Chlorophyll a determination

Chlorophyll a determination

Source: Plymouth Marine Laboratory

Required:

Clean water sampler, e.g. Niskin bottle

Filtration system with 25mm or 47mm filter holders

50mm Petri plates

47mm or 25mm GF/F filters

Filter forceps (flat)

Dispensor for 90% buffered acetone solution

Homogenizer for GF/F filters

90% buffered (MgCO₃) acetone

Wash bottle with 90% buffered acetone

Glass bottle for acetone waste

Centrifuge tubes

Spatula

Lint-free tissues (e.g. Kimwipes) for cleaning cuvettes

HCl  1 N

Sampling

1. Filter  water sample (e.g. 1000 ml) through 47mm (or 25mm) GF/F filter at relatively low vacuum pressure (<250 mm Hg). Smaller volumes may be adequate, e.g. 10 to 100ml, depending upon predicted chlorophyll concentration.

2. Fold filter in half (sample side inwards) place in labeled petri-dish and freeze until required.

Chlorophyll extraction

1. For natural seawater and more delicate species (e.g Phaeocystis; Isochrysis ) : place filter in bottom of centrifuge tube, add 10 ml 90% acetone then refrigerate (4°C) for at least 16, but not more than 24 hours [1]. Gently invert tube several times before decanting off into fluorometer tube or spectrophotometer cuvette (see further on for fluorometer or spectrophotometer protocols).

2.  For more robust species: place filter at the bottom of homogeniser tube, add 2 - 4 ml  taken from a measured volume of 10 ml of  90% Acetone. Homogenise at low speed for approximately 20 seconds.

a) Pour into centrifuge tube. Scrape out any remaining bits of filter with spatula and add these to centrifuge tube. Rinse homogeniser tube with remaining 6-8 ml 90% acetone and add this to the centrifuge tube,. Cap centrifuge tube and store in refrigerator for between 16 and 24 hours. Clean off homogeniser head and tube between samples.

b) Remove samples from fridge and centrifuge for 5 mins at 3,000 to 4,000 rpm (if centrifuge has cooling facility then set this at 5 - 10 °C).

Note: Throughout storage and analysis of samples, exposure to light (especially strong sunlight) should be avoided or at least minimised.

Fluorometric determination[2]

If possible, use a fluorometer (e.g. Turner Designs TD700) that does not require the acid addition to. If not possible, measure sample fluorescence before and after addition of 2 drops (~100 ul) of 1N  HCl to the cuvette.

Chlorophyll a (µg litre) = (FA (RB - RA)*Ve (ml))/Vf (ml)   

FA = calibration factor of fluorometer (calculated as µg litre)

RB = fluorescence reading before addition of 1N HCL

RA = fluorescence reading after addition 1N HCL

Ve = Vol.of acetone extract (ml)

Vf = Vol. of sample filtered (ml)                                                            

Spectrophotometric determination

Use wavelength 750nm to measure turbidity and 663nm for chl a, using bandwidth of 1 nm. Ensure digital display knob is set on absorbance!

Measuring sequence: read absorbance at 750nm (E750), 663nm (E663), 750nm; add 2 drops HCl (leave 30 sec), read at 663nm (E663a)

Rinse cuvette with 90% acetone between samples.

Chlorophyll a (µg litre) =( 26.7((E663 - E750) - (E663a - E750)) x vol.extract (ml))  / (vol filtered (litres) x path length (cm) )

E663 = abs. prior to HCL

E663a  = abs. after addition of HCL

Quality Assurance

Sampling QA

• Keep the samples cool and in the dark.

• For chlorophyll a it is recommended that the sample is filtered immediately after sampling or, at least, as soon as possible thereafter to avoid deposition of cells. If storage is unavoidable, the filters should be deep frozen (< -20 oC).

Spectrophotometric or fluorometric chlorophyll a analysis QA

• The analysis should follow ISO 10260; departure from this has to be documented, and

evidence of comparability of the data provided.

• The samples/filters and the chlorophyll a extracts should be handled in subdued light.

• Avoid evaporation of the extraction solvent during extraction and measurement procedures.

• The measurements should be done immediately after clearing the extracts; the preference is for equipment for measuring the whole spectrum (800–350 nm) for easier checking of shifting of the chlorophyll peaks.

• Validate the spectrophotometer and the fluorometer at least once a year, or when changes of the equipment are required.

• Calibrate the equipment with a certified reference material, if possible; use control charts.

Reference

Strickland J.D.Hand Parsons T.R. 1968. A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada (Ottawa 1968) Bulletin 167.