile organic compounds, J. Chromatogr. A 1239 (2012) 72–77. [4] S. Hoffmann, L.G. Blomberg, J. Buijten, K. Markides, T. Wännman, Gas chromatographic—mass spectrometric analysis of compounds generated upon thermal degradation of some stationary phases in capillary gas chromatography, J. Chromatogr 302 (1984) 95–106. [5] S. Fujimoto, H. Ohtani, S. Tsuge, Characterization of polysiloxanes by highresolution pyrolysis-gas chromatography-mass spectrometry, Fresenius Z. Anal. Chem. 331 (1988) 342–350. [6] K. Grob Jr., H. Neukom, Z. Li, Introduction of water and water-containing solvent mixtures in capillary gas chromatography: III. Water-resistant deactivation of uncoated precolumns, J. Chromatogr 473 (1989) 401–409. [7] S. Varaprath, R.G. Lehmann, Speciation and quantitation of degradation products of silicones (silane/siloxane diols) by gas chromatography mass spectrometry and stability of dimethylsilanediol, J. Environ. Polym. Degrad. 5 (1997) 17–31. [8] J.N. Lee, C. Park, G.M. Whitesides, Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices, Anal. Chem. 75 (2003) 6544–6554.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Evaluation of hydrolysis and alcoholysis reactions in gas chromatography/mass spectrometry inlets Guomin Ai ∗ , Tong Sun, Xiuzhu Dong State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing 100101, China
a r t i c l e
i n f o
Article history: Received 5 March 2014 Received in revised form 9 May 2014 Accepted 11 June 2014 Available online 19 June 2014 Keywords: Hydrolysis Alcoholysis Septum particles GC inlet liner Methanol Ethanol
a b s t r a c t During gas chromatography/mass spectrometry (GC–MS) analyses using water and methanol as injection solvents, hydrolysis reactions after injecting water control and alcoholysis reactions after injecting methanol control or ethanol into a GC–MS system were observed and studied. Two dominant hydrolysis/alcoholysis product series were detected, and were identified as being HO (CH3 )2 Si OR and HO (CH3 )2 Si O (CH3 )2 Si OR, where R = H, methyl, or ethyl, when pure water, methanol and ethanol were injected. The chemical structures of the reaction products were cross-checked by injecting H2 O/D2 O and H2 O/MeOH/EtOH, and comparable EI mass fragmentation patterns were found. The water and alcohols injected reacted with silicones in septum particles which accumulated in the injection port liner after numerous injections, and both hydrolysis and alcoholysis reaction products gradually increased in concentration as the number of injections increased. Potential interferences from hydrolysis or alcoholysis reactions should be paid attention to, evaluated or eliminated when water or methanol was used as the GC or GC–MS solvent, and especially when underivatized methanol or ethanol was subject to GC and GC–MS analysis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Silicones have been used extensively as gas chromatographic (GC) stationary phases [1,2]. In addition, most septa used in GC instruments are made of silicone rubber materials, such as Agilent BTO® septa [3]. This type of silicone rubber-based septum may be prone to coring or shedding particles into the injection port liner after repeated injections, and this behavior is related to the inlet temperature and physical interactions with the syringe needle [3]. There has been much research into the thermal degradation of GC stationary phases, especially the degradation of silicones [1,4,5]. The formation of cyclic siloxanes from the thermal degradation of GC septum is also occasionally mentioned in such studies [4]. In addition to the thermal degradation of such GC stationary phases and GC septa, the re-activation of deactivated GC columns when water or alcohols such as methanol are introduced has been also observed based on the impairment of chromatographic performances that can be restored by resilylating the newly formed silanol groups on the column wall [6]. However, to the best of
our knowledge, almost no hydrolysis or alcoholysis products of GC stationary phases resulting from water or alcohols injection have been identified and reported in the literature, and no information is available on the occurrence of hydrolysis or alcoholysis reactions between the silicones that is used in the GC inlet and the polar solvents (e.g., water, methanol and ethanol) that are injected into GC or GC–MS systems. This short communication describes a study of the hydrolysis and alcoholysis reactions that occur in a GC inlet system when water and alcohols (methanol and ethanol) are injected, respectively. We started to acquire fundamental knowledge on the hydrolysis and alcohol hydrolysis products that may affect analytical performance and pose potential problem in identification of compounds in sample. The use of water and methanol as GC and GC–MS solvents, and the popular application of GC and GC–MS techniques for analyzing underivatized methanol and ethanol (such as distilled spirits), provided motivation for this study. 2. Experimental 2.1. Materials, reagents and chemicals
∗ Corresponding author. Tel.: +86 10 64807429; fax: +86 10 64807429. E-mail addresses: [email protected], [email protected] (G. Ai). http://dx.doi.org/10.1016/j.chroma.2014.06.036 0021-9673/© 2014 Elsevier B.V. All rights reserved.
