H. Jansen

Miniaturization of solid-phase reactors for on-line post-column derivatization in narrow-bore liquid chromatography

SummaryThe use of solid-phase reactors for post-column derivatization in narrow-bore HPLC (1.0mm i.d. analytical columns) is evaluated. Two systems are described, viz. for the determination of N-methylcarbamate pesticides and for that of urea and ammonia. The solid-phase reactor is packed with a strong anion exchange resin and urease immobilized on silica, respectively, to effect the catalytic hydrolysis of the solutes eluting from the analytical column. In both systems, the hydrolysis product is reacted with o-phthalaldehyde followed by fluorescence monitoring.Analytical data are presented and band broadening from various parts of the reaction detector system is discussed. An on-line trace enrichment procedure via a micro precolumn is descried for the trace level determination of N-methylcaramates in surface water samples.

Fluorescence enhancement for aflatoxins in HPLC by post-column split-flow iodine addition from a solid-phase iodine reservoir

SummaryA method for post-column derivatization of the highly carcinogenic aflatoxins with iodine has been developed. It involves splitting of the mobile phase used for the reversed phase HPLC separation. One part flows through the injection valve and the C18 analytical column to achieve the separation. The other part flows through a column packed with solid iodine. The iodine-containing solution is recombined with the flow coming from the analytical column. The derivatization reaction takes place in a knitted open tubular reactor maintained at 60 °C. Detection is done by fluorescence measurement. Due to the low solubility of iodine in the mobile phase, the iodine solid-phase column can be used for very long periods of time before refilling is necessary. The analytical system consists of only one pump and therefore gives the opportunity to carry out low-cost post-column reaction detection. The method yields reproducible results, a linear response over at least two orders of magnitude and detection limits of about 1 ppb, both for standard solutions and for peanut butter samples.

Determination of urea and ammonia using ion-pair liquid chromatography with on-line post-column derivatization in an enzymatic solid-phase reactor

Abstract A quantitative method of analysis for urea and ammonia is described, which is based on ion-pair high-performance liquid chromatography with on-line post-column derivatization on immobilized urease. In the urease solid-phase reactor, urea is quantitatively converted into ammonia, which reacts with o-phthalaldehyde and is detected by fluorescence monitoring. The method is sensitive, highly specific and easy to use, and has a linear range of ca. 1.5 orders of magnitude for urea and ca. 2.5 orders of magnitude for ammonia. Detection limits are 0.4 ng (3·10−7 M) and 0.3 ng (9·10−7 M) for urea and ammonia, respectively. The method is used for the determination of the urea and ammonia content of samples from an urea plant and in wastewater samples.

Stereoselective determination of L-amino acids using column liquid chromatography with an enzymatic solid-phase reactor and chemiluminescence detection

A stereoselective post-column reaction detection system for eight L-amino acids that makes use of a reactor packed with immobilized L-amino acid oxidase is described. The combination of the selectivity of the enzyme and the selectivity of the peroxyoxalate chemiluminescence detection used provides an extremely selective detection system. The detection system gives linear responses over two orders of magnitude and detection limits at the 0.35.10(-6)-3.0.10(-6) M level. The method was used for the determination of selected L-amino acids in urine and beer.

Parallel column ion exchange for post-separation pH modification in liquid chromatography: application to barbiturates and miniaturization

A new approach to the UV detection of barbiturates in high-performance liquid chromatography is demonstrated. The analytical system comprises an anion-exchange column inserted parallel to the injection valve and analytical column. One part of the acetate-containing mobile phase flows through the injection valve and analytical column to achieve the separation, the other part flows through the anion-exchange column where the acetate ions cause the release of hydroxide ions from this column. Finally, the alkaline stream from the anion-exchange column is recombined with the analytical column effluent. This results in an alkaline medium which can be favourable for many detection processes. A series of barbiturates, which show enhanced UV detectability at 254 nm in alkaline solution, was chosen to demonstrate the potential of such a method. Applications of this principle to the analysis of urine and plasma samples are described. In the system only one pump is needed for the separation and the post-column pH modification. A critical comparison between conventional scale and narrow-bore systems is made.

Derivatization of some polymers with o-phthalaldehyde prior to high-performance size-exclusion chromatography

Abstract The well known OPA ( o -phthalaldehyde) derivatization commonly used in aqueous media has been modified on order to be able to carry out the reaction with various polymeric materials in tetrahydrofuran, a solvent very often used in size-exclusion chromatography. The modified OPA reaction leads to stable fluorescent derivatives of the investigated analytes. The derivatization procedure has three important advantages: (i) the selectivity of the analytical method is greatly improved since only compounds having a primary amino group are detected; (ii) the sensitivity is increased by at least two orders of magnitude compared to ultraviolet or refractive index detection; (iii) the chromatography is facilitated due to a conversion of the primary amino groups.

In-column and post-column photochemical fluorescence enhancement in packed capillary liquid chromatography

Abstract A photochemical reaction detection system coupled with a 320-μm I.D. packed fused-silica HPLC analytical column is described. This reaction detection system does not require the addition of reagent, hence it is suitable for combination with a miniaturized analytical column. As a model system, the separation and detection of some dansylated chlorophenols was chosen. In the photochemical reactor, the main reaction product is 5-dimethylaminonaphthalene-1-sulphonic acid (dansyl-OH), which is determined by fluorescence monitoring. Both the photochemical reaction after the column and in the end part of the column (‘in-column’) were investigated and a comparison between the two approaches was made. In both cases, band broadening due to the photochemical reaction detection system was less than 0.1 μl. In-column detection of the photochemical reaction product was investigated in order to study the possibility of carrying out separation, derivatization and detection in the analytical column. An increase in...