Postcolumn reaction

Wetai Ion Analysis. We have reported a sensitive trace-metal analysis based upon HPLC separation of p-aminophenyl EDTA chelates and fluorescence detection by postcolumn reaction with fluorescamine (23). An application of the pyridone chemistry already discussed leads to a fluorescent-labeled EDTA (VIII). 

Holland, L.A. and Lunte, S.M., Postcolumn reaction detection with dualelectrode capillary electrophoresis-electrochemistry and electrogenerated bromine, Anal. Chem. 71, 407, 1999. 

Many IC techniques are now available using single column or dual-column systems with various detection modes. Detection methods in IC are subdivided as follows [838] (i) electrochemical (conductometry, amper-ometry or potentiometry) (ii) spectroscopic (tJV/VIS, RI, AAS, AES, ICP) (iii) mass spectrometric and (iv) postcolumn reaction detection (AFS, CL). The mainstay of routine IC is still the nonspecific conductometric detector. A significant disadvantage of suppressed conductivity detection is the fact that weak to very weak acid anions (e.g. silicate, cyanide) yield poor sensitivity. IC combined with potentiometric detection techniques using ISEs allows quantification of selected analytes even in complex matrices. The main drawback... 

Nishiyama and Kuninori [65] described a combination method of assay for penicillamine using HPLC and postcolumn reaction with 6,6 -dithiodi(nicotinic acid). Thiols were separated by HPLC on a reversed-phase column (25 cm x 4.6 mm) packed with Fine Sil 08-10, with 33 mM KH2PO4 (adjusted to pH 2.2 with H3PO4) or 33 mM sodium phosphate (pH 6.8) as the mobile phase. Detection was by postcolumn derivatization with 6,6 -dithiodi(nicotinic acid), and measurement of the absorbance of the released 6-mercaptonicotinic acid was made at 344 nm. The detection limit for penicillamine was 0.1 nmol. A comparison was made with a... 

Hydrogen peroxide [26-33] and analytes converted to hydrogen peroxide via either an enzymatic [34-52] or a photochemical postcolumn reaction [53-55]... 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Igawa, M., J. W. Munger, and M. R. Hoffmann, Analysis of Aldehydes in Cloud- and Fogwater Samples by HPLC with a Postcolumn Reaction Detector, Environ. Sci. Technol, 23, 556-561 (1989). 

Quantitation of the amino acids proceeds through the postcolumn reaction with ninhydrin at 120-135°C, yielding a purple complex for the primary amino acids (absorbance measured at 570 nm) and a yellow complex for the amino acids proline and hydroxyproline (absorbance at 440 nm). 

As described above, the amino acids are separated at a constant flow on a high-resolution cation-exchange column using buffer and temperature gradients. The postcolumn reaction with the ninhydrin reagent is carried out at 135°C and the absorbances of the reaction products are read at both 570 and 440 nm. Amino acids are identified by comparing their retention time and 570/440 ratio with that of authentic reference substances (see Fig. 2 .2). 

When one is deciding what column geometry is optimal for trace analysis with unlimited sample volume, two additional points should be evaluated. First, to what extent does the analysis require accurate and reproducible injections Strict performance specifications may eliminate microbore columns from consideration. The accuracy and reproducibility of injection systems that deliver 0.1-, 0.2-, and 0.5-/xL samples have not been adequately characterized. Second, if the analyte of interest requires postcolumn derivatization, construction of a postcolumn reaction system that is compatible with the exceedingly small band volumes characteristic of microbore columns may be extremely difficult, but not impossible. Apffel et al. (28) developed and evaluated both packed-bed and open tubular postcolumn reactors for use with 1-mm i.d. analytical columns. Catecholamines were postcolumn derivatized with o-phthal-aldehyde and detected spectrofluorometrically. The 5-/zm particle... 

Precolumn derivatization is often inadequate for dirty samples. In these cases, application of a postcolumn reaction detection system will often suffice. Deelder et al. (44) and van der Wal (45) have examined different configurations for postcolumn reactors and defined optimal selections on the basis of reaction time and type and effect on resolution and sensitivity. Both studies preferred the packed-bed reactor to the open tubular reactors when conventional column geometries were employed for separation, that is, 4.6 mm i.d. X 15 or 25 cm. 

Nondek et al. (46) reported an innovative approach to the analysis of N-methylcarbamates in river water using postcolumn reaction detection. Separation of the underivatized N-methylcarbamates was carried out on a reversed-phase column hooked directly to a bed reactor packed with Aminex A-28, a tetraalkylammonium anion-exchange resin. The packed bed catalytically base-hydrolyzed the carbamates and... 

