Laser-induced fluorescence polarization

Fig. 8 Schematic diagram showing capillary electrophoresis separation with laser-induced fluorescence polarization detection. (From Ref. 48.)...

L Ye, C Le, JZ Xing, M Ma, R Yatscoff. Competitive immunoassay for cyclosporine using capillary electrophoresis with laser induced fluorescence polarization detection. J Chromatogr B 714 59-67, 1998. 

Fu, H., Guthrie, J. W., Le, X. C. (2006). Study of binding stoichiometries of the human immunodeficiency virus type 1 reverse transcriptase by capillary electrophoresis and laser-induced fluorescence polarization using aptamers as probes. Electrophoresis 27, 433-441. 

In addition to wavelength and time-resolved fluorescence techniques, polarization fluorescence can yield important information about an analyte." This is especially true when differentiating between chiral compounds. Combining, for example, a fluorescently tagged antibody immunoassay with polarization detection allows for very sensitive detection limits of chiral enantiomers. Laser-induced fluorescence polarization (LIFP) has been used to detect concentrations as low as 0.9 nM of an antibody-boimd cyclosporine A (an immunosuppressive drug) in human blood. A conventional single channel fluorescence detector can be easily modified to perform such measurements, simply by adding the appropriate polarization filters. 

Laser-induced fluorescence polarization (LIFP) has been used as a detection technique in immunocapillary electrophoresis analysis of haptens. Polarization of fluorescence depends on the molecular size of the molecule. Small molecules exhibit low fluorescence polarization. LIFP was used to distinguish the peak of the fluorescent hapten-Ab complex from the peak of the excess of free fluorescent hapten in competitive assays [124]. Besides, the dependence of LIFP on molecular size has made it possible to perform quantitation without separating free from bound labeled hapten [125]. An attempt to use LIFP to quantitate staphylococcal enterotoxin A (28 kDa) did not show an increase of polarization upon formation of the Ag-Ab complex due to the high molecular weight of the Ag [126]. Formation of the complex could be identified by measuring the LIFP peak while varying the Ag/Ab ratio. 

Altkorn R and Zare R N 1984 Effects of saturation on laser-induced fluorescence measurements of population and polarization Annual Review of Physical Chemistry ed B S Rabinovitch, J M Schurr and H L Strauss (Palo Alto, CA Annual Reviews)... 

The pump and probe pulses employed may be subjected to a variety of nonlinear optical mixing processes they may be prepared and characterized by intensity, duration, spectral band width, and polarization. They may arrive in the reaction chamber at a desired time difference, or none. The probe pulse may lead to ionizations followed by detections of ions by mass spectrometry, but many alternatives for probing and detection have been used, such as laser-induced fluorescence, photoelectron spectroscopic detection, absorption spectroscopy, and the like. 

FIGURE 1. Polarization of laser induced fluorescence of 0H(X n) photodissociated from H2O in the 145-185 nm region. The absorption transition moment is perpendicular to the molecular plane corresponding to the transition A P-j-XlA]. The dissociated 0H(x2n) is also in the molecular plane, since the induced fluorescence intensity of OH is preferentially polarized along the Z axis (25) perpendicular to the molecular plane. The OH radical rotates on the H2O plane XY plane) after dissociation. The unpaired p-orbitals of excited H2O and of dissociated OH are perpendicular to the molecular plane. 

At the same time it is possible to obtain sufficiently interesting information on the dynamics of collisions by means of technically simpler measurements of the polarization of laser-induced fluorescence at excitation of molecules in bulk. A short account of such investigations is given in [288]. 

Let us now consider the following experimental scheme (see Fig. 3.14). Let the level J" be populated by fluorescence in the cycle J" —> J —> J", that is, in a three-state cycle of so-called A-configurations, where state J has greater energy than J" and J. To probe the polarization of the angular momenta created on level J" laser-induced fluorescence in the second cycle J —> J[ — J f may be used (see the broken lines in Fig. 3.14). 

Auzinsh, M.P. (1991). The influence of optical pumping of molecules on the polarization of laser induced fluorescence, Latvian Journal of Physics and Technical Sciences, 1, 3-10. 

Laser-Induced Fluorescence. Laser-induced fluorescence (Lif) provides, much as does ir spectroscopy, fingerprints of different organic molecules, which can be quantified by measuring fluorescence intensities. Selectivity is excellent, as both pump and fluorescence frequencies can be individually chosen for optimum performance, and it can be improved with measurements of fluorescence lifetimes and polarization behavior. The enhanced null-background sensitivity can achieve single-atom or single-molecule detection (256—258). Lif has important applications in gas analysis (259) and combustion and plasma diagnostics (260). 

V. Sivaprakasam and D. K. Killinger, Effect of polarization and geometrical factors on quantitative laser induced fluorescence-to-Raman intensity ratios of water samples and a new calibration technique, J. Opt. Soc. Am. B 20,1980 (2003). 

FIGURE 4 Indirect laser-induced fluorescence detection of 19 amino acids by microchip electrophoresis. Separation buffer 1.0 mM sodium carbonate, 0.5 mM fluorescein, and 0.2 mM CTAOH at pH 10.3. Separation condition /eff 5.5 cm, 15 s injection at 417 V/cm (reversed polarity), 183 V/cm separation voltage, sample amino acid concentrations of 0.4 mM in 1.0 mM sodium carbonate and 0.2 mM CTAOH. Reprinted with permission from [66]. Copyright 2000, The American Chemical Society. 

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