Calculation of Peak Purity in HPLC
The calculation of peak purity in HPLC (High-Performance Liquid Chromatography) is typically performed using a photodiode array (PDA) detector, which can detect absorbance at multiple wavelengths simultaneously.
Steps :
Collect Spectral Data Across the Peak: During HPLC analysis, a PDA detector collects the UV-visible spectrum at different points across the peak (start, apex, and end of the peak). These points are compared to see if the spectra change.
Compare Spectra Across the Peak: Similarity Analysis: The software compares the spectra at different points across the peak to check for any significant variation in absorbance or spectral shape.
- Purity Angle vs. Purity Threshold: The software calculates the “purity angle” and “purity threshold” for each peak.
- Purity Angle: A numerical representation of the variation in spectral data across the peak.
- Purity Threshold: A reference value derived from the noise in the baseline. This value represents the maximum allowed variation for a peak to be considered pure.
Determine Peak Purity: The comparison between the purity angle and purity threshold helps in determining the purity of the peak:
- Purity Angle < Purity Threshold: The peak is considered pure (i.e., it contains a single component).
- Purity Angle > Purity Threshold: The peak may be impure, indicating co-elution of two or more components.
Visualize Purity Plot: Software generates a purity plot, which shows the spectra at different points across the peak. This plot can be visually inspected to confirm the presence of overlapping peaks.
- If the spectra are consistent across the peak, the plot will confirm that the peak is pure.
- If the spectra show variation, it indicates the presence of an impurity. Calculation
Example:
Collect Spectral Data: Suppose we collect spectral data at three points: the start, the apex (top), and the end of the peak. The software will overlay these spectra to check for consistency.
Purity Angle Calculation: The software computes the purity angle based on the difference between the spectra at each point.
Compare Purity Angle and Purity Threshold:
- If the purity angle is 0.5 and the purity threshold (based on baseline noise) is 1.0, then: Purity Angle (0.5) < Purity Threshold (1.0) → The peak is considered pure.
- If the purity angle is 1.2 and the purity threshold is 1.0, then: Purity Angle (1.2) > Purity Threshold (1.0) → The peak is considered impure, suggesting possible co-elution.
Purity Angle: A calculated value representing the variation in spectra across the peak.
Purity Threshold: A reference value indicating the allowed level of variation, usually based on baseline noise.
Peak Purity Plot: A visual representation of the spectra comparison across the peak.
Tools and Software: The calculation of peak purity is usually done using HPLC software like Waters Empower, Agilent ChemStation, or Shimadzu LabSolutions.
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