Chlorophyll (spectrophotometric analysis)
Method adapted from Lesley Clementson CSIRO (April, 2002)
Sampling
Water samples collected for chlorophyll analysis from the field and from cultures are filtered through 47 mm and 25 mm GF/F filters respectively. For field samples, if filtering directly from the Niskin bottle, the filtrate must be collected so the volume of water filtered can be measured. The pressure used during filtering should be low (@ 5 mm Hg) to prevent cell breakage. Filtering and then handling of filters should be performed under dimmed lighting.
| Volume to filter | Filter size | |
|---|---|---|
| Field Sample | no less than 2 L and preferably 4 L or more | 47 mm GF/F |
| Culture Sample | Typical coloured culture may only need 25 mL | 25 mm GF/F |
After filtering, remove the filter from its holder (with the vacuum still applied, if possible, to remove as much moisture as possible from the filter) and place in a cryo-tube. Label each tube with cruise number, station number, depth and pigment number. (When filtering directly from the Niskin bottle under pressure rather than vacuum, the excess moisture in the filters must be removed before freezing. This can be done by using a second pump that can apply a vacuum to the filter holder or by folding the filter in half and blotting dry with white paper towel.
Analysis
All glassware is cleaned in dilute Extran solution, rinsed three times with MQ water and once with acetone (AR). Scissor blades are wiped clean on tissues between samples.
Frozen filters are cut into halves and placed in a clean 10 mL centrifuge tube. 3 mL of 100% acetone (see note on solvents below) is added to the tube. Cover tube with parafilm and vortex for 30 seconds before placing the tube in an ice-water bath whilst the filter and acetone are sonicated for 15 minutes. The filter and acetone are then stored for at least 18 hours at 4°C. After this time, 0.2 mL MQ water is added to each tube (solvent » 90:10 acetone : water) and the filter and solvent sonicated for another 15 minutes. Solvent and filter are then transferred quantitatively to a Biorad column (see figure x) containing a small GF/F filter acting as a plug. The sample tubes are rinsed with 2 x 0.5 mL of acetone/water (90:10) which is quantitatively added to the Biorad column. Each Biorad column is fitted into a centriguge tube and centrifuged for 5 minutes at 5000 rpm. The filtrate is stored in the cool and dark (small foam esky) just prior to analysis. The absorbance of the filtrate is measured using a U.V./visible spectrophotometer with 10 mm path length optical glass cells (40 mm cells can be used if the colour of the extracts is very pale). Absorbance is read at wavelengths of 750, 664, 647 and 630 nm. The absorbance at 750 nm is subtracted from the absorbance at each of the other three wavelengths and substituted into the following equations:
[chl. a]extract = 11.85A664/l – 1.54A647/l – 0.08A630/l
[chl. b]extract = 21.03A647/l – 5.43A664/l – 2.66A630/l
[chl. c]extract = 24.52A630/l – 1.67A664/l – 7.60A647/l
A = corrected absorbance.
l = path length in cm.
The concentration of each chorophyll in the sample in µg/L is obtained by the following equation:
[chl.x]sample = [chl.x]extract * (v/V)
v = volume of extract in mL.
V = volume of seawater filtered in L.
The total concentration of chorophyll in the sample in µg/L is obtained by the following equation:
[chl.]total = [chl.a]sample + [chl.b]sample + [chl.c]sample
Solvents
Solvents used for phytoplankton pigment extraction are many and varied. Dimethyl formamide has the highest extraction efficiency but is particularly toxic and therefore difficult to handle. Methanol is another highly efficient extractor, particularly for hard to extract species such as some cyanobacteria. Unfortunately accurate equations to determine chlorophyll a, b and c are not available for methanol pigments. Acetone (either 90 or 100%) is therefore regarded as the most suitable solvent for a broad range of marine phytoplankton species and is widely used for both field samples and cultures. If in doubt, protocols in Jeffrey et al, 1997 detail the suitability of various solvents for a range of different classes and species of microalgae (full ref below).
“Phytoplankton pigments in oceanography: Guidelines to modern methods. 1997. Ed. S. W. Jeffrey, R. F. C. Mantoura, S. W. Wright. Unesco Publishing”