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Chapter 12. The Determination of Reactive Silicate in Sea Water
1.0 Scope and field of application
This procedure describes a method for the determination of reactive silicate in seawater.
This method is suitable for the assay of oceanic concentrations between 0.1 mmol l -1 to
140 mmol l -1 . This method is a modification of Strickland and Parsons (1968).
2.0 Definition
The silicate concentration of seawater is given in units of mmol kg -1 in seawater.
3.0 Principle of Analysis
The determination of reactive silicate is based on the method of Strickland and Parson
(1968). A seawater sample is allowed to react with ammonium molybdate under
conditions which result in the formation of silicomolybdate, phosphomolybdate and
arsenomolybdate complexes. A reducing agent of metol and oxalic acid is added and
silicomolybdate is reduced to a silicomolybdous acid with a blue color, the absorbance of
which is measured spectrophotometrically.
4.0 Apparatus
Spectrophotometer
5.0 Reagents
5.1 Molybdate reagent: Dissolve 4.0 g of reagent quality ammonium paramolybdate,
(NH 4 ) 6 Mo 7 O 24 4H 2 O in 300 ml of deionized water. Slowly add 12.0 ml concen-trated
HCl and mix. Make up volume to 500 ml with deionized water. This solution
is stable for many months if stored refrigerated in a polyethylene bottle. Discard if a
white precipitant forms.
5.2 Metol-sulphite solution: Dissolve 6g of anhydrous sodium sulphite, Na 2 SO 3 , in 500
ml of deionized water and then add 10 g metol. When the metol has dissolved filter
the solution through a No. 1 Whatman filter paper and store it in a glass bottle. This
solution should be replaced monthly.
5.3 Oxalic acid solution: Shake 50 g of reagent grade oxalic acid dihydrate with 500 ml
of deionized water. Store the solution in a glass bottle and decant the solution from
the crystals for use.
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5.4 Sulfuric acid solution: Dilute 250 ml of concentrated sulfuric acid to 500 ml using
deionized water. Cool the solution and store in a glass bottle.
5.5 Reducing reagent: Mix 100 ml of metol-sulphite solution with 60 ml of oxalic acid
solution. Slowly add 60 ml of the 50% sulfuric acid solution and make the solution
to a volume of 300 ml with deionized water. This solution should be prepared for
immediate use.
6.0 Sampling
6.1 Samples are collected on deeper casts after the oxygen, CO 2 and salinity samples
have been drawn. Shallow samples (upper 250 m) are collected on the gases cast
after oxygen, CO 2 , DOC and salinity samples are drawn. An in-line filter (0.8 mm
Nuclepore filter) is connected to the Niskin bottle. The spigot is opened and three
sets of samples are collected from the water that passes through the filter by gravity
filtration. Each bottle is rinsed three times and then filled just below the shoulder.
Care must be taken to avoid overfilling of samples to be frozen. These bottles are
transferred to a freezer (- 20C) and kept frozen until analysed.
6.2 Contamination is a major problem with nutrient samples, especially near the surface
where the ambient concentrations are low. All the nutrient bottles are rigorously
cleaned before use. The cleaning begins by a wash with a detergent (Aquet) followed
by a rinse with 10% HCl, three rinses with deionized water and a final rinse with de-ionized
water.
6.3 Prolonged storage of samples is not advised, even if frozen. Sufficient water should
be sampled for washing purposes.
7.0 Procedures
7.1 Sample analysis
7.1.1 All glassware should initially be washed in chromic-sulfuric acid and rinsed
well with de-ionized before and after each subsequent use.
7.1.2 Sample solutions should be stored at a temperature between 18 and 25.
7.1.3 Add 10 ml of the molybdate reagent to a dry 50 ml measuring cylinder fitted
with a stopper.
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7.1.4 Pipette 25 ml of the sample into the cylinder, stopper, mix the solutions and
allow the mixture to stand for 10 minutes.
7.1.5 Add the reducing reagent rapidly to make the volume 50 ml and mix immedi-ately.
7.1.6 Allow the solution to stand for 23 hours then measure the extinction at 810
nm.
7.2 Reagent blank determination
7.2.1 The reagent blank is determined using open ocean surface seawater as a sam-ple.
Follow the exact procedure outlined in section 7.1. Repeat.
7.2.2 A reagent blank should not exceed 0.01 on a 1 cm cell or 0.1 on a 10 cm cell
and should be determined for each batch of samples.
