Experiment 6

Experiment 6: Introduction to Spectroscopy



Experimental objectives: 

  • Gain introduction to Visible Spectroscopy

  • Learn how to use a visible-near IR spectrophotometer to measure absorption spectra



Learning objectives: 

  • Understand what the λmax of a spectrum is and why it is important 

  • Understand the relationship between absorbance of light, chemical nature, path length, and analyte concentration



Background/Theory

Interaction of light with matter

  • Spectroscopy: The study of interactions between light and matter

  • Equation for energy of a photon: Ephoton = hv = hc

  • Energy of an emitted/absorbed photon: Ephoton = ΔE = Efinal - Initial

  • When light is absorbed a molecule can be raised to an excited state and when light is emitted a molecule in an excited state can return to the ground state

  • Elements absorb a few distinct wavelengths while molecules absorb many more wavelengths



Absorption spectrum

  • Y axis = Absorbance 

  • X axis = wavelength

  • White substances = reflects all wavelengths of the visible spectrum 

  • Black substances = absorbs all wavelengths of visible light 

  • Color is a result of all the wavelengths not absorbed by the substance



Quantitative vs Qualitative analysis:

Qualitative analysis: measurement of photon energy emitted or absorbed 

Quantitative analysis: measurement of the number of photons of light emitted or absorbed 

 ↳ this method also known as spectrometry or spectrophotometry



Absorption of light by a substance

  • Incident light (I0) = the intensity of light incident on the sample

  • Transmitted light (IT) = the intensity of light passing through the sample

  • l = pathlength (distance that light travels through the sample)

  • Transmittance =  fraction of light that passes through a sample

  • %Transmittance (%T) = % of photons that pass through the sample

  • A = Absorbance - related to how many photons get absorbed

  • Important Equations to know:

    • %T = It, λ/ I0, λ

    • A = log10 [I0, λ/It, λ] = log10[100% / %T ]



Analyzing data/observations:

  • What does Calibration do?

    1.  It provides a known standard to compare results. Calibration compares the spectra of the sample to the spectra of what was used to calibrate the spectrometer

  • What is the relationship between max wavelength and color?

    1. As the color darkens there is a decrease in max wavelength

  • What is the relationship between Amax and pathlength?

    1. They are directly proportional, as the pathlength increases, Amax increases and vice versa.

  • How does molar absorptivity vary with different wavelengths and dyes?

    1. Molar absorptivity is different for different wavelengths and dyes

  • What is the benefit of using the value from the slope rather than using the calculated value from one set of data?

    1. The slope values are derived from the linear equation, reducing errors and increasing accuracy because they are based on theoretical values.

  • In the Amax v. Concentration graph, what does the slope represent?

    1. Relationship between absorbance and concentration with a constant pathlength

  • Extensive vs. Intensive properties

    1. Extensive property depends on the amount of the substance while intensive property does not depend on the amount

    2. Absorbance, Molarity, & Molar absorptivity = intensive property

    3. Pathlength = extensive property

  • Is the relationship between absorbance and pathlength true for high and low concentrations?

    1. True for low concentrations but not for high concentrations

  • What are the units of absorbance?

    1. Absorbance has no units

  • When the amount of incident light is equal to the amount of transmitted light?

    1. All the light has been fully transmitted. No light is being absorbed or reflected.  

  • Important relationship: Wavelength and energy are inversely proportional

  • Why do you think molecular/atomic energies are often reported in eV?

    1. It's a convenient, small unit that matches the energy scales of electron transitions, photon interactions, and atomic processes.



Procedure:

  • Why is it important that the 5mm and 2mm cuvettes are held vertically when in the spectrometer?

  1. It allows the light to pass through the cuvette

  • If the solvent was ethanol instead of water, what would you calibrate the spectrum with?

    1. You would calibrate the spectrum with ethanol (you always calibrate the spectrum with the solvent)

  • How does the spectra of ethanol and methanol differ from the spectrum of water?

    1. Following calibration with water, the spectra of ethanol and methanol peaks at all wavelengths compared to the spectra of water



Common sources of error

  • Incorrect calibration of the spectrometer

  • Impurities in sample

  • Air bubbles in cuvette

  • Improper placement of cuvette in the spectrometer