9/13/11 1 Lecture 04 – Sep 14 Chem 4101 – Fall 2011 UV Absorption Spectrometry - II 1- Molecular features and UV absorption 2- Experimental Procedures 3- Examples of UV Absorption methods -Sections 14B, 14C, 14D (exc. 14D-3), 14F Suggested exercises: 14-2, 14-8, 14-11, 14-15 Lecture 04 – Sep 14 Chem 4101 – Fall 2011 Chromophores in organic compounds - I Table 14-1, Section 14B-1 Lecture 04 – Sep 14 Chem 4101 – Fall 2011 Chromophores in organic compounds - II Table 14-2, Section 14B-19/13/11 2 Lecture 04 – Sep 14 Chem 4101 – Fall 2011 Solvent Cutoff wavelengths Table 14-3, Section 14C-1 Lecture 04 – Sep 14 Chem 4101 – Fall 2011 Quantitative Analysis – Establish relationship between Absorbance and Concentration Section 14D-2 Solvent selection - Solubility of analytes - Wavelength cutoff - pH - Ionic strength Analyte and standards - Maximum absorption - Molar absortivity - Matrix interferences? - NO – External standards (Section 1D-2) - YES - Standard addition (Section 1D-3) BLANK - Everything present in the sample (i.e. matrix), except for the analytes Lecture 04 – Sep 14 Chem 4101 – Fall 2011 Example 14-1, Standard Addition Method (Duplicate in your own excel sheet) Nitrate Determination, Griess reaction Nitrate + sulfanilimide + N-(1-naphthyl)ethylenediamine product (~ 550 nm) Procedure: a. 5-mL aliquots of sample pipetted into FIVE 50-mL volumetric flask. b. Each flask received 0.00, 2.00, 4.00, 6.00, and 8.00 mL of 10.00 µM nitrate c. Each flask received the same amount of color forming reagents d. Each flask filled to the mark, mixed and let sit for 30 min at room temperature. Question: Calculate the nitrate concentration in the sample and its standard deviation Sections 14D-2, 1D-3, Appendices a1-b3, a1-D Answers : Nitrite concentration in the original sample (Cell B14) = 2.7709 µM Standard deviation of this concentration (Cell 21) = 0.2517 µM Report as 2.8 ± 0.3 µM nitrate (Consider the number of significant figures!)9/13/11 3 Lecture 04 – Sep 14 Chem 4101 – Fall 2011 Example 14-1, Spreadsheet Figure 14-8, Section 14D-2 Lecture 04 – Sep 14 Chem 4101 – Fall 2011 UV-VIS Analysis of Mixtures (On your own, explore calculations needed) A1 = εM,λ1bCM + εM,λ1bCN A2 = εM,λ2bCM + εM,λ2bCN (eqs. 14-2 and 14-3) Section 14-D2, Figures 14-9 Lecture 04 – Sep 14 Chem 4101 – Fall 2011 UV-VIS Kinetic measurements (enzymes, substrates, activity) (On your own, explore calculations needed for Michaelis Menten Equations) Section 14-F, Figures 14-15 € E + Sk1 → ESk2 → P + Ek-1 (Eq. 14-5) € d[P]dt=k2[Eo][S]KM+ [S](Eq.
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