UV-Vis Spectrophotometer

Overview of UV-VIS Spectrophotomer:

A spectrophotomer measures how much light passes through (transmission of light)

Another fancy term for light that you may often encounter is electromagnetic radiation (EMR)

When light passes through a chemical system (such as a solution or gas), some light is absorbed by the analyte

  • This occurrence results in radiant power decreasing (Radiant power is defined as the energy per second per unit area of the light beam)
  • Absorbance is related to the concentration of molecules able to absorb light
  • Absorbance is also dependent on Beer’s Law (A = Ԑbc). Note that absorbance (A) is unitless, pathlength (b) is usually in centimeters, and concentration (c) is in molarity. Therefore, to make A dimensionless, the molar absorptivity (Ԑ) must have dimensions that cancel pathlength and concentration’s units. That is Ԑ is ­cm-1M-1 in this case.

Transmittance and absorbance are related:

Spectrophotomers are equipped with detectors (usually highly sensitive photopmultiplier tube(PMT) detectors) that directly measure the transmittance of EMR, which can be used to calculate the absorbance of light by some math:

Transmittance is how much light passes through the system (T = P/P­o)

Absorbance is how much light is absorbed by the system (A = -logT)

Combining the two equations, we can make absorbance as a function of transmittance (A = -log(P/Po) or A = -logT

Limitations of UV-VIS Spectrophotometry:

It is important to know the limitations of UV-VIS or any analytical technique, so that we are not blindly measuring the wrong chemical system and making incorrect analysis.

The limitations of spectrophotometry (in general) is due its dependence on Beer’s Law. That is:

  • Beer’s Law works for monochromatic radiation passing through a dilute solution (this is approximately equal to or less than 0.01M). This ensures the absorbing analyte does not participate in concentration dependent equilibrium. High concentrations result in the solute molecules being too close to each other and changing the molar absorptivity, in addition, the solute becomes the solvent.
    • Concentration dependent chemical equilibrium means that the absorptivity changes with concentration
  • Molar absorptivity of the analyte will change depending on the solvent it is put in.
  • Other molecules (can be non-absorbing molecules) may interact with the absorbing analyte, which changes their absorptivity
  • Instrument errors, such as stray light can reach the detector
  • Temperature fluctuations can change molar absorptivity

General Procedure for Analysis using Spectrophotometry:

We perform background correction in our analysis to remove extraneous noise from other absorbing species in the chemical system that we are not interested in measuring.

  • This involves having a “blank” sample containing all the compounds in the sample except the analyte itself.
  • The blank spectrum will be used to deduct unnecessary peak absorbances from the actual sample or standard spectrum.

Because many environmental and chemical variables can change the absorbance of the analyte, both quantitative and qualitative analysis involves the use of known standards.

  • In qualitative analysis, we usually look for peaks in absorbance characteristic only to the analyte of interest
  • In quantitative analysis, we use known standards to make a calibration curve of concentrations against absorbance. Ideally, we want the curve to be as linear as possible, to get a y=mx + b equation, where y is the absorbance and x is the concentration
  • After the calibrations are done, the unknown analyte is run to collect its spectrum. The absorbance of interest (y-value) is plugged into our calibration curve equation in order to solve for concentration (x-value)
  • This way, we can figure out the concentration of our unknown analyte.

Other Notes:

  • UV-VIS spectrophotometry only emits EMR in the ultraviolet and visible spectrum. Therefore, the species has to be able to absorb and undergo electronic excitation within the specified wavelength (about 190-900nm).
  • Cuvettes usually are made from 3 types of materials: glass, plastic, and quartz
    • Quartz is the best type of cuvette because the container does not absorb in the UV-VIS region
    • However, if cost is an issue, some opt for the cheaper glass cuvettes
    • Some chemicals also stain and ruin quartz cuvettes. Therefore, a plastic cuvette might be a benefit
    • Just be aware that plastic and glass cuvettes absorb UV radiation and would only be useful in the VIS region.

Chemstack's Shimadzu UV-2401PC

A few details about the instrument:

It is a double beam Uv-Vis capable of measuring wavelength from 190 nm to 900 nm
Resolution: 0.1 nm
wavelength accuracy: 0.3nm (at slit width of 0.2nm)
Variable scanning speed (fast, middle, slow, very slow)
Detector: Photomultiplier
This instrument was made in the late 1990’s


  1. Harris, Daniel C. Exploring Chemical Analysis. 5th ed., W.H. Freeman and Company, 2013.