Reactor, post column

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. 

Fig. 2.4p shows three types of post-column reactor. In the open tubular reactor, after the solutes have been separated on the column, reagent is pumped into the column effluent via a suitable mixing tee. The reactor, which may be a coil of stainless steel or ptfe tube, provides the desired holdup time for the reaction. Finally, the combined streams are passed through the detector. This type of reactor is commonly used in cases where the derivatisation reaction is fairly fast. For slower reactions, segmented stream tubular reactors can be used. With this type, gas bubbles are introduced into the stream at fixed time intervals. The object of this is to reduce axial diffusion of solute zones, and thus to reduce extra-column dispersion. For intermediate reactions, packed bed reactors have been used, in which the reactor may be a column packed with small glass beads. 

Consider a solute which is detected by derivati-sation, using a post-column reactor of the type shown in Fig. 2.4p(i). 

Other methods used to characterize and identify DOP involve bioassays with Chlorella to study the biological availability and biouptake of the HMW SRP fraction (4, 6). These bioassays indicate that the algal growth responds similarly to HMW SRP and to PO. A preference for PO 3- was detected, and not all of the reactive HMW fraction was used. Enzymatic assays used by Herbes et al. (13) tentatively identified inositol hexaphosphate as part of the DOP. Using an anion-exchange HPLC system with a phosphorus-specific post-column reactor, Minear and co-workers (15,16) possibly have detected inositol hexaphosphate, DNA, and nucleotide fragments in lake waters. 

Of the platinum-based drugs, cisplatin or cf -diarnmincdichloroplatinum (II) has been the most studied in treatments of cancerous tumours. Quantities of the drug administered in treatments must be carefully controlled because of appearances of side effects, primarily nephrotoxicity and nausea in patients. In some studies, ultrafilterable cisplatin, or free platinum in blood serum or plasma has been differentiated from platinum bound to proteins (Goel et al., 1990). HPLC has been used extensively in separations of intact cisplatin from other species. An anion-exchange column was connected to a post-column reactor and a UV-spectrophotometer for measurements of cisplatin concentrations in plasma and urine (Kizu et al., 1995). The detection limit was 20 nmol dm-3. Modes of action... 

Mayer determined acetylcholine and choline by enzyme-mediated liquid chromatography with electrochemical detection [195]. The two compounds were separated by passing the eluted fractions through a post-column reactor containing immobilized Acetylcholineesterase and choline oxidase. In the presence of either compound, the dissolved oxygen was converted into hydrogen peroxide, which was detected amperometrically at a platinum electrode. This method was used to determine choline in rat brain homogenates. 

Reversed-phase packing post column reactor 3 cm x 2.1 mm of Aquapore AX 300. 20 mM Tris acetate (pH 7)-l mM-tetramethyl-ammonium chloride-2pM octanesulfonate containing 2% of acetonitrile. Electrochemical Tissue extracts, AChE and ChO are absorbed and immobilized on the post column [148]... 

The reservoir can also act as a post-column reactor. The appropriate reagent is dissolved in the mobile phase and will react immediately with any solute eluted from the column to form a derivative that can be sensed by the detector. Using the integral detector as a post-column reactor, however, can only be successful if the derivatives are not... 

Post-column derivatization does not merely require the selection of the most appropriate reagent to react with the solute to render it detectable, but also involves the modification of the chromatographic system to allow the reaction to take place prior to entering the detector. This necessitates the insertion of a post column reactor between the column and the detector. Such a reactor can easily interfere with the resolution obtained from the column and consequently the reactor system must be designed with some care to... 

A diagram of a post-column reactor system is shown in figure 9. 

The post-column reactor consists essentially of four parts, a reagent reservoir, a pump, mixing T and a reaction chamber. The pump should provide a pulse-free flow of reagent as there is little resistance downstream to the detector and most detectors are flow sensitive to a greater or lesser extent. Preferably, either a syringe pump should be employed or if a reciprocating pump is used, it should be fitted with... 