Methanol (LC/MS grade) was purchased from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA). Water (LC/MS grade) and absolute
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Fig. 1. GC–MS chromatograms of the separate injection of pure water (a), ethanol (b) and methanol (c), 0.2 L each, in split (1/5) mode. Reaction products eluted at 9.263 and 11.002 min from 100% methanol injection, 9.542 and 11.195 min from 100% ethanol injection, and 13.792, 18.107, 22.018, and 23.765 min from 100% H2 O injection were detected.
ethanol (HPLC grade) were purchased from Duksan Pure Chemicals Co. Ltd. (Ansan, South Korea). D2 O (99.990% D) was purchased from Sigma-Aldrich (Steinheim, Germany).
3. Results and discussion
2.2. GC–MS analysis
When pure water, methanol or ethanol was injected into the GC–MS system, four product peaks (eluted at 13.792, 18.107, 22.018, and 23.765 min) were found from water, two peaks (eluted at 9.263 and 11.002 min) were found from methanol, and two peaks (eluted at 9.542 and 11.195 min) were found from ethanol, as shown in Fig. 1. The retention times of all the product peaks were constant during all of the experiments. However, none of the product peaks were detected when either air injections or empty injections (manually started GC–MS runs without an injection) were performed. Therefore, we concluded that the peaks were for products of reactions between water or alcohol and the organic matter in the GC–MS (specifically in the GC) system. These reaction products were identified (or speculatively identified), and the positions in the GC system where the reactions took place were identified, as described below.
GC–MS analyses were performed on an Agilent GC–MS system (7890A gas chromatograph and 5975C mass-selective detector; Agilent Technologies, Santa Clara, CA, USA) equipped with a DBWAX column (30 m long, 250 m i.d., 0.25 m film thickness; Agilent Technologies). The inlet liner was an Agilent MS-certified split/splitless liner (part no. 5188–6576; 4.0 mm i.d., 870 L volume, single taper, glass wool positioned in the middle of the liner). The septum was an Agilent BTO® septum (part no. 5183–4757; 11 mm diameter with “CenterGuide”). The GC conditions were as follows: split injection (injector temperature 230 ◦ C, split 1/5 was used to place as much sample onto the column as possible); oven temperature, programmed from 35 ◦ C (held for 3 min) to 47 ◦ C at 5 ◦ C/min, then to 100 ◦ C at 25 ◦ C/min, then to 145 ◦ C at 2.5 ◦ C/min, and then to 200 ◦ C (held for 5 min) at 25 ◦ C/min; the post-injection dwell time, 0.04 min; carrier gas and flow, He 1.0 mL/min; interface temperature, 160 ◦ C. The injection volume was 0.2 L. Under these conditions, the relative compounds to this work were (with elution times, in minutes, in brackets) methanol (3.8), ethanol (4.6), water (6.8), products 1 (9.3) and 2 (11.0) (from pure methanol injections), products 1 (9.5) and 2 (11.2) (from pure ethanol injections), products 1 (13.8), 2 (18.1), 3 (22.0), and 4 (23.8) (from pure water injections). The MS was used in electron impact (EI) ionization mode, with electron energy of 70 eV, an ion source temperature of 230 ◦ C, and a quadrupole temperature of 150 ◦ C. Data were acquired in full-scan (m/z 10–500) mode. Solvent delay of 12.0, 8.3, 9.0 and 8.55 min were used when pure water (and D2 O), methanol, ethanol, and a mixture of methanol and ethanol (1:1, v/v) were injected, respectively. Data were acquired and analyzed using Enhanced ChemStation (Version E.02.00.493, Agilent Technologies). In order to measure changes in the responses of the reaction products as the number of injections increased, water and the mixture of methanol and ethanol (1:1, v/v) were alternately injected. For reducing the sample run time, a shorter GC oven program was run from the 32nd to the 59th, the 64th to the 129th, and the 134th to the 179th injections. The shorter GC oven program was: 100 ◦ C, increased at 25 ◦ C/min to 200 ◦ C, which was held for 3 min. A solvent delay of 6.8 min (the total short run time was 7 min) was used, so that no analytes were detected during these sample runs.
