EPA-NERL: 445.0: Chlorophyll and Pheophytin in Algae by Fluorescence

Summary
Analytes
Revision
Data and Sites

Official Method Name

In Vitro Determination of Chlorophyll a and Pheophytin a in Marine and Freshwater Algae by Fluorescence

Current Revision

Revision 1.2, September 1997

Media

WATER

Instrumentation

Fluorescence Spectroscopy

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

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.

Scope and Application

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.

Applicable Concentration Range

Not available.

Interferences

(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 +/- 3^oC in subdued light conditions. During storage, samples must be kept at -20 to -70^oC in the dark, to prevent degradation.

(E) Turbidity: Samples must be clarified by centrifugation prior to analysis.

Quality Control 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.

Sample Handling

Samples are collected using a pump or grab sampler. Enough water should be collected to concentrate phytoplankton on three or more filters. Four liters may be required for ocean water (where phytoplankton density is usually low), whereas one liter is generally sufficient for lake, bay, and estuarine water. The method provides detailed information on the procedure for collecting samples. Once collected and filtered, the sample may be stored 2-4 hours on ice in the dark, but should be stored at -20 or -70^oC as soon as possible. Filters frozen at -20^oC may be stored as long as 3.5 weeks without significant loss of chlorophyll a.

Maximum Holding Time

3.5 weeks at -20^oC

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	0.050 ug/L	N/A	15.00 % RSD (ML)			1576.00 ug/L
Pheopigments of Chlorophyll(N-55812) Pheo a Pheopigments of Chlorophyll	0.060 ug/L	N/A	N/A

Precision Descriptor Notes:	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.
Detection Level Note:	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.

Precision Descriptor Notes:

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.

Detection Level Note:

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	PDF/Link
Revision 1.2, September 1997

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EPA-NERL: 445.0: Chlorophyll and Pheophytin in Algae by Fluorescence

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