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on Advances in Water Pollution Research, Tokyo, 0. Jaag (ed). pp. 61-82. Struclaneyer, B. E., L.A. Peterson, and F. Hsi-Mei Tai. 1969. Ef- fects of copper on the composition and anatomy of tobacco. Agron. J. 61:932-936. Suttle, N. F., and C. F. Mills. 1966. Studies of the toxicity of copper to pigs-zinc and iron on copper toxicosis. Br. J. Nutr. 20:135, 149. Swaine, D. J. 1955. The trace element content of soils. Commonw. Bur. Soil Sci. Tech. Commun. No. 48. Herald Printing Works, York, England. 167 pp. Swaine, D. J., and R. L. Mitchell. 1960. Trace element distribution in soil profiles. J. Soil Sci. 11 (2):34 7-368. Thornton, I., W. J. Atkinson, J. S. Webb, and D. B. R. Poole. 1966. Geochemical reconnaissance and bovine hypocuprosis in County Limerick, Ireland. Irish J. Agric. ReL 5:280-283. Thornton, 1., R.N. B. Moon, and J. S. Webb. 1969. Geochemical reconnaissance of the lower Lias. Nature 221:457-459. TJpton, I. H., P. L. Stewart, and P. G. Martin. 1966. Trace elements in diets and excreta. Health PhyL 12:1683-1689. Copper and Molybdenum 19 Titcomb, J. W. 1914. The use of copper sulphate for the destruction of obnoxious rashes in ponds and lakes. Trans. Am. Fish. Soc. 44:20-26. Turekian, K. K., and K. H. Wedepohl. 1961. Distn'bution of the ele- ments in some major units of the earth's crust. Geol. Soc. Am. Bull. 72:175-191. Underwood, E. J. 1971. Trace elements in human and animal nutri- tion (3rd ed.). Academic Press, New York. 543 pp. Van Campen, D. R., and Earl Gross. 1965. Influence of ascorbic acid on copper absorption by ratL J. Nutr. 95:617. Webb, J. S., and W. J. Atkinson. 1965. Regional geochemical recon- naissance applied to some agricultural problems in county Lime- rick, Eire. Nature 208:1056-1059. Webb, J. S.,l. Thornton, and W. K. Fletcher. 1968. Geochemical re- connaissance and hypocuprosis. Nature 217:1010-1012. Wedepohl, K. H. 1970. Geochemical data on sedimentary carbonates and carbonate rocks and their facies and petrogenic evaluation. Verb. Geol. Bundesanst. 4:692-705. (In German.) Chem. Abstr. 75(7):71 (1971).
X Analytical Methods GEORGE H. MORRISON, Co-Chairman JAMES 0. PIERCE, Co-Chairman William H. Allaway, Ernest E. Angino. Helen L. Cannon, Roger Jorden. Joe Kubota, Herbert A. Laitinen, Hubert W. Lakin The acquisition of reliable and relevant analytical data is a critical procedure in studies involving the environment in relation to health and disease. A clear defmition of the overall environmental problem to be solved must be estab- lished before the experimental design is formulated and the study is initiated. Active communication must exist between field and laboratory personnel to ensure agreement and un- derstanding of goals. To best achieve this communication, the analyst must be accepted as an integral part of the in- vestigative team. Because of the essential role of analysis, an obvious need exists for research in the development of analytical methods; but, because such research is not necessarily mission-oriented, funding is very difficult to obtain. If analysis is to keep pace with the increasing demands of environmental studies, inde- pendent funding of basic analytical research is a necessity. All trace analytical methods consist of three steps: sam- pling, chemical or physical pretreatment, and instrumental measurement. In this chapter, analytical methods for each of the metals under consideration by this workshop are re- viewed. Rather than to explore in depth the specifics of each analytical method, the purpose of the Methods and Sampling Work Group was to investigate and summarize current research and knowledge. In the next chapter, the status of sampling, sample preparation, and storage are reviewed in connection with analysis. 