Post columns

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Post-column in-line photochemical derivatization permits fluorescence detection of the common aflatoxins Bl, B2, Gl, and G2 (60). Chromatographic evidence indicates that photolysis causes the hydration of the nonfluorescent Bl and Gl components to B2a and G2a components, respectively. Analysis of naturally contaminated com samples show no interfering peaks and permits the deterrnination of 1 and 0.25 ppb for Bl and B2, respectively. 

Chromatographic methods including thin-layer, hplc, and gc methods have been developed. In addition to developments ia the types of columns and eluents for hplc appHcations, a significant amount of work has been done ia the kiads of detectioa methods for the vitamin. These detectioa methods iaclude direct detectioa by uv, fluoresceace after post-column reduction of the quiaone to the hydroquinone, and electrochemical detection. Quantitative gc methods have been developed for the vitamin but have found limited appHcations. However, gc methods coupled with highly sensitive detection methods such as gc/ms do represent a powerful analytical tool (20). 

APPLICATION OF EUROPIUM-SENSITIZED LUMINESCENCE FOR POST-COLUMN DETERMINATION OF OXYTETRACYCLINE IN BOVINE MILK... 

The proposed chemical system could also been exploited as a post-column detection system for the HPFC detenuination of these dmgs. 

Fig. 2.34. Arrangement of post-column and incolumn imaging energy filters [2.173],...

EC, electrochemical detection Flu, fluorescence detection MS, mass specu-omeu-ic detection pre-Flu, fluorescence detection after pre-column derivatization post-Flu, fluorescence detection after post-column derivatization UV, UV absorbance detection. 

Figure 11.2 (a) LC-LC system with post-column reaction detection for the determination... 

K. Eanbeck-Vallen, J. Carlqvist and T. Nordgren, Determination of ampicillin in biological fluids by coupled-column liquid cliromatography and post-column derivatiza-tion , 7. Chromatogr. 567 121-128 (1991). 

An alternative way of eliminating water in the RPLC eluent is to introduce an SPE trapping column after the LC column (88, 99). After a post-column addition of water (to prevent breakthrough of the less retained compounds), the fraction that elutes from the RPLC column is trapped on to a short-column which is usually packed with polymeric sorbent. This system can use mobile phases containing salts, buffers or ion-pair reagents which can not be introduced directly into the GC unit. This system has been successfully applied, for example, to the analysis of polycyclic aromatic hydrocarbons (PAHs) in water samples (99). 

Derivatization techniques are divided into pre-column and post-column techniques. Post-column derivatization is especially useful to enhance the detection of compounds, whilst pre-column derivatization is the method of choice for enan-tioseparations via derivatization. 

In liquid chromatography, in contrast to gas chromatography [see Section 9.2(2)], derivatives are almost invariably prepared to enhance the response of a particular detector to the substance of analytical interest. For example, with compounds lacking an ultraviolet chromophore in the 254 nm region but having a reactive functional group, derivatisation provides a means of introducing into the molecule a chromophore suitable for its detection. Derivative preparation can be carried out either prior to the separation (pre-column derivatisation) or afterwards (post-column derivatisation). The most commonly used techniques are pre-column off-line and post-column on-line derivatisation. 

Pre-column off-line derivatisation requires no modification to the instrument and, compared with the post-column techniques, imposes fewer limitations on the reaction conditions. Disadvantages are that the presence of excess reagent and by-products may interfere with the separation, whilst the group introduced into the molecules may change the chromatographic properties of the sample. 

Post-column on-line derivatisation is carried out in a special reactor situated between the column and detector. A feature of this technique is that the derivatisation reaction need not go to completion provided it can be made reproducible. The reaction, however, needs to be fairly rapid at moderate temperatures and there should be no detector response to any excess reagent present. Clearly an advantage of post-column derivatisation is that ideally the separation and detection processes can be optimised separately. A problem which may arise, however, is that the most suitable eluant for the chromatographic separation rarely provides an ideal reaction medium for derivatisation this is particularly true for electrochemical detectors which operate correctly only within a limited range of pH, ionic strength and aqueous solvent composition. 

Chemical derivatization can be carried out before the separation (pre-column derivatization) or after the separation and before detection (post-column derivatization). If derivatization is carried out prior to separation, then a phase system must now be selected to separate the... 

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

In post-column derivatization the chromatographic system is modified to allow the reagent to mix with the column eluent, give the reaction mixture sufficient time to complete and finally pass the reaction mixture to the detector. 

A Chromatograph System Including a Post-Column Reactor... 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

When FAB is utilized for FC-MS, often known as continuous-flow FAB, a matrix material is added to the HPFC eluent, either pre- or post-column, and this mixture continuously flows to the tip of a probe inserted into the source of the mass spectrometer where it is bombarded by the atom beam (Figure 3.3). 

Since droplet formation is a particular problem with aqueous mobile phases, continuous post-column solvent extraction, in which the solutes are extracted into an immiscible organic mobile phase, has been proposed [4]. The mobile phase reaching the belt thus becomes totally organic in nature and much more easily removed. The major disadvantage of this approach is the possible loss of analyte during the extraction procedure. 

Reference has been made to the problems associated with the presence of highly involatile analytes. Many buffers used in HPLC are inorganic and thus involatile and these tend to compromise the use of the interface, in particular with respect to snagging of the belt in the tunnel seals. The problem of inorganic buffers is not one confined to the moving-belt interface and, unless post-column extraction is to be used, those developing HPLC methods for use with mass spectrometry are advised to utilize relatively volatile buffers, such as ammonium acetate, if at all possible. 

The matrix, which in most reported applications appears to be glycerol, may either be incorporated directly into the mobile phase pre-column or added postcolumn. If added to the mobile phase, its effect on the separation must be considered, while if added post-column, significant peak broadening may be observed. 

The effect of the variation in mobile phase composition can be overcome by the use of post-column on-line extraction to remove water from the mobile phase and thus produce a 100% organic mobile phase and this is also likely to bring about an increase in overall sensitivity. 

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