DAYTON E. CARRITT Department of Oceanography, The Johns Hopkins University, Baltimore, Maryland
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analytical chemist's job in oceanography, as elsewhere, is to design analytical procedures, and to perform analyses. In oceanography his sample usually is sea water-a material that is chiefly water, but contains either dissolved or suspended substances representing probably all of the blocks in the preManhattan District periodic table, some of the postManhattan District blocks, as well as a wide variety of organic compounds, the latter usually in extremely low concentrations. For variety, he frequently must analyze bottom muds and oozes, fish, algae, antifouling paints, and in recent times the debris left by the successor of the Manhattan District. The cruise of H.M.S. Challenger (1873-76) has been noted by the authors of previous papers to be a turning point in the development of modern oceanography. The analyses performed by Dittmar on samples collected during this cruise gave a much firmer foundation than had existed previously for the proposition of "the constancy of relative proportionsn-a notion that says that so far as sea water is a solution of the major dissolved constitnents, the only observable changes that can be noted from one sample t o another is in the water content. As a consequence of this, the analysis for a single major constituent provides information about all the other major constituents and specifies the density at known temperatures. This is an appealing notion-a single analysis gives answers that otherwise would require several, thereby at least satisfying the human desire to obtain the greatest return for a given expenditure of time and effort. CONSTANCY OF RELATIVE PROPORTIONS
Despite the appeal and the practical advantages of the "constancv of relative uro~ortions." it mav be . . Presented as part of the Symposium on Chemistry of the sea before the Division of Chemical Educ&m at the 131st Meeting of the American chemical Society, ~ i ~~ ~ ~1957. i i , l contri. , bution No. 34 from the Chesapeake Bay Institute.
VOLUME 35, NO. 3, MARCH, 1958
instructive to examine the basis for the belief that the proposition is valid. Lyman and Abel (1958) have just noted that the ocean is "well shaken together" and that Maury observed "that the relative proportions of dissolved salts in the ocean are everywhere virtually the same." In a sense we are asking here "how well shaken is 'well shaken'?", or, "what is meant hy virtually?" On purely logical grounds the proposition of "constancy of relative proportions" cannot he valid without some modifying clause in its statement. For, as soon as we recognize processes that preferentially remove or add elements, we are forced to conclude that differences in relative composition must exist. We need then only inquire into the magnitude of the differences and over what time intervals and distances the differences can be detected with existing analytical techniques. An obvious qualification is t o preface our statement xith "within the limits of accuracy and precision of existing analytical methods." Such a qualification not only emphasizes the empirical nature of our conclusion, it also opens the way t o more quantitative qualifications such as a statement of the departure from constancy of the ratios Na+ t o C1-, SO,-- t o C1-, etc., when improved analytical techniques will permit better measures of the ratios. My purpose in pointing out a possible limitation to a rather well-established idea in oceanography is not to suggest that the foundations of the science are about to crumble, rather, I use it as a means of emphasizing the connection between analytical chemistry and the science of oceanography. In the light of our knowledge of analytical and inorganic chemistry, let ns look a t the substances involved. Thev ~" are six ions: rhloride. sodium. sulfate. magnesium, calcium, and potassium. ' In an "&eragel; sea water sample their approximate concentrations will be: chloride, 19,000 mg./kg. of sea water; sodium, 10,500; magnesium, 1200; sulfate sulfur, 880; calcium, ~~~~
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400; and potassium, 380. All other dissolved constituents will be less than 100 mg./kg. From the viewpoint of the analytical chemist these six elements spell trouble insofar as analyses of high precision and accuracy are concerned. Sodium and potassium are a notorious pair, as are calcium and magnesium. Dittmar's (1844) report of his analyses of the Challager samples, 77 in number, is interesting not only because they are the most comprehensive set of analyses of sea water in existence and therefore the basis of our faith in the "constaucy of relative prw portions," but also are rather humbling when viewed by analytical chemists. Today, some 75 years later, we are unable to do much better. Very briefly his methods were as follows: he determined halides by the Volhard method and on the basis of replicate analyses of synthetic samples claimed a precision of one part in ten thousand. Today when high precision and accuracy are required we use the same method with only slight modification. Sulfate was determined gravimetrically with BaSOl as the weighing form. His analyses of synthetic samples were about one part per thousand high, but he considered a correction of this order of magnitude unnecessary. As we would expect, calcium and magnesium gave him trouble. Calcium he determinod gravimetrically by single oxalate precipitation and ignition to calcium oxide, and from the calcium oxalate filtrate he precipitated magnesium ammonium phosphate and ignited it to pyrophosphate. After completing about a third of his analyses, he suspected coprecipitation of magnesium along with the calcium oxalate. He therefore combined all of his calcium oxide residues and twice repeated the calcium oxalate precipitation with analysis for magnesium in the filtrates. He thus was able to obtain an empirical correction for both the calcium and magnesium and he applied this constant L'corrector" to all of his analyses. If calcium and magnesium gave him trouble, potassium and sodium gave him great trouble. He finally adopted a modification of the Finkener process for potassium; conversion of all salts to sulfates, precipitation of chloroplatinates, elution of soluble salts, and reduction of the residual potassium chloroplatinate to metallic platinum with hydrogen. There being no suitable method for sodium, he determined it essentially by difference, using the results from all previous analyses and the weight obtained from the conversion of all salts t o sulfates, as the basis for his calculatiou. As noted above, Dittmar's analyses are the only "complete" analysis of a world-wide set of ocean samples on record. Workers following Dittmar have been content to determine the ratio of pairs of constituents. Professor Thompson, the author of the first paper of this Symposium, has made contributions to this part of the science. For the most part, studies of this sort have been with what we would call classical analytical methods. Thompson's recent determination of the calcium to chloride ratio utilized flame photometric measures of calcium and is one of very few studies which have not depended upon some modification of well-known gravimetric or volumetric procedures.