The limitations of electrochemical detection can be overcome in some cases by the use of pre- or postcolumn reactions. There are numerous examples of these reactions, and only a few are covered here. Several reviews that cover this area more thoroughly have been published [37,38]. 

Postcolumn reactions in LCEC can take several forms, most of which are illustrated in Figure 27.18. These include transformation of the analyte by chemical, electrochemical, photochemical, and enzymatic methods. 

Figure 27.18 Common configuration for postcolumn reactors with electrochemical analysis. (A) LC-chemical reaction-EC. Postcolumn addition of a chemical reagent (for example, Cu2+ or an enzyme). (B) LC-enzyme-LC. Electrochemical detection following postcolumn reaction with an immobilized enzyme or other catalyst (for example, dehydrogenase or choline esterase). (C) LC-EC-EC. Electrochemical generation of a derivatizing reagent. The response at the second electrode is proportional to analyte concentration (for example, production of Br2 for detection of thioethers). (D) LC-EC-EC. Electrochemical derivatization of an analyte. In this case a compound of a more favorable redox potential is produced and detected at the second electrode (for example, detection of reduced disulfides by the catalytic oxidation of Hg). (E) LC-hv-EC. Photochemical reaction of an analyte to produce a species that is electrochemically active (for example, detection of nitro compounds and phenylalanine). Various combinations of these five arrangements have also been used. [Reprinted with permission from Bioanalytical Systems, Inc.]...

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

Fig. 6 Typical ion-exchange separation of amino acids followed by postcolumn reaction with ninhydrin. Separation was achieved employing Beckman 6300 amino acid analyzer with cation-exchange column. Three sodium buffers were used with a stepwise elution scheme as supplied/recommended by the manufacturer. Detection was the sum of 440-nm and 570-nm absorbance. Standard three-letter abbreviations for amino acids are used also, CA = cysteic acid, tau = taurine, and nle = norleucine. Data was supplied by Stephen D. Smith, Ross Products Division of Abbott Laboratories, Columbus, OH.

The earliest approach to amino acid analysis involved postcolumn reaction. This scheme offers several advantages compared to precolumn reaction. First, it simplifies the sample preparation necessary. Often, precolumn derivatizations require sample cleanup steps to eliminate sample... 

An advantage to precolumn derivatization is that there are no limitations with respect to reaction kinetics. Of course, rapid reaction is always desirable, but need not be the principal factor when choosing a derivatizing reagent. This means that a wide variety of chemistries are available for derivatization. However, it is important that very stable derivatives be formed. The limited stability of OPA derivatives is the reason why OPA is most commonly employed for postcolumn reaction. If it is to be used in the precolumn fashion, very well-controlled and automated (mechanized) sample/reagent handling immediately prior to injection must be employed. 

Biotin and biotin analogs Infant formula Protein precipitation using concentrated hydrochloric acid neutralization with 6 M NaOH lipid extraction with n-hexane Precolumn Microsorb C18 (15 X 4.6 mm, 5 jam Rainin). Analytical Microsorb C18 (250 X 4.6 mm, 5 /zm Rainin). Isocratic 100 mM phosphate buffer, pH 7.0 + methanol (80 + 20, v/v). 0.4 ml/min. Postcolumn reaction system UV absorbance at 220 nm followed by streptavidin-fluorescein isothiocyanate (2.0 mg/L) knitted open tubular reaction system (10.0 m x 0.5-mm ID) at a flow rate External standardization. 184 Linear range = 0.08-1.00 fjM biotin. LoD = 0.02 /zM or 97 pg biotin at SNR = 3. Repeatability CV 3.5% for biotin in infant formula. 

A rapid preseparation technique was developed for the extraction of SAL from various chicken tissues using the irradiation of the sample in EtOH-2-PrOH for 9 s in a common household microwave oven. The extract was analyzed without further cleanup and detected via postcolumn reaction with DMABA at 86°C. Recoveries ranged between 87% and 100% (105). 

The same derivatization was applied to the HPLC determination of STR and DHS in milk. The comparison of HPLC and ELISA methods was also performed for DIHS. After removal of fat by extracting a milk sample with oxalic acid and centrifuging, proteins were precipitated with TCA. The supernatant was treated by SPE on a Cl8 column. The cartridge was washed with water, and the analytes were eluted with ion-pair in MeCN. The eluate was reconcentrated by evaporating and dissolving in water. Postcolumn reaction took place at 65°C. Recoveries were dependent on the concentration level and the batch of SPE columns used, and independent of the fat content and homogenization. The sample cleanup was not sufficient for the analysis of cheese. The DIHS concentrations of incurred samples determined by ELISA were higher than those obtained by the LC method (107). 

HPLC with postcolumn reaction with ninhydrin (125°C). 

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