Process post column detection

The key to successful post-column detection in CE-ICP-MS is to establish efficient transport of nanolitre amounts of sample, without affecting the separation process. The first hyphenation of CE to ICP-MS was accomplished using a conventional concentric nebuUser in combination with a conical spray chamber. It was evident from this first publication that interface development was challenging. The fundamental requirements for hyphenation of CE to ICP-MS can be summarised as follows ... 

In the second case monitoring is performed on the column outlet with the main objective of identifying when the individual fractions elute. Post column detection is a very importemt part of analytical chromatographs where sophisticated diode array detectors may be utilized, and base line correction and peak area calculations are used for concentration determination of individual fractions. In contrast, in process chromatography, post column detection tends to be used purely qualitatively and may even be absent. 

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. 

The application of the fluorescence derivatization technique in an HPLC method involves utilization of a post column reaction system (PCRS) as shown in Figure 3 to carry out the wet chemistry involved. The reaction is a 2-step process with oxidation of the toxins by periodate at pH 7.8 followed by acidification with nitric acid. Among the factors that influence toxin detection in the PCRS are periodate concentration, oxidation pH, oxidation temperature, reaction time, and final pH. By far, the most important of these factors is oxidation pH and, unfortunately, there is not one set of reaction conditions that is optimum for all of the PSP toxins. The reaction conditions outlined in Table I, while not optimized for any particular toxin, were developed to allow for adequate detection of all of the toxins involved. Care must be exercised in setting up an HPLC for the PSP toxins to duplicate the conditions as closely as possible to those specified in order to achieve consistent adequate detection limits. 

Merit The main merit of post-column-on-line derivatization is that ideally the separation and detection processes can be optimized individually. 

It is used in IC systems when the amperometric process confers selectivity to the determination of the analytes. The operative modes employed in the amperometric techniques for detection in flow systems include those at (1) constant potential, where the current is measured in continuous mode, (2) at pulsed potential with sampling of the current at dehned periods of time (pulsed amperometry, PAD), or (3) at pulsed potential with integration of the current at defined periods of time (integrated pulsed amperometry, IPAD). Amperometric techniques are successfully employed for the determination of carbohydrates, catecholamines, phenols, cyanide, iodide, amines, etc., even if, for optimal detection, it is often required to change the mobile-phase conditions. This is the case of the detection of biogenic amines separated by cation-exchange in acidic eluent and detected by IPAD at the Au electrode after the post-column addition of a pH modiher (NaOH) [262]. 

Although an excellent detector for PAEis, the fluorometer is not widely used in environmental analysis, as the number of environmental pollutants with fluorescent spectra is limited. The sensitivity and selectivity of the fluorometer are also used in the A-methyl carbamate pesticide analysis (EPA Method 8318). These compounds do not have the capacity to fluoresce however, when appropriately derivatized (chemically altered), they can be detected fluorome-trically. The process of derivatization takes place after analytes have been separated in the column and before they enter the detector. This technique, called post column derivatization, expands the range of applications for the otherwise limited use of the fluorometer. 

Fig. 1.22. Direct injection of aluminium processing solution. Conditions Supelcosil LC-18-DB column gradient programme at a flow-rate of 1.0 ml/min from 1.05 M HIBA to 0.4 M HIBA over 10 min. and held at 0.4 M for 5 min. modifier, 1-octanesulphonate at 0.01 M (A), eluents at pH 4.5 (B), eluents at pH 3.8 detection at 658 nm after post-column reaction with Arsenazo III sample injected, 50 pi sample dilution, (A) 10 ml to 100 ml,...

The CUSAL-HPLC couple has been combined additionally with pre- or post-column derivatization. Thus, pre-column derivatization was used for the determination of colistin A and B in feeds following USAL, the analytes were derivatized with o-phthaldialdehyde/2-mercaptoethanol and separated by HPLC for fluorimetric detection [48]. The experimental set-up used is depicted in Fig. 4.1 OA. Another application of CUSAL-HPLC is the determination of A/-methylcarbamates in soils and food [49] (see Fig. 4.10B), where the analytes were also derivatized with o-phthaldialdehyde after separation for fluorescence-based monitoring. A number of steps of the process including leaching, filtration, solid-phase extraction, liquid chromatographic separation, post-column derivatization and fluorescence detection were performed on-line, all in an automated manner. 

Of the two detection techniques mentioned above, fluorescence is preferred in general because it suffers from fewer operational difficulties. Many compounds do not display a native fluorescence however, this can be overcome if the analyte can be converted by chemical reaction into a fluorescent compound. This process is known as derivatisation and can be accomplished by either derivatising the sample prior to injection (precolumn) or after chromatographic separation (post-column). 

An important constituent in copper pyrophosphate baths is nitrate, which enhances the maximum permissible current density [31]. Fig. 8-30 shows the respective chromatogram with the separation of nitrate and orthophosphate. The latter is the hydrolysis product of pyrophosphate that is formed during the plating process. The main component pyrophosphate may also be separated on a latexed anion exchanger. It is detected after complexation with ferric nitrate in a post-column reaction by measuring the light absorption (see Section 3.3.5.2). 

After elution from the column, the eluate carrying the concentrate zone may either be transported directly to the detector or be further processed to produce a detectable species before being transponed to the detector. The former case refer to systems where the analyte can be determined without transformation, e.g. in AAS. ICPES or selective electrode potentiometric measurements. The latter category include most applications in spectrophotometry, fluorimetiy and chemilumine.scence, which usually require a post column reaction. In both cases, minimization of dispersion in the conduits between the column and detector is essential for obtaining better enrichment factors. 

The most widely used method for ion chromatography detection is to treat or choose the eluent prior to detection to make the eluent ions less detectable and/or make the sample ions more detectable. The most common example of this is chemical suppression used in conductometric detection. The suppressor is really a chemical reactor that reacts with the post-column eluent stream and changes the ionic counterion for the eluent and for the sample peaks. In its most common form, sodium or potassium ions are removed from the stream and hydronium ions are added in an exchange process. This makes the background signal less... 

Many chemicals are not detected using a fluorescence detection scheme unless they are chemically derivatized with a fluorescent label. A reaction may be done before a separation is performed, but there are advantages if it is done after the separation. In chromatography this is known as post-column reaction. This is a chemical reaction performed "on-the-fly" in the sample stream as it moves towards a detector, and it must create a fluorescent product only when sample is present. One of the most common reagent used for post-column derivatization is o-phthaldialdehyde (OPA), which reacts with primary amines to create a fluorescent product. In electrophoresis this process might be called an in-capillary reaction, since due to the electric field the separation... 

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