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Why do you think the column oven is maintained at approximately 30°C?
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Figure 11.5 Schematic diagram of a typical high performance liquid chromatograph. Reproduced by permission of Mr E. Ludkin, Northumbria University, Newcastle, UK.
This is to prevent changes in the retention times of the compounds, both between and during chromatographic runs. The temperature of 30° C is arbitrarily fixed to be just above ambient room temperature. It can obviously be adjusted depending on the ambient air temperature.
Samples (10-20 Rl) are injected, via a fixed-volume loop connected to a six-port injection valve (Figure 11.6), onto the column and after separation are detected. The most common detector used for HPLC is the ultraviolet-visible spectrometer (available as a single-wavelength unit or with a photodiode array which allows multiple wavelength detection), although a range of more specialized detectors are also available, e.g. fluorescence, electrochemical, refractive index, light-scattering or chemiluminescence. Recently, the introduction of low-cost bench-top liquid chromatograph-mass spectrometers has made the use of this universal detector, with the capability of mass spectral interpretation of unknowns, more readily available.
An HPLC system can be operated in two different modes. What are they?
The HPLC system can be operated in either the isocratic mode, i.e. with the same mobile phase composition throughout the chromatographic run,
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Load position Inject position
Figure 11.6 Schematic diagrams of a typical injection valve used for high performance liquid chromatography: (a) load position; (b) inject position.
or by gradient elution, i.e. the mobile phase composition varies with respect to the run time. The choice of gradient or isocratic operation depends largely on the number of analytes to be separated and the speed with which the separation is required to be achieved.
11.2.2 Other Techniques
Infrared (IR) spectroscopy is regularly used for the identification of compounds, often in conjunction with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). However, it can also be used for the quantitative analysis of environmental compounds, e.g. BTEX, in a sample extract.
What is the basis of infrared spectroscopy?
This technique is concerned with the energy changes involved in the stretching and bending of covalent bonds in molecules.
Infrared spectra are represented in terms of a plot of percentage transmittance versus wavenumber (cm-1). In its most common form, infrared spectroscopy makes use of Fourier transformation, a procedure for interconverting frequency functions and time or distance functions. Fourier-transform IR (FTIR) spectroscopy allows the rapid scanning of spectra, with great sensitivity, coupled with
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simplicity of operation. FTIR spectra can be obtained for solid, liquid or gaseous samples by the use of an appropriate sample cell. Spectra of liquid samples are normally obtained by placing the pure, dry sample between two sodium chloride discs (plates) and placing this in the path of the IR radiation. Solid samples can be prepared as either a Nujol® mull (where a finely ground solid is mixed with a liquid paraffin) and placed between two sodium chloride discs or as a KBr disc (where finely ground powder is mixed with potassium bromide and pressed as a pellet). These sample preparation techniques are all used for qualitative analysis. For quantitative work in environmental analysis, typically a sample extract, the liquid extract is placed in a solution cell which is then positioned in the path of the IR radiation. For example, the analysis of BTEX in a suitable extract can be determined by observing the IR spectrum at approximately 3000 cm-1 (C-H stretching frequencies occur at >3000 cm-1 in unsaturated systems while at < 3000 cm-1 C-H stretching frequencies occur for CH3, CH2 and CH groups in saturated systems). By recording the percentage transmittance (signal) for a range of standards and plotting a graph of concentration versus signal, unknown concentrations can then be determined. Care should be taken to ensure that a suitable solvent has been used to extract the sample. After all, the FTIR spectra will display signals for C-H groups in solvents, as well as those of the sample extracts. In this context, a standard EPA method exists for the FTIR analysis of total recoverable petroleum hydrocarbons in environmental samples (EPA Method 8440) after supercritical fluid extraction using CO2 only (EPA Method 3560). Other suitable solvents include ‘Freon-113’ and carbon tetrachloride, although the use of these is no longer recommended. The manufacture of ‘Freon-113’ is no longer possible under the Montreal Protocol on Substances that Deplete the Ozone Layer (1990), while carbon tetrachloride is a known carcinogen and also has ozone-depleting properties. For these reasons, the preferred choice, after supercritical CO2, is tetrachloroethene (or perchloroethylene), C2Cl4,