Stopped-flow scanning

Peak height ratios for different detectors or, e.g., ultraviolet detection at several wavelengths, can be used. Another method is to observe the change in retention times upon derivatization of the sample either by chemical or enzymatic methods. With continuous wavelength detectors stopped flow scanning of ultraviolet or fluorescence spectra is a possibility. Isotopic labelling can be used and, finally, fractions can be collected and characterized by, e.g., spectroscopic methods. 

Physical Methods. Vitamins D2 and D exhibit uv absorption curves that have a maximum at 264 nm and an (absorbance) of 450—490 at 1% concentration (Table 8). The various isomers of vitamin D exhibit characteristically different uv absorption curves. Mixtures of the isomers are difficult to distinguish. However, when chromatographicaHy separated by hplc, the peaks can be identified by stop-flow techniques based on uv absorption scanning or by photodiodearray spectroscopy. The combination of elution time and characteristic uv absorption curves can be used to identify the isomers present in a sample of vitamin D. 

Flow injection analysis (FIA) (Ruzicka and Hansen), since 1975 In continuous flow, stopped flow or with merging zones (FIA scanning or intermittent pumping) Adapted voltammetric electrodes Membranes for Partial dialysis Membrane amperometry (Clark) Differential techniques (Donnan) Computerization, including microprocessors Special measuring requirements in plant control (to avoid voltage leakage, etc., Section 5.5)... 

Flow injection analysis (F1A). In this technique, introduced by Ruzicka and Hansen, a small amount of sample is injected into a liquid flow (see Fig. 5.16), which apart from being automated is normally continuous, but can include the use of stopped-flow, merging zones extraction techniques in addition to FIA scanning and methods based on intermittent pumping89. The principles of FIA and the versions just mentioned will now be briefly discussed on the basis of the excellent review of Ruzicka and Hansen89 in order to understand the appli-cational possibilities of electrochemical detection in this technique. 

Fig. 35 Proton spectra obtained from the stopped-flow experiment. Above acetal 4. Below acetal 5. In each case 16 scans, relaxation delay 1 sec...

Atienza et al. [657] reviewed the applications of flow injection analysis coupled to spectrophotometry in the analysis of seawater. The method is based on the differing reaction rates of the metal complexes with 1,2-diaminocycl-ohexane-N, N, N, A/Metra-acetate at 25 °C. A slight excess of EDTA is added to the sample solution, the pH is adjusted to ensure complete formation of the complexes, and a large excess of 0.3 mM to 6 mM-Pb2+ in 0.5 M sodium acetate is then added. The rate of appearance of the Pbn-EDTA complex is followed spectrophotometrically, 3 to 6 stopped-flow reactions being run in succession. Because each of the alkaline-earth-metal complexes reacts at a different rate, variations of the time-scan indicates which ions are present. 

As might be expected, the problem of obtaining spectra of a reacting system increases as the time resolution involved decreases. The spectral changes associated with a reaction may be constructed by wavelength point-by-point measurements. The method, although tedious and costly on materials, is still used. However rapid-scan spectrophotometry, linked to stopped-flow, is now more readily available and reliable. Two systems are used, shown schematically in (3.29) and (3.30). An example of its use is shown in Fig. 3.9. Rapid scan... 

Similarly, the spectrum of a mixture of Fe(tpps)H20 and Fe(tpps)(OH) can be measured by rapid scan/stopped-flow at various pH s within a few milliseconds after generation (Fig. 3.9). In this short time, dimerization is unimportant so that the spectrum of Fe(tpps)OH can be measured and the pAi of Fe(tpps)H20 estimated. 

A number of stopped-flow systems are commercially available. Three of the most used are manufactured by Atago Bussan (formerly Union Giken), Japan Dionex (formerly Durrum), USA and Hi-Tech Scientific, UK. These also manufacture rapid scan spectrophotometers, multimixer, temperature-jump and flash photolysis equipment. 

