Fluorescence arrays, analytes

Despite their importance, gas chromatography and liquid chromatography cannot be used to separate and analyze all types of samples. Gas chromatography, particularly when using capillary columns, provides for rapid separations with excellent resolution. Its application, however, is limited to volatile analytes or those analytes that can be made volatile by a suitable derivatization. Liquid chromatography can be used to separate a wider array of solutes however, the most commonly used detectors (UV, fluorescence, and electrochemical) do not respond as universally as the flame ionization detector commonly used in gas chromatography. 

Though we and others (27-29) have demonstrated the utility and the improved sensitivity of the peroxyoxalate chemiluminescence method for analyte detection in RP-HPLC separations for appropriate substrates, a substantial area for Improvement and refinement of the technique remains. We have shown that the reactions of hydrogen peroxide and oxalate esters yield a very complex array of reactive intermediates, some of which activate the fluorophor to its fluorescent state. The mechanism for the ester reaction as well as the process for conversion of the chemical potential energy into electronic (excited state) energy remain to be detailed. Finally, the refinement of the technique for routine application of this sensitive method, including the optimization of the effi-ciencies for each of the contributing factors, is currently a major effort in the Center for Bioanalytical Research. 

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. 

Fig. 6 (a) Schematic illustration of a flow cytometer used in a suspension array. The sample microspheres are hydrodynamically focused in a fluidic system and read-out by two laser beams. Laser 1 excites the encoding dyes and the fluorescence is detected at two wavelengths. Laser 2 is used to quantify the analyte, (b) Scheme of randomly ordered bead array concept. Beads are pooled and adsorbed into the etched wells of an optical fiber, (c) Scheme of randomly-ordered sedimentation array. A set of encoded microspheres is added to the analyte solution. Subsequent to binding of the analyte, microparticles sediment and assemble at the transparent bottom of a sample tube generating a randomly ordered array. This array is evaluated by microscope optics and a CCD-camera. Reproduced with permission from Refs. [85] and [101]. Copyright 1999, 2008 American Chemical Society... 

Cross-reactive sensing arrays were developed to detect odors and vapors in an artificial nose manner. Solvatochromic dyes such as Nile Red are adsorbed on the surface or embedded into various polymeric or porous silica beads. The beads respond to analyte vapor by a change in fluorescence maxima or/and intensity due to changes of polarity inside the bead. A portable instrument and preliminary field test for the detection of petroleum products was recently described [106]. 

The potential for using multiplexed single-photon counting to acquire fluorescence lifetime data from a distributed array of optical fiber sensors, each sensing a different analyte, has recently been demonstrated by Birch etal.(39,47) Figure 12.9 illustrates such a network to be used in conjunction with the arrangement shown in Figure 12.6. 

The characteristics that discourage the use of RPLC for preparative isolation of bioactive proteins favor its use as an analytical tool for studying protein conformation. Chromatographic profiles can provide information on conformational stability of a protein and the kinetics of folding and unfolding processes. Information about solvent exposure of certain amino acid residues (e.g., tryptophan) as a function of the folding state can be obtained by on-line spectral analysis using diode array UV-vis detection or fluorescence detection. 

Dams R, Benijts T, Lambert WE, De Leenheer AP. 2002. Simultaneous determination of in total 17 opium alkaloids and opioids in blood and urine by fast liquid chromatography-diode-array detection-fluorescence detection, after solid-phase extraction. J Chromatogr B Analyt Technol Biomed Life Sci 773(1) 53-61. 

Rushing and Hansen (504) described a sensitive analytical procedure for screening and confirmation of residues of malachite green, gentian violet, and their leuco analogs in catfish and trout tissues using liquid chromatographic separation and electrochemical, diode array, and fluorescence detection in series. 

The variety of detection modes available for HPLC analysis that provide additional information about the eluent as it exits the column greatly facilitates unknown characterization. The majority of analytical methods for phenolic compounds includes HPLC with spectrophotometric-based detection techniques (UV absorption, fluorescence, photo diode array—PDA) as well as HPLC with electrochemical detection. 

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