Figure 1. Whisky extraction chromatograms obtained using EG-Silicone, Acrylate, and PDMS Twisters, non-polar column separation. 5 mL whisky sample (20 % EtOH (v/v), 1:1 dilution with water), 1000 rpm for 1 hour at room temperature. Peak identification: 1. Phenol; 2. Ethyl hexanoate; 3. o-Cresol; 4. p-Cresol; 5. Phenethyl alcohol; 6. o-Ethylphenol; 7. 2,4- Xylenol; 8. Ethyl octanoate; 9. Octanoic acid; 10. Ethyl decanoate; 11. Decanoic acid; 12. Ethyl dodecanoate; 13. Dodecanoic acid.
Peak
No.
Compound Extracted Ion [m/z] Peak area
EG-Silicone
Peak area
PA
Peak area
PDMS
1 Phenol 94 1.1E+07 2.3E+06 6.0E+04
2 Ethyl hexanoate 88 1.8E+06 2.8E+05 7.6E+06
3 o-Cresol 108 1.5E+07 1.7E+06 1.8E+05
4 p-Cresol 108 1.1E+07 1.4E+06 1.6E+05
5 Phenethyl alcohol 91 2.4E+07 7.9E+06 1.6E+06
6 o-Ethylphenol 107 9.3E+06 6.1E+05 2.1E+05
7 2,4-Xylenol 107 1.7E+07 1.3E+06 4.3E+05
8 Ethyl octanoate 88 1.5E+08 4.0E+06 1.4E+08
10 Ethyl decanoate 88 2.3E+08 1.2E+07 2.3E+08
12 Ethyl dodecanoate 88 1.1E+08 5.0E+06 7.0E+07
Table 1. Peak Areas of marked peaks obtained from extraction using three different Twister types.
Whisky standards Log KO/W EG-silicone PA PDMS
Phenol 1.46 5.7 4.2 3.0
o-Cresol 1.95 9.8 5.2 1.5
cis-Whisky
lactone
2.00 6.1 1.7 24.3
Eugenol 2.27 29.5 3.3 32.9
Table2.Recovery in % for selected whisky standard substances obtained with three types of Twisters.
Compound class PDMS EG-Silicone
Phenols and aromatic compounds 14 40
Fusel alcohols 10 10
Fatty acids 11 11
Aliphatic acid ethyl esters 15 15
Other esters 22 22
Lactones 1 2
Acrolein derivates 7 7
Terpenes and norisoprenoids 6 7
Miscellaneous 6 12
Total 92 126
Table 3. Whisky compounds extracted by SBSE using the PDMS and EG-Silicone Twister respectively.
Figure 2. SBSE-TD-GC/MS chromatogram, polar column separation, resulting from an EG-Silicone Twister extraction of a 5 mL whisky sample diluted 1:1 with water (20 % EtOH v/v). Sample extracted for one hour at room temperature and 1000 rpm.
Peak
No.
Proposed
Identity
Peak
No.
Proposed
Identity
1 Ethyl octanoate 12 d-Nerolidol
2 Ethyl nonanoate 13 Octanoic acid
3 Ethyl decanoate 14 o-Ethylphenol
4 1-Decanol 15 2,4-Xylenol
5 Phenethyl acetate 16 p-Ethylphenol
6 Ethyl dodecanoate 17 Capric acid
7 Guaiacol 18 Farnesol
8 Phenethyl alcohol 19 Lauric acid
9 Trans Whisky lactone 20 Vanillin
10 o-Cresol 21 Ethyl vanillate
11 p-Ethylguaiacol 22 Myristic acid
Table 4. Tentatively identified compounds found in Scotch whisky by Twister extraction and GC/MS analysis using a ZB-FFAP Column.
Figure 3. Multivitamin juice chromatogram obtained from EG-Silicone and PDMS Twisters, non-polar column separation. 10 mL sample, 1000 rpm, 1 hour, room temperature.
Peak
No.
Proposed
Identity
Peak
No.
Proposed
Identity
1 Formic acid 21 Hydroxymethyl-furfurole (HMF)
2 Acetic acid 22 Eugenol
3 Furfural 23 trans-Caryophyllene
4 Furfural alcohol 24 alpha-Humulene
5 Isoamyl acetate 25 Valencene
6 2-Hydroxycyclopent-2-en-one 26 Elemicin
7 alpha-Pinene 27 Nerolidol
8 3-Methyl-2,5-Furandion 28 Methoxyeugenol
