Light and Color Principles

White Light

• White light is a combination of different wavelengths of light known as polychromatic light
• When white light passes through a prism, filter or diffraction grating it can be broken down into a visible spectrum
• Infrared wavelengths are not visible to the human eye. These are below the visible light spectrum 750 nm
• Visible light ranges between 400 - 750 nm

Transmitted or Absorbed

• When light strikes an object, some wavelengths are transmitted and some are absorbed
• The transmitted or reflected light determines the color we see

Colored Solutions & Light

• When white light is shone at a colored solution, certain energies of light are transmitted, and other energies are absorbed.
• Transmittance is the fraction of light that passes through a sample
• Absorbance is the fraction of light absorbed by the sample
• Absorbance is calculated by -log (T). Where T = It/Io.
• Io = Intensity of incident light
• It = Intensity of transmitted light
• T = Transmittance
• A = Absorbance

Transmittance & Absorbance

• Transmittance decreases with increasing absorbance
• Absorbance decreases with increasing transmittance

Beer-Lambert Law

• States that there is a linear relationship between absorbance and the concentration of a solution
• The concentration of a substance in a solution is directly proportional to the amount of light absorbed, or inversely proportional to the logarithm of the transmitted light.
• Beer Lambert law equation: A = ε b C
• A = Absorbance
• ε = Molar absorptivity coefficient
• b = Path length
• C = Concentration of the solute

Clinical Methodologies

• Quantitative techniques are used to measure the concentration of unknown solutions
• The methodologies used in analytical instruments are based on traditional technologies like photometry, spectrophotometry, ion-selective electrodes, electrophoresis, nephelometry, immunoassays, mass spectrometry, fluorescence, and molecular diagnostic techniques.

Absorbance Spectrophotometry

• The process of measuring the concentration of an unknown solution using the properties of light energy (electromagnetic spectrum)
• The concentration of an unknown solution can be determined by comparing its absorbance to a known standard
• The intensity of the color is directly proportional to the concentration of the substance.

Calibration Standards

• Standards are chemicals that contain a known concentration of an analyte.
• They provide a reference to determine unknown concentrations or calibrate analytical instruments.

Wavelength

• Every chemical compound absorbs, transmits, or reflects light of a specific wavelength or range of wavelengths
• Light visible to the human eye is referred to as the visible light spectrum
• The visible light spectrum ranges from 400 (violet) – 750 (red) nm

Beer's Law

• Absorbance is directly proportional to the concentration of a solution, when the path length and the molar absorptivity coefficient are constant (A ~ C).

Absorbance Spectrophotometry Uses

• Determine the quantity of various solutes in solution
• Can be found in many analytical instruments
• Range from visible light to UV.

Limitations

• The solution needs to be colored to use this technique

Spectrophotometer

• A device that measures the light transmitted by a solution to determine the concentration of an absorbing substance.
• A quantitative measurement is dependent on two main factors:
  • The color itself
  • The intensity of the color

Spectrophotometer Components

• Light Source
  • Different lamps are used for different spectral ranges: - Tungsten-Halogen lamp is used for visible light - Xenon & Mercury lamp is used for UV & visible light
    • Deuterium lamp is used for UV light
• Collimator
  • A lens that concentrates the light
  • May not be used in every spectrophotometer
• Monochromator
  • Produced by a prism or diffraction grating to split white light into separate colors of the visible spectrum (wavelengths)
  • When we change the wavelength on the instrument, this component is changed
• Wavelength Selector (Slit)
• Selects a specific color (wavelength) - Remains stationary
• Sample Cell/ Cuvette
  • A vessel that holds the solution
• Detector (Photocell)
• Converts light energy into an electrical signal
• Amplifier
  • Amplifies the electrical signal from the detector
  • Photomultiplier & photodiodes amplify low intensity light
• Meter
  • Displays the data from the detector
  • The results are printed out and displayed on the screen.

How the Spectrophotometer Works

• The light source illuminates the sample cell
• The light transmits through the solution
• The transmitted light is measured by the detector
• The detector converts the light energy into an electrical signal and displays the result on the meter

Green Dye Determination

• Absorption Curve:
  • Determines the wavelength of maximum absorbance of a known standard
    • Greatest sensitivity for the method
    • Minimizes interferences
  • Procedure:
    • Set the spectrophotometer to the starting wavelength (ex. 400nm)
    • Insert a blank solution into the cuvette holder and press zero absorbance. The display should read 0.000.
    • Take an aliquot of the known standard (green dye solution) and place in the cuvette holder. - Note the absorbance reading (ex: 0.581). Record the absorbance value.
    • Repeat at the next wavelength increment.
• An absorption curve is plotted using the absorbance readings from different wavelengths

Relationship Between Colors

• Max Absorbance represents the complementary color wavelength of the colored solution being tested - If the maximum absorbance is 630nm Red, the solution will appear Green. - If the maximum absorbance is 520nm Green, the solution will appear Red.

Explore the fascinating properties of light with this quiz on white light and color perception. Understand how white light can be separated into a spectrum and how different materials interact with light. Test your knowledge on transmittance, absorbance, and the visible spectrum.