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The particulate organic carbon content is defined as µg POC /l
seawater.
The particulate organic nitrogen content is defined as
µg PON /l seawater.
and the organic nitrogen into N
gas; these are separated on a column and measured by thermal conductivity.
and N by TCD gas chromatography can
be used. None of the commercially available analyzers is particularly
recommended.
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Hydrochloric acid (HCL: reagent grade)
Acetanilide, EDTA (reagent grade) or other non
hydrophile substances with a C:N ratio close to those of the samples are
used as standards.
- General Precautions
- Sample Acquisition
- Filtration
- Storage of Samples
During the entire procedure of sampling, filtration, storage, fuming and analysis, care must be taken to avoid contamination by both organic and inoranic forms of carbon and nitrogen. Acid cleaning and rinsing with deionized water of water samplers, filtration bottles, filtration device and storage containers is recommended. During storage, particularly when deep frozen, samples must not be exposed to other sources of volatile carbon and nitrogen.
A representative aliquot of the water from the sampler or the contents of the entire sampler is taken. In the former case homogeneity of the entire sample must be maintained; e.g., by gentle agitation prior to the taking of an aliquot. In the latter case all water must be taken from the bottle including the dregs. Sample size is chosen in accordance to the sensitivity of the analyzer and in situ concentrations of the respective environment under investigation (0.5 to 2 l in the euphotic zone, 5 to 10 l in deep water). For deep water sampling it is preferred to take duplicates or triplicates to achieve good numbers rather than daily sampling of one sample of discrete depths.
Prescreening of water samples may exclude mucilagious or chain-forming algae and large faecal pellets and strings. If prescreening is necessary due to zooplankton and/or infrequent large particles a 200 micron mesh should be used and this must be reported in the protocols. Removal of such particles from the filters is also recommended. From sediment trap samples a liquid or freeze dried aliquot can be used.
Glass fiber filters (GF/F) or quartz fiber filters are used. The latter have the advantage of not melting in the C/N analyzer furnace tube. Filters are precombusted in a muffle furnace for 4 hours at 500°C. With increasing sample size, retention of smaller particles on fiber filters is increased. Therefore, the sample size should be kept as constant as possible to maintain a consistent filter loading. A pressure differential of less than 0.5 atm should be used in pressue or vacuum filtration. To avoid cell lysis filters must not run dry. Generally salt residues do not cause a problem, therefore filters should not be rinsed with deionized water. Rinsing with filtered seawater is useful when mucilagious particles are sticking to the filtration device.
Samples can be either kept deep frozen at -20°C or in a desiccator after drying at 60°C for 5 hours. When drying filters in an oven care must be taken to avoid contamination from volatile organics. Samples from sediment traps can be stored as dried powder. Storage time is nearly unlimited. In the case of deep-freezing samples are desiccated before analysis.
- Acidification of Filters
- Standards
- Blanks
In certain regions and seasons filter loads may contain carbonate minerals amounting to a considerable portion of total particulate carbon, e.g. coccolithophorid blooms, foraminifera, coral reef environments. Removal of inorganic carbon can be done by fuming the dried filters with concentrated hydrochloric acid for 24 hrs. Thereafter filters are dried again. When rinsing the filters with acid, losses in particulate organic carbon and nitrogen of up to 40 % have been observed. In the case of acid rinsing of filters the rinse should be collected and analyzed for dissolved organic carbon and nitrogen. When low amounts of carbonate minerals in the samples are expected, acid can be added directly to the sample capsule of the analyzer. Whether samples were acidified or not and what mode of acidification was used must be reported in the protocols.
The standard should be not hydrophilic and should have a C/N ratio close to that of the samples. Pure substances such as acetanilide and EDTA, are easily analyzed and may not fully account for the instruments performance for samples with more refractory components. A reference standard containing more refractory components should be prepared and used by the JGOFS community. Standards should be run every 20 to 40 samples to check the drift of the analyzer.
The machine blank is usually included by the analyzer calibration. A blank for filters, filtration, handling and storage is best achieved by refiltering the same volume of filtered seawater as for the samples through a precombusted filter and thereafter follow exactly the same procedure as for the samples. This should also account for filtration artefacts such as adherence of dissolved organic matter to the fibers and formation of colloids during filtration. One blank should be run for every 10 samples.
- Accuracy
- Precision
- Quality control
The exact nature of organic particles on the filters is unknown. Therefore the accuracy must be regarded as 100 % provided that the analyzer is adequately calibrated and an appropriate standard is used.
Precision is defined as the replicability of a homogeneous standard or sample material. Precision for the standard has to be within ±5 %. The limit of detection varies somewhat depending on the instrument used. Samples should contain at least 10 micrograms of nitrogen and 15 micrograms of carbon.
Between 10 and 20 % of the samples should be run as triplicates. The coefficient of variation of replicate samples should be not higher than ±10--15 %. Operation of the machine should mainly follow the manual of the manufacturer. If changes are introduced these should be discussed with the manufacturer and harmonized with the colleagues using the same model.