80 ANALYTICAL METHODS FOR IODINE AND FLUORINE Iodine and fluorine are not common contaminants of the materials used in sampling tools (e.g., stainless steel, poly- ethylene). However, Teflon and some stopcock greases are very inert perfluoro compounds often used in the handling and processing of samples. If particles of these materials become mixed with the sample and are not removed before the measurement step, appreciable contamination will re- sult when analysis is performed with neutron activation or some total-destruction method. Such contamination is, however, unimportant if a method measuring the fluoride ion (F-) activity is used, e.g., ion-selective electrodes. Because dilute solutions of most elements tend to adsorb on container surfaces, it is advisable either to adsorb the iodide ion (I-) or F- from natural waters on anion resins or to freeze-dry the sample in the container in which the measurement will be made, as in neutron- or photon- activation methods. No definitive study of loss or contamination in materials containing iodine and fluorine has yet been done for the sample-processing step. Acidic solutions of fluoride contain hydrofluoric acid (HF), which itself may be lost (finite va- por pressure above the solution) or may attack the silicon of flasks and tubing to yield adsorbed or readily volatilized
silicon fluorides, depending on the temperature. High- temperature ashing of fluorine-containing compounds is best done under alkaline conditions (Bussmann and Hanni, 1967), preferably in the presence of some species that com- bines strongly with fluoride (e.g., calcium). Oelschlager (1968) has reported that ashing in a muffle furnace lined with fireclay brick can yield positive errors (e.g., +390 per· cent at 550 C) unless the furnace interior is lined with nickel or platinum. The production of HF has been used to advantage (Whar- ton, 1962) in the removal of interfering ions. As shown in the accompanying schematic, the HF produced diffuses through the polyethylene membrane of a Conway dish into a sodium hydroxide (NaOH) receiving solution in which r can then be determined by a number of techniques. Sealing Well HCI04 (perchloric acid)+ Sample+ Wetting Agent Pharmaceuticals and organic compounds have been suc- cessfully submitted to oxygen-flask combustion to free organically bound fluorine. The nickel combustion bomb is still the universal method for sample decomposition, whereas sodium biphenyl reagent is a milder treatment to free fluorine from the carbon-fluorine bond. Whereas fluorine in biological systems remains almost exclusively in its inorganic form, iodine is often distributed among inorganic and several organic forms; hence, sample treatment should have in it the option of separating these various forms before measurement. If total iodine present, regardless of form, is sought, then destructive methods such as the Parr-bomb oxygen flask or combustion tube may be used immediately (Y aka tan and Tuckerman, 1966). Preseparations must be made if further chemical charac- terization of the forms of iodine present is to be accom- plished. Recourse to destructive oxidative methods must nevertheless subsequently be made to perform elemental analysis. The inorganic I- may be separated from protein-bound iodine by passing the sample through a Sephadex G-25 column (V eselsky et al, 1970) or simply by precipitation with the silver ion ( Ag ~ if the sample is such that the pre- cipitate can be isolated and removed. Other methods in· volve short-term electrophoresis or protein separation with trichloracetic acid (ChCCOOH), the latter being least satis· factory (Hocman et al., 1966). Major volatility problems occur with acidic or oxidizing Analytical Methods 81 solutions because mixed halides may be formed under these conditions. As with fluorine, this volatility can be put to good use, e.g., in some radiochemical separations (Palomares et al., 1970). The absorbing solutions (as with the oxygen- combustion methods) usually contain NaOH or arsenite to ensure that I- is the only form present. The methods of measurement for iodine and fluorine are summarized in Tables 29 and 30. ANALYTICAL METHODS FOR LITHIUM Several analytical methods have been used for the determi- nation of lithium in water. Among these are emission spec- trography (usually preceded by evaporative concentration of the sample), flame photometry, atomic absorption spec- trophotometry, and wet chemistry. Of these, atomic ab- sorption seems to be preferable in most applications. It is simple, rapid, reasonably accurate, and, in the hands of a good technician, will reproduce well. Although atomic absorption is generally free from spectral interferences, it is still subject to chemical interferences, which tend to be- come more critical with increasing solution concentration. This problem warrants consideration when lithium analy- ses are