RECENT REFINED MEASUREMENTS
I n thinking about this problem of departures from the "constaucy" rule and of methods that are sufficiently refined to measure small differences in each of the major dissolved constituents, I have for some time been intrigued by the idea that if each of the cations could be separated from all others, essentially giving a beaker containing all the sodium from a sea water sample, another beaker containing all the calcium, etc., most of our analytical difficulties would be eliminated, for analyses of a solution of a single salt is a relatively simple matter. This idea, of course, is as old as chemistry itself and is the basis for all gravimetric procedures. The problem is how to get all of one and none of the others. The use of ion exchange resins immediately suggests itself as a possible means of achieving this end. Starting with this idea of complete separation prior t o determination, Carpenter has made what appears to be a break-through in the field of precision analysis of natural waters. He has been concerned primarily with the determination of magnesium and calcium. Using a combination of several of the newer developments in chemistry he devised a method that will routinely give results for both calcium and maguesium with a precision of considerably better than a part per thousand. Starting with a 10-milliliter sample, all divalent cations are taken up on an ion exchange column, Do~vex 50, with controlled particle size and crosslinkage, and in the ammonium form. Preferential elution of first magnesium and then calcium is achieved by using a reagent that forms tight enough complexes with the element to be removed so that it can competc with the element-resin equilibria, and in addition, the stability constants of each of the element-complex equilibria are different enough to effect separation of the elements during elution. These obviously are the specifications one would write for an ion exchange chromatographic system. Carpenter found that acetylacetone (pentane-dione-2.4), a 6-diketone, has the required properties: the logarithm of the formation constant of the magnesium complex is 9.12, of the calcium complex about 1.2 and of the strontium complex, unknown, but much less than for the calcium complex. In addition, acetylacetone decomposes on heating to form acetone and acetic acid, thus allowing complete removal of the complexing agent after the separations have been achieved. Carpenter's method of terminal analysis is a good example of the use of modern analytical techniques in oceanography. Having calcium, for example, now separated from all interfering elements greatly increases the kinds of procedures that can be used for final analysis. He developed a spectrophotometric titration, by weight, using copper standardized EDTA (ethylene diaminetetraacetic acid) and ammonium purpurate as an indicator. The precision that can be obtained by such a procedure is remarkable. Eight analyses gave standard deviation of 0.02'30. Carpenter's work is mentioned here primarily because it demonstrates a somewhat new look a t the old problem of the analyses of plentiful substances, the major constituents of sea water. It represents the use JOURNAL OF CHEMICAL EDUCATION
of ion exchange resins, chromatographic separations, compleximetric and spectrophotometric titrations, and in the published version, a rather well-developed theoretical treatment of the systems employed. The oceanographic significance of Carpenter's work is to be found in the results of calcium analyses he performed on a series of samples taken in the Bahamas, in a region in which active calcium carbonate precipitation occurs. This, then, is a process that preferentially removes one of the major constituents-a process that must lead t o differences in the relative composition of samples. His analyses show definite reduction in the ratio Ca++ to C1- in samples taken in the regions of precipitation. Thus, we now have a tool that will permit us to trace ocean water that has passed through the region, and will provide a measure of the rate of mixing between this tagged water and unaffected ocean waters.