3.1. Reaction product peaks caused by water/alcohol injection
3.2. Identification of the hydrolysis/alcoholysis products We acquired EI mass spectra of the water injection reaction products eluted at 18.11 and 22.02 min (a2 and a3 in Fig. 2), the methanol injection products eluted at 9.26 and 11.00 min (a1 and a2 in Fig. 3), and the ethanol injection products eluted at 9.54 and 11.20 min (b1 and b2 in Fig. 3). We found two reaction product series with m/z differences of 14 between the fragment ions, which were m/z 77 (a2 in Fig. 2), m/z 91 (a1 in Fig. 3) and m/z 105 (b1 in Fig. 3), and m/z 151 (a3 in Fig. 2), m/z 165 (a2 in Fig. 3) and m/z 179 (b2 in Fig. 3), from water, methanol and ethanol injection, respectively. The difference in the m/z ratios (14) is simply the difference in the molecular mass between water, methanol and ethanol, indicating the possibility that there were two kinds of hydrolysis and alcoholysis reactions. The fragment ion schemes for the six reaction products mentioned above were proposed and their chemical structures were identified and are shown in Fig. 4a. These results were confirmed using D2 O injections (b2 and b3 in Fig. 2). The chemical structures of the six hydrolysis/alcoholysis products were cross-checked using H2 O/D2 O injections and H2 O/MeOH/EtOH injections, which gave comparable mass fragmentation patterns. In the case of water injection, the two minor reaction products eluted at 13.8 and 23.8 min were speculatively identified, and we did not take any more measures to confirm their chemical structures. Their proposed chemical structures and EI fragmentation patterns are shown in Fig. 4b.
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Fig. 2. EI mass spectrum of water injection reaction products (a1–a4) and deuterated water injection reaction products (b1–b4). The products eluted at 13.78, 18.15 and 22.01 min were deuterated when 100% D2 O was injected. It is not certain whether the product eluted at 23.70 min was deuterated though m/z 207 was not 2 H-labeled, because this product could be formed from two pathways (Fig. 4b).
It is well known that hexamethylcyclotrisiloxane (D3) is the main thermal degradation product of polydimethylsiloxane [2,4], which gives a discernible peak at m/z 207 in EI mode. However, according to our results (described in Section 3.2), the formation of hexamethyl-1, 5-trisiloxanediol (trimerdiol) through the hydrolysis of PDMS is also reasonable. Trimerdiol and D3 give similar mass fragmentation patterns in EI mode [7], therefore, D3 and trimerdiol that was produced from H2 O and D2 O hydrolysis
cannot be distinguished from each other in EI mode (a4 and b4 in Fig. 2). For the other minor water injection reaction product that eluted at 13.8 min, we deduced that this product was created by a reaction between reactive hydrogen atoms (from water detained in the analytical systems) and PDMS, with one H atom being deuterated in the product molecule (a1 and b1 in Fig. 2). The proposed fragmentation schemes for the H2 O and D2 O injection reaction products are
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Fig. 3. EI mass spectrum of reaction products from the injection of methanol (a1–a2) and of ethanol (b1–b2).
