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HPLC Summary

Please see below for the following protocols for HPLC use

  • Pigment Calibration

    • Steps to determine pigment concentration from pigments Chl a and b, beta-carotene, and lutein. This step is necessary when DAD lamp is changed and pigment values don't come in the same ranges as previous calibration. Please note: this is not a systemic calibration, this calibration will change the fitted lines for pigment concentration.
    • Current pigment equations (Last update - 10.18.2022):
      • Neoxanthin -> \(Area = 97.757x + 0\)
      • Violaxanthin -> \(Area = 106.41x + 0\)
      • Antheraxanthin -> \(Area = 118.11x + 0\)
      • Lutein -> \(Area = 121.43x +0\)
      • Zeaxanthin -> \(Area = 127.79x + 0\)
      • Chlorophyll b -> \(Area = 37.353x +0\)
      • Chlorophyll a -> \(Area = 20.297x + 0\)
      • Beta-carotene -> \(Area = 129.05x + 0\)

  • Pigment Extraction

  • Run Samples

    • Protocol to run samples on the HPLC (High Performance Liquid Chromatography) machine.
    • File Template

  • ImageJ

    • Determine leaf area of needles and leaves (for leaves that cannot be hole punched).
    • Leaf Area Template

  • Export & Process Data

    • Steps to export pigment area and concentration. Use an R script to compile csv files to include pigment concentration values and metadata.
    • Path to script: LAB-cavender\1-Important Lab Information\R Scripts\HPLCimport.R
    • SampData Template
Last update: October 20, 2022