1.3 Instrumentation

Pollutants are usually present at trace levels between the microgram/1 (ppb) and nanogram/1 (ppt) range, requiring the utmost sensitivity from analytical instrumentation, especially since the detection limits of most methods are very close to the concentration limits set by regulatory bodies. It is therefore important to choose the correct procedure and instrument configuration based on the compounds to be detected and the required detection limits. Gas chromatographs (GC) and high pressure liquid chromatographs (HPLC) have traditionally been the most commonly used instruments for routine environmental analysis. More recently, capillary GC/mass spectroscopy (CGC/MS) has become an essential tool in pollutant monitoring, while other hyphenated techniques, for example capillary GC/atomic emission detection (CGC/AED), capillary GC/Fourier transform infrared (CGC/FTIR) and HPLC/MS are appearing in an increasing number of routine laboratories. The potential benefits of thin layer chromatography (TLC) have been outweighed by the tedious nature of the technique. Additional techniques including supercritical fluid chromatography (SFC) and capillary electrophoresis (CE) are outlined in the chapter "Future trends".

1.3.1 Gas chromatography

Gas chromatography (GC) is by far the most widely used technique in the analysis of organics in water. The 500 and 600 series of the US Environmental Protection Agency's methods for analysis of organic pollutants in drinking and waste water respectively, prescribe predominantly gas chromatography, initially with packed columns and more recently with fused silica capillary columns.

1.3.1.1 Capillary columns

Fused silica columns with i.ds of 0.20 to 0.75mm and lengths of 30 to 105m are the work horses of environmental analysts. For optimal environmental chromatography, FSOT columns with different film thickness of methyl silicone, methyl 5% phenyl silicone and methyl 50% phenyl silicone are commercially available. The Hewlett-Packard Environmental Catalogue describes recommended columns for the different classes of pollutants in water.

1.3.1.2 Sample introduction systems

If the heart of the capillary GC system is the column, then sample introduction is often its Achilles' heel! GC inlets have capabilities, strengths and weaknesses that we have to recognise and understand in order to avoid making sample introduction a stumbling-block in environmental analysis. Inlet systems can be divided into two groups, universal inlets and selective inlets. Universal inlets such as split/splitless, cool on-column and temperature programmed vaporiza­tion (PTV), all introduce the complete sample into the column whereas in selective injection, only a fraction, albeit a well-defined fraction, enters the column. Data obtained with selective inlets, for example purge and trap, headspace, etc., are generally much more accurate and precise than those obtained with universal inlets since the fractions presented to the column contain only volatile compounds and the techniques themselves are fully auto­mated. Most problems in environmental analysis are encountered with universal inlets, a category unfortunately including the popular splitless vaporizing inlet. Using a splitless injection port effectively means that there is an interface, 'the vaporizer' before the column. The dictum, "no interface is the best interface", which normally refers to the column outlet to spectroscopic method coupling, is also valid for the inlet of the column. Prerequisites for proper sample intro­duction are twofold; the sample must enter the column without alteration, for example sample discrimination or degradation and the inlet system should not contribute to band-broadening. The first prerequisite is best met by applying cool on-column injection, in which a 'plug' of liquid is introduced directly into the column (no interface) where vaporization takes place. Cool on-column injection is however hardly ever applied in routine laboratories due to several drawbacks, the most important of which is loss of chromatographic perfor­mance after the analysis of a few 'real' samples! As an example, if the extract of a waste water sample obtained by continuous liquid-liquid extraction with dichloromethane and Kuderna-Danish enrichment is introduced directly into the column, contamination occurs very quickly. The extract contains non-volatile materials which are retained on the first few centimetres of the column. With splitless injection, the pre-column vaporizer (liner) prevents non-volatiles from entering the column but suffers from other drawbacks, including discrimination and decomposition. Discrimination may be prevented by using fast automated injection and decomposition may be diminished by reducing the residence time of the solutes in the liner by means of electronic pressure control. The present state-of-the-art in splitless injection is illustrated in Figure 1.6, showing the analysis of PCBs with а 5ц1 pressure-pulsed splitless injection with retention gap.

