Enhanced separation of hop oils by flow-modulated GC×GC–TOF MS with Tandem Ionisation

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Courtesy of SepSolve Analytical

Beer contains hundreds of organic ingredients, with concentrations spanning many orders of magnitude, and including monoterpenes (C10) and sesquiterpenes (C15). These are aroma-active hydrocarbons found in the essential oils of various plants, including hops, and provide much of the characteristic ‘bitterness’ of the finished beer.

Of the greatest importance for beer is the monoterpene β-myrcene, and the sesquiterpenes caryophyllene, β-farnesene and α-humulene. However, there are hundreds of other terpenes that may also be present and have an impact on the final aroma and flavour – and a number of factors can affect levels of these compounds, including seasonal variations, packaging, storage and ageing. This makes robust quality control essential.

Brewing not only uses hops directly, but can also use the extracted essential oils. These hop oils also have uses in alternative medicine, such as the treatment of anxiety, insomnia and other sleep disorders, making it even more important that their content is fully characterised.

Comprehensive two-dimensional gas chromatography coupled with mass spectrometry (GC×GC–MS) has become the technique of choice for the separation of complex oils. The enhanced separation capacity offered by the coupling of two columns of different selectivity, combined with highly sensitive mass spectral identification, provides a high-performance approach to sample characterisation.

The key component in the GC×GC system is the modulator – the device that samples and re-injects the first-column effluent on to the second column in narrow bands to ensure that the first-dimension separation is retained and that the short second-dimension column does not become overloaded.

Thermal modulation is the most commonly used technique, but this often requires expensive liquid cryogen and can make it difficult to achieve precise replication of results across multiple instruments. An alternative approach is flow-modulation, which avoids the inconvenience and expense of liquid cryogen, and offers much better between-sample and between-instrument repeatability. This study investigates the application of flow-modulated GC×GC–MS to enhance the separation of hop oils. The modulator used is the Insight from SepSolve Analytical, which allows separation of volatiles ranging from C1 to C40 (and above), the flexibility to change the loop volume in method optimisation, and additional options including heart-cutting, splitting for simultaneous detection and backflushing.

In addition, we examine the benefit offered by the use of Tandem Ionisation [1] technology, which simultaneously acquires both conventional 70 eV spectra for library matching and low-eV spectra for added confidence in analyte identity.

Experimental
Sample preparation: 10% (v/v) dilutions of two hop oils were prepared in hexane.

GC×GC: Injector: Split/splitless; Liner: Single taper with wool, 4 mm (i.d.); Carrier gas: Helium, constant-flow at 0.6 mL/min; Injection volume: 0.5 μL; Split: 100 : 1; Temperature: 280°C; Septum purge: On, 1 mL/min. 2D column set: 1st dimension: BPX5, 20 m × 0.18 mm × 0.18 μm; 2nd dimension: DB1701, 2 m × 0.25 mm × 0.15 μm. Temperature program: Main oven: 40°C (1 min), 3°C/min to 260°C (10 min). Modulator: Insight flow modulator (SepSolve Analytical); Loop dimensions: 0.53 mm i.d. × 230 mm (loop volume: 50 μL); Fill time: 3600 ms; Flush time: 200 ms; PM: 3.8 s.

TOF MS: Instrument: BenchTOF-Select (Markes International); Filament voltage: 1.7 V; Ion source: 300°C; Transfer line: 280°C; Mass range: m/z 40–300; Data rate: 50 Hz; Tandem Ionisation: Simultaneous acquisition of 70 eV and 12 eV data.

Software: Image processing: GC Image (GC Image, LLC).

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