being made on brine, serum, and other biological fluids. Techniques for compensating or eliminating these interferences can be found in Ramirez-Munoz (1968), Angino and Billings (1967), and Slavin (1968). Because lithium is associated with other alkaline metals, tests and corrections for sodium, potassium, and cesium interferences are extremely critical. If analyses of ash or solid samples are preferred or man- datory, then either emission spectrography or flame pho- tometry can be used, provided that a highly trained opera- tor, familiar with the limitations of each method, is available. In summary, aU things being equal, atomic absorption is the preferred method for lithium analyses. The development of a method for routinely and accurately determining lith- ium at levels below 100 p.g/1 in biological fluids, especially urine, is badly needed. ANALYTICAL METHODS FOR CADMIUM~ ZINC, AND LEAD Table 31, prepared with the cooperation of R. C. Vogel, of the Environmental Analytical Laboratory of the University of Illinois, compares the limits of detection of several ana- lytical methods for the three subject elements. These limits are intended to reflect the current status, rather than the ultimate possible limits reachable by using special testing. As an example, extraction procedures have been coupled with atomic absorption to render the method applicable to natural waters. The methods fall into two groups, solution techniques
82 THE RELATION OF SELECTED TRACE ELEMENTS TO HEALTH AND DISEASE TABLE 29 Methods of Measurement of Iodine Amount or Technique Material Concentration Atomic emission Synthetic 2ng Thyroid, freeze lpg dried Spectrophotometry Organically bound 1 Serum bound I Fluorescence Synthetic 1-10 pg Titration Urine Ion-selective-electrode Idealized system 3 pg/1 Electrochemistry Idealized system 0.1 ~o&M 2 X 10-6% Gas chromatography Idealized system Neutron-activation Aerosol particles Active charcoal ftlter Protein bound 1- General biologicals 4 X 10-4 ppm Blood Photo-activation Seawater Hair, blood, wood, 0.2pg tobacco Catalytic Blood Soil General biologicals Spark-source mass Human hair 0,03 1-'8 spectrometry X-ray fluorescence Pharmaceuticals and solid-sample techniques. In the former, illustrated by atomic absorption or anodic stripping voltammetry, the detection limit experimentally determined is a concentration detection limit (Osteryoung and Oster- Detail Comment (Reference) Graphite resistance furnace Poor precision in nanogram region (L'vov and Khartsyzov, 1969) RF Plasma excitation No pretreatment but organic matrix makes sample hard to control (Talmi and Morrison, 1972) 0 2 Combustion with indirect determination (Fukamauchi and ldeno, 1967) via Cul2 Technicon autoanalyzer method Accepts only samples near nor- malcy, failing for grossly ab- normal ones (Simpson, 1967; Crowley and Jensen, 1965) Fluorescence quenching of 2',7' bis (ace- (Colovos eta/., 1970) toxymercuri) fluorescein CaO Ignition, steam distillation Th4+ (Mestitzova and J avorslca, titration alizarin inductor 1965) Hg2+-EDTA methylthymol blue indicator (Nomura, 1970) Precipitate impregnated membrane (Rechnitz eta/., 1966) Flow through cell with Ag/Agl electrode (Bardin and Tolstousov, 1970) Cathodic stripping voltammetry-stationary (Perchard eta/., 1967) Hgdrop Cathodic stripping using graphite electrode (Brainina and Sapozhnikova, 1966) I Converted to 21 ethanol Simultaneous determination of each ofl, Br, a (Ruessel, 1970) Impactor with particle size resolution (Loucks eta/., 1969) Volatilization separation with scintillation (Bock and Strecker, 1968) counting Inorganic I removed with Sephadex G-25 (Veselsky eta/., 1970) I Adsorbed on anion exchanger and counted (Dahl eta/., 1964) X-ray covnting to remove Na interference (Pillay and Miller, 1969) Freeze-dried samples (Wilkniss and Linnenbom, 1968) (Anderson eta/., 196 7) Ce4+/As3+ Redox reaction catalyzed by I (Hoch and Lewallen, 1969) Fe(SCN)J'NaN02 Reaction catalyzed by I (Proskuryakova, 1969) Ce4+/As +Redox reaction catalyzed by I (Hoch eta/., 1964) (Yurachek eta/., 1969) 241 Am Source (Schiller eta/., 1967) young, 1972). From this, the absolute detection limit can be derived. In the other group, illustrated by spark-source mass spectrometry or electron-probe analysis, the absolute detection limit is experimentally determined by the amount
