Spectroscopy Fundamentals

• Spectroscopy studies the absorption and emission of light and other radiation by matter.
• It involves splitting light into its constituent wavelengths, forming a spectrum.
• The definition of spectroscopy now includes the study of interactions between particles based on collision energy.
• Spectrometry measures the interactions between light and matter, including radiation intensity and wavelength.
• Spectrometry is a method for studying and measuring specific spectra and is used for spectroscopic analysis of sample materials.

Spectroscopy vs. Spectrometry

• Spectroscopy studies the interaction between matter and radiated energy.
• It examines the absorption characteristics or behavior of matter when exposed to electromagnetic radiation and does not generate results.
• Spectrometry is a method to quantitatively measure the spectrum and is a practical application for quantifying absorbance, optical density, or transmittance.

Spectrometers

• Spectrometers measure the wavelength and frequency of light to identify and analyze atoms in a sample.
• In their simplest form, spectrometers act like a sophisticated form of diffraction.
• Light from a source is passed through a diffraction grating and onto a mirror.
• As light disperses and is reflected it then becomes a wavelength that we can detect and quantify, being characteristic of its atomic composition.
• All spectroscopic techniques shine electromagnetic radiation onto a sample and observe its response and the result gets recorded as a function of radiation wavelength creating a spectrum.
• When a molecule absorbs light, energy transfers from the ray of light to the molecule.
• Light is absorbed if the frequency of the light matches the frequency at which molecules vibrate, otherwise, it passes through unaltered.
• Inert molecules appear colored based on how they modify light.
• Different objects absorb some wavelengths and reflect others.
• If white light passes through a yellow solution, all colors except yellow are absorbed.

Spectroscopic Techniques

• Absorption spectroscopy
• Astronomical spectroscopy
• Atomic absorption spectroscopy
• Circular dichroism spectroscopy
• Electrochemical impedance spectroscopy (EIS)
• Electron spin resonance (ESR) spectroscopy
• Emission spectroscopy
• Energy dispersive spectroscopy
• Fluorescence spectroscopy
• Fourier-transform infrared (FTIR) spectroscopy
• Gamma-ray spectroscopy
• Infrared (IR) spectroscopy/ Vibrational spectroscopy
• Magnetic resonance spectroscopy
• Mass spectroscopy
• Molecular spectroscopy
• Mossbauer spectroscopy
• Nuclear magnetic resonance (NMR) spectroscopy
• Photoelectron spectroscopy
• Raman spectroscopy
• UV spectroscopy
• Ultraviolet and visible (UV/Vis) spectroscopy
• X-ray photoelectron spectroscopy

Spectrophotometry

• Spectrophotometry measures how much a substance absorbs or transmits light.
• It is used widely for quantitative analysis in science and engineering.
• It's used in biochemistry to determine enzyme-catalyzed reactions and to examine blood or tissues for clinical diagnosis.
• Variants include atomic absorption and emission spectrophotometry, used for routine measurements in hospitals, petrochemical, food, water quality control labs, and chemical/biological plants.
• UV-Visible spectrophotometer: uses light over the ultraviolet range (185-400nm) and visible range (400-700nm)
• IR spectrophotometer: uses light over the infrared range (700-1300nm)

Spectrophotometer Structure

• A spectrophotometer measures the amount of light absorbed by a sample.
• It has two devices: a spectrometer and a photometer.
• A spectrometer produces, disperses, and measures light, creating a desired range of wavelengths.
• A collimator (lens) transmits a straight beam of light (photons) through a monochromator (prism) which splits it into several component wavelengths (spectrum)
• A wavelength selector (slit) transmits only the desired wavelengths.
• A photometer is a photo detector that measures light intensity.
• After the desired wavelength range passes through the sample in a cuvette, the photometer detects absorbed photons and sends a signal to a galvanometer or a digital display.
• The spectrophotometer technique measures light intensity as a function of wavelength.
• It diffracts a light beam into a spectrum of wavelengths, detecting intensities with a charge-coupled device
• It then displays the results as a graph on the detector and the display device.
• A prism or grating splits the incident beam into different wavelengths inside spectrophotometers.
• Specific wavelengths can be manipulated to fall on the test solution by suitable manipulations and the range of wavelengths of the incident light can be as low as 1 to 2nm
• The spectrophotometer measures the absorption spectrum of a compound, that is, the absorption of light by a solution at each wavelength.

Spectrophotometer Components

• Radiant energy source
  • Materials that can be excited to high energy states by a high-voltage electric discharge or electrical heating function as radiant energy sources.
• Monochromator
  • Breaks polychromatic radiation into component wavelengths.
  • Resolves polychromatic radiation into individual wavelengths and isolates these wavelengths into narrow bands.
• Prisms
  • Disperses polychromatic light from the source into constituent wavelengths.
  • Two types of prisms are used in commercial instruments: 600 cornu quartz prism and 300 Littrow Prism.
• Gratings
  • Often used in monochromators of spectrophotometers operating in ultraviolet, visible, and infrared regions.
• Transport vessels (cuvettes)
  • Holds the sample
  • Samples studied in the ultraviolet or visible region are placed in cuvettes.
  • Cuvettes for the visible region are made of ordinary glass or sometimes Quartz.
• Photosensitive detector and readout system
  • Most detectors depend on the photoelectric effect and the current is proportional to the light intensity.
  • Radiation detectors generate electronic signals proportional to the transmitter light.
  • These signals are translated into an easy to interpret format.
  • This is accomplished using amplifiers, ammeters, potentiometers, and potentiometric recorders.

