SPECTROPHOTOMETRY

Cards (27)

  • Spectrophotometry
    • a technique used in chemistry and biology to measure the concentration of substances in a sample
    • measures the amount of light absorbed or transmitted by the sample at different wavelengths
  • HOW DOES IT WORK?
    1.) Spectrophotometers use a light source to generate a beam of light, which passes through a sample.
  • HOW DOES IT WORK?
    2.) The amount of light absorbed or transmitted by the sample at different wavelengths is detected by a photodetector.
  • HOW DOES IT WORK?
    3.) A spectrophotometer then produces a spectrum or graph of absorbance or transmittance versus wavelength.
  • Absorbance
    • measure of the amount of light absorbed by the sample
  • Absorbance
    • A= -log(T)
  • The absorbance is directly proportional to the concentration of the absorbing substance in the samples .
  • Transmittance
    • measure of the amount of light that passes through a sample and reaches the detector
  • Transmittance
    • T= 100% - A
  • Transmittance is the complement of absorbance.
  • BEER'S LAW
    • A = ε x c x l
    A = Absorbance
    ε = molar absorptivity
    c = concentration of sample
    l = path length
  • BEER’S LAW
    • this states that the concentration of a substance is directly proportional to the amount of light absorbed
  • Light Source
    • Source of Light
  • Monochromator
    • Isolation of individual wavelengths of light
  • Sample Cell/Cuvet
    • Holds samples, designed to ensure that light beam passes through the sample evenly
  • Photodetector (Photomultiplier Tube)
    • Convert the transmitted radiant energy into an equivalent amount of electrical energy
  • Display/Data Output
    • Shows the measurement results, including absorbance or transmittance values at the selected wavelength
  • Entrance Slit
    • prevents stray lights from entering
    • controlled light
  • Exit Slit
    • isolates the desired wavelength from the monochromator
  • Blank
    • used in spectrophotometry to account for background signals or interferences
  • Blank
    • Used to measure background signal from solvent, reagents, or sample matrix
  • Blank
    • Distilled water is the most commonly used
  • Blank
    • Reagent Blank - a solution that contains all the reagents used in the analysis, but no sample
  • Standard
    • solution that contains a known concentration of the analyte of interest
  • Concentration of Atom = (Absorbance of Unknown/Absorbance of Standard) x (Concentration of Standard)
  • CONCENTRATION - Directly Proportional - ABSORBANTS
  • CONCENTRATION & ABSORBANTS - Inversely Proportional - TRANSMITTANCE