ample, repeated attempts to conduct precision pipetting, with one hand and while being seasick, would suggest to most of us either a change of profession or a modification of the procedure. Fortunately, the latter alternative has been taken and as a result a benchmounted syringe, connected directly to the reagent bottle, and fitted with a spring driven plunger that operates between mechanical stops, has been devised for routine pipetting; it can be operated with one hand. Gadgeteering of this sort is common, some of it rather ingenious and directly applicable to operation in any laboratory, seagoing or shore-based. In general, analytical techniques that terminate in either titrimatric or spectrophotometric measurements, with a minimum number of operations between sampling and terminal measurement, can be adapted t o use a t sea. . ANALYSIS FOR TRACE SUBSTANCES
ANALYSIS
The studies noted above were all conducted in mellequipped, shore-based laboratories. To see the analysts who did the work in their laboratories it would be impossible t o distinguish them from thousands of other analytical chemists-there would be no indication that these men were chemical oceanographers. The point I have in mind to make here is suggested by a comment made by a student upon returning from a rather stormy cruise. His first comment upon hitting the beach was that not only was Dittmar the first chemical oceanographer, he probably was the smartest -he stayed ashore and had his samples brought t o him. Chemical operations on a research vessel are very different in many respects from those done ashore. Many operations that are routine in a shore-based laboratory cannot be performed a t sea. Precision weighing obviously cannot he done. However, an electronic balance has been developed that functions well in the "several milligram" range, under conditions that mould make common instruments useless. So far as instrumentation of analytical procedures is concerned "going to sea" restricts the choice of equipment, primarily because of ship's motion-not only the motion produced by sea and swell, but also the much higher frequency motion produced by vibration from propulsion engines and generators-because of high humidity and wide variations in temperature, and unless rather expensive precautions are taken because of relatively poor voltage and frequency control of a.-c. power. Once the effects from these three sources (motion, humidity and temperature, and poor power regulation) are overcome, instrumentation problems are in the clear. Nevertheless, even though instruments call be made to function, there still is a major problem before chemical measurements can be made in an efficientmanner. This is the problem arising from the effects of ship's motion on the chemical oceanographer. The old saying, "one hand for the ship and one for yourself," applies t o all who go t o sea-captains, chemists, and cooks. Furthermore, chemical oceanographers appear t o be just as susceptible to seasickness as captains or cooks. The effects of ship's motion on the analyst and on his equipment frequently combine in such a way as to produce rather drastic modifications in commonplace operations. For exVOLUME 35, NO. 3, MARCH, 1958
The minor dissolved constituents (the trace substances) in sea water present a rather different set of problems to the analytical chemist than do the major constituents. We can recognize three classes of trace substances in the oceans: (1) inorganic, (2) organic, and (3) dissolved gases. Thanks to the relatively simple colorimetric techniques that have been developed for the determination of dissolved inorganic phosphate and for nitrate and nitrite we have a fairly welldeveloped picture of the broad biochemical cycles of phosphorus and fixed nitrogen in the sea. These substances truly are trace substances; phosphate occurs in the range up to approximately 3 microgramatoms per liter, and nitrate up to 25 or 30 microgramatoms per liter. A modification of the familar molybdenum blue reaction, using stannous chloride as a reducing agent, permits the determination of phosphate with a precision of several hundredths of a microgramatom per liter. Nitrate is determined colorimetrieally using strychnidine as the color-producing reagent. Fortunately, these procedures can be made to function adequately at sea. With the exception of silicate, dissolved atmospheric oxygen, and carbon dioxide, our knowledge of the distributions of the remaining trace substances and of their geobiochemical significance in the sea is meager. As an example of the kinds of interrelations that await study, we can speculate on a possible role of cobalt, using information obtained in biochemical and nutritional studies as the basis for our speculations. First, it should be noted that there are only a few reliable determinations for cobalt in sea water. Richards in commenting on the "State of Our Knowledge of Trace Elements in the Oceans," noted that no information is available for cobalt in the deep oceans and only a few determinations have been done in surface waters. It appears t o be in the range of 0.001 to 0.0001 p.p.m. (1.0 to 0.1 pg. per liter). On the speculative side, the following sequence seems reasonable. We know that vitamin Biz contains an atom of cobalt per molecule of the vitamin. Biz is a natural product, and when it is present in the sea the cobalt requirement of the producer organisms must have been met. Furthermore, in vitro studies have shown the essentiality of B12for the growth of certain classes of phytoplankton organisms: no BI2,no growth. This specificity is SO