7.3 Standardization
7.3.1 Primary silicate standard: Dissolve 0.9403g dried Na 2 SiF 6 in deionized
water and make up to 1 liter with DIW. 1 ml = 5 mmol. Store in a dark
polypropylene bottle. As with nitrite, ultra pure sodium fluosilicate is diffi-cult
to obtain. It may be advisable to compensate for these impurities.
7.3.2 Working standards of concentrations of 50, 25, 10 and 5 mM are prepared by
diluting 10 ml, 5 ml, 2 ml and 1 ml of primary silicate standard respectively
to 1000 ml in open ocean surface seawater. Carry out the exact procedure as
described in section 7.2 These solutions should be stored in plastic beakers
and used within a few hours.
8.0 Calculation and expression of results
8.1 Standardization Factor, F
8.1.1 Subtract the absorbance of the reagent blanks from the absorbance values of
the standards. Perform a linear regression of the silicate concentration and
the corrected extinction values. The slope of the line is the standardization
factor, F. The value of F is typically 100. If a 10 cm cell is used, the F factor
may be assumed to be equal to 0.1 x F (1 cm) .
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8.1.2 F is a function of the salinity of the seawater samples. Between salinities of
25 and 35, the variation may be neglected. The factor Fs at a salinity of S is
related to F by:
This correction should be used when the salinity varies more than 10 from a
value of 28.
8.2 Concentration of reactive silicate (mmol/1) = F  corrected extinction
where:
Corrected absorbance= sample absorbance - blank absorbance
F = standardization factor
9.0 Notes
The silicate and molybdate must combine before the reducing agent is added. Ten minutes
is allowed for this reaction. The reducing solution must be added within 30 minutes or else
changes in the isomeric form of the silicomolybdate complex will occur.
The sample should be added to the acid molybdate solution instead of the reverse. The
prevents unwanted isomeric forms of the silicomolybdate complex.
The time required for the full color development varies with the amount of silicate present
in the sample. With a concentration of less than 50 mM, 1 hour is sufficient. For amounts
exceeding 75 mM, at least 3 hours should be allowed.
10.0 References
Strickland, J.D.H., and Parsons, T.R. (1968). Determination of reactive silicate. In: A
Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada,
Bulletin 167, 6570.
F s
F 1 0.003S + ( ) 
1.08 ---------------------------------------- - =
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Chapter 13. Measurement of Algal Chlorophylls and Carotenoids by HPLC
1.0 Scope and field of application
Many individual algal pigments or pigment combinations and ratios are taxon-specific.
Therefore, analysis of the chlorophylls and carotenoids present in a seawater sample can
reveal the taxonomic composition of natural algal populations. This technique allows for
the rapid separation of important phytoplankton pigments with detection limits for
chlorophylls and carotenoids (using absorbance spectroscopy as analyzed by HPLC) on
the order of 1 ng (Bidigare, 1991). The HPLC method described here is a modified version
of Wright et al. (1991), provided by Bidigare (in press). Scientists who employ this or
other methods to measure pigments should make themselves aware of the current and
historical issues that surround these techniques and make appropriate decisions about
specific methodologies for their application based on the scientific requirements and
constraints of their individual programs.
2.0 Definition
The concentration of all pigments is given as ng kg -1 in seawater.
3.0 Principle of Analysis
The reverse phase high performance liquid chromatography method described here
separates all the phytoplankton pigments listed below in order of polarity upon passage
through a column. The most polar pigments are removed earlier than the less polar
pigments.
Chlorophyllide b
Chlorophyllide a
Chlorophyll c 3
Chlorophyll c 1 + c 2 and Chlorophyll Mg 3,8DVP a 5
Peridinin
19' - Butanoyloxyfucoxanthin
Fucoxanthin
19' - Hexanoyloxyfucoxanthin
Prasinoxanthin
Pyrophaeophorbide a
Diadinoxanthin
Alloxanthin
Diatoxanthin
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Lutein
Zeaxanthin
Chlorophyll b
Chlorophyll a
Phaeophytin b
Phaeophytin a
a Carotene
b Carotene
Picoplanktonic prochlorophytes are abundant in tropical and subtropical seas and oceans.
They contain divinyl-chlorophyll a and divinyl-chlorophyll b (more appropriately called
8-desethyl, 8-vinyl Chlorophyll), both co-eluting with normal chlorophyll a and b with
this reverse phase liquid chromatography technique.
