The absorbance method

Though a continuous scale of absorbance values may be obtained, it is not very reliable outside the 0.2-0.8 range and the coefficient of variation tends to increase significantly outside this range. The absorbance values are not linearly proportional to the titres. 

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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... 

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. 

In addition, a fluorescence method has also been described, leading to a detection limit more than 10 times lower than that of the absorbance method. A further reduction, down to femtomolar concentrations, can be achieved by coupling the hexokinase procedure to a bioluminescent indicator reaction using firefly luciferase. 

This relationship results in a sigmoidal plot of absorbance versus pH centered on the pKa value. In comparison to fluorescence, the absorbance method is simple... 

The absorbance at 280 nm is convenient for monitoring protein concentration because it is broad, relatively insensitive to protein conformation and not overly prone to background interference. In contrast, the protein backbone absorbs strongly in the far-UV region (below 240 nm), but these absorbance bands are highly dependent on protein conformation and very susceptible to backgrovmd interference (many molecules, such as salts and common buffer excipients, absorb significantly in the far-UV). Use of the absorbance method assumes that the... 

The final colour in the Lowry method is a result of two reactions. The first is a small contribution from the biuret reaction of protein with copper ions in alkali solution. The second results from peptide-bound copper ions facilitating the reduction of the phos-phomolybdic-tungstic acid (the Folin reagent) which gives rise to a number of reduced species with a characteristic blue colour. The amino acid residues which are involved in the reaction are tryptophan and tyrosine as well as cysteine, cystine and histidine. The amount of colour produced varies slightly with different proteins. In this respect it is a less-reliable assay than the biuret method, but it is more reliable than the absorbance method since A280 may include contribution from other species, and also the absorption of a given residue is dependent on its environment within the protein. 

Using this concept, Burdett developed a method in 1955 to obtain the concentrations in mono-, di- and polynuclear aromatics in gas oils from the absorbances measured at 197, 220 and 260 nm, with the condition that sulfur content be less than 1%. Knowledge of the average molecular weight enables the calculation of weight per cent from mole per cent. As with all methods based on statistical sampling from a population, this method is applicable only in the region used in the study extrapolation is not advised and usually leads to erroneous results. 

Adaptations of this method have been proposed in order to take into account the band displacement due either to substitution on the aromatic ring, or to chains of different lengths. The variations consist, instead of measuring the absorbance at maximum absorption, of an integration of the absorbance curve over a specified range (Oelert s method, 1971). More exact, this method is used less often mainly because the Brandes method is simpler... 

In the standard method, the metal enclosure (called the air chamber) used to hold the hydrocarbon vapors is immersed in water before the test, then drained but not dried. This mode of operation, often designated as the wet bomb" is stipulated for all materials that are exclusively petroleum. But if the fuels contain alcohols or other organic products soluble in water, the apparatus must be dried in order that the vapors are not absorbed by the water on the walls. This technique is called the dry bomb" it results in RVP values higher by about 100 mbar for some oxygenated motor fuels. When examining the numerical results, it is thus important to know the technique employed. In any case, the dry bomb method is preferred. 

Nitrogen is the most widely used absorbent (at 77 K) for the BET method and has been employed almost universally. Argon is more suited to the measurement of microporous zeolites. Krypton may be used for the... 

Ammonia may be estimated by dissolving the gas in a known volume of standard acid and then back-titrating the excess acid. In a method widely used for the determination of basic nitrogen in organic substances (the Kjeldahl method), the nitrogenous material is converted into ammonium sulphate by heating with concentrated sulphuric acid. The ammonia is then driven off by the action of alkali and absorbed in standard acid. 

The beat method of drying, if time permits, is to place the crystals in a desiccator containing an appropriate substance (usually anhydrous calcium chloride, silica gel, or concentrated sulphuric acid) to absorb the solvent. More eflicient and more rapid drying is obtained with the aid of a vacuum desiccator (see Section 11,38 and Fig. 77, 38, 1). 

Quantitative analysis. Spectroscopic analysis is widely used in the analysis of vitamin preparations, mixtures of hydrocarbons (e.y., benzene, toluene, ethylbenzene, xylenes) and other systems exhibiting characteristic electronic spectra. The extinction coefficient at 326 mp, after suitable treatment to remove other materials absorbing in this region, provides the best method for the estimation of the vitamin A content of fish oils. 

