Chlorophyll & phaeopigments protocol#

In-house contacts: Anette Wold

Status: To be checked

- Get a hold of the photo referred to below?

Sampling & filtration#

  • Collect water from Niskin bottles into 2L clean dark containers.

  • Filter ca. 50 - 2000 ml (depending on biomass, a light colour on the filter is enough, from each depth through 25 mm GF/F filters.

If no time for extraction: - Filters are placed in extraction tubes (10 ml PP-tubes) and frozen as cold as possible (liquid nitrogen, dryshipper or -80°C) immediately after filtering (if immediate analysis onboard is impossible). Wrap samples in aluminium foil.

Extraction#

  • Work as dark as possible.

  • Fold the filter once and place it in Chl a extraction vial.

  • Add 5 ml methanol to the vial using a dispenser, put a lid on the vial and cover with aluminium foil.

  • Extract it for approx. 12 hours in a refrigerator.

  • Turn on Turner Design fluorometer at least 10 min before taking the first measurement.

  • Transfer the sample to a clean borosilicate cuvette and dry the cuvette on the outside.

  • Place the cuvette in the cuvette holder of the fluorometer and wait until readings have stabilized. Press * button on fluorometer (see picture below), it will first show Delay, then Average and finally Done on the fluorometer display. Read the value on the fluorometer. This is the Rb value (Reading before acid addition) to get the total chlorophyll.

  • Take the cuvette out of the cuvette holder and add 2 drops of 5% HCl, cover the cuvette with parafilm and mix it gently 3 times. Read the value on the fluorometer. This is the Ra value (reading after acid addition) to get the phaeopigment concentration.

  • Wash cuvette with clean methanol between every sample and let it dry.

  • Before start, use a methanol blank to check that the cuvette is clean and that the fluorometer is zero for methanol.

Calculation#

See the file Chl_a_calibr_TEMPLAT.xlsx for calculations

::

Chl a [mg/m3] = Fd*Tau*(Rb-Ra)*volume methanol*(dilution factor/volume filtered)

::

Phaeopigment [mg/m3] =

    Fd*Tau*((Rb/Ra*Ra)-Rb)* volume methanol*(dilution factor/volume filtered)

Result is presented as:

::

Chl a [mg/m3]

::

Phaeopigment [mg/m3]

Calibration#

Standard:

  • Chlorophyll a (1mg)

  • Sigma (C 6144 from Anacystis nidulans algae)

Solvent: 100% Methanol

Equipment:#

  • Erlemeyerflask 1000ml x1

  • Erlemeyerflask 100ml x2

  • Erlemeyerflask 50ml x1

  • Erlemeyerflask 20ml x2

  • Erlemeyerflask 10ml x8

  • Fintip pipette 1 ml

  • Glass/plastic pipette & Pileusball: 1-5ml (1-10ml)

Not able to use Finntip pipette to suck up standard 2 from the erlemeyerkolbe because it does not get deep enough.

NB: One could transfer the standard from Erlenmeyer colbes to beakers and use Finntip pipette instead of long glass pipettes

Standard:#

  • Standard 1: (1000 µg/l): Dissolved 1 mg Chl a in 1000 ml methanol

  • Standard 2: (10µg/l) Diluted 1 ml of standard 1 in 99 ml methanol

The chlorophyll standard is small particles. Make sure that everything is transferred from the vial containing the standard to the erlemeyerflaske. Use a pipette and methanol in order to rinse both the vial and the lid.

Fill up the erlemeyerflask approx. 1/2 full and add the standard. Stir well and leave for 1-2 hrs to make sure all the particles are dissolved. Stir and shake several times. You can see that the particles are dissolving (green whirl). Add the last amount with methanol and use a pipette to be accurate.

Cover the standards in aluminium foil.

Dilution series:#

  • Series from standard 2: 0.5, 1, 2, 4, 5, 6, 8 (µg/l)

  • Series from standard 1: 10, 20, 100, 200 (µg/l)

Can also add more concentrations (e.g. 50 & 150 ml) to be more precise at the high concentrations.

Fluorometer readings:#

  • Note down which cuvette you are using: - Borosilicat = slightly green at the edge - Quarts = more transparent).

(We have been using the borosilicat.)

Calculations:#

::

Rb/Ra

::

Tau= (Rb/Ra)/((Rb/Ra)-1)

::

Fd=Chl a concentration/Rb