Endpoint Detection Methods

Electrochemical endpoint detection methods provide a number of advantages over classical visual indicators. These methods can be used when visual methods of endpoint detection cannot be employed because of the presence of colored or clouded solutions and in the case of detection of several components in the same solution. They are more precise and accurate. In particular, such methods provide increased sensitivity and are often amenable to automation. Electrochemical methods of endpoint detection are applicable to most oxidation-reduction, acid-base, and precipitation titrations, and to many complex-ation titrations. The only necessary condition is that either the titrant or the species being titrated must give some type of electrochemical response that is indicative of the concentration of the species. 

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Numerous approaches have been proposed for use in CMP for in situ EPD. They include optical, electrical, and acoustic sensing. Given the benefits of EPD, it is no surprise that many of these methods have been awarded patents. Some of these methods, most notably current sensing, have been developed to become commercially viable products while others remain laboratory curiosities. For a review of in situ endpoint detection methods up to early 1998, see the work of Bibby and Holland [68]. 

Some method of signaling is required to indicate when the amount of titrant generated is equivalent to the amount of unknown present, and all of the endpoint detection methods used in volumetric titrimetry are, in principle, applicable to coulometric titrations. A list that covers most of the published coulo-metric titration procedures is given in Table 25.2. It is beyond our scope here to describe any of these in detail because each of these methods is a subject for discussion in its own right. Discussions of the equations for a number of types of titration curves are found in texts by Lingane [15], Butler [16], and Laitinen and Harris [17]. 

Different experimental approaches are possible with the same endpoint detection method. For example, the titration curve can be plotted and the endpoint determined graphically. First and second derivative curves can be plotted or the derivatives obtained electronically. Another approach is to titrate to a predetermined endpoint signal. This technique is very useful with coulometric titrations, and many examples, especially those involving potentiometric endpoint detection, are found in the literature. The most widely applicable way... 

Table 25.2 Selected Endpoint Detection Methods for Coulometric Titrations...

However, real-time detection requires access to a special real-time PCR cycler, which is able to detect the increase/decrease of added fluorescence labels during DNA amplification. Although these machines are more and more common for quantitative DNA analysis, their availability in clinical laboratories is still limited. Therefore, the following subsections also include a detailed overview of the classical approaches to quantitative (I)PCR amplificate, analysis which exchanges less demanding PCR equipment for additional hands-on time. The sensitivity of real-time or end-point IPCR detection is quite similar. A comparison of the influence of different endpoint detection methods to the overall sensitivity of IPCR is given in Fig. 5. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

The use of dual-polarized electrodes was first suggested more than 70 years ago 2 the subject has been reviewed thoroughly by two more recent publications.3,4 Almost all modem commercial pH meters have provision for imposing a polarizing current of either 5 or 10 nA to make possible measurements by dual-polarized electrode potentiometry. Such a provision is included because dual-polarized potentiometry is by far the most popular endpoint detection method for the Karl Fischer determination of water. For this titration a combination of reagents is used, including iodine the response curve is similar to that of Figure 4.3b. In practice the response is many times more sensitive than... 

As with amperometric titrations, to have straight-line portions of the titration curve dilution corrections must be made because the response is directly dependent on the concentration of the ionic species. Also, the important data are taken before and after the equivalence point rather than precisely at the equivalence point. The general conditions for effective conductometric measurements of solutions are discussed in Chapter 5 and are directly applicable when the system is used as the endpoint detection method. A particularly complete review of the subject has been presented.8... 

Kimura N, Sakata F, Takahashi T. Polishing endpoint detection method.US patent... 

The approved variations [14] in the Karl Fischer method include volumetric titration methods to either a visual (excess iodine or addition of an indicator) or volta-metric endpoint detection method. The visual or voltametric endpoint methods usually require 30-40 mg of sample for analysis for freeze-dried biological products containing from 1.0% to 3.0% residual moisture. Coulometric Karl Fischer instruments generate the iodine from potassium iodide for water titration at the electrodes. Only 10-20 mg of freeze-dried sample is required for accurate analysis. 

Both DIE and TT are subject to the same restrictions as in classical calorimetry. If TT is used simply as an endpoint detection method, the restrictions also apply, but to a much lesser degree. 

The use of amperometry as an endpoint detection method has been widely adopted for the titration of substances at the millimole per liter level, with excellent analytical precision ( 1% RSD at 0.01 mmol 1 level). The majority of these titrations involve the formation of precipitates, while others involve complexometric and redox titrations. At the millimole per liter level, the accuracy of this method is better than that achievable with other electroan-alytical methods and as good as those of spectro-photometric titration. 

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