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• Beer’s Law - the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light
• Beer's Law: Or unknown concentration is directly proportional to the intensity of the color of the solution (not applicable to things we read at 340 nm, which tend to be colorless)
• Instrument must be "blanked" or calibrated; this tells the instrument how much light is equal to 0% absorbance or 100% transmittance
• When the instrument is being "blanked," reagent itself will absorb some light or water and cuvette will absorb some light. This must be accounted for by setting absorbance of blank to 0%
• When the actual patient sample is measured, the absorbance should be less, as the reagent absorbance is subtracted
• the incident light in spectrophotometry is the beam of light that is hitting the sample
• in the sample - interferences include hemolysis and icterus
• depending on the wavelength of light, hemolysis may interfere with all wavelengths
• hemolysis will appear reddish
• icterus - will affect range of wavelengths, but not entire spectrum
• icterus appear greenish-brown
• %T is the percentage of light that makes it through the sample
• %T is what's actually measured by the photodetector
• the graph of percent transmittance vs concentration of the unknown is a logarithmic curve
• logarithmic - 3 is 10x stronger than 2
• %T is measured directly by spectrophotometer
• Absorbance's graph produces a linear curve when absorbance is plotted against concentration
• absorbance must be calculated by spectrophotometer, as photodetector only detects transmitted light
• The greater the absorbance, the greater the concentration
• the greater the percent transmittance, the lower the concentration
• I= transmitted light or light detected with sample
• Io= incident light or amount of light detected with blank
• for every analyte we are measuring, it has its own molar absoptibity
• molar absorptivity is defined as the fraction of a specific wavelength of light absorbed by a given molecule
• A=ExBxc

  E and b are constants, therefore A is proportional to the concentration
• spectrophotometer quality control

  • wavelength accuracy - wavelength on selector matches wavelength actually transmitted (incident light)
• wavelength accuracy is checked by using blanks of known maximum absorbance
• stray light - wavelengths of light outside the band transmitted by monochromator

  • we select a wavelength and expect all wavelengths of light to be within band pass
  • if outside band pass and light still transmitted to photomultiplier when using blanks of known maximum absorbance, this may cause stray light
• stray light can be caused by

  • scratches on the optics
  • dust particles in the light path
  • prism may be scratched and cause irregular transmittance
  • diffraction grating may have imperfection
• to fix stray light

  • blow air in spectrophotometer
  • check for stray light using cut off filters rather than blank of maximum absorbance
• cut off filter - filter blocks all light within bandpass
• linearity - change in concentration results in a straight-line calibration curve
• how to check linearity on analyzer

  • using series of diluted colored solutions
• linearity on analyzer

  • series of diluted colored solution
  • disadvantages: pipettes must be extremely precisely calibrated; very little room for error and technique must be good
• linearity of analyzer

  • most facilities purchase standards that are already prepared
  • can only check absorbances of the spectrophotometer
• most linearity is applied to analytes - use commercially prepared stock solution, create dilutions, and measure concentrations, and interpolate unknown concentration

  e.g. glucose
• interferences of spectrophotometric reading include hemolysis and icterus and lipemia
• turbidimetry is a technique and not an instrument
• turbidimetry uses a spectrophotometer to determine the amount of light that is blocked
• turbidimetry refers to the light that is blocked in a sample; even if it appears clear or colorless to the naked eye, spectrophotometer reads light that is blocked

  e.g glucose hexokinase measures absorbance of light by NADH