UV-vis spectrophotometric methods

Using the UV/VIS spectrophotometric method, the equilibria of Scheme 12 have been investigated93 as a model of the bromonium tri- and pentabromides, the intermediates of the reactions of olefin bromination. 

Many of the UV-VIS spectrophotometric methods (shown in Tables 12.3 and 12.6) have been automated by using flow analyzers. Thus, nitrite and nitrate,50,82 ammonium,50,83 orthophosphate,50,84,85 silicates,50,86 chloride,50,87 cyanide,50,88 and sulfate50,89 are measured by CFA and FIA. Oxygen is measured by iodometric titration51,90 and electrochemical methods91 (Table 12.7). Other dissolved gasses (Table 12.2) are measured by ISE-based gas sensors. 

The UV-vis spectrophotometric methods require validation of the method for the analysis of pharmaceutical compounds. Once the method is developed, data regarding precision, accuracy, linear response behavior, limit of quantitation, limit of detection, selectivity, and ruggedness are generated. These terms are very well-defined in many compendial monographs and in ICH, USP, and FDA guidelines. These are not addressed further. However, it should be emphasized that without appropriate validation data and reasonable understanding of how the method results will be affected by minor day-to-day variation of experimental parameters, routine generation of acceptable data may be difficult. 

Garland, J. A. Curtis, H. J. (1981). Emission of iodine from the sea surface in the presence of ozone. J. Geophys. Res., Vol.86 (C4), pp 3183-3186, ISSN 0148-0227 George, M., Nagaraja, K S., Natesan Balasubramanian, N. (2011). Spectrophotometric Determination of Iodine Species in Table Salt,Pharmaceutical Preparations and Sea Water., Eurasian JAnal Chem., Vol.6, No.2, ppl29-139, ISSN 1306-3057 Gilfedder, B.S., Althoff, F., Petri, M. Biester, H. (2007). A Thermo Extraction-UV/Vis Spectrophotometric Method For Total Iodine Quantification In Soils And Sediments. Analytical And Bioanalytical Chemistry, Vol.389, No(7-8), pp. 2323-2329, ISSN 1618-2642... 

Although considered a basic technique, ultraviolet-visible (UV-vis) is perhaps the most widely used spectrophotometric technique for the quantitative analysis of pure chemical substances such as APIs in pharmaceutical analysis. For pharmaceutical dosage forms that do not present significant matrix interference, quantitative UV-vis measurements may also be made directly.114,115 It is estimated that UV-vis-based methods account for 10% of pharmacopoeia assays of drug substances and formulated products.116... 

Procedure 1 We select several HA-type acids, for which pKa values of <7.5 are expected, and determine their pKa and K HAJ) accurately by method(s) other than potentiometry. If the selected acid is a nitro-substituted phenol that has no tendency to homoconjugate (p. 71), we dissolve various amounts of it in the solvent and measure the UV/vis spectrophotometric absorption for the phenolate anion formed by dissociation. For the conductimetric determination of pKa and K HA])), see Section 7.3.2. 

Another type of method is based on the spectrophotometric properties of Cr (VI), sometimes used as standard solution [38]. Moreover, the spectrophotometric determination of Cr (VI) is well known in the field of water examination, as it can be used for COD alternative measurement [39] (see Chapter 4). Two other UV-visible spectrophotometric methods have been proposed. The first one [40], designed for natural water, uses the peak height measurement at 372 nm, for a basified sample (pH > 9). The peak height is calculated from the absorbance values at three wavelengths (310, 372 and 480 nm), taking into account a very simple third-degree-polynomial interference signal. 

Fluorimetric methods have not been used as widely as ultraviolet-visible (UV-Vis) spectrophotometric ones, although they are much more sensitive detection requires only 1% of the amount of pigment necessary in the latter method [70,71]. With this technique, chlorophylls or their derivatives have been detected quantitatively in picomole amounts [72-77]. Very low amounts of chlorophylls a and b can also be determined from their mixtures, as the fluorescence of one is independent of the other [78]. 

The oldest application of solvent extraction in spectrophotometric determinations uses extraction from the original aqueous solution and subsequent back-extraction into a second aqueous phase. Here the extractant provides only separation or concentration, as in the case of Np(IV) and Pu(IV) determination [16]. However, as only a few element species (e.g., Mn04, Cr04 ) are capable of absorbing light in the UV-VIS range, usually all spectrophotometric methods are based on reactions of analytes with... 

A common technique for measuring the values has been to employ species that produce anions with useful ultraviolet (UV) or visible (vis) absorbances and then determine the concentrations of these species spectropho tome trie ally. Alternatively, NMR measurements could be employed, but generally they require higher concentrations than the spectrophotometric methods. A hidden assumption in Eq. 5 is that the carbanion is fully dissociated in solution to give a free anion. Of course, most simple salts do fully dissociate in aqueous solution, but this is not necessarily true in the less polar solvents that are typical employed with carbanion salts. For example, dissociation is commonly observed for potassium salts of carbanions in DMSO because the solvent has an exceptionally large dielectric constant (s = 46.7) and solvates cations very well, whereas dissociation occurs to a small extent in common solvents such as DME and THE (dielectric constants of 7.2 and 7.6, respectively). In these situations, the counterion, M+, plays a role in the measurements because it is the relative stability of the ion pairs that determines the position of the equilibrium constant (Eq. 6). 

