Colorimetric end-point

Settle [2] has described the evolution of analytical instrumentation over five generations. First-generation instruments consisted of simple devices such as burettes and balances, in which the analyst obtained data, point by point, through manual and visual interaction with the device. Fig. 8.2. After manual manipulations, analysts recorded values associated with visually significant effects, such as colorimetric end-points. The analyst was totally involved in all aspects of determination. 

Among the high number of immunoassay techniques, the enzyme-linked immunosorbent assays (ELISAs) combined with a colorimetric end point measurement are the most widely used. These techniques have also been introduced on the market as PCBs ELISA kits by many companies (see Table 25.2). 

In colorimetric end-point measurement the reflection of the dyed slide at a defined wavelength after a definite incubation time is measured. The concentration of the analyte can be obtained by the following equation ... 

Colorimetric end-point (two-wavelengths) measurement The formed dye is measured at two different wavelengths ... 

Individual phenols by HPLC Total monohydric phenols by distillation with colorimetric end point Ostracod chronic ecotoxicity test on a soil sample... 

The glucose can be determined enzymatically with a colorimetric end point or titrimetrically commercial kits based on enzymatic hydrolysis of starch and measurement of glucose are currently available. 

In the colorimetric, end-point, Reitman-Frankel method, the 2,4-dinitrophenylhydrazone derivative of pyruvate is measured. (The 2,4-dinitrophenylhydrazone derivative of 2-oxoglutarate is also present but this is not as chromogenic.) This method is now little used. 

It is certainly clear that a coulometric titration, like any other type of titration, needs an end-point detection system in principle any detection method that chemically fits in can be used, be it electrometric, colorimetric, photoabsorptionmetric, etc. for instance, in a few cases the colour change of the reagent generated (e.g., I2) may be observed visually, or after the addition of a redox, metal or pH indicator the titration end-point can be detected photoabsorptiometrically by means of a light source and photocell combination. Concerning the aforementioned coulometric titration of Fe(II), it is... 

The above system of directly sensing a process stream without more is often not sufficiently accurate for process control so, robot titration is preferred in that case by means of for instance the microcomputerized (64K) Titro-Analyzer ADI 2015 (see Fig. 5.28) or its more flexible type ADI 2020 (handling even four sample streams) recently developed by Applikon Dependable Instruments20. These analyzers take a sample directly from process line(s), size it, run the complete analysis and transmit the calculated result(s) to process operation (or control) they allow for a wide range of analyses (potentiometric, amperometric and colorimetric) by means of titrations to a fixed end-point or to a full curve with either single or multiple equivalent points direct measurements with or without (standard) addition of auxiliary reagents can be presented in any units (pH, mV, temperature, etc.) required. 

More recently, a colorimetric-based LAL procedure has been devised. This entails addition to the LAL reagent of a short peptide, susceptible to hydrolysis by the LAL clotting enzyme. This synthetic peptide contains a chromogenic tag (usually para-nitroaniline, pNA) which is released free into solution by the clotting enzyme. This allows spectrophotometric analysis of the test sample, facilitating more accurate end-point determination. 

Clinical chemistry Enzyme rate assays, colorimetric assays, end-point assays, immunoassays Upstone, 2000 ... 

Titratable Acidity. The possibility of carboxylic acid group generation from excessive oxidation of corn starch was monitored by titratable acidity (TA). A 0.01 N NaOH solution was used to titrate a dilute aqueous starch suspension (20 mL of a 5% w/v sample) for the presence of acidic functional groups, using phenolphthalein as the indicator dye. An unreacted starch sample was also titrated to yield a sample blank value. TA values were expressed as mL of base required to reach the colorimetric phenolphthalein and end-point. 

It is based on the addition of Mn2+ solution, followed by die addition of a strong alkali to die sample in a glass-stoppered bottle. Dissolved 02 rapidly oxidizes an equivalent amount of the dispersed divalent manganous hydroxide precipitate to hydroxides of higher valence states. In die presence of iodide ions in an acidic solution, the oxidized manganese reverts to die divalent state, with die liberation of a quantity of iodine equivalent to die original dissolved 02 content. The iodine is then titrated with a standard solution of thiosulfate. The titration end point can be detected visually with a starch indicator, or by potentiometric techniques. The liberated iodine can be determined colorimetrically. 

A suitable solvent for dissolution of the compound is glacial acetic acid. The quantitative titration is performed after addition of approximately 10 ml of 15% mercuric acetate in glacial acetic acid to complex the hydrochloride anion. The end point is determined potent-iometrically or colorimetrically, utilizing crystal violet indicator. One equivalent of the compound is titrated under the conditions described. 

