Post-separation techniques

This approach has been used extensively for amino acid analysis using low-pressure ion-exchange chromatography and post-column ninhydrin reaction. Spraying, dipping and vapour-treatment techniques are well known as post-separation reactions in TLC, but these are considered only briefly since the majority of them are not quantitative. While the problems of pre-separation techniques are quite similar for TLC and HPLC, they differ considerably for post-separation reactions. 

The advantages of post-column procedures are summarized below  

The limitations of this mode of operation can be quite numerous  

These and other technological difficulties may explain why only few applications of this derivatization mode have appeared in the literature to date. However, a substantial increase in work in this area may be expected in the near future arising from the knowledge of automatic analysis systems. 

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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. 

With the development of HPLC, a new dimension was added to the tools available for the study of natural products. HPLC is ideally suited to the analysis of non-volatile, sensitive compounds frequently found in biological systems. Unlike other available separation techniques such as TLC and electrophoresis, HPLC methods provide both qualitative and quantitative data and can be easily automated. The basis for the HPLC method for the PSP toxins was established in the late 1970 s when Buckley et al. (2) reported the post-column derivatization of the PSP toxins based on an alkaline oxidation reaction described by Bates and Rapoport (3). Based on this foundation, a series of investigations were conducted to develop a rapid, efficient HPLC method to detect the multiple toxins involved in PSP. Originally, a variety of silica-based, bonded stationary phases were utilized with a low-pressure post-column reaction system (PCRS) (4,5), Later, with improvements in toxin separation mechanisms and the utilization of a high efficiency PCRS, a... 

Kessler et al. developed the Difference and Sums of Traces within Cosy Spectra (DISCO) technique for the extraction of couplings from the crosspeaks of two-dimensional (COSY) spectra. This post-processing technique combined selected cross-sections of these peaks in order to extract the active coupling , i.e., /ab from the cross-peak of A and B. After such combination, the active coupling was the separation of the in-phase and anti-phase DISCO peaks. DISCO requires accurate scaling of the constituent spectra and resolved cross-peaks. An alternative and simpler method for the accurate measurement of vicinal couplings from COSY spectra has been presented by Kim and Prestergard. ... 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

Deleuze et al. used the same approach for the synthesis and functionalization of emulsion-derived polymeric foams [119]. Alternatively, a post-synthesis grafting method recently developed in our group offers access to high-capacity functionalized monolithic systems. Such high capacity monoliths are vital to various applications such as catalysis, extraction of environmental contaminants, extraction of biochemicals for either pharmaceutical or clinical purposes or, more generally, separation techniques [100]. With these systems, amounts of grafted monomers can exceed 1 mmol/g [94]. 

The post-bombardment processing of the activated sample may follow either a nondestructive assay of the radioactivity in the sample (gamma-ray scintillation spectrometry is used most often for this) or a chemical processing of the sample prior to the radioactivity assay. Techniques involving either precipitation, electrodeposition. solvent extraction, and ion exchange or some combination of these form the basts of the radio-chemical separation techniques used in activation analysis. 

Odor-active components in cheese flavor, many of which are derived from milk lipids, can be detected using GC-olfactometry (GC-O). GC-0 is defined as a collection of techniques that combine olfactometry, or the use of the human nose, as a detector to assess odor activity in a defined air stream post-separation using a GC (Friedich and Acree, 1988). The data generated by GC-0 are evaluated primarily by aroma extract dilution analysis or Charm analysis. Both involve evaluating the odor activity of individual compounds by sniffing the GC outlet of a series of dilutions of the original aroma extract and therefore both methods are based on the odor detection threshold of compounds. The key odourants in dairy products and in various types of cheese have been reviewed by Friedich and Acree (1988) and Curioni and Bosset (2002). 

Henglein and Weller made significant progress [14-16] using CdS colloids prepared by controlled predpitation methods [17-22]. However, to obtain highly monodispersed nanopartides, post-preparative separation techniques such as size exdusion chromatography [20] and gel electrophoresis have been employed [21]. Gel electrophoresis was found to be superior to other separation techniques. 

Increasing reproducibility of available separation techniques and sensitivity and affordability of mass spectrometers, as well as the desire and need to automate the identification process, have caused peptide mass fingerprinting and MS/MS sequencing to gain importance and to become the method of choice for many proteomics laboratories. Several tools are available to assist users in the interpretation of mass spectrometry data. Peptldent (http //www.expasy.org/tools/peptident.html) on the ExPASy server follows the concept of the other tools from the ExPASy proteomics suite, in that it takes into account annotation available in the SWISS-PROT/TrEMBL database, in particular as post-translational modifications and processing are concerned. The user can paste peptide masses (monoisotopic or average) into the Peptldent form, but peptide mass data can also be uploaded from a file on the user s local computer. Supported file formats are .pkm ... 

As far as separation techniques are concerned, they can be implemented on automatic continuous analysers or robot stations as ancillary modules (dia— lysers, ion exchangers, liquid-liquid extractors). As stated in Chapter 12, chromatographic processes —particularly column (HPLC and GC), but occasionally also planar chromatographic purposes— are commonly the subject of automation. A conventional chromatograph furnished with a system for sequential introduction of samples —which can even be partially treated in a continuous fashion before of after column separation (derivatlration and post-column techniques)— markedly resembles continuous flow analysers. Gas and liquid chromatographs are often used as separative-determinative modules in robot-stalons. 

Other NAA methods involve the chemical separation of interfering ions (Blotcky et al., 1976) in these the short half-life (2.24 minutes) of AI makes post-irradiation separations a problem. Pre-irradiation separation techniques have potential problems of contamination and losses during the separation phase. 

Published assay methods typically employ a post-assay separation technique followed by either fluorescence or radiochemical detection [29, 31, 34], making high-throughput screening for this particular protein target imprachcal. 

Today, the use of sophisticated and large capacity computers with faster and more wide ranging ADCs and DACs allows con tlete conttol of all the mass spectrometers functions, acquisition of data in several forms, and powerful post acquisition processing to automatically search the enormous data files for relevant information. A high degree of automation is possible including conttol of any hyphenated separation technique. The mass spectrometer operator can load the samples and then process the information at a comfortable work station away from the noisy laboratory as the instrument carries out the analyses. The scope of the computer s abilities will be exenplified by the exanples given as apphcations. 

There is now a ground-swell of interest which has reached the academic community. It is hoped that this review will be helpful in providing background and guide posts for future development and application of this rediscovered separation technique. 

In particular, the project is associated with a 25 tonnes per day COj capture plant, in which the flue gas is at atmospheric pressure and, apart from CO2 (about 11-12%), contains also other gases such as N2, O2, H2O, SO and NOj as well as particulate matter. For post-combustion CO2 capture, the project considers both the existing commercial separation techniques (solvent absorption with amino acid and potassium carbonate solvents) and also novel capture options, such as membrane and adsorption. With respect to the membranes, the programme includes the evaluation of the performance of module configurations under real flue gas conditions, and with respect to the membrane plant, two process options are available ... 

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