Spectrophotometric end-point

Discussion. Salicylic acid and iron(III) ions form a deep-coloured complex with a maximum absorption at about 525 nm this complex is used as the basis for the photometric titration of iron(III) ion with standard EDTA solution. At a pH of ca 2.4 the EDTA-iron complex is much more stable (higher stability constant) than the iron-salicylic acid complex. In the titration of an iron-salicylic acid solution with EDTA the iron-salicylic acid colour will therefore gradually disappear as the end point is approached. The spectrophotometric end point at 525 nm is very sharp. 

A prevailing problem with neuroactive steroid analysis by HPLC is the lack of sufficient chromaphores or fluorophores within their chemical structures to allow suitable spectrophotometric end-point detection such as with UV/VIS or fluorescence with adequate sensitivity. The multitude of structural isomers of the metabolites also decreases the applicability of RP-HPLC since the chromatographic profiles become very complex with co-eluting peaks. Due to these inherent problems, it is often necessary to derivatize this group of compounds prior to chromatographic separation and suitable end-point detection to allow their direct determination at physiological concentrations. 

H.V. Malmstadt, Rapid and accurate automatic titration of calcium and magnesium in dolomites and limestones. Use of EDTA titrant and automatic derivative spectrophotometric end-point termination, Anal. Chim. Acta 19 (1958) 563. 

An automated two-phase titration may be conducted to a spectrophotometric end point. Various researchers have demonstrated this technique with specially made glass vessels or by using membranes to separate the aqueous and organic phases. Nowadays, the manufacturers of automatic titrators supply equipment and optimized conditions to permit this type of titration (1,29). Usually, the vessel is stirred after each increment of titrant, then the phases are allowed to separate and the transmittance of the organic phase is deter-... 

Two-phase titration may be conducted to a spectrophotometric end point, observing the change in absorbance of either the aqueous or organic phase (40,96-98). For example, cationic surfactants can be titrated with picrate ion, while monitoring the UV absorbance (k = 400 nm) of either phase. As titrant is added, the absorbance of the organic phase increases linearly from zero to a maximum. It remains at the maximum value after the end point, which is determined by extrapolation of the two linear portions of the titration plot. If the absorbance of the aqueous phase is monitored, its absorbance increases linearly after the end point, as additional picrate ion is added but not extracted into the organic phase. 

In acid-base titrations the end point is generally detected by a pH-sensitive indicator. In the EDTA titration a metal ion-sensitive indicator (abbreviated, to metal indicator or metal-ion indicator) is often employed to detect changes of pM. Such indicators (which contain types of chelate groupings and generally possess resonance systems typical of dyestuffs) form complexes with specific metal ions, which differ in colour from the free indicator and produce a sudden colour change at the equivalence point. The end point of the titration can also be evaluated by other methods including potentiometric, amperometric, and spectrophotometric techniques. 

F. Alternative methods of detecting the end point. In addition to the visual and spectrophotometric detection of end points in EDTA titrations with the aid of metal ion indicators, the following methods are also available for end point detection. 

General discussion. In a spectrophotometric titration the end point is evaluated from data on the absorbance of the solution. For monochromatic light passing through a solution, Beer s Law may be written as ... 

Discussion. In acid solution arsenic(III) can be oxidised to arsenic(V) and antimony(III) to antimony(V) by the well-established titration with a solution of potassium bromate and potassium bromide (Section 10.133). The end point for such determinations is usually observed indirectly, and very good results have been obtained by the spectrophotometric method of Sweetser and Bricker.23 No change in absorbance at 326 nm is obtained until all the arsenic)III) has been oxidised, the absorbance then decreases to a minimum at the antimony(III) end point at which it rises again as excess titrant is added. 

This reaction was studied spectrophotometrically by monitoring the absorbance at 830 nm, where Pu02+ absorbs. The paired values of time and absorbance are presented for one experiment in Table 2-4. Figure 2-5 shows the data treatment according to Eq. (2-35). Nonlinear least-squares analysis gives k = (9.49 0.22) X 102 L mol-1 s-1 and a calculated end point absorbance of 0.025 0.003. 

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. 

The routine determination of TCAs has often utilized RP-HPLC with some form of spectrophotometric or, in some cases, electrochemical detection. These systems include UV/VIS, fluorescence, or amperomet-ric/coulometric end-point detection. The TCAs are usually lipophilic strong bases (possess high pKa values) and therefore isolated as free bases by alteration of the biological matrix to pH 12 followed by extraction into an organic solvent. 

