Creating a Standard Curve 1. What is a Standard Curve? Graph that plots known concentrations of a substance against the corresponding measurement of an analytical signal (e.g., absorbance, fluorescence). It is used to determine the concentration of unknown samples. 2. Steps to Create a Standard Curve 1. Prepare standard solutions: • Use a stock solution of known concentration. • Perform serial dilutions to create a range of standard concentrations. 2. Measure the signal for each standard: • Use a spectrophotometer, fluorometer, or other analytical instrument to measure the signal for each standard. 3. Plot the data: • On the x-axis, plot the known concentrations of the standard solutions. • On the y-axis, plot the corresponding measured signals. 4. Fit a trendline: • Use linear regression to determine the equation of the line (y = mx + c). • Ensure the R2 value (coefficient of determination) is close to 1 for accuracy. 5. Determine unknown concentrations: • Measure the signal for unknown samples. • Use the equation of the trendline to calculate the concentration of unknowns. 3. Example: Absorbance vs. Concentration of Dye Data: Concentration (mg/mL) Absorbance (A) 0.0 0.00 0.2 0.10 0.4 0.19 0.6 0.30 0.8 0.39 1.0 0.50 Plotting the Standard Curve: 1 Standard Curve: Absorbance vs. Concentration Dat Trendlin 0.5 Absorbance (A) 0.4 0.3 0.2 0.1 0 −0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 Concentration (mg/mL) Analysis: • The trendline equation is: y = 0.5x where y is the absorbance and x is the concentration. • To calculate the concentration of an unknown sample with an absorbance of 0.25: x= 0.25 y = = 0.5 mg/mL 0.5 0.5 4. Tips for Creating Accurate Standard Curves • Use precise pipetting techniques to prepare standard solutions. • Ensure the instrument is calibrated before taking measurements. • Include replicates for each standard concentration to improve reliability. • Use a wide range of concentrations to capture the full dynamic range. 2