EPA Method 447

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EPA Method 447:
Chlorophyll a and b in phytoplankton by HPLC/UV. Official Name: Determination of Chlorophylls a and b and Identification of Other Pigments of Interest in Marine and Freshwater Algae Using High Performance Liquid Chromatography with Visible Wavelength Detection.

A water/phytoplankton sample is ground using a mechanical grinder, and chlorophyll is extracted from phytoplankton with 90% acetone over 2-24 hours. The slurry with the phytoplankton is centrifuged to clarify the solution. An aliquot of the supernatant is filtered. The concentrations of chl a and b in the filtrate are measured using a high performance liquid chromatography (HPLC) system equipped with a ultraviolet/visible (UV/Vis) detector. NOTE: The HPLC/UV system is calibrated using chl a and b standards that have be spectrophotometrically quantified using EPA Method 446.0.

This method determines chlorophylls a (chl a) and b (chl b) in marine and fresh water phytoplankton.

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

(A) Extracted contaminants: Contaminants that are co-extracted with analytes, and which absorb at 440 nm, can interfere.(B) Storage and sample preparation conditions: Keep samples cold (-20 to -70 degrees Celsius) and away from light during storage and sample preparation to prevent degradation.(C) Prolonged extraction: Prolonged extraction may lead to pigment degradation.(D) Turbidity: Samples must be filtered to prevent fouling the HPLC column.

QC Requirements:
Each laboratory must maintain a formal quality control program. At a minimum, this program must include an initial demonstration of laboratory capability and the continued analysis of laboratory reagent blanks, field replicates, QC samples, and calibration checks as a continuing check on performance.

Maximum Holding Time:
3.5 weeks at -20o



Not available.

For Chl a, precision was calculated using 10 observations in 5 laboratories of a 1.384 mg/L sample of chl a collected from 100 mL of filtered sea water (see Table 8 of the method). Additional data, including species-specific data, are available in the method.

Pooled Estimated Detection Limit, based on data from nine laboratories. Additional data are available in the method.

Revision Number:
Version 1.0, September 1997