Protocol for extraction of chlorophyll a

Joan Bunbury, Autumn 2006

Chlorophyll a is a common measure of the phytoplankton biomass within a lake. Typically, 1 litre of lake water is collected and filtered in the field using a filtering apparatus and Whatman GF/C glass microfibre filters (42.5 mm diameter, 0.45 µm pore size). The filters are folded in half, wrapped in foil, labeled with lake name, date, and volume filtered, and immediately frozen. We put them in a Ziploc bag between two ice packs, and this works very well at keeping them frozen until we can get them to a freezer at the end of the day.

We use dimethyl sulfoxide and 90% acetone to extract the chlorophyll and measure absorbances using a spectrophotometer (Burnison, 1980).

Chlorophyll a is a common measure of the phytoplankton biomass within a lake. Typically, 1 litre of lake water is collected and filtered in the field using a filtering apparatus and Whatman GF/C glass microfibre filters (42.5 mm diameter, 0.45 µm pore size). The filters are folded in half, wrapped in foil, labeled with lake name, date, and volume filtered, and immediately frozen. We put them in a Ziploc bag between two ice packs, and this works very well at keeping them frozen until we can get them to a freezer at the end of the day.

We use dimethyl sulfoxide and 90% acetone to extract the chlorophyll and measure absorbances using a spectrophotometer (Burnison, 1980).

Chemicals

Take appropriate precautions – see MSDS sheets
Acetone
Dimethyl Sulfoxide (DMSO)

Equipment

• Heater of some sort, we use a water bath, other labs have block heaters
• Seven 5 ml glass vials with screw cap numbered 1 to 7
• Seven 15 ml plastic centrifuge tubes numbered 1 to 7
• Filtering apparatus:
  125 ml glass ehrlenmeyer
  filtering apparatus (funnel with rubber stopper, mesh filter, graduated glass tube, and clamp)
  pump
  stand to hold ehrlenmeyer
• Squeeze bottle for 90% acetone
• Graduated cylinder to make 90% acetone
• Brinkman dispenser for DMSO
• Tweezers
• Glass stir rod
• Filters (Whatman 934-AH glass microfibre filters, 25 mm diameter)
• Spectrophotometer and cuvettes

Procedure

1. Make 90% acetone (we use 900 ml acetone, 100 ml distilled water)
2. Set water bath to 56°C and let heat up for 1 hour so it is ~55°C
3. Take 7 filters out of the freezer
4. Set up fume hood with filtering apparatus, acetone, DMSO, tweezers, stir rod, pump
5. Roll up filter and stick it in vial #1, add 4 ml DMSO, put in water bath for 10 minutes
6. Be sure to log sample on log sheet with vial #, time and original sample name, from here on sample will be associated with vial #
7. Put vial #2 in after 5 minutes and continue in order of vials until you reach #7 (this is how many samples the spec will hold)
8. Put filter on filter apparatus, put tube on top, clamp
9. Pour in the heated vial and the filter, rinse vial with acetone, pour that in
10. Squish filter around to get the chlorophyll out being careful not to rip the filter paper underneath
11. Pump out filtrate, rinse tube, pump again, take apart apparatus and throw out filter papers
12. Pour filtrate into 15 ml centrifuge tube that corresponds to vial #
13. Rinse ehrlenmeyer once with acetone, discard acetone
14. Note volume of filtrate, should be around 13 ml
15. Cap sample until ready to go to spec
16. Once you have 7 samples prepared, go to spec
17. Take another 7 samples out of the freezer
18. Fill one cuvette with 90% acetone and put in cell #1. This is your reference. NOTE: when you remake 90% acetone you need to change your reference, so check that you have enough 90% acetone before you start a set of 7.
19. Pour sample #1 into a cuvette, wipe clear sides so there are no drips to affect the reading, put in cell 2 spot; put #2 in cell 3 spot, etc.
20. Check reference is 0 at all wavelengths, then measure chlorophyll samples at appropriate wavelengths (750, 665, 664, 647, 630) and enter on log sheet.

Data Analysis

Compute the chlorophyll a of each sample using the different wavelengths. 750 µm is the background and must be subtracted from the other absorbances in the equation (so E 664 is in fact 664-750). 665 µm is used only for comparison to 664 µm, which is the value included in the equation.

The equation is as follows:

chlorophyll a = 11.85 E 664 – 1.54 E 647 – 0.08 E 630 (Jeffrey, 1975)

Of course, you must also include the original sample volume and the volume noted during processing. We do this by multiplying the above equation by ¼ * cuvette volume/(sample volume/1000).

We'd like to thank Frances Pick in the Biology Department at the University of Ottawa and Paul Hamilton at the Canadian Museum of Nature for help in preparing this protocol.

References

Burnison, B. K., 1980. Modified Dimethyl Sulfoxide (DMSO) Extraction for Chlorophyll Analysis of Phytoplankton, Canadian Journal of Fisheries and Aquatic Sciences 37: 729-733.

Jeffrey, S. W.& G. F. Humphrey, 1975. New Spectrophotometric Equations for Determining Chlorophylls a , b , c 1 , and c 2 in Higher Plants, Algae and Natural Phytoplankton, Biochemie Physiologie Pflanzen 167: 191-194.

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