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Appendix D
5/20/2009
1
Chesapeake Biological Laboratory
University of Maryland Center for Environmental Science
Nutrient Analytical Services Laboratory
Fluorometric Determination of Chlorophyll α in waters and sediments
of Fresh/Estuarine/Coastal Areas
.
1.
SCOPE and APPLICATION
1.1
This is an acetone extraction method to determine chlorophyll
α
in water
and sediments.
1.2
A Method Detection Limit (MDL) of 0.90 µg/L total chlα, 1.01 µg/L
phaeophytin, 0.82 µg/L active chlα, and 1.17
µ
g/L non-acid chlα was
determined using 3X the standard deviation of 7 replicates.
1.3
The quantitation limit for chlα is dependent upon sample volume.
1.4
This procedure should be used by analysts experienced in the theory and
application of chlorophyll analysis. Three months experience with an
experienced analyst, certified in the analysis using the fluorometer, is
required.
1.5
This method can be used for all programs that require fluorometric
analysis of chlorophyll α.
1.6
This procedure conforms to EPA Method 445.0
2.
SUMMARY
2.1
Chlorophyll
α
is extracted from the cells using a 90% solution of
acetone. The samples are refrigerated in the dark from 2 to 24 hours
(over night is preferable). After the appropriate time, the samples are
allowed to come to room temperature, and then centrifuged to separate the
sample material from the extract. The extract is analyzed on a
fluorometer. To determine phaeophytin and active chlα, the extract is then
acidified using 5% HCl, and reread. The concentrations are then
calculated
.
3.
DEFINITIONS
3.1
Acceptance Criteria – Specified limits placed on characteristics of an
item, process, or service defined in a requirement document. (ASQC)
3.2
Accuracy – The degree of agreement between an observed value and an
accepted reference value. Accuracy includes a combination of random
error (precision) and systematic error (bias) components which are due
to sampling and analytical operations; a data quality indicator. (QAMS)
3.3
Aliquot – A discrete, measured, representative portion of a sample taken
for analysis. (EPA QAD Glossary)
3.4
Analytical Range – The analytical range is dependent on the volume of
water filtered and the volume of acetone used in the extraction.