Analytical Methods 83 of sample actually involved in the analysis. From this quan- tity, the concentration detection limit can be derived. biological samples, followed by dissolution. Dry ashing is commonly used for plant materials, and wet ashing for ani- mal materials. Soil or plant extracts can be directly ana- lyzed (Hwang et al .⢠1971 ). The two classes of methods may broadly be called trace methods and micro methods, and each has a natural class of samples to which it lends itself. Thus, ion-probe analysis is a micro method, ideally suited to the identification of a small particle or to studying heterogeneity, but poorly suited to examining a dilute and homogeneous sample. For a wide variety of plant, animal, and soil samples not requiring the ultimate in sensitivity, and in which only one or two elements are to be determined, atomic absorption spectrometry is the method of choice. It requires ashing of Atomic absorption, however, is not sensitive enough for water analysis. Rather than resort to concentration or extrac- tion techniques, anodic stripping voltammetry, using thin- layer mercury-on-graphite electrodes, is used directly. This method has the added advantage of allowing a degree of speciation, in the sense that labile metal ions can be deter- mined in the samples as received, whereas "bound" metal ions can be determined after acidification of the samples. TABLE 30 Methods of Measurement of Fluorine Technique Atomic absorption Spectrophotometry Fluorescence Titration Ion-selective electrodes Electrochemistry Gas chromatography Photon-activation Catalytic Isotope Dilution Material Synthetic General biological Natural waters General biological Idealized system Idealized system Soft tooth deposits Human milk Natural waters Synthetic samples General biologicals (wine, saliva, bone) Hair, blood, wood, tobacco Dental enamel Amount or Concentration Detail 1 o-3 -10-s g Measure depression of Mg absorbance 10-9 g Prior separation by HF diffusion 0 2 combustion and absorption of F in NaOH Technicon autoanalyzer La-alizarin development Th·Alizarin fluorescence quenching by F- Comment (Reference) Oflimited use due to other anions having same effect (Bond and O'Donnell, 1968) (Tusl, 1970) (Van Gogh, 1966) (Otan and Riley, 1966) (Taves, 1968) Titrate with ((C6 Hs)4SbhS04 and measure (Orenberg and Morris, 1967) endpoint with F- electrode IOJ&M 0.2-2 mg/1 O.S mg/ml 10 J&g 10-9 g Not counting interferences, EMF found to be explainable in terms of only F, HF, HF2 concentrations Study of effects of pH and interfering ions, e.g., in buffers Modification for small sample volumes (SO J&}) LaF 3 Solid state membrane electrode Separation by HF diffusion Fat, protein, and casein removed by centrifugation Rotating AI electrode continuous amperometry Conductimetric titration with Th(N03)4 Formation of fluorotrimethyl silane Liver esterase-ethyl butyrate reaction inhibited by F- (Srinivasan and Rechnitz, 1968) (Bock and Strecker, 1968) (Durst and Taylor, 1967) (Lingane, 1967) (Birkeland, 1970) (Simpson and Tuba, 1968) (Morrow and Henry, 196 7) Not a real trace method (Israel eta/., 1966) (Fresen et al., 1968) (Andersen et a/ .⢠196 7) Weak point is titration of butyric acid with NaOH to determine turnover rate (Hoch and Lewallen, 1969) Errors depend on form of F used to cause isotope ex- change (Dirks and Cox, 1967)
00 ~ TABLE 31 Limits of Detection of Analytical Methods Used in Trace Analysis4 Concentration Detection Limits of the Element in a Sample, ppb Absolute Detection Limits, ng Aqueous Solutionsb Biological Materialsc Geological MateriaJsd Analytical Method Cd Pb Zn Cd Atomic absorption Nebulizer-flamee 3 30 3 3 Nonflamef 0.002 O.o2 0.002 0.02 Emission spectroscopyK 0.7 10 I 3.5 Anodic stripping voltammetrye 0.05 0.1 0.1 0.05 Atomic fluorescence Nebulizer-flamee 0.001 10 0.02 0.001 Nonflamef 0.00003 0.01 0.00005 0.0003 Spark-source mass spectroscopy [ approx. 0.1-1 [ Neutron-activationh 5 1000 10 5 Electron-probe [ approx. 10-3 I Ion-probe [ approx. 