Fluorescent detection, instrument laser

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Fluorescent detection technology applicable to biochips is evolving rapidly, resulting in detection instruments that are more powerful, user-friendly, and less expensive. Most systems employ photomultiplier tube (PMT) technology in conjunction with multiple colors, lasers, and a variety of filters. It is essentially a fluorescent microscope that... 

Finally, we note that future instrument for lifetime-based sensing and imaging can be based on laser diode light sources. At present it is desirable to develop specific probes which can be excited from 630 to 780 nm, the usual range of laser diodes. The use of such probes will allow us to avoid the use of complex laser sources, which should result in the expanded use of fluorescence detection in the chemical and biomedical sensors. 

Samples are introduced into the capillary by either electrokinetic or hydrodynamic or hydrostatic means. Electrokinetic injection is preferentially employed with packed or monolithic capillaries whereas hydrostatic injection systems are limited to open capillary columns and are primarily used in homemade instruments. Optical detection directly through the capillary at the opposite end of sample injection is the most employed detection mode, using either a photodiode array or fluorescence or a laser-induced fluorescence (LIF) detector. Less common detection modes include conductivity [1], amperometric [2], chemiluminescence [3], and mass spectrometric [4] detection. 

Ulfelder KJ (1994) Quantitative capillary electrophoretic analysis of PCR products using laser-induced fluorescence detection. Applications Information Bulletin A-1774. Beckman Instruments, Inc., Fullerton, CA. 

The architecture of the individual instruments used to detect atoms and small free radicals by atomic and molecular resonance fluorescence or by laser-induced fluorescence is shown in Figure 13, but is discussed in detail elsewhere. Briefly, a nacelle, hollow through the core from nose to tail with an impeller in the anterior section, provides for the laminar flow of stratospheric air around and through the instrument. Detection of trace species is carried out at one (or. more) optical axes within the nacelle. A major subset of the important stratospheric radicals can be detected using the configuration shown in Figure 13. 

A CEC instrument basically consists of a system for injection (pressure driven or electrokinetic), a column in which the separation takes place, a detector and a high voltage supply (Fig. 16.1). The most commonly used detector so far has been UV with transmission through the capillary outside of the packed bed. Laser induced fluorescence detection has been employed in several studies. Also, mass-spectrometry has been used. Normally, isocratic CEC is performed, but approaches to gradient CEC have been reported [29]. However, special equipment must be employed in most cases. 

The so-called micro-total analytical systems (/tTAS) can integrate sample handling, separation, and detection on a single chip [9]. Postcapillary reaction detectors can be incorporated as well [10]. Fluorescence detection is the most common method employed for these chip-based systems. A commercial instrument (Agilent 2100 Bioanalyzer) is available for DNA and RNA separations on disposable chips using a diode laser for LIF detection. In research laboratories, polymerase chain reaction (PCR) has been integrated into a chip that provides size separation and LIF detection [11]. 

Capillary electrophoresis can be also applied for DNA sequencing. For this purpose, multicapillary (array) instruments with laser-induced fluorescence detection are being developed. Detailed descriptions of these methods is beyond the scope of this entry. 

Galle B, Grennfelt P. 1983. Instrument for polycyclic aromatic hydrocarbon analysis of airborne particules by capillary gas chromatography with laser induced fluorescence detection. J Chromatogr 279 643-648. 

Laser excitation for fluorescence detection has received much research interest, but as of yet there is no commercially available instrument. Fluorescence intensity increases with excitation intensity, and it is generally assumed that laser excitation would then offer improved limits of detection. However, as Yeung and Synovec have shown, various types of light scattering, luminescence from the flow cell walls, and emission from impurities in the solvent all increase with source intensity as well, yielding no net improvement in signal-to-noise ratio (53). Where laser excited fluorescence may prove useful is for the design of fluorescence detectors for microbore packed and open tubular LC columns, where the laser source can be focused to a small illuminated volume for on-column detection. 

Fluorescence detection is often used in liquid chromatography. It has a low detection limit, but sometimes it does not cover the requirements of some compounds. To improve the detection limit, visible diode laser-induced fluorescence has been used as a detection system in liquid chromatography.208 Spectra are automatically corrected for differences in excitation efficiency in the red region of the spectrum. The automation of all components of analytical instruments improves the reliability of the analytical information. 

LIF detection is the most sensitive optical method so far, but is hard to miniaturize in order to satisfy the ultimate goal of a microfluidic chip that assembles all analytical processes within one micrometerscale microstructure. Therefore, how to achieve the miniaturization of fluorescence detection on microdevices is becoming an active field for lab-on-a-chip research. Several examples demonstrate recent advances in miniaturized LIF detection on the microchip. In 2005, Renzi et al. designed a hand-held microchip-based analytical instrument that combines fluidic, optics, electrical power, and interface modules and integrates the functions of fluidics, microseparation, lasers, power supplies etc., into an... 

A detector, for example, a UV absorbance detector, through which the solution flows, is placed near or at one end of the capillary. A focused beam is passed through the capillary and may be collected by an optical fiber coupled to a photomultiplier tube. The short pathlengths (10 to 100 /rm) involved make sensitive detection a challenge. But the small peak volumes, often less than 1 nL, lead to very low detection limits, even with moderately sensitive detectors (i.e., the solute is concentrated in a very small volume). The use of laser sources, especially for fluorescence detection, has pushed detection limits to zeptomoles (10"- mol) A capillary electrophoresis instrument is shown in Figure 21.19. 

In CE, the principle detection schemes are spectrometric and electrochemical. Fluorescence is easy to implement (especially off-chip), is extremely sensitive, which is useful since the sample volumes are typically very small, and is well understood. However, some compounds may need to be fluorescently labelled . This can be done prior to, during or after separation. Renzi et al. from Sandia National Laboratories have reported a handheld microanalytical instrument for CE analysis of proteins using laser-induced fluorescence detection ". The fused silica chip is 2 X 2 cm and features on-chip sample introduction, inlet port filters and a 10 cm separation column. Nanomolar concentrations of fluores-camine-labelled proteins were detected. 

In addition to adsorption, issues to be addressed for the routine use of microchips for clinical analysis include the development of lower cost, compact instrumentation for detection on microchips. Shrinivasan et al. detailed the development of a miniaturized LIF detector applicable for fluorescent detection of DNA on microchips, which could also be applied to proteins with the necessary fluorescent tags. By replacing the argon-ion laser with a diode laser, the cost and power requirements were significantly decreased, with only a small loss in sensitivity. Integration of fluid flow and mixing... 

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