Measurement by spectrophotometry

Minor levels of titanium are conveniently measured by spectrophotometry, eg, by the 410-nm absorbance of the yellow-orange peroxide complex that develops when hydrogen peroxide is added to acidic solutions of titanium. 

Collagen determination. In order to calculate the amount of collagen in a sample, hydroxyproline was measured in acid hydrolyzates (Jamall et al., 1981). After oxidation, hydroxyproline reacts with p-dimethyl-aminobenzaldehyde to give a red colour, which is measured by spectrophotometry. For bovine dentin, collagen mass was estimated as 8.0 x the hydroxyproline value as calculated from previous data (Volpin and Veis, 1973). To calculate the molar quantities of collagen, a molecular mass of 300 kDa was used. 

Many analytes listed in Table 1 have been measured spectrophotometri-cally in seawater for some time, including many metal ions and some gases, although spectrophotometry is the preferred method for only a minority. Some analytes, like alkanes, are spectrophotometrically silent, or do not form colored complexes with other reagents. Similarly, individual nuclides cannot be distinguished by classical spectrophotometry, and many of the other analytes, such as halogenated pesticides and metal alkyls, are more easily determined by other methods, such as gas chromatography with electron capture detection, or emission spectroscopy. Indeed, many of the analytes, such as zinc or copper, are present at trace levels and are not measurable by spectrophotometry. 

For a method to be used to follow the course of a chemical research, the method given in Ref 33 might be used to advantage. In this method RDX is detd in HMX in concns of 0.05 to 5% by Thin-Layer Chromatography on alumina coated plates. The RDX is recovered from the subscrate by extraction with acetonitrile and is measured by spectrophotometry at 228 millimicrons. See also Analytical Procedures under RDX in this Volume Note This Section was written in collaboration with E.F. Reese of PicnArsn)... 

Copper speciation in water was investigated by means of spectrophotometry of Cu+-bathocuprine complex formation (Bjoerklund and Morrison, 1997). The complex was separated by means of solid-phase extraction on PTFE-supported octadecyl (Qg) bonded silica discs. The discs provided rapid filtration and contributed low blanks. After filtration, the copper complex was eluted, and the copper concentration was measured by spectrophotometry. Total copper concentrations in the samples were measured after UV irradiation. The bathocuprine-available copper detection limits (for 500 ml samples) were 0.4 and 3.8 mg dm-3 copper for pure and polluted water, respectively. 

OH- solutions in DMSO-water mixtures. For acetone, the ionisation ratios were measured by spectrophotometry, but in other cases an indirect kinetic method was used. This latter is based on monitoring the rates of detritiation of a standard labelled carbon acid (HS1) both in the presence and absence of a second acid HS2 (ketone). The dissociation of HS2 brings about a decrease in hydroxide ion concentration, and, since the rate of detritiation of HS1 is proportional to [OH ], the consequent decrease in rate can be related to [(S2) ]/[HS2]. Data listed in Table 7 exhibit large variations with structure, far larger than those expected from ionisation rate constants if an enolate-like transition state were assumed (see p. 34). 

Copigmentation is driven by hydrophobic vertical stacking between the anthocyanin and the copigment to form tt-tt complexes from which water is excluded. The flavylium cation as well as the quinonoidal base are planar hydrophobic structures and can be involved in such complexes whereas the hemiketal form cannot. The association thus results in displacement of the anthocyanin hydration equilibrium from the colorless hemiketal to the red flavylium form that can be easily measured by spectrophotometry. 

The berkelium (IV) extraction coefficients have been determined by stripping solvents previously loaded with tetravalent cerium and berkelium in the presence of sodium bismuthate. Sodium bismuthate has been found to be an efficient oxidizing agent for trivalent cerium. Because of its small solubility it does not affect the distribution coefficients of tetravalent cerium. These two properties have been demonstrated by comparing the distribution coefficients of cerium (IV) measured by spectrophotometry with those of cerium oxidized by sodium bismuthate and measured by beta counting of the cerium isotope tracer. The data are summarized in Table I and indicate no real difference in the distribution coefficients of cerium obtained by these two methods when using trilaurylmethylammonium salts-carbon tetrachloride as solvent. 

RNA can be isolated by a number of extraction methods. The Chomczynski method works well with tumor tissue samples because the multiple extraction steps in the protocol help eliminate the higher levels of protein found in tissue samples (24). The RNA Stat-60 (Tel-Test, Inc., Friendswood, TX), kit works well with cell lines. The key is intact RNA accurately measured by spectrophotometry (see Note 1). 

Total calcium is most frequently measured by spectrophotometry using metaUochromic indicators or dyes. Of the metaUochromic indicators that change color on selectively binding calcium, o-cresolphthalein complexone (CPC) (3, 3"-bis [ [bis-(carboxymethyl)amino] -methyl] -5", 5"-dimethylphenolphthalein) (Figure 49-3) and arsenazo III are most widely used. These methods, although less accurate and reproducible than atomic absorption spectrophotometry, have been easier to automate on chemistry analyzers. 