10-6-10-7 I Aame emissione 4 X 102 10 2 X 103 4 X 102 aNumbers apply to materials to which the analytical method is applicable without in· terferences. Variations will occur between different matrices. For practical minimum reporting limits, multiply the numbers by 10. bcatculated on a weight per volume basis. Includes water, biological fluids, or other aqueous solutions of less than S percent salt content . ccatculated on the basis of a biological sample of 1 percent ash content. Analytical solutions are S percent total salt content. - dcatculated on the basis of a sample of 100 percent ash content. Analytical solutions, where applicable, are S percent total salt content. Pb Zn Cd Pb Zn Cd Pb Zn 30 3 0.6 6 0.6 60 600 60 0.2 0.02 0.004 0.04 0.004 0.4 4 0.4 50 5 0.35 5 0.5 35 500 50 0.1 0.1 0.01 0.02 O.o2 1 2 2 10 0.02 0.0002 2 0.004 0.02 200 0.4 0.1 0.0005 0.00006 0.02 0.0001 0.006 2 0.01 approx. 0.1-1 I [ approx. 1-10 I [ approx. 102-103 I 1000 10 10 0.05 10 0.10 5 1000 10 [ approx. 105-106 I approx. 105-106 I [ approx. 103-1 04 I [ approx. 103-104 I 2 X 103 80 2 4 X 102 8 X 103 200 4 X 104 eon the basis of a 1-ml sample volume. !on the basis of a 100-1.11 volume of aS percent solution of the umple. gOn the basis of 20 mg of Ignited solids, or 200 1.11 of a S percent solution of the sample. hon the basis of an equivalent samf'e of 1 ml or 1 g irradiated 1 h in a thermal neutron flux of 1.8 X 1012 n em- s- 1.
As another electrochemical technique, pulse anodic strip- ping, using a hanging mercury drop electrode, appears competitive with the thin-layer mercury electrode. Both are more demanding of operator skill than atomic absorp- tion, and both require addition of a reagent, which must be carefully purified to assure negligible blanks. The anodic stripping voltammetry method gives a simultaneous response to the three subject elements plus copper (Shuman and Woodward, 1973). Emission spectroscopy is used especially for multi· element analysis, for scanning for the presence or absence of elements, and for historical sample analysis in which spectrographic plates may be stored for later re-evaluation. Electron-probe microanalysis, and scanning electron microscopy with nondispersive x-ray emission, are espe· cially useful for looking at spatial distribution of elements in biological tissue and in looking at the association of elements. By this method, for example, the presence of lead phosphate crystals in com leaves heavily contaminated by lead has been proved. Especially difficult situations are represented by soil samples and by natural water samples, largely because the goal is to analyze soils and water for "available metal." Natural water samples contain suspended or colloidal ma- terial that may represent a substantial portion of the heavy- metal content. A fdtration procedure, although admittedly somewhat arbitrary, is generally used, and both fdtrate and precipitate are analyzed separately. Two separate labora- tories are involved in analysis of the fdtrate, and both are therefore given portions of the same fdtrate to assure com- parability of the data. ANALYTICAL METHODS FOR SELENIUM AND TELLURIUM Selenium in Biological Materials As most of the selenium in plants appears to be present as selenomethionine, and this is utilized by animals, measure- ment of the total selenium in plants is a valid measure of the nutritional value of the plant in respect to selenium. Selenium levels in whole blood have frequently been used to characterize the selenium status of animals and people. Some of the selenium in fish meal is evidently of limited nutritional value, but no analytical scheme for differenti· ating between effective and ineffective forms of selenium in animal diets has been proposed. Most of the selenium measurements currently being made utilize the methods of Allaway and Cary (1964), Watkin- son (1966), or variants of them. This method involves de- struction of the organic matrix with a mixture of nitric acid (HN03 ) and perchloric acid (HC104 ) followed by measurement of the fluorescence of the selenium complex with 2,3-diaminonapthalene. The method is satisfactory Analytical Methods 85 with most biological materials; the possible exception is urine. Neutron-activation methods based on counting 75Se are highly regarded as referee methods but are too slow and expensive for routine work. Strictly instrumental neutron-activation procedures, utilizing short half-life iso· topes of selenium, such as the metastable 77mse, have not yet yielded consistent results on low-selenium materials. X-ray fluorescence is satisfactory for biological samples containing more than 5 ppm selenium. The earlier