Diode array detection, DAD

The resolution of a multicomponent system involves the description of the variation of measurements as an additive model of the contributions of their pure constituents [1-10]. To do so, relevant and sufficiently informative experimental data are needed. These data can be obtained by analyzing a sample with a hyphenated technique (e.g., HPLC-DAD [diode array detection], high-performance liquid chromatography-DAD) or by monitoring a process in a multivariate fashion. In these and similar examples, all of the measurements performed can be organized in a table or data matrix where one direction (the elution or the process direction) is related to the compositional variation of the system, and the other direction refers to the variation in the response collected. The existence of these two directions of variation helps to differentiate among components (Figure 11.1). 

DAD Diode-array detection SDS Sodium dodecyl sulphate... 

Abbreviations CZE capillary zone electrophoresis MECC or MEKC micellar electrokinetic chromatography CGE capillary gel electrophoresis CIEF capillary isoelectric focusing CEC capillary electrochromatography DAD Diode-array detection... 

AES, atomic emission spectrometry AP(C)I, atmospheric pressure (chemical) ionization CGC, capillary gas chromatography DAD, diode array detection ESI, electrospray ionization FI, fluorescence detection ICP, indcutively coupled plasma LIE, laser-induced fluorescence Nl, negative ion NMR, nuclear magnetic resonance PFPD, pulsed flame photometric detector SRM, selected reaction monitoring. 

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is now considered as a perfect complement to HPLC-DAD (diode array detection) and gas chromatography (GC)-MS for the general unknown screening of drugs and toxic compounds. 

A powerful tool now employed is that of diode array detection (DAD). This function allows peaks detected by UV to be scanned, and provides a spectral profile for each suspected microcystin. Microcystins have characteristic absorption profiles in the wavelength range 200-300 nm, and these can be used as an indication of identity without the concomitant use of purified microcystin standards for all variants. A HPLC-DAD analytical method has also been devised for measurement of intracellular and extracellular microcystins in water samples containing cyanobacteria. This method involves filtration of the cyanobacteria from the water sample. The cyanobacterial cells present on the filter are extracted with methanol and analysed by HPLC. The filtered water is subjected to solid-phase clean-up using C g cartridges, before elution with methanol and then HPLC analysis. 

LC-MS-MS was also the method of choice for the analysis of UV filters in solid matrices. Both LC and UPLC have been applied in three out of the four methods available for the determination of UV filters in sludge. Separation was performed on C8 and C18 LC-chromatographic columns (Zorbax, Eclipse, Vydac, and Purosphere) using binary gradient elution of mobile phases consisting of water/ methanol or water/acetonitrile. MS-MS detection was performed in SRM with ESI and atmospheric pressure photoionization (APPI) in both positive and negative modes. For each compound, two characteristics transitions were monitored. In addition to MS and MS-MS, diode array detection (DAD) was occasionally applied to the determination of OT. Spectra were recorded between 240 and 360 nm and discrete channels at 310 nm. 

The greatest disadvantage of all detector systems such as, e.g. FID, UV, diode array detection (UV-DAD), FL, refractory index (RI), light scattering detector (LSD) or conductivity, applied in combination with GC, LC or CZE, is that they only provide an electric signal at the detector. The retention time alone of standard compounds, if available, is not sufficient for a reliable identification. LC separation of surfactant-containing extracts may often result in non-reproducible retention... 

Capillary electrophoresis with diode array detection. Ultraviolet spectra (190-350 nm) were recorded after separation of fractions III, IV, and V-2 by capillary electrophoresis with diode array detection (CE-DAD) in 30 mM phosphoric acid buffer pH 2.3 and pH 7.2. Separations were carried out with a Hewlett-Packard 3D GE equipped with a diode array detector. Samples were introduced under pressure (30 mbar, 10 sec) into a fused silica capillary (effecfive lengfh 8 cm, total 64.5 cm, ID 50 pm, OD 375 pm) followed by separafion af 30 kV. 

Fernandez et al. [27] applied HPLC with diode array detection (DAD) to the determination of heroin, methadone, cocaine and metabolites in plasma after mixed-mode SPE. Analytes were separated using a RP8 column (250 mm x 4.6 mm i.d., 5 jam particle size) and acetonitrile-phosphate buffer pH 6.53 as mobile phase with elution in the gradient mode. The method, which provides a LLOQ of 0.1 j,g/mL for all compounds, was successfully applied to 21 plasma samples from fatal overdoses. 

With the introduction of diode-array technology in the 1980s, a further dimension is now possible because coupled LC-UV with diode array detection (DAD) allows the chromatographic eluent to be scanned for UV-visible spectral data, which are stored and can later be compared with a library for peak identification. This increases the power of HPLC analysis... 

Polychromatic detection. The most sophisticated detectors (e.g. DAD diode-array detectors) either allow the wavelength to be changed during the course of the analysis or simultaneously record the absorbance at several wavelengths. These detectors can be used not only to record the chromatogram but also to provide spectral information that can be used to identify the compounds. This is called specific detection (cf. 11.14). 

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