Absorbance standard addition

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. 

When possible, a quantitative analysis is best conducted using external standards. Unfortunately, matrix interferences are a frequent problem, particularly when using electrothermal atomization. Eor this reason the method of standard additions is often used. One limitation to this method of standardization, however, is the requirement that there be a linear relationship between absorbance and concentration. 

Figure 6.2. (a) The effects of salinity on the sensitivity of standard additions of ammonia in laboratory mixed waters ( ) and in waters from the Tamar estuary (A) expressed as percentage of response in river water. For comparison, the salt error curves reported by Loder and Gilbert [3] are also shown (... and —, respectively), (b) Contribution of reactive index and organic absorbance to the optical blacks in the Chemlab Colorimeter. = River water-seawater mixture, o = De-ionized water-seawater mixture. Source [2]... 

In certain circumstances the matrix, defined as everything except the analyte, contributes significantly to the absorbance of a sample and is also highly variable. One method that can be used to improve results is the method of standard additions. The basic idea is to add standard to the analyte so that the standard is subjected to the same matrix effects as the analyte. This method assumes that the system obeys the Beer-Lambert Law. 

The standard addition method is widely employed in AAS. In this case, two more aliquots of the sample are transferred to volumetric flasks. The first, is diluted to volume, and the absorbance of the solution is measured. The second, receives a known quantity of analyte, whose absorbance is also measured after dilution to the same volume. Likewise, data for other standard additions may also be obtained. 

If a plot between absorbance and concentration reveals a linear relationship, which may be accomplished by several stepwise standard additions, the following expressions hold good, namely ... 

Weight of contents of 20 capsules = 10.556 g Weight of capsule content analysed = 0.1025 g Capsule eontents were dissolved in methanol and adjusted to 100 ml Concentration of aspirin standard solution = 50.4 mg/100 ml Absorbance of sample at 299 nm in 0.1 M NaOH without standard addition = 0.488... 

The difference between the absorbance with standard addition and that without represents the absorbance due to a 0.504 mg/100 ml solution of aspirin. 

Campins P, Bosch F, Verdu J, Molins C (1994) Study of the behaviour of the absorbent blanks in analytical procedures by using the H-point standard additions method (HPSAM). Talanta 41 39-52. 

The contribution of flow analysis to improving the performance of atomic spectrometry is especially interesting in the field of standardisation. FIA can provide a faster and reliable method to relate the absorbance, emission or counts (at a specific mass number) to the concentration of the elements to be determined. In fact, flow analysis presents specific advantages to solving problems related to the sometimes short dynamic concentration ranges in atomic absorption spectrometry, by means of on-line dilution. The coupling of FI techniques to atomic spectrometric detectors also offers tremendous possibilities to carry out standard additions or internal standardisation. 

The instrument yields the absorbance by ratioing the transmitted intensities in the presence and absence of sample. Linearity is only observed for weak concentrations (typically below 3 ppm). The methods used, comparable to those used in molecular absorption spectrophotometry, involve classical protocols methods using a calibration curve or standard additions, as long as the range of concentrations stays within the linear conditions of absorbance. 

The slope of the standard addition curve is 0.018 8 absorbance units/ppm. If, instead, Sr is added to distilled water, the slope is 0.030 8 absorbance units/ppm. That is, in distilled water, the absorbance increases 0.030 8/0.018 8 = 1.64 times more than it does in aquarium water for each addition of standard Sr. We attribute the lower response in aquarium water to interference by other species that are present. The absolute value of the jc-intercept of the standard addition curve, 7.41 ppm, is a reliable measure of Sr in the aquarium. 

The concept of order applies across the analytical field (recall the discussion of kinetics in Chapter 2). Order is also applied in classifying chemical sensors. When only one physical parameter constitutes the output of the sensor and is correlated with concentration, we call it a first-order sensor. An example is optical sensing of a component at one fixed wavelength. The concentration of the unknown sample is then obtained from the calibration curve (Fig. 10.1a) against absorbance, or by a standard addition method. For nonlinear sensors it is possible to use a linearization function /. 

