Ulla Lund

Reversed-phase ion-pair partition chromatography of phenylacetic, mandelic and pyridinecarboxylic acid derivatives

Phenylacetic, mandelic and pyridinecarboxylic acids of physiological interest have been separated by ion-pair partition chromatography with 1-pentanol as stationary phase and tetrabutylammonium as counter ion in the aqueous mobile phase. The stability of the chromatographic system is very high and the support is spontaneously re-coated with stationary phase. Untreated plasma samples can be injected in relatively large volumes without serious loss of separating efficiency. A venting system was found efficiently to reduce disturbing early peaks on injection of biological samples.

High-performance liquid chromatography on dynamically modified silica

Abstract High-performance liquid chromatography on silica using eluents containing cetyltrimethylammonium (CTMA) bromide was investigated, and adsorption isotherms were determined for two silica packings of different pore diameter and surface area. It was found that about one CTMA ion was adsorbed per square nanometer of the silica surface at pH 7.5 and at a concentration of 6 m M CTMA bromide in 50% of methanol. Fourteen different silica packings were compared using a test mixture, and thirteen were found to exhibit the same selectivity towards the test mixture, which included acids, bases and non-ionic compounds, thus providing a chromatographic system that is largely independent of the origin of the column material. The retention mechanisms for the five test compounds are discussed.

High-performance liquid chromatography on dynamically modified silica: III. Modification of silica with long-chain and symmetrical quarternary ammonium compounds☆

Abstract The influence of the nature of quaternary ammonium compounds on retention in high-performance liquid chromatography on dynamically modified silica was investigated. Adsorption isotherms were determined on bare silica (LiChrosorb Si 60) for four alkyltrimethylammonium bromides and two symmetrical tetraalkylammonium bromides, each containing 15–21 carbon atoms. It was found that only the long-chain quaternary ammonium ions are adsorbed on to the silica surface in appreciable amounts and that the affinity increasing number of carbon atoms in the alkyl chain. The maximum amount that can be adsorbed per gram of silica is of the same order of magnitude for each of the four long-chain quaternary ammonium compounds. This amount, however, is reached at lower concentrations in the eluent the longer is the alkyl chain. The retention of five test compounds was determined over the whole concentration range investigated for each of the modiflying agents. The surfactant concentration that causes maximum retention for four of five test compounds coincides with its critical micellar concentration. The retention mechanisms and the influence of the type of the modifying agent on selectivity are discussed, and compared with published results on related experiments on chemically bonded stationary phases.

High-performance liquid chromatography on dynamically modified silica. IV: Effects of variations of pH, ionic strength and buffer composition in eluents containing cetyltrimethylammonium bromide

The influence of pH changes as well as variation of ionic strength and buffer composition on the retention of selected test solutes in high-performance liquid chromatography on dynamically modified silica were investigated. Chromatography was performed on columns of bare silica (LiChrosorb Si 60) using eluents consisting of mixtures of methanol and aqueous phosphate buffers and containing cetyltrimethyl-ammonium (CTMA) bromide. With increasing pH the amount of CTMA ions adsorbed on to the silica surface rose, thus increasing the retention due to reversed-phase partition. It was shown that apart from the adsorption of CTMA on to silica, ion exchange plays only a minor role in the retention of cations, except for hydrophobic quaternary ammonium compounds. An increase in the ionic strength of the phosphate buffer caused a decrease in the retention of most ionic test solutes. This effect was shown to be due to interaction with the phosphate ions in the eluent and not to a decrease in the amount of CTMA adsorbed. The addition of inorganic salts, with anions different from the buffer anion, had no effect on retention apart from ionic strength effects. The addition of alkane-sulphonates was shown to lead to a selective decrease in the retention of anionic solutes. The nature of the buffer cation was shown greatly to influence retention and selectivity.

Occurrence of low- and high-chlorinated phenols in municipal sewage before and after passing through biological treatment plants

Four biological treatment plants for municipal sewage, situated at different locations, were examined for their chlorophenol content and their ability to degrade these compounds. A routine method for the analysis of both low- and high-chlorinated phenols was developed, based on gas chromatography with electron-capture detection (GC-ECD). The method was evaluated by comparing the GC-ECD results with those obtained by combined gas chromatography-mass spectrometry with selected-ion monitoring of the molecular ions of the specific phenols. The total phenol content, determined by GC-ECD showed a good correlation with the phenol number, obtained by using the 4-aminoantipyrine method.

Screening of Pollution from a Former Municipal Waste Dump at the Bank of a Danish Inlet

An investigation of the potential pollution from a former municipal waste dump also containing chemical waste has been performed. The dump is situated at the bank of the inlet Holbaek Fjord, Denmark. The investigation has involved hydrogeological investigations of the dump and surroundings and chemical analyses and ecotoxicological tests of leachate from the dump. The investigations have shown that the water flow from the dump is towards the inlet, and that no ground water resources are threatened. Analyses have shown the presence of approx. 40 compounds in the leachate, of which pentachlorophenol and 2,3,4,6-tetrachlorophenol are thought to involve the highest environmental risk. Acute toxic effects have been shown on approx. 1/3 of the samples of leachate and it has been shown that this effect is removed upon dilution by a factor of 5. It was concluded that the impact of the toxic substances in the leachate on the inlet should be further investigated.