shown in Fig. 4b. Repeatedly injecting samples onto a GC system can generate surfaces containing reactive hydrogen atoms, especially if moisture is present in the analytes [7], so the formation of H [(CH3 )2 SiO]n H is likely. 3.3. Influence of the number of injections on the hydrolysis and alcoholysis products Changes in the sizes of the hydrolysis and alcoholysis product signals as the number of injections increased were investigated. The injection order was water and then the methanol and ethanol mixture. Both the sizes of the hydrolysis and alcoholysis reaction products gradually increased with increasing number of injections. For water injection, the peak areas of extracted ion at m/z 77 for hydrolytic products (eluted at 18.11 min) of the 30th, the 62nd, the 132nd, the 182nd injection were 2.95, 5.12, 8.64 and 10.58 times that of the 4th injection, respectively. In the case of the injection of the methanol and ethanol mixture, the peak areas of extracted ion at m/z 91 for methanol hydrolysis products (eluted at 9.26 min) of the 31st, the 63rd, the 133rd, the 183rd injection were 1.58, 2.08, 2.93 and 3.35 times, and the peak areas of extracted ion at m/z 105 for ethanol hydrolysis products (eluted at 9.54 min) were 1.68, 2.22, 3.28 and 3.78 times that of the 3rd injection, respectively. These results showed that interferences from hydrolysis and alcoholysis reaction products should be monitored periodically throughout a series of runs, not only at the beginning or end. The increase in the hydrolysis and alcoholysis product signals when water and alcohols were used as the injection solvents might affect the necessary inlet maintenance time intervals (because of accumulation of septum particles in GC inlet liner and their reaction activity), in addition to the usual consideration of the ability of the septum to maintain the inlet pressure. 3.4. Interpretation of the reaction positions and mechanisms When both the GC septum and inlet liner were replaced with new ones the amounts of hydrolysis and alcoholysis products
dropped significantly. These significant decreases in the sizes of the reaction product peaks support the conclusion that the injection port is the main reaction site in the GC system rather than the main site being the GC column. When only the septum was replaced there were almost no changes in the hydrolysis or alcoholysis product signal sizes. Therefore, we concluded that the instant reactions between water or alcohol and the septum or septum particles instantly shed during that injection were not responsible for the hydrolysis and alcoholysis reactions, and that the reactions mainly occurred in the inlet liner. It is widely accepted that GC septa degrade relative to injector operating temperature and also based on the physical interactions with the syringe needle [3]. The interactions between the septum and needle may cause the shedding of particles that then accumulate in the inlet liner [3]. The reactions of water, methanol or ethanol with the septum particles accumulated in the injection port liner after repeated injections, therefore, appeared to contribute to the hydrolysis and alcoholysis reactions. The mechanism for the reactions studied appears to be that the silicones in the septum particles are first desilylated, and HO–PDMS is formed, then the HO–PDMS is subjected to further hydrolysis and alcoholysis reactions (Fig. 4). We deduced that the sizes of the hydrolysis and alcoholysis product signals was positively related to both the amount of water/alcohol injected onto the GC column and of the HO–PDMS accumulated in the GC inlet liner. In this work, a low split ratio (1/5) was chosen and relatively strong reaction product signals were detected. Therefore, GC/GC–MS run with splitless injection would produce more effects, and with split injection using normal split ratios (1/20–100) would produce less effects than this (using split ratio of 1/5), respectively. In fact, When 0.2 L of the mixture of 1% methanol and 1% ethanol (v/v) in acetone or acetonitrile with a split ratio of 1/20 (relatively high concentrations of methanol and ethanol for the routine GC–MS analyses in scan mode) was injected, no alcohol hydrolysis products were detected in scan mode (data not shown). When 0.2 L of 2% (v/v) Chinese liquor samples (containing about 45% ethanol in water, v/v) in acetonitrile, acetone or water, with a split ratio of 1/20, was injected, the amounts of
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Fig. 4. The proposed reaction pathways of injected water, methanol and ethanol with PDMS in GC inlet. (a) The main products from water hydrolysis and alcohol hydrolysis of PDMS were identified based on mass spectra (Figs. 2 and 3), D2 O injection, and the m/z difference of 14 among the fragment ions of H2 O, methanol and ethanol injection. The main (alcohol) hydrolysis products were HO (CH3 )2 Si OR and HO (CH3 )2 Si O (CH3 )2 Si OR, where R = H, methyl, or ethyl, when pure water, methanol and ethanol were injected and subjected to GC–MS analysis, respectively. (b) Products eluted at 13.8 min and 23.8 min were speculatively identified.
