LIF detection

L-pyrenyldiazomethane to form stable, highly fluorescent L-pyrenyhnethyl monoesters (87). These esters have been analy2ed in human blood by ce combined with lif detection. To mimini e solute adsorption to the capillary wall, they were coated with polyacrjiamide, and hydroxypropyl methylceUulose and dimethylfoTTnamide were used as buffer additives to achieve reflable separations. Separation was performed in tris-citrate buffer, pH 6.4, under reversed polarity conditions. The assay was linear for semm MMA concentrations in the range of 0.1—200 p.mol/L. 

Table III. Detection Limits of 0PA/2-ME and NDA/CN Derivatized Amino Acids Using Conventional and Laser-Induced Fluorescence (LIF) Detection...

In LIF detection systems, excitation power may be increased up to six orders of magnitude compared to CF detection. Most LC-LIF detection concerns under-ivatised polynuclear aromatic hydrocarbons (PAHs) and fluorescing dyes (e.g. polymethines). Because only a limited number of analytes possess native fluorescence, derivatisation of the analyte before detection is normally required in trace analysis of organic solutes by means of LIF detection. LIF detection in HPLC was reviewed... 

Pre-column derivatization with either 134 or 135 followed by CZE and LIF detection was proposed for amino acids. The amino group of the analyte displaces the succinyloxy moiety of the reagent yielding a carboxamide325. See also Section IV.D.3.C for other acylating reagents derived from A-hydroxysuccinimide (95 and 96). 

When compared to fluorescence detectors for HPLC, the design of a fluorescence detector for CE presents some technical problems. In order to obtain acceptable sensitivity, it is necessary to focus sufficient excitation light on the capillary lumen. This is difficult to achieve with a conventional light source but is easily accomplished using a laser. The most popular source for laser-induced fluorescence (LIF) detection is the argon ion laser, which is stable and relatively inexpensive. The 488-nm argon ion laser line is close to the desired excitation wavelength for several common fluorophores. The CLOD for a laser-based fluorescence detector can be as low as 10 12 M. 

Stalberg, O., Westerlund, D., Rodby, U. B., and Schmidt, S. (1995). Determination of impurities in remoxipride by capillary electrophoresis using UV-detection and LIF-detection — principles to handle sample overloading effects. Chromatographia 41, 287—294. 

Another fluorescent dye for use with LIF detection/ 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQCA), has been recently developed for the CE-SDS method. It is a fluorogenic dye. Sample preparation is simpler than other traditional fluorescent labeling procedures, and the assay is easy to perform. 

A third type of detector is the intrinsic or native fluorescence detector that utilizes native fluorescence properties of amino acids. The sensitivity of this detector is between UV/PDA and LIF detection. The advantage of this technique over pre-labeling is that there is no pre-labeling step required therefore, the sample preparation is relatively simple, and the sensitivity is improved over UV/LIF. However, the intrinsic fluorescence detection relies on the presence of Tryptophan (Try), Tyrosine (Tyr), Phenylalanine (Phe), and this detector has just become commercially available. 

FIGURE I CE-SDS separations of non-reduced and reduced preparations of a 5-TAMRA SE-labeled rMAb sample. Electrophoretic conditions were as follows Bio-Rad Biofocus 3000 instrument with LIF detection, effective length 19.4 cm, total length 30 cm, 50-pm ID, 375-pm OD uncoated fused-silica capillary both anode and cathode buffers were the Bio-Rad CE-SDS running buffer. The samples were injected at a constant electric field of 4l7V/cm for 20s and electrophoresed at 625 V/cm (21.2 pA) and 20 C. 

FIGURE 4 Effect of sample preparation on the fragmentation of an rMAb observed in (A) SDS-PAGE and (B) CE-SDS with LIF detection. SDS-PAGE lanes (Lane I) molecular weight standards bovine serum albumin at (Lane 2) 8 ng and (Lane 3) 2 ng (Lane 4) rMAb control after alkylation with (Lane 5) iodoacetic acid and (Lane 6) iodoacetamide. (See color plate 4.)... 

The response function and the associated analytical merits for absorption spectroscopic techniques (e.g., NIR, UV-vis and infrared) are determined by the optical path length, detector gain, signal averaging and spectral resolution. The LIF detection performance is also governed by these parameters but is also influenced by critical parameters associated with the excitation source (e.g., optical power, pulse rate, etc.) as previously discussed. ... 

Of all the detection methods applied to the lab-on-a-chip, the most popular by far has been laser-induced fluorescence (LIF) detection. Direct LIF detection benefits... 

An ultraviolet (UV) monitor is most commonly used in CE experiment. Such interaction studies using the ACE method can also be hampered by the inadequate sensitivity of UV detection. Fluorescence labeling and laser-induced fluorescence (LIF) detection have been employed to enhance the sensitivity of this method, as shown by the mobility-shift assay of fluorescence-labeled sugar caused by the interaction with the lectin, concanavalin A (74). When fluorescent dyes are employed for labeling, LIF detection provides several hundred times more sensitivity than UV detection. 

Monoclonal antibody-binding affinity was determined by microchip-based capillary electrophoresis with LIF detection (33). The mixing was carried out off-chip, and the on-chip separations were performed in less than 60 s (Fig. 9). A Scatchard plot analysis resulted in an affinity constant for the monoclonal anti-BSA antibody to fluorescently labeled BSA (BSA ) of... 

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