447.0-1

Method 447.0

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

Elizabeth J. Arar

Version 1.0

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Version 1.0 September 1997 447.0-2

METHOD 447.0

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

1.0 Scope and Application

1.1 This method provides a procedure for

determination of chlorophylls a (chl a) and b (chl b) found

in marine and freshwater phytoplankton. Reversedphase high performance liquid chromatography (HPLC)

with detection at 440 nm is used to separate the pigments

from a complex pigment mixture and measure them in

the sub-microgram range. For additional reference, other

taxonomically important yet commercially unavailable

pigments of interest are identified by retention time.

1.2 This method differs from previous descriptions of

HPLC methods in several respects. Quality

assurance/quality control measures are described in

Sect. 9.0, sample collection and extraction procedures

are described in Sect. 8.0 and reference chromatograms

of pure pigments and reference algae are provided.

This method has also been evaluated in a multilaboratory

study along with EPA Methods 445.0 and 446.0.

Estimated detection limits, precision and bias are reported

in Section 13. 

 

Chemical Abstracts Service

Analyte Registry Number (CASRN)

 

Chlorophyll a 479-61-8

Chlorophyll b 519-62-0

 

1.3 Instrumental detection limits (IDLs) of 0.7 ng chl

a, and 0.4 ng chl b in pure solutions of 90% acetone were

determined by this laboratory. Method detection limit

(MDL) determinations were made by analyzing seven

replicate unialgal samples containing the chl a and b.

Single-laboratory MDLs were chl a - 7 ng and chl b - 4 ng.

A multilaboratory estimated detection limit (EDL) (in mg/L

of extract is reported in Section 13.

1.4 Most taxonomically important pigments are not

commercially available, therefore, a laboratory must be

willing to extract and purify pigments from pure algal

cultures to quantify and qualitatively identify these very

important pigments. This method contains

chromatographic information of select pure pigments

found either in marine or freshwater algae. The

information is included to aid the analyst in qualitatively

identifying individual pigments and possibly algal species

in natural samples.

1.5 This method uses 90% acetone as the extraction

solvent because of its efficiency for extracting chl a from

most types of algae. (NOTE: There is evidence that

certain chlorophylls and carotenoids are more thoroughly

extracted with methanol or dimethyl sulfoxide.) Using (1-3) (4)

high performance liquid chromatography (HPLC),

Mantoura and Llewellyn found that methanol led to the (5)

formation of chl a derivative products, whereas 90%

acetone did not. Bowles, et al.(3) found that for chl a 90%

acetone was an effective solvent when the steeping

period was optimized for the predominant species

present.) 

1.6 One of the limitations of visible wavelength

detection is low sensitivity. It may be preferable to use

fluorometry or HPLC with fluorometric detection if

(6-8) (913)

high volumes of water (>4 L) must be filtered to obtain

detectable quantities of chl a or b. 

1.7 This method is for use by analysts experienced in

handling photosynthetic pigments and in the operation of

HPLC or by analysts under the close supervision of such

qualified persons.

2.0 Summary of Method

2.1 The HPLC is calibrated with a chl a and b

solution that has been spectrophotometrically quantified
 447.0-3 Version 1.0 September 1997

according to EPA Method 446. Chlorophyll-containing reagents, or apparatus. For this method the LRB is a

phytoplankton in a measured volume of sample water are blank filter that has been extracted as a sample.

concentrated by filtration at low vacuum through a glass

fiber filter. The pigments are extracted from the 3.6 Material Safety Data Sheet (MSDS) -- Written

phytoplankton into 90% acetone with the aid of a information provided by vendors concerning a chemical's

mechanical tissue grinder and allowed to steep for a toxicity, health hazards, physical properties, fire, and

minimum of 2 h, but not exceeding 24 h, to ensure reactivity data including storage, spill, and handling

thorough extraction of the pigments. The filter slurry is precautions.

centrifuged at 675 g for 15 min (or at 1000 g for 5 min) to

clarify the solution. An aliquot of the supernatant is 3.7 Method Detection Limit (MDL) -- The minimum

filtered through a 0.45 μm syringe filter and 50 to 200 μL

is injected onto a reversed-phase column. Following

separation using a ternary gradient, concentrations are

reported in μg/L (ppb) or mg/L (ppm) in the whole water

sample. This method is based on the HPLC work of

Wright, et. al.(9)

3.0 Definitions

3.1 Calibration Standard (CAL) -- A solution

prepared from dilution of a stock standard solution. The

CAL solution is used to calibrate the instrument response

with respect to analyte concentration or mass.

3.2 Calibration Check Standard (CALCHK) -- A

mid-point calibration solution that is analyzed periodically

in a sample set to verify that the instrument response to

the analyte has not changed during the course of

analysis.

 

3.3 Field Replicates -- Separate samples collected

at the same time and placed under identical

circumstances and treated exactly the same throughout

field and laboratory procedures. Analyses of field

replicates give a measure of the precision associated with

sample collection, preservation and storage, as well as

with laboratory procedures.

3.4 Instrument Detection Limit (IDL) -- The

minimum quantity of analyte or the concentration

equivalent that gives an analyte signal equal to three

times the standard deviation of a background signal at the

selected wavelength, mass, retention time, absorbance

line, etc. 

3.5 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water or other blank matrices that are treated

exactly as a sample including exposure to all glassware,

equipment, solvents, reagents, internal standards, and

surrogates that are used with other samples. The LRB is

used to determine if method analytes or other

interferences are present in the laboratory environment,

concentration of an analyte that can be identified,

measured and reported with 99% confidence that the

analyte concentration is greater than zero.

