Detectors direct spectrophotometric

Besides the spectrophotometric detectors seen in HPLC based on absorbance or fluorescence of UV/Vis radiation, another type of detector based on electrolyte conductivity can be used. This mode of detection measures conductance of the mobile phase, which is rich in ionic species (Fig. 4.6). The difficulty is to recognise in the total signal the part due to ions or ionic substances present in the sample at very low concentrations. In a mobile phase loaded with buffers with a high conductance, the contribution of ions due to the analyte is small. In order to do a direct measurement, the ionic loading of the mobile phase has to be as low as possible and the cell requires strict temperature control (0.01 °C) because of the high dependence of conductance on temperature. Furthermore, the eluting ions should have a small ionic conductivity and a large affinity for the stationary phase. 

Figure 8. Renaturation of low molecular weight urokinase observed in samples collected from continuous flow refolding. UK was injected in 9.3 M urea into 2.0 M urea, 20 mM Bis-Tris, pH 7.8 buffer with a gradient from 2.5 mM reduced glut-athione (GSH) to 2.5 mM oxidized glutathione (GSSG). Upper panel Data for GSSG concen-tration are from direct absorbance measurements at the secondary UV detector data for GSH were determined by DTNB titration of collected fractions. Lower panel urokinase activity in collected fractions measured by spectrophotometric assay using S-2444 (12) in a Molecular Devices titerplate reader.

Direct detection of anions is also possible, providing a detector is available that responds to some property of the sample ions. For example, anions that absorb in the UV spectral region can be detected spectrophotometrically. In this case, an eluent anion is selected that does not absorb (or absorbs very little). 

The discussion of UV-VIS detectors for use in ion chromatography is divided into two parts (a) the direct monitoring of column effluents and (b) post-column derivati-zation with subsequent spectrophotometric measurement. 

The area of a peak, without taking into account this specific parameter, renders the direct calculation of concentration unfeasible by a simple check of the chromatogram. Spectrophotometric detectors are examples of selective detection. For compounds that do not possess a significant absorption spectrum it is possible to perform derivatization of the analytes prior to detection. 

Exploitation of this approach is straightforward in flow analysis the sample zone is handled, stopped inside the detector for a pre-set time interval and then directed towards waste. The initial and final measurements of the analytical signal are considered. The blank value is directly compensated by this approach and lag-phase effects become of minor concern, as demonstrated in the spectrophotometric determination of ethanol in whole blood [359]. The samples were injected without prior treatment other than dilution with a buffer solution and the stop period was 50—70 s. Sensitivity could be varied by selecting other stop periods. 

FI manifolds for column separation and preconcentration in spectrophotometry are diverse, and there is hardly one which may be considered typical. However, the reader may refer to the manifold used in the determination of boron just mentioned (63]. Another interesting contribution by Novikov et al.[ll] combined ion-exchange column preconcentration with on-line solvent extraction followed by spectrophotometric detection. The eluate from the column preconcentration was released into an on-line liquid-liquid extraction system. An advantage of this approach is that interferences from Schlieren effects are avoided, since the eluate does not flow directly to the detector. Selectivity and sensitivity are also enhanced due to the combination of two separation procedures. The system has been used successfully for the determination of lead in alloys, soil leachates and sea water. 

Sample ions that absorb sufficiently in the UV or visible spectral region may be detected by direct spectrophotometry. Indirect spectrophotometric detection is commonly used for ions that do not absorb. An absorptive reagent is added to the BGE, and this gives a peak in the direction of reduced absorbance when a sample ion passes through the detector. The absorbing reagent, which is sometimes called a visualization reagent, should have a mobility that matches those of the sample ions as closely as possible. Chromate is often used for the indirect detection of anions and a protonated amine cation, such as benzylamine, for detection of cations. 

All modern automated analytical systems are wet chemical analysers based on the Continuous Flow Analysis (CFA, Technicon) introduced by Weichart (1%3) and Grasshoff (1964). For seawater analysis a continuous stream of sample water is taken either from sample bottles or from a direct seawater intake. All operations such as the addition of reagents, heating, dialysing or phase transfer are performed in a closed tubing system between the inlet and flow-through cell of the detector system (usually a spectrophotometric cell). 

The limited number of well functioning, classical or spectrophotometric methods is available for measuring fluoride ion concentration in different samples. Therefore, after the invention of lanthanum fluoride crystal-based ISE [15], its use as a detector in standardized methods becomes almost general. For example, the Environmental Protection Agency (ERA) METHOD 9214 [44] is for measuring the concentration of fluoride ions in water samples as well as in soil extracts. It is a direct potentiometric method using the ion-selective fluoride electrode and the conventional or double junction reference electrode. The lower limit of detection is 0.025 mg dm. Fluoride concentrations from 0.025 to 500 mg dm can be measured. 

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