Fluorescence detected CD

Analogous g-values may be defined for the degree of circular polarization in emission [or circularly polarized photoluminescence (CPPL)] and circularly polarized electroluminescence (CPEL), eg. gCppL = 2(JL - 1R)/(1L + 1r), where IL and IR denote the intensity of left- and right-handed circularly polarized emission, respectively. CPPL should not be confused with fluorescence-detected CD. 

Figure 6. Experimental arrangement for measuring fluorescence-detected CD. From ref. [27].

More esoteric experimental procedures which can be applied to complex molecules include, for example, picosecond time-resolved circular dichroism. A spectrometer for this purpose has been described and used to observe carbon monoxide elimination from excited carbon raonoxymyoglobulin.Fluorescence detected CD has also been used to observe riboflavin in capillary electrophoresis. 

CD instruments using other basic designs have been constructed. VCD instrumentation using both dispersive and Fourier transform designs are described in several reviews.Fluorescence-detected CD (FDCD) ... 

Fluorescence-detected CD has also been described and discussed (Schles-singer et al, 1975 Tinoco and Turner, 1976 Brittain and Richardson, 1977 Richardson, 1979a, pp. 189-217 Tinoco, 1979, pp. 57-85 Brittain, 1980). For fast kinetic studies (millisecond range) of biological molecules, prototype stopped-flow CD spectrophotometers have been developed and applied (Nitta et al, 1977). 

The nonreactive base structures of cyanine dyes (or carbocyanines) have been used for many years as components in photographic emulsions to increase the range and sensitivity of film and also in CD-R and DVD-R optical disks to record digital information. A major innovation came when Ernst et al. (1989) and Waggoner et al. (1993) recognized that cyanine dyes would make excellent labels for fluorescence detection, and for this reason, they synthesized reactive dye derivatives, which then could be covalently attached to proteins and other molecules. 

Mackey, D.3., 1983b. Metal-organic complexes in seawater- an investigation of naturally occurring complexes of Cu, Zn, Fe, Mg, Ni, Cr, Mn and Cd using high performance liquid chromatography with atomic fluorescence detection. Mar. Chem., 13 169-180. 

Chiral separation of FITC-labeled amino acid enantiomers was performed on a glass chip using fluorescent detection. Analysis time ranged from 75 s for the most basic amino acids to 160 s for the most acidic ones. y-CD was used as the chiral selector [627]. Chiral separation of amino acids in extraterrestrial samples or meteorites were also performed [610,628],... 

Warner and co-workers justify the elaborate optical/detection system and the time commitment required per analysis on the basis of the additional sensitivity available using fluorescence detection, and on the multidimensional CD information available. For applications in which two, or more CD active fluorophores may be present, the ability to provide both an excitation and emission FDCD profile for the sample may allow differentiation of the individual components without pre-separation. Replacement of the mechanical mechanism for prism movement with an electro-optical device may improve both the SNR and reduce the time required per sample. These improvements will greatly facilitate general application of this multidimensional approach to FDCD measurements. 

In summary, the simple but versatile stopped-flow technique, coupled with optical detection (typically absorbance, fluorescence, or CD), has long been the primary source of kinetic insight into fast reactions. A major strength of stopped-flow methods is that they can be readily combined with a wide range of detection methods. As the need to understand molecular mechanisms increases, stopped-flow methods will continue to play an important role. 

The analysis of PAHs by fluorescence detection HPLC is often the water analysts first introduction to HPLC. The analysis of the WHO six PAHs (section 11.8.1.1) namely fluoranthene, benzo[6]fluoranthene, henzo[k]fluoranthene, benzo[a]pyrene, benzo[g/j/ perylene and indeno[l,2,3-cd]pyrene was carried out using fixed excitation and emission E wavelengths. With the advent of relatively cheap variable wavelength programmable fluorescence detectors, the detectors can be optimised for each separate PAH with a resultant lowering of detection limit. Ultratrace determination of PAHs down to 180fg of benzo[a]pyrene was reported as early as 1983. 

Because all the rules that apply to absorbance detection apply equally well to CD, it is convenient to think of CD as a modified form of absorption spectrophotometry. Spectra are temperature- and pH-dependent non-linear correlations of signal versus concentration are commonplace and are produced for the same reasons, such as chemical equilibria, polychromatic radiation, stray light, etc. Fluorescence emission CD (FDCD) spectroscopy is observed whenever an analyte meets all... 

Similarities between CD and absorbance methods are also found between CD and fluorescence and CD and circularly polarized luminescence (CPL). Three prerequisites are needed to produce FDCD and CPL activities. Intense emission signals normally associated with fluorescence are attractive because limits of detection are lowered considerably. FDCD finds more uses as a chromatographic detection device. A CD signal is usually induced by some kind of molecular complexation reaction. Association can be with a simple molecule or with an aggregate of molecules, such as chiral micelles, which are known to be fluorescence enhancers. In cases of color induction combined with fluorescence induction, FDCD can lead to even higher levels of selectivity among analytes that have been derivatized by the same color reagent. 

Fluorescence-detected circular dichroism (FDCD) is a chiroptical technique in which the spectrum is obtained by measuring the difference in total luminescence obtained after the sample is excited by left- and right-circularly polarized light. For the FDCD spectrum of a given molecular species to match its CD spectrum, the luminescence excitation spectrum must be identical to the absorption spectrum. 

Huang CJ, Harootunian A, Maher MP, Quan C, Raj CD, McCormack K, et al. Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential. Nat Biotechnol 2006 24 439-46. 

Figure 5.15. Reversed-phase gradient elution separation of a mixture of polycyclic aromatic hydrocarbons using time programmed fluorescence detection. Compounds 1 = naphthalene 2 = acenaphthene 3 = fluorene 4 = phenanthrene 5 = anthracene 6 = fluoranthene 7 = pyrene 8 = benz(a)anthracene 9 = chrysene 10 = benzo(b)fluoranthene 11 = benzo(k)fluoranthene 12 = benz(a)pyrene 13 = dibenz(a,h)anthracene 14 = benzo(g,h,i)perylene 15 = indeno(l,2,3-cd)pyrene and 16 = coronene.

Since polypeptides are chiral polymers, the chromophore arrangement shows some chirality. Therefore, additional information on the chromophore arrangement in the ground state, as well as in the excited state, may be obtained from chiroptical spectroscopy, such as circular dichroism (CD), circularly polarized fluorescence (CPF) [43], and fluorescence-detected circular dichroism (FDCD) [44). 

The original reported use of lasers in CE with fluorescence detection was described by Gassmann et in which laser-induced fluorescence (LIE) was used to distinguish and measure the chiral enantiomers in mixtures of dansylated amino acids. Since that time, lasers have become commonplace in CE systems with fluorescence detection. The most common types of lasers used have been the He-Cd, Ar-ion, and He-Ne lasers, as they are relatively inexpensive and have emission lines that match commonly used fluorescent reagents, such as o-phthalaldehyde (OPA) (325 nm) and fluorescamine (354 nm). 

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