Dando acceso al CI pretendido

The partition of analytes between the carrier gas and the stationary phase is highly dependent on temperature. GC ovens contain an electric heating element on which the column is mounted. The heat from this element is distributed in the oven uniformly as air circulation driven by a powerful fan to ensure an even temperature throughout the oven. A temperature sensor inside the oven allows oven temperature control. Typical GC ovens should operate over a fairly wide temperature range and can be quickly and precisely heated to the preferred temperature varying from –100 to 450 ^oC at a rate of 0.1 to 50 ^oC/min [13].

2.1.6. GC Detectors

Once the components of a mixture are separated using gas chromatography, they must be detected as they exit the GC column. Detectors can be grouped either on the basis of physical detection mechanisms like ionization, bulk physical properties, optical and electrical detectors, or based on the nature of the response. Detectors are broadly classified as universal, selective, or specific. Universal (non-selective) detectors respond to all chemicals differing from the carrier gas. Flame ionization (FID) and thermal conductivity (TCD) are typical examples of universal detectors. Selective detectors respond to certain compounds which have common chemical and physical properties. Detectors falling in this category include atomic emission (AED), electron capture (ECD), flame photometric (FPD), and photo ionization (PID) detectors. On the contrary specific detectors respond only to one compound. In addition to selectivity, detectors can be grouped according to their response to the concentration of analytes as mass flow and concentration dependent detectors [3,13,14]. The most important detector that provides an extra dimension of information is the mass spectrometer (MS). The mass to charge ratios (m/z) of ions resulting from breakdown of compounds are measured by mass spectrometry, which is therefore very useful for compound identification. This detector has been used extensively in the current study and will be discussed briefly.

After the determination of mass to charge ratio (m/z) of an electron, J.J. Thomson performed his first MS experiment with hydrogen, and latter with carbon, nitrogen and oxygen atoms in 1912. A few years later Thomson’s student, F.W. Aston discovered the two isotopes ²⁰Ne and ²²Ne, which consequently led to the discovery of 212 naturally occurring isotopes. From these results, Aston formulated the so-called

“Whole Number Rule”, which states that when expressed in atomic weight units, the atomic weights of isotopes are very nearly whole numbers, and the deviations found in samples of elements are due to the presence of several isotopes with different weights. The use of a mass spectrometer as a detector in gas chromatography was developed in 1957 by J.C. Holmes and F.A. Morrell [3,15]. Gohlke described the direct introduction of GC effluent into a mass spectrometer in 1959, and four years latter, in 1963, detailed GC-MS analysis of natural products was reported [5].

A typical mass spectrometer consists of an ion source, mass analyzer, and a detector.

Once an analyte passes through the transfer line into the ion source of the MS, it is ionized and fragmented. The produced fragments are separated based on their m/z ratios and measured. The most common and perhaps standard form of ionization used in GC technology is electron impact ionization (EI). The molecules enter into the MS where they are bombarded with free electrons emitted from a filament (70 eV). The electrons bombard the molecules causing hard ionization that fragments the molecule.

2.1.6.1. Quadrupole mass spectrometry (qMS)

Mass spectrometers are distinguished based on the type of mass analyzer. The most common type of mass analyzer associated with gas chromatography (GC) is the quadrupole (qMS, Figure 2.5.). A quadrupole mass analyser is a device which uses the stability of ion trajectories in an oscillating electric field to separate ions according to their m/z ratios. It is composed of four parallel hyperbolic circular rods in a square array. Those rods opposite to each other are electrically connected to a radio frequency (RF) and direct current (DC) and a voltage with opposite polarity (+/-) is applied to adjacent rods. Ions are accelerated along the z-axis between the rods out of the ion source. These ions encounter forces in the x and y axes resulting in oscillation away from the rods. When the oscillation is too large, the ions strike the rods and are lost without reaching the detector [9,14,16-19].

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d e

a

b c

d e

a

b c

Figure 2.5. Schematic shows basic components of a quadrupole mass spectrometer: a) transfer line, b) ion source, c) focusing lenses d) quadrupole mass analyzer, and e) electron multiplier. (Adapted from [17,20])

A qMS can be operated both in scan mode, where scanning of all possible fragment ions within the specified range (eg. 35 – 350 amu) takes place, and in selected ion monitoring (SIM) mode, where only pre-selected ions will be detected. When the MS is running in the former mode, it is used as a universal detector, on the other hand the latter mode acts as a selective detector. The advantages of SIM over scan mode includes lower detection limit (10³ fold compared to scan mode) as the instrument is only looking at a small number of fragments (e.g. three fragments) during each scan.

In SIM mode more of the ions of interest reach the detector within a given time and since only a few mass fragments are being monitored, matrix interferences are low.

The fewer ions used in the SIM can result in ambiguous identification, hence it is important to confirm the identity of the analyte by comparing the ratio of the ions from the various mass fragments [16-18].

2.1.6.2. Time-of-flight mass spectrometry (TOFMS)

Time-of-flight mass spectrometry (TOFMS) was first proposed by Stephens in 1946 but only became available commercially in 1955 after the design of the instrument improved by Wiley and McLaren [17,21]. Since it was perceived as having low sensitivity and resolution, TOF was never utilized widely in many major areas of mass spectrometry [21]. However, in the 1980s the demands for rapid mass scanning capabilities and wide mass ranges have sparked renewed interest in TOFMS. This can be attributed to developments in data acquisition techniques, which allowed the fast and efficient collection of large amounts of data [22,23]. In addition, developments in mass spectrometric techniques such as laser and plasma desorption, laser ionization and surface analysis [17,21] have also played important roles in the advancement of TOFMS. These techniques require the ability of TOFMS in handling an unlimited

mass range [21]. The invention of the matrix-assisted laser desorption/ionization TOF (MALDI-TOF) has opened new applications for biomolecules as well as synthetic polymers and polymer-biomolecule conjugates, which also contributed to the expansion of TOFMS [17].

A time-of-flight mass analyzer possesses a simple design used to separate ions based on their migration times. TOFMS operates on the principle that a packet of ions, with different m/z ratios but equal energy or momentum, when projected into a constant electric field will separate according to their m/z ratios (Figure 2.6.). Ions are formed in a short source (accelerating) region (s). A positive voltage (V) is applied to the backing plate imposing an electric field (E = V/s) across the source region, which accelerates all the ions with the same kinetic energy (KE). Then the ions pass through a much longer drift (field free) region (D), where they spend most of their time and separated according to their velocities before reaching the detector. Since ions with the same KE are produced, those with lower m/z arrive first followed in succession by those of higher m/z (Figure 2.6.) [17,20,21].

Electron Filament

Ion Source

Region Accelerating Region

Drift (field free)

Region Detector

s D

E_D = 0 GC column

inlet

Electron Filament

Ion Source

Region Accelerating Region

Drift (field free)

Region Detector

s D

E_D = 0 GC column

inlet

Figure 2.6. Basic components of time-of-flight mass spectrometry. (Adapted from [20,21])

In addition to the ability of handling a complete spectrum with unlimited mass range, TOFMS is capable of the highest data acquisition rate of any mass spectrometer.

TOFMS data can be used for spectral deconvolution of overlapping mass spectra to yield pure chromatographic peak profiles for accurate identification or/and quantification [17,21,24]. Due to its fast data acquisition rate, TOFMS is one of the very few detectors that are fully compatible with comprehensive two-dimensional gas

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30 chromatography (GC × GC), in providing sufficient data density for an accurate definition of the narrow peaks [25].