3.8 Quality Control Sample (QCS) -- A solution of

method analytes of known concentrations that is used to

fortify an aliquot of LRB or sample matrix. Ideally, the

QCS is obtained from a source external to the laboratory

and different from the source of calibration standards. It

is used to check laboratory performance with externally

prepared test materials.

4.0 Interferences

4.1 Any compound extracted from the filter or

acquired from laboratory contamination that absorbs light

at 440 nm may interfere in the accurate measurement of

the method analytes. 

4.2 Proper storage and good sample handling

technique are critical in preventing degradation of the

pigments.

4.3 Precision and recovery for any of the pigments is

related to efficient extraction, i.e. efficient maceration of

the filtered sample and to the steeping period of the

macerated filter in the extraction solvent. Precision

improves with increasing steeping periods, however, a

drawback to prolonged steeping periods is the possibility

of pigment degradation. The extracted sample must be

kept cold and in the dark to minimize degradation. 

4.4 Sample extracts must be clarified by filtration

through a 0.45 μm filter prior to analysis by HPLC to

prevent column fouling.

4.5 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 C or o

 -70 C to prevent rapid degradation. o
Version 1.0 September 1997 447.0-4

5.0 Safety

5.1 Each chemical used in this method should be

regarded as a potential health hazard and handled with

caution and respect. Each laboratory is responsible for

maintaining a current awareness file of Occupational

Safety and Health Administration (OSHA) regulations

regarding the safe handling of the chemicals specified in

this method. A file of MSDS also should be made

(15-18)

available to all personnel involved in the chemical

analysis.

5.2 The grinding of filters during the extraction step of

this method should be conducted in a fume hood due to

the volatilization of acetone by the tissue grinder.

6.0 Apparatus and Equipment

6.1 Centrifuge, capable of 675 g.

6.2 Tissue grinder, Teflon pestle (50 mm X 20 mm)

with grooves in the tip with 1/4" stainless steel rod long

enough to chuck onto a suitable drive motor and 30-mL

capacity round-bottomed, glass grinding tube.

6.3 Filters, glass fiber, 47-mm or 25-mm nominal

pore size of 0.7 μm unless otherwise justified by data

quality objectives. Whatman GF/F filters were used in this

work.

6.4 Petri dishes, plastic, 50 X 9-mm, or some other

solid container for transporting and storing sampled

filters.

6.5 Aluminum foil.

6.6 Laboratory tissues.

6.7 Tweezers or flat-tipped forceps.

6.8 Vacuum pump or source capable of maintaining

a vacuum up to 6 in. Hg (20 KPa).

6.9 Labware -- All reusable labware (glass,

polyethylene, Teflon, etc.) that comes in contact with

chlorophyll solutions should be clean and acid free. An

acceptable cleaning procedure is soaking for 4 h in

laboratory grade detergent and water, rinsing with tap

water, distilled deionized water and acetone.

6.9.1 Assorted Class A calibrated pipets.

6.9.2 Graduated cylinders, 500-mL and 1-L.

6.9.3 Volumetric flasks, Class A calibrated, 10-mL, 25-

mL, 50-mL, 100-mL and 1-L capacity.

6.9.4 Glass rods or spatulas.

6.9.5 Pasteur Type pipets or medicine droppers.

6.9.6 Filtration apparatus consisting of 1 or 2-L filtration

flask, 47-mm fritted glass disk base and a glass filter

tower.

6.9.7 Centrifuge tubes, polypropylene or glass, 15-mL

capacity with nonpigmented screw-caps.

6.9.8 Polyethylene squirt bottles.

6.9.9 Amber 2-mL HPLC autosampler vials with screw

or clamp caps.

6.9.10 Glass syringe, 1 or 2-mL capacity.

6.9.11 HPLC compatible, low-volume, acetone resistant

glass fiber or PTFE syringe filters.

6.10 Liquid Chromatograph

6.10.1 This method uses a ternary gradient thus

requiring a programmable gradient pump with at least

three pump inlets for the three different mobile phases

required. A Dionex Model 4500 chromatograph equipped

with a gradient pump, UV/VIS detector (cell path length,

6 mm, volume 9 μL) and PC data analysis (Dionex AI450

software, Version 3.32) system was used to generate

data for this method. Tubing was made of polyether ether

ketone (PEEK). A Dionex degas module was used to

sparge all eluents with helium. 

6.10.2 Helium or other inert gas for degassing the

mobile phases OR other means of degassing such as

sonication under vacuum.

6.10.3 Sample loops of various sizes (50-200 μL).

6.10.4 Guard Column -- A short column containing the

same packing material as the analytical column placed

before the analytical column to protect it from fouling by

small particles. The guard column can be replaced

periodically if it is noticed that system back pressure has

increased over time.
 447.0-5 Version 1.0 September 1997

6.10.5 Analytical Column -- A C18 reversed-phase the graduated cylinder and transfer to the flask or bottle

column with end capping. A J.T. Baker 4.6 mm X 250 containing the water. Mix, label and store.

mm, 5 μm pore size column was used to generate the

data in this method.

6.10.6 A visible wavelength detector with a low volume

flow-through cell. Detection is at 440 nm.

6.10.7 A recorder, integrator or computer for recording

detector response as a function of time.

6.10.8 Although not required, an autosampler

(preferably refrigerated) is highly recommended.

7.0 Reagents and Standards

7.1 Acetone, HPLC grade, (CASRN 67-64-1). 

7.2 Methanol, HPLC grade, (CASRN 67-56-1).

Prepare ELUENT A, 80% (v/v) methanol/20% 0.5 M

ammonium acetate, by adding 800 mL of methanol and

200 mL of the 0.5 M ammonium acetate (Sect. 7.5) to an

eluent container.

