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HPLC - Pigment Calibration Protocol

Author

Cathleen Lapadat and Anna Schweiger

Goal

Protocol to extract pigments from spinach and determine range and calculate dilution series for each pigment needed. Measure pigment dilution with UV-VIS Spectrophotometer and run same dilutions on HPLC to match concentrations of each pigment – Neoxanthin, Violaxanthin, antheraxanthin, zeaxanthin, chlorophyl b, chlorophyl a, and beta-carotene.

Supplies/Equipment Needed

Notes

Pigment extraction from Spinach. Extract for chlorophyll a, b and beta-carotene. Highly concentrated Lutein was purchased from Sigma and is stored in -80 freezer

  • Spinach
  • Long TLC plates
  • Microcentrifuge tubes
  • Chemical for TLC extraction
  • Pipettor
  • Pipet tips
  • Vortex mixer

Spectrophotometer supplies

  • Vials
  • Cooler
  • Ice
  • Pipettor
  • Pipet tips
  • Cuvette
  • UV-VIS Cary Spectrophotometer on 4th floor lab (Finlay and Cotner labs)

TLC Extraction

Spectrophotometer Use

  1. Determine range and calculate dilution series for each pigment needed

  2. Dilute standard using 90% acetone

    1. in standard vial
    2. add acetone with syringe (record amount added), mix with syringe
    3. take out amount needed
    4. freeze rest

  3. Let equilibrate in the dark cold-room for 5 min

    1. keep standards cool and dark

  4. Start dilution series

    1. dilute high concentration from standard vial until level 1 (highest conc) reached
    2. record all values
    3. make dilution series for each pigments until measurable with UV-VIS Spec (Cary, Cotner Lab: absorbance below 2, above 2 not accurate)
    4. confirm concentration

  5. Continue dilution series

    1. e.g. 10 steps for each pigment
    2. use level 1 as starting point (check if amount is enough)
    3. for all levels: UV-VIS Spec needs ~1.5 ml minimum
    4. pigments can be combined (mix for 10 step, saves amounts)
    5. calculate higher concentrations based on dilution series

  6. Determine peak areas using HPLC

  7. Measure dilutions in UV-VIS Spec

    1. for absorbances below 2 (above 2 not accurate)

  8. Calculate concentration using extinction coefficients from literature

  9. Calculate calibration equations for NEO, VIO, ANT, ZEA (no standards purchased) based on normalized coefficients and LUT area units see Del la Rivas et al. 1989.

Notes: - 5-8 points in calibrated range, plus 2 x 4 points at tails of sigmoidal distribution - ideal: to have 2 internal standards each run to check if calibration is still valid - calibrations are solvent specific (and method specific): stay with old method if possible to allow for re-analysis of last old data, if separation needs to be increased: increase transition time between solvents A and B - standards should ideally only used for the specified pigment, not for multiple pigments

References

Javier de las Rivas, Anunciación Abadía, Javier Abadía, A New Reversed Phase-HPLC Method Resolving All Major Higher Plant Photosynthetic Pigments , Plant Physiology, Volume 91, Issue 1, September 1989, Pages 190–192, https://doi.org/10.1104/pp.91.1.190

HPLC Refer to “HPLC - Run Samples” for method to run HPLC

Last update: October 20, 2022