Optical detection systems absorbance

Optical Detection on Centrifugal Microfluidic Lab-on-a-Disc Platforms, Fig. 2 Portable, lab-on-a-disc-based ELISA system with an absorbance optical detection system [6]. (a) Photograph and (b) schematic of the blood analyzer system. A detector module is installed to perform absorbance detection, (c) Top and bottom plate of the disc... 

While the alkyl chain distribution is determined on a non-polar RP8 and RP18, EO homologue distribution is determined using a polar phase. AEOs are not UV-absorbing species, so they cannot be directly determined by HPLC followed by standard optical detection systems (UV and FL), unless suitable derivatives are prepared [2], Because of this, methods based on liquid chromatography-mass spectrometry [77-79] are currently considered as the benchmark procedure that gives sufficiently high selectivity and sensitivity. 

Figure 13.8. Analytical ultracentrifuge equipped with a UV-vis optical detection system. The sliding slit at the photomultiplier allows positional recording of the absorbance along the cell.

Figure 13.11. Results for a moving-boundary ultracentrifuge experiment using different optical detection systems and a double-sector cell. Part (a) is a graphical representation, (b) is the result of an uv photograph, (c) is a plot of absorbance versus distance (from b), id) is a photograph obtained with Schlieren optics, (e) is an interference diagram obtained using Rayleigh optics, and (f) is another interference diagram, obtained with Lebedev optics.

Label-free optical detection systems can be divided into two main categories absorbance-based and scattering-based detection systems. Both types of systems utilize a (Hie-dimensirmal scanning approach. 

AUC systems are available commercially with several optical systems. The ProteomeLab XL-I model (Beckman Coulter, Inc.) is equipped with two optical detection systems. A Rayleigh interference system, driven by a 660 nm laseg measmes concentration by monitoring refractivity, and a 190-800 nm xenon-lamp system measmes sample absorbance. An XLA version, equipped with absorbance-optics only, is also available. 

AUC-SV measures the rate at which proteins sediment in a rapidly spinning rotor. The rate of sedimentation is expressed as a sedimentation coefficient, which depends on both molecular mass and shape. Proteins form concentration gradients, known as boundaries, as they sediment toward the base of a sector-shaped centrifuge cell. An optical detection system based on absorbance, Rayleigh interference, or fluorescence is used to record the movanent of the boundaries at regular intervals of time. The data from an AUC-SV experiment are then used to calculate a continuous distribution of sedimentation coefficients known as c(5). The resulting c(s) distribution provides a measure of... 

A number of other laser spectroscopic techniques are of interest but space does not permit their discussion. A few specialized methods of detecting laser absorption worthy of mention include multiphoton ionization/mass spectrometry (28), which is extremely sensitive as well as mass selective for gas-phase systems optically detected magnetic resonance (29) laser intracavity absorption, which can be extremely sensitive and is applicable to gases or solutions (30) thermal blooming, which is also applicable to very weak absorbances in gases or liquids (31) and... 

Monochromatic detection. A schematic of a monochromatic absorbance detector is given in Fig. 3.12. It is composed of a mercury or deuterium light source, a monochromator used to isolate a narrow bandwidth (10 nm) or spectral line (i.e. 254 nm for Hg), a flow cell with a volume of a few pi (optical path 0.1 to 1 cm) and a means of optical detection. This system is an example of a selective detector the intensity of absorption depends on the analyte molar absorption coefficient (see Fig. 3.13). It is thus possible to calculate the concentration of the analytes by measuring directly the peak areas without taking into account the specific absorption coefficients. For compounds that do not possess a significant absorption spectrum, it is possible to perform derivatisation of the analytes prior to detection. 

The chemical species, whether they are charged positively or negatively, usually migrate towards the cathode (cf. 8.3.2). A detection system is placed near the end of the capillary. In the UV mode, for example, the capillary is inserted in the optical path between the source and the photodetector. This allows measurement of the absorbance of the solution while avoiding dead volumes. Electrochemical detection is conducted in a similar way microelectrodes are placed within the capillary. 

As described above, recent advances in accelerator technology have enabled the production of very short electron pulses for the study of radiation-induced reaction kinetics. Typically, digitizer-based optical absorbance or conductivity methods are used to follow reactions by pulse radiolysis (Chap. 4). However, the time resolution afforded by picosecond accelerators exceeds the capability of real-time detection systems based on photodetectors (photomultiplier tubes, photodiodes, biplanar phototubes, etc.) and high-bandwidth oscilloscopes (Fig. 8). Faster experiments use streak cameras or various methods that use optical delay to encode high temporal resolution, taking advantage of the picosecond-synchronized laser beams that are available in photocathode accelerator installations. 

Because CE uses online optical detection, artifacts can result in the form of system peaks. These often originate from the sample or the interfaces between the sample and the separation buffer because any species that absorbs at the detection wavelength wfll generate a response. This differs from protein slab gel electrophoresis where detection specificity is governed by a protein specific stain. It is not uncommon, for example, for buffer species present in the sample but not in the separation buffer to generate system peaks. However, clinical serum protein electrophoresis provides one example where artifacts are elimmated by CE. 

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