marked with some organisms, Euglena graeilis, for exam~le,that the organism can be used in a bio-assay for B;~. We have here then a possible sequence of events in which cobalt appears as an essential link and possibly a limiting factor in the growth and development of some of the organisms that form the base of the food web. I t certainly follows that unless the cobalt requirement of the Biz producing organisms is met, there can be no growth of the organism having a BIPrequirement. Two problems are obvious here for the analytical chemists: cobalt and vitamin Biz. I n many ways the problems presented by these two substances are typical of analytical problems presented by many of the substances in the inorganic and organic classes noted above. These substances are present in the submicrogram-per-liter range and appear to be biologically significant in the same concentration range. In most csses, direct analysis is not possible a t these extreme dilutions and in the presence of the many interfering substances present in sea water. . Obviously, two functions must be performed before determination. A technique of concentrating the test substance and of separating it from interference must be devised. Generally speaking, concentration factors of a t least 1000 must he achieved before most of the usual methods of terminal analysis can be applied. Carrier or sweeper techniques have been used with limited success in other similar situations and may possibly find application to the sea water problem. We have had fair success in the analysis of brackish water by using a column packed with cellulose acetate as the support for dithizone in CCln for the collection and concentration of Zn,++I%++, Cu++,Co++, and Mn++. Concentration factors of 10,000 have been achieved with such a column. The dithizone is nsed here merely as a collector, and terminal analysis has been polarographically for the Zn++, Pb++, Cu++, and Co++, and colorimetrically for Mn++. 'IIigh molecular weight organic substances can be obtained by dialysis to remove the inorganic salts, followed by low temperature evaporation t o concentrate the residues. Again with brackish waters, we have nsed these techniques followed by chromatographic techniques to separate components, and by spectrofluormetric or colorimetric analysis to characterize the separated components. Starting with 20 liters of sample we have found as many as nine components on chromatograms. None of the components have as yet been identified. GROWTH OF PURE CULTURES
The examples noted above, in which analytical problems exist which involve trace substances, are parts of broader biological problems, primarily the nutrition of marine organisms. There is one more example in this category that should be mentioned. It has to do with the culturing of marine microorganisms in the laboratory. Although this is primarily a biological problem it now appears that many intriguing analytical problems will have to be solved before many of the obvious advantages of this kind of experimentation can be more fully exploited in the solution of marine biological problems.
The basic problem is this: Although the broad, qualitative aspects of the food web in the oceans are clear, we have only very meager information about the several parts of most marine geobiochemical systems. We know for example, as on land, that the plant life of the seas (the phytoplankton) turns inorganic constituents into living organic matter through the photosynthetic process. The phytoplankton then form the base of the marine food web. The phytoplankton are eaten by small animals (the zooplankton), the smaller zooplankton by larger zooplankton, bacteria break down dead organic stuffs-in all a continual degradation of the energy that entered the system as solar radiation used in photosynthesis. We are now asking questions about the parts of this complicated system. Some oceanic regions are more productive than others.. We would like to know what controls the fixation of energy, the growth of the phytoplankton populations, and the transfer of this energy from one trophic level to the next. One approach to this problem is through the study of the nutritional requirements of the producer organisms, the phytoplankton. If we know what the organisms require we will then be able to examine the natural environment to see if these requirements are met. Attempts are now being made to grow marine phytoplankton in pure culture-a single species of organism, in a bacteria free environment-to grow these pure cultures in a completely defined medium, that is, in an environment in which the kinds and quantities of dissolved and suspended substances are known to the experimenter. In such a system nutritional requirements and toxicity levels can be studied by noting changes in reproduction, growth, and composition of the organisms, produced by changes in media constituents. It has frequently been noted that as the purity of commercially available chemicals has increased, during the last 25 to 50 years, the number of failures to obtain growth in cultures has also increased, In other words the "c.P." chemicals of tweuty-five years ago contained traces of impurities, not present (or at much lower levels) in the same chemicals today; and these traces, present in the media but not known to be there by the media makers, satisfied the requirements of the organisms. Media makers now need both organic and inorganic chemicals with extremely high purity specifications. In a few cases where requirements are known, growth is affected at the parts-per-billion level. To meet specifications of this kind will require the application of some of the finest precision tools and techniques we can muster in inorganic and organic analytical and preparative chemistry. BIBLIOGRAPHY LYMAN, J., AND R. B. ABEL,J. CHEM.EDUC., 35, 113 (1958). DITTMAR, W., Report an researches into the composition of ocean water, oolleoted by H. M. S. Challenger. Challenger Repts., Phvsics and Chem., Vol. 1, 1884. CARPENTER, J. H., "A Study of Some Major Cations in Natural Wstera." Ph.D. thesis, The Johns Hapkins University, Baltimore, Maryland, 1957. CARPENTER, J. H., "The Determination of Caleium in Natural Waters." Limnology and Oceanography, 2 , 271 (19578). RICHARD?, F. A,, Geochim. el Cosmochim. Acla, 10, 241 (1956)
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