methods, based on isolation of selenium by distillation of SeBr4 , lack the sensitivity needed for studying selenium-deficient situa- tions and are more tedious than the Watkinson method. Fluorometric methods utilizing oxygen-flask combustion are now used primarily for referee purposes. Selenium in Geologic Materials Selenium can be determined in geologic materials with ade- quate sensitivity and precision by neutron-activation anal- ysis (Brunfelt and Steinnes, 1967; Kiesl, 1969). Several recent fluorometric methods of satisfactory sen- sitivity and precision are available for determining selenium in soils, rocks, and lake sediments (Levesque and Vendette, 1971; Wiersma and Lee, 1971; Molloy, 1967; and Wells, 1967). Tellurium Until very recently, no analytical methods with sufficient sensitivity were available for ~etermining tellurium in soils, rocks, and biological materials. Neutron-activation analysis and atomic absorption spectrometry offer opportunities to reach downward to between a few and 100 ppb of tellurium (Abu-Samra and Leddicotte, 1969; Nakagawa and Thomp· son, 1968; Davis eta/., 1969; and Spitz, 1971). Catalytic methods give good sensitivity but lack adequate precision (Lakin and Thompson, 1963; Hubert, 1971 a,b ). More adequate analytical methods must be developed before the geochemistry and biochemistry of tellurium can be known. ANALYTICAL METHODS FOR CHROMIUM Many methods are available for the determination of chro- mium, some of which have been carefully evaluated by Beyermann ( 1962). Colorimetric methods involve forma- tion of colored complexes with reagents such as diphenyl- carbazide. The diphenylcarbazide complex has been exten- sively studied and can be used to determine 3.5 ng chromium. The standard of the method is approximately 3 percent for 100 ng. Although fairly sensitive, the method is subject to interference from other ions, which may account for some of the discrepancies in the reports of chromium levels that appear in the literature. Flame photometry has been used
86 THE RELATION OF SELECTED TRACE ELEMENTS TO HEALTH AND DISEASE for the determination of chromium, although this method is less sensitive (by a factor of I 0) than others, even with the use of special organic solvents (Woehlbier et aL. 19S9). Others have obtained a limit of detection of S ppb in bio- logical material (Pickett, 1967). Emission spectrography is a very specific method, sensitive to 0.1 ng, particularly when used with the modern modifications (Gordon, 196S). X-ray emission spectrography is not quite sensitive enough for use with biological samples unless preceded by con- siderable concentration of the metal (Woehlbier et al .⢠19S9). Polarography has been used to study the interaction be- tween chromium, insulin, and mitochondrial membranes (Christian et al., 1963). However, the method is not sensi- tive enough for determination of chromium in biological material, because its limit of detection is approximately SO ng (Mertz, 1969). A rapid polar technique has been published for measuring several metals, including chro- mium, in sewage and industrial wastes at concentrations of S ppm or greater (Butts and Mellin, 19S 1 ). Bioamper- ometry is sensitive to 1 ng, but the method is subject to considerable interference by other ions present in the reac- tion mixture (Woehlbier et al., 19S9). Neutron-activation analysis has been used for determination of chromium in biological material (Coleman et al. , 1967) and in air filters. The detection limit is dependent on the length of irradia- tion and the flux of thermal neutrons-the longer their- radiation time and the higher the flux, the lower the detec- tion limit. For example, a S-hour irradiation at l.S X 1013 neutrons/cm2 /s gives a detection limit of 20 ng for air samples done nondestructively. Separation should improve the detection limit by reducing the background radiation. Gas-liquid chromatography of metal complexes of hexa- fluoroacetylacetonates or related compounds appears to be a very promising new tool, with a sensitivity to 0.01 ng (Aue, 1967; Sievers et al., 1967). A method using trifluoro- acetylacetone with a sensitivity of 1 ng/100 ml has been applied to the determination of chromium in serum (Savory et al .