9 5-Methyl-2-furfural 29 alpha-Cubebene
10 beta-Myrcene 30 Myristic acid
11 delta-3-Carene 31 Nootkatone
12 D-Limonene 32 8-Hydroxy-6-methoxy
13 Isoamylbutyrate 33 9-Hexadecenoic acid
14 gamma-Terpinene 34 Palmitic acid
15 alpha-Terpinolene 35 Limetin
16 Linalool 36 Xanthotoxin
17 Apple oil 37 Linoleic acid
18 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 38 Isopimpinellin
19 4-Terpineol 39 Squalene
20 alpha-Terpineol    
Table 5. Identified compounds found in multivitamin juice by Twister extraction and GC/MS analysis using a ZB-5 Column.
Peak
No.
Compound Extracted Ion [m/z] Peak area
EG-Silicone
Peak area
PDMS
7 alpha-Pinene 93 1.7E+05 3.9E+05
10 beta-Myrcene 93 1.4E+06 2.6E+06
11 delta-3-Carene 93 2.8E+06 4.7E+06
12 D-Limonene 68 1.3E+07 1.8E+07
16 Linalool 71 1.5E+06 4.3E+05
19 4-Terpineol 71 4.3E+05 2.7E+05
20 alpha-Terpineol 59 1.5E+06 4.3E+05
27 Nerolidol 69 5.2E+05 4.8E+05
Table 6. Peak area responses of Terpenes resulting from Twister extractions.
Figure 4. Sauvignon Blanc chromatogram profiles obtained from EG-Silicone-, PDMS- and PA Twister extractions of 5 mL samples for one hour at 1000 rpm, non-polar column separation.
Peak
No.
Proposed
Identity
Peak
No.
Proposed
Identity
1 2-Methyl-butanol 16 2,3-Dihydro-3,5-6-methyl-4H-pyran-4-one
2 3-Methyl-butanol 17 Octanoic acid
3 2,3-Butanediol 18 Ethyl octanoate
4 Ethyl butanoate 19 Phenethyl acetate
5 Furfural 20 Ethyl dl-malate
6 1-Hexanol 21 Nonanoic acid
7 Isoamyl acetate 22 Malic acid
8 trans-4-Hydroxymethyl-2-methyl-1,3-dioxolane 23 Methyl-2,3-dihydroxybenzoate
9 cis-4-Hydroxymethyl-2-methyl-1,3-dioxolane 24 Capric acid
10 Citraconic anhydride 25 Ethyl decanoate
11 Hexanoic acid 26 p-Hydroxyphen-ethyl alcohol
12 Ethyl hexanoate 27 2,4-Di-tert-butylphenol
13 1-Hexyl acetate 28 Methyl-2,5-dihydroxybenzoate
14 Glycerine 29 Lauric acid
15 Phenethyl alcohol 30 Ethyl laurate
Table 7. Tentatively identified compounds extracted from white wine using different Twisters and separated on a ZB-5 GC Column.
Figure 5. Sauvignon blanc chromatogram profiles obtained from EG-Silicone and PDMS Twister extractions of 5 mL samples for one hour at 1000 rpm, polar column separation.
Peak
No.
Proposed
Identity
Peak
No.
Proposed
Identity
1 1-Hexyl acetate 16 Octanoic acid
2 1-Hexanol 17 Nonanoic acid
3 Ethyl octanoate 18 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one
4 Acetic acid 19 Decanoic acid
5 Furfural 20 2,4-Di-tert-butylphenol
6 trans-4-Hydroxy-methyl-2-methyl-1,3-dioxolane 21 Glycerine
7 2,3-Butanediol 22 2,3-Dihydrobenzofuran
8 Ethyl decanoate 23 Lauric acid
9 cis-4-Hydroxy-methyl-2-methyl-1,3-dioxolane 24 Hydroxymethylfurfurole (HMF)
10 Clorius 25 Malic acid
11 5-Methyl-2-furfural 26 Myristic acid
12 Phenethyl acetate 27 p-Hydroxyphenethyl alcohol
13 Hexanoic acid 28 Palmitic acid
14 Phenethyl alcohol 29 Ethyl-3-(4-hydroxyphenyl)-propenoate (Z or E)
15 Ethyl dl-malate    
Table 8. Tentatively identified compounds extracted from white wine using different Twisters and separated on a ZB-FFAP Column.
Acknowledgment

