Techniques for Measuring Absorbance

Frequency-Modulation Spectroscopy. Frequency-modulation spectroscopy (tins) is a high sensitivity null-background infrared technique for measuring absorbances down to 10 with fast acquisition speeds. Fms involves frequency-modulating a laser source at COq to produce a carrier frequency having sidebands at cJq where is an integral multiple of the modulation frequency. Dye lasers and many other single-line sources can... 

When bubbler systems are used for collection, the gaseous species generally undergoes hydration or reaction with water to form anions or cations. For example, when SOj and NH3 are absorbed in bubblers they form HSO3 and NH4, and the analytical techniques for measurement actually detect these ions. Table 13-1 gives examples of gases which may be sampled with bubbler systems. 

Apparatus. The course of reaction was followed by periodically measuring the volume of oygen absorbed at constant pressure, and by analysis. The apparatus and techniques for measuring oxygen uptakes have already been described (3). Spectrophotometric investigation of complexes in solution was carried out using a Unicam SP800 spectrophotometer. 

A common technique for measuring the values has been to employ species that produce anions with useful ultraviolet (UV) or visible (vis) absorbances and then determine the concentrations of these species spectropho tome trie ally. Alternatively, NMR measurements could be employed, but generally they require higher concentrations than the spectrophotometric methods. A hidden assumption in Eq. 5 is that the carbanion is fully dissociated in solution to give a free anion. Of course, most simple salts do fully dissociate in aqueous solution, but this is not necessarily true in the less polar solvents that are typical employed with carbanion salts. For example, dissociation is commonly observed for potassium salts of carbanions in DMSO because the solvent has an exceptionally large dielectric constant (s = 46.7) and solvates cations very well, whereas dissociation occurs to a small extent in common solvents such as DME and THE (dielectric constants of 7.2 and 7.6, respectively). In these situations, the counterion, M+, plays a role in the measurements because it is the relative stability of the ion pairs that determines the position of the equilibrium constant (Eq. 6). 

In 1967, Ritchie reported a direct technique for measurement of albumin and immunoglobulins by the development of antigen-antibody precipitates in liquid media. In this method turbidity produced by the antigen-antibody complex was determined by measuring absorbance at 420 nm with a spectrophotometer (Rl). 

Earlier techniques for measuring cytosolic free Ca2+ (1-2) such as the luminescent photoprotein aequorin, the absorbance dye arsenazo III and Ca2+-sensitive microelectrodes, all required microinjection or impalements, and were therefore applied mainly to giant cells. Later, photoproteins have been loaded with various reversible permeabilization procedures (3), but the largest expansion in the range of cell types in which Ca2+ signals can be quantified has come from the development of new fluorescent indicators that can be loaded using hydrolyzable esters. Currently four fluorescent indicators are used frequently quin-2, fura-2, indo-1 and fluo-3. 

Fio. 5. (A) Use of FRET technique for measuring HIV protease activity. The polypeptide was Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Lys(DABCYL)-Arg. Because a simple system which could be commercialized was desired (fluorescence means HIV activity, no fluorescence means no HIV activity), the nonfluorescent acceptor was beneficial because it does not contribute signal after cleavage. This system also has the attribute that detailed information about the dyes and their energy transfer properties are not needed. (B) Good overlap between donor emission (maximum at 490 nm, excitation at 340 nm) and acceptor absorbance (maximum of 28,000 cm" ). (From Molecular Probes Catalog. )... 

During the last ten years a new, very sensitive detection technique for measuring small absorptions, cavity ring-down spectroscopy (CRDS), has been developed and gradually improved. It is based on measurements of the decay times of optical resonators filled with the absorbing species [6.31]. We can understand its general principle as follows ... 

The total absorption intensity of the spectrum is a function of the concentration ofMossbauernuclei in the absorber and the cross-sections of the nuclear processes involved. This absorption intensity, together with the signal-to-noise ratio of the detection system and the total number of counts, determine the quality of the Mossbauer spectrum and the accuracy with which information can be obtained from it. It should be noted that Mossbauer spectroscopy is not generally an appropriate technique for measuring the total concentration of a certain nuclide within a system because it is usually relatively insensitive to small concentrations and because the absorption intensity depends on a number of other factors which may be difficult to quantify. However, relative concentrations of different chemical forms of the Mossbauer nuclide can frequently be obtained, such as the relative concentration of the element in different oxidation states. 

Two techniques for measurement of absorbed hydrogen concentration in thin films are described the quartz crystal microbalance (QCM) and an adaptation of the volumetric technique. Their relative advantages and limitations are discussed. 

The second method is DMA. What is DMA DMA is a technique for measuring the modulus and damping factor of a sample [1]. The modulus is a measure of how stiff or flimsy a sample is and the amount of damping a material can provide is related to the energy it can absorb see Glossary for definitions. DMA is commonly used on a variety of materials, for example thermoplastics, thermosets, composites and biomaterials. The samples may be presented in a variety of forms including bars, strips, discs, fibres and films. Even powders can be tested when suitable containment is arranged. 

Another example of a teclmique for detecting absorption of laser radiation in gaseous samples is to use multiphoton ionization with mtense pulses of light. Once a molecule has been electronically excited, the excited state may absorb one or more additional photons until it is ionized. The electrons can be measured as a current generated across the cell, or can be counted individually by an electron multiplier this can be a very sensitive technique for detecting a small number of molecules excited. 

Separate sample blanking requires an additional analytical channel, and is therefore wasteflil of both reagents and hardware. An alternative approach that is used on several automated systems, eg, Du Pont ACA, BM-Hitachi 704, Technicon RA-1000, is that of bichromatic analysis (5) where absorbance measurements are taken at two, rather than one, wavelength. When the spectral curves for the interference material and the chromogen of the species measured differ sufficiently, this can be an effective technique for reducing blank contributions to assay error. Bichromatic analysis is effective for blanks of both the first and second type. 

Chromatography A technique for separating a sample material into constituent components and then measuring or identifying the compounds by other methods. As an example separation, especially of closely related compounds, is caused by allowing a solution or mixture to seep through an absorbent such as clay, gel, or paper. Result is that each compound becomes adsorbed in a separate, often colored layer. 

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