7.3 Acetonitrile, HPLC grade, (CASRN 75-05-8).

Prepare ELUENT B, 90% (v/v) acetonitrile/10% water, by

adding 900 mL of acetonitrile and 100 mL of water (Sect.

7.7) to an eluent container.

7.4 Ethyl acetate, HPLC grade, (CASRN 141-78-6).

ELUENT C, 100% ethyl acetate.

7.5 Ammonium acetate, ACS grade (CASRN 631-

61-8). Prepare a 0.5 M solution by dissolving 38.54 g in

approximately 600 mL of water in a 1-L volumetric flask.

After the ammonium acetate has dissolved, dilute to

volume with water.

7.6 Chl a free of chl b and chl b substantially free of

chl a may be obtained from a commercial supplier such

as Sigma Chemical (St. Louis, MO). 

7.7 Water -- ASTM Type I water (ASTM D1193) is

required. Suitable water may be obtained by passing

distilled water through a mixed bed of anion and cation

exchange resins.

7.8 Aqueous Acetone Solution -- 90% acetone/10%

ASTM Type I water. Carefully measure 100 mL of the

water into the 1-L graduated cylinder. Transfer to a 1-L

flask or storage bottle. Measure 900 mL of acetone into

7.9 Chlorophyll Stock Standard Solution (SSS) --

Chl a (MW = 893.5) and chl b (MW = 907.5) from a

commercial supplier is shipped in amber glass ampules

that have been flame sealed. The dry standards must be

stored at -20 or -70EC in the dark. Tap the ampule until

all the dried pigment is in the bottom of the ampule. In

subdued light, carefully break the tip off the ampule.

Transfer the entire contents of the ampule into a 25-mL

volumetric flask. Dilute to volume with 90% acetone: (1

mg in 25 mL = 40 mg chl a/L) and (1 mg in 25 ml = 40

mg chl b/L), label the flasks and wrap with aluminum foil

to protect from light. When stored in a light- and air-tight

container at -20 or -70 C, the SSS is stable for at least six o

months. Dilutions of the SSS should always be confirmed

spectrophotometrically using EPA Method 446.

7.10 Laboratory Reagent Blank (LRB) -- A blank

filter that is extracted and analyzed just as a sample filter.

The LRB should be the last filter extracted of a sample

set. It is used to assess possible contamination of the

reagents or apparatus.

7.11 Quality Control Sample (QCS) -- Since there

are no commercially available QCSs, dilutions of a stock

standard of a different lot number from that used to

prepare calibration solutions may be used.

8.0 Sample Collection, Preservation and

Storage

8.1 Water Sample Collection -- Water may be

obtained by a pump or grab sampler. Data quality

objectives will determine the depth and frequency at

(21)

which samples are taken. Healthy phytoplankton,

however, are generally obtained from the photic zone

(region in which the illumination level is 1% of surface

illumination). Enough water should be collected to

concentrate phytoplankton on at least three filters so that

precision can be assessed. Filtration volume size will

depend on the particulate load of the water. Four liters

may be required for open ocean water where

phytoplankton density is usually low, whereas 1 L or less

is generally sufficient for lake, bay or estuary water. All

apparatus should be clean and acid-free. Filtering should

be performed in subdued light as soon as possible after

sampling since algal populations, thus pigment

concentrations, can change in relatively short periods of

time. Aboard ship filtration is highly recommended.
Version 1.0 September 1997 447.0-6

Assemble the filtration apparatus and attach the vacuum 9.2 Initial Demonstration of Performance

source with vacuum gauge and regulator. Vacuum (Mandatory)

filtration should not exceed 6 in. Hg (20 kPa). Higher

filtration pressures may damage cells and result in loss of 9.2.1 The initial demonstration of performance is used

chlorophyll. Care must be taken not to overload the to characterize instrument performance (IDLs) and

filters. Do not increase the vacuum during filtration. laboratory performance (MDLs, extraction proficiency,

Prior to drawing a subsample from the water sample

container, gently stir or invert the container several times 9.2.2 Instrumental Detection Limit (IDL) -- After a low

to suspend the particles. Pour the subsample into a level calibration (Sect. 10), prepare a standard solution

graduated cylinder and accurately measure the volume. that upon injection into the chromatograph yields an

Pour the subsample into the filter tower of the filtration absorbance of 0.002-0.010. If using an autosampler,

apparatus and apply a vacuum (not to exceed 20 kPa). variable volumes may be injected and the micrograms

Typically, a sufficient volume has been filtered when a

visible green or brown color is apparent on the filter. Do

not suck the filter dry with the vacuum; instead slowly

release the vacuum as the final volume approaches the

level of the filter and completely release the vacuum as

the last bit of water is pulled through the filter. Remove

the filter from the fritted base with tweezers, fold once

with the particulate matter inside, lightly blot the filter with

a tissue to remove excess moisture and place it in the

petri dish or other suitable container. If the filter will not

be immediately extracted, wrap the container with

aluminum foil to protect the phytoplankton from light and

store the filter at -20 C or -70 C. Short term storage (2 to o o

4 h) on ice is acceptable, but samples should be stored

at -20EC or -70 C as soon as possible. o

8.2 Preservation -- Sampled filters should be stored

frozen (-20 C or -70 C) in the dark until extraction. o o

8.3 Holding Time -- Filters can be stored frozen at 

-20 C for as long as 31⁄2 weeks without significant loss of o

chl a. 