The principle behind volumetric determination of nonionics is the already mentioned ability of polyethers to form complexes with large cations, and in particular with Ba2+, in the presence of large anions, such as tetraphenylborate, tetraiodobismuthate, anions of heteropolyacids, and some others. Usually, either a colorimetric or potentiometric method of end-point detection is used. 

Indirect spectrophotometric methods are in most cases based on the formation of ion pairs that are extractable into organic solvents. These methods are often used in combination with volumetric and gravimetric methods, as many precipitated surfactant complexes can be dissolved in the appropriate solvents and analyzed colorimetrically. The spectrophotometric determination of the end-point in two-phase titration is often carried out. 

Twenty analytical units for Individual determination of as many analytes. In addition to independent reagent and diluent streams, heaters, dia-lysers, etc., they are provided with their own detector, usuaiiy photometric (for both end-point and kinetic determinations), and occasionally potentio-metric (ISE). In colorimetric determinations each analytical unit posseses one or several flow-cells and a suitable filter. 

Additional experiments with compleximetric, redox, or precipitation-based titrations have been designed using the same concepts as described above, employing colorimetric indication of the end point. Pertinent details can be found in Ref. 183, in Chapter 2.4.4 (Fig. 2.23), Chapter 4.9... 

These methods are suitable for both AChE and BuChE and are applicable to any substrate because hydrolysis of an ester always results in acid formation. The increase in acid concentration can be measured poieniiometrically by continuous titration with sodium or potassium hydroxide at a constant pH (pH-stat method) (Jeasen-Holm et a ., 1959), or one can measure the decrease in pH after a selected time of assay ApH). The ApH method is an end point method introduced by Michel (1949), and it is used for routine or field measurements, pH can be measured colorimetrically with a pH indicator, which is a simple protocol. 

The oxidizing agents used include gold chloride (potentiometric determination of the end point of the extremely slow oxidation) (Karrerefal, 1938) ceric sulfate (determination of the end point of the almost immediate reaction, with diphenylamine as indicator) (Kofler, 1941) lead tetraacetate (Kofler, 1941) but mostly ferric chloride in presence of a Fe -complex-forming compound, such as a,a -dipyridyl (Emmerie and Engel, 1938). The red color produced in this reaction is used for colorimetric determination. 

The standard iodometric method for peroxide value requires a sample of 5 g for peroxide values below 10, and about 1 g for higher peroxide values. The sensitivity of this method is about 0.5 meq/kg, and can be improved by determining the iodine starch-end point colorimetrically, or the liberated iodine electrometrically by reduction at a platinum electrode maintained at constant potential. 

Titrations constitute a major application of quantitative calculations and these receive a chapter to themselves later. Allied to this is the topic of color-change indicators used to signal end points in titrations, and also used for colorimetric pH determination. These indicators are intensely colored substances whose color changes with gain or loss of protons. The intense color ideally allows use of a quantity so small that only a negligible fraction of acid and base involved in the titration is used to change the color. 

The formaldehyde formed in a periodate reaction is determined gravimet-rically with Dimedon, or colorimetrically with chromotropic acid, after the excess oxidant is destroyed with ethylene glycol, stannous chloride, sodium arsenite, or sodium bisulfite 221), The formic acid is generally titrated directly. Titration to pH 6.0 will measure all the formic acid present. Titration to higher alkalinities will usually measure any formic acid bound as an ester 222) but also other acidic products the end-point may be indefinite. Sucrose will give 80 to 90 % of the theoretical formic acid, and maltose and lactose similarly low values. Both formaldehyde and formic acid are attacked only extremely slowly by periodate, but light apparently accelerates an oxidation to carbon dioxide and water 223),... 

Although all of the work reported here has involved either pH or electrometric titrations, much work has been done on coulometric generation of reagents and colorimetric determination of end points. This work is still in progress. Thus far none of the industrial problems encountered have required these techniques. 

A new instrument for automatic colorimetric and fluorimetric titrations is described. Titrant delivery is automatically stopped at the end point by the use of a microammeter with a built-in relay which stops a motor-driven buret equipped with digital read-out or a gravity driven buret equipped with solenoid control. Monochromatic light is used to excite fluorescence, which is received at 90° by a photomultiplier tube with the microammeter in its anode circuit. Light transmitted through the titration vessel is detected by a barrier-layer photocell, the output of which is fed to the microammeter. 

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