Chlorophyll Extraction Methods:

Chlorophyll Extraction & Fluorometry Methods
Part I: Extract Chlorophyll
Remove all Chl samples from liquid nitrogen (keep in foil)
Arrange Chl samples in chronological order by date and sample ID
Record sample date and sample ID on data sheet
Assign each sample a number (1,2,3…)
Turn off lights
Label a 15 mL centrifuge tube with the first sample number (1)
Remove Chl filter from foil using forceps and push it to the bottom
of the centrifuge tube
8. Add 7 mL of 90% acetone and cap
a. 7 mL usually works well for offshore samples with 25 mm
GF/F filters. Sometimes the extraction volume is increased:
larger filters, estuarine samples, bloom conditions.
9. Vortex. Make sure Chl filter is submerged.
10.Place tube in a dark drawer
11.Repeat this process for all samples, being sure to accurately label
each tube
12.Keep samples in the freezer for 24 hours to allow for complete Chl
Part II: Fluorometry
1. Remove samples from refrigerator 1-2 hours before analysis,
allowing them to warm up to room temperature.
a. Keep samples in the dark during this time – in a drawer or
under foil, etc.
b. Fluorometric response is a function of temperature.
2. Turn off lights
3. Turn on fluorometer (Turner Designs - Aquafluor)
4. Make sure fluorometer is on channel B (‘B’ will be displayed in
lower left corner of the screen)
Run Blanks and QC standards:
5. Put ~3 mL of 90% acetone in a culture tube (12x75mm)
6. Use a Kimwipe to remove any smudges from the tube
7. Place tube in fluorometer and press ‘read’
8. Record the number displayed in the ‘FLB’ column of the data
9. Using a Eppendorf Repeater pipette, add 100µl of 10% HCl to the
10.Press ‘read’ and record the number displayed in the ‘FLA’ column
of the data sheet
11.Dump liquid (Acetone,HCl) in ‘Hazardous Waste’ container
12.Throw away used culture tubes in glass disposal
13.Repeat this process twice (total of 3 blanks)
14.Repeat process with QC standards.
a. There are three QC standards in the lab freezer – they are in
brown HDPE bottles.
Run Solid Standard:
15.Remove the sample chamber insert and place the solid standard in
the fluorometer, with the tall side at 12:00
16.Press ‘read’ and record the number displayed in the FLB column
corresponding with the sample label ‘STD 12’
17.Repeat for 3:00, 6:00 and 9:00 orientations.
Run Chl Samples:
18.Place Chl samples (in centrifuge tubes) in the Eppendorf centrifuge
19.Spin the samples at 1500 rpm for 5 minutes.
20.Remove the tubes from the centrifuge and place them in a rack
21.Using a disposable transfer pipette, transfer ~3mL of liquid from
centrifuge tube #1 to a glass culture tube
22.Remove smudges with a Kimwipe and place the culture tube in the
23.Press ‘read’ on the fluorometer and record the number displayed in
the FLA column of the data sheet
24.Using an Eppendorf Repeater pipette, add 100μl of 10% HCl to the
culture tube
25.Press the ‘read’ button and record the number displayed in the FLB
column of the data sheet (This number should be LESS than the
FLA number)
26.Pour all liquid (from centrifuge and culture tubes) into the
‘Hazardous Waste’ container
27.Throw glass culture tube in glass disposal
28.Throw centrifuge tube, transfer pipette and Kimwipe in the trash
29.Repeat this process for each Chl sample
Run 3 Blanks and 4 STDs
30.Run 3 blanks and 4 STDs as outlined previously
Clean Up & Follow Up
31.Turn off and put away fluorometer
32.Cover and put away ‘Hazardous Waste’ container
33.Return remaining 10% HCl to the original container and wash the
pipette tip
34.Enter data into the Chl spreadsheet on the computer
 When transferring the Chl solution from the centrifuge tube to the
culture tube, take the liquid from the top of the centrifuge tube to
avoid filter particles in the sample
 If the fluorometer turns off, simply turn it back on, make sure it’s still
on channel B and proceed
 If the FLA reading is greater than the FLB reading, rerun the sample
with the remaining solution