FLUOROMETRIC ANALYSIS OF CHLOROPHYLL A. AND PHEOPIGMENTS


SUMMARY: Concentrations of chlorophyll a and pheopigments, considered an index of the phytoplankton standing stock, are measured by fluorometry after particulate sample concentration by vacuum filtration and extraction in 100% acetone.


1. Principle

Historically chlorophyll a (chl a) has been used as a measure of phytoplankton standing stock. Also, when related to photosynthetic carbon production, the concentration of chl a and distribution in the water column allows one to estimate an index of the efficiency of phytoplankton in harvesting light.

Fluorescence is produced by atoms or molecules that, under exited state, return to their state of minimum energy (or ground state) by losing energy in the form of light. The absorption of blue light by chlorophylls and pheopigments produces emission of red light. In a sample, the fraction of light transformed is proportional to the amount of pigment. Hence, by knowing the amount of incident light and by measuring the light emitted at a second wavelength, it is possible to estimate the concentration of these pigments in the sample.

2. Sampling, Filtration, Extraction and Storage

2.1. Seawater samples are collected from 5, 25, 45, 60, 75, 85, 95, 105, 115, 125, 150, 175 and 200 m using 12-liter Niskin bottles attached to the rosette sampler and from the primary productivity cast (see Chapter 14) using Go-Flo bottles attached to a Kevlar line.
2.2. A 500 ml sample from each bottle is collected in clean 4-liter polyethylene bottles and stored in the dark. The polyethylene bottles are rinsed three times with 100-200 ml of sample before the collection of the final sample.
2.3. Filtration and storage
2.3.1. As soon as possible, triplicate 100 ml subsamples from the 12-liter Niskin bottles from 30, 95 and 125 m are filtered through 25 mm GF/F filters. For the remaining depths of the same cast a single 100 ml sample is filtered.
2.3.2. Triplicate 100 ml samples from each Go-Flo bottle are filtered as in Chapter 12, section 2.3.1.
2.3.3. Immediately following filtration, the filter is transferred to a glass screw cap tube containing 5 ml of cold (-20 °C) 100% acetone. Then the tubes are wrapped in aluminum foil and stored at -20 °C to prevent photodegradation of pigments.
2.4. Sample Analysis
2.4.1. Samples are brought to room temperature 2 hours before the analysis and the fluorometer is allowed to stabilize for 30 minutes before the samples are analyzed.
2.4.2. The external standard, prepared with reagent grade chl a (see Chapter 12, section 3), is read before other samples.
2.4.3. Samples are analyzed from deepest to shallowest depth. Each time the door of the fluorometer is changed, the dial is brought to the "zero" setting using 100% acetone as a blank.
2.4.4. Each sample is acidified by adding 2 drops of HCl (1 M) to the sample in the cuvette. The new reading must be stable before recording the value and is made using the same door used for the reading before the acidification.
2.4.5. The fluorometer door used, the readings before and after acidification and the dilution factor are recorded in the chlorophyll log book.

3. Standard Preparation and Analysis

3.1. A chl a standard stock is prepared every four months using commercially-available chl a (SIGMA) and 100% acetone. The stock is wrapped in aluminum foil and stored at -20 °C.
3.2. To determine the concentration of the stock, a 50 ml sample is brought to room temperature avoiding exposure to the light. An absorbance spectrum from 350-750 nm is obtained using a scanning spectrophotometer and the concentration is calculated considering an extinction coefficient of 88.15 (l g-1 cm-1) for the absorbance at 662 nm.
3.3. Using the same stock sample, three dilutions (1/10 each) are prepared gravimetrically using 100% acetone and read in the fluorometer. During this step, chl a must be kept under dim light.
3.4. A calibration of the fluorometer is performed every 6 months following the procedure described by Strickland and Parsons (1972).

4. Data Reduction and Calculations

4.1. Concentrations of chl a and pheopigments are calculated using the following equations:

chl a = (T/(T-1))*(Rb-Ra)*Fd*volex/volfilt

phaeo = (T/(T-1))*((T*Ra)-Rb)*Fd*volex/volfilt

where: chl a = concentration of chl a (mg m-3)
phaeo = concentration of pheopigments (mg m-3)
T = acidification coefficient (Rb/Ra average obtained during the calibration of the fluorometer)
Rb = reading before acidification
Ra = reading after acidification
Fd = door factor (µg/(ml* reading units))
volex = volume of extraction (ml)
volfilt = volume filtered (l)

5. Equipment/Supplies

6. Reagents

7. References