HPLC Pigment methods - Process 6

Robert Bidigare
University of Hawaii

References:
      1. Wright et al (Mar. Ecol. Prog. Ser. 1991, 77:183-196)
      2. Latasa et al (Mar. Chem. 1996, 51:315-324) - estimates of
                        chl_a1, chl_a2, chl_b1 and chl_b2

Methodology notes for the P6 pigment data:

I) A comparison of the TURNER-determined chlorophyll a concentrations with
the HPLC-determined TOTCHLA concentrations (monovinyl chlorophyll a +
divinyl chlorophyll a + monovinyl chlorophyllide a;  units = ng Chl a
equivalents/L) was performed for Process Cruise #6 (TTN-053).  While a
slope of approximately of 1 was obtained (see below), there was a
significant amount of scatter in the cross-plot (r = 0.938).  This scatter
was probably caused in part by the variable presence of Chl accessory
pigments which interfere with the fluorometric method.  Thus, we recommend
that whenever possible use the HPLC pigment data and not the TURNER pigment
data.  If HPLC data is not available for a given cast, we further recommend
that you use the following equation to transform the TURNER data into
HPLC-equivalent concentrations (cf., Babin, M., A. Morel, H. Claustre, A.
Bricaud, Z. Kolber and P.G. Falkowski.  1996.  Nitrogen- and
irradiance-dependent variations of the maximum quantum yield of carbon
fixation in eutrophic, mesotrophic and oligotrophic marine systems.
Deep-Sea Research, in press).

Results of geometric mean regression analyses (reduced major axis):

Y = HPLC TOTCHLA (monovinyl chlorophyll a + divinyl chlorophyll a +
monovinyl chlorophyllide a), units = ng Chl a equivalents/L

X = TURNER chlorophyll a (it is necessary to convert the Turner Chl a
concentrations in the Arabian Sea data base from mg/m3 to ng/L by
multiplying concentrations by 1000)

Process Cruise #6 (TTN-053)

HPLC TOTCHLA = TURNER*(1.052) - 56.474 (r = 0.938, n = 479)


II)     Accessory pigment quantification

(1) Although most pigment peaks were clearly identifiable based on
retention time, a few of the minor pigments presented difficulties.  The
presence of lutein, in particular, was difficult to determine
unambiguously.  Lutein concentrations are therefore specified only where
that peak is clearly evident, although it may be present in trace
quantities in other samples as well.

(2) The peak identified as alloxanthin agreed very well with a concurrently
run standard for approximately the first half of the samples run.  However,
in samples run later, what appears to be the same peak does not agree well
with the known alloxanthin retention time.  Since samples were run in
random order and because there is no particular reason for the sudden
absence of the pigment, we continued to identify this peak as alloxanthin
throughout the remainder of the data set.  The appearance of the peak as a
doublet in some instances suggests the possibility of pigment alteration
(e.g., a trans to cis isomerization). The change in agreement between
sample and standard alloxanthin peaks also corresponds with a change in the
HPLC column, though this does not appear to have affected the comparability
of other peaks with their standards.

(3) Chlorophyll b occasionally appeared as a split peak, or with a very
close shoulder on the main peak (either leading or trailing).  This may
have resulted from either a partial separation of monovinyl and divinyl
chlorophyll b, or from the coelution of some other pigment with chlorophyll
b.  In all cases where this occurred, both peaks (or peak with shoulder)
were included in the area used to calculate chlorophyll b concentrations.