UV-Absorption

The ultraviolet (UV) absorption HPLC detector is basically a UV spectrophotometer that measures a flowing solution rather than a static solution. It has a light source, a wavelength selector, and a phototube like an ordinary spectrophotometer. The cuvette is a flow cell, through which the column effluent flows. As the mobile phase elutes, the chromatogram traces a line at zero absorbance, but when a mixture 

FIGURE 13.8 The HPLC variable-wavelength UV detector—a UV spectrophotometer with a flow cell. A peak appears when a mixture component that absorbs the set wavelength elutes from the column. 

While a UV absorption detector is fairly sensitive, it is not universally applicable. The mixture components being measured must absorb light in the UV region in order for a peak to appear on the recorder. Also, the mobile phase must not absorb an appreciable amount at the selected wavelength. 

Nanostructured Ti02 particles (particle size 5-50 nm) are used as sunscreens in the cosmetic industry. Nanosized Ti02 is an effective absorber of UV-B (280-320 nm) and UV-A radiation (320-400 nm). Because ofits small particle size, itappears transparent [2.70]. Intensive research work is in progress worldwide aimed at utilizing the photoactivity of Ti02. Titanium dioxide catalyzes the decomposition of organic compounds in wastewater [2.71]. 

Aromatic amino acids such as phenylalanine, tyrosine and tryptophan absorb in the UV-range of the spectrum with absorption maxima at 200-230 nm and 250-290 nm (Fig. 1.4). Dissociation of the phenolic HO-group of tyrosine shifts the absorption curve by about 20 nm towards longer wavelengths (Fig. 1.5). 

The free ester is released from its salt by the action of alkali. A mixture of free esters can then be separated by distillation without decomposition. Fractional distillation of esters is the basis of a method introduced by Emil Fischer for the separation of amino acids  

Free amino acid esters have a tendency to form cyclic dipeptides or open-chain polypeptides  

Absorption readings at 280 nm are used for the determination of proteins and peptides. Histidine, cysteine and methionine absorb between 200 and 210 nm. 

For this method care must be taken to ensure that the water sample is not cloudy since this can give erroneous results. Any cloudiness which may be present must therefore be eliminated by filtration, e.g. using a membrane filter with an average pore size of 0.45 pm, before determination is carried out. When comparative measurements are carried out, centrifuging can also be carried out before determination. 

It should be borne in mind that nitrate ions also show absorption in the UV range.  

The further below 260 nm analysis is carried out, the stronger this influence will be. For this reason it is recommended that determination be carried out at the mercury line of 254 nm. The recording of an absorption spectrum can also be used to obtain further information. This method can be supplemented by determining absorption in the visible wavelength range at the mercury line of 436 nm. 

Equipment for filtration via membrane filter and under vacuum. 

Spectrophotometer or filter photometer with which the 254 nm mercury line and also, if necessary, the 436 nm mercury line can be utilized for measurement. 

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This characteristic is used to analyze aromatics in gas oil cuts an example of a UV absorption spectrum is shown in Figure 3.5. 

The specific nature of UV absorption for certain structures when combined with the high sensitivity of the method enables trace quantities (— 1 ppm) of molecules in a matrix transparent to UV beams to be analyzed. Benzene in cyclohexane is an example. 

This same principle, as indicated earlier, is used in atomic absorption spectroscopy and UV absorption. 

Burdett, R.A., L.W. Taylor and L.C. Jones Jr (1955), Determination of aromatic hydrocarbons in lubricating oil fractions by far UV absorption spectroscopy , p. 30. In Molecular Spectroscopy Report Conf. Institute of Petroleum, London. 

We now proceed to some examples of this Fourier transfonn view of optical spectroscopy. Consider, for example, the UV absorption spectnun of CO2, shown in figure Al.6.11. The spectnuu is seen to have a long progression of vibrational features, each with fairly unifonu shape and width. Wliat is the physical interpretation of tliis vibrational progression and what is the origin of the width of the features The goal is to come up with a dynamical model that leads to a wavepacket autocorrelation fiinction whose Fourier transfonn... 

Figure Al.6.11. Idealized UV absorption spectrum of CO2. Note the regular progression of intemiediate resolution vibrational progression. In the frequency regime this structure is interpreted as a Franck-Condon...

Glanzer K, Quack M and Tree J 1977 High temperature UV absorption and recombination of methyi radicais in shock waves 16th Int. Symp. on Combustion (Pittsburgh The Combustion institute) pp 949-60... 

