Dataset: pigments

   PI:              Dan Repeta
   of:              Woods Hole Oceanographic Institution
   dataset:         Pigment concentrations, water bottle casts
   project/cruise:  North Atlantic Bloom Experiment cruises

Methodology:

CHLOROPHYLL-A (Repeta, WHOI)

For shipboard extraction of suspended particulate matter, 0.5-1 L of seawater
is filtered through a 47 mm Gelman A/E or 47 mm Whatman GF/F glass fiber
filter under low vacuum. The filter is immediately placed in a glass grinding
tube at 0° Centigrade, 2 mL of acetone and 0.1 mL of the internal
standard (8'-Ethyl-apocarotenoate or canthaxanthin in acetone) added,
and the sample ground at high speed for 2 min. The sample is centrifuged
for 2-4 min, and analyzed by high pressure liquid chromatography (HPLC).
If the sample is to be stored for future analysis, the filter is folded,
heat sealed in a plastic bag, and rapidly frozen to -20° Centrigrade.

For shipboard analysis of suspended particulate matter samples, we
use a 10 cm reverse-phase ODS column (adsorbosphere HS, 3µm) eluted
with a linear gradient of 0-100% B (A=20/80 0.5N NH4Ac(aq)/MeOH; B=20/80
acetone/MeOH) over 10 min at 1.5 mL/min. For more detailed analysis,
we use a 15 cm column and a 30 min gradient. All samples were analyzed
by diode array spectroscopy (350-700nm) and fluorescence detection.

Individual components were quantified by normaling the data to the
internal standard to correct for evaporative losses, differences in
water retention by filters, and nonquantitative transfers during handling,
then correcting the data for individual compounds by using an appropriate
detector calibration factor. We calibrate our system by isolating pure
pigments, making quantitative standards, and carrying out a series of
analyses over the calibration range of interest. Depending on the pigment's
spectral properties, and the wavelength of detection, calibration factors
for individual pigments may differ by up to a factor of 5. For calibration
in the field, concentrated solutions of standards are prepared in benzene,
and 1 mL aliquots dispensed to 10 mL volumetric flasks. The flasks are
flushed with N2, capped, frozen at -20° Centigrade. Randomly chosen
standards are stored in the laboratory for spectroscopic analysis at
a later date. The remaining flasks are stored on shipboard until use.
For calibration, the standards are returned to room temperature and
acetone or methanol added to 10 mL. Aliquots are then analyzed by HPLC.
In the US JGOFS program, over 30 replicate standards were prepared and
analyzed daily in triplicate showed a SD of <10% by HPLC analyses. Detectors
were calibrated daily for the internal standard and twice weekly for
chlorophyll-a. Calibration factors for major xanthophylls were determined
at least one time per week.