The titration cycle starts with first a homogenizing period which allows dissolution of sohd samples, flushing with an inert gas, or appHcation of a chemical reaction. The sample may also be heated to a predetermined temperature. Next, a precise volume of reagent or reagents is dispensed if required. While the sample is being stirred a titration can be performed, either to a relative or an absolute end-point, or incrementally with or without equihbrium detection. Several titration modes are available, including potentiometric, amperometric, voltammetric and spectrophotometric. 

Although changes in pM can readily be followed by physical means (e.g. potentiometrically), following colour changes associated with the formation and dissociation of metal coordination complexes visually or spectrophotometrically is a more versatile and convenient procedure. The serendipitous discovery of so-called metallochromic indicators made by G. Schwarzenbach (1945) led immediately to the introduction of murexide (50) as an indicator in calcium titrations and initiated the search for indicators for other metal-EDTA systems. It will be realized that the chosen metal indicator must be considerably less stable than the metal-EDTA complex, but not so weak as to dissociate appreciably in the vicinity of the end-point when the concentration of free metal... 

The specific enzyme to be used in an EIA is determined according to a number of parameters including enzyme activity and stability (before, during, and after conjugation), cost and availability of the enzyme substrate, and the desired end point of the EIA, such as color. Most EIAs utilize a colored end point which can be readily determined both visually and spectrophotometrically. Table 1 lists a number of enzymes which have been used in immunoassays and their substrates. 

EDTA was determined in the urine of patients treated with Na2CaEDTA upon formation of its Fe(II) complex with 2,4,6-tripyridyl-s-triazine at pH 4.5 [30], Concentrations ranging from 0.1-15.7 pM of EDTA in urine could be accurately determined. EDTA can be standardized by spectrophotometric titration with electrolytically pure Cu(II) at pH 5 without an indicator [31]. The break of the titration curve obtained at 700 nm is used as the end-point of the titration. 

Two procedures have been adopted to prevent oxidation of excess unreacted iodide by molecular oxygen. Either unreacted iodide is com-plexed with cadmium (Buege and Aust, 1978), or spectrophotometric measurements are made in air-free stoppered cuvettes and development of I3 is followed continuously. The second procedure allows reliable detection of the reaction end point and identifies any oxygen contamination (Hicks and Gebicki, 1979), but it is not applicable to routine measurements of many samples as the time taken for complete reduction of the hydroperoxide is prohibitive. Methods are therefore described which are based only on the procedure utilizing cadmium. 

The scope of the present treatment does not include details of the various instrumental methods for the detection of EDTA titration end points. Nevertheless, we may mention spectrophotometric detection methods, which are of two types. The first is based on instrumental observation of the color changes of metal ion indicators. The second is based on the absorption of radiation in the visible or ultraviolet regions of the spectrum by the metal-EDTA complex. For example, MgY shows appreciable absorbance at a wavelength of 222 nm, whereas the reagent HjY ... 

Potentiometric titrations of the acid with base reveal two distinct end points a sharp one at about pH 5 and a less distinct one at pH 10. Spectrophotometric data... 

Spectrophotometric Methods Measurement of UV/visible absorption can also be used to detennine the end points of titrations (see Section 26A-4). In these cases, an instrument responds to the color change in the titration rather than relying on a visual detennination of the end point. 

Spectrophotometric titration [29-32] consists in repeated measurement of an absorbance which changes in the course of titration of the sample solution. The use of this method depends on the existence of a linear relationship between the absorbance measured and the concentration of the absorbing substance in the solution being titrated. The course of the titration is represented graphically by two intersecting straight lines. To find the titration end-point it is necessary to determine the absorbance at two points before and two points behind it. The graphs are drawn in the system of A (absorbance) versus v (volume of titrant solution). To increase the accuracy of determination, corrections are made for the dilution caused by the addition of the titrant solution. 

Ultraviolet-visible spectrophotometry has also been applied to titrimetry. In this case the variation in the absorbance of the analyte with addition of titrant is used to obtain a spectrophotometric profile from which titration end points and/or equilibrium constants, etc., can be determined. This has been applied to the whole range of titrations in which a chromophore is generated. These include acid-base, redox, and complexometric titrations. 

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. 

Other investigators have examined the potential antioxidant properties of habenariol [251] particularly to inhibit copper-induced lipid peroxidationof human low density lipoprotein (LDL). However, in both kinetic and end-point spectrophotometric assays, it was found to be less effective than a-tocopherol. 

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