(20)

9.0 Quality Control

9.1 Each Laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability and the continued

analysis of laboratory reagent blanks, field replicates,

QCSs, and CALCHKs as a continuing check on

performance. The laboratory is required to maintain

performance records that define the quality of the data

generated.

and analyses of QCSs) prior to sample analyses.

(μg) injected calculated by multiplying the known

concentration (μg/μL) of the standard by the volume

injected (μL). A practical starting point may be to inject

0.05 μg (that would be a 50 μL injection of a 1.0 mg/L

standard solution) and reduce or increase the mass

injected according to the resulting signal. Avoid injecting

really small volumes (< 10 μL). After the quantity of

pigment has been selected, make three injections and

calculate the IDL by multiplying the standard deviation of

the calculated mass by 3.

9.2.3 Method Detection Limit (MDL) -- At least seven

natural phytoplankton samples known to contain the

pigments of interest should be collected, extracted and

analyzed according to the procedures in Sects. 8 and 11,

using clean glassware and apparatus. Mass of the

pigment injected into the chromatograph should be 2 to

5 times the IDL. Dilution of the sample extract solution to

the appropriate concentration or reducing the volume of

sample injected may be necessary. Calculate the MDL

(in micrograms) as follows.(19)

 MDL = (t) X (S)

where, t = Student's t-value for n-1 degrees of freedom at

the 99% confidence level. t = 3.143 for six degrees of

freedom.

S = Standard deviation of the replicate analyses.

9.2.4 Quality Control Sample (QCS) -- When beginning

to use this method, on a quarterly basis or as required to

meet data quality needs, verify instrument performance

with the analysis of a QCS (Sect. 7.11). If the determined

value is not within +10% of the spectrophotometrically

determined value, then the instrument should be

recalibrated with fresh stock standard and the QCS

reanalyzed. If the redetermined value is still

unacceptable then the source of the problem must be

identified and corrected before continuing analyses.
 447.0-7 Version 1.0 September 1997

9.2.5 Extraction Proficiency -- Personnel performing 10.3 Program the pump with the following gradient:

this method for the first time should demonstrate

proficiency in the extraction of sampled filters (Sect. 11.1). Time Flow %1 %2 %3 Condition

Fifteen to twenty natural samples should be obtained 0.0 1.0 100 0 0 Injection

using the procedure outlined in Sect. 8.1 of this method. 2.0 1.0 0 100 0 Linear Gradient

Sets of 10 samples or more should be extracted and 2.6 1.0 0 90 10 Linear Gradient

analyzed according to Sect. 11. The percent relative 13.6 1.0 0 65 35 Linear Gradient

standard deviation (%RSD) should not exceed 15% for 20.0 1.0 0 31 69 Linear Gradient

samples that are at least 10X the IDL. 22.0 1.0 0 100 0 Linear Gradient

9.3 Assessing Laboratory Performance 30.0 1.0 100 0 0 Equilibration

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) -- The

laboratory must analyze at least one blank filter with each 10.4 The first analysis is a blank 90% acetone solution

sample batch. The LRB should be the last filter followed by calibration. Calibrate with at least three

extracted. LRB data are used to assess contamination concentrations, covering no more than one order of

from the laboratory environment. LRB values that exceed magnitude, and bracketing the concentrations of

the IDL indicate contamination from the laboratory samples. If an autosampler is used, variable volumes

environment. If the LRB value constitutes 10% or more ranging from 10 - 100% of the sample injection loop

of the analyte level determined in a sample, fresh volume are injected to give a calibration of detector

samples or field replicates must be analyzed after the response versus mass of pigment. If doing manual

contamination has been corrected and acceptable LRB injections, variable solution concentrations are made and

values have been obtained. a fixed sample loop volume is injected for standards and

9.3.2 Calibration Check Standard (CALCHK) -- The versus mass or detector response versus concentration

laboratory must analyze one CALCHK for every ten

samples to verify calibration. If the CALCHK is not +10%

of the spectrophotometrically determined concentration,

then the instrument must be recalibrated. 

10.0 Calibration and Standardization

10.1 Allow the visible wavelength detector (440 nm) to

warm up for at least 15 min before calibration. Prepare

ELUENTS A - C and degas by sparging with an inert gas

for 10 minutes or sonicating under vacuum for 5 minutes.

Prime the pump for each eluent taking care to remove all

air that may be in the liquid lines. Equilibrate the column

for ten minutes with 100% of ELUENT A. 

10.2 Remove the SSS from the freezer and allow it to

come to room temperature. Add 1 mL of the SSS to a

10-mL volumetric flask and dilute to 10 mL with 90%

acetone. Prepare the chl a and b separately and

determine the concentrations according to EPA Method

446 using the monochromatic equations for chl a

determination. After the concentration of the SSS is

determined, add 1 mL of the chl a SSS plus 1 mL of the

chl b SSS to a separate 10-mL flask and dilute to volume.

Store the calibration standard in a light tight glass bottle.

 

25.0 1.0 100 0 0 Linear Gradient

Flow is in mL/min.

samples. Calibration can be either detector response

(mg/L or μg/L). Linearity across sensitivity settings of the

detector must be confirmed if samples are analyzed at a

different sensitivity settings from that of the calibration. 

10.5 Construct a calibration curve of analyte response

(area) versus concentration (mg/L in solution) or mass

(μg) of pigment and perform a linear regression to

determine the slope and y-intercept. A typical coefficient

of determination is > 0.99. 

10.6 Calibration must be performed at least weekly

although it is not necessary to calibrate daily. Daily midpoint CALCHKs must yield calculated concentrations

+10% of the spectrophotometrically determined

concentration.

