SPECTROPHOTOMETRY

• 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