EPA-NERL: 446.0: Chlorophylls and Pheopigments in Phytoplankton by Spectrophotometry

Summary
Analytes
Revision
Data and Sites

Official Method Name

In Vitro Determination of Chlorophylls a, b, c1 + c2 and Pheopigments in Marine and Freshwater Algae by Visible Spectrophotometry

Current Revision

Revision 1.2, September 1997

Media

WATER

Instrumentation

Spectroscopy (Colorimetry; Photometry)

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 absorbance of the supernatant liquid is measured at 4 wavelengths to determine turbidity, and chlorophylls a, b, and c1 + c2. Pheopigment-correct chl a can be determined by using absorbance measurements from an acidified and non-acidified sample. Absorbance values are entered into a set of equations to that utilize the extinction coefficients of the pure pigments in 90% acetone to simultaneously calculate the concentrations of the pigments in a mixed solution.

Scope and Application

This method determines chlorophylls a, b and c1 + c2, and pheopigments of chlorophyll a found in marine and freshwater phytoplankton.

Applicable Concentration Range

Interferences

(A) Absorbing materials, generally: Any material that absorbs near the measured wavelengths can interfere with measurements. High absorption at 750 nm (>= 0.005 AU) indicates a poorly clarified solution, requiring centrifugation or filtration prior to analysis.

(B) Spectral overlap of pigments: The relative amounts of chlorophyll a, b, and c1 + c2 vary with the taxonomic composition of the phytoplankton. Due to the spectral overlap of the chlorophylls and pheopigments, over- or underestimation of the pigments is inevitable in solutions containing all of these pigments. Knowledge of taxonomic composition of samples and proper analytical technique can reduce errors, as discussed extensively in the method.

(C) Steeping: The amount of sample steeping can effect precision and recovery by effecting extraction of target pigments and of interfering substances.

(D) Temperature and light: All photosynthetic pigments are light and temperature sensitive. Work must be performed in subdued light and all standards, QC materials, and filtered samples must be stored in the dark at -20 or -70^oC to prevent rapid degradation.

Quality Control Requirements

Each laboratory using this method is required to implement a formal quality control (QC) program. The minimum requirements of this program consist of an initial demonstration of performance, continued analysis of laboratory reagent blanks, field replicates and QC 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 6 analytes associated with it.

Analyte	Detection Level	Bias	Precision	Spiking Level
Chlorophyll a(479-61-8) Chl a Chlorophyll a	0.080 mg/L	N/A	9.80 % RSD (SL)	1.64 mg/L
Chlorophyll b(519-62-0) Chl b Chlorophyll b	0.093 mg/L	N/A	24.43 % RSD (SL)	0.09 mg/L
Chlorophyll c1(18901-56-9) Chl c1 Chlorophyll c1	N/A	N/A	N/A
Chlorophyll c1 + c2(N-55801) Chl c1 + c2 Chlorophyll c1 + c2	N/A	N/A	9.65 % RSD (SL)	0.29 mg/L
Chlorophyll c2(27736-03-4) Chl c2 Chlorophyll c2	N/A	N/A	N/A
Pheopigments of Chlorophyll(N-55812) Pheo a Pheopigments of Chlorophyll	0.085 mg/L	N/A	40.53 % RSD (SL)	0.17 mg/L

Precision Descriptor Notes:	For chl a, precision was calculated using trichromatic calculations, with 36 observations in 18 laboratories of a 1.636 mg/L chl a sample collected from 100 mL of filtered sea water (see Table 11 of the method). For chl b and chl c1+c2, precision was calculated from data generated at a single lab by analyzing seven replicates of a sample from Raritan Bay using a final extraction volume of 10 mL, 24 steeping, and trichromatic equations. Modified monochromatic equations were used to calculate precision of pheo a (see Table 4 of the method). Additional data, including species-specific data, are available in the method.
Detection Level Note:	Instrument Detection Limits (IDLs) were determined using 90% acetone solution with a 1 cm glass cells. IDLs and other detection limits can be lowered by using cells with longer path lengths.

Precision Descriptor Notes:

For chl a, precision was calculated using trichromatic calculations, with 36 observations in 18 laboratories of a 1.636 mg/L chl a sample collected from 100 mL of filtered sea water (see Table 11 of the method). For chl b and chl c1+c2, precision was calculated from data generated at a single lab by analyzing seven replicates of a sample from Raritan Bay using a final extraction volume of 10 mL, 24 steeping, and trichromatic equations. Modified monochromatic equations were used to calculate precision of pheo a (see Table 4 of the method). Additional data, including species-specific data, are available in the method.

Detection Level Note:

Instrument Detection Limits (IDLs) were determined using 90% acetone solution with a 1 cm glass cells. IDLs and other detection limits can be lowered by using cells with longer path lengths.

Revision	PDF/Link
Revision 1.2, September 1997

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EPA-NERL: 446.0: Chlorophylls and Pheopigments in Phytoplankton by Spectrophotometry

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