11.0 Procedure

11.1 Extraction of Filter Samples

11.1.1 For convenience, a 10-mL final extraction volume

is described in the following procedure. A smaller

extraction volume may be used to improve detection

limits. 
Version 1.0 September 1997 447.0-8

11.1.2 If sampled filters have been frozen, remove them 11.2.1 Draw into a clean syringe 2-3 times the injection

from the freezer but keep them in the dark. Set up the loop volume and inject into the chromatograph. If using

tissue grinder and have on hand laboratory tissues and an autosampler, load the sample tray, prepare a

wash bottles containing water and acetone. Workspace schedule and begin analysis. A typical analyses order

lighting should be the minimum that is necessary to read might be: (1) blank 90% acetone, (2) CALCHK, (3) 10

instructions and operate instrumentation. Remove a filter samples, (4) CALCHK, (5) QCS.

from its container and place it in the glass grinding tube.

You may also tear the filter into smaller pieces and push 11.2.2 If the calculated CALCHK is not +10 of the

them to the bottom of the tube with a glass rod. With a spectrophotometrically determined concentration then

volumetric pipet, add 3 mL of the aqueous acetone recalibrate with fresh calibration solutions.

solution (Sect. 7.6) to the grinding tube. Grind the filter

until it has become a slurry. (NOTE: Although grinding

is required, care must be taken not to overheat the

sample. Good judgement and common sense will help

you in deciding when the sample has been sufficiently

macerated.) Pour the slurry into a 15-mL screw-cap

centrifuge tube and, using a 7-mL volumetric pipet, rinse

the pestle and the grinding tube with the aqueous

acetone. Add the rinse to the centrifuge tube containing

the filter slurry. Cap the tube and shake it vigorously.

Place it in the dark before proceeding to the next filter

extraction. Before placing another filter in the grinding

tube, use the acetone and water squirt bottles to

thoroughly rinse the pestle, grinding tube and glass rod.

To reduce the volume of reagent grade solvents used for

rinsing between extractions, thoroughly rinse the grinding

tube and glass rod with tap water prior to a final rinse with

ASTM Type I water and acetone. The last rinse should

be with acetone. Use a clean tissue to remove any filter

residue that adheres to the pestle or to the steel rod of the

pestle. Proceed to the next filter and repeat the steps

above. The last filter extracted should be a blank. The

entire extraction with transferring and rinsing takes

approximately 5 min. Approximately 500 mL of acetone

and water waste are generated per 20 samples from the

rinsing of glassware and apparatus. 

11.1.3 Again, shake each tube vigorously before placing

them to steep in the dark at 4 C. Samples should be

o

allowed to steep for a minimum of 2 h but not to exceed

24 h. Tubes should be shaken at least once, preferably

two to three times, during the steeping period to allow the

extraction solution to have maximum contact with the filter

slurry.

11.1.4 After steeping is complete, centrifuge samples for

15 min at 675 g or for 5 min at 1000 g. Draw

approximately 1 mL into a glass syringe, attach a 0.45 μm

syringe filter, filter the extract into an amber autosampler

vial, cap and label the vial. Protect the filtered samples

from light and heat. If using a refrigerated autosampler,

chill to 10 C. o

 

11.2 Sample Analysis

12.0 Data Analysis and Calculations

12.1 From the chl a or b area response of the sample,

calculate the mass injected or concentration (CE) of the

solution that was analyzed using the calibration data.

Mass injected must be converted to concentration in

extract by dividing mass by volume injected (μL) and

multiplying by 1000 to give concentration in mg/L

(mg/L = μg/mL). Concentration of the natural water

sample may be reported in mg/L by the following

formula:

CE X extract volume (L) X DF 

sample volume (L)

where:

 CE = concentration (mg/L) of pigment in extract.

 DF = any dilution factors.

 L = liters.

12.2 LRB and QCS data should be reported with each

data set.

13.0 Method Performance

13.1 Single Laboratory Performance

13.1.1 An IDL was determined by preparing a mixed chl

a (0.703 ppm) and chl b (0.437 ppm) standard. The

injected mass yielded 0.004 AU for chl a (0.035 μg) and

0.003 AU for chl b (0.022 μg). Seven replicate 50 μL

injections were made and the standard deviation of the

calculated concentration was multiplied by three to

determine an IDL. The IDL determined for chl a was 0.76

ng and 0.44 ng for chl b. The %RSDs for chl a and chl b

was 0.45 and 0.67, respectively.

13.1.2 MDLs for chl a and chl b were determined by

spiking seven replicate filtered samples of Pycnacoccus,

extracting and processing according to this method. An
 447.0-9 Version 1.0 September 1997

injection volume of 100 μL yielded an MDL for chl a of 7.0

ng and 4.0 ng for chl b. The RSDs were 5.1% for chl b

and 4.7 % for chl a.

13.1.3 Recoveries of chl a and chl b from filtered

samples of phaeodactylum were determined by spiking

three filters with known amounts of the pigments,

extracting, processing and analyzing the extraction

solution according to the method, along with three

unspiked filtered samples (to determine the native levels

in the algae). The spiked levels were 1.1 ppm chl a and

0.53 ppm chl b in the 10 mL extraction volume. Chl a

was 87% recovered and chl b was 94% recovered.

13.1.4 Figures 1-7 are chromatograms of seven

reference unialga cultures processed according to this

method.

13.1.5 Table 1 is a list of pure pigments with retention

times obtained using this method. Purified pigments were

prepared under contract to EPA by Moss Landing Marine

Laboratory, Moss Landing, CA.

