Absorbance method

Methods for improving the precision of absorption measurements (a) high-absorbance method (b) low-absorbance method and (c) maximum precision method. Abbreviations Sa = sample St = standard. 

Turbidimetry and Nephelometry. In contrast to classical absorbance methods, immunoassay reactions frequently involve agglutination in which the optical scatter signal of the agglutinated particles is measured by turbidimetric or nephelometric means. The principles of light scattering as it relates to analytical methods is discussed in reference 6. 

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

There are five categories of protein assay colorimetric assays, direct absorbance methods, fluorescence methods, amino acid analysis, and custom quantitation methods. A brief summary of the principles, advantages, and limitations of these methods follows. 

The direct absorbance methods require only a protein-specific extinction coefficient to deliver an accurate protein concentration. These methods typically require minutes to perform and require only a spectrophotometer and a good quantitative... 

In addition to the direct absorbance methods, colorimetric methods are suited for relatively pure proteins as purification progresses. They are accurate if calibrated from a standard curve of the test protein reference sample and fast if automated. However, they are not as simple to perform as direct absorbance methods. Hence they are not as suitable for production as direct absorbance methods. The relative simplicity of colorimetric methods makes them more suited to automated formulation and stability studies and total-protein assays of complex mixtures. Microtiter plate versions of colorimetric assays allow for automation and consumption of relatively small sample sizes while requiring little specialized equipment or training. 

The first assay to be employed for protein concentration is the Bradford assay, a commercially available colorimetric assay used to quantitate the total extracted protein. Amb a 1 is approximately 1% of the total protein extracted from ragweed pollen hence the Bradford assay does not reflect Amb a 1 concentration. However, at this step of the production process, the protein concentration is used to calculate final yields and not to make time-dependent or expensive decisions. Hence the nonspecific Bradford assay is ideal. A simpler direct absorbance method is not suitable due to the presence of a nonprotein chromophore in the ragweed extract. 

The Amb a 1 concentration of the final purified intermediate bulk is determined by an absorbance method chosen for its precision, accuracy, and simplicity. Because Amb a 1 bulk intermediate will now be conjugated to 1018 ISS (and the number of linked 1018 ISS affects the activity of the resulting AIC), it is essential to quantitate the Amb a 1 concentration accurately and precisely. A significant over- or underestimation of protein concentration will result in an over- or underestimation of the heterobifunctional linker required to activate the protein for coupling to 1018 ISS. The absorbance method, more dependent on well-calibrated instrumentation than lab technique, was chosen because it is an easy procedure to transfer to the production site. Dilution skills are the only requirement for robust performance of a well-developed and validated absorbance method. Hence a contract manufacturing site could readily quantitate Amb a 1 without the... 

The most commonly used absorbent method in use today is solid-phase microextraction (SPME Fig. 18.1c) [7-9]. In this method, an inert needle is coated with an absorbent (Table 18.1). The absorbent-coated needle may be placed above a food product, or in the food product. Depending upon the type of coating placed on the needle, volatiles with an affinity for the absorbent will migrate from the food matrix to the needle coating and be absorbed there. 

Traina, S. J., J. Novak, and N. E. Smeck. 1990. An ultraviolet absorbance method of estimating the percent aromatic carbon content of humic acids. Journal of Environmental Quality 19 151... 

The TLM detection method has been applied to detect labeled amino acids. For instance, after separation in a conventional capillary, DABSYL-labeled amino acids were detected in a Pyrex TLM detector chip. The LOD was 4.6 x l() 8 M, as compared to the LOD of 5.2 X 10-6 M obtained by the conventional absorbance method [343]. TLM has also been applied for the analysis of metal ions such as Co [319,734], Ni [735], Pb [736], Fe [734], and of organic molecules such as o-toluidine after its oxidation [737],... 

In CE detection is performed on-column, so the detection cell is defined by the diameter of the capillary. Because sensitivity is proportional to the path length, sensitivity of absorbance methods is limited. Thus, capillary... 

Bacterial colonies were suspended in 0.9% NaCl until a turbidity comparable to MacFarland s standard was achieved. Thereafter, the effectiveness of selected experimental agents was assayed at various concentrations using serial dilutions and absorbance methods at 600 nm with a bacterial stock of 1 x 108 bacteria/ml. 

Allen, J.M., Balcavage, W.X., Ramachandran, B.R., Shrout, A.L. (1998) Determination of Henry s law constants by equilibrium partitioning in a closed system using a new in situ optical absorbance method. Environ. Toxicol. Chem. 17, 1216-1221. 

