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HPLC - Pigment Extraction Protocol

Author

Anna Schweiger and Cathleen Lapadat

Needs update: name of filter funnel and check method parameters name

Goal

Protocol to extract pigments (chlorophyll-carotene-xanthophyll) from samples and run on HPLC.

Extraction Template

Extraction Template

Supplies/Equipment Needed

Chemicals (ALL CHEMICALS NEED TO BE HPLC GRADE)
Keep chemicals for maximum of 5 days (including HPLC water)

Solvent A

  • Acetonitrile
  • Methanol
  • Tris

Solvent B

  • Methanol
  • Ethyl Acetate

Wash vial

  • Isopropanol

Each run

  • Acetone

Supplies for chemical preparation

  • Glass Vacuum filter holder
  • Silver clip (to clasp filter and funnel together)
  • Whatman Anodisc 47mm membrane filter
  • Vacuum Volumetric flask
  • Vacuum pump
  • Silicone tube
  • Beaker
  • Stir bar

For extraction (Refer to “” for list of product ids of supplies)

  • Vials with caps
  • Centrifuge tubes
  • Filters
  • Needles
  • Syringe
  • Tips
  • Glass beads

Steps

Solvent Preparation

Solvent A

80 - Acetonitrile
10 - Methanol (MeOH)
10 - Tris

  1. Mix tris base (powder) with HPLC grade H2O: can be stored for approx. 2 weeks

    1. 3.035 g Tris + 0.25 L H2O (only if very large amount of solvent A needed within 5 days: 12.14 g Tris + 1 L H2O)
    2. Weigh Tris on precision scale and combine with H2O in beaker
    3. Use magnetic stirbar and stirplate and for 5 minutes on ‘5’, don’t heat
    4. Vacuum filter Tris through 0.02 µm Anodisc membrane filters

  2. For 1 liter solvent A combine in solvent bottle or beaker

    1. 800 ml Acetonitrile
    2. 100 ml MeOH
    3. 100 ml Tris
      1. Use magnetic stirbar and stirplate and for 5 minutes on ‘5’, don’t heat
Solvent B

68 - MeOH
32 – Ethyl Acetate

  1. For 1 liter solvent B combine in solvent bottle or beaker

    1. 680 ml MeOH
    2. 320 ml Ethyl Acetate

  2. Use magnetic sirbar and stirplate to stir entire solvent mixture for 5 minutes on ‘5’

Note: this method uses very little of Solvent B. It is recommended to only make half a liter.

Method parameters
  • 2 ml/min

  • Stop: 25 min

  • 12 min 0% B (100% A)

  • 4 min 100% B (Gradient from A to B)

  • 2 min 100% B (100% B)

  • 1 min 0% B (Gradient from B to A and 100% A until stoptime)

HPLC grade H2O
  1. Filter nanopore filtered water (is filtered next to the sink in the back of the lab, stored in big plastic containers) through 0.02 µm Anodisc membrane filters (Anodisc 47mm, Whatman) using a vacuum pump. Max. 4 l should be filtered with each disc.

Note: The filter membrane is fragile, only touch the filter on the outer polypropylene ring, the blue separation disks are NOT the filter (the filter is white).

Pigment Extraction

IMPORTANT:

  • The samples need to remain cool and dark. Place tubes always on ice and cover. Cool them after centrifuging for additionally 1 min.
  • Work under low light, max. 20-25.

  • Ensure pigments of samples are completely extracted—if the samples are white, they are completely extracted. If you still see green pigment in the sample after grinding, regrind an additional 10 seconds at a time (only regrind for the not fully extracted samples).

  • Place six 3mm silica balls inside 1.5 mL Amber Eppendorf Micro-centrifuge tubes.

  • Weigh sample and record initial mass.

    1. Avoid from sampling the midrib vein
    2. Refer below for details of conifer preparation.
    3. When extracting sample from an individual for the first time, measure a hole punch for percent moisture.
      1. Measure wet weight. Label and wrap sample in aluminum foil.
      2. Store in oven for at least two nights.
      3. Remove from oven. Let cool (important as heat/warmth disturbs the accuracy of the scale)
      4. Weigh for dry weight.

  • Place sample in tubes.

  • Add 200 µL 80% acetone: 160 µL acetone + 40 µL H2O. 1.

  • Grind Sample using Beadbeater (May Lab) for 25 seconds at 1750 RPM. (formerly: Mini-Beadbeater for 10 seconds)

  • Micro-centrifuge for 2 minutes at 14,000 RPM (use centrifuge in the cold room)

  • Extract supernatant and place in another 1.5 Micro-centrifuge tube

    1. If you still see pigment in the sample, grind only the not fully extracted samples for an additional 10 seconds.

  • Re-suspend pellet using 600 µL 100% Acetone. 2

  • Micro-centrifuge samples for 2 minutes at 14,000 RPM

  • Extract supernatant and place in same tube as in Step 7

  • Measure amount of extracted supernatant using a syringe, record

  • Filter sample using syringe filter and place in HPLC vial: if you have enough sample (> 0.2 ml) discard first few drops passing the filter before placing the rest in the vial)

  • Keep vials cool and dark until all samples have been prepared and HPLC is ready (stable pressure, stable lamp)

  • Place vials in HPLC and run

For 1:1 Dilution:

  1. Add an additional 200 µL 80% Acetone.

  2. Add an additional 600 µL 100% Acetone.

Notes:

• Test samples: Reuse syringes, syringe filters and needles after rinsing with Acetone

• Real samples: • Always use new needles • Syringe can be rinsed with Acetone, use 1 for each sequence (ca. 15 samples) • Use 1-2 syringe filter for each sequence (ca. 15 samples) if not contaminated

Mass based estimates: dry weight

• Determine wet weight of additional hole punches from the same sample as in step 1 (use 1 hole punch for analysis, keep 2 spares in freezer, weigh the rest) • Place into coin envelopes and put into drying oven at 65 degrees for 48 h • Weigh (dry mass) and calculate calibration (samples should be similar, avoid veins!) • Determine dry mass for analyzed samples based on equation

Area based estimates: content

• Calculate disc area: 2015 -> 28.27 mm2 • If no disc: use mm grid as weighing paper and take picture • Image J for area calculations

Pigment extraction of Conifers

  1. For conifer needles, cut about an inch of a needle and weigh (ideally keep it consistent to ~0.005g.

  2. To calculate area of the sample--keep in weigh boat, take a picture of the needle. Follow the below points for the picture:

    1. The whole weigh boat is in the picture.
    2. The widest part of the needle is showing.
    3. Take the picture straight on (ensure that it’s not at an angle).
    4. Nothing else is in the picture including small specks. Avoid shadows as much as possible.
    5. Name the picture with the individual ID.

  3. Needles don’t grind as well in the tubes. To ensure full extraction, cut the needle into small pieces into the tube before the grinding step.

  4. Continue with the extraction process.

  5. Refer to “HPLC imageJ” to analyze the area of the samples.

Note: this method can be used for broad leaf samples when a hole punch is not possible.

Last update: October 20, 2022