13.1.6 Table 2 contains single lab precision data for

seven reference algal suspensions.

13.2 Multilaboratory Testing - A Multilaboratory

validation and comparison study of EPA Methods 445.0,

446.0 and 447.0 for chlorophyll a was conducted in 1996

by Research Triangle Institute, Research Triangle Park,

N.C. (EPA Contract No. 68-C5-0011). There were 8

volunteer participants in the HPLC methods component

that returned data. The primary goals of the study were

to determine estimated detection limits and to assess

precision and bias (as percent recovery) for select

unialgal species, and natural seawater.

13.2.1 The term, pooled estimated detection limit (pEDL), is used in this method to distinguish it from the EPA

defined method detection limit (MDL). The statistical

approach used to determine the p-EDL was an adaptation

of the Clayton, et. al. method that does not assume

(21)

constant error variances across concentration and

controls for Type II error. The approach used involved

calculating an estimated DL for each lab that had the

desired Type I and Type II error rates (0.01 and 0.05,

respectively). The median DLs over labs was then

determined and is reported in Table 3. It is referred to as

Pooled-EDL (p-EDL).

The p-EDL was determined in the following manner.

Solutions of pure chlorophyll a in 90% acetone were

prepared at three concentrations (0.11, 0.2 and 1.6 ppm)

and shipped with blank glass fiber filters to participating

laboratories. Analysts were instructed to spike the filters

in duplicate with a given volume of solution and to

process the spiked filters according to the method. The

results from these data were used to determine a p-EDL

for each method. Results (in ppm) are given in Table 3.

The standard fluorometric and HPLC methods gave the

lowest p-EDLs while the spectrophotometric

(monochromatic equations) gave the highest p-EDLs.

13.2.2 To address precision and bias in chlorophyll a

determination for different algal species, three pure

unialgal cultures (Amphidinium, Dunaliella and

Phaeodactylum) were cultured and grown in the

laboratory. Four different “concentrations” of each

species were prepared by filtering varying volumes of the

algae. The filters were frozen and shipped to participant

labs. Analysts were instructed to extract and analyze the

filters according to the respective methods. The “true”

concentration was assigned by taking the average of the

HPLC results for the highest concentration algae sample

since chlorophyll a is separated from other interfering

pigments prior to determination . Pooled precision data

are presented in Tables 4-6 and accuracy data (as

percent recovery) are presented in Table 7. No

significant differences in precision (%RSD) were observed

across concentrations for any of the methods or species.

It should be noted that there was considerable lab-to-lab

variation (as exhibited by the min and max recoveries in

Table 7) and in this case the median is a better measure

of central tendency than the mean.

In summary, the mean and median concentrations

determined for Amphidinium carterae (class

dinophyceae) are similar for all methods. No method

consistently exhibited high or low values relative to the

other methods. The only concentration trend observed

was that the spectrophotometric method-trichromatic

equations (SP-T) showed a slight percent increase in

recovery with increasing algae filtration volume.

For Dunaliella tertiolecti (class chlorophyceae) and

Phaeodactylum tricornutum (class bacillariophyceae)

there was generally good agreement between the

fluorometric and the spectrophotometric methods,

however, the HPLC method yielded lower recoveries with

increasing algae filtration volume for both species. No

definitive explanation can be offered at this time for this

phenomenon. A possible explanation for the

Phaeodactylum is that it contained significant amounts of

chlorophyllide a which is determined as chlorophyll a in

the fluorometric and spectrophotometric methods. The

conventional fluorometric method (FL-STD) showed a

slight decrease in chlorophyll a recovery with increasing

Dunaliella filtration volume. The spectrophotometric-
Version 1.0 September 1997 447.0-10

trichromatic equations (SP-T) showed a slight increase in

chlorophyll a recovery with incresing dunaliella filtration

volume. The fluorometric and athe spectrophotometric

methods both showed a slight decrease in chlorophyll a

recovery with increasing Phaeodactylum filtration volume.

Results for the natural seawater sample are presented in

Table 8. Only one filtration volume (100 mL) was

provided in duplicate to Participant labs.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

USEPA has established a preferred hierarchy of

environmental management techniques that places

pollution prevention as the management option of first

choice. Whenever feasible, laboratory personnel should

use pollution prevention techniques to address their waste

generation (e.g., Sect. 11.1.2). When wastes cannot be

feasibly reduced at the source, the Agency recommends

recycling as the next best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research

institutions, consult Less is Better: Laboratory Chemical

Management for Waste Reduction, available from the

American Chemical Society's Department of Government

Relations and Science Policy, 1155 16th Street N.W.,

Washington D.C. 20036, (202) 872-4477.

15.0 Waste Management

15.1 The U.S. Environmental Protection Agency

requires that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

waste identification rules and land disposal restrictions.

For further information on waste management consult

The Waste Management Manual for Laboratory

Personnel, available from the American Chemical Society

at the address listed in the Sect. 14.2.

16.0 References

1. Holm-Hansen, O., "Chlorophyll a determination:

improvements in methodology," OIKOS, 30

(1978), pp. 438-447.

2. Wright, S.W. and J.D. Shearer, "Rapid extraction

and HPLC of chlorophylls and carotenoids from

marine phytoplankton," J. Chrom., 294 (1984),

pp. 281-295.

3. Bowles, N.D., H.W. Paerl, and J. Tucker,

"Effective solvents and extraction periods

employed in phytoplankton carotenoid and

chlorophyll determination," Can. J. Fish. Aquat.

Sci., 42 (1985) pp. 1127-1131.