Since all of the chalcogenometalates have strong and characteristic absorption bands in the UV-vis region, the reactions in which they are formed and decomposed can be readily followed by spectrophotometric methods. When hydrogen sulfide is passed into an aqueous solution of an oxometalate the electronic spectrum changes, the bands of all the species M04 S (n = 1-4 M = V, Mo, W, Re) appearing in succession (cf Figure 3 of Chapter 16.1). 

In principle, any property of a reacting system which changes as the reaction proceeds may be monitored in order to accumulate the experimental data which lead to determination of the various kinetics parameters (rate law, rate constants, kinetic isotope effects, etc.). In practice, some methods are much more widely used than others, and UV-vis spectropho-tometric techniques are amongst these. Often, it is sufficient simply to record continuously the absorbance at a fixed wavelength of a reaction mixture in a thermostatted cuvette the required instrumentation is inexpensive and only a basic level of experimental skill is required. In contrast, instrumentation required to study very fast reactions spectrophotometrically is demanding both of resources and experimental skill, and likely to remain the preserve of relatively few dedicated expert users. 

These are now probably the most widely used methods in kinetic and mechanistic studies, and include a wide range of spectral frequencies radio frequencies (NMR, ESR), IR and UV-vis. Appropriate instrumentation which is easily adapted for kinetics is readily available in most research laboratories it is usually easy to use, and the output easily interpreted. Spectrophotometric methods are also widely used for the determination of equilibrium constants [25]. However, before deciding upon a spectrophotometric technique, the following experimental aspects must be considered carefully. 

Table 1. The effect of 1 mM NaF + 20 pM A1C13 on the acetylcholinesterase activity (AChE) in freshly prepared intact RBC and in hemolysate of patients with AD (mean age 72.5 5.1 years), age-matched healthy controls (AM-HS) (72.1 1.6 years), and the group of young healthy subjects (YS) (35.9 8.5 years). Whole venous blood samples were drawn from each subject after overnight fasting., always at 07 30 AM. Red blood cells (RBC) were isolated from the blood of patients with AD, AM-HS, and YS by centrifugation [68], RBC AChE activity was evaluated in intact freshly prepared RBC or hemolyzate following the spectrophotometric method [45] with modifications. Buffer was Tris-HCl, pH 7.5 in the solution of 154 mmol L 1 NaCl, acetylthiocholine iodide was a substrate. Measurement of enzymatic activity was performed in fluorimeter polystyrene cuvettes for 3 min (UV/VIS spectrophotometer Shimadzu, Japan). The effects of 1 mmol L-1 NaF in the presence of 20 pmol L 1 A1C13 were measured. Data are expressed in percentage of the AChE activity in the absence of aluminum and fluoride ions. No differences between the AChE activity were found between the investigated groups...

In the first spectrophotometric method [22] the measurements were carried out using an Agilent 8453 model UV-Vis spectrophotometer with a DAD. The spectra were recorded from 225 to 475 nm. The quantitative analysis were performed at 376 nm for the zero-order derivative UV spectrophotometric method by measuring the height of the peak from zero and at 302 nm, and 281 nm for the first-order derivative UV... 

Lakshmi et al. [26] developed two new simple, accurate, and economic spectrophotometric methods, in the UV/Vis region, for fhe deferminafion of paracetamol and lornoxicam in bulk and in tablet formulations. 

Although TLC-MS (mass spectrometry) has been shown to be technically feasible and applicable to a variety of problems, thin-layer chromatography is generally coupled with spectrophotometric methods for quantitative analysis of enantiomers. Optical quantitation can be achieved by in situ densitometry by measurement of UV-vis absorption, fluorescence or fluorescence quenching, or after exctraction of solutes from the scraped layer. The evaluation of detection limits for separated enantiomers is essential because precise determinations of trace levels of a d- or L-en-antiomer in an excess of the other become more and more important. Detection limits as low as 0.1% of an enantiomer in the other have been obtained. 

Spectrophotometric analyses are the most common method to characterize proteins. TTie use of ultraviolet-visible (UV-VIS) spectroscopy is t rpically used for the determination of protein concentration by using either a dye-binding assay (e.g., the Bradford or Lowry method) or by determining the absorption of a solution of protein at one or more wavelengths in the near UVregion (260-280 nm). Another spectroscopic method used in the early-phase characterization of biopharmaceuticals is CD. 

Nowadays, spectrophotometry is regarded as an instrumental technique, based on the measurement of the absorption of electromagnetic radiation in the ultraviolet (UV, 200-380 nm), visible (VIS, 380-780 nm), and near infrared region. Inorganic analysis uses UV-VIS spectrophotometry. The UV region is used mostly in the analysis of organic compounds. Irrespective of their usefulness in quantitative analysis, spectrophotometric methods have also been utilized in fundamental studies. They are applied, for example, in the determination of the composition of chemical compounds, dissociation constants of acids and bases, or stability constants of complex compounds. 

Spectrophotometric methods of identification and determination of substances are based on the existence of relationships between the position and intensity of absorption bands of electromagnetic radiation, on the one hand, and molecular structure on the other. Electronic spectra result from changes in the energy states of electrons [o, ti, and free electron pairs (n)] in a molecule as a result of absorption in the UV-VIS region. The changes depend on the probability of electronic transitions between the individual energy states of the molecule. The number of absorption bands, and their positions, intensities and shapes are the spectral parameters utilized in qualitative and quantitative chemical analyses [1-3]. 

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