The reagents used in post-column derivatization will be specific for the solute of interest and will often be the same, or similar to those used in pre-column derivatization. Reagents that produce derivatives that absorb in the visible light range are popular, as relatively simple detectors can be employed. In general, derivatization, particularly post-column derivatization, is only used as a last resort to provide sensitivity or selectivity, which is difficult or impossible to obtain by any other means. Post-column reactors complicate an already complex instrument, render it more difficult to operate and make it more expensive. In addition, however well the post-column reactor is designed it will inevitably impair, to some extent, the separation achieved by the column. 

Nelson et al. used the oxidation of morphine to pseudomorphine 1n a post-column reactor. For the post-column derivatizing reagent, a solution of 50 mg potassium ferric cyanide in 250 ml of 4 M ammonia was used. Methanol - 0.1 M aqueous potassium bromide (12.5 87.5) was used as mobile phase for the separation of the alkaloids on a octadecyl stationary phase. The fluorescence was measured at 432 nm after excitation at 323 nm. 

RSP,methylreserpate Post-column reactor for Phenyl bonded phase not ACN-0.005M NaH.PO. buffer(pH 6) ... 

In connection with the development of a post-column reactor, Gfeller et al. used a stationary phase with chemically bonded diol groups, which allowed the use of exclusively aqueous mobile phases (0.01 M, pH 3, phosphate buffer). A series of alkaloids was separated, viz. dihydroergotamine and bromocryptine. 

Imanari et al. has reported a spectrophotometric detection of many inorganic anions using a post-column reactor [42]. A stream of ferric perchlorate, which is essentially colorless, is mixed with the column effluent. The ferric perchlorate is colorless because perchlorate is a poor complexing anion, but most anions will complex the iron and form colored species that can be detected at 330-340 nm (Table 4.3). A similar detection method works for ions such as orthophosphate, pyrophosphate, nitrilo-triacetic acid (NTA), and ethylenediaminetetraacetic acid (EDTA) [46]. 

F9. Fulton, J. A., Schlabach, T. D., Kerl, J. E., and Toren, E. C., Jr., Dual-detector-post-column reactor system for the detection of isoenzymes separated by high-performance liquid chromatography. ]. Chromatogr. 175, 283-291 (1979). 

The possibility of separation and analysis of enzymes by CZE has been explored. Banke, et al. separated alkahne proteases from crude fermentation broth, and collected fractions from CZE for enzyme analysis. CZE was also used to monitor the progress of an enzyme reaction [23]. Konse, et al. reported modification of a microtitre plate assembly which was used to coUect fractions on polyvinylidene difluoride (PVDF) membranes. Fractions blotted onto the PVDF membranes were then subsequently analyzed by a sequencer [24]. Emmer and Roeraade described an on-hne micro-post column reactor which they used in conjunction with on-column detection. By using the two detector system, they were able to rapidly monitor enzyme activity in samples. Through careful optimization of conditions in the reactor, the loss of efficiency at the point of detection through the reactor capiUary was minimal [25]. 

Brinkman, U.A.T. Frei, R.W. Lingeman, H. Post-column reactors for sensitive and selective detection in high-performance liquid chromatography Categorization and applications. J.Chromatogr, 1989, 492, 251-298 [post-column reaction review]... 

Fig. 4.7.7. Chromatography of a crude yeast extract on Cibacron Blue F3G-A silica with on-line monitoring of hexokinase (HK) and 3-phosphoglycerate kinase (PGK) activity. Sample applied at first arrow crude yeast extract (2 pi) column irrigant, 0.1 M Tris-HCI buffer (pH 7.3) containing O.S mM EDTA, 5 mM MgClj and O.S mM 2-mercaptoethanol flow rates, I ml/min, both pumps post-column reactor temperature, 40 0.1°C assay mix temperature, 0°C temperature of analytical column, 22°C eluents (400 pi), as indicated by the arrows UV-detector (290 nm), 0.32 AUFS enzyme activity detector, 0.5 AUFS. Reproduced with permission from Ref. 126.

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