Standardization single-point

Example showing how an improper use of a single-point standardization can lead to a determinate error in the reported concentration of analyte. 

A multiple-point standardization presents a more difficult problem. Consider the data in Table 5.1 for a multiple-point external standardization. What is the best estimate of the relationship between Smeas and Cs It is tempting to treat this data as five separate single-point standardizations, determining k for each standard and reporting the mean value. Despite its simplicity, this is not an appropriate way to treat a multiple-point standardization. 

In a single-point standardization, we assume that the reagent blank (the first row in Table 5.1) corrects for all constant sources of determinate error. If this is not the case, then the value of k determined by a singlepoint standardization will have a determinate error. 

Effect of a Constant Determinate Error on the Value of k Calculated Using a Single-Point Standardization... 

Table 5.2 demonstrates how an uncorrected constant error affects our determination of k. The first three columns show the concentration of analyte, the true measured signal (no constant error) and the true value of k for five standards. As expected, the value of k is the same for each standard. In the fourth column a constant determinate error of +0.50 has been added to the measured signals. The corresponding values of k are shown in the last column. Note that a different value of k is obtained for each standard and that all values are greater than the true value. As we noted in Section 5B.2, this is a significant limitation to any single-point standardization. 

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. 

Assume that p-xylene is the analyte and that methylisobutylketone is the internal standard. Determine the 95% confidence interval for a single-point standardization, with and without using the internal standard. 

External Standard. In this approach, related substance levels are determined by calculation using a standard curve. The concentration of related substance is determined by the response (i.e., peak area of individual related substance) and the calibration curve. A reference standard of the drug substance is typically used in the calibration. Therefore, a response factor correction may be required if the response of related substance is very different from that of the drug substance. A single-point standard curve (Figure 3.4) is appropriate when there is no significant v-intercept. Otherwise, a multipoint calibration curve (Figure 3.5) has to be used. Different types of calibration are discussed in Section 3.2.3. 

A single point calibration may be used instead of a working calibration curve for quantitation by either external or internal standard method, if the response from the single point standards produces a response that deviates from the sample extract response by no more than 20%. The solvent for preparing calibration standards should preferably be the same one used to make the final sample extract. Hexane, isooctane, or methyl-feri-butylether is an appropriate solvent for the analysis of chlorinated pesticides by GC-ECD. 

We use the method of standard additions when it is difficult or impossible to duplicate the sample matrix. In general, the sample is spiked with a known amount or amounts of a standard solution of the analyte. In the single-point standard addition method, two portions of the sample are taken. One portion is measured as usual, but a known amount of standard analyte solution is added to the second portion. The responses for the two portions are then used to calculate the unknown concentration, assuming a linear relationship between response and analyte concentration (see Example 8-8). In the multiple additions method, additions of known amounts of standard analyte solution are made to several portions of the sample, and a multiple additions cahbration eurve is obtained. The multiple additions method gives some... 

Single-point standard addition methods are inherently more risky than multiple-point methods. There is no check on linearity with single-point methods, and results depend strongly on the reliability of one measurement. 

The single-point standard addition method was used in the determination of phosphate by the molybdenum blue method. A 2.00-mL urine sample was treated with molybdenum blue reagents to produce a species absorbing at 820 nm, after which the sample was diluted to 100 mL. A 25.00-mL aliquot of this solution gave an absorbance of 0.428 (solution 1). Addition of 1.00 mL of a solution containing 0.0500 mg of phosphate to a second 25.0-mL aliquot gave an absorbance of 0.517 (solution 2). Use these data to calculate the concentration of phosphate in milligrams per milliliter of the specimen. 

In the direct UV method, compounds are dissolved in DMSO stock solution at 10 mg/mL. A small volume is added to an aqueous buffer and mixed. If the target concentration exceeds the solubility of the compound, the insoluble material will precipitate. The solution is allowed to settle for certain period of time (e.g. overnight) and is then filtered to remove the precipitate. The concentration of the supernatant is determined by using a UV plate reader and the solubility is derived against a single point standard (Avdeef, 2001). 

In the simplest case with a single-point standardization, the pH meter standardization control is used to adjust any deviation of potential of the electrode pair from the ideal Nernst response. If all electrodes produced the same potential for a known buffer solution... 

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