Precolumn method

This was suggested by Groszek [51] for measuring the amount of solute adsorbed on to a solid surface using a flow microcalorimeter and is described in detail later. 

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Krause et al. (44) extensively studied the derivatization with DBS, proposed by Chang et al. (76), since DBS reacts with both primary and secondary amines and amino acids at room temperature, leading to stable products detectable in the visible region with high sensitivity. In particular they developed a method for the analysis of BAs, together with the AA, in cheese, seasonings, rice wine, and ham. The deproteinized samples (by ultrafiltration or acidification with TCA) were derivatized with an automated precolumn method and analyzed by RP-HPLC-UV. The DBS derivatization was successively applied to study the occurrence of BAs and free AA in raw-milk cheese (detection at 436 nm). They found that the proportions of free AA and BAs relative to the total amount fell into three broad groups that were independent of the type and ripeness of the cheese (77). 

Although there is an increasing trend toward precolumn derivatization methods, there are several advantages to an ion-exchange separation with postcolumn derivatization. The first is that there is little sample preparation protein hydrolysates can be injected directly into the column for analysis by LC. Unlike precolumn methods, it is not necessary to separate reaction side-products from the reaction mixture instead, they become a source of background. With postcolumn methods there is more sample-to-sample consistency because of the robustness of the ion-exchange separation and the fact that the reaction time is determined by the column size. 

When detection limits are inadequate for the levels of analyte present in the prepared sample, some form of sample preconcentration becomes necessary. The most widely used approach to trace enrichment in IC involves the use of a precolumn designed to trap trace levels of solutes from a large volume of sample. The precolumn method is popular because it is simple and convenient to apply, is amenable to automation, offers high enrichment factors, and is less prone to sample contamination effects than other methods. 

OPA precolumn derivatization is probably the most commonly used precolumn method. OPA precolumn derivatized amino acids are detected with fluorescent detectors using the same wavelengths used for postcolumn OPA. The derivatized sample can also be loaded directly on the reversed-phase column, allowing automation of the derivatization reaction with an antosampler. The sample, however, does need to be evaporated to dryness after hydrolysis for removal of the acid. The removal of the acid needs to be complete, usually requiring redissolving and redrying or strongly buffered to an alkaline pH. 

In almost all studies of adsorption from solution it is necessary to measure the concentration of the solution before and after adsorption. A variety of analytical methods may be used to measure such changes in concentration, including the Langmuir trough, gravimetric, colorimetric, titrimetric, interferometry arid precolumn methods. 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

When a first column of a very short length (and therefore a low selectivity) is used (this is especially suitable for multiresidue methods), we talk about an on-line precolumn (PC) switching technique coupled to LC (PC-LC or solid-phase extraction (SPE)-LC). This is particulary useful for the enrichment of analytes, and enables a higher sample volume to be injected into the analytical column and a higher sensitivity to be reached. The sample is passed through the precolumn and analytes are retained, while water is eliminated then, by switching the valve, the analytes retained in the precolumn are transferred to the analytical column by the mobile phase, and with not just a fraction, as in the previous cases. 

Figure 13.11 Column-switcliing RPLC trace of a surface water sample spiked with eight chlorophenoxyacid herbicides at the 0.5 p-g 1 level 1, 2,4-dichlorophenoxyacetic acid 2, 4-chloro-2-methylphenoxyacetic acid 3, 2-(2,4-diclilorophenoxy) propanoic acid 4, 2-(4-cliloro-2-methylphenoxy) propanoic acid 5, 2,4,5-trichlorophenoxyacetic acid 6, 4-(2,4-dichlorophenoxy) butanoic acid 7, 4-(4-chloro-2-methylphenoxy) butanoic acid 8, 2-(2,4,5-tiichlorophenoxy) propionic acid. Reprinted from Analytica Chimica Acta, 283, J. V. Sancho-Llopis et al., Rapid method for the determination of eight chlorophenoxy acid residues in environmental water samples using off-line solid-phase extraction and on-line selective precolumn switcliing , pp. 287-296, copyright 1993, with permission from Elsevier Science.

One of the first examples of the application of reverse-phase liquid chromatography-gas chromatography for this type of analysis was applied to atrazine (98). This method used a loop-type interface. The mobile phase was the most important parameter because retention in the LC column must be sufficient (there must be a high percentage of water), although a low percentage of water is only possible when the loop-type interface is used to transfer the LC fraction. The authors solved this problem by using methanol/water (60 40) with 5% 1-propanol and a precolumn. The experimental conditions employed are shown in Table 13.2. 

J. V. Sancho-Elopis, E. Hernandez-Hernandez, E. A. Hogendoorn and P. van Zoonen, Rapid method for the determination of eight clilor ophenoxy acid residues in envhon-mental water samples using off-line solid-phase excaction and on-line selective precolumn switching . Anal. Chim. Acta 283 287-296(1993). 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Larsen, B. R. and West, F. G., A method for quantitative amino acid analysis using precolumn o-phthaladehyde derivatization and high performance liquid chromatography, /. Chromatogr. Sci., 19, 259, 1981. 

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