4. Shoaf, W.T. and B.W. Lium, "Improved extraction

of chlorophyll a and b from algae using dimethyl

sulfoxide," Limnol. and Oceanogr., 21(6) (1976)

pp. 926-928.

5. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid

determination of algal chlorophyll and carotenoid

pigments and their breakdown products in

natural waters by reverse-phase high

performance liquid chromatography," Anal.

Chim. Acta., 151 (1983) pp. 297-314.

6. Yentsch, C.S. and D.W. Menzel, "A method for

the determination of phytoplankton chlorophyll

and phaeophytin by fluorescence," Deep Sea

Res., 10 (1963), pp. 221-231.

7. Strickland, J.D.H. and T.R. Parsons, A Practical

Handbook of Seawater Analysis, Bull. Fish. Res.

Board Can., 1972, No.167, p. 201.

8. USEPA Method 445.0, "In vitro determination of

chlorophyll a and pheophytin a in marine and

freshwater phytoplankton by fluorescence,"

Methods for the Determination of Chemical

Substances in Marine and Estuarine

Environmental Samples, EPA/600/R-92/121.

9. Wright, S.W., S.W. Jeffrey, R.F.C. Mantoura,

C.A. Llewellyn, T. Bjornland, D. Repeta, and N.

Welschmeyer, "Improved HPLC method for the

analysis of chlorophylls and carotenoids from

marine phytoplankton," Mar. Ecol. Prog. Ser.,

77:183.
 447.0-11 Version 1.0 September 1997

10. Brown, L.M., B.T. Hargrave, and M.D. 20. Weber, C.I., L.A.Fay, G.B. Collins, D.E. Rathke,

MacKinnon, "Analysis of chlorophyll a in and J. Tobin, “A Review of Methods for the

sediments by high-pressure liquid

chromatography," Can. J. Fish. Aquat. Sci., 38

(1981) pp. 205-214.

11. Bidigare, R.R., M.C. Kennicutt, II, and J.M.

Brooks, "Rapid determination of chlorophylls and

their degradation products by HPLC," Limnol.

Oceanogr., 30(2) (1985) pp. 432-435.

12. Minguez-Mosquera, M.I., B. Gandul-Rojas, A.

Montano-Asquerino, and J. Garrido-Fernandez,

"Determination of chlorophylls and carotenoids

by HPLC during olive lactic fermentation," J.

Chrom., 585 (1991) pp. 259-266.

13. Neveux.J., D. Delmas, J.C. Romano, P. Algarra,

L. Ignatiades, A. Herbland, P. Morand, A. Neori,

D. Bonin, J. Barbe, A. Sukenik and T. Berman,

"Comparison of chlorophyll and pheopigment

determinations by spectrophotometric,

fluorometric, spectrofluorometric and HPLC

methods," Marine Microbial Food Webs, 4(2),

(1990) pp. 217-238. 

14. Sartory, D.P., "The determination of algal

chlorophyllous pigments by high performance

liquid chromatography and spectrophotometry,"

Water Research, 19(5), (1985), pp. 605-10.

15. Carcinogens - Working With Carcinogens,

Department of Health, Education and Welfare,

Public Health Service, Center for Disease

Control, National Institute for Occupational Safety

and Health, Publication No. 77-206, 1977.

16. "OSHA Safety and Health Standards, General

Industry," (29 CFR 1910), Occupational Safety

and Health Administration, OSHA 2206, revised

January 1976.

17. Safety in Academic Chemistry Laboratories,

American Chemical Society publication,

Committee on Chemical Safety, 3rd Edition,

1979.

18. "Proposed OSHA Safety and Health Standards,

Laboratories," Occupational Safety and Health

Administration, Federal Register, July 24, 1986.

19. Code of Federal Regulations 40, Ch.1,

Pt.136, Appendix B.

Analysis of Chlorophyll in Periphyton and

Plankton of Marine and Freshwater Systems,”

Oho State University, Grant No. NA84AA-D00079, 1986, 54 pp.

21. Clayton, C.A., J.W. Hine, and P.D. Elkins,

“Detection Limits within Specified Assurance

Probabilities,” Analytical Chemistry, 59(1987), pp.

2506-2514.
Version 1.0 September 1997 447.0-12

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Table 1. Pure Pigments and Retention Times

PIGMENT RETENTION TIME

19' butanoyloxyfucoxanthin 8.13

2,4-divinylpheoporphrin a5 8.60

Peridinin 8.69

Fucoxanthin 8.75

19' hexanoyloxyfucoxanthin 8.90

Neoxanthin 10.07

Chlorophyll C3 10.27

Chlorophyll C2 10.40

Prasinoxanthin 11.20

Violaxanthin 12.00

Diadinoxanthin 15.20

Chlorophyll b 15.60

Myxoxanthophyll 17.00

Aphanaxanthin 17.20

Chlorophyll a 17.80

Monadoxanthin 17.93

Lutein 18.00

Alloxanthin 18.07

Nostaxanthin 18.70

Diatoxanthin 19.07

Zeaxanthin 19.40
 447.0-13 Version 1.0 September 1997

Table 2. Single Lab Precision for Seven Pure Unialgal Cultures

Algae Chlorophyll a Chlorophyll b

Pycnacoccus provasolii N 3 3

(1)

Mean (mg/L) 2.15 1.47 (2)

STD DEV 0.114 0.065

% RSD 5.31 4.45

Rhodomonas salina N 3 3

(1)

Mean (mg/L) 4.0 ND(3)

STD DEV 0.014 ND

% RSD 0.28 ND

Selenastrum N 3 3

capricornitum

(1)

Mean (mg/L) 4.25 0.483

STD DEV 0.199 0.058

% RSD 4.68 12.01

Amphidinium carterae N 3 3

(1)

Mean (mg/L) 2.38 ND

STD DEV 0.176 ND

% RSD 7.40 ND

Dunaliella tertiolecti N 3 3

(1)

Mean (mg/L) 6.68 1.42

STD DEV 0.635 0.0412

% RSD 9.51 2.90

Emiliania huxleyi N 3 ND (1)

Mean (mg/L) 1.03 ND

STD DEV 0.008 ND

% RSD 0.79 ND

Phaeodactylum N 3 ND

tricornutum

(1)

Mean (mg/L) 1.09 ND

STD DEV 0.072 ND

% RSD 7.07 ND

(1) N = Number of filtered samples.

