Absorbance calibration

Evaluate Cd-U against aqueous or individual standard addition, dependent on instru-ment.Determine standards and samples in duplicate on two different days. Blank firings of water and diluted nitric acid must give zero absorbance. Calibration curves must consist of at least 4 points. Typical cun/es are dependent on the instrument used. Co-determine two (certified) reference samples for every 20 samples. 

Wavelength and absorbance calibration can be checked using standard filters. However, even with a well-calibrated instrument and properly prepared samples, things can go wrong. Look at the graph in Figure 2.11, which shows the actual measured absorbance at 280 nm for a series of standard protein solutions of different concentrations. 

The lodlmetrlc method was, itself, calibrated by quantitative iodine liberation using excess iodide and standard lodate solution, prepared using dried iodate as a primary standard. Ozone in the gas phase was measured by UV absorbance, calibrated against the wet methods by absorbing the gas in reagent solution contained in the reactor. 

An illustrative example generates a 2 x 2 calibration matrix from which we can determine the concentrations xi and X2 of dichromate and permanganate ions simultaneously by making spectrophotometric measurements yi and j2 at different wavelengths on an aqueous mixture of the unknowns. The advantage of this simple two-component analytical problem in 3-space is that one can envision the plane representing absorbance A as a linear function of two concentration variables A =f xuX2). 

Equations 10.4 and 10.5, which establish the linear relationship between absorbance and concentration, are known as the Beer-Lambert law, or more commonly, as Beer s law. Calibration curves based on Beer s law are used routinely in quantitative analysis. 

According to Beer s law, a calibration curve of absorbance versus the concentration of analyte in a series of standard solutions should be a straight line with an intercept of 0 and a slope of ab or eb. In many cases, however, calibration curves are found to be nonlinear (Figure 10.22). Deviations from linearity are divided into three categories fundamental, chemical, and instrumental. 

Chemical Limitations to Beer s Law Chemical deviations from Beer s law can occur when the absorbing species is involved in an equilibrium reaction. Consider, as an example, an analysis for the weak acid, HA. To construct a Beer s law calibration curve, several standards containing known total concentrations of HA, Cmt, are prepared and the absorbance of each is measured at the same wavelength. Since HA is a weak acid, it exists in equilibrium with its conjugate weak base, A ... 

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. 

Substituting the unknown s absorbance into the calibration expression gives the concentration of Fe in the waste stream as 1.48 ppm. 

Sensitivity The sensitivity of a molecular absorption analysis is equivalent to the slope of a Beer s-law calibration curve and is determined by the product of the analyte s absorptivity and the pathlength of the sample cell. Sensitivity is improved by selecting a wavelength when absorbance is at a maximum or by increasing the pathlength. 

The following absorbances were obtained for a set of Cu calibration standards... 

Linear regression of the calibration standards gives the relationship between absorbance and concentration as... 

Accuracy When spectral and chemical interferences are minimized, accuracies of 0.5-5% are routinely possible. With nonlinear calibration curves, higher accuracy is obtained by using a pair of standards whose absorbances closely bracket the sample s absorbance and assuming that the change in absorbance is linear over the limited concentration range. Determinate errors for electrothermal atomization are frequently greater than that obtained with flame atomization due to more serious matrix interferences. 

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. 

Calculate the absorbance of each solution when Pstray is 5% of Pq, and plot Beer s law calibration curves for both sets of data. Explain any differences between the two curves. (Hint Assume that Pq is 100). 

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. 

In the DPD colorimetric method for the free chlorine residual, which is reported as parts per million of CI2, the oxidizing power of free chlorine converts the colorless amine N,N-diethyl-p-phenylenediamine to a colored dye that absorbs strongly over the wavelength range of 440-580 nm. Analysis of a set of calibration standards gave the following results... 

In a quantitative flow injection analysis a calibration curve is determined by injecting standard samples containing known concentrations of analyte. The format of the caK-bration curve, such as absorbance versus concentration, is determined by the method of detection. CaKbration curves for standard spectroscopic and electrochemical methods were discussed in Chapters 10 and 11 and are not considered further in this chapter. 

A calibration curve of absorbance versus parts per million P04 gives the following calibration relationship. 

Deming and Pardue studied the kinetics for the hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase. The progress of the reaction was monitored by measuring the absorbance due to p-nitrophenol, which is one of the products of the reaction. A plot of the rate of the reaction (with units of pmol mL s ) versus the volume, V, (in milliliters) of a serum calibration standard containing the enzyme yielded a straight line with the following equation... 

One of the most effective ways to think about optimization is to visualize how a system s response changes when we increase or decrease the levels of one or more of its factors. A plot of the system s response as a function of the factor levels is called a response surface. The simplest response surface is for a system with only one factor. In this case the response surface is a straight or curved line in two dimensions. A calibration curve, such as that shown in Figure 14.1, is an example of a one-factor response surface in which the response (absorbance) is plotted on the y-axis versus the factor level (concentration of analyte) on the x-axis. Response surfaces can also be expressed mathematically. The response surface in Figure 14.1, for example, is... 

The proportionality between the concentration of chromophores and the measured absorbance [Eqs. (6.8) and (6.9)] requires calibration. With copolymers this is accomplished by chemical analysis for an element or functional group that characterizes the chromophore, or, better yet, by the use of isotopically labeled monomers. 

Composition The law of mass aclion is expressed as a rate in terms of chemical compositions of the participants, so ultimately the variation of composition with time must be found. The composition is determined in terms of a property that is measured by some instrument and cahbrated in terms of composition. Among the measures that have been used are titration, pressure, refractive index, density, chromatography, spectrometry, polarimetry, conduclimetry, absorbance, and magnetic resonance. In some cases the composition may vary linearly with the observed property, but in every case a calibration is needed. Before kinetic analysis is undertaken, the data are converted to composition as a function of time (C, t), or to composition and temperature as functions of time (C, T, t). In a steady CSTR the rate is observed as a function of residence time. 

Procedure To an aliquot of the sample solution containing 12.5 - 305 p.g of platinum(IV) were added 5 ml of hydrochloric acid - sodium acetate buffer of pH 2.1, 1 ml of O.IM Cu(II) sulphate solution, and 3.0 ml of 0.5% propericiazine solution. The solution was diluted to 25 ml with distilled water, mixed thoroughly, and the absorbance measured at 520 nm against a reagent blank solution after 10 min. The platinum concentration of the sample solution was determined using a standar d calibration curve. 

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