The authors would like to thank Dr. Kevin MacNamara, Irish Distillers,
Pernod-Ricard for his kind support.

Authors
Yunyun Nie, Dr. Eike Kleine-Benne,
GERSTEL GmbH & Co. KG,
Eberhard-Gerstel-Platz 1,
D-45473 Mülheim an der Ruhr, Germany

Twister extraction

All good things come in threes

A novel Ethylene Glycol- (EG) and Silicone based combined sorbent phase has been developed for stir bar sorptive extraction (SBSE) using the GERSTEL Twister® with the aim of improving recovery of analytes across a wide polarity range. In this article, the performance of EG-Silicone, polyacrylate (PA), and polydimethylsiloxane (PDMS) Twisters is investigated to determine their usefulness in generating qualitative flavor profiles of beverages such as whisky, white wine, and multivitamin juice.

Stir bar sorptive extraction (SBSE) is based on principles similar to solid phase micro-extraction (SPME). Both techniques rely on partitioning of analytes between a sorbent phase and a liquid sample phase, resulting in extraction and concentration of the analytes in the sorbent phase depending on the partitioning coefficient. Following sample extraction, the coated stir-bar is thermally desorbed in a flow of carrier gas, releasing and transferring the analytes to a GC/MS system. The most widely used Twister phase is polydimethylsiloxane (PDMS), which is non-polar. It has been reported that the extraction efficiency of the PDMS based Twister can be up to 250 times higher than for PDMS based SPME fibers [1] due to the much larger sorbent phase volume, improved phase ratio and improved phase contact during extraction, all of which enable more efficient extraction and extraction of larger volumes. Successful applications of SBSE include extraction and analysis of VOCs, SVOCs, PAHs, pesticides, and offodors in water; drugs of abuse such as Tetrahydrocannabinol (THC), barbiturates and benzodiazepines; phthalates and various metabolites in biological fluids; flavor compounds, preservatives, thrichloroanisole, pesticides, and fungicides in food and beverages [2,3]. For polar compounds with an octanol-water partition coefficient (Ko/w) lower than 10,000 (logKo/w) < 4), it has been found that recoveries gradually decrease with decreasing Ko/w when using PDMS based Twisters. Among the more hydrophilic solutes are, for example, polar pesticides, alcohols, esters, and phenolic compounds. Although recoveries could successfully be improved for many polar pesticides by adding 30 % NaCl (w/w) into the water sample, the salting-out technique does not necessarily help for all polar compounds and there has increasingly been demand for a Twister with a more polar phase. Two new Twisters with more polar phases are now available from GERSTEL: The Polyacrylate (PA) Twister and the Ethylene Glycol (EG) Silicone Twister. These new Twisters extract several classes of polar compounds more efficiently than the PDMS Twister due to their polar nature. In addition, the EG-Silicone Twister, since it is silicone based, will also efficiently extract non-polar compounds.

