UV-vis spectrophotometric measurements

Other illustrative examples of carbanionic ion-pair dissociation/aggregation are lithium triphenyhnethide, which exists as a tight ion pair in diethyl ether and as a solvent-separated ion pair in tetrahydrofuran, as shown by UV/Vis spectrophotometric measurements [287], and lithium 10-phenylnonafulvene-10-oxide, which exists as a tight ion pair (2b) in tetrahydrofuran solution and as a solvent-separated ion pair (3b) when hexamethylphosphoric triamide or dimethyl sulfoxide are added ( H and NMR measurements) [288]. 

The current study utilizes two complementaiy techniques to monitor the behaviour of the BZ system in the absence and presence of additives CO2 evolution rate measurements (6), which reflects the rate of the overall reaction, and UV-vis spectrophotometric measurements. 

UV-vis spectrophotometric measurements. The changes in the Ce(IV) absorbance at 350 nm have been recorded with a computer-controlled Beckman... 

We passed then to a particular olefin, adamantylideneadamantane, whose reaction with Br2 had been shown to stop at the stage of bromonium ion formation because of steric hindrance to backside nucleophilic attack. An UV-Vis spectrophotometric study (ref. 10) has shown that the complicated equilibrium reported in Scheme 4 is immediately established on mixing the olefin and Br2 in DCE. Equilibrium (1) could be isolated by working at low Br2 and ten to hundred fold higher olefin concentrations. A Scott plot followed by a NLLSQ refinement of the data gave a Kf = 2.89 x 10 (4.0) M-l. It is worth noting that conductimetric measurements showed the non-ionic nature of the 1 1 adduct, consistent with a CTC intermediate, but not with a bromonium-bromide species. 

While the control resins were deep red in color due to the presence of soluble porphyrin complexes, the methacrylate resins obtained after removal of the polyethylene-supported catalysts varied from light yellow to nearly water-white (APHA < 25). UV-Vis spectrophotometric analysis of the yellow resins indicated an absorption signal for the cobalt porphyrin complex Soret band (wavelength of cobalt(ll) porphyrin species appears at -415 nm free porphyrin ligand is formd at -423 tun). Resin samples that visttally appear as water-white show little or no porphyrin species present in the spectrum. Measured catalyst activity and PDl of the polyethylene-supported porphyrin complexes are in the expected range for soluble porphyrin CCT catalysts (PDl = M /Mn - 1.2- 2.0)." The screening resrrlts clearly... 

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. 

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. 

Because a chlorophyll molecule contains a closed circuit of ten conjugated double bounds to absorb light, spectrophotometric (UV-Vis) and fluorometric measurements are satisfactory to identify and estimate amounts of chlorophyll a and chlorophyll b, usually the only ones present in fresh plant extracts. The basis of numerous spectrophotometric determinations reported in literature is that chlorophylls strongly absorb at 500 to 700 nm in the visible region and show a large typical band around 400 nm. 

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... 

Figure 14.3. Sample holders for various spectrophotometric measurements. (A) UV-Vis cells (B) infrared ATR plate and stand-plate behind which B is attached when in use (C) simple KBr pellet maker using the two bolts and the center dye (D) a sample tube for NMR spectroscopy. 

Trypsin inhibition The ability of the various trypsin inhibitors Allium extracts) to prevent tiypsin hydrolysis of BAPNA is measured spectrophotometrically (405 nm, = 9.96 cm pmoT ) [11] at 25°C, with a Jasco UV-Vis spectrometer by time course measurement of AAbs. One trypsin unit hydrolyzes 1.0 pmol of A-ft-benzoyl-DL-arginine /i-nitroanilide (BAPNA) per minnte at pH 7.8 and 25°C and one Trypsin Inhibitor Unit (TIU) will decrease the activity of 2 trypsin units by 50%. 

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. 

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... 

Carbonized coir pith was prepared from dried coir pith powder (2S0-500pm) using a muffle frunace at 700°C for 1 h. Adsorption experiments were carried out by agitating 300 mg of carbon with 50 nil of dye solution of desired concentration and pH at 200 rpm, 35°C in a thermostated rotary shaker (ORBITEK, Chennai, India). Dye concentration was estimated spectrophotometrically by monitoring the absorbance at 660 nm using UV-Vis spectrophotometer (Hitachi, model U-3210, Tokyo). pH was measured using pH... 

Noise in UV-vis spectrophotometry refers to uncertainties caused in the measurement of the absorbance signal. Essentially, there are two sources of noise. One isdependent on the source intensity (Schott noise) and the other independent of it. The effect of noise in spectrophotometric measurement can be significantly reduced if the concentration of the analyte is adjusted such that measured absorbance is between 0.3 and 1.2 absorbance units. A significant source of noise in double-beam instruments arises when sample and reference cells are not positioned properly. Minor imperfections in the cells cause reflections and scattering... 

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. 

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. 

The quality of the measuring instrument has a strong influence on the reliability of the results obtained. The standard spectrophotometric apparatus used in modem analytical laboratories is very different from the equipment used in the initial period of application of this technique. The progress in the development of spectrophotometric apparatus up to the middle of the 1980s has been described [9-11]. The trends in the development of UV-VIS spectrophotometry, with special consideration to improved detection, and modern methods of data treatment, have been discussed [12]. The following discussion will be devoted to apparatus installed in most analytical laboratories. 

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 amount of catalase was determined spectrophotometrically by the decrease in the concentration of the catalase in the solution after the adsorption. The spectrophotometric measurements were carried out on a Specord UV VIS (Carl Zeiss, Jena, Germany). The amount of the catalase in the solution was determined on the basis of a calibration graph (Fig.l-b) for the maximum at Koil = 280nm. The value of the extinction coefficient was 8280 = 0.9x10 l.morVcm. ... 

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. 

The actual acquisition of the data for determining complexation capabilities similarly employs a variety of experimental techniques. In addition to the powerful spectrophotometric methods now available, most often NMR and/or UV-Vis spectrometry (picrate salts are frequently used in complexaton measurements because of the advantage provided by the color of the picrate anion), various other techniques are also being used. Mass spectrometry, for example,... 

Figure 9-11. (A) Structure of 3-nitrotyrosine. Bond distances (A) are taken from the crystallographic structure of free NT [103]. (B-C) Spectrophotometric titration of Y3NT by UV/Vis absorption spectroscopy. Absorption spectra (B) and plot of the absorbance values at 430nm of Y3NT (2mL, 48(xM) as a function of pH (C). Measurements were carried out in 2-mM citrate-borate-phosphate buffer, at the indicated pH. Fitting data points yields a pKa value of 6.74 + 0.02 for NT.

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