Post-column reagent

DHAA ELD (A, =350nm =430 nm) post column reagent o-phenyldiamine ED... 

X4 i=520nm) post column reagent avidin-FITC in phosphate buffer solution... 

Improvements in separations ability offered by the Dionex HPIC-CS5 column, which exhibits both anion and cation exchange capacity, have allowed the determination of ten metal ions in a single injection with a pyridine-2,6-dicarboxylic acid (PCDA) eluent (Fig. 2.3). The post-column reagent used in the detection scheme was 4-(2-pyridylazo) resorcinol. 

Figure 1. Fluorescence (top, A ciiation = 332 nm, Kn im = 442 nm) and UV (bottom) (254 mn) chromatograms of SFA (500 mg/L) with an injection volume of 80 xL. Eluent and post-column reagent (0.0 mM Cu ) flow rates were 0.7 mL/min. and 0.1 mL/min., respectively.

Figure 3. Quenching of SFA fluorescence chromatograms with addition of Cu. Post-column reagent concentration of Cu " is listed above corresponding chromatogram. Conditions are the same as listed in figure 1.

Table I lists binding parameters for SFA based on total Cu concentration, correcting for the dilution of the Cu ion when post-column reagent stream was mixed with the column eluent. While these values show that this data treatment works well for SFA, binding parameters for WFA showed negative values for Cl and /res, indicating that the quenching behavior of WFA is not fit by the model on which the data treatment is based. The fact that quenching of the WFA is not modelled well may be a result of the fundamental assumption of solution equilibrium not being met (2).

The complicated nature of the instrumentation required for these experiments may also result in artifacts which may cause the fluorescence quenching to not conform to the model. Fluctuations in the proportioning of solutions A and B in the post-column reagent pump may result in error in the total Cu concentration reported as well as variation in concentration of Cu " in the post-column system throughout an experiment. Likewise, fluctuations in pump flow rate of both the eluent and post-column reagent pumps may also effect the total Cu concentration in the post-column system. Since each experiment was conducted over nine to twelve hours, temperature of the system and the fluorescence detector lamp output may also have contributed to variations in fluorescence quenching. A decreased run time for the chromatographic method and a decrease in the number of sample runs for each experiment may help to reduce this artifact. 

Pyridine dicarboxylic acid (PDCA) was used as a complexing agent for Cr to form anionic Cr(PDCA)2. The sample pH is critical to the separation. Optimum results were reported at pH 6.8. Both Cr(III) and Cr(VI) were detected by UV/VIS detection and diphenylcarbohydrazide as a post-column reagent. The method was found to be applicable for plating baths and waste-water analysis. Geddes and Tarter... 

Maeda M, Tsuji A. Chemiluminescence with lucigenin as post-column reagent in high-performance liquid chromatography of corticosteroids and p-nitrophenacyl esters. J Chromatogr, 1986 352 213-29. 

Fig. 11.2.9. Amino acid analysis using a conventional analyser. Chromatographic conditions colunm, Dionex DC-6A (300x4.6 mm I.D.) mobile phase, sodium citrate, three-buffer programme detection, post-column reagent ninhydrin (absorbance 1 full scale). Peaks 1, cysteic acid 2, aspartic acid 3, methionine sulphone 4, threonine 5, serine 6, glutamic acid 7, proline 8, glycine 9, alanine 10, half cystine 11, valine 12, methionine 13, isoleucine 14, leucine 15, Y-leucine 16, tyrosine 17, phenylalanine 18, ammonia 19, lysine 20, histidine 21, arginine. Reproduced from Beckman information catalogue, with permission.

High-sensitivity detection of metals, silica, and other UV-absorbing compounds using either post-column reagent addition or direct detection. 

Detectors should have variable wavelength capabiUty. The UV range 190 to 370 nm is used most often for direct detection. The visible wavelength region is used quite often with the post-column reagents. Metal separations with PAR detection are... 

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