Experimental

Samples: Scotch whisky (40 % EtOH v/v); white wine, sauvignon blanc (13 % EtOH v/v) and multivitamin juice.
Instrumentation. The TD-GC/MS analysis was performed using a Thermal Desorption Unit (TDU) combined with a MultiPurpose Sampler (MPS) and a Cooled Injection System (CIS 4) programmed temperature vaporization (PTV) type inlet (all GERSTEL). An Agilent 6890N gas chromatograph with a 5975B inert XL (triple axis) mass selective detector (MSD) was used. The entire analysis system was operated under MAESTRO software control integrated with Agilent ChemStation software using one integrated method and one integrated sequence table.
The more polar PA- and EG-Silicone Twisters do retain some water during extraction of aqueous samples, but excess water can be eliminated prior to GC/MS analysis by operating the TDU in solvent vent mode. In this mode, water is evaporated at low initial temperature, for example at 30-40 °C and ambient pressure (0 kPa) for a pre-determined time before the temperature ramp for the thermal desorption starts. As a result, the introduction of water into the GC/MS system is avoided or significantly reduced. An alternative way to reduce water background is to leave the Twisters exposed to a dry atmosphere for approximately 15 minutes.
In this work, we used the TDU solvent vent mode for water removal since it is an automated process, which delivers more reproducible and reliable results. Extraction of aqueous samples using an EG-Silicone or a PA Twister is performed in exactly the same way as with a PDMS Twister. Aqueous sample was transferred into a 10 mL headspace vial. The Twister was added and the vial was sealed with a screw cap. The extraction was performed at room temperature for 60 min while stirring at 1000 rpm on a multiple position magnetic stirrer. After the extraction had been completed, the Twister was removed from the sample with a magnetic rod and briefly rinsed with HPLC grade water. After carefully drying it with a lint-free tissue, the Twister was stored in a 1.5 mL vial. The Twister was finally placed in a TDU glass liner and the liner stored on an MPS sample tray for GC/MS analysis.

Analysis conditions
TDU: 40 mL/min solvent vent (0.5 min)
EG-Silicone and PA Twister:
40 °C (0.5 min); 120 °C/min;
220 °C (5 min)
PDMS-Twister:
40 °C (0.5 min); 120 °C/min;
270 °C (5 min)
KAS: split 1:10
-100 °C (0.5 min); 12 °C/s;
300 °C (5 min)
Polar separation
Column: 15 m ZB-FFAP (Phenomenex)
di = 0.25 mm, df = 0.25 μm
Pneumatics: He, constant flow = 1.4 mL/min
Oven: 50 °C (2 min); 5 °C/min;
60 °C; 10 °C/min;
165 °C; 20 °C/min;
250 °C (5 min)
Non-polar separation
Column: 30 m ZB-5 (Phenomenex)
di = 0.25 mm, df = 0.25 μm
Pneumatics: He, constant flow = 1.2 mL/min
Oven: 60 °C (2 min); 5 °C/min;
200 °C; 10 °C/min;
300 °C (5 min)

