EPA-NERL: 447.0: Chlorophyll a and b in phytoplankton by HPLC/UV

Summary
Analytes
Revision
Data and Sites

Official Method 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.

Current Revision

Version 1.0, September 1997

Media

WATER

Instrumentation

High Performance Liquid Chromatography with Ultraviolet Detection

Method Subcategory

Biochemical

Method Source

EPA-NERL

Citation

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

Brief Method Summary

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.

Scope and Application

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

Applicable Concentration Range

Not available.

Interferences

(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.

(D) Turbidity: Samples must be filtered to prevent fouling the HPLC column.

Quality Control 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.

Sample Handling

Water may be obtained by a pump or grab sampler. All apparatus should be clean and acid-free. Filtering should be performed in subdued light as soon as possible after sampling; aboard ship filtration is highly recommended. If the filter will not be immediately extracted, then wrap the container with aluminum foil to protect the phytoplankton from light and store the filter at -20^oC or -70^oC. Short term storage (2 to 4 h) on ice is acceptable but samples should be store at -20^oC or -70^oC as soon as possible in the dark until extraction.

Maximum Holding Time

3.5 weeks at -20^o

Relative Cost

Unknown

Sample Preparation Methods

This method has 2 analytes associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
Chlorophyll a(479-61-8) Chl a Chlorophyll a	0.081 mg/L	N/A	15.40 % RSD (SL)			1.38 mg/L
Chlorophyll b(519-62-0) Chl b Chlorophyll b	N/A	N/A	N/A

Precision Descriptor Notes:	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.
Detection Level Note:	Pooled Estimated Detection Limit, based on data from nine laboratories. Additional data are available in the method.

Revision	PDF/Link
Version 1.0, September 1997

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EPA-NERL: 447.0: Chlorophyll a and b in phytoplankton by HPLC/UV

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