Analytical diode array detectors

Some analytical instruments produce a table of raw data which need to be processed into the analytical result. Hyphenated measurement devices, such as HPLC linked to a diode array detector (DAD), form an important class of such instruments. In the particular case of HPLC-DAD, data tables are obtained consisting of spectra measured at several elution times. The rows represent the spectra and the columns are chromatograms detected at a particular wavelength. Consequently, rows and columns of the data table have a physical meaning. Because the data table X can be considered to be a product of a matrix C containing the concentration profiles and a matrix S containing the pure (but often unknown) spectra, we call such a table bilinear. The order of the rows in this data table corresponds to the order of the elution of the compounds from the analytical column. Each row corresponds to a particular elution time. Such bilinear data tables are therefore called ordered data tables. Trilinear data tables are obtained from LC-detectors which produce a matrix of data at any instance during the... 

Polar or thermally labile compounds - many of the more modern pesticides fall into one or other of these categories - are not amenable to GC and therefore LC becomes the separation technique of choice. HPLC columns may be linked to a diode-array detector (DAD) or fluorescence detector if the target analyte(s) contain chromophores or fluorophores. When using a DAD, identification of the analyte(s) is based on the relative retention time and absorption wavelengths. Similarly, with fluorescence detection, retention time and emission and absorption wavelengths are used for identification purposes. Both can be subject to interference caused by co-extractives present in the sample extract(s) and therefore unequivocal confirmation of identity is seldom possible. 

There is a recent trend towards simultaneous CE separations of several classes of food additives. This has so far been applied to soft drinks and preserved fruits, but could also be used for other food products. An MEKC method was published (Lin et al., 2000) for simultaneous separation of intense sweeteners (dulcin, aspartame, saccharin and acesulfame K) and some preservatives (sorbic and benzoic acids, sodium dehydroacetate, methyl-, ethyl-, propyl- and isopropyl- p-hydroxybenzoates) in preserved fruits. Ion pair extraction and SPE cleanup were used prior to CE analysis. The average recovery of these various additives was 90% with good within-laboratory reproducibility of results. Another procedure was described by Frazier et al. (2000b) for separation of intense sweeteners, preservatives and colours as well as caffeine and caramel in soft drinks. Using the MEKC mode, separation was obtained in 15 min. The aqueous phase was 20 mM carbonate buffer at pH 9.5 and the micellar phase was 62 mM sodium dodecyl sulphate. A diode array detector was used for quantification in the range 190-600 nm, and limits of quantification of 0.01 mg/1 per analyte were reported. The authors observed that their procedure requires further validation for quantitative analysis. 

Microprocessor based analytical equipments is no longer an uncommon phenomenon towards the modernization, automation, and above all the ease of function and handling of sophisticated devices, for instance a microprocessor scans the array of diodes many times a second in a diode array detector a microprocessor does the temperature programming of a constant temperature chamber of HPLC unit. 

Every analyst who has access to CE equipment and ESI-MS may take this new alternative into consideration when appropriate. When is it appropriate If an analytical problem can be solved using affinity capillary electrophoresis and a more universal and specific detection or structural information is required than is accessible with the help of conventional UV diode array detectors, consider ACE/MS. 

Riordon JR. Diode array detectors for HPLC— High performance across the spectrum. Analytical Chemistry 72, 483A--187A, 2000. 

It is also possible to monitor a broad range of wavelengths without interrupting the flow in the column. This results in the full UV spectrum of the eluting mobile phase and provides a good means of identification of the analytes (Fig. 3.14). Such diode-array detectors (DAD) can be used in the gradient elution mode. Actually, they are frequently used in automated routine analysis. 

Analytes are identified on the basis of retention time compared to standards and with the addition of the suspected compound to the sample (55). The diode array detector has been used recently as an additional aid in the identification of sweeteners and the determination of peak purity (56). Quantification is performed by the internal or external standard method on the basis of peak height or area. 

Specificity with real samples Use samples with analytes. Check peak purity with a diode-array detector and/ or a mass selective detector. Run the sample under different chromatographic columns and/or with different columns. 

Spectroscopic detectors measure partial or complete energy absorption, energy emission, or mass spectra in real-time as analytes are separated on a chromatography column. Spectroscopic data provide the strongest evidence to support the identifications of analytes. However, depending on the spectroscopic technique, other method attributes such as sensitivity and peak area measurement accuracy may be reduced compared to some nonselective and selective detectors. The mass spectrometer and Fourier transform infrared spectrometer are examples of spectroscopic detectors used online with GC and HPLC. The diode array detector, which can measure the UV-VIS spectra of eluting analytes is a... 

HPLC Agilent 1200 equipped with diode array detector (DAD). Analytical column Zorbax ... 

The coulometric array detection mimics the diode array detector if two peaks are eluted together, they can be electrochemically and spectrophotometrically resolved and quantified. While a diode array detector relies on the different UV-Vis spectra of various compounds and characterizes analytes on the basis of their retention times and spectroscopic features, the coulometric array detector takes advantage of the variability of the voltammograms for diverse analytes and typifies them based on retention time and reaction potential. 

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... 

Fig. 5.8. (A) General scheme of a dynamic focused microwave-assisted extractor. (B) Experimental set-up used to integrate microwave-assisted extraction with the subsequent steps of the analytical process. (1) Leaching step CT controller, MO microwave oven, S sample, R condenser, WR water reservoir, TCPP two-channel piston pump, ER extract reservoir, SV switching valve. (2) Clean-up/preconcentration step M methanol, A air, B buffer, PP peristaltic pump, F filter, EL elution loop, MC mini-column, R retention direction, E elution direction, 1V1-1V3 injection valves, W waste. (3) Individual separation-detection step HPIV high-pressure injection valve, AC analytical column, DAD diode array detector, SR solvent reservoirs.

Dongjin Pyo also reported some other procedures The extraction by SFE was performed with a Beckman 116 pump (System Gold programmable solvent module 126), a 15 cm X 10 mm I.D. ODS column and Agilent HPLC 1100 series diode-array detector. Methanol/0.05 M phosphate buffer (52 48), pH 3, was used as a mobile phase at a flow rate of 2 mL/min. The analytical results obtained from real lake samples indicate the viability of the method for the determination of microcystins in real samples. 

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