For determination of protein solubility, after the solution phase is separated from the solid phase, the amount of protein in the solution phase is usually measured by spectrophotometry using an absorbance at 280 nm. After subtracting the blank signal from that of the protein, protein concentration is calculated using a predetermined extinction coefficient as follows ... 

Spot Removal. The spot may be removed from the paper by cutting or from a plate by scraping off the adsorbent containing the spot. The substance can be eluted or extracted from the strip or plate, and then handled as any other sample solution (e.g., measured by spectrophotometry, polarography, etc.)... 

Carbon dioxide in acidified blood samples is transferred through a hydrophobic micro-porous membrane and dissolved in an acceptor stream of basic buffer solution containing an acid-base indicator. The absorbance change is measured by spectrophotometry. 

See Table I for conditions of film preparation Measured by spectrophotometry Measured by chelate titration... 

Enzyme inhibition The measurement of the rate (v) of products/reactants formation can be measured by spectrophotometry the dissociation constant Xj coiTesponds to the concentration of inhibitor at which the observed constant ( obs) is half its maximum value Bo ine cyclophilin [9]... 

Fig. 1. Thermodegradation of plasmid DNA in the presence of various KCl concentrations. Left Thermodegradation time course measured by spectrophotometry as in ref. 5. Right DNA sample was taken from the above experiment after 1 hr of incubation at 107° in 2 M KCl and run on agarose neutral gel with plasmid control as described.

In continuous-flow analysis, CO2 gas is released by acidification of the analytical portion. It diffuses across a silicon-rubber membrane into an alkaline hydrogen carbonate buffer. The gas is converted into hydrogen carbonate and hydrogen ions this causes a pH change, which is monitored by a pH indicating dye. The color change of the dye is measured by spectrophotometry and is proportional to total CO2 of the sample. 

Dye-binding methods use the ability of dyes to bind to charged amino acid residues of the proteins. The proteins may be separated as previously described and then treated with the dye. Dyes used include amido black and orange G. On binding to the protein they form an insoluble complex that may be removed by centrifugation. The amount of residual color in the supernatant is measured by spectrophotometry and calibrated by reference to the Kjeldahl method. 

Early analytical methods developed to assay heroin and its metabolites in biological fluids and tissues utilized indicator-dye, paper chromatography (PC), and TLC techniques. Indicator-dye techniques, based upon the reaction of organic bases with colored organic acids such as methyl orange to form complexes soluble in organic solvents, were used to quantify heroin and metabolites. The colored complexes were subsequently measured by spectrophotometry. 

Other film dosimeters. Colorless PVC foils are used at electron accelerators to monitor the irradiation process and the accelerator parameters (like scan width and beam spot). In these materials unsaturated chemical bonds form upon irradiation and the optical absorption of these new species can be measured by spectrophotometry at 395 nm in the dose range of 0.5-60 kGy (Artandi and Stonehill 1958). It is important to mention, however, that due to various factors (environmental effects on the response, dose rate effects, batch-to-batch variation, etc.) these films should not be considered and used as dosimeters, but only as dose indicators. 

In these assays, the initial reaction product is colourless, but a second enzyme is included to convert this into a product measurable by spectrophotometry. For example, for an ATPase assay, the product ADP can be used to drive NADH oxidation (measurable by A340) in the presence of phosphoenolpyruvate + pyruvate kinase (which generate pyruvate in the presence of ADP) and lactate dehydrogenase (which converts NADH to NAD in the presence of p30uvate). 

The most specific method for sialic acid determination is possible with enzymes. Using acylneuraminate pyruvate-lyase (EC 4.1.3.3), sialic acids are cleaved to acylmannosamines and pyruvate. By coupling this reaction to lactate dehydrogenase the reduction of pyruvate allows monitoring of the pyruvate concentration and drives the lyase reaction, otherwise an equilibrium reaction, to completion (Brunetti et al. 1963). The concentration of sialic acid can be measured by spectrophotometry or fluorimetry of the amount of NADH oxidized on completion of the reaction, or by measurement of the initial rate of NADH oxidation. A less sensitive alternative is determination of N-acylmannosamine using colorimetric methods. Brunetti et al. (1962) utilized the Morgan-Elson reaction with dimethyl-aminobenzaldehyde for this purpose. 

Arum italicum L spadices were collected in woods near Paris and mitochondria were isolated at various developmental stages and purified on sucrose gradient, according to (1). Cytochrome oxidase and malate dehydrogenase activities were measured by spectrophotometry in a 300 mOsm medium. Tlie ratio of the activities with or without Triton X-100 (0,2%) give an estimation of outer membrane permeability to cytochrome c or of inner membrane permeability to NADH. and thus of mitochondrial integrity. Lipids were extracted and analysed according to (3) and separated by TLC... 

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