A Brain glycolipids (gl), (500 ng, and glycoprotein (gp), 100 ng, were added to AKR spleen cells, and resulting PFC were enumerated in a lawn of C3H thymocytes. Culture medium (m) was added to control cultures. Antigens are identified by Thy-1.2 (C3H) or Thy-I.I (AKR) according to the mice from which they were derived. Control cultures were absorbed before addition to cultures with anti-Thy-1.2 (a-1.2) or anti-Thy-1.1 (a-I.l) antisera. Values are the means and standard errors of five cultures (3). 

The method of standard addition should be performed to achieve accurate results. The method involves spiking an equal volume of standard solutions, at least three different concentrations to equal volumes of reagent grade water and sample aliquots, respectively. The absorbance is recorded and plotted in y-axis against the concentration in x-axis. The linear curve is extended through the y-axis. The distance from the point of intersection on the x-axis to the origin is equal to the concentration of the metal in the sample. An example is illustrated below in Figure 1.8.1. 

Exact matrix matching is not always feasible for example, the precise matrix composition may be unknown for various reasons. In such a case the standard additions method may be employed. The sample is spiked with at least two additions of known amounts of determinant in such a way that the matrix is not significantly altered, and the absorbance of spiked and unspiked samples is measured compared to that of aqueous standards, as shown in Figure 1. By extrapolation back to the negative extension of the concentration axis, the unknown concentration may be calculated. 

Subsequent to optimisation of the heating programme, the instrument should be calibrated with aqueous standards. When a linear and reproducible calibration curve is obtained, a series of standard additions on the samples should be performed in order to elucidate whether a matrix interference is operating. This is evident when the analyte absorbance is enhanced or depressed in the sample matrix compared to the pure standard. Often however, interference is indicated by the shape of the analyte absorbance peak differing in the sample relative to that in the standard or when the analyte absorbance in the sample is irreproducible or spurious. 

Although it is possible to measure directly Mn in body fluids at normal concentrations of 18—180 nmol 1 I (1—lOpgl-1) it is difficult to achieve a precision of better than 0.10 RSD. The elimination of the considerable molecular absorption interferences requires strict control of ETA ashing temperatures and a good background correction system, and the variable condensed phase matrix interferences from the inorganic constituents necessitates the use of standard additions for calibration [64], Even this approach may not yield a viable method due to curvature of the calibration graph at very low absorbances, particularly with a diluted blood matrix [65],... 

Another standardization technique which is widely used in AAS is standard addition. It is especially useful for samples where the matrix is diflBcult to reproduce, such as samples decomposed by fusion. In this method, a known quantity of the element to be determined is added to a portion of the sample, preferably at the start of the decomposition procedure. This known amount then serves as the standard when the absorbances of the two solutions are compared. Because of the rather prevalent curvature of absorbance vs. concentration plots, it is usually necessary to run three standards as well as the unspiked sample. Thus, even if one could use a single multiple element spike as the solution for standard addition, this would quadruple the work load for sample decomposition and analysis. If such a solution were added after decomposition, it would require splitting the quantitatively diluted solution, spiking, and rediluting, and would quadruple the work load for analysts. 

The standard addition method, mostly chosen for spectrometric analysis, involves adding different increments of a standard solution containing the analyte to aliquots of the same size of the sample. Each solution is then diluted to a fixed volume before measuring the absorbance values. 

The method of standard additions may take one of several forms. If the matrix interference is constant and small, the sample is diluted to a known volume and the absorbance is measured. Then multiple volumes of a reference standard of the analyte at a known concentration are added and diluted to the same volume. The absorbance of each of the sample thus prepared is measured. From a plot of volume added versus absorbance and by extrapolation to zero volume of addition, the concentration of the analyte can be determined. Fig. 4 shows a plot of calculated absolute weight of a drug added to a fixed volume of solution as a function of measured absorbance. The x coordinate... 

Flameless atomic absorption spectroscopy using the heated graphite furnace is a sensitive method for analyzing environ-mental samples for trace metals. High salt concentrations cause interference problems that are not totally correctable by optimizing furnace conditions and/or using background correctors. We determined that samples with identical ratios of major cations have trace metal absorbances directly related to their Na and trace metal concentrations. Equations and curves based on the Na concentration, similar to standard addition curves, can be calculated to overcome the trace element interference problem. Concentrations of Pb, Cd, Cu, and Fe in sea water can be simply (ind accurately determined from the Na concentration, the sample absorbance vs. a pure standard, and the appropriate curve. 

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