Chapter 7 Flashcards
Explain why quantitative UV-Vis spectroscopy is based on Beer’s Law
Beer’s Law states that absorbance is directly proportional to analyte concentration in a sample. This allows for determination of concentration by measuring the absorbance at a specific wavelength.
How does an increase in analyte concentration affect the absorbance of a solution?
Absorbance increases proportionally with analyte concentration. Higher concentration means more photons are absorbed, reducing the transmitted light intensity.
What are the primary causes of deviation from Beer’s Law?
High analyte concentration, chemical equilibrium shifts, Polychromatic light, and stray light interference
Why do you use absorbance rather than transmittance for calculating analyte concentration?
Absorbance is directly proportional to concentration, while transmittance is not. The relationship between absorbance and transmittance is logarithmic, making absorbance more reliable for linear calibration curves.
Describe the purpose of using calibration curves in quantitative spectroscopy
Calibration curves establish the relationship between absorbance and concentration for a given analyte. They help correct for instrumental variations, matrix effects, and nonlinearities in real samples.
What factors should be considered when selecting a wavelength for UV-Vis analysis?
Choose the wavelength with maximum absorbance for highest sensitivity. Avoid regions where other sample components absorb. Ensure the chosen wavelength follows Beer’s Law over that required concentration range.
What are the advantages of fluorescence spec over absorption spec?
Higher sensitivity (detects lower concentrations), lower background noise (due to emission signal being separate from excitation light), molecular specificity (fluorescence is selective for certain compounds)
What is the role of a monochromator in a spectrophotometer?
Isolates a specific wavelength range from a broader light source spectrum. It ensures the spectrophotometer uses monochromatic light, improving accuracy and selectivity.
Why is it important to measure absorbance within an optimal range (typically 0.2-0.8 absorbance units)?
Minimizes error from instrumental noise, ensures linearity in Beer’s Law, Absorbance values >1.0 suffer from higher uncertainty, and values <0.2 are too small for precise quantification
What factors affect the sensitivity of fluorescence measurements?
Quantum yield, excitation wavelength, instrument sensitivity, background fluorescence
