EPA Method 445

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EPA Method 445:
Chlorophyll and Pheophytin in Algae by Fluorescence. Official Name: In Vitro Determination of Chlorophyll a and Pheophytin a in Marine and Freshwater Algae by Fluorescence

Chlorophyll-containing phytoplankton in a measured volume of sample water are concentrated by filtration at low vacuum through a glass fiber filter. The pigments are extracted from the phytoplankton in 90% acetone with the aid of a mechanical tissue grinder and are allowed to steep 2-24 hours. The resulting slurry is centrifuged to clarify the solution, and the fluorescence of the supernatant liquid is measured before an after acidification. Sensitivity calibration factors, previously determined on solutions of pure chlorophyll a of known concentration are used to calculate the concentration of chlorophyll a and pheophytin a in the sample extract.

This method determines low levels of chlorophyll a (chl a) and its magnesium-free derivative, pheophytin a (pheo a), in marine and freshwater phytoplankton using fluorescence detection. Phaeophorbides present in the sample are determined collectively as pheo a.

Methods for Determination of Chemical Substances in Marine and Estuarine Matrices - 2nd Edition (EPA/600/R-97/072)

(A) Fluorescing materials, generally: Any substance in the sample that fluoresces in the red region of the spectrum may interfere.(B) Spectral overlap of pigments: The relative amounts of chlorophyll a, b, and c vary with the taxonomic composition of the phytoplankton. Chlorophylls b and c may significantly interfere with chlorophyll a and pheophytin a measurements depending on the amounts present. Knowledge of the taxonomy of the algae under consideration will aid in determining if the spectrophotometric method using trichromatic equations (EPA Method 446.0) or an HPLC Method (EPA Method 447.0) is more appropriate.(C) Quenching: Quenching effects are observed in highly concentrated solutions of chlorophylls or carotenoids. Minimum sensitivity settings on the fluorometer should be avoided; samples should be diluted instead.(D) Temperature and light: Fluorescence is temperature and light sensitive. All samples and standards should be measured at an ambient temperature which does not vary by more than +/- 3oC in subdued light conditions. During storage, samples must be kept at -20 to -70oC in the dark, to prevent degradation.(E) Turbidity: Samples must be clarified by centrifugation prior to analysis.

QC Requirements:
Each laboratory using this method is required to use a formal quality control program. At a minimum, this program should include an initial demonstration of laboratory capability and the continued analysis of laboratory reagent blanks, field duplicates, and quality control samples as a continuing check on performance.

Maximum Holding Time:
3.5 weeks at -20oC



Not available.

Precision and accuracy values were generated using data from the conventional fluorometric method with pheophytin a correction (as opposed to using special interference filters). Precision was calculated using 15 observations in 8 laboratories of a 1.576 mg/L chlorophyll a sample collected from 100 mL of filtered sea water (see Table 9 of the Method). Additional data, including species-specific data, are available in the method.

Instrument Detectio Limits (IDLs) determined using a 90% acetone solution. Method detection limits using mixed assemblages of algae provide little information because the fluoresence of other pigments interferes in the fluoresence of chlorophyll a and pheophytin a.

Revision Number:
Revision 1.2, September 1997

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