Spectrophotometry, Measurements in Solution

Abstract In this chapter the reader can find an overview on the UV-vis absorption spectroscopy technique. A very brief introduction on the nature and formation of the electronically excited states is functional to the following discussion on the characteristics, and interpretation of UV-vis absorption spectra. The typical instrumentation is also schematically described, and a particular emphasis is devoted to the possible qualitative and quantitative information that can be obtained with this kind of measurements. Practical indications to obtain accurate and reliable experimental results were introduced with the aim to help the readers that will approach for the first time this fundamental experimental technique. The chapter ends with the discussion of a few examples, taken from the literature, with the aim to make clear the investigative great potentialities of this spectroscopy in different fields. 

Molecules can be promoted to their excited states if exposed to suitable perturbations , in particular when appropriate energy to be absorbed is provided, as discussed in Chap. 1. The light-matter interaction is the easiest way to obtain an excited state. The energy gained by a molecule when it absorbs a photon (or better, the energy transported by photons) causes an electron to be promoted to a higher 

Department of Chemistry G. Ciamician , University of Bologna, Via Selmi 2, 

Ceroni (ed.). The Exploration of Supramolecular Systems and Nanostructures by Photochemical Techniques, Lecture Notes in Chemistry 78, 

Electronic transitions in molecules need energies correlated to wavelengths that are in the range of the ultraviolet (UV) and visible spectral regions. The absorption of a UV or visible photon by a molecule induces changes in the distribution of the electrons surrounding the nuclei, and in the forces between the atomic nuclei of a molecule. As a result, molecules in electronically excited states often have very different chemical and physical properties than their electronic ground states. 

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First, let us consider batch mixing processes, as exemplified by ordinaiy laboratory practice in solution kinetics. A portion of one solution (say, of the substrate) is added by pipet to a second solution (containing the reagent) in a flask, the flask is shaken to achieve homogeneity, and then samples are withdrawn at known times for analysis, or the solution is subjected to continuous observation as a function of time, for example, by spectrophotometry. For reactions on a time scale (measured by the half-life) of hours or even several minutes, the time consumed in these operations is a negligible portion of the reaction time, but as the half-life of the reaction decreases, it becomes necessary to consider these preliminary steps. Let us distinguish three stages ... 

In the literature (2), the possible formation of plutonyl bicarbonate complex is discussed. In order to verify whether we are dealing with bicarbonate or carbonate complexes, the Pu(IV) solutions prepared in NaHC03 and Na2C03 solutions are examined by spectrophotometry. The absorption spectra measured up to 900 nm show no visible difference for both solutions. For this reason it is believed that the Pu(IV) ion forms carbonate complexes irrespective of carbonate or bicarbonate ions present in solution. 

Applications Applications of UV/VIS spectrophotometry can be found in the areas of extraction monitoring and control, migration and blooming, polymer impregnation, in-polymer analysis, polymer melts, polymer-bound additives, purity determinations, colour body analysis and microscopy. Most samples measured with UV/VIS spectroscopy are in solution. However, in comparison to IR spectroscopy additive analysis in the UV/VIS range plays only a minor role as only a limited class of compounds exhibits specific absorption bands in the UV range with an intensity proportional to the additive concentration. Characteristic UV absorption bands of various common polymer additives are given in Scheirs [24],... 

In order to determine the stability constants for a series of complexes in solution, we must determine the concentrations of several species. Moreover, we must then solve a rather complex set of equations to evaluate the stability constants. There are several experimental techniques that are frequently employed for determining the concentrations of the complexes. For example, spectrophotometry, polarography, solubility measurements, or potentiometry may be used, but the choice of experimental method is based on the nature of the complexes being studied. Basically, however, we proceed as follows. A parameter is defined as the average number of bound ligands per metal ion, N, which is expressed as... 

The Department of the Environment UK [155] has described a number of alternative methods for the determination of total oxidised nitrogen (nitrate and nitrite) in aqueous solution, while specific methods for nitrate and nitrite are also included. Among the methods for total oxidised nitrogen, one is based on the use of Devarda s alloy for reduction of nitrate to ammonia, and another uses copperised cadmium wire for reducing nitrate to nitrite, which is determined spectrophotometrically. Nitrate may also be determined spectrophotometrically after complex formation with sulfosalicylic acid or following reduction to ammonia, the ammonia is eliminated by distillation and determined titrimetrically. Other methods include direct nitrate determination by ultraviolet spectrophotometry, measurements being made at 210 nm, and the use of a nitrate-selective electrode. Details of the scope, limits of detection, and preferred applications of the methods are given in each case. 

The products of oxidation (alcohol, ketone, acid) lower the concentration of active complexes and, in addition, form complexes with a mixed ligand sphere with lower catalytic activity (kdi >kd2). The values of equilibrium constants Ain (Lmol-1) measured spectrophotometri-cally in a decane solution for cupric stearate + product are given below [70],... 

