02.Calibration and validation of Instrumental Methods

Calibration of Instrumental Methods

  • Focus: Validation of Analytical Methods

  • Presenter: Assoc. Prof. Gonca BİLGE ÖZEL

  • Department: Food Engineering

  • Contact: gonca.bilge@yeditepe.edu.tr

Calibration

  • Definition: Determines the relationship between analytical response and analyte concentration.

  • Chemical Standards: Used to establish calibration; almost all analytical methods require calibration.

  • Exceptions: Gravimetric methods and some coulometric methods do not rely on calibration with chemical standards.

Standard Calibration Weights for Analytical Balances

  • Importance of standard weights in ensuring balance accuracy.

  • Ensures reliable measurements in calibration.

Chemical Standards

  • Utilized in the calibration of instruments to deliver valid results.

Calibration Curve

  • Example: Linear equation demonstrating calibration curve characteristics.

  • Equation: y = 0.00467x + 0.024 with R² = 1

  • Description: Graph illustrates the relationship between concentration (mg/l) and analytical response.

Coefficient of Determination (R²)

  • Definition: Measures the goodness of fit for calibration data.

  • Formula: R² = 1 - (RSS/SSR)

    • Residual Sum of Squares (RSS) = Σ(yi – yi^)²

    • Sum of Squares (SSR) = Σ(yi,avg – yi^)²

Calibration Techniques

  1. External Standard Method

  • Known solutions used for calibration, prepared separately from samples.

  • Suitable when no interferences from matrix components exist.

  1. Internal Standard Method

  • A constant quantity of internal standard is added to the sample and other standards.

  • Useful for compensating sample loss during preparation.

  1. Standard Addition Method

  • Standard is added directly to aliquots of analyzed samples to enhance accuracy.

External Standard Method

  • Characteristics:

    • Calibration standards should include at least one blank and multiple non-zero standards.

    • Signals from the blank are adjusted to reflect the analyte signal accurately.

  • Process:

    • Calibration curve is plotted to predict unknown analyte concentrations based on measured signals.

    • Calibration should not be extrapolated beyond the measured range.

Internal Standard Calibration

  • Definition: Involves comparing signal responses of analytes against an internal standard.

  • Benefits:

    • Mitigates variabilities in sample injection and analysis conditions.

  • Process:

    • Measure the relative response of the internal Standard(S) and analyte(X); derive concentration from signal ratios.

Standard Addition Method

  • Definition: A method where known amounts of standard are added directly to sample aliquots for accurate concentration determination.

  • Example: Involves sequentially diluting samples and measuring detector responses to compute unknown concentrations.

Method Validation

  • Importance: Ensures testing systems are suitable for specific evaluations and generates legitimate data.

  • Standards: Defined by organizations like ASTM, AOAC, and AACC.

Parameters for Method Validation

  1. Accuracy: The closeness of measurement to the actual value.

  2. Precision: Consistency of results (repeatability & reproducibility).

  3. Specificity/Selectivity: Ability to measure an analyte accurately amid interferences.

  4. Range: Concentration limits where the method performs reliably.

  5. Linearity: Relationship between analyte concentration and response over a specified range.

  6. Stability: Ability of the analyte to remain unchanged over time during storage/preparation.

  7. Detection Limit (LOD) and Quantification Limit (LOQ): The smallest analyte concentrations detectable or quantifiable with accuracy.

Linearity

  • Defined: Linear response to analyte concentration across a specified range.

  • Method: Use standards to generate a linearity graph (Concentration vs. Peak Area Response).

Accuracy and Precision Examples

  • The balance between accuracy and precision is crucial for analytical method validation.

Selectivity/Specificity

  • Importance: Accurately measuring the analyte amid potential interferences.

  • Method: Analyze chromatographic blanks to ensure no analyte interference exists.

Range

  • Definition: The interval where the method exhibits accuracy and precision.

Stability

  • Importance: Analytes must remain stable through all analytical processes.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

  • LOD: The lowest concentration detectable but not quantifiable.

  • LOQ: The smallest amount quantifiable with accuracy.

  • Formulae:

    • LOD = 3σb / m

    • LOQ = 10σb / m

    • Where σb is the standard deviation of the blank, and m is the slope of the calibration line.

Ruggedness vs. Robustness

  • Ruggedness: Consistency of results regardless of testing conditions (different lab, instruments, etc.).

  • Robustness: Method's ability to withstand minor changes during execution.

Conclusion

  • Importance of rigorous calibration and validation in instrumental methods in food analysis.