GCXGC-qMS with Total Flow Modulation for Volatile secondary metabolites analysis from organically-grown apples by HS-SPME
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1 GCXGC-qMS with Total Flow Modulation for Volatile secondary metabolites analysis from organically-grown apples by HS-SPME Users Meeting RIVA DEL GARDA 20 May 2014 Eligio Sebastiani - SRA Instruments SpA - sebastiani@srainstruments.com Gianluca Stani - SRA Instruments SpA - stani@srainstruments.com Fabio Villanelli - SRA Instruments SpA - fvillanelli@ortelli.com Luca Calamai - CISM-Universita degli Studi di Firenze - Luca.calamai@unifi.it
2 From 1D-GC to 2D-GC 1D-chromatography Single dimension chromatogram Dimensionality= selettivity criterion
3 Heart-cut 2D-chromatography One or more fractions from first dimension are diverted to a second column. Chromatogram from 1st dimension column Chromatogram from 2nd dimension column
4 Comprehensive 2D-gas chromatography Chromatogram from 1st dimension column Annotation: GCXGC repeated periodical heartcuts are separated in a second column. Each component undergoes all separation steps Chromatogram from 2nd dimension column Ortogonalità Comprensività
5 2 dimension 2 dimension IINTERACION 2 dimension Principle of orthogonal separation Two columns with opposite separation mechanisms: For example: 1 dimension: apolar column Separation by compounds volatility 2 dimension: polar colomn Separation by chemical affinity to stationary phase VOLATILITY GCXGC 1 dimension 1 dimension 1 dimension Alkanes Alkenes Aromatic Hydrocarbons
6 Principle of GCxGC Gain in sensitivity by 3-5 fold StructuraliInformazioni on volatility/polarity (orthogonal separation) Large peak capacity n 2D = n 1 x n 2 1 X e Y same volatility P i 0 X + Y different polarity ɣ i ȣ 2 3 X + Y x y x x y y y x y x
7 Signal Reconstruction in a GCxGC image 1 dimension: conventional GC 2 dimensione: Fast GC t r = 1 t r + 2 t r 2 t r ( P mod ) < < 1 t r
8 GC Image Software Creates 2D image from Data Files -.ch,.ms,.uv,.gcd,.qgd,.cdf,.csv,.txt Baseline subtraction - Removes baseline noise Blob detection - reveals 2D peaks Matches template -
9 Thermal Modulator Setup within a 7890 Agilent GC Split/Splitless Injector FID Detector Cold Jet Hot Jet Loop Modulator Oven 2 Column 1 Column 2 Main Oven
10 Capillary Flow Technology Modulator Advantages: No need of cryogenic fluids Simple engeneering and management No maintenance Allows the modulation of volatili compounds (e.g. air) Inexpensive Minimal space occupied in GC oven Simple switching to conventional 1D chromatography
11 GCXGC-MS system GC 7890B with split/splitless injector, FID and MSD PCM C FID atm H2 H2 1 ml/min 17.3 ml/min modulation time 2.0 sec Injezion time 0.2 sec 25 mt x 0.25mm ID HP-5 MS 5 mt x 0.25mm ID HP-INNOWAX 19 ml/min Split Union Tee 1.7 ml/min MSD vacuum
12 FID / MS sensitivity with 1:11post colomn split Correct modulation on both signals
13 How can the analytical condition be improved for a better sensitivity in GC-MS? 1) Avoid splitting before MSD detector 2) Decrease column flow to MSD 3) Avoid hydrogen as the carrier gas We report preliminary results using He as carrier gas and total column flow to MSD (no splitting)
14 GCXGC-qMS system GC 7890B with inert split/splitless, FID and MSD Quadrupole Mass Spectrometer Detector 5977A Ion Extractor CFT Flow Modulator Gerstel MPS2 liquid, headspace and SPME autosampler Agilent MSD Chemstation and Zoex GC Image
15 SRA_GCXGC_MS_TotalFlow-Solution GC 7890B with inert split/splitless, FID and MSD PCM He.0.3 ml/min He Modulation period 2.0 sec Inject time 0.2 sec J. De Zeeuv, et al (2009). J. Sep. Sci. 32, mt x 0.18mm ID HP-5 MS 5 mt x 0.32mm ID HP-INNOWAX 5ml/min MSD vacuum
16 Bio Diesel Sample
17 Fuji apple (HS-SPME GCxGC)
18 Volatile compounds profile of apples (HS-SPME GCxGC) Instrument setup: Agilent 5977 Ion Extraction MSD, Total flow to MS, scan rate amu/sec, Helium carrier gas Modulation period 2 sec, inject time 0.45 sec Comun 1 st dimension: 20m, 0.2 mm i.d. x 0.2 d.f. Column 2 st dimension: 5m, 0.32 mm i.d. x 0.2 d.f. Experimental conditions: SPME sampling,pdms/dvb/carboxen 1cm fibre Incubation: 5 min, 1 g apple purea, 1 g NaCl, 250 mg ascorbic acid 1 ml phosfate buffer phh sorption time 2 min at 40 C; Desorption time 2 min
19 HS-SPME GCxGC profile comparisons Fuji Granny Smith
20 LoD with Naftalene-D 8 Extract Ion m/z 136 (N-D 8 ) S/N 115 (scan rate amu/sec) liquid splitless injection Brown signal: 100 pg Blue signal : 50pg i Red signal: 5pg
21 Standard liquid with SPME Dilution 1:100
22 Modulated Signal of Octanoic Acid from 100 ug/l to 0.01 ug/l (HS-SPME on 5 ml of liquid sample)
23 l Signal to noise ratio (SNR) calculated on a diluited standard mix (1:10.000) LoD by HS-SPME on liquid sample (total amount in sample vial) 3-Methyl, 1-Butanol Benzaldehyde 1-Hexanol, 2-ethyl Benzaldehyde, 4-ethyl Octanoic acid, ethyl ester Benzaldehyde, 2,4-dimethyl gamma-octalactone delta-octalactone 10pg 0.75pg 0.45pg 3 pg 0.75pg 3 pg 3.8pg 0.36pg
24 Calibration on liquid standard by HS-SPME (5 scales)
25 Calibration on standard solution at 5 scales (SPME)
26 Conclusions GCxGC with flow modulator can be conveniently coupled to MSD in total flow A 2 D fingerprinting characteristic of different apple cultivar can be developed A quantitation approach at ppb level is possible in HS- SPME GC x GC
27 Aknowledgements Agilent technologies SRA instruments CISM, Universityà degli Studi di Firenze
28 Thank you for your attention
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