Chlorophyll a Size Fractionation


The size distribution of total chlorophyll a is determined by filtering water though filters of differing pore sizes, extracting these in acetone and analyzing fluorometrically. Using size fractionation of chlorophyll a and taxon-specific pigments (chlorophylls and carotenoids) from HPLC analysis, the samples analyzed within the California Current Ecosystem are used to develop a metric for phytoplankton community structure that can be used to monitor its state and changes thereof over time.


The original description of the method was written Nov 2004 by R. Goericke, S. Dovel and M. Roadman. Changes to the method are listed below.



1. Principle

For the size fractionation of chlorophyll a, a 10 meter depth sample of seawater is collected, filtered in parallel through filters with five different pore sizes (GF/F filters, 1 µm, 3 µm and 8 µm, and 20 µm pore sizes), and these are extracted in 90% acetone for 24 hours in the dark at -20°C for standard fluorometric analysis. Each size treatment has 3 replicates.

2. Sampling Collection

Water for size fractionation of chlorophyll a is sampled from a 10m depth on Line 83 and 87 with scattered inshore stations within the CalCOFI and SCCOOS stations located in the CCE. A seawater sample (about 4.5 – 5 liters) is collected from the niskin bottle in a 10 liter carboy

3. Filtration and Sample Extraction


Set up filtration system for filtering 3 replicates of each filter pore size (GF/F, 1 µm, 3 mm and 8 mm polycarbonate filters, and 20 µm Nitex filters).


Mix the seawater sample in the carboy to get a homogeneous sample and then measure out fifteen samples of 100ml to 200ml with a graduated cylinder. If the CTD fluorescence is high or the water is turbid, use 100 ml per sample.


Filter samples under low vacuum pressure (30-40 mm Hg).


Place filter into a labeled 13 x 100 mm test tube, fill with 7mls of 90% acetone (submerging filter), cap and gently mix.


Store in a -20°C freezer for 24 hours.

4. Fluorometer Analysis


An hour before analysis, the fluorometer is turned on and the samples are removed from the freezer, and placed in the dark to come to room temperature. The fluorometer is left on for the rest of the cruise.


A blank value is recorded both at the beginning and end of analysis using a test tube filled with 7mls of the 90% acetone used for extraction (this blank is not overly critical since it only affects values of pheopigments, not values of Chl a).


Measuring samples:

  1. Gently mix sample and carefully remove filter using a spatula or forceps.
  2. Using a Kimwipe, remove condensation and fingerprints from test tube and place tube slowly into fluorometer, replacing light cap.
  3. Once the number has stabilized record the initial sample reading (Fo). Remove light cap and carefully add two drops of 1N HCl to test tube, wait for value to stabilize and record the final sample reading (Fa).

4a. Fluorometer Analysis Dilution

When Chlorophyll values are too high and machine readings saturate, the sample needs to be diluted!


Fill an empty vial with 7 ml 90% acetone from the automatic dispenser.


Remove 1 ml of acetone with an automatic pipette. (Note that acetone has a high vapor pressure and this will push liquid out of the pipette unless the air inside the pipette has been equilibrated with acetone fumes. To achieve this, take acetone from the vial into the pipette tip and place it back into the vial. Do this a couple of times until no more acetone drips from the pipette tip after it has been drawn into the tip.)


Remove 1 ml of extract from the sample vial, pipette it into the vial that now has 6 ml acetone, cap and mix the contents.


Read the vial in the fluorometer as described above in 4.

5. Calculations

Since each filter is filtered in triplicate, the mean of the Chl a concentration for each treatment, e.g. Chl (>1µm), is calculated and is given in µg/l seawater.

Chl (µg/l) = (F * (tau/tau-1) * (Fo-Fa) * VolExt)) / Volfil


FMean fluorescence response factor for sensitivity, average of pre and post cruise calibrations Chlorophyll a standard used (µg /(ml*Fl))
TauMean acid ratio of pure Chl a (Fo / Fa for pure Chl a), average from pre and post cruise calibrations (Fl)
FoFluorescence signal of sample before acidification (Fl)
FaFluorescence signal of sample after acidification (Fl)
VolExtThe volume of 90% acetone that was added to the test tubes (ml)
VolfilSeawater Sample volume filtered (liter)
BlankFor value obtained at the beginning and end of each set of samples analyzed

Chlorophyll concentrations in specific size classes are calculated by:

E.g. Chl (1-3µm) = Chl (>1µm) - Chl (>3µm)

Then changed into a percentage

Chl (<1µm)% = 100 * (Chl (GFF) - Chl (>1µm)) / Chl (GFF)
Chl (1-3µm)% = 100 * (Chl (>1µm) - Chl (>3µm)) / Chl (GFF)
Chl (3-8µm)% = 100 * (Chl (>3µm) - Chl (>8µm)) / Chl (GFF)
Chl (8-20µm)% = 100 * (Chl (>8µm) - Chl (>20µm)) / Chl (GFF)
Chl (>20µm)% = 100 * Chl (>20µm) / Chl (GFF)

Size classes:

Total Chl in the sample and
Chl a <1µm, 1-3µm, 3-8µm, 8-20µm and > 20 µm.

6. Equipment/Supplies

  • 10L PVC bottle (productivity-clean)
  • Sample tubing and 10 L Carboy with spigot
  • Graduated Cylinders
  • 10 ml bottle top dispenser
  • Vacuum filtration system
  • Forcep, spatula, Kimwipes, test tube racks, Aluminum foil, bottle dropper
  • Disposable Borosilicate Glass Tubes with Polypropylene Screw Cap, O.D. x L: 13 x 100mm (Fisher Scientific Cat. No. 14-962-26D)
  • Whatman GF/F, 25mm (Fisher Scientific Cat. No. 1825 025)
  • Whatman 1 µm pore size, Polycarbonate, 25mm filter (Fisher Scientific Cat. No. 09-300-64)
  • GE Osmonics 3 µm pore size, Polycarbonate, 25mm filter (Fisher Scientific Cat. No. 09-732-25)
  • Whatman 8 µm pore size, Polycarbonate, 25mm filter (Fisher Scientific Cat. No. 09-300-66)
  • 20 µm mesh size Nitex 25mm filter (Obtain bulk mesh from tow net supplier, die cut to 25mm)
  • -20°C Freezer for sample storage
  • Turner Designs 10-AU Fluorometer, fitted with a red-sensitive photomultiplier tube (PMT)

7. Reagents

  • Acetone (90% and 100%)
  • Double Distilled water (ddH2O)
  • 1N Hydrochloric acid (Fisher Scientific Cat. No. SA 48-1)

8. References

  • Arar, E.J. and G.B. Collins. 1997. In Vitro Determination of Chlorophyll-a and Pheophytin-a in Marine and Freshwater Phytoplankton by Fluorescence. Environmental Monitoring and Support Laboratory. U.S. EPA Method 445.0.
  • Herbland, A., A. Le Bouteiller, and P. Raimbault. 1985. Size structure of phytoplankton biomass in the equatorial Atlantic Ocean. Deep-Sea Research 32: 819-836.
  • Jeffrey, S.W. and G.F. Humphrey. 1975. New Spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae, and natural phytoplankton. Biochemie und Physiologie der Pflanzen 167: 191-194.
  • Li, W.K.W. 1986. Experiment approaches to field measurements: methods and interpretation. Canadian Bulletin of Fisheries and Aquatic Sciences 214: 251-286.
  • Turner Designs Model 10-AU-005 Field Fluorometer User’s Manual/November 1992 (P/N 10-AU-075).