More recent determinations of by the more direct method of observing changes in the absorbance of the solution at 290 nm gave values which were not in very good agreement with these earlier ones (10 kjl mol i s i at 4-0, lo-o and 25-0 °C was i6-o, 30-0, and 95-120, respectively). The reaction was first order in the concentration of nitric acid ([HNO3] = 0-04-0-2 mol 1 at 25 ° C) and thus first-order overall. 

Quantitative Analysis for a Single Analyte The concentration of a single analyte is determined by measuring the absorbance of the sample and applying Beer s law (equation 10.5) using any of the standardization methods described in Chapter 5. The most common methods are the normal calibration curve and the method of standard additions. Single-point standardizations also can be used, provided that the validity of Beer s law has been demonstrated. 

Two additional methods for determining the composition of a mixture deserve mention. In multiwavelength linear regression analysis (MLRA) the absorbance of a mixture is compared with that of standard solutions at several wavelengths. If Asx and Asy are the absorbances of standard solutions of components X and Y at any wavelength, then... 

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. 

Standardizing the Method Equations 10.32 and 10.33 show that the intensity of fluorescent or phosphorescent emission is proportional to the concentration of the photoluminescent species, provided that the absorbance of radiation from the excitation source (A = ebC) is less than approximately 0.01. Quantitative methods are usually standardized using a set of external standards. Calibration curves are linear over as much as four to six orders of magnitude for fluorescence and two to four orders of magnitude for phosphorescence. Calibration curves become nonlinear for high concentrations of the photoluminescent species at which the intensity of emission is given by equation 10.31. Nonlinearity also may be observed at low concentrations due to the presence of fluorescent or phosphorescent contaminants. As discussed earlier, the quantum efficiency for emission is sensitive to temperature and sample matrix, both of which must be controlled if external standards are to be used. In addition, emission intensity depends on the molar absorptivity of the photoluminescent species, which is sensitive to the sample matrix. 

In the process of performing a spectrophotometric determination of Ee, an analyst prepares a calibration curve using a single-beam spectrometer, such as a Spec-20. After preparing the calibration curve, the analyst drops the cuvette used for the method blank and the standards. The analyst acquires a new cuvette, measures the absorbance of the sample, and determines the %w/w Ee in the sample. Will the change in cuvette lead to a determinate error in the analysis Explain. 

Brown and Lin reported a quantitative method for methanol based on its effect on the visible spectrum of methylene blue. In the absence of methanol, the visible spectrum for methylene blue shows two prominent absorption bands centered at approximately 610 nm and 660 nm, corresponding to the monomer and dimer, respectively. In the presence of methanol, the intensity of the dimer s absorption band decreases, and that of the monomer increases. For concentrations of methanol between 0 and 30% v/v, the ratio of the absorbance at 663 nm, Asss, to that at 610 nm, Asio, is a linear function of the amount of methanol. Using the following standardization data, determine the %v/v methanol in a sample for which Agio is 0.75 and Ag63 is 1.07. 

As noted, this procedure is adapted from a standard spectrophotometric method. The instructions for the spectrophotometric method indicate that the absorbance should be measured 5-10 min after adding the ascorbic acid. Why is this waiting period necessary in the spectrophotometric method, but not necessary in the FIA method ... 

The reduction of the yellow-colored Mo(VI) complex to the blue-colored Mo(V) complex is a slow reaction. In the standard spectrophotometric method, it is difficult to reprodudbly control the amount of time that reagents are allowed to react before measuring the absorbance. To achieve good precision, therefore, the reaction is allowed sufficient time to proceed to completion before measuring the absorbance. In the FIA method, the flow rate and the dimensions of the reaction coil determine the elapsed time between sample introduction and the measurement of absorbance (about 30 s in this configuration). Since this time is precisely controlled, the reaction time is the same for all standards and samples. 

In the FIA method we measure the absorbance before the reduction of the yellow-colored Mo(VI) complex is complete. For this reason, the absorbance for any standard solution of phosphate will always be smaller when using the FIA method. This means that the FIA method is less sensitive, and higher concentrations of phosphate are necessary. 

Onc-Factor-at-a-Timc Optimization One approach to optimizing the quantitative method for vanadium described earlier is to select initial concentrations for ITiOz and 1T2S04 and measure the absorbance. We then increase or decrease the concentration of one reagent in steps, while the second reagent s concentration remains constant, until the absorbance decreases in value. The concentration of the second reagent is then adjusted until a decrease in absorbance is again observed. This process can be stopped after one cycle or repeated until the absorbance reaches a maximum value or exceeds an acceptable threshold value. 

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