Experimental access to the probabilities P(E ,E) for energy transfer in large molecules usually involves teclmiques providing just the first moment of this distribution, i.e. the average energy (AE) transferred in a collision. Such methods include UV absorption, infrared fluorescence and related spectroscopic teclmiques [11. 28. 71. 72, 73 and 74]. More advanced teclmiques, such as kinetically controlled selective ionization (KCSI [74]) have also provided infonnation on higher moments of P(E ,E), such as ((AE) ). 

With M = He, experimeuts were carried out between 255 K aud 273 K with a few millibar NO2 at total pressures between 300 mbar aud 200 bar. Temperature jumps on the order of 1 K were effected by pulsed irradiation (< 1 pS) with a CO2 laser at 9.2- 9.6pm aud with SiF or perfluorocyclobutaue as primary IR absorbers (< 1 mbar). Under these conditions, the dissociation of N2O4 occurs within the irradiated volume on a time scale of a few hundred microseconds. NO2 aud N2O4 were monitored simultaneously by recording the time-dependent UV absorption signal at 420 run aud 253 run, respectively. The recombination rate constant can be obtained from the effective first-order relaxation time, A derivation analogous to (equation (B2.5.9). equation (B2.5.10). equation (B2.5.11) and equation (B2.5.12)) yield... 

Subsequently, the recombination of methyl radicals was studied by the high-temperature UV absorption of the methyl radicals near 216 mn [5455 and 56]. 

Figure B2.5.7. Oscilloscope trace of the UV absorption of methyl radical at 216 mn produced by decomposition of azomethane after a shock wave (after [M]) at (a) 1280 K and (b) 1575 K.

Bensasson R V, Bienvenue E, Dellinger M, Leach S and Seta P 1994 Cgg in model biological systems. A visible-UV absorption study of solvent-dependent parameters and solute aggregation J. Phys. Chem. 98 3492-5000... 

Endo-exo product mixtures were isolated using the following procedure. A solution of cyclopentadiene (concentration 2-10" M in water and 0.4 M in oiganic solvents) and the dienophile (concentration 1-5 mM) in the appropriate solvent, eventually containing a 0.01 M concentration of catalyst, was stirred at 25 C until the UV-absorption of the dienophile had disappeared. The reaction mixture (diluted with water in the case of the organic solvents) was extracted with ether. The ether layer was washed with water and dried over sodium sulfate. After the evaporation of the ether the... 

On the basis of the studies described in the preceding chapters, we anticipated that chelation is a requirement for efficient Lewis-acid catalysis. This notion was confirmed by an investigation of the coordination behaviour of dienophiles 4.11 and 4.12 (Scheme 4.4). In contrast to 4.10, these compounds failed to reveal a significant shift in the UV absorption band maxima in the presence of concentrations up to one molar of copper(ir)nitrate in water. Also the rate of the reaction of these dienophiles with cyclopentadiene was not significantly increased upon addition of copper(II)nitrate or y tterbium(III)triflate. 

The desired pyridylamine was obtained in 69 % overall yield by monomethylation of 2-(aminomethyl)pyridine following a literature procedure (Scheme 4.14). First amine 4.48 was converted into formamide 4.49, through reaction with the in situ prepared mixed anhydride of acetic acid and formic acid. Reduction of 4.49 with borane dimethyl sulfide complex produced diamine 4.50. This compound could be used successfully in the Mannich reaction with 4.39, affording crude 4.51 in 92 % yield (Scheme 4.15). Analogous to 4.44, 4.51 also coordinates to copper(II) in water, as indicated by a shift of the UV-absorption maximum from 296 nm to 308 nm. 

Many pharmaceutical compounds contain chromophores that make them suitable for analysis by UV/Vis absorption. Products that have been analyzed in this fashion include antibiotics, hormones, vitamins, and analgesics. One example of the use of UV absorption is in determining the purity of aspirin tablets, for which the active ingredient is acetylsalicylic acid. Salicylic acid, which is produced by the hydrolysis of acetylsalicylic acid, is an undesirable impurity in aspirin tablets, and should not be present at more than 0.01% w/w. Samples can be screened for unacceptable levels of salicylic acid by monitoring the absorbance at a wavelength of... 

The fluorescent emission for quinine at 450 nm can be induced using an excitation frequency of either 250 nm or 350 nm. The fluorescent quantum efficiency is known to be the same for either excitation wavelength, and the UV absorption spectrum shows that 250 is greater than 350- Nevertheless, fluorescent emission intensity is greater when using 350 nm as the excitation wavelength. Speculate on why this is the case. 

