Measurement and Use of Absorbance Readings

Measurement and Use of Absorbance Readings

Section 1: Understanding Absorbance

  • Absorbance: An optical characteristic of solution which measures the amount of light absorbed at a particular wavelength.
  • Spectrophotometer: An instrument used to measure the absorbance of a solution by passing a beam of light through it and measuring the intensity of light that emerges.

Section 2: Preparing the Sample

  • Sample Preparation: Generally involves diluting the sample with a suitable solvent to ensure that the absorbance measurement falls within the range of the spectrophotometer.
  • Cuvettes: Small, transparent vessels used to hold the sample in the spectrophotometer. Ensure they are clean and clear of smudges to avoid interference with light transmission.

Section 3: Taking Absorbance Readings

  • Calibration: Essential first step of using a spectrophotometer. It usually involves setting the equipment to zero absorbance (A=0) using a blank sample that contains only the solvent.
  • Selecting Wavelength: Choose a wavelength that corresponds to maximum absorbance (peak wavelength) for the substance being analyzed.
  • Recording Absorbance: Place the prepared sample into a cuvette and insert into the spectrophotometer. Record the absorbance value or values, depending on the nature of the analysis.

Section 4: Analysing Absorbance Readings

  • Beer-Lambert Law: The mathematical relationship between absorbance and concentration. It states that absorbance (A) is equal to specific absorbance (a) times concentration (c) times path length (l): A = acl.
  • Calculating Concentration: If path length and specific absorbance are known, the concentration of the sample can be calculated from the measured absorbance.
  • Potential Errors: Possible errors include incorrect sample preparation, inappropriately zeroed machine, or imprecise readings. Repeating readings and checking preparations can minimize these errors.

Section 5: Safety and Care in Using Spectrophotometer

  • Safety: Always handle the spectrophotometer with care to avoid damaging the instrument. Ensure your hands are clean before use to avoid smudging the cuvette or interior of the machine.
  • Maintenance: Regularly clean the cuvette and calibrate the spectrophotometer for accuracy. Follow the manufacturer’s instructions for this process.