4.0 Apparatus and Reagents
4.1 Filtration System and Whatman 47 mm GF/F filters
4.2 Liquid nitrogen and freezer for storage and extraction
4.3 Glass centrifuge tubes for extraction, 15 ml
4.4 High pressure liquid chromatograph capable of delivering three different solvents at
a rate of 1 ml/minute.
4.5 High-pressure injector valve equipped with a 200 mL sample loop.
4.6 Guard Column (50 x 4.6 mm, ODS-2 C18 packing material, 5 mm particle size) for
extending life of primary column.
4.7 Reverse phase HPLC Column (250 x 4.6 mm, 5 mm particle size, ODS-2 Spherisorb
column).
4.8 Absorbance detector capable of monitoring ar 436 nm, or preferably, an on-line
diode array spectrophotometer.
4.9 Data recording device: strip chart recorder or, preferably, an electronic integrator or
computer equipped with hardware and software for chromatographic data analysis.
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4.10 Glass syringe, 500 ml
5.0 Eluants
Eluant A (80:20, v:v, methanol: 0.5 M ammonium acetate, aq., pH=7.2), eluant B (90:10,
v:v, acetonitrile:water), and eluant C (ethyl acetate). Use HPLC-grade solvents, measure
volumes before mixing. Filter eluents through a solvent-resistant 0.4 mm filter before use
and de-gas with helium.
The gradient program is listed in Table 13-1.
6.0 Sample Collection and Storage
Water samples are collected from niskins into clean polyethylene bottles with Tygon 
tubing. Samples are immediately filtered through 47 mm GF/F filters using polycarbonate
in-line filter holders (Gelman) and a vacuum of less than 100 mm Hg. Filters are folded in
half twice and wrapped in aluminum foil, labeled, and stored in liquid nitrogen (to avoid
formation of degradation products) until on-shore analysis.Alternatively, filters can be
immediately placed in acetone for pigment extraction if analysis is to be carried out
onboard ship. Samples collected for HPLC analysis can also be used in the measurement
of chlorophyll a and phaeopigments by fluorometric analysis.
Filtration volume will vary with sampling location. For oligotrophic waters, 4 liters are
filtered, whereas in coastal regions a smaller volume (0.5-1.0 liters) may be appropriate. In
this case, a 25 mm GF/F filter is recommended.
7.0 Procedure
7.1 After removal from liquid nitrogen, the pigments are extracted by placing the filters
in 5.0 ml 100% acetone. For 47 mm GF/F filters, 0.8 ml of water is retained on the
filter, adjusting the final extraction solution to 86% acetone and the final extraction
volume to 5.8 ml. In order to correct for any errors introduced by evaporation or
experimental losses, 100 ml of an internal standard (canthaxanthin in acetone, Fluka)
is added to each sample which elutes after zeaxanthin and before chlorophyll b. The
samples are covered with Parafilm to reduce evaporation, sonicated (0C, subdued
light) and allowed to extract for 4 hours in the dark at -20 o C. Following extraction
samples are vortexed, filters are pressed to the bottom of the tube with a stainless
steel spatula, and centrifuged for 5 minutes to remove cellular debris. External stan-dards
are also run before each sample set for daily HPLC calibration.
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The addition of 5.0 ml acetone for pigment extraction is necessary to completely
submerge 47 mm GF/F filters in 15 ml centrifuge tubes. However, this volume can
be altered depending on the sizes of the filter and the extraction tube.
7.2 The HPLC system is setup and equilibrated with solvent system A at a flow rate of 1
ml/min.
7.3 Samples and standards are prepared for injection by mixing a 1 ml aliquot of the pig-ment
extract with 300 ml of distilled water in a 2 ml amber vial. Shake and allow to
equilibrate for 5 minutes prior to injection.
7.4 Approximately 500 ml of a sample is injected into the 200 ml sample loop and the
three-step solvent program initiated is on closure of the injection valve. The chro-matogram
is then collected on a recording device.
7.5 The identities of the peaks from the sample extracts are determined by comparing
their retention times with those of pure standards and algal extracts of known pig-ment
composition. Peak identities can be confirmed spectrophotometrically by col-lecting
eluting peaks from the column outlet.