Spectrophotometer Applications

• Detection of substance concentrations

• Detection of impurities

• Structure elucidation of organic compounds

• Monitoring dissolved oxygen content in freshwater and marine ecosystems

• Characterization of proteins

• Detection of functional groups

• Respiratory gas analysis in hospitals

• Molecular weight determination of compounds

• Visible and UV spectrophotometers identify classes of compounds in both pure and biological states.

• Spectrophotometers produce a variety of wavelengths and different compounds absorb best at different wavelengths.

• Transmittance (T) is the fraction of light that passes through the sample and can be calculated once the intensity of light passing through the cuvette is known.

• Transmittance, T = I/Io

  • It is the light intensity after the beam of light passes through the cuvette and Io is the light intensity before the beam of light passes through the cuvette.

• Absorbance, A = -log(T) = -log (Io/I)

  • Transmittance indicates how much light reaches the spectrophotometer.
  • Absorbance indicates how much light is absorbed by the solute's chemicals.

• Beer-Lambert Law: A = εcl = log(Io/I)

  • States that there is a linear relationship between the absorbance and the concentration of a sample.
  • c = concentration (mol/liter)
  • l = length of light path through the cell (cm)
  • ε = molar absorption coefficient (L mol-¹ cm-¹)

Preparing Samples for Spectrophotometry

• Turn on the spectrophotometer and allow it to warm up for at least 15 minutes for accurate readings.
• Carefully clean cuvettes with deionized water, avoiding scratches, especially if they are made from glass or quartz.
• Load proper volume of sample into cuvette (less than milliliter), and remember that quartz cuvettes are designed for use in UV-visible spectrophotometry
• When handling the cuvette, avoid touching the sides the light will pass through and if you do accidentally touch these clear sides, use a kimwipe to carefully clean it
• If using a pipette, use a new tip for each sample to prevent cross-contamination.
• Prepare a control solution as a blank-the blank’s dye must be of the same volume as the solution in the same kind of container
• Before placing the cuvette into the spectrophotometer you want to make sure it is as clean as possible to avoid interference from dirt or dust particles.
• With a lint free cloth remove any water droplets or dust that may be on the outside of cuvette.

Running the Experiment

• Set the specific wavelength of light and set the desired specifications.
• Use a single wavelength of light (monochromatic color) and note that the experiment will always be different than the color of the test sample
• Calibrate the machine with the blank:
• On an analog spectrophotometer: Place the blank into the cuvette holder and shut the lid. There will be a screen with a needle that moves based on the intensity of light detection and you can use the adjustment knob to calibrate the machine
• Digital spectrophotometers operate in mostly the same way; they will just have a digital readout and adjustment knobs.
• When you remove the blank, the calibration will still be in place and the absorbance from the blank, if any, will automatically be subtracted out of your measurements
• Be sure to use a single blank per session so that each sample is calibrated to the same blank. For instance, if you blank the spectrophotometer then analyze only some of samples and blank it again, the remaining samples would be inaccurate. You would need to start over.
• With the blank removed ensure the needle should stay at 0 (zero) or the digital readout should continue is read 0
• If the needle or readout is not 0, repeat the calibration steps with the blank. If you continue to have problems, seek assistance or have the machine looked at for maintenance.
• Remove the blank and place the experimental sample into the machine; Record the values of % transmittance and/or absorbance.
• The absorbance is also known as the optical density (OD).
• The more light that is transmitted, the less light the sample absorbs
• Repeat the reading for each individual sample at least 3 times and average them together. This ensures a more accurate readout.
• Run your experiment across successive wavelengths of light

Analyzing Absorbance Data

• Calculate the transmittance and absorbance of the sample; The transmittance (T) is found by dividing the intensity of the light that passed through the sample solution with the amount that passed through the blank: T = I/Io, where
• Io is the light intensity before the beam of light passes through the cuvette
• I is the light intensity after the beam of light passes through the cuvette
• The absorbance (A) is expressed as the negative of the base-10 logarithm (exponent) of the transmittance value: A = -log10T.
• Plot the absorbance values versus the wavelengths on a graph.
• For each wavelength of light tested, plot the maximum absorbance values which identifies the compounds making up the test substance and their proportions.
• Compare the spectrum with the plots on a graph, and you can identify contaminants if there are 2 clear peaks on the graph instead of the expected one.

Atomic Absorption Spectroscopy

• Atomic absorption is an analytical technique utilizing the principle of spectroscopy for Quantitative determination of chemical elements.

• In this process, free electrons generated in an atomizer absorbs radiation of different wavelengths and absorb UV or visible light, causing the electrons to transfer to higher energy orbits

• The absorption spectrum is released, which is detected by the photodetectors and the amount of photon (radiation) absorbed results in an absorption spectrum which can then be measured in terms of absorbance

• Liquid mix with spirit gets vaporized by acetylene and lamp sets the wavelength as light source. Sample passes through detector to read absorbance of solvent bank A graph is formed when plotting for concentration of the molecules in the sample.

Atomic Emission Spectroscopy

• Atomic emission spectroscopy is a method of chemical analysis that uses the intensity of light emitted from a flame, plasma, arc, at a particular wavelength to determine the quantity of an element in a sample.
• In this process when electrons or compounds are heated either on a flame or by an electric heater, they emit energy in the form of light
• The light emitted from the compound is passed into a spectrometer then disperses the light into separate wavelengths. Each element emits a set of discrete wavelengths that is characteristic to it based on its electronic structure, and from these wavelengths.
• The sample gets heated to cause excitation of radiation
• Radiation is passed through spectrometer to detect wavelengths in spectroscope.

Uses of Atomic Emission Spectroscopy

• Agriculture and environmental analysis
• Industrial analysis
• Detection of metals and alloys