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Chlorophyll Measurements
with the
10-AU Digital Fluorometer
Chlorophyll
a and Pheophytin a (extracted, Optical Kit 10-037R)
EPA
Method 445.0 is a standard method for measuring extracted chlorophyll
a and pheophytin a in marine and fresh water algae by fluorescence.
It requires extraction with 90% acetone, measurements before and after
acidification, and some fairly simple calculations to arrive at the chlorophyll
a and pheophytin a concentrations. Method 445.0 is detailed
and straightforward. (If high concentrations of pure chlorophyll b
are present, see the next section, Optical Kit 10-040R.)
A known concentration
of pure chlorophyll a (as a standard) is required at least the
first time you calibrate the instrument. For greatest accuracy, however,
we recommend that you periodically (once every few months), use a known
concentration of pure chlorophyll a in 90% acetone to recalibrate
your instrument. (Liquid Primary and Solid Secondary Chlorophyll a
standards are available from Turner Designs).
The only
way to obtain pheophytin a concentration is to use the above
EPA Method 445.0
to calculate it.
Chlorophyll
a (extracted, non-acidification, Optical Kit 10-040R)
The Welschmeyer
method is a new, simplified way to measure chlorophyll a without
the need for acidification. It accurately measures chlorophyll a
even in the presence of chlorophyll b and pheophytin. However,
you cannot obtain a pheophytin a measurement with this procedure.
Using this method, you extract your samples according to EPA Method 445.0,
but skip the acidification step.
Again, you
still need to calibrate the instrument the first time using a known concentration
of pure chlorophyll a in 90% acetone.
Secondary
Standard for Extractive Measurements
We recommend
the 10-AU Secondary Solid Standard, P/N 10-AU-904, which is stable and
fluoresces in the same wavelengths as chlorophyll. This product contains
high-level and low-level standards in one housing. No special storage
conditions are required and stability is guaranteed for two years.
To use our
secondary standard for extracted chlorophyll a analysis, first
calibrate your instrument using a known standard concentration of chlorophyll
a. Next, insert your solid secondary standard and note the readings
for both high and low standard positions. These two readings can then
be used for future analyses to check for instrument drift. The reading
can also be used to adjust data if drift occurs until the instrument can
be recalibrated with primary chlorophyll standards again.
In Vivo
Measurements (Optical Kit 10-037R or 10-096R)
For monitoring
algae, in vivo chlorophyll a fluorescence is a good indicator
of algal levels, since all algae contain chlorophyll a.
If you wish
to measure in vivo chlorophyll you can use one of the two methods
below. For greatest accuracy of in vivo measurements, you should
take samples regularly for extraction to correlate with the in vivo
readings.
Please note
that there is no need to use purified chlorophyll a to calibrate
for in vivo studies because extracted chlorophyll a in solvent
fluoresces differently than chlorophyll a in living cells (in
vivo).
1. Standard
Method using traditional chlorophyll filters (Optical Kit 10-037R).
In this method, use the 10-050R excitation filter, the 10-051R emission
filter, the 10-032 1ND reference filter, and the 10-045 Daylight White
Lamp.
This filter
set-up will read chlorophyll a and some chlorophyll b.
The calibration
method is the same whether using flow through or test tube samples.
2. Chlorophyll
a in the presence of high blank, humic substances, or chlorophyll
b (in vivo) (Optical Kit 10-096R). If you are going
to use the in vivo method and background (blank) is very high
due to high concentrations of humic substances and/or chlorophyll b-containing
algae, we recommend the following filter setup: 10-050R excitation filter,
the 034-0395R emission filter (680 nm interference filter), the 10-032
1ND reference filter, and the 10-089 Blue Mercury Vapor Lamp. Note that
the 10-045 Daylight White Lamp is sufficient unless you want to measure
very low levels of chlorophyll.
This filter
setup should help reduce interference from these substances.
Procedure
for In vivo Measurements
The following
is one suggested way of doing an in vivo study. Note that in this
method you are not calibrating at first with a standard of known concentration.
This is something that is done later. Refer to the 10-AU User's Manual
for details.
1. Place
a typical water sample in a 25 mm test tube or start it flowing through
the instrument. On the MED range, if possible, adjust the basic operating
level using the sensitivity knob to FS% between 40-50% (screen 3.2).
2. Next,
set your unknown sample to an arbitrary fluorescent value (i.e. 100)
on 10-AU screen 2.2 and calibrate on screen 2.3. Take a grab sample
immediately afterward for later extraction. You can start taking
readings of samples now, but will have to do ratio calculations after
you determine the actual concentration through extraction.
NOTE: You
can actually use this method without calibrating. For example, if your
grab sample reads 37 on the 10-AU and later, extraction shows it actually
contains 6 ppb of chlorophyll a, you can do a simple ratio calculation
to arrive at the actual concentration of unknowns: i.e., 6/37 x reading
of unknown = actual concentration of unknown.
3. Following
EPA 445.0, extract the grab sample taken in step 2. Calibrate your fluorometer
with a known chlorophyll a standard. Then, determine the actual
concentration of your grab sample.
4. To estimate
the concentration of unknowns, simply do a ratio calculation using the
extracted and corresponding in vivo data.
For example
(the numbers are for example ONLY!):
If your grab
sample used for calibration in step 2 was 12 ppb with its reading set
to [100] and your unknown sample of water reads 21, you would calculate
the actual concentration of the unknown by:
Actual
Concentration of Calibrated Sample Water
|
X |
10-AU
Reading of Unknown |
= |
Actual
Concentration of Unknown |
| 10-AU
Reading of the Calibration Sample Water |
or 12 ppb/100
x 21 = 2.52 ppb (m g/L)
Secondary
Standard for In vivo Measurements
At the present,
no solid secondary standard exists for use with flow cells. An alternative
to solid standards in this sampling mode is the use of Rhodamine WT dye.
Concentrations of the dye in the high ppb or low ppm concentration range
will result in fluorescence signals equal to natural chlorophyll concentrations.
However, a preliminary correlation study is required to determine exactly
what dye concentration corresponds to a specific chlorophyll concentration.
NOTE: We do not recommend coproporphyrin because it is prepared in an
acidic solution. Thus, it should not be used in the flow cell as it can
damage the seals, etc. For your safety, use caution when handling
acidic solutions.
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