Automated nutrient analysis

Automated methods not only increase the amount of chemical data obtainable during a cruise, they also avoid some of the main pit falls of manual analytical procedures. Most of the standard manual analytical methods include multiple handling of samples in open vessels while adding reagents or during titration, etc. The resulting risk of sample contamination, though tolerable in a clean laboratory, becomes incalculable under the somewhat provisional conditions in a ship s laboratory even if the floor is not in constant motion. 

However, it seems necessary to state that automation does not mean progress per se. While the sources of errors in the application of approved manual analytical methods are well known and most of the occurring faults are obvious to the operator, malfunctions of an automated system might pass completely unnoticed by the operator as long as the data do 

The large amount of data produced by an automated analytical system has to be processed by an on-line computer. Owing to the miniaturization of analytical and electronic components multi-channel nutrient analysers may be designed as rather compact and handy units, even fit to be used on small vessels. However, they are very complex and by no means switch on-measure-switch off machines. Well trained personnel, skilled in analytical chemistry, mechanics and data processing, and a great deal of experience is required to reliably operate nutrient analysers. Automated analysis is most profitable in every day routine measurements of large sample numbers. A few occasional nutrient samples, however, are better analysed by manual methods. 

Since the introduction of automated chemical nutrient analysis a number of methods for the determination of different compounds in seawater and modifications of the methods have been developed. With few exceptions the continuous flow method of the Technicon AutoAnalyzer is the fundamental principle of these methods. 

In addition to the determination of the traditional nutrients, flow methods have been reported for a number of other components using either flow spectrophotometers or other types of detectors. Flow methods can be easily combined in multi-variable systems. Despite the occasionally low accuracy, they can provide useful additional information about the analysed samples. As an example, consider conventional water sampling in an estuary using a small vessel and sample bottles on a hydrographic wire. To confirm that a sample has really been taken at the depth indicated by the cable length and sampler position on the cable, it is necessary to measure the salinity. Instead of using sample water for separate salinity determinations with a salinometer (increasing the required sampler size), a small conductivity cell in the flow analyser would provide this information. 

When coloured solutions have been obtained by suitable chemistry, these are pumped through colorimeters similar in principle to those used in spectrophotometric analysis. Light of the most suitable wavelength is obtained from incandescent lamps and interference filters, and the transmission of a solution (100% for a blank down to about 10% for samples) is measured by a pen recorder from which the concentration of a nutrient can be evaluated after suitable standardization. 

All this equipment is conveniently mounted on the upper surface of a large box in which can be housed much of the wiring and tubing. Reagent bottles and bottles for standards and blanks, eta, can be sunk in circular wells shown as circles in Fig. 1. The reagents are mixed in coils and tubing fastened to the platforms.  

Nutrient analysis has been designed around three main approaches. Procedures A, B, and C, which represent compromises in sensitivity according to the concentrations of nutrients to be expected in various circumstances. 

Procedure A utilizes the full sensitivity of all methods and is generally used when measuring the concentration of nutrients in surface waters, whilst the ship is underway. Occasionally in areas of high coastal upwelling, where surface nitrate concentrations exceed 7 /big-at N/liter, the sensitivity may be too great, necessitating the use of Procedure B. 

Procedure B is designed for work with a profiling hose to obtain nutrient profiles to 100-200 m. A modified Procedure B may be used where the maximum concentration of nitrate does not exceed about 15 jug-at N/liter. 

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Loder TC, Gilbert PM (1976) Blank and salinity corrections for automated nutrient analysis of estuarine and sea waters. University of New Hampshire Contribution UNH-59-JR101 to Technicon International Congress December 13-15... 

Whitledge T.E., MaUoy S.C., Patton C.J., and Wirick C.D. 1981. Automated nutrient analysis in seawater. Upton, NY Department of Energy and Environment (Brookhaven National Laboratory Formal Report No. 51398), 216 pp. 

Flow injection analysis is a rapid method of automated chemical analysis that allows for quasi-continuous recording of nutrient concentrations in a flowing stream of seawater. The apparatus used for flow injection analysis is generally less expensive and more rugged than that used in segmented continuous flow analysis. A modified flow injection analysis procedure, called reverse flow injection analysis, was adopted by Thompson et al. [213] and has been adapted for the analysis of dissolved silicate in seawater. The reagent is injected into the sample stream in reverse flow injection analysis, rather than vice versa as in flow injection analysis. This results in an increase in sensitivity. 

TTHE BENEFITS OF AUTOMATING NUTRIENT ANALYSES IN SEAWATER have been recognized and utilized for several decades. Automated analyses allow samples to be processed faster and generally with better precision and accuracy than is possible with most manual methods. The only technique that was available at a reasonable cost for the automated analysis of seawater nutrients, until recently, was segmented continuous-flow analysis (CFA). Segmented CFA is characterized by the use of air bubbles to segment the liquid in the reaction tube so that dispersion of the sample is limited. 