In the reverse direction, a proton may be effective by aiding ring-opening directly or via a reactive protonated species. It may intervene with the ring-opened species. A splendid example of these effects is shown in the acid hydrolysis of ferrioxamine B (9). Four stages can be separated and the kinetics and equilibria have been characterized by stopped-flow and rapid-scan spectral methods. 

The aquated iron(III) ion is an oxidant. Reaction with reducing ligands probably proceeds through complexing. Rapid scan spectrophotometry of the Fe(III)-cysteine system shows a transient blue Fe(lII)-cysteine complex and formation of Fe(II) and cystine. The reduction of Fe(lII) by hydroquinone, in concentrated solution has been probed by stopped-flow linked to x-ray absorption spectrometry. The changing charge on the iron is thereby assessed. In the reaction of Fe(III) with a number of reducing transition metal ions M in acid, the rate law... 

Time-shcing is a variation of the stop-flow mode where the flow is stopped in a time-dependent manner (e.g. every 15 s) for the whole chromatographic run, in a sense the ultimate approach in slow-flow. When the flow is stopped, sufficient scans can be made to give the desired level of detection before moving on to the next time-slice. The whole process can be automated through software control. There is no requirement for an UV detector, and the approach has been used to identify non-UV-active components in drug substances [48]. The peak purity of... 

Optical Spectroscopy General principles and overview, 246, 13 absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes, 246, 19 circular dichroism, 246, 34 bioinorganic spectroscopy, 246, 71 magnetic circular dichroism, 246, 110 low-temperature spectroscopy, 246, 131 rapid-scanning ultraviolet/visible spectroscopy applied in stopped-flow studies, 246, 168 transient absorption spectroscopy in the study of processes and dynamics in biology, 246, 201 hole burning spectroscopy and physics of proteins, 246, 226 ultraviolet/visible spectroelectrochemistry of redox proteins, 246, 701 diode array detection in liquid chromatography, 246, 749. 

Later, Smith and coworkers succeeded in measuring rate constants of the reaction of MeLi with a carbonyl compound at various reagent concentrations with a stopped-flow/rapid scan spectroscopic method, and demonstrated that the reaction also exhibited a fractional kinetic order . Thus, the reaction of 2,4-dimethyl-4 -methylmercaptobenzophenone with MeLi in diethyl ether at 25 °C showed one-fourth order in MeLi in the concentration range of MeLi between 3.9 mM and 480 mM (Figure 1). The rate constant was 200 7 M s . Under these conditions, the monomer was considered the reactive species that exists in equilibrium with the tetramer. Addition of LiBr or Lil depressed the reaction rate but did not change the kinetic order. The same... 

The analysis of pharmaceutical agents in preparations has been one of the most important applications of modem RPC. A simple gradient maker to be used on the low pressure side of the pump for use in gradient elution of pharmaceuticals has been described (560). For the detection and identification of pharmaceuticals, micro internal reflection infrared spectroscopy (561) and ultraviolet scanning spectroscopy with stopped flow (562) were also employed. 

The homogeneous catalysis method is suitable to measure rate constants over a very wide range, up to the diffusion limit. The lower limit is determined by interferences, such as convection, which occur at very slow scan rates. It is our experience that, unless special precautions are taken, scan rates below lOOmV/s result in significant deviations from a purely diffusion-controlled voltammetric wave. For small values of rate constants (down to 10 s ), other potentiostatic techniques are best suited, such as chronoamperometry at a rotating disk electrode UV dip probe and stopped-flow UV-vis techniques. ... 

The latter two processes are slow as a result of which the detection of the protonated carbonato complex, and determination of its protonation constant by stopped-flow, rapid scan spectrophotometry were possible in some cases, van Eldik et al. (83) interpreted the initial spectral changes (shift of absorption maxima and the isosbestic points) of ( -cis-[Co(edda)C03l and [Co(nta)COa] at [H+]=0.05 M due to the formation of the protonated carbonato complexes, p-cis-[Co(edda)C03H] and [Co(nta)C03H], Repetitive rapid scan spectral measurements at [H + ] = 2.0 M, however, led them to the identification... 

---