En la espada, comprobará el recorrido total y el recorrido residual de la punta de arresto:

3.5.1 THERMAL DESORPTION SAMPLER

Sample introduction to the GC-MS is performed using a Series 1 Unity thermal desorption platform (Markes International, UK). The Unity system employs a two- stage thermal desorption process which comprises a glass refocusing cold-trap that has an internal diameter of 2 mm and is packed with identical adsorbent to that of the sample tube used for the breath sample collection described in Section 3.3.3. The cold-trap is commercially available from Marks International and is a general- purpose hydrophobic cold-trap. The cold-trap refocusing system is electrically cooled by a Peltier system that is capable of generating temperatures down to -10°C.

Figure 18 illustrates the thermal desorption inlet process. When a sample is placed into the sample oven the Unity system performs a leak-test to ensure that there will be no loss of sample during the desorption process. On successful completion of the leak-test the system purges the sample tube with the carrier gas (helium) before any heat is applied to the tube, this process removes any air in the sample tube that may cause damage to the sorbents through oxidation from residual oxygen when the tube is heated to high temperatures. The primary desorption flow through the sample tube

(45 cm3 min-1) is higher than the flow of the GC capillary column (2 cm3 min-1). The sample tube is then heated rapidly to a high-temperature (300°C) for 5 mins. The primary desorption-flow is directed through the cold-trap (-10°C) where the VOC analytes freeze and concentrate into a small volume in the cold-trap adsorbent. The gas-flow to the cold-trap is then reversed and directed into the capillary column of the GC-MS system at a rate of 2 cm3min-1. The cold-trap is then heated rapidly to 300°C causing desorption of the concentrated VOCs onto the head of the analytical capillary column. The operating parameters for the thermal desorption process used in this work are summarised in Table 1.

Table 1 Thermal desorption parameters for the Series 1 Unity Thermal

Desorption

Parameter Setting

Tube desorption 300 oC for 5 minutes Desorption flow 45 ml min-1 Splitless

Cold trap General purpose hydrophobic Trapping Temperature -10 oC

Trap desorption 300 oC for 5 minutes Trap desorption flow 2.0 ml min-1 Splitless Split ratio splitless

Figure 18 Overview of the thermal desorption process in the Unity thermal desorption platform. (A) Sample pre purge, He flows through the sample tubes at ambient temperature to purge the sample tube of any oxygen. (B) Sample cold trapping, the sample tube is heated desorbing the analytes off the surface of the adsorbent in the tube in to the Helium flow and is refocused on to the cold trap. (C) Sample introduction, the helium flow supplied to the cold-trap is reversed and diverted to the analytical column. The cold-trap is then heated rapidly desorbing the sample analytes.

(A)

(B)

(C)

Sample

Cold trap

GC

3.5.2 GAS CHROMATOGRAPHY MASS SPECTROMETRY

Figure 19 Schematic diagram of TD-GC-MS, (A) thermal desorption sampler, (B) Heated transfer line, (C) capillary column, (D) Gas Chromatograph, (E) Mass spectrometer heated transfer line and (F) Ion trap mass spectrometer.

Chromatographic separation of VOC analytes from exhaled breath samples was performed with a Varian 3800 GC equipped a 60 m x 0.25mm x 0.25µm capillary column with a 5% phenyl 95% dimethyl arylene siloxane stationary phase (DB5-MS, Agilent technologies). The analytical column was connected directly to the outlet transfer-line of the thermal desorber and was supplied with a constant flow (2 cm3 min-1) of helium carrier gas through the instrument’s pneumatic flow controller. The oven’s initial temperature was 40 oC with no hold-time, the oven temperature was then increased at a rate of 5 oC min-1 to 300oC. The final hold-time was 8 min bringing the total run-time to 60 min.

Detection of VOC analytes was performed with a Varian 4000 ion-trap mass spectrometer. The spectrometer uses a 3-D ion-trap mass analyzer with an electron ionization (EI) source. The system was configured in internal ionization mode, which allows both EI and positive chemical ionization (PCI) to be performed in separate time segment in the same analytical run. The column effluent-flow enters the ionization source though a heated transfer line set to 300oC, located between the spectrometer and GC oven. In addition to the column effluent flow the spectrometer also employs a helium buffer gas flow at 4 cm3 min-1, which is directed into the trap and causes the ions to adopt a more compact orbit. This buffering produces higher

resolution when the ions are ejected from the mass analyzer. After ejection, the ions strike a conversion dynode, which begins the signal multiplication process performed by an electron multiplier. A comprehensive list of instrumental conditions is given in Table 2.

Table 2 Gas chromatography conditions (Varian 3800)

Parameter Setting

Column DB 5 MS 60 m x 0.25 mm id x 0.25 μm Initial oven temperature 40 oC Hold for 0 min

Oven temperature ramp 300 oC at 5 oC min-1 Hold for 8 min Total analysis time 60 min

Carrier gas Helium Carrier gas flow 2.0 ml min-1

Mass spectrometer conditions (Varian 4000)

Parameter Setting

Trap temperature 150 oC Manifold temperature 50 oC Transfer line temperature 300 oC EI scan mode EI Auto EI scan frequency 2 Hz