YSI 600DW-B Sonde - Manual (Page 234)

Principles of Operation Section 5
is not perfect and a minor interference on chlorophyll readings from suspended solids may result.
Laboratory experiments indicate that a suspension of typical soil measured with a YSI 6026 sensor will
have a turbidity interference characterized by a factor of about 0.03 µg/L per NTU. For example, the
turbidity of the water must be above 100 NTU to produce an apparent chlorophyll reading equal to 3 µg/L.
In very cloudy water, the user may wish to use the independently-determined turbidity value and the above
compensation factor to correct measured chlorophyll values using, for example, a spreadsheet.
LIMITATIONS OF
IN VIVO CHLOROPHYLL MEASUREMENTS
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 interferences 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 interferences 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 semiquantitative calibration, the user needs a
“substitute” standard such as Acridine Orange (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 provide an accuracy specification for chlorophyll
measurement made with in vivo fluorometers and therefore no accuracy specification is quoted for the YSI
6025.
YSI Incorporated Drinking Water Monitoring Systems Operation Manual 5-21