Scotch Whisky

The EG-Silicone Twister is especially well suited for extraction of polar compounds which form hydrogen bonds as hydrogen donors, for example, phenols and similar substances. In figure 1, a comparison of three chromatograms from three extractions of a whisky using different Twisters is shown. The EG-Silicone Twister extraction provided the best recovery for phenols, ethyl esters and fatty acids from whisky. It is clearly seen that the EG-Silicone Twister extracts more compounds, and in greater amount. In table 1, peak areas are listed for the annotated compounds shown in the chromatograms. The peak areas that result from the EG-Silicone Twister extraction are an order of magnitude higher than the compound peaks obtained using the PA or PDMS Twisters for almost all compounds. Due to its polydimethylsiloxane basis, the EG-Silicone Twister also has high affinity for nonpolar analytes like long carbon-chain ethyl esters and acids. When comparing the chromatograms from the EG-Silicone- and PDMS Twister extractions, it becomes clear that the EG-Silicone Twister extraction (top chromatogram) results in the same number of peaks in the region after 25 minutes, but the peaks are significantly larger and recoveries significantly better. Table 2 shows the extraction efficiency (recovery in %) for selected whisky components: phenol, o-cresol, cis-whisky lactone and eugenol, obtained with three types of Twisters from spiked water samples. The highest recovery for phenol and o-cresol was obtained using an EG-Silicone Twister: 5.7 % and 9.8 %, respectively. The PDMS Twister gave high extraction efficiency for non-polar compounds like lactone and eugenol: 24.3 % and 32.9 %, respectively. In direct comparison with the PA Twister, the EG-Silicone Twister provided higher sensitivity for whisky lactone (6.1 %) and eugenol (29.5 %). These results prove that the EG-Silicone Twister extracts phenolic substances very efficiently and that it is also highly suitable for many non-polar compounds. As is clearly seen in table 3, a significantly larger number of phenols and aromatic compounds were extracted from the whisky sample with the EG-Silicone Twister than with the PDMS Twister. The total number of extracted compounds is 126 for the EG-Silicone Twister and 92 for the PDMS Twister. For the other compounds classes, both Twisters extract a similar number of compounds, but the EG-Silicone Twister generally gives better recovery.
In order to achieve better separation of polar compounds from the whisky sample, a ZB-FFAP column was subsequently used. The resulting chromatogram is shown in figure 2, the whisky profile was obtained based on extraction with an EGSilicone Twister. Table 4 lists the proposed compound names identified with the mass spectral database (Wiley 6N). All identified peaks have a hit quality higher than 80.
The plausibility of the identification was checked against literature to ensure that the reported compounds were known to be present in whisky. Using the polar column, the peaks for acids, phenols and other polar compounds show a better peak shape. Many important whisky compounds (vanillin, ethyl vanillate, etc.), which were covered by broad co-eluting acid peaks when using the ZB-5 non-polar column, were now well separated and could easily be identified.

Multivitamin Juice

Extraction of multivitamin juice or of other fruit juices is often negatively influenced by fruit pulp, which blocks analyte access to the extraction phase and/or hinders phase separation following the extraction. In contrast, the presence of fruit pulp has no effect on the SBSE extraction process for multivitamin juice. A 10 mL sample was directly dispensed into a 10 mL vial, the Twister was added and the sample stirred for 1 hour at 1000 rpm. Both EG-Silicone- and PDMS Twisters were used for the extraction.
As can be seen in the chromatograms in figure 3, the EG-Silicone Twister extracts more compounds than the PDMS Twister and with better recovery. The peaks obtained using the EG-Silicone Twister are significantly larger. In the chromatogram obtained with EG-Silicone Twister, 39 peaks were clearly identified. Nine compounds were not at all found or identified using the PDMS Twister: formic acid, acetic acid, furfural, furfural alcohol, 2-hydroxycyclopent-2-enone, 3-methyl-2,5-furandione, 5-methyl-2-furfural, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one and Hydroxymethylfurfurole (HMF). Most of these compounds are furfurals and derivatives of furan. Moreover, the peaks for these nine compounds were very large using EG-Silicone Twister extraction, for example furfural (No. 3) and HMF (No. 21). Some important terpenes in the multivitamin juice were extracted by both Twisters, these are listed in table 6. Eight terpenes were selected and their peaks integrated based on extracted ion chromatograms (EICs). The EIC masses used and the resulting peak areas are also listed in table 6. It can be seen that EG-Silicone- and PDMS Twisters have similar extraction efficies for the terpenes judging by the very similar peak areas obtained using the two Twisters. For more polar alcohol-terpenes linalool, 4-terpineol, alpha-terpineol, and nerolidol, the EG-Silicone Twister does provide better recovery than the PDMS Twister; conversely, for monoterpenes like alpha-pinene, betamyrcene, delta-3-carene and d-limonene the PDMS Twister gives better recovery.

