Postcolumn reaction detection, HPLC

Singer et al. 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. 

Wetai Ion Analysis. We have reported a sensitive trace-metal analysis based upon HPLC separation of p-aminophenyl EDTA chelates and fluorescence detection by postcolumn reaction with fluorescamine (23). An application of the pyridone chemistry already discussed leads to a fluorescent-labeled EDTA (VIII). 

Reaction detectors are a convenient means of performing online postcolumn derivatization in HPLC. The derivative reaction is performed after the separation of the sample by the column and prior to detection in a continuous reactor. The mobile phase flow is not interrupted during the analysis and reaction, although it may be augmented by the addition of a secondary solvent to aid the reaction or to conform to the requirements of the detector. Reaction detectors are finding increasing application for the analysis of trace components in complex matrices where both high detection sensitivity and selectivity are needed. Many suitable reaction techniques have been published for this purpose [641-650]. 

Nishiyama and Kuninori [65] described a combination method of assay for penicillamine using HPLC and postcolumn reaction with 6,6 -dithiodi(nicotinic acid). Thiols were separated by HPLC on a reversed-phase column (25 cm x 4.6 mm) packed with Fine Sil 08-10, with 33 mM KH2PO4 (adjusted to pH 2.2 with H3PO4) or 33 mM sodium phosphate (pH 6.8) as the mobile phase. Detection was by postcolumn derivatization with 6,6 -dithiodi(nicotinic acid), and measurement of the absorbance of the released 6-mercaptonicotinic acid was made at 344 nm. The detection limit for penicillamine was 0.1 nmol. A comparison was made with a... 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Fig. 2 Configuration used for postcolumn derivatization detection in HPLC. E = eluent P = HPLC pump IV = injection valve CC = chromatographic column R = pump reagent RC = reaction coil D = detector.

Aqueous samples analyzed by HPLC using a postcolumn reaction detector, formaldehyde separated on a reversed phase C-l 8 column derivatized with 3-methyl-2-benzothiazolinone hydrazone and detected at 640 nm (Igawa et al., 1989). (The method was developed for cloud and fogwater analysis.)... 

Because the reaction temperature markedly influences the rates of formation and degradation of the fluorescent adduct, its precise control is an essential factor for the reproducibility of the postcolumn fluorescence detection. Therefore preheating of both the eluent and OPA reagent to a constant temperature of 40°C is required before their mixing, and these was achieved by insertion of preheater tubes for both the eluent and OPA reagent into the hne. As described in the section HPLC System and Conditions, the preheater tubes, as well as colunms, resistor tube, and the reactor tube were placed in a column oven maintained at 40°C. 

Problems are often found in many analytical methods due to the complex nature of the mixture and the lack of adequate detection means, thus leading to poor quantitation techniques. For the routine separation of a broad range of surfactants, high-performance liquid chromatography (HPLC) appears to be the most cost-effective [7-18]. Ultraviolet (UV) and fluorescence detectors are commonly used in HPLC analysis of surfactants because of their compatibility with separation techniques requiring gradient elution. However, these detectors have two inherent limitations (a) the detector response is dependent on molecular structure (i.e., degree of aromaticity and type of substitution) and (b) only species with a chromophore can be detected. To overcome those limitations, postcolumn reaction detectors, based on extraction of fluorescent ion pairs, were introduced for on-line detection of alkylsul-... 

Figure 7.11. HPLC analysis of carbamates according to U.S. EPA Method 531.1 using postcolumn reaction and fluorescence detection. Reprinted with permission from reference 25.

HPLC methods are rarely applied to the analysis of short-chain organic acids due to their poor UV-absorbance and their nonfluorescent character. Only high concentrations can be measured directly by HPLC in combination with UV, diode array, or fluorescence detection. To enhance method sensitivity, organic acids may be derivatized in pre- or postcolumn reactions. Although an abundance of derivatization methods for various compounds is available, especially for... 

Clarkin, C.M., Minear, R.A., Kim, S. and Elwood, J.W. (1 992) An HPLC postcolumn reaction system for phosphorus-specific detection in the complete separation of inositol phosphate congeners in aqueous samples. Environmental Science and Technology 26, 199-204. 

The fluorimetric method of Bates and Rapoport [8], based on the oxidation of PSP toxins in alkaline conditions to form fluorescent derivatives, was incorporated into a detection method with the PSP toxins separated in a chromatographic column by Buckley et al. [17]. This method set the basis for the development of a high pressure liquid chromatography with postcolumn reaction system that was subsequently improved to achieve a better toxin separation and adequate sensitivity [18]. Sullivan et al. [ 19] evaluated its applicability to shellfish toxicity monitoring, by comparing the results obtained by the HPLC method and the standard Association of Official Analytical Chemists (AOAC) mouse bioassay. They found, in general, a good correlation between the two methods. However, Cl and C2 toxins could not be separated and individually quantified. Further improvements and modifications... 

Fig. 1 Flow diagram of an HPLC system with postcolumn iodine-azide reaction detection. A, Ion and ion-pair chromatography, and B, reversed-phase chromatography 1, mobile phase 2, pump 3, injection valve 4, HPLC column 5, mixing tee 6, sodium azide solution 7, iodine solution 8, reaction tube 9, thermostat 10, LC spectrophotometer 11, recorder or computer as a recorder 12, acetonitrile 13, postcolumn reaction module 14, temperature control system 15, bus Sat/In module 16, water.

Kawamoto and coworkers used HPLC and post-column derivatization with 2,6-dibromoquinone-4-chlorimide to quantify PN in pharmaceutical products (88). The condensation product shows strong absorption in the visible spectrum (650 nm) detection has been reported to be almost seven times more sensitive than UV (250 nm) detection of PN. The main advantage of this method is selectivity other B vitamins or caffeine (often present in vitamin preparations) were shown not to interfere in PN quantiflcation. The disadvantage of the method is that two postcolumn reaction pumps with reaction coils are required to deliver reagents to the column eluate. Furthermore, only PN can be analyzed by this method, while most modem quality control programs tend to optimize vitamin analyses by simultaneous analysis of several vitamin compounds. 

Most of the detection modes used in HPLC have been demonstrated for capillary electrophoresis (absorbance, fluorescence, conductivity, electrochemical, radioactivity, mass spectrometry, postcolumn reaction). However, of these, only absorbance, fluorescence, conductivity, and mass spectrometry are available for automated CE systems. 

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