Absorption or refractive index

Here, we describe the advantages o CBNFs for biochemical sensing applications. The biochemical sensor is shown to exhibit high spectral resolution and excellent sensitivity to changes in the absorption or refractive index of its... 

Figure 3 Instrument setup of whole-column imaging detection for CIEF. Absorption or refractive index gradient mode with camera (I) placed in the direction of illuminated light fluorescence mode with camera (II) placed vertically to the direction of illuminated light. (Reproduced from Mao, Q., Pawliszyn, J J. Biochem. Bio-phys. Meth. 39, 1999, 93-110, with permission of Elsevier Science Publishers.)...

On adaptation of known analytical methods to HPLC, this problem is invariably the first to be encountered. For the most utilized method of detection in HPLC, UV absorption or refractive index (RI), this adaptation was carried out at an early stage of the development of HPLC and no significant problems were encountered on reducing the cell volume in these devices to less than ID ul. A typical UV absorption cell used in present-day LC detectors has a cylindrical shape with a length (direction of the light beam) of 10 ram and a diameter of 1 mm, with a volume of about 8 rl. With these dimensions, the noise level can be kept at the same level as, or may even be less than that in classical large-volume measurements. 

Asphalt contains many different compounds that vary not only in molecular, or particle, size but also in UV absorptivity or refractive index. Figure 17 shows the relation between detector response per unit mass and apparent molecular size for some asphalts (12). Neither detector is uniform, as a mass detector would be. The UV detector is much less uniform than the RI detector. This is mainly because paraffinic hydrocarbons, known as saturates, which comprise roughly 10-20% of a typical asphalt, are very weak absorbers of UV light, and the aromatic components in the asphalt are strong UV absorbers. Consequently, a... 

The spectrophotometer measures the transmission and, if an absorption measurement is carried out, converts the transmission into absorbance using these equations. This conversion works fine for samples where there is no reflection, either specular or diffuse, as is the case for nonturbid solutions. However, for films there is invariably some reflection, which is often quite large, particularly for films of high dielectric constant (or refractive index) materials, such as PbS and PbSe. Additionally, if the films are not completely transparent, then scattering introduces an extra element of reflection. Therefore, to measure the real absorption of a film, a reflection measurement must also be carried out and correction for this reflection made. The correction will be approximate and depends on the nature of the film itself. However, that most commonly used is... 

The most common LC detectors are based on ultraviolet absorption (UV), refractive index (RI) changes, or molecular fluorescence emission. All three are very common phenomena, and the detectors used in LC are, with few exceptions, modifications of existing technology and not based on new principles. This was not the case with GC detectors, many of which were invented specifically for GC. In fact, many of the GC detectors, such as the FID, electron capture, and TID, have been adapted to LC as well. 

Optical biosensors can be defined as sensor devices which make use of optical principles for the transduction of a biochemical interaction into a suitable output signal. The biomolecular interaction on the sensor surface modulates the light characteristics of the transducer (i.e., intensity, phase, polarization, etc.), and the biosensing event can be detected by the change in diverse optical properties such as absorption, fiuorescence, luminescence or refractive index, among others. 

Several detectors connected in series can be used for simultaneous effluent detection, and there exist also the so-called combined detectors which measure the light absorption at two wave lengths, absorption plus fluorescence or absorption plus refractive index, in the same measuring cell. 

The total concentration of protein may be measured by nitrogen, or by specific gravity or refractive index. The method is generally applicable to sera and to serum protein solutions. Care should be taken to check the possibility of absorption by nonprotein constituents of serum which in some diseases such as cirrhosis of the liver may be considerable. The presence of such substances is shown by a marked fall in absorption of the serum after dialysis. 

The mobile phase consists of one or more solvents that are pumped through the chromatographic system, resulting in the separation of analytes. Mobile phases may also contain modifiers. Examples of frequently used solvents include hexane, methanol, 2-propanol, acetonitrile (ACN), and water. Examples of modifiers include tri-fluoroacetic acid, acetic acid, or formic acid. In general, the composition of the mobile phase should be kept simple. Factors that influence the choice of mobile phase include the solubility of the sample in the mobile phase, the polarity of the mobile phase, ultraviolet absorption wavelength, refractive index, and viscosity of the solvents. The purity of the solvents in the mobile phase is also important because the region of UV that is used for the detection of lipids (200-215 nm) must be free of interferences. For phospholipids, the most popular solvent systems are transparent to UV in the range of 200-215 nm they include... 

If the object has an exactly sinusoidal variation of absorption, thickness or refractive index in one dimension, diffracted beams appear only when d sin (/) = A (i.e. m = 1). This is important because of Fourier s Theorem, which states that any (single valued) function of a variable x can be expanded as a sum of sines and cosines of multiples of x. Thus any phase or intensity variation in the sample can be considered as a sum of sinusoidal variations of different wavelength, each giving a certain intensity at a single characteristic angle 0. The intensity at a point in the diffraction pattern corresponds to the strength of a variation of some sample property... 

Numerous methods based on the correlation of other physical properties with protein content have been described. Some of them are so much less favorable than specific gravity or refractive index that they do not warrant extended discussion here. Others, such as electrophoresis (160, 161) and infrared absorption spectroscopy (162) are to be considered at this time as being primarily research methods until wider distribution of information and equipment is available. The interesting technique of spreading the protein on a liquid surface and measuring the area as a means of quantitative analysis was described by Hooft (163) and by Gorter and associates (164). The viscosity of formalin treated serum... 

Detectors can be broadly classified into two types. Bulk Property detectors which function by measuring some bulk physical property of the column eluent (e.g. dielectric constant or refractive index) and Solute Property detectors which function by measuring a physical and/or chemical property that is characteristic of the solute only (e.g. UV absorption). This classification is not completely precise, for example, the UV detector, which is usually classed as a solute property detector, when used with an ethyl acetate-heptane solvent mixture as the mobile phase will give a constant background signal due to UV absorption by the ethyl acetate. Furthermore, any fluctuation in ethyl acetate content of the mobile phase will appear as noise on the detector output. It follows that the UV detector, although a solute property detector, behaves as a hybrid between a bulk property detector and a solute property detector under some conditions of use. 

Multi-functional detectors monitor the column eluent by the measurement of more than one physical or chemical property simultaneously, employing a single sensing cell. To date, three bifunctional detectors and one trifunctional detector have been described. The three bifunctional detectors have combined UV absorption and fluorescent detection, UV absorption and electrical conductivity detection and UV absorption and refractive index detection. The latter uniquely combines a bulk property detector with a solute property detector producing, at least in theory, the nearest approach to a universal detector. The trifunctional detector incorporates UV absorption, electrical conductivity and fluorescence functions. Multi-functional detection provides detector versatility and a means of confirmir solute identity. Such detectors have to be designed, so that the performance specifications are not seriously compromised, and the cell and eluent conduits do not contribute significantly to peak dispersion. 

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