What is the protein content of a sample that was diluted 50-fold and had an absorbance of 0.30, as measured by ultraviolet-visible (UV-Vis) spectrophotometry?

Calculation of Protein Content Using UV-Visible Spectrophotometry

The protein content of the sample is 125 mg/mL (option d).

Understanding the Problem

This question requires calculating the protein concentration of a sample using UV-visible spectrophotometry data. We need to:

1. Determine the concentration from the calibration curve using the measured absorbance
2. Account for the dilution factor to find the original sample concentration

Step-by-Step Solution

Step 1: Find the Concentration from the Calibration Curve

• The diluted sample has an absorbance of 0.30
• Using the calibration curve (which relates absorbance to concentration), we can determine that an absorbance of 0.30 corresponds to a protein concentration of 2.5 mg/mL in the diluted solution

Step 2: Account for the Dilution Factor

• The original sample was diluted 50-fold
• To calculate the original concentration:
  • Original concentration = Diluted concentration × Dilution factor
  • Original concentration = 2.5 mg/mL × 50 = 125 mg/mL

Explanation of the Method

UV-visible spectrophotometry is commonly used for protein quantification 1, 2. The technique works by:

• Converting proteins into colored complexes using specific reagents
• Measuring the absorbance of these complexes at an appropriate wavelength
• Comparing the absorbance to a calibration curve created with standard protein solutions of known concentrations

This approach is widely used because it's sensitive, reproducible, and relatively simple to perform 3, 4. The Beer-Lambert law forms the theoretical basis for this calculation, relating absorbance to concentration:

Concentration = Absorbance / (extinction coefficient × path length)

Common Pitfalls to Avoid

• Failing to account for the dilution factor when calculating the original concentration
• Using a calibration curve outside its linear range
• Not properly blanking the spectrophotometer
• Using inappropriate wavelengths for the specific colorimetric method
• Buffer components interfering with the assay 2

The spectrophotometric determination of protein concentration is a fundamental technique in biochemistry and molecular biology laboratories, allowing for accurate quantification of proteins in various samples 5.