Prepare the standards the concentration and volume of

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Prepare the Standards The concentration and volume of the stock solution added should be chosen to increase the concentration of the unknown by about 30% in each succeeding flask.
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x s s x s s x x x x s s s s x x t s s t x x V C V C C kV C kV A ) C kV , b C , x kV ax (a b y C kV C kV V C bV V C bV A bC A 0 C x : unknown concentration
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Limits to Beer’s Law Chemical Deviations absorbing undergo association, dissociation or reaction with the solvent Instrumental Deviations non-monochromatic radiation stray light
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Limits to Beer’s Law Chemical Deviations high concentration particles too close Average distance between ions and molecules are diminished to the point. Affect the charge distribution and extent of absorption. Cause deviations from linear relationship.
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Limits to Beer’s Law Chemical Deviations chemical interactions monomer-dimer equilibria, metal complexation equilibria, acid/base equilibria and solvent-analyte association equilibria The extent of such departure can be predicted from molar absorptivities and equilibrium constant. (see p561 ex 21-3)
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Limits to Beer’s Law Instrumental Deviations non-monochromatic radiation
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Limits to Beer’s Law Instrumental Deviations Stray light (P o ' + P o ") A m = log -------------- (P' + P")
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Electronic Spectroscopy Ultraviolet and visible
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Where in the spectrum are these transitions?
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Why should we learn this stuff? After all, nobody solves structures with UV any longer! Many organic molecules have chromophores that absorb UV UV absorbance is about 1000 x easier to detect per mole than NMR Still used in following reactions where the chromophore changes. Useful because timescale is so fast, and sensitivity so high. Kinetics, esp. in biochemistry, enzymology. Most quantitative Analytical chemistry in organic chemistry is conducted using HPLC with UV detectors One wavelength may not be the best for all compound in a mixture. Affects quantitative interpretation of HPLC peak heights
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Uses for UV, continued Knowing UV can help you know when to be skeptical of quant results. Need to calibrate response factors Assessing purity of a major peak in HPLC is improved by “diode array” data, taking UV spectra at time points across a peak. Any differences could suggest a unresolved component. “Peak Homogeneity” is key for purity analysis. Sensitivity makes HPLC sensitive e.g. validation of cleaning procedure for a production vessel But you would need to know what compounds could and could not be detected by UV detector! (Structure!!!) One of the best ways for identifying the presence of acidic or basic groups, due to big shifts in for a chromophore containing a phenol, carboxylic acid, etc.
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