As an example, I will use the determinations of DOC and DON, partly because the discussions are very recent and partly because they are very familiar to me. The question of the accuracy of measurements of DOC in seawater has been disputed at least since the publications of Putter [62] and Krogh [63] the early work has been reviewed at length in an earher pubhcation [64]. While a variety of wet oxidation methods [65] were proposed for marine samples, the use of persulfate [35] provided the first real approach to a standard method. Persulfate oxidation, however, hke all purely chemical oxidations, was a batch process, and not easily automated. A number of workers proposed photo oxidation using ultraviolet (UV) hght [66-70], and automated analytical systems which produced data almost in real time were soon constructed [71-73]. Commercial units soon appeared, but many of the units in the field were jerry-built, constructed out of parts scavenged from discarded autoanalyzers formerly used for nutrient analysis. 

At the time of the first edition of this book the automated chemical analysis of nutrients, introduced into oceanography by Weichart (1963) and Grasshoff 19M), was still a relatively new technique but now it is state-of-the-art in most oceanographic laboratories. 

The standard setups for nutrient analysis are either manual or automated air-segmented-flow-analysis versions of a wet chemical treatment of water samples to convert the desired nutrient into a coloured compound with high molar absorptivity in a suitable range of the light spectrum. Flow-injection analysis is based on identical chemical procedures but requires different reagent recipes. A more detailed introduction into automated flow-analysis is presented in Section 10.3 at the end of this chapter. 

Bioprocess Control An industrial fermenter is a fairly sophisticated device with control of temperature, aeration rate, and perhaps pH, concentration of dissolved oxygen, or some nutrient concentration. There has been a strong trend to automated data collection and analysis. Analog control is stiU very common, but when a computer is available for on-line data collec tion, it makes sense to use it for control as well. More elaborate measurements are performed with research bioreactors, but each new electrode or assay adds more work, additional costs, and potential headaches. Most of the functional relationships in biotechnology are nonlinear, but this may not hinder control when bioprocess operate over a narrow range of conditions. Furthermore, process control is far advanced beyond the days when the main tools for designing control systems were intended for linear systems. 

Matrix effects in the analysis of nutrients in seawater are caused by differences in background electrolyte composition and concentration (salinity) between the standard solutions and samples. This effect causes several methodological difficulties. First, the effect of ionic strength on the kinetics of colorimetric reactions results in color intensity changes with matrix composition and electrolyte concentration. In practice, analytical sensitivity depends upon the actual sample matrix. This effect is most serious in silicate analysis using the molybdenum blue method. Second, matrix differences can also cause refractive index interference in automated continuous flow analysis, the most popular technique for routine nutrient measurement. To deal with these matrix effects, seawater of... 

In planning the introduction of automation into an analytical laboratory, it is important not only to consider all stages of the analysis, but also the wider context within which the laboratory serves the organization of which it is a part. Examples of laboratories that have engaged in effective planning are the Laboratory of the Government Chemist, London the Nutrients Composition Laboratory at USDA, Beltsvillle, Maryland, USA and Shell Development Company, Seal Hollow, Houston, Texas, USA. 

Flow injection analysis (FIA) is a robust method for automating complex chemical analyses (Ruzicka and Hansen, 1988). It is relatively simple and can be adapted for use with a variety of detectors, including spectrophotometers, fluorometers, mass spectrometers, and electrochemical analyzers. It has been used on board ships to determine dissolved nutrients (Johnson et al., 1985) and trace metals (Sakamoto-Arnold and Johnson, 1987 Elrod et al., 1991). Unsegmented continuous flow analysis (CFA) systems based on the principles of FIA can operate in situ over the entire range of depths found in the ocean (Johnson et al., 1986a, 1989). 

Flow-injection analysis (FIA) is a technique for automating chemical analyses. The principles of FIA are reviewed here. Methods for applying FIA to the anayses of nitrate, nitrite, phosphate, silicate, and total amino acids in seawater are examined. Analyses of other nutrients, metals, and carbonate system components are also discussed. Various techniques to eliminate the refractive index effect are reviewed. Finally, several examples of the application of FIA to oceanographic problems are presented. 

N.J. Blundell, P.J. Worsfold, H. Casey, S. Smith, The design and performance of a portable, automated flow injection monitor for the in-situ analysis of nutrients in natural waters, Environ. Int. 21 (1995) 205. 

FIA analyzers or FIA components. One company produces a series of instruments that are flow injection systems with atomic absorption spectrometric detection dedicated to determination of mercury. Some companies produce flow injection analyzers for a large number of ions. One supplier has an analyzer that comprises three separate units a basic analytical module, an automatic sample module, and a data capture module, all these units being completely automated. The instrument is capable of analyzing nutrients, ions, and metals. It offers a wide analytical choice using ion-selective electrodes (ISEs), chemiluminescence, or fluorescence. With analysis speeds up to 120 samples per hour and detection limits down to parts per billion levels, this flow injection analyzer performs determinations well compared with other techniques. 

As the outlines of methods are nearly identical for manual analysis and automated air-segmented-flow-analysis (with regard to chemistry, sample pretreatment, preparation of standards and reagents), we have combined the analytical procedures for each nutrient. 

Subsamples for automated analysis should be taken directly from the water samplers into subsample bottles of the desired volumes, which can be acconunodated by the autosampling device of the nutrient analyser. 

Though manual determinations of nutrients are still in use, the majority of samples are analysed by automated flow-analyses. The oxidation procedures for the determination of total nitrogen, phosphorus and silicon as presented in the previous editions of this book, have thus been adapted to subsequent flow-analysis of inorganic nutrients without modifications to the manifolds or reagents. 

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