Chapter 19: Q3P (page 484)
When are isosbestic points observed and why?
When spectra of two compounds with a constant total concentration cross at any wavelength, the isosbestic points are observed.
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Here is an immunoassay to measure explosives such as trinitrotoluene (TNT) in organic solvent extracts of soil. The assay employs a flow cytometer, which counts small particles (such as living cells) flowing through a narrow tube past a detector. The cytometer in this experiment irradiates the particles with a green
laser and measures fluorescence from each particle as it flows past the detector.
1. Antibodies that bind TNT are chemically attached to 5mmdiameter latex beads.
2. The beads are incubated with a fluorescent derivative of TNT to saturate the antibodies, and excess TNT derivative is removed. The beads are resuspended in aqueous detergent.
3. 5mlof the suspension are added to 100mlof sample or standard. TNT in the sample or standard displaces some derivatized TNT from bound antibodies. The higher the concentration of TNT, the more derivatized TNT is displaced.
4. An aliquot is injected into the flow cytometer, which measures fluorescence of individual beads as they pass the detector. The figure shows median fluorescence intensity 6 standard deviation. TNT can be quantified in the ppb to ppm range.
Draw pictures showing the state of the beads in steps 1, 2, and 3and explain how this method works.
Iodine reacts with mesitylene to form a complex with an absorption maximum at 332 nm in CCl4 solution:
(b)Spectrophotometric data for this reaction are shown in the table.Becausewe can say that [mesitylene][mesitylene]tot. Prepare a graph ofversusand find the equilibrium constant and molar absorptivity of the complex.
Two ways to analyze a mixture. Figure 19-5 shows the spectrum of the indicator bromothymol blue adjusted to several pH values. The spectrum at pHis that of the pure blue form and the spectrum at pH 1.8is that of the pure yellow form. At other pHvalues, there is a mixture of the two forms. The total concentration isand the path length isin all spectra. For the purpose of calculation, assume that there are more than two significant digits in concentration and path length. Absorbance at the dots on three of the curves in Figure 19-5 is given in the table.
(a) Prepare a spreadsheet like Figure 19-3 to use absorption at all six wavelengths to findin the mixture. Comment on the sum.
(b) Fromin the mixture, and fromfor HIn,calculate theof the mixture. (This calculation is the source of pH labels in the figure.)
(c) Use Equations 19-6 at the peak wavelengths ofto findin the mixture. Compare your answers to those in (a). Which answers, (a) or (c), are probably more accurate? Why?
What is the advantage of a time-resolved emission measurement with Eu3+versus measurement of fluorescence from organic chromophores?
Explain what is done in flow injection analysis and sequential injection. What is the principal difference between the two techniques? Which one is called “lab-on-a-valve”?
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