The connection of a retention gap and better sample preparation are of great importance when using cool on-column injection for routine analysis. There are several reasons to recommend the installation of a retention gap before the analytical column. Firstly, its focusing effects (solvent effect, cryofocusing and stationary phase focusing) suppress band broadening which usually occur when large aliquots of apolar solvents or small aliquots of polar solvents are injected and secondly, they retain non-volatiles in the same manner as the splitless injection port liner. When chromatographic performance deteriorates, the retention gap may simply be washed with a suitable organic solvent or the first few centimetres removed. Connecting retention gaps with press fit connectors is not always easy but good results can normally be obtained by softening the polyimide layer with a hairdryer or by applying a small layer of methylpyrroli-done to the polyimide coating before making the connection. In the past, wide bore precolumns (retention gaps) were required when automatic injectors were used together with 0.25 and 0.32 mm i.d. capillary columns but nowadays, auto­mated cool on-column systems for injection into 0.25 and 0.32 mm i.d. columns are commercially available.

1.3.1.3 GC detectors

GC detectors are usually classified on the basis of their response or selectivity, universal detectors responding to every component in the mobile phase, selective detectors to a related group of substances and specific detectors to a single or limited number of components with like chemical characteristics. Since the differentiation between selective and specific detectors is often confusing, they have been combined under the heading selective detectors. Another classification is destructive versus non-destructive, the latter allowing a variety of detectors to be coupled in-series. The most important characteristics for practical work are sensitivity, dynamic range, linear range, detector response factors and selectivity. A brief description of the detectors most commonly used for monitoring pollutants in water samples follows in Table 1.1, which lists their selectivity, minimum detectability and linear range.

NAME	TYPE	SELECTIVITY	MINIMUM	LINEAR
			DETECTABILITY	RANGE
FID	UNIVERSAL	C-H	lOpg C/sec.	107
ECD	SELECTIVE	compounds capturing	0.2pg Cl/sec.	10^4
		electrons e.g. halogens
NPD	SELECTIVE	NandP	lpg N/sec.	10^4
			5pg C/sec.
PID	SELECTIVE	aromatics	107
ELCD	SELECTIVE	halogens and S, N	lpg Cl/sec.	10^6
			5pg S/sec.	10^4
MS	UNIVERSAL	characteristic ions	1 ng full scan mode	10^6
			lpg ion monitoring mode
AED	UNIVERSAL	any element	0.2 - 50pg/sec.	10^4
			depending on element
FTIR	UNIVERSAL	molecular vibrations	1 ng (strong absorber)	10^3

Table 1.1: Characteristics of common GC Detectors.

Flame ionization detector

The flame ionization detector (FID) is a universal, sensitive detector working on the following principle. Carrier gas is an excellent electrical insulator whose conductivity increases considerably due to the ions that are generated when organic molecules in the carrier gas are exposed to a flame. An electrode, positioned a few millimetres above the flame, measures the electric conductivity of the flame between itself and the jet tip assembly. The FID responds proportio­nally to the number of carbon atoms in the molecule with only small differences in response between various compound classes. Its operational simplicity, fast response, excellent stability, 10⁷ linear dynamic range, 10pg C/sec sensitivity, insensitivity to small changes in carrier gas, hydrogen and air flows and univer­sal response to organic compounds have made the FID the most common GC detector in use today. The FID's universal response however, may be a handicap when performing target compound analysis of complex mixtures and a more selective detector may be required to minimize the number of interfering peaks. Another point worth remembering is the FID's relatively poor response to compounds with low carbon content and additional heteroatoms such as the trihalomethanes.

