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2004 Fluorometric Determination
of Chlorophyll a
Arctic LTER Protocol
References-method is adapted from EPA Method 445.0, Arar, J and G. B. Collins. In Vitro Determination of Chl a and Pheo a in Marine and Freshwater Algae by Fluorescence. Revision 1.9, 1997 http://www.epa.gov/nerlcwww/m445_0.pdf
Pre-collection Prep/materials - 15ml graduated Vulcan Tubes or similar-note make sure they are PP and not polystyrene (Falcon# 352096 or 352196) - 90% Buffered Acetone-in 1L volumetric add 100ml DI, 1mg MgCO3, and fill to volume - with 100% acetone, invert to mix - 47mm Whatman GFF 0.7um filters - Amber Nalgene Bottles (PP, HDPE) 1L or 500ml - Cooler with ice (for extraction) - Forceps - Gelman or similar vacuum filtration tower - 1000ml plastic graduated cylinder - 0.1N HCL (8.5ml 12.1N HCl, fill to volume 500ml with DI) - 100-1000uL Pipette - 13x100mm Borosilicate Glass Tubes - Van Dorn or similar for field collection
Field CollectionWater samples are collected using a Van Dorn or similar collection device and stored in 1L Amber Nalgene Bottles (PP or HDPE). Most samples are filtered in the lab within 1hr of collection. If stored for over an hour they are placed in a cooler with ice or similar dark, cool container.
Lab Filtering
Filter water from amber bottles
through Gelman filtration
set-up.
1. Using forceps
place pre-combusted Whatman GF/F
47 mm (0.7um pore) glass fiber
filter onto frit; attach upper
reservoir to filter holder.
2. Rinse graduated
cylinder a few times with a
small amount of sample water.
3. Measure 300 ml of
sample (200 ml from 500 ml
bottles) in graduated cylinder.
Pour into filter reservoir.
Turn on vacuum pump, be sure not
to allow pressure differential
to exceed .3 ATMs, to minimize
damage to delicate organisms and
don’t let filter pull dry
(slowly release vaccum as final
volume of water is filtered).
3.
Place filter into 10ml of 90% acetone (1mg MgCO3/l) in centrifuge tubes
(Vulcan Tubes). Make
sure filter is completely
immersed in acetone, cap
tightly and place in light
tight box while processing,
then move to cooler with ice
for extraction. These
filters will be analyzed for
Chlorophyll a content
after a 16-18 hour
extraction period.
4.
Tubes should be inverted/mixed at least once during extraction, but allow
sufficient time (>4hours)
for particles to settle
before reading.
Reading Samples on Turner
Designs10-AU Fluorometer
-TD-10AU should already be
calibrated and ready for use,
for more information regarding
calibration, lamp/filter
arrangements, etc. refer to
users manual or Fluorometer use
protocols. Do not change
settings or parameters without
consulting I. Washbourne or C.
Crockett.
*Safety Precautions, chronic
exposure to acetone can cause
health problems (see MSDS).
Always wear gloves, eye
protection, and make sure
snorkel hood is working
properly.
1.
Verify TD fluorometer is on, if not press red button at front of unit and
allow to warm up for 20
minutes.
2.
Make sure windows are covered and other light sources subdued
3.
Print out spreadsheet (see below) or make your own for recording
fluorescence values
4.
Place chlorophyll solid standard in fluorometer, replace black cap, wait
2 minutes for reading to
stabilize, press [*] to read
average and record values
for both the high and low
std. To switch between
the two simply turn 180
degrees till solid slides
into place. If
readings deviate from mean
std. readings by more than
10%, the unit may need
recalibration (see Lakes or
Nutrient RA).
5.
Read and record resting fluorescence with no tube in fluorometer.
It should be around 0.328.
This will help you catch
problems associated with
changes in lamp intensity.
A second check is a clean
glass tube which has a
fluorescence around 3.32.
6.
Read and record fluorescence of two acetone blanks (90% acetone w/MgCO3)
in 13x100 glass tubes
7.
Reading samples-Carefully transfer extract from Vulcan tube to a clean
13x100mm borosilicate tube
(leave last 0.5ml of
acetone, filter, and any
particles in the Vulcan
tube).
8.
Wipe exterior of glass tube with kimwipe/tissue to remove any
fingerprints or residual
acetone.
9.
Place tube into fluorometer, replace black cap
10.
Allow reading to stabilize (about 5-10 seconds), press [*] and read
average fluorescence value,
record preacidification
value (Rb).
11.
Using calibrated pipette, transfer 300ul 0.1N HCL into the sample you
just read, mix by inversion
(place Saran wrap over end,
hold with thumb) or by using
disposable glass pipette to
“mix”
12.
Let stand for 2 minutes (its easiest to work out a system where you read
a row unacidified, then
acidify and let stand while
reading 2nd row,
acidify, etc, etc)
13.
Repeat steps 7-10, record post acidification fluorescence (Ra)
14.
After recording both readings, dump contents of glass tube into acetone
waste container, rinse with
small amount of DI
15.
Tubes are usually disposed of in broken glass container box. You
could double rinse and then
dry for reuse, but trace HCL
left on tubes could
contaminate the next run.
16.
When finished with samples rerun and record low and high chl. solid
standard values (let
readings stabilize about 2
minutes)
17.
Make sure all glass tubes are rinsed and disposed of properly, place cap
on acetone waste bottle,
clean up kimwipes, pipettes,
acid, etc. Leave
fluorometer on for next user
18.
Remove filters from Vulcan tubes dispose into filter bin, rinse tubes
with DI, refill with 10ml
90% acetone, place back in
dark box until next
chlorophyll run.
19.
See equations below to determine chlorophyll a and pheophytin a
concentrations
Chlorophyll
a
C
E, C = Chlorophyll a
concentration in the extract
(usually 10ml)
C E, C = Fs(r/r-1)(Rb-Ra)
Where:
C E, C
= corrected chlorophyll a
concentration (ug/L) in the
extract solution analyzed
Fs = response factor for the sensitivity setting S
r = the before to after acidification ration of a pure
chl a solution (Rb/Ra)
Rb = fluorescence of sample extract before acidification
Ra = fluorescence of sample extract after acidification
C S, C =
corrected chl a
concentration (ug/L) in the
whole water sample
C S, C ={(C E, C
x extract volume in L x
Dilution Factor)/(sample volume
in L)}
Pheophytin
a
PE
= pheophytin a concentration (ug/L)
in the sample extract
PE
= Fs (r/r-1) (rRa
- Rb)
PS
= pheophytin a
concentration (ug/L) in the
whole water sample
PS
= (PE x extract
volume in L x dilution
factor)/(sample volume in L)
Note: if no dilution took place,
then DF equals 1
Helpful Websites/Notes
Turner Designs Fluorometer 10-AU
(see links at bottom of this
page for PDFs of FAQ, background
info, scope of applications,
etc_
http://www.turnerdesigns.com/t2/instruments/10au.html
Quick Start Manual for 10-AU
http://www.turnerdesigns.com/t2/doc/manuals/10au_manual.pdf
Frequently asked questions about
Fluorometry (Good Background
Info)
Chlorophyll a Determination
by Fluorescence-Arctic Lakes
Spreadsheet
Name___________________
Date___________
Resting Fluorescence
(no tube) ______
start of run
______ end of run
Chlorophyll Solid Standard-mean early season 2004 High
49.11, Low 7.95
_______high start of run ______low
start of run
______high end of run
_____low end of run
Blanks (90% acetone) _____
start of run
_____ start of run
_____ end of run
_____ end of run
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