Description of Method. The water-soluble vitamins Bi (thiamine hydrochloride), B2 (riboflavin), B3 (niacinamide), and Be (pyridoxine hydrochloride) may be determined by CZE using a pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer or by MEKC using the same buffer with the addition of sodium dodecyl-sulfate. Detection Is by UV absorption at 200 nm. An Internal standard of o-ethoxybenzamide Is used to standardize the method. 

Acryhc polymers are fairly iasensitive to normal uv degradation siace the primary uv absorption of acryhcs occurs below the solar spectmm (59). The iacorporation of absorbers, such as i9-hydroxyben2ophenone [117-99-7] further improves the uv stabihty (59). Under normal use conditions acryhc polymers have superior resistance to degradation and show remarkable retention of their original properties. 

Bromine Monofluoride. Bromine monofluoride is red to red-brown (4) and is unstable, disproportionating rapidly into bromine and higher fluorides. Therefore, the measurement of its physical properties is difficult and the values reported in Table 1 are only approximate. The uv-absorption spectmm is available (25). 

Bromine Trifluoride. Bromine trifluoride is a colorless Hquid. The commercial grade is usually amber to red because of slight bromine contamination. The molecule has a distorted T stmeture (26). Infrared spectral data (26—30), the uv-absorption spectmm (31), and vapor pressure data (32) may be found in the Hterature. 

Bromine Pentafluoride. Bromine pentafluoride is a colorless Hquid having the molecular stmeture of a tetragonal pyramid (5). The index of refraction is 1.3529 (33). Infrared spectra (13,34), the uv-absorption spectmm (35), and vapor pressure data (11) are all available. 

Chlorine Monofluoride. Chlorine monofluoride is a colorless gas that condenses to a Hquid with a slight yeUow cast and free2es to a white soHd. The infrared spectmm of gaseous chlorine monofluoride and the Raman spectmm of the Hquid have been studied (36). The uv-absorption spectmm (37) and vapor pressure data are also available (11). 

Chlorine Pentafluoride. Chlorine pentafluoride is a colorless gas at room temperature. The ir and Raman spectra of the Hquid and gas phase have been studied (34,39). The uv absorption spectmm (45) and vapor pressure data may be found in the Hterature (18). 

Optical Properties and Radiation Effects. Within the range of wavelengths measured (uv, visible, and near-ir radiation), Teflon PFA fluorocarbon film transmits slightly less energy than FEP film (29) (Table 6). In thin sections, the resin is colorless and transparent in thicker sections, it becomes translucent. It is highly transparent to it radiation uv absorption is low in thin sections. Weather-O-Meter tests indicate unlimited outdoor life. 

In order to make an efficient Y202 Eu ", it is necessary to start with weU-purifted yttrium and europium oxides or a weU-purifted coprecipitated oxide. Very small amounts of impurity ions, particularly other rare-earth ions, decrease the efficiency of this phosphor. Ce " is one of the most troublesome ions because it competes for the uv absorption and should be present at no more than about one part per million. Once purified, if not already coprecipitated, the oxides are dissolved in hydrochloric or nitric acid and then precipitated with oxaflc acid. This precipitate is then calcined, and fired at around 800°C to decompose the oxalate and form the oxide. EinaHy the oxide is fired usually in air at temperatures of 1500—1550°C in order to produce a good crystal stmcture and an efficient phosphor. This phosphor does not need to be further processed but may be milled for particle size control and/or screened to remove agglomerates which later show up as dark specks in the coating. 

Alkylation of the amino group to a mono- or dialkyl form strengthens the uv absorption and also increases the wavelength of the fluorescent light two alkyl groups are more effective than one and ethyl groups are more powerflil than methyl groups. 

Colorimetric and Fluorimetric Analysis. The functional groups of amino acids exhibit Htde absorption of uv light from 210 to 340 nm where uv absorption spectrometry is most conveniently conducted. Thus color or fluorescence formation reactions are employed for amino acid detection (128). 

Aminophenols have been detected in waste water by investigating uv absorptions at 220, 254, and 275 nm (87). These compounds can also be detected spectrophotometricaHy after derivatization at concentrations of 1 part per 100 million by reaction in acid solution with /V-(1-napbtby1)etby1enediamine [551-09-7] (88) or 4-(dimethylainino)ben2aldehyde [100-10-7] (89), and the Schiff base formed can be stabilized in chloroform by chelation to increase detection limits (90). 

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