Vacuum pump figure 12.2 Scheme of the TEA.
allowing the detection in the range of picomols. A linear response is observed over a wide range of concentrations with two to three orders of magnitude. Because of the high selectivity and sensitivity of TEA, it is possible to analyze samples in the presence of many coeluting compounds that do not interfere with NOC. This advantage leads to a reduction of time in the clean-up procedures.
Various applications of GC-TEA for the determination of volatile A^-nitrosamines have been reported in the literature, some of them being included in Table 12.1.
The TEA has also been interfaced to HPLC equipment. The advances in technology for interfacing reversed-phase HPLC with TEA have resulted in the development of two interfaces, a KI/HOAc postcolumn reaction interface161 and a UV photolysis-based interface.125 Both interfaces are based on reactions which involve liberation of nitric oxide (NO gas) from the A^-nitroso moiety rather than from pyrolysis as performed in the typical GC-TEA mode. The liberated NO (gas) and LC solvent is then swept by a flow of carrier gas into a series of cold traps which remove the LC mobile phase and the residual vapors. The NO (gas) survives the cold traps, enters the TEA detection cell where it is mixed with ozone gas. The resulting chemiluminescence is detected by means of a sensitive photomultiplier tube. These two interfaces allow high sensitivity (1 to 10 ng of total compound injected) and also a high selectivity for a variety of A^-nitroso compounds. However, these suffer from several limitations such as the impossibility of using either an aqueous carrier solvent, which might seriously affect the baseline stability, or inorganic buffers, which might result in solid residues accumulating in the pyrolyzer.
Billedeau et al.129 developed an HPLC-TEA interface utilizing a particle beam type of instrumentation developed initially for interfacing HPLC to MS. The interface incorporates a thermospray (TSP) vaporiser, a desolvatation chamber, a counter flow gas diffusion cell for reducing the LC effluent in a dry aerosol, and a single-stage momentum separator to form a particle beam of the nonvolatile analyte. The high solvent removal efficiency of this interface has made possible HPLC-TEA analysis with reversed-phase solvents without the need for solvent venting162 or cryogenic trapping techniques125,161 currently being used as alternatives to HPLC-TEA interfaces.
The nitrite ion can be detected spectrophotometrically after the separation of nitrosamines by HPLC followed by photolytic or chemical denitrosation. Postcolumn formation of an azo dye by the reaction of nitrite with a Griess-type reagent allows its spectrophotometric detection at 546 nm.156,175 The kinetics and mechanisms of the Griess reaction have been extensively studied.176
Singer et al.177 developed a specific method in which a postcolumn reaction detection system is used for HPLC. This system is useful for those compounds which can be hydrolyzed in a dilute acidic solution to give the nitrite ion. This method involves the use of the Griess reagent in the postcolumn reactor for production of chromophores from A^-nitrosamines. The theoretical detection limit for this method was reported to be 0.5 nmol. However, owing to the slow reaction kinetics of some nitroso compounds, this technique requires both an air segmentation system and a high-temperature reactor.
Based upon the previous procedure, Bellec et al.126 described a method for the separation and detection of volatile and nonvolatile A^-nitrosamines with colorimetric detection by a Griess reagent of the nitrite generated by the cleave of nitroso compounds with a postcolumn photohydrolysis-UV photoreactor.156 The yield of the photohydrolysis depends upon pH and time of exposure under UV light. The detection limits reported were 8 pmol for A^-dialkylnitrosamines and 20 pmol for Af-nitrosamines bearing two phenyl groups.
These methods are in general based on their photolytic or chemical denitrosation, and the subsequent derivatization of the resulting nitrite ion or the corresponding amine.
Lee and Field178 reported a selective fluorescence detection method for the determination of some N-nitrosamines after a postcolumn reaction. The nitrosamines eluted from the column are first hydrolyzed to produce the nitrite anion, which is then oxidized with Ce4+ to give the fluorescent Ce3+. The detection limit for this method is at the ppb level. A more sensitive fluorimetric method has been developed based upon the reaction of nitrite with 2,3-diaminonaphthalene to form the highly fluorescent product 1-(H)-naphthotriazole.179 About 10 nmol/l of nitrite can be detected by this procedure.
Among the more sensitive methods for the determination of N-nitrosamines are the ones based on the denitrosation of N-nitroso compounds by hydrobromic-acetic acid,158'180 and the subsequent detection of the liberated secondary amines via fluorescence derivatization. Precolumn or postcolumn derivatization has been used for the determination of N-nitrosamines. The most commonly used fluorescent derivatization reagents are listed in Table 12.4.
Peroxyoxalate chemiluminescence detection has been shown to be a highly sensitive detection method184-186 for the determination of N-nitrosamines and secondary amines64,127 in combination with reversed-phase HPLC. Fentomole limits of detection can be obtained with conventional instrumentation. The principle of the reaction is illustrated in Figure 12.3.
Fu et al.64 have reported the determination of six nitrosamines by HPLC combined with a sensitive postcolumn bis(2-nitrophenyl) oxalate-hydrogen peroxide chemiluminescent detection. The sample was first denitrosated with hydrobromic acid-acetic acid to produce the corresponding secondary amines, which were then subjected to reaction with dansyl chloride to form fluorescent dansyl derivatives. The reaction mixtures were separated on a C18 column with a mobile phase consisting of acetonitrile-water-ethanol (pH 6.2 with oxalic acid) containing 3.0 mmol/l of imidazole added as a catalyst for the chemiluminescence reaction. The sensitivity of this method was 120 times greater than that of fluorescence detection and four orders of magnitude greater than that of UV-Vis spectrophotometry detection. The detection limits with this procedure at a signal-to-noise ratio of four were between 0.31 and 1.20 pg.
This method was applied simultaneously to the determination of A^-nitrosamines and the corresponding secondary amines in environmental water samples.127 The method combines solidphase extraction using a mini activated carbon column, followed by elution with acetone, concentration of the extracts by denitrosation, and fluorescent derivatization.
The development in the determination of many classes of nonvolatile nitrosamines depends on the development of detectors suitable for the trace analysis of ionizable, ionic, macromolecular, and thermally unstable A^-nitroso compounds. In particular, the development of detectors compatible with reversed-phase liquid chromatographic conditions is receiving special attention. Among others, the electrochemical detectors are attractive because of their high sensitivity and their ability to operate in different aqueous and mixed aqueous-organic eluents.
Some interesting classes of nitrosamines are decomposable by warm strong acids or by photolytic cleavage in alkaline media so that nitrite or nitrous acid is produced. They can be electrochemically detected as such or after further reaction. The availability of a voltametric detector for flowing solutions equipped with solid electrodes allows the direct anodic detection of the NO2 species produced by postcolumn photolysis.132 The Ce(IV) and iodide in acid medium have been investigated as postcolumn reagents for the oxidation and voltametric detection of the
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