Ultraviolet/visible diode-array detection

Optical Spectroscopy General principles and overview, 246, 13 absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes, 246, 19 circular dichroism, 246, 34 bioinorganic spectroscopy, 246, 71 magnetic circular dichroism, 246, 110 low-temperature spectroscopy, 246, 131 rapid-scanning ultraviolet/visible spectroscopy applied in stopped-flow studies, 246, 168 transient absorption spectroscopy in the study of processes and dynamics in biology, 246, 201 hole burning spectroscopy and physics of proteins, 246, 226 ultraviolet/visible spectroelectrochemistry of redox proteins, 246, 701 diode array detection in liquid chromatography, 246, 749. 

Ion-pair HPLC (IP-HPLC) and reversed-phase HPLC (RP-HPLQ have been used to determine folate (Wilson and Horne 1984, 1986), most commonly in conjugation with fluorescence, ultraviolet and UV-visible (UV-vis) spectrometry (Vahteiisto et al. 1996) or electrochemical detection (BCD) systems (Kohashi et al. 1986 Lucock et al. 1989). The pteridine ring structure of the folate can be determined on the basis of its retention time determined by HPLC and spectral characteristics via the diode array detection (DAD) system (Selhub 1989). 

The most commonly-used detectors are those based on spectrophotometry in the region 184-400nm, visible ultraviolet spectroscopy in the region 185-900nm, post-column derivativisation with fluorescence detection (see below), conductivity and those based on the relatively new technique of multiple wavelength ultraviolet detectors using a diode array system detector (described below). Other types of detectors available are those based on electrochemical principles, refractive index, differential viscosity and mass detection. 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the usual method of choice for the separation of anthocyanins combined with an ultraviolet-visible (UV-Vis) or diode-array detector (DAD)(Hebrero et al., 1988 Hong et ah, 1990). With reversed-phase columns the elution pattern of anthocyanins is mainly dependent on the partition coefficients between the mobile phase and the Cjg stationary phase, and on the polarity of the analytes. The mobile phase consists normally of an aqueous solvent (water/carboxylic acid) and an organic solvent (methanol or acetonitrile/carboxylic acid). Typically the amount of carboxylic acid has been up to 10%, but with the addition of a mass spectrometer as a detector, the amount of acid has been decreased to as low as 1 % with a shift from trifluoroacetic acid to formic acid to prevent quenching of the ionization process that may occur with trifluoroacetic acid. The acidic media allows for the complete displacement of the equilibrium to the fiavylium cation, resulting in better resolution and a characteristic absorbance between 515 and 540 nm. HPLC separation methods, combined with electrochemical or DAD, are effective tools for anthocyanin analysis. The weakness of these detection methods is a lack of structural information and some nonspecificity leading to misattribution of peaks, particularly with electrochemical... 

These methods of obtaining complete ultraviolet-visible spectra can be very time-consuming. However, rapid scanning spectrophotometers based on optical diode array detectors are now eormnereially available and are capable of recording complete spectra in very short time intervals (1 second or less). They can therefore be used to obtain spectra of separated components without stopping eluent flow or to detect at several wavelengths simultaneously. 

Detection systems for GC are chosen for their sensitivity and selectivity for a particular class of VOCs. Detectors for GC include FID, the BCD, the photoionization detector (PID), the pulsed discharge detector (PDD), and the reduction gas detector (RGD). A variety of mass spectrometers can also be interfaced with a GC for confirmation of molecular structure and quantitation. Singlewavelength ultraviolet-visible detectors (190 to 600 nm) and diode array detectors are used to detect carbonyls as their 2,4-dinitrophenylhydrazone derivatives. The absorption maxima for aliphatic carbonyls, aromatic carbonyls, and dicarbonyls are near 360 nm, 385 to 390 nm, and 415 to 430 nm, respectively. Formic, acetic, and pyruvic acid are detected by ion conductivity. 

The most common method of detection in HPLC exploits the ultraviolet and visible regions of the electromagnetic spectrum (EM see Figure 5.1) in order to detect the analytes of interest. The detectors employed that utilise these regions of the EM spectrum are the ultraviolet (UV) and diode array detectors. However, other, more specific detectors can be used for specialised applications, such as conductivity and refractive index (not discussed here), and with the introduction of hyphenated techniques, mass spectrometry has become a widely used detector method. 

Detection by a diode array detector can be used to confirm the identity of an additive. This detector is usually coupled with a liquid chromatograph to record the ultraviolet-visible spectra for each of the chromatographic peaks so that they can be compared with the spectra of the pure compounds. Moreover, the purity of each of the chromatographic peaks can be checked. 

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