Absorbance spectroscopy

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

Duffy NW, Peter LM, Wang RL, Lane DW, Rogers KD (2000) Electrodeposition and characterisation of CdTe films for solar ceU applications. Electrochim Acta 45 3355-3365 Duffy NW, Peter LM, Wang RL (2002) Characterisation of CdS/CdTe heterojunctions by photocurrent spectroscopy and electrolyte electroreflectance/absorbance spectroscopy (EEA/EER). J Electroanal Chem 532 207-214 (see also references therein). 

Spectrofluorometry presents sensitivity and selectivity greater than the absorbance spectroscopy, being more suitable for chlorophyll estimates in the nmol range and for residual amounts of derivatives in food products. Absorbance spectroscopy is satisfactory for concentrations > 1 xMP Spectrofluorometry is also more accurate for a wide range of chlorophyll a-to-chlorophyll b ratios, but it is less accurate when applied to complex sample matrices because of unpredictable quenching effects. 

As far as we know, this is the first molecular probe that includes two different types of reporter units activated upon on a specific stimulus. The other option to achieve dual detection would be to use two separate probes. However, in this case there could be a problem of competitive catalysis (circumstances in which the Km of the two substrate is not identical). In our probe, 6-aminoquinoline and 4-nitrophenol, detected by fluorescence and absorbance spectroscopy, respectively, were used as reporter units. Due to the synthetic flexibility of our approach, other reporter molecules with different types of functional groups, like amine or hydroxyl, can be linked to our molecular probe. The two assays must be orthogonal to each other, in order to prevent disturbances in the detection measurement. Another advantage of the probe is the aqueous solubility... 

The basic measurements of absorbance spectroscopy are actually 70 and I that determine the transmittance. The uncertainty in the measurement... 

Based on minimizing the photometric error, what range of absorbances is optimal for absorbance spectroscopy What is the relative dynamic range of absorbance measurements ... 

Tao P. and Sosnick T. R. Intermediates and kinetic traps in the folding of a large ribo-zyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity. Nat Struct. Biol. (1997) 4(11) 931-938. 

Many conventional OFCD exploit pH indicators as molecular probes.03-19 The pH indicators allow the detection of a chemical species by measuring the generation or uptake of hydrogen ions in chemical reactions. Some indicators such as p-nitrophenol,3) Congo Red,04 bromophenol blue,05 and others06-19 have been used successfully. Unfortunately, not all chemicals can be detected in this fashion and alternate methods of detection are necessary. Additionally, the use of absorbance spectroscopy by itself may not allow low detection limits since many molecules can interfere with absorbance measurements. 

Transport properties were determined by mounting the membrane between the two halves of a U-tube permeation cell [108]. The feed halfcell contained 5 ml of an aqueous solution (5 mM) of the molecule to be transported (the permeant molecule) the permeate half-cell initially contained 5 ml of pure water. The transport of the permeant molecule into the permeate half-cell was monitored by periodically assaying (via UV absorbance spectroscopy) the permeate solution. These membranes showed reproducible fluxes for periods of at least 10 days. 

Fig. 14. Effects of temperature on the absorbance of hemopexin and the N-domain of hemopexin. The unfolding of hemopexin and N-domain in 25 mM sodium phosphate, pH 7.4, was examined using absorbance spectroscopy (N. Shipulina et al., unpublished). The second derivative UV absorbance spectra of the protein moieties were used to follow protein unfolding and the Soret and visible region spectra to monitor the integrity of the heme complexes, as done with cytochrome 6502 (166). The ferri-heme complex is more stable than the apo-protein moiety, but the is slightly lower than that assessed by DSC, indicating that changes in conformation occur before thermodynamic unfolding. Reduction causes a large decrease in heme-complex stabihty, which is proposed to be a major factor in heme release from hemopexin by its cell membrane receptor, and addition of 150 mM sodium chloride enhanced the stabihty of ah forms of hemopexin.

Riley DJ, Tull EJ (2001) Potential modulated absorbance spectroscopy an investigation of the potential distribution at a CdS nanoparticle modified electrode. J Electroanal Chem 504 45-51... 

