Postcolumn addition, of NaOH

Figure 3.217 Separation of various monosaccharides on CarboPac PA1. Eluent 1 mmol/L NaOH flow rate 1 mL/min detection pulsed amperometry on a gold working electrode after postcolumn addition of NaOH...

Wine was analyzed by Hughes and Johnson (1983) and carbohydrates and alcohols were determined. Cation-exchange chromatography with a water mobile phase was used for the separation, and postcolumn addition of NaOH was necessary for PAD detection. Both dry and sweet wines were analyzed, and as expected, the dry wine had much less fructose and dextrose than the sweet wine. A peak for sorbitol was not observed for any of the wines tested. Because sorbitol is not naturally present in grapes, its presence in wine would indicate adulteration of the wine with other fruit juices. 

FIGURE 10.9 Chromatogram of the ohgosaccharide profile of a pure honey sample. Chromatographic conditions Two CarhoPac PAl colunms (Dionex) connected in series gradient elution was used with NaOH as the primary eluent. Postcolumn addition of NaOH was used to minimize baseline drift. PAD at an Au electrode was used. (Reprinted from Swallow, K.W. and Low, N.H., J. Assoc. Off. Anal. Chem., 77(3), 695-702, 1994. With permission.)... 

Figure 1 Expanded chromatograms from Dionex ion chromatograph of enzyme hydrolysates of corn and potato starch, the use of a single enzyme, amyloglucosidase (AMG), and a mixture of AMG, a-amylase (a-A), and pullulanase (PU), to show the effect on hydrolysis of limit dextrin. Anion exchange column AS6, with pulsed amperometric detection. Gradient flow solvents (1) ISOmmolT NaOH and (2) ISOmmoll NaOH + 500mmol I NaOOCCHs. Postcolumn addition of 0.3mmoll NaOH.

Postcolumn addition of a sodium hydroxide concentrate is necessary to raise the eluent pH to about 13, a value that is optimal for pulsed amperometric detection. The addition of NaOH can be done pneumatically via a metal-free manifold connected to a reaction coil of corresponding dimension in which the column effluent and the NaOH concentrate are mixed. Alternatively, a pump with a corresponding pulse damper can be used. 

Not only is the postcolumn addition of concentrated NaOH of significance for the electrochemical detection of amino sugars but it also allows the separation... 

The pulsed amperometric detector (PAD) developed by Johnson and co-workers using an Au or Pt electrode has permitted the direct detection of aliphatic alcohols including carbohydrates, amines, and sulfur compounds. Fouling of these electrodes is prevented by application of both positive (to eliminate sample adsorption) and negative (to reduce any metal oxide) reactivation step potentials on the order of 100 ms before resetting the potential for detection of the analyte. The analytical current is usually sampled near the end of the detection potential pulse to permit decay of the charging current. The oxidation of these aliphatic compounds such as carbohydrates is facilitated in basic solution at about pH 12, so postcolumn addition of 0.1 Af NaOH or the use of a polymeric column with a basic mobile phase is required. Detection limits of alcohols and carbohydrates are at the 10 ppb level. Alka-nolamines, amino acids, and sulfur compounds other than sulfonic acids and sulfones can also be detected. 

Fluorescence detection is not nearly as widely used as UV detection since most carbamates possess no native fluorescence. However, the structure of these pesticides contains a V-methyl substituted urethane with variations in the ester moiety. The common methylamine functionality allows the detection of compounds via a two-stage postcolumn reaction. Carbamates in the column effluent are first hydrolyzed with NaOH at a high temperature to form methylamine, which is converted into a fluorophore compound by addition of 6>-phthaldehyde (OPA) and... 

HPLC analysis of APG is carried out with C8 or Cl8 columns by use of a refractive index detector or a conductivity detector after the addition of 0.3 mol/1 NaOH to the eluate in a postcolumn reactor. ... 

Figure 7 Application of HPLC to study vitamin Bg metabolism in human lymphocytes. HPLC conditions Column Partisil 10 ODS-3 (Whatman, Clifton, NJ, USA). Mobile phase Isocratic elution with 0.05 mol/L KH2PO4 buffer, pH = 2.9, containing 3% acetonitrile, at flow rate 1.1 mL/min. Detection Fluorescence, excitation 367 nm, emission 478 nm. Postcolumn aUcalinization of mobile phase to pH 12.0 was achieved by addition of 4% NaOH solution. Peaks 1 = PLP semicarbazone 2 = PL semicarbazone. PLP and/ or PL concentrations were determined using an external standard (A 300 nmol/L of PLP and PL, respectively) and are expressed per mg of lymphocyte protein (A) standards, (B) lymphocyte extract, (C) quantitation of PL kinase activity using added PL as a substrate.

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