The main attraction of fluorescence detection for HPLC is that for strongly fluorescent molecules, it can offer limits of detection two or three orders of magnitude lower than UV absorbance methods. The reason for this lies in the difference in the nature of the measurements. In a UV absorbance detector the photodetector is constantly illuminated at a high... 

Similarities between CD and absorbance methods are also found between CD and fluorescence and CD and circularly polarized luminescence (CPL). Three prerequisites are needed to produce FDCD and CPL activities. Intense emission signals normally associated with fluorescence are attractive because limits of detection are lowered considerably. FDCD finds more uses as a chromatographic detection device. A CD signal is usually induced by some kind of molecular complexation reaction. Association can be with a simple molecule or with an aggregate of molecules, such as chiral micelles, which are known to be fluorescence enhancers. In cases of color induction combined with fluorescence induction, FDCD can lead to even higher levels of selectivity among analytes that have been derivatized by the same color reagent. 

R132 Assmann, G., Brinkers, H., Schulte, H. and Carstensen, C.A. (1989). Comparison of the reflectance method (Reflotron reflectance photometer) with the absorbance method (automatic analysers) for the determination of cholesterol. J. Clin. Chem. Clin. Biochem. 27, 961-966. 

Serum alkaline phosphatase, which is an important metabolic indicator, is generally quantitated by absorbance methods. Alkaline phosphatase catalyzes the dephosphorylation of NADP+ and a variety of other substrates in vivo, but in vitro, the synthetic substrate / -nitrophenyIphosphate (Eq. 3.20) can be used. 

Fluorescence. The use of molecular fluorescence spectroscopy for the quantitation of enzyme reaction products has resulted in detection limits that are several orders of magnitude lower than those achieved by standard absorbance methods. At low analyte concentrations, fluorescence emission intensity is directly proportional to concentration, and its value depends on both the molar absorptivity of the analyte at the excitation wavelength, and the fluorescence quantum yield of the analyte, under the assay conditions. 

The HPLC methods cannot be used to routinely monitor the other PAHs because the instrumentation is rather sophisticated and data interpretation and analysis requires a high level of expertise. Instead simple UV absorbance methods are used to measure the absorbance at wavelengths characteristic of certain key PAHs. These include 305 nm for coronene, 335 nm for pyrene, 345 nm for dibenzo[cdJm]perylene, and 495 nm for dinaphtho[2,l,8,7-defg 2, r8, 7 -opqr]pentacene. Tracking of each of these absorbances from process startup allows process engineers to follow the buildup of the PAHs and gain an idea of the chemistry going on within the process. 

The use of modem analytical methods has led to the determination of the PAHs which are produced in catalytic hydrocrackers. A variety of HPLC-DAD, fluorescence, and UV absorbance methods were developed to determine the occurrence of the PAHs. These PAHs result from a small number of reactions. These are either a new ring forming through two-or four-carbon addition or the condensation of pyrene, coronene, or ovalene. The latter reactions result in very large PAHs which cause process problems because of their low solubilities. Their production rates (and eventual precipitation in the process streams) can be monitored through the use of UV absorbance and fluorescence spectrometries. A synchronous-scanning fluorescence method was developed to monitor the production of dicoronylene during process operation. The results of these analyses can then be used to determine process performance. 

As with turbidimetric assays, many of the direct UV absorbance assays are set up to determine kinetic solubility. However, the UV absorbance method also lends itself well to thermodynamic solubility determination by extending the period of sample agitation prior to filtration to 24 h or more. This offers a number of advantages. The solubility data generated are less dependent on the physical form of the initial material precipitated from DMSO and are much closer to thermodynamic solubility values determined later in discovery and in early development. As such, it gives more consistent solubility data through the discovery phase and enables a better quality early assessment to be made of the likely difficulties or otherwise of progressing a lead series into development. 

Protein content, particularly in urine or cerebrospinal fluid, may also be estimated by methods based on precipitation using sulfosalicylic acid (an anionic protein precipitant) or heat. The turbidity, which is a measure of protein concentration, can be quantitated by spectropho-tometric absorbance methods or light scattering analysis. Absorbance of a hydrophobic indicator dye that binds to protein and changes color is also used. 

UV absorbancy method Thermal denaturation method Tm value (°C)... 

Table 5.2 Calibration of an absorbance method for the analysis of calcium in milk...

---