Overview
This lecture covers how to create a calibration curve for quantitative analysis using instruments like HPLC, including step-by-step preparation of standards by volumetric and gravimetric methods, and their comparison.
Purpose of Calibration Curves
- Calibration curves relate known standard concentrations to instrument responses (e.g., absorbance) for quantifying unknown samples.
- Unknown concentrations are estimated by matching their instrument response to the calibration curve.
Example Calculation
- If standards of 100 ppm and 200 ppm give responses of 1,000 and 2,000 units, a 1,500-unit unknown is likely 150 ppm.
- Calibration curves enable interpolation between known points.
Designing a Calibration Curve
- A 5-point calibration curve might use concentrations: 10, 25, 50, 100, 200 ppm.
- Start with a "neat" (pure) standard to prepare a stock solution, usually very concentrated (e.g., 2,000 ppm).
Required Materials
- Neat standard, 100 mL volumetric flask (stock solution), several 10 mL volumetric flasks, transfer pipette, spatula, weigh boats, balance, pipette and tips.
Preparing the Stock Solution
- Weigh ≈200 mg of neat standard, dissolve in 100 mL flask to make 2,000 ppm stock.
- Use water; if using other solvents, consider their density.
Serial Dilution for Calibration Points
- Use stock solution to prepare standards: e.g., 1 mL stock into 10 mL for 200 ppm, 0.5 mL for 100 ppm, etc.
- Each dilution is vortexed and labeled.
Gravimetric vs. Volumetric Preparation
- Gravimetric: weigh each solution at every step for higher accuracy.
- Volumetric: rely on flask and pipette volumes; faster but slightly less accurate.
HPLC Injection and Results
- Both gravimetric and volumetric curves can yield accurate results.
- Gravimetric prep showed higher accuracy (R² = 0.999 vs. 0.998).
Key Terms & Definitions
- Calibration Curve — Graph relating instrument response to known standard concentrations.
- Neat Standard — Pure sample used to make a stock solution.
- Stock Solution — Highly concentrated solution from which standards are prepared.
- Volumetric Preparation — Using measured volumes (flasks, pipettes) to dilute standards.
- Gravimetric Preparation — Using measured weights to prepare solutions for greater accuracy.
Action Items / Next Steps
- Practice preparing both volumetric and gravimetric calibration curves.
- Review the procedure for serial dilution and standard preparation.
- Read instrument-specific guidelines for calibration curves on your assigned system.