Electron capture detector

The electron capture detector (ECD) is frequently used for the determination of halogen-containing compounds such as organochloro pesticides, PCBs, dioxins and furans, trihalomethanes etc. The principle of this detector is based on the decrease of conductivity caused when electrons are captured by specific analytes. The detector uses a low intensity radioactive source, usually a ⁶³Ni foil, to generate high energy electrons. In the presence of a gas such as nitrogen or argon/methane, these electrons generate ions and thermal electrons, principally responsible for the conductivity in the ECD ionization chamber. When a halo-genated organic compound enters the ionization chamber, the thermal electrons are captured and the conductivity diminishes, generating the detector signal. Even though this approach is very practical for direct analysis of clean samples such as drinking and ground water, difficulties arise with surface and waste samples where the presence of many organic compounds can interfere with the ECD's response. Proper sample preparation and clean-up are therefore of the utmost importance.

Nitrogen phosphorus detector

An adaptation of the FID, the nitrogen phosphorous detector (NPD) is a selective detector that makes use of a rubidium glass bead that when heated in a flame selectively increases the ionization efficiency of nitrogen and phosphorous containing organic molecules (herbicides, pesticides, insecticides, fungicides).

Photoionization detector - Electrolytic conductivity detector in-series The serial connection of a non-destructive photoionization detector (PID) with an electrolytic conductivity detector (ELCD) is recommended by the EPA for the analysis of purgeable aromatics and halocarbons. In the PID, compounds are excited by photons emitted from a UV lamp and the resultant charged particles measured between two electrodes. Selectivity depends upon the lamp used, a 10.2 eV lamp providing, for example, selective and sensitive signals for aromatics. For halogen detection by ELCD, the column effluent is reduced with hydrogen in a nickel reaction tube at 850° С to produce gaseous acid which is in turn dissolved in n-propanol, the change in solvent conductivity producing the detector signal. Although this combination is quite powerful, European labora­tories prefer the selectivity of a mass spectrometer for this application.

Unfortunately, selective detectors can provide misleading information on the nature of eluting solutes and all too often, positive responses from ECDs are interpreted as eluting pesticides, PCBs, chlorophenols etc. For this reason, dual column systems are recommended to avoid false positives when working with selective detectors. Increasingly, the detection of environmental pollutants will be performed by spectroscopic detectors that allow selective recognition of the separated compounds. Today, hyphenated systems such as CGC/mass spectro-scopy (CGC/MS), CGC/Fourier transform infrared spectroscopy (CGC/FTIR) and CGC/atomic emission detection (CGC/AED) are amongst the most powerful analytical techniques available, providing sensitive and selective quantitation of target compounds along with structural elucidation and identification of unknowns. Their applicability and ease of use was greatly increased by the introduction of fused silica wall coated open tubular columns whose low flow rates obviated the need for special interfaces, allowing columns to be directly connected to the different spectrometers. The introduction of relatively inexpensive bench-top hyphenated systems has allowed many laboratories to acquire such instrumentation, increasing their applicability still further.

CGC/MS, CGC/FTIR and CGC/AED are usually operated as stand-alone units but the non-destructive character of FTIR makes CGC/FTIR/MS an attractive proposition that has recently become available commercially, with software that allows the simultaneous recording of infrared and mass spectra from the eluting compounds. In principle CGC/FTIR/MS/AED is also possible by using an open split interface for the CGC/FTIR/MS combination, taking the split-line flow directly into the AED. Within the framework of this booklet it is impossible to discuss all the hyphenated techniques in detail so only their main characteristics and features are presented. We refer the interested reader to papers cited in the bibliography, where the fundamental aspects of CGC/MS, CGC/FTIR and CGC/AED are discussed.

Mass selective detector

The mass selective detector (MSD), a very popular bench-top model quadrupole mass spectrometer, may be viewed as a special kind of ionization detector for gas chromatography. Organic compounds entering the mass spectrometer are ionized in the ionization chamber (ion source) by either electron impact ioni­zation (El) or chemical ionization (CI). In electron impact ionization, the mole­cules are bombarded with electrons of 70eV emitted from a rhenium or tungsten filament. A molecular ion (M^+o) is formed with a sufficient amount of energy accumulated in its bonds to dissociate into typical fragment ions, radicals and neutral species. As low ion source pressures are employed, reactions in the ion source are unimolecular and association between molecule and fragment ions do not occur. A disadvantage of electron ionization is that the energy in the molecule is often high enough to cause complete fragmentation, obliterating the molecular ion. Chemical ionization is a so-called soft ionization technique that ionizes molecules via a gas-phase ion-molecule reaction, causing less fragmentation. In chemical ionization, a reagent gas such as methane is intro­duced into the ion source at a relatively high source pressure, where it is ionized by electrons to form the ions which interact with the sample molecules. Since these reactions are low in energy, abundant quasi molecular ions, most often M^+o+l are observed. Figures 1.7. and 1.8. show the electron impact and the chemical ionization spectra of dibutylphtalate and carbaryl respectively.