Figure 5.11 Diffuse Reflectance Absorbance Spectroscopy Taking In Chemometiics (DRASTIC), a FT-IR-based method for rapid screening for metabohtes. Different concentrations of ampicillin (ranging from 0 to 5 mg/mL) were mixed with a constant amount E. coli cells, dried and analyzed by FT-IR (Winson et at, 1997).

Winson, M.K., Goodacre, R., Woodward, A.M., Timmins, E.M.Jones, A., Alsberg, B.K. et al. (1997) Diffuse reflectance absorbance spectroscopy taking in chemometrics... 

Proof of Grafting. The presence of the lignin-PS graft copolymer in the extract was shown by absorbance spectroscopy, as shown in Figure 4. In Figure 4(a) a dilute solution of the extract in toluene shows an absorbance... 

Long-pathlength spectrometer. [Adapted from W. Yao. R. H. Byrne, artdR. D. Walerbury. Determination of Nanomolar Concentrations of Nitrite and Nitrate Using Long Path Length Absorbance Spectroscopy Environ. Sd. TechnoL 1998, 32,2646.]... 

This work is also notable in that Cole et al. performed detailed aggregation studies of the heme MPPIX using UV-vis and fluorescence spectroscopies to detect the formation of 71-71 hetero-metalloporphyrin assemblies under assay conditions. By employing UV-vis absorbance spectroscopy, the aggregation of porphyrin and metalloporphyrin systems may be examined. The in vitro assay system used for hemozoin... 

E. Stem and K. Timmons, Electronic Absorbing Spectroscopy in Organic Chemistry (Russian translation), Mir, Moscow, 1974. 

Vibrational spectroscopy, too, has been used to study supercritical fluid systems. Buback reviewed (59) this area however, much of his discussions are on fluid systems that are well removed from ambient conditions or difficult to handle easily (e.g., H20, HC1). In an early report, Hyatt (21) used IR absorbance spectroscopy to determine the influence of several solvent systems, including COz, on the vibrational frequencies ( ) of solute molecules. Specifically, he studied the vc=o of acetone and cyclohexanone and vs.H of pyrrole. The goal of this work was to determine the suitability of supercritical fluids as reaction solvent. Hyatt concluded that the ketones experienced an environment similar to nonpolar hydrocarbons in COz and that there were no differences between liquid and supercritical CO2. In contrast, the pyrrole studies indicated that the solvent strength of CO2 was between ether and ethyl acetate. This apparent anomalous result was a manifestation of the, albeit weak, degree of pyrrole hydrogen bonding to CO2. 

In this paper, we present a preliminary analysis of the steady-state and time-resolved fluorescence of pyrene in supercritical C02. In addition, we employ steady-state absorbance spectroscopy to determine pyrene solubility and determine the ground-state interactions. Similarly, the steady-state excitation and emission spectra gives us qualitative insights into the excimer formation process. Finally, time-resolved fluorescence experiments yield the entire ensemble of rate coefficients associated with the observed pyrene emission (Figure 1). From these rates we can then determine if the excimer formation process is diffusion controlled in supercritical C02. 

Fabbricino, M., and Korshin, G. V. (2005). Formation of disinfection by-products and applicability of differential absorbance spectroscopy to monitor halogenation inchlorinated coastal and deep ocean seawater. Desalination 176, 57-69. 

Korshin, G. V., Benjamin, M. M., Chang, H.-S., and Gallard, H. (2007). Examination of NOM Chlorination reactions by conventional and stop-flow differential absorbance spectroscopy. Environ. Sci. Technol. 41, 2776-2781. 

Wolk, J., Spaid, M., Jensen, M., MacReynolds, R., Steveson, K., Chien, R., Ultraviolet absorbance spectroscopy in a 3-dimensional microfluidic chip. Micro Total Analysis Systems, Proceedings 5th pTAS Symposium, Monterey, CA, Oct. 21-25, 2001, 367-368. 

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