In
Vivo
Chlorophyll Calibration Procedure for the Aquafluor Handheld Fluorometer
The
following procedure details how to calibrate the Aquafluor Handheld
Fluorometer (Model 8000-001) for in-vivo
chlorophyll a
sampling. The procedure uses the optional accessory Solid Standard
to provide convenience, and excellent repeatability.
Users
wanting to correlate the in vivo calibration with extracted
chlorophyll a data should see the Extracted Chlorophyll a
Calibration section below.
Background
Information on Calibration Considerations for in vivo Chlorophyll
a Measurements
In
vivo chlorophyll analysis is the fluorescent detection of chlorophyll
a in algal and cyanobacterial cells in water. In this technique,
the excitation light from the fluorometer passes through untreated
sample water and excites chlorophyll a within the cells.
Environmental conditions; presence of interfering compounds; cellular
physiology; morphology; and light history all can influence the
relationship between in vivo fluorescence and the concentration
of chlorophyll a. These factors cause in vivo fluorescence
to be a semi-quantitative tool. Despite its semi-quantitative nature,
in vivo fluorescence data can supply valuable information
on the spatial and temporal distribution of chlorophyll concentrations
quickly and easily.
Using
a Secondary Standard
When
you calibrate the Aquafluor as described below, you are assigning
a value (Cal Standard Value) equal to the fluorescent intensity
of the Solid Secondary Standard calibration accessory. Keep in mind
that the Cal Standard Value you assign will only be a relative value
compared to the actual chlorophyll a concentration being
measured.
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The
Full Scale value is calculated using data from the AquaFluor
for "% of Full Scale of Standard" and the Calibration
Standard Value
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The
Solid Standard simulates the in vivo fluorescence of a
10 µg/L marine diatom culture which will have a different
fluorescence to the natural culture being monitored, hence why extraction
is the preferred method when best possible accuracy is required.
In
summary, the Secondary Standard enables you to 1) determine if the
Aquafluor has drifted since the last calibration, and 2) consistently
set the Aquafluor to indicate the same concentration.
Blank
Solution Considerations
The
Aquafluor calibration reads a Blank solution and automatically subtracts
it from the sample readings (zero point). The best "true"
blank is the natural water that has been filtered through a GF/F
or membrane filter in order to remove the algal cells but to retain
the dissolved components. However, distilled water can be used for
the Blank since the in vivo readings are semi-quantitative.
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Extracted
Chlorophyll a Calibration:
The
Aquafluor Model 8000-001 is designed for in vivo chlorophyll
detection by exciting the natural water sample at 460 nm.
This Model should not be used for extracted samples because
solvents produce a matrix shift that puts the Chlorophyll
a fluorescence peak at 430 nm. You will also find the
plastic cuvettes are not compatible with most solvents. Refer
to the products section of our web page for Fluorometer models
that are equipped for reading extracted Chlorophyll samples.
To
obtain quantitative chlorophyll a results, the in
vivo fluorescence data must be correlated with extracted
chlorophyll a data that can be obtained through the
extraction and measurement of the pigment from grab samples
on a Lab fluorometer, spectrophotometer or HPLC.
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Calibration
Procedure
Equipment
required
A) Aquafluor fluorometer Model 8000-001, (in-vivo chlorophyll model)
B) The Solid Secondary Standard (PN 8000-950)
Procedure
A) Press the <ON/OFF> button. The instrument
will turn on and count down for 5 seconds.
B)
Press the <A/B> button to choose the CHL (chlorophyll)
channel
C)
Press the <STD VAL> button. Use the 
(up) and 
(down) arrows to adjust the standard value to the desired value.
Holding either button down will activate a faster scrolling of the
value.
D)
Press <ENT> to accept the value and to return to the
Home screen.
E)
Press the <CAL> button and Press <ENT>
to start the calibration.
F)
Insert your blank and press <ENT>. The Aquafluor will
average the blank reading for 10 seconds.
G)
Insert the Solid Secondary standard and press <ENT>.
The reading will be averaged for 10 seconds.
| Note
- Do not Blank without a liquid sample because it can result
in a falsely high blank value. Fill the Cuvette at least 2/3rds
full to insure the sample meniscus is above the light path.
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H)
Press <ENT> when the calibration is complete to accept
the calibration. If <ENT> is not pressed within 10
seconds, you will be asked if you want to abort the calibration.
Press the
(up) or
(down)
arrow to abort or accept the calibration respectively. In addition,
the calibration can be aborted at any time by pressing <ESC>
I)
The calibration settings are then stored in the Aquafluor and can
be viewed by pressing the <DIAG> and <ENT>
keys. The %FS- STD value should be significantly higher than the
%FS-Blank value, (>3:1) to provide a good Signal to Blank ratio
for best results. The Aquafluor will turn itself off after a short
period to save battery power. All the settings and calibrations
are retained in non-volatile memory.
J)
Remove the Solid Secondary standard and keep it in a clean and dry
location.
K)
For operating the Turbidity Channel, use an appropriate turbidity
standard solution and perform the calibration. The Turbidity channel
comes with the factory calibration default of 100 NTU. Turbidity
standards can be obtained at APS Analytical Standards, refer to
this web site, http://www.apsstd.com
L)
You are now ready to read your samples by inserting the sample and
pressing the <READ> button.
M)
The readings can be stored for later download by activating the
Logging function. See the User Manual for details.
Click here to see a video of this calibration procedure
For
more detailed information on in vivo and extracted chlorophyll
analysis, please visit our website in the Application Notes section.