Often the ionization method of choice for quantitative mass spectroscopy using ion-monitoring, chemical ionization is also helpful in elucidating the molecular ion of unknowns. After ionization, the positive ions are accelerated and sepa­rated in the mass analyser by tuning the voltage on the quadrupole rods. An ion of unit mass will only pass the rods and reach the detector at a specific voltage, therefore, to detect all ions, the voltages must be varied in time, this cycling of the voltage being referred to as a scan. This means that if we would like to detect all ions between mass 20 and mass 400 within one second (a one second scan time), the voltages are at the exact value for each ion to pass for only 1/380 of a second. This is called the full scan mode and is used to identify compounds from their fragmentation patterns. Data are acquired by continuous repetitive scanning of the column eluate, the chromatogram being in fact a plot of the total ion current in function of time or scan number. All recorded spectra or scans are stored in the computer where they can be recalled and compared with spectral libraries. The stored mass scans can be further processed, using a technique called mass chromatography to provide the selectivity required to identify specific compounds or compound classes. Mass fragmentography or selected ion-monitoring (SIM) on the other hand is a method to quantify target compounds with a high degree of selectivity and sensitivity. In this mode of operation, the voltages on the quadrupole rods are adjusted stepwise to detect only 2, 3 or 4 ions. The time the rods 'dwell' on a particular voltage is far longer than in full scan mode, allowing a greater number of ions of a particular mass to pass through to the detector, resulting in enhanced sensitivity. With modern CGC/MS systems picogram concentration levels are easily quantified when operating in the selected ion-monitoring mode. An interesting feature of SIM is that internal standards can be selected which chemically, physically and chromatographically behave exactly the same as the compounds to be measured. In the case of PAH analysis for example, the best internal standards are deuterium labeled PAHs.

Atomic emission detector

A relative newcomer, the atomic emission detector (AED) is designed specifically for capillary gas chromatography. Using the element specific AED, detection levels in the order of O.lpg/sec for organometallics, 0.2pg/sec for carbon (more sensitive than the FID!), lpg/sec for sulphur and 15pg/sec for nitrogen are possible. The power of the technique lies in its supreme selectivity, with the ability to detect all elements selectively. Unlike ECD, AED allows the analyst to differentiate between different halogenated compounds, for example fluoro, chloro and bromo components or to perform multi-element analyses by simply preselecting the atoms to be monitored. In AED, the solutes eluting from the column are atomized in a high energy source where the resulting atoms, in the excited state, emit light as they return to the ground state. The various wavelengths of the emitted light are dispersed in a spectrometer and measured using a photodiode-array. Each element has its own typical emission spectrum, the emission lines commonly occurring in clusters with constant relative intensities within the cluster. The introduction of diode-array technology enables the simultaneous multi-wavelength detection which lies at the heart of the tech­nique. Quantitative treatment of the data obtained in a multi-element analysis allows the calculation of empirical formulae, yielding information which is complementary to and helpful in the interpretation of mass spectral data.

Quantitation is considerably simplified, as calibration of the detector is no longer dependent on the type of components to be quantified, allowing non-toxic compounds to be used to quantify toxic chemicals. Figure 1.9. illustrates the sensitivity of the AED for organometallic compounds. Organolead halides are derivatized with butylmagnesium bromide into tetra-alkylated solutes which are amenable to CGC analysis. By selecting wavelength 406nm, high selectivity and sensitivity (lOpg per compound) is guaranteed.

