YSI ADV6600 - Manual (Page 131)

Section 9. Principles of Operation
ADV6600
Y S I
Environmental
Page 121
As noted above, the measurement of chlorophyll from in vivo fluorescence measurements will
always be less reliable than determinations made on molecular chlorophyll that has been extracted
from the cells using the procedures described in Standard Methods. This section describes some of
the known problems with in vivo chlorophyll measurement.
Interferences from other fluorescent species
The analytical methods described in Standard Methods for chlorophyll involve disruption of the
living organisms present in suspension, followed by extraction of molecular chlorophyll into a
homogeneous solution in an organic solvent. Acidification of the extract helps to minimize the
interference caused by a number of other, non-chlorophyll species. In addition, readings can be
taken at various wavelengths on a spectrophotometer to differentiate between the various forms of
chlorophyll (a, b, c) and pheophytin a.
In contrast to this fairly controlled situation, all in vivo sensors operate under whole-cell,
heterogeneous conditions where the sensor will measure, at least to some degree, everything which
fluoresces in the region of the spectrum above 630 nm when irradiated with 470 nm light.
Therefore, the sensor is really quantifying overall fluorescence under these optical conditions, rather
than chlorophyll specifically. While it is probable that most of the fluorescence is due to suspended
plant and algal matter and that much of the fluorescence from this biomass is due to chlorophyll, it
is impossible to exclude interference from other fluorescent species using the approach described
above.
Note that in vivo fluorometers usually cannot differentiate between the different forms of
chlorophyll.
Lack of calibration reagents
The usual reagents which are used for the calibration of fluorometric measurements for chlorophyll
after extraction into organic solvents are purchased as “purified chlorophyll a” from chemical
supply vendors such as Sigma. These standards are not soluble in aqueous media and, even if they
were, their fluorescence is unlikely to be the same as when the chlorophyll is present in the whole
living cell. Therefore, for even a semi-quantitative calibration, the user needs a “substitute”
standard such as rhodamine WT (see above) to provide a method for estimating the sensitivity of the
sensor. Field readings based on this type of calibration will provide only an estimate of chlorophyll
in environmental water where the measurement is taken on whole cell suspensions in vivo. The
calibration standard that provides the best measure of accuracy for in vivo chlorophyll sensors is a
portion of a phytoplankton suspension that has been analyzed for chlorophyll by the extractive
procedure. We recommend the use of this procedure and further recommend that the phytoplankton
suspension be taken from the site being monitored so that the species producing the fluorescence in
the standard are as close as possible to the field organisms. To truly assess data reliability in a long
term monitoring study, grab samples should be taken periodically, e.g. weekly, and analyzed in the
laboratory as the study progresses. These data can then be used to “postcalibrate” the readings
logged to the instrument during the study, perhaps using a spreadsheet for the simple mathematical
treatment. In any case, getting quantitative chlorophyll data from any in vivo fluorometric sensor is
much more difficult than with most other environmental sensors. For this reason, it is difficult to