Chapter 4.1. Types of liquid column chromatography

Publisher Summary This chapter discusses several types of liquid column chromatography. In adsorption chromatography, the retention of the solute is a consequence of the interaction with the surface of the solid adsorbent. The adsorbent surface has a rigid structure making this type of chromatography uniquely useful for separations of geometric and structural isomers with molecular weights up to about 1000. In liquid-liquid partition chromatography, the distribution of solutes takes place between two immiscible solvents. In normal phase (straight phase) chromatography the more polar liquid—often water rich—is the stationary phase, whereas the opposite is true in reversed phase partition. The stationary phase may be situated on a variety of supports depending on the polarity of the stationary phase. Partition chromatography is used for separation of solutes with molecular weights up to a few thousands, and is a powerful tool in the separation of series of homologs. In size exclusion chromatography, the solid support is a porous polymer with a controlled pore size, and the solute molecules are separated according to their size in solution. The larger molecules are mostly excluded and thus, they have the shortest retention times. The size exclusion may be performed in aqueous systems (gel filtration), where water soluble macromolecules can be separated, or in nonaqueous systems (gel permeation). By proper calibration, the method can also be used for determination of molecular weight or molecular weight distribution.

Chapter 4.2. Instrumentation

Publisher Summary This chapter focuses on the equipments required in liquid column chromatography (LC), which ranges from a simple and inexpensive glass column eluted by gravity flow to a sophisticated, computerized high-performance liquid chromatograph. A high-performance LC (HPLC) procedure is built from three parts—namely, the column or column system, the solvent delivery system, and the detection device. The column is the most important part of an LC system. The separation of solutes takes place in the column, and it is the dimensions of the column and the column packing material, together with the nature of the mobile phase that determine the efficiency of the separation. An HPLC column is an extremely efficient filter as any particular materials or strongly retained impurity in the sample injected remains on the top of the column. To prevent a fast deterioration of the analytical column, a cheaper precolumn may be installed. The precolumn may be discarded or repacked after a certain number of sample injections. The solvent delivery system consists of—a solvent reservoir, an in-line filter and a pumping system. The reservoir may be as simple as the flask in which the solvent is delivered. In case of detectors, there seems to be no limitation on the kind of detectors used for HPLC. Even detectors designed for use in gas phase analyses have been applied to HPLC. The principles of the detectors used for routine analysis are based on absorption of light in the ultraviolet and visible spectrum, refractive index or fluorescence.

Chapter 4.3. Detection

Publisher Summary This chapter discusses the importance of choosing an appropriate detector and the use of proper methods for the enhancement of detection. A good liquid chromatographic detector should meet the following requirements. It should have (1) high sensitivity; (2) a predictable selectivity; (3) a wide linear dynamic range; (4) give a response—that is independent of the mobile phase; (4) have low volumes of detector cell and connecting tubing; (5) have a fast response; and (6) be nondestructive. The ultraviolet detector is probably the most common used detector. . This is available as a fixed wavelength detector and as a variable wavelength spectrophotometer. The fixed wavelength detectors often produce less noise than the variable wavelength detectors and they are much cheaper. The variable wavelength detector is more versatile, and it provides the possibility of detection at the most favorable wavelength for the given sample, which is not always possible with a fixed wavelength detector. The fluorescence detector is of great utility in biochemical analysis. It can be used forcompounds that have native fluorescence—for example, indoles, catecholamines, porphyrins, or else derivatization can be used to produce or enhance fluorescence.

Chapter 4.4. Adsorption and partition chromatography

Publisher Summary This chapter discusses adsorption and partition chromatography. Adsorption chromatography is based on liquid–solid partition—that is, a partition of solute molecules between the liquid mobile phase and active sites on the surface of the solid stationary phase. The solid support used is most often silica or alumina, the former being far more widely used. During the chromatographic process solute molecules are adsorbed to active sites on the support surface in competition with solvent molecules. The active sites consist of hydroxyl groups (for silica silanol groups, Si-OH) and the affinity to them is greater the more polar the molecules, and the greater their ability to form hydrogen bondings. The surface structures of different brands of silica are largely identical, and thus their selectivity towards various mixtures of solutes does not exhibit great variation. Separation by liquid–liquid partition chromatography is the result of the difference in the distribution between two immiscible liquid phases of the individual components of a mixture. The liquid stationary phase is coated on a solid support, which is ideally inert. As the support relatively large pore silica (10–50 nm pore diameter) is most often used. Coating the stationary phase onto the support may be performed in various ways. The solvent evaporation technique (as in the preparation of GC column materials) may be used, provided that the stationary phase is non-volatile and that the particle size of the support exceeds 20 μm.