White Wine (Sauvignon Blanc)

EG-Silicone-, PA- and PDMS Twisters were used to extract a broad range of volatile compounds and generate a flavor profile of the white wine. Subsequently, the extraction results for the three Twister types were compared. PA- and PDMS Twisters can be added directly to the wine sample without modifying the sample. Prior to extraction with EG-Silicone Twister, the wine sample needed to be neutralized to pH 3.6 in order to avoid break-down of the Twister phase. Chromatograms were obtained using both ZB-5 (non-polar) and ZB-FFAP (polar) columns, the tentatively identified wine compounds are listed in Table 7. Except for the oven programs and column flow rates used, all conditions for TDU, CIS, and MSD were the same for all analyses performed.
A stacked view comparison of chromatograms from extractions using different Twisters is shown in figure 4. It can be seen that the EG-Silicone Twister extracts a larger number of individual substances (30 tentatively identified peaks) from wine than the PDMS and PA Twisters. Substances like furfural, cis- and trans-4-hydroxymethyl-2-methyl-1,3-dioxolane, glycerin, malic acid, methyl 2,3-dihydroxybenzoate are only found in the EG-Silicone- and PA Twister based chromatograms. Furthermore, most peaks are much larger in the EG-Silicone Twister-based chromatogram than in the PA Twister-based chromatogram. The EG-Silicone Twister extracts acids much more efficiently from wine than the PDMS Twister, see peaks No. 11, 17, 21, 22, and 24 as well as alcohols like 2,3-butanediol (No. 3), 1-hexanol (No. 6), and Phenethyl alcohol (No.15).
PDMS Twisters, conversely, extract larger amounts of esters compared to EG-Silicone Twister (see No. 4, 7, 12, 13, 18, 25). To achieve better resolution and separation of polar compounds extracted from the wine, a polar column was also used. As can be seen in figure 5, the polar column produced sharp acid peaks and enabled the separation of several key polar compounds that were covered by big co-eluting ester peaks in the chromatogram produced on the non-polar column. Although a different column was used, the quantitative results and determined compound identities obtained from EG-Silicone- and PDMS Twister extractions were in good agreement. Some polar acids, alcohols, as well as other polar compounds could be extracted only using the EG-Silicone Twister. Additionally, 5-methyl-2-furfural (No. 11) and Hydroxymethylfurfurole (HMF) (No. 24 ), p-Hydroxyphenethyl alcohol (No. 27) and ethyl 3-(4-hydroxyphenyl)-propenoate (Z or E) (No. 29) were found only when combining EG-Silicone Twister extraction with separation on a polar column (Table 8).

CONCLUSION

The novel EG-Silicone Twisters and PA Twisters presented in this work enable higher extraction efficiency than traditional PDMS Twisters for polar compounds in samples like whisky, multivitamin juice and white wine. For compounds like phenols, furans, alcohols, and acids, use of EG-Silicone Twister results in the best extraction efficiency and shows better performance than the PA Twister. For non-polar compounds such as terpenes and ethyl esters etc., EG-Silicone Twisters, due to their dimethylsiloxane base, provide extraction efficiencies similar to those achieved using PDMS Twisters. By using both the EG-Silicone- and the PDMS Twister in a sequential SBSE process, an overall analyte profile of non-polar and polar organic compounds in a sample can be obtained. The pH value of the sample is a critical point for the EG-Silicone Twister. In water-based standards, the optimum pH range was found to be from 3.5 to 10.0, for wine samples from 3.6 to 7.0. Like the PDMS Twister, the extraction using the EG-Silicone Twister is easy to perform. Only a few instrumental parameters have to be adjusted. Additionally operating the TDU in solvent vent mode is important when desorbing EG-Silicone- and PA Twisters in order to remove excess water that is retained due to their polar nature. This is needed to eliminate water from the GC/MS system.

Literature

[1] Frank David, Bart Tienpont,Pat Sandra, Stir-bar sorptive extraction of trace organic compounds from aqueous matrices, LCGC North America, 21: 21-27 (2003)
[2] Kevin Mac Namara, Michelle Lee, Albert Robbat Jr., J.Chromatogr. A 1217 (2010) 136
[3] Kevin Mac Namara, Dagamara Dabrowska, Meike Baden, Norbert Helle, LC/GC Chromatography, Sep. 2011