7.6 Calibration: The HPLC system is calibrated with pigment standards obtained com-mercially
(chlorophylls a and b, and -carotene can be purchased from Sigma
Chemical Co., and zeaxanthin and lutein from Roth Chemical Co.) and/or by prepar-ative
scale HPLC (collecting and purifying HPLC fractions and placing in standard
solvents) standards. Concentrations of pigment standards should be determined
using a monochromator-based spectrophotometer in the appropriate solvents prior to
the calibration of the HPLC system. The recommended extinction coefficients for
most of the common algal pigments are provided in Table 13-2 (Bidigare 1991). Pig-ment
standard concentrations are calculated as follows:
where:
Cs = pigment concentration (mg l -1 )
A max = absorbance maximum (Table 2)
A 750 nm = absorbance at 750 nm to correct for light scattering
E = extinction coefficient (L g -1 cm -1 , Table 2)
l = cuvette path length (cm)
C s
A max A 750nm  ( )
E l  ----------------------------------------- - 1000mg
1g ------------------- -  =
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Standards stored under nitrogen in the dark at -20C are stable for approximately
one month.
After determining the concentrations of the pigment standard they are injected onto
an equilibrated HPLC system to calculate standard response factors (RF). Response
factors are calculated as weight of standard injected (determined spectrophotometri-cally)
divided by the area of the pigment standard plus areas of structurally related
isomers, if present.
8.0 Calculation and expression of results
Concentration of the individual pigments in the sample are calculated using the following
formula:
where:
C i = individual pigment concentration (ng per liter)
A = integrated peak area
RF = standard response factor
IV = injection volume
EV = extraction volume with internal standard correction
SV = sample volume
The units of ng kg -1 can be obtained by dividing this result by the density of the seawater.
9.0 References
Bidigare, R. (1991). in Spencer and Hurd (eds.). The analysis and characterization of
marine particles. American Geophysical Union, Washington D.C.
Herbland, A., A. Le Bouteiller, and P. Raimbault. (1985). Size structure of phytoplankton
biomass in the equatorial Atlantic Ocean. Deep-Sea Res. 32:819-836.
Holm-Hansen, O., and B. Riemann. (1978). Chlorophyll a determination: improvements
in methodology. Oikos, 30: 438-447.
Wright, S.W., S.W. Jeffrey, F.C. Mantoura, C.A. Llewellyn, T. Bjrnland, D. Repeta, and
N. Welschmeyer (1991). Improved HPLC method for the analysis of chlorophylls and
carotenoids from marine phytoplankton. Mar. Ecol. Prog. Ser. 77:183-196.
C i A ( ) RF ( )  1
IV ----- -      EV ( )  1
SV ------ -      =
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Table 13-1. HPLC solvent system program.
Time Flow Rate %A %B %C Conditions
0.0 1.0 100 0 0 Linear gradient
2.0 1.0 0 100 0 Linear gradient
2.6 1.0 0 90 10 Linear gradient
13.6 1.0 0 65 35 Linear gradient
18.0 1.0 0 31 69 Hold
23.0 1.0 0 31 69 Linear gradient
25.0 1.0 0 100 0 Linear gradient
26.0 1.0 100 0 0 Hold
34.0 1.0 100 0 0 Inject
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Table 13-2:
Pigment Wavelength (solvent) E 1cm(L g -1 cm -1 )
Chlorophyll a 664 nm (90% acetone) 87.67
Chlorophyll b 647 nm (90% acetone) 51.36
Chlorophyll c 1 +c 2 631 nm (90% acetone) 42.6
Chlorophyllide a 664 nm (90% acetone) 128.0
Fucoxanthin 449 nm (EtOH) 160.0
19 - Hexanoyloxyfucoxanthin 447 nm (EtOH) 160.0
19 - Butanoyloxyfucoxanthin 446 nm (EtOH) 160.0
Lutein 445 nm (EtOH) 255.0
Zeaxanthin 450 nm (EtOH) 254.0
Prasinoxanthin 454 nm (EtOH) 160.0
Alloxanthin 453 nm (EtOH) 262.0
Peridinin 472 nm (EtOH) 132.5
Diadinoxanthin 446 nm (EtOH) 262.0
Diatoxanthin 449 nm (EtOH) 262.0
b Carotene 453 nm (EtOH) 262.0
Phaeophorbide a 665 nm (90% acetone) 69.8
Phaeophytin a 665 nm (90% acetone) 49.5