Diode array detector range

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. 

Figure 3.7 Diode array detector. Light from the lamp passes through the flow cell and to a holographic reflectance grating and the resulting spectrum is focused on the diode array. Detectors frequently have a spectral range of 190-800 nm and can offer bandwidths as low as 1.0 nm.

A diode array detector is well suited to achieve these goals. Full range spectra should be collected for the method development samples and evaluated with two-dimensional or three-dimensional visualization to determine the best detection wavelength. 

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. 

Ostermeyer (87) described the rapid and sensitive determination of acesulfame, saccharin, aspartame, SA, and BA in fishery products. Aspartame is determined directly in the aqueous extract of samples. For the other additives, cleanup of the extract is performed by anion-exchange chromatography. Ostermeyer used a Nucleosil 100-5 C lg (Macherey-Nagel) (250 X 4 mm, 5 /xm ) column and 0.02 mol/L KH2P04 acetonitrile as mobile phase and 219 nm detection using a diode array detector. Recoveries of spiked samples at concentrations of 50-400 mg sweetener/kg and 50-4000 mg preservative/kg were in the range 84-102% for all analyzed substances (87). The HPLC conditions for the determination of OAs are summarized in Table 5. 

The major difficulty in analyzing OPPs in fatty samples has to do with the wide polarity range for both pesticides and lipids present in the matrix. Normal-phase HPLC is an adequate technique for cleaning up this type of sample using silica gel and modifiers with different polarity. In fact, an automated sample-cleanup system based on normal-phase HPLC using a silica gel column has been reported efficiently to clean up and fractionate chlorpyriphos, chlorpyriphos methyl, and their metabolites in molluscs. The system presents several advantages The procedure is fully automated, from the injection of the extract to the collection of fractions, which are injected directly into the GC system, and a diode array detector (DAD) allows online monitoring of the elution of lipids (68). 

Owing to the varied structures of various food dyes, they can often be differentiated from one another by their characteristic ultraviolet/visible absorbance spectra. Using HPLC coupled with a diode array detector (HPLC-DAD) it is possible to collect a compound s absorbance spectrum as it elutes from the HPLC column, which greatly assists in identification. At Reading Scientific Services Ltd (RSSL) this type of detector is routinely used in a range of analyses of such substances as patulin, a mycotoxin found in apple juice, and in the analysis of colours and vitamins, which allows a more certain assignment of a particular peak to a specific compound to be made. 

The need for a reverse-field polarity power supply is at least two-fold 1) It permit a complete spectral analysis of the substance under study. By reversing field polarity, the substance zones can be run forward and backward in front of the detector as many times as needed. Incremental changes as small as 1- or 2-nm in wavelength can be used to maximize instrumental sensitivity, thus allowing coverage of the entire spectral range. In fact, this feature provides the same functions as a diode array detector, albeit somewhat slower. Proteins and peptides have almost identical spectral characteristics, however, when other functional groups are attached to it, is possible to observe more than one maximal absorbance peak. For example,... 

The main problem in HPLC is the peak assignment. EC MS systems are available but are far from being standard equipment. Some help may be expected from a diode-array detector, which allows the measurement of the UV-Vis absorption spectrum, for example, in the range 200-800 nm, on-line during chromatographic separation. Unfortunately the absorption spectra of organic polysulfanes are not very specific. The measurement of absorption spectra rmder static conditions cannot be recommended since polysulfanes rapidly decompose or react with the solvent on UV irradiation. 

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