Fluorescence suppression

Quantitation may be done crudely on the spots separated by planar chromatographic techniques such as TLC or slab gel electrophoresis (see Chapter 13). One might compare the optical density, the fluorescence, or the degree of stationary phase fluorescence suppression by the unknown spot to a series of standards of known concentration. In contrast, the electrical signals from the variety of detectors used in various column chromatography instruments can be precisely, reproducibly, and linearly related to the amount of analyte passing through the detector cell. If aU parameters of injection, separation, and detection are carefully controlled from run to run and especially if appropriate quantitative ISs are incorporated in the procedure, accuracy and precision better than 1% may be attained. 

An alternative approach to improving the sensitivity of the Raman technique with potential for environmental applications arose in 1974 [11] with the discovery of surface-enhanced Raman spectroscopy (SERS) in which detection limits can typically be lowered by a factor of approximately 10 -10 relative to NRS, with SERS having the advantage of fluorescence suppression over the RRS. (Considerable research has been conducted for determining the sources of enhancement, and the reader is referred to the reviews of Vo-... 

Online fluorescence suppression in modulated Raman spectroscopy. Anal. Chem., 82, 738 -745. 

Praveen, B.B. et al. (2012) Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis. 

Although reduced guest fluorescence had been reported in previous studies, it was always described and explained in terms of bimolecular quenching by the CD hosts. To emphasize that the effect of CD on 7-MC is an environmental polarity effect, and does not involve energy transfer to the CD as quenchers, we proposed the term fluorescence suppression to describe this observed effect. This corresponds nicely to the term fluorescence enhancement commonly used to describe the opposite (and much more common) polarity effect. 

Chromatographic methods, notably hplc, are available for the simultaneous deterrnination of ascorbic acid as weU as dehydroascorbic acid. Some of these methods result in the separation of ascorbic acid from its isomers, eg, erythorbic acid and oxidation products such as diketogulonic acid. Detection has been by fluorescence, uv absorption, or electrochemical methods (83—85). Polarographic methods have been used because of their accuracy and their ease of operation. Ion exclusion (86) and ion suppression (87) chromatography methods have recently been reported. Other methods for ascorbic acid deterrnination include enzymatic, spectroscopic, paper, thin layer, and gas chromatographic methods. ExceUent reviews of these methods have been pubHshed (73,88,89). 

As discussed in the previous chapter, the Phen residue in APh-x forms the CT complex with MV2 + in aqueous solution [76]. Interestingly, the CT formation is suppressed in the poly(A/St/Phen)-MV2+ system in spite of the Phen fluorescence being quenched by MV2 + very effectively. This fact indicates that it becomes very less likely for the Phen moiety to come into a face-to-face contact with MV2+, while the fluorescence from the compartmentalized Phen residue can be quenched effectively via a collision-less ET to MV2 +. ... 

Similarly to the methods used to characterize natural chlorophylls, RP-HPLC has been chosen by several authors to identify the individual components in Cn chlorophyllin preparations and in foods. The same ODS columns, mobile phase and ion pairing or ion suppressing techniques coupled to online photodiode UV-Vis and/or fluorescence detectors have been used. ° ... 

I. PRODUCTION OF ANTIBIOTICS. The production of secondary metabolites with antimicrobial properties has long been recognized as an important factor in disease suppression (see Chap. 7). Metabolites with biocontrol properties have been isolated from a large number of rhizosphere microorganisms, including the fluorescent pseudomonads (Table 2). Further discussion is not given here since this is the subject of recent reviews (122,123). 

G. W. Xu and D. C. Gross, Selection of fluorescent pseudonionads antagonistic to Erwinia curotovota and suppressive of potato seed piece decay. Phytopathology 76 414 (1986). 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

The acetone-sensitized photodehydrochlorination of 1,4-dichlorobutane is not suppressed by triplet quenchers (20), but the fluorescence of the sensitizer is quenched by the alkyl chloride (13). These observations imply the operation of a mechanism involving collisional deactivation, by the substrate, of the acetone excited singlet state (13,21). This type of mechanism has received strong support from another study in which the fluorescence of acetone and 2-butanone was found to be quenched by several alkyl and benzyl chlorides (24). The detailed mechanism for alkanone sensitization proposed on the basis of the latter work invokes a charge-transfer (singlet ketone)-substrate exciplex (24) and is similar to one of the mechanisms that has been suggested (15) for sensitization by ketone triplets (cf. Equations 4 and 5). 

Fluorescence measurement using this probe does not require a fluorescence quencher or washing process to suppress the fluorescence emission from nonbinding probes and nonspecific binding probes, which would be advantageous for the detection of mRNAs with poly(A) tracts in cells. 

Fluorescence lifetime imaging Multi-point calibration, minimum resolvable differences, and artifact suppression. Cytometry 43, 248-60. 

Siemering, K. R., Golbik, R., Sever, R. and Haseloff, J. (1996). Mutations that suppress the thermosensitivity of green fluorescent protein. Curr. Biol. 6, 1653-63. 

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