Fourier transform infrared spectroscopy

In the combination CGC/Fourier transform infrared spectroscopy (FTIR), IR spectra of the eluting peaks are recorded as they emerge sequentially from the chromatographic column. The eluate is introduced into a light pipe, where compounds absorb radiation at well defined frequencies, i.e. frequencies characteristic of the bonds within a molecule. The absorption is sensed and recorded, sensitivity depending on the functional groups within a molecule, strong IR absorbers producing a good spectrum from 1 ng. Today's computerized FTIRs allow recorded spectra to be compared with a library of spectra, aiding in compound identification, while monitoring specific wavelengths allows the elucidation of classes of compounds e.g. aldehydes, ketones, alcohols, ethers etc. IR spectra are complementary in nature to MS spectra, especially for the elucidation of isomers like dichlorobenzenes, dinitrotoluenes etc., for which MS is not very informative. The on-line combination CGC/FTIR/MS is a very powerful tool for the identification of unknowns.

In water pollution control, the compounds detected are not always on the priority pollutants lists, and therefore correct identification for this type of screening is of utmost importance. For such analysis capillary gas chromato-graphy/mass spectrometry (CGC/MS) is the analytical technique of choice although the mass spectral libraries currently available will not always give the correct answer for an unknown compound. For adequate qualitative analysis, chromatographic retention data have to be combined with spectroscopic information from other hyphenated techniques. Mass spectrometry, Fourier transform infrared spectrometry and atomic emission detection all produce complementary information, leading to the correct identification of compounds which otherwise could not be correctly identified by a single technique.

The following is an example of how CGC/MS, CGC/FTIR, and CGC/AED were applied to the analysis of a contaminated water sample. 250 ml of an industrial waste water sample was extracted twice with 20 ml dichloromethane at pH7, the extracts combined, concentrated to 1 ml and analysed by CGC/MS, CGC/FTIR and CGC/AED. The CGC/MS full scan total ion-current (TIC) chromatogram of the extract is presented in Figure 1.10. The chromatogram shows the presence of a dominant peak a and an homologous series of other compounds. One of the homologues giving rise to peak b has been identified, the other peaks in the series are marked with asterisks. The compound generating peak с was also identified, most of the other peaks corresponding to n-alkanes. Chromatograms from CGC/AED analysis for carbon-, oxygen-, nitrogen-, and sulphur-containing compounds are presented in Figure 1.11.

The sulphur-containing compounds were present at levels below the detection limits of MS and FTIR and could therefore not be identified. The data relevant to the identification of the compounds generating peaks a, b, and с are summarized in Tables 1.2., 1.3. and 1.4.

Compound a was identified by MS as 2,3-diethyl-2,3-dimethylsuccinic acid dinitrile and the identification was supported by an FTIR library search, which indicated the presence of nitrile. The mass and infrared spectra generated by the compound and the results of the respective library searches are presented in Figure 1.12., CGC/AED results (Figure 1.11) are strongly indicative of the presence of nitrogen.

The compound generating peak b and its homologues were identified by both CGC/MS and CGC/FTIR as a series of ethoxylated alcohols (Figure 1.13.). The elemental composition and the presence of oxygen were clearly demonstrated by the AED results (Figure 1.11). The structure proposed by both MS and FTIR library search is, however, incorrect and the compound is probably an ethoxylated alcohol containing three ethylene oxide (EO) units. To elucidate the structure fully, it would be necessary to perform GC/MS with chemical ionization to provide molecular weight information but for environmental purposes, it is sufficient to know that the compound is a non-ionic surfactant.

Compound с was identified by MS as oleamide (Figure 1.14.) and results from FTIR analysis confirmed the presence of an amide function (absorption band at 1731cm"¹). Comparison with standards (measurement of retention indices) indicated however, that the identification was incorrect and manual interpretation of the MS spectrum showed the compound to be erucamide (C22H43NO - MW 337). This conclusion was confirmed by AED-nitrogen detection where two peaks were found, the second corresponding to c, the first, to oleamide. This example illustrates well that unknown compounds can only be identified with a high degree of certainty by combining chromatographic and spectro-scopic data.