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hydrolysis products decreased markedly when using acetonitrile or acetone as a solvent for diluting liquor sample replacing water (data not shown). PDMS swells more when it is in contact with the nonpolar solvents, and swells less when it is exposed to the polar solvents [2,8]. Attention should be paid to the potential uncertainty and unpredictability of hydrolysis and/or alcoholysis reactions in a GC inlet liner because they could be affected by different solvents or by substances that act as catalysts. 4. Conclusions The work presented here clearly demonstrates that the hydrolysis or alcoholysis of silicones can occur in the GC injection port when water or alcohol (methanol or ethanol) is repeatedly injected into a GC system, and that these reactions form reactive silanol groups (R Si OH, where the H atom comes from water or the alcohol hydroxyl group). The reactive silanol groups formed can then be subject to further hydrolysis or alcoholysis reactions to mainly form two series of products, which have been identified as HO (CH3 )2 Si OR and HO (CH3 )2 Si O (CH3 )2 Si OR, where R = H, methyl, or ethyl, when pure water, methanol, or ethanol is injected. Although the observed hydrolysis and alcohol hydrolysis of silicones came as no surprise, no direct (alcohol) hydrolysis products of the silicones used in GC systems have previously been identified. To our knowledge, this is the first report of hydrolysis and alcoholysis reactions in GC inlets. These reaction products can potentially interfere with compounds eluting from the GC column and cause problems in identifying compounds of interest. Such a case is commonly encountered when water and methanol are used as solvents in GC and GC–MS analyses. The potential reaction product peaks from injected water/alcohol should be separated from the analyte peaks. It would, therefore, be better if such solvents were avoided or used minimally in GC and GC–MS analyses, if possible. Analysts should be aware of the potential for the yield of hydrolysis or alcoholysis products increasing with the number of injections. In addition, the potential for alcoholysis reactions occurring in routine
methanol or ethanol determinations using GC, GC–MS, or GC–IRMS methods should be paid attention to, monitored and evaluated. Presently, the magnitude of alcohol hydrolysis effects on quantitative GC, GC–MS analysis of and GC–IRMS isotope ratio analysis of the alcohols themselves has been unknown yet. Acknowledgements This research was supported by the Technical Innovation Program on Development of Instrumental Function of the Chinese Academy of Sciences (No. YG2011024, entitled “Establishment and application of analytical platform for microbial metabolic fluxes based on GC–IRMS”), and by the National Natural Science Foundation of China (No. 30621005). References [1] S. Schmidt, S. Hoffmann, L.G. Blomberg, Gas—chromatographic massspectrometric analysis of compounds generated upon thermal degradation of some stationary phases in gas-chromatography-Part II, HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 8 (1985) 734–740. [2] S. Seethapathy, T. Górecki, Applications of polydimethylsiloxane in analytical chemistry: a review, Anal. Chim. Acta 750 (2012) 48–62. [3] J. Westland, K. Organtini, F.L. Dorman, Evaluation of lifetime and analytical performance of gas chromatographic inlet septa for analysis of reactive semivolatile organic compounds, J. Chromatogr. A 1239 (2012) 72–77. [4] S. Hoffmann, L.G. Blomberg, J. Buijten, K. Markides, T. Wännman, Gas chromatographic—mass spectrometric analysis of compounds generated upon thermal degradation of some stationary phases in capillary gas chromatography, J. Chromatogr 302 (1984) 95–106. [5] S. Fujimoto, H. Ohtani, S. Tsuge, Characterization of polysiloxanes by highresolution pyrolysis-gas chromatography-mass spectrometry, Fresenius Z. Anal. Chem. 331 (1988) 342–350. [6] K. Grob Jr., H. Neukom, Z. Li, Introduction of water and water-containing solvent mixtures in capillary gas chromatography: III. Water-resistant deactivation of uncoated precolumns, J. Chromatogr 473 (1989) 401–409. [7] S. Varaprath, R.G. Lehmann, Speciation and quantitation of degradation products of silicones (silane/siloxane diols) by gas chromatography mass spectrometry and stability of dimethylsilanediol, J. Environ. Polym. Degrad. 5 (1997) 17–31. [8] J.N. Lee, C. Park, G.M. Whitesides, Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices, Anal. Chem. 75 (2003) 6544–6554.