Phytoplankton use pigments to absorb energy from the sun to drive photosynthesis. Chlorophyll-a is used as the primary light harvesting molecule while other accessory pigments, such as chlorophyll-b, -c, and carotenoids assist by expanding the light absorption capability of the organism, therefore increasing efficiency and adaptability (Bidigare et al., 2002).
Many individual algal pigments or combinations and ratios are taxon-specific. Therefore, pigment composition from seawater samples can be used to separate major algal groups and result in chemotaxonomic characterization. These analyses can be used to determine phytoplankton community structure and physiological state of the autotrophic assemblage (Wallerstein et al., 1999; Bidigare et al., 2002)
The methodology described here is based on the Protocols for the Joint Global Ocean Flux Study (JGOFS) Core Measurements (BATS, 1997), and describes the use of high performance liquid chromatography (HPLC) for the rapid separation of phytoplankton pigments with detection limits for chlorophylls and carotenoids on the order of one nanogram (Bidigare, 1991). This HPLC method was adopted as BATS protocol in July 1994 (BATS 70 cruise). This method uses less solvent and gives improved peak separation and better resolution at lower concentrations.
Field sampling
Discrete samples are collected monthly using Niskin bottles at the Bermuda Atlantic Time-series Study site from depths ranging from the surface to 250 meters. Expeditious sampling and contamination prevention protocols were used to obtain the best possible samples, since the physico-chemical and biological parameters of the Niskin water become increasingly altered with time spent on deck, especially if in full sunlight. Sea water is filtered using glass fiber filters (GF/F) with standard pore size of 0.7 microns and the filtered samples then flash frozen and stored at -80ºC until analysis.
HPLC pigment measurements
Water samples were analyzed for the concentration of various phytoplankton pigments following the method of Wright et al. (1991) as modified by Dr. Robert Bidigare. Pigments present in a seawater sample can be separated by high-performance liquid chromatography (HPLC) based on differences in polarity. Pigment polarities determine how the pigments interact with the solid phase (column) and the mobile phase (solvent) within the HPLC. Pigments that are less polar will be more attracted to the non-polar stationary phase and take longer to pass through than more polar pigments, thus a temporal separation is achieved. The length of time it takes for the pigment to elute is known as the retention time. Under comparable conditions, pigment retention times are consistent and can therefore be used for identification when compared to a reference. The retention times are determined using a diode array detector (DAD), which detects absorption across a range of wavelengths in the UV-Vis portion of the spectrum. The separated pigments are transported by the flowing mobile phase to the detector, where the solution passes through a flow cell and is dispersed by a diffraction grating. Photodiode arrays detect the light intensity for each wavelength, which is converted to an electrical signal, resulting in a visible peak on a chromatogram. Concentration is proportional to the area of the peak, and can be calculated using calibration factors determined from known standards (aka response factor), in addition to other parameters such as volume of water sampled and dilution factors.
The HPLC currently in use is an Agilent 1100 series. The results from two different wavelengths are reported in this method, 436nm and 450nm. All pigments except divinyl chlorophyll-a produce a signal at 436nm. However, divinyl and monovinyl chl-a cannot be separated at 436nm due to a similar detector response, so 450nm is also used to try and separate mono and divnyl chl-a since divnyl absorbs at this wavelength and monovinyl does not.
Fluorometric measurements
In addition to the HPLC measurements, pigment samples from BATS are also analyzed using fluorometric techniques. Fluorescence is the physical property of compounds to absorb light energy and instantaneously re-emit light at a different wavelength to the absorbed light. Fluorescent compounds, such as chlorophyll-a, have characteristic absorption and emission wavelengths. In fluorometry, a sample is excited at the appropriate absorption wavelength and the intensity of the emitted light is measured using a photodiode detector to give a raw fluorescence recorded value that is proportional to concentration. When compared to reference standards, the raw fluorescence measurements are used to calculate the concentration of the fluorescent material in the sample.
Prior to January 2020, fluorometry was performed on the Turner 10-AU fluorometer, whereas samples are currently analyzed using the Trilogy Fluorometer at BIOS. Data from the fluorometer is compared to the chlorophyll-a data from the HPLC which allows an extra quality control method to ensure data from both methods are similar. This data is being released as part of the BATS dataset since fluorometry is often used instead of HPLC in the oceanographic community for chlorophyll analysis.
Additional information
Additional details on methods, standardization, and calibration can be found in the BATS methods document (Protocols for the Bermuda Atlantic Time-series Study Core Measurements)
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