Of course, not all dissolved ions produce colored solutions, and therefore not all ions in solution can be quantified by colorimetry. Noncolored solutions can sometimes, however, be converted to colored solutions by introducing chromophore species which complex with (i.e., attach themselves to) the target ion to produce a colored solution, which may then be measured by UV/visible colorimetry. An important archaeological example of this is the determination of phosphorus in solution (which is colorless) by com-plexation with a molybdenum compound, which gives a blue solution (see below). The term colorimetry applies strictly only to analytical techniques which use the visible region of the spectrum, whereas spectrophotometry may be applied over a wider range of the electromagnetic spectrum. 

The main advantage of fluorescence techniques is their sensitivity and measurements of nanogram (10—9 g) quantities are often possible. The reason for the increased sensitivity of fluorimetry over that of molecular absorption spectrophotometry lies in the fact that fluorescence measurements use a non-fluorescent blank solution, which gives a zero or minimal signal from the detector. Absorbance measurements, on the other hand, demand a blank solution which transmits most of the incident radiation and results in a large response from the detector. The sensitivity of fluorimetric measurements can be increased by using a detector that will accurately measure very small amounts of radiation. 

Boron may be analyzed by various instrumental methods, such as atomic absorption (AA) and atomic emission spectrophotometry (ICP/AES). Individual isotopes at an exceedingly trace concentration in solution phase may be measured by ICP/MS. The later method should be preferred over the AA techniques. 

Cadmium in acidified aqueous solution may be analyzed at trace levels by various instrumental techniques such as flame and furnace atomic absorption, and ICP emission spectrophotometry. Cadmium in solid matrices is extracted into aqueous phase by digestion with nitric acid prior to analysis. A much lower detection level may be obtained by ICP-mass spectrometry. Other instrumental techniques to analyze this metal include neutron activation analysis and anodic stripping voltammetry. Cadmium also may be measured in aqueous matrices by colorimetry. Cadmium ions react with dithizone to form a pink-red color that can be extracted with chloroform. The absorbance of the solution is measured by a spectrophotometer and the concentration is determined from a standard calibration curve (APHA, AWWA and WEF. 1999. Standard Methods for the Examination of Water and Wastewater, 20th ed. Washington, DC American Public Health Association). The metal in the solid phase may be determined nondestructively by x-ray fluorescence or diffraction techniques. 

Elemental compostion Ce 25.56%, H 1.47%, N 20.44%, 0 52.53%. The aqueous solution of the compound may be analyzed for Ce by AA or ICP spectrophotometry. Also, the solution may be measured for NH4 ion by ammonium ion-selective electrode and the NO3 ion by nitrate ion-specific electrode, ion chromatography or cadmium-reduction colorimetry. For all these measurements, the solution may require sufficient dilutions. For quantitation, its solution may be standardized by titration with a reducing agent such as sodium oxalate in the presence of iron and ferroin indicator. 

In 1C, the election-detection mode is the one based on conductivity measurements of solutions in which the ionic load of the eluent is low, either due to the use of eluents of low specific conductivity, or due to the chemical suppression of the eluent conductivity achieved by proper devices (see further). Nevertheless, there are applications in which this kind of detection is not applicable, e.g., for species with low specific conductivity or for species (metals) that can precipitate during the classical detection with suppression. Among the techniques that can be used as an alternative to conductometric detection, spectrophotometry, amperometry, and spectroscopy (atomic absorption, AA, atomic emission, AE) or spectrometry (inductively coupled plasma-mass spectrometry, ICP-MS, and MS) are those most widely used. Hence, the wide number of techniques available, together with the improvement of stationary phase technology, makes it possible to widen the spectrum of substances analyzable by 1C and to achieve extremely low detection limits. 

If the concentration of a solution prepared for fluorescence measurement is too high, some of the light emitted by the sample as fluorescence will be reabsorbed by other unexcited molecules in solution. For this reason, fluorescence measurements are best made on solutions with an absorbance of less than 0.02 at their maximum, i.e. solutions of a sample 10-100 weaker than those which would be used for measurement by UV spectrophotometry. 

This type of extraction is employed in the BP assay of Cyclizine Lactate Injection the injection is diluted with dilute H2SO4 and then neutral and acidic excipients are extracted with ether. The solution is basified and the cyclizine is extracted into ether leaving the lactate ion, which would not have extracted during the initial ether extraction step, behind in the aqueous layer. For convenience in measurement by UV spectrophotometry and in order to carry out volumetric dilution of the extract, cyclizine is then back extracted into dilute H2SO4 and subjected to further dilution. 

The quantitation of substances separated by TLC may be carried out in several ways. The most common method is to remove the spot from the plate, elute the compound from the adsorbent and measure the concentration of the compound in solution by spectrophotometry, fluorimetry, etc. The elution process has been significantly improved and facilitated with the Eluchrom instrument developed by Sandoz and marketed by Camag (see Fig.3.6). This instrument permits direct elution from the plates via small PTFE cups in a continuous flow-through mode without the necessity of removal of the adsorbent and with the minimum requirement of solvent (usually less than 1 ml). The measuring instruments used are those available for classical solution analysis. A discussion of these instruments is beyond the scope of this book. 

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