⢠1968). At present, atomic-absorption spectroscopy is the most widely used procedure. The sensitivity of this method can be increased to approximately 6 ng by use of organic solvents, such as methylisobutylketone, which at the same time serve to extract hexavalent chromium from its aqueous solution. The method therefore requires diges- tion and oxidation of the sample (Mertz, 1969; Feldman et al., 1967; Feldman and Purdy, 196S). The concentration of chromic acid mists in air can be estimated by a direct- field method described by Ege and Silverman ( 1947; Silver- man and Ege, 1947, 1949). This is a spot-test method using phthalic anhydride and S-diphenylcarbazide. Because the hexavalent form of chromium is considered more toxic than the trivalent form, it would frequently be useful to determine the concentration of hexavalent chro- mium in addition to the total chromium. This is impossible with all methods that require oxidation to the highest oxi- dation state for solvent extraction or for formation of a colored complex (Woehlbier et al., 19S9). Omission of the oxidation steps, however, would result only in the determi- nation of any naturally occurring hexavalent chromium. A parallel assay with the oxidation step included would give the total chromium content of the sample; the differ- ence between the two would be a measure of the trivalent form (Mertz, 1969). The precision of a particular method near the detection limit is very poor ( 100 percent or greater error). The pre- cision improves, reaching a maximum of l-S7 percent about SO-l 00-times above the detection limit. Detection limits are constantly being lowered by advancements in technology and improvement in the methods of sample preparation. Care must be taken to avoid erroneous results caused by interferences, especially near the detection limits. Other ions may form colored complexes with the diphenylcar- bazide and have some absorbency at the wavelength of the chromium diphenylcarbazide. In neutron-activation analy- sis, another element could have a gamma energy at the same place as the chromium; this can be checked by looking at the half-life of the 320-keV peak. With gas-liquid chro- matography, it is possible for two compounds to have the same retention times on a column; it is therefore a good idea to use two columns of different polarity, so that re- tention times will change. At low levels of chromium, using atomic absorption spectroscopy, the edge of some other ion's absorption may give a signal that could be mistaken for chromium. A recent publication by Hambidge (1971) described an application of arcing in a static argon atmosphere, using a refined excitation chamber for the determination of chro- mium in biological media. These techniques were applied to the trace analysis of ashed biological material, especially to the measurement of nanogram quantities of chromium in blood, hair, and urine. The mean relative standard devia- tion for quantities of chromium, ranging from 1-7 ng in 0.2 ml aliquots of serum, was 6 percent. The method of sample preparation involved ashing by oxidizing the organic material in the oxygen plasma of a low-temperature asher. Preliminary data indicate this method to be potentially excellent, especially as a research tool of chromium in bio- logical tissue. The greater sensitivity reported for this method over conventional atomic absorption spectropho- tometry is a significant advantage in the measurement of very low levels of chromium in the nanogram range (Ham- bidge, 1971). Another recent advance reported in the literature for the analysis of chromium in plants and other biological materials involves a refmement of preconcentration techniques, with the subsequent determination of chromium by atomic ab- sorption spectrophotometry. This method employs a wet- digestion procedure, isolation of chromium by solvent ex- traction of the chromium chelate of 2, 4-pentanedione into
chloroform. The chloroform is evaporated, and the chro- mium is taken up in 4-methyl-2-pentanon (Cary and Allaway, 1971 ). Although this procedure is rather rigid and time consum- ing, recoveries were 95 percent, with very good sensitivity and precision, when interlaboratory comparisons were performed. The authors report that this method allows for estimation of 4 ng of chromium per milliliter of solution, and interferences are minimal (Cary and Allaway, 1971). REFERENCES Iodine and Fluorine Andersen, G