Liquid chromatography detector capabilities

Many nonvolatile and thermally labile allelochemicals can be well separated by liquid chromatography (LC). Identification of the separated components on-line by mass spectrometry (MS) is of great value. Fused-silica LC columns of 0.22 mm ID packed with small-particle material are used in the described LC/MS system. The shape of the column end allows direct connection to a electron impact ion source of a magnetic sector mass spectrometer. Separations by LC are reported and LC/MS mass spectra are shown for monoterpenes, diterpene acids, phenolic acids and cardiac glycosides. The LC/MS system provides identification capability and high-efficiency chromatography with a universal detector. 

High-performance liquid chromatography (HPLC) is one of the premier analytical techniques widely used in analytical laboratories. Numerous analytical HPLC analyses have been developed for pharmaceutical, chemical, food, cosmetic, and environmental applications. The popularity of HPLC analysis can be attributed to its powerful combination of separation and quantitation capabilities. HPLC instrumentation has reached a state of maturity. The majority of vendors can provide very sophisticated and highly automated systems to meet users needs. To provide a high level of assurance that the data generated from the HPLC analysis are reliable, the performance of the HPLC system should be monitored at regular intervals. In this chapter some of the key performance attributes for a typical HPLC system (consisting of a quaternary pump, an autoinjector, a UV-Vis detector, and a temperature-controlled column compartment) are discussed [1-8]. 

James and Martin (49) reported on visual and automatic titration methods capable of detecting microgram quantities of acids and bases. This type of detection has the distinction of being the first type of detector used in gas-liquid chromatography. Electronically modified designs for improving the automation of the detector are possible. 

LIQUID CHROMATOGRAPHY. An analytical method based on separation of the components of a mixture in solution by selective adsorption. All systems include a moving solvent, a means of producing solvent motion (such us gravity or a pump I, a means ol sample introduction, a fractionating column, and a detector. Innovations in functional systems provide the analytical capability for operating in three separation modes (1) liquid-liquid partition in which separations depend on relative solubilities of sample components in two immiscible solvents (one of which is usually water) 12) liquid-solid adsorption where the differences in polarities nf sample components and their relative adsorption on an active surface determine tile degree ol separation (2) molecular size separations which depend on the effective molecular size of sample components ill solution. 

Apparatus (See Chromatography, Appendix IIA.) Use a high-performance liquid chromatograph operated at room temperature with a 10-p.m particle size, 30-cm x 4-mm (id), C18 reverse-phase column (jxBondapak C18 column, Waters Corp., 34-T Maple Street, Milford, MA 01757, or equivalent). Maintain the Mobile Phase at a pressure and flow rate (typically 2.0 mL/min) capable of giving the required elution time (see System Suitability in High-Performance Liquid Chromatography). Use an ultraviolet detector that monitors absorption at 254 nm (0.2 to 0.1 AUFS range). 

Detectors capable of continuously measuring the density of the flowing liquid have been designed by Kratky et al. [146]. They are based on the measurement of frequency oscillations of a quartz tube through which the eluate flows. The oscillation frequency depends on the tube mass and, thus, for the given eluent, on the concentration and density of the solute. Their application to size exclusion chromatography has been described by Trathnigg and Jorde [147]. Kirkland applied such a detector for FFF [148]. 

Derivatives may also be used to m e the molecule capable of detection by selective detectors. The reaction may be carried out during extraction (e.g. extractive methylation), on the dry residue (e.g. silylation), or during injection (e.g. methylation). If a compound requires derivatisation in order to reduce its polarity, the use of high pressure liquid chromatography should be considered instead of GLC, assmning that sensitivity is not the limiting factor. 

Electrochemical detectors are based upon the volta-metric oxidation or reduction of separated analytes at a micro- or thin-film electrode. A number of pharmacologically active compounds that are aldehydes, ketones, or quinones (such as doxorubicin), or nitro compounds (such as nitrofurantoin) are amenable to reduction at a mercury or platinum electrode electron-rich indole derivatives and catecholamines can be oxidized at these electrodes. An important condition that must be fulfilled for electrochemical detection to be practicable is that the mobile phase must be capable of conducting an electrical current. This makes electrochemical detection particularly useful in reversed-phase liquid chromatography, where buffered water mixed with one or more organic cosolvents is usually the mobile phase. 

The diode array detector (DAD), which arose from the analyst s needs to reduce data observations times in chromatography, has become a powerful tool in a research environment and in the quality assurance laboratory. Diode array adds a new dimension of analytical capability to liquid chromatography because it allows qualitative information to be obtained beyond simple identification by retention time. 

An important feature of modern high-performance liquid chromatography (HPLC) is its excellent quantitation capability. HPLC can be used to quantify the major components in a purified sample, the components of a reaction mixture, and trace impurities in a complex sample matrix. The quantitation is based on the detector response with respect to the concentration or mass of the analyte. In order to perform the quantitation, a standard is usually needed to calibrate the instrument. The calibration techniques include an external standard method, an internal standard method, and a standard addition method. For cases in which a standard is not available, a method using normalized peak area can be used to estimate the relative amounts of small impurities in a purified sample. 

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