(2) Mean concentration in extract solution.

(3) ND = none detected.
Version 1.0 September 1997 447.0-14

TABLE 3. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLL A METHODS(1)

_____________________________________________________________

Method N p-EDL (mg/L) (2) (3) (4)

FL -Mod 8 0.096 (5)

FL - Std 9 0.082

HPLC 4 0.081

SP-M 15 0.229

SP-T 15 0.104

_____________________________________________________________

(1) See Section 13.2.1 for a description of the statistical approach used to determine p-EDLs. 

(2) FL-Mod = fluorometric method using special interference filters.

 FL-Std = conventional fluorometric method with pheophytin a correction.

 HPLC = EPA method 447.0

 SP-M = EPA method 446.0, monochromatic equation.

 SP-T = EPA method 446.0, trichromatic equations.

(3) N = number of labs whose data was used.

(4) The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.

(5) Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are

 unrealistically high.
 447.0-15 Version 1.0 September 1997

TABLE 4. Measured Chlorophyll a (mg/L) in Dunaliella Samples

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

HPLC 5 5 0.172 0.064 36.8

 10 5 0.276 0.074 26.8

 50 5 0.757 0.344 45.4

100 5 1.420 0.672 47.3

___________________________________________________________________________________

(1) Not all participants labs followed the EPA method exactly.

 

(2) N = Number of volunteer labs whose data was used.
Version 1.0 September 1997 447.0-16

TABLE 5. Measured Chlorophyll a (mg/L) in Amphidinium Samples

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

HPLC 5 5 0.104 0.043 56.8

 10 5 0.172 0.083 37.5

 50 5 0.743 0.213 17.4

100 5 1.394 0.631 14.5

___________________________________________________________________________________

(1) Not all participants labs followed the EPA method exactly.

(2) N = number of volunteer labs whose data was used.
 447.0-17 Version 1.0 September 1997

TABLE 6. Measured Chlorophyll a in Phaeodactylum Samples

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

HPLC 5 5 0.193 0.074 38.4

 10 5 0.317 0.114 36.1

 50 5 1.024 0.340 33.2

100 5 1.525 0.487 29.9

___________________________________________________________________________________

(1) Not all participants labs followed the EPA method exactly.

(2) N = number of volunteer labs whose data was used.
Version 1.0 September 1997 447.0-18

TABLE 7. Minimum, Median, and Maximum Percent Recoveries by Genera, Method, and Concentration Level

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

Amphidinium Minimum FL-MOD 70 73 75 76

FL-STD 66 91 91 90

HPLC 82 85 87 88

SP-M 36 48 68 64

SP-T 21 63 71 70

Median FL-MOD 105 112 105 104

FL-STD 109 107 111 109

HPLC 102 106 112 105

SP-M 99 101 101 101

SP-T 95 96 106 107

Maximum FL-MOD 121 126 143 146

FL-STD 156 154 148 148

HPLC 284 210 131 116

SP-M 141 133 126 125

SP-T 115 116 119 117

Dunaliella Minimum FL-MOD 162 159 157 156

FL-STD 179 171 165 164

HPLC 165 109 64 41

SP-M 120 188 167 164

SP-T 167 169 166 165

Median FL-MOD 206 246 227 223

FL-STD 250 228 224 210

HPLC 252 177 89 80

SP-M 240 247 247 243
Table 7. Cont’d.

 447.0-19 Version 1.0 September 1997

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

SP-T 225 244 256 256

Dunaliella Maximum FL-MOD 295 277 287 288

FL-STD 439 385 276 261

HPLC 392 273 172 154

SP-M 342 316 296 293

SP-T 291 283 283 283

Phaeodactylum Minimum FL-MOD 216 183 157 154

FL-STD 189 220 223 219

HPLC 150 119 84 75

SP-M 161 138 156 160

SP-T 203 195 216 244

Median FL-MOD 292 285 250 245

FL-STD 296 263 254 254

HPLC 225 203 114 90

SP-M 287 274 254 253

SP-T 286 281 277 274

Maximum FL-MOD 357 337 320 318
Version 1.0 September 1997 447.0-20

Table 8. Chlorophyll a Concentrations in mg/L Determined in Filtered Seawater Samples

Method Con. No. Obs No. Labs Mean Std. Dev RSD (%) Minimum Median Maximum (1)

FL-MOD 100 14 7 1.418 0.425 30.0 0.675 1.455 2.060

FL-STD 100 15 8 1.576 0.237 15.0 1.151 1.541 1.977

HPLC 100 10 5 1.384 0.213 15.4 1.080 1.410 1.680

SP-M 100 38 19 1.499 0.219 14.6 0.945 1.533 1.922

SP-T 100 36 18 1.636 0.160 9.8 1.250 1.650 1.948

All Methods 100 113 57 1.533 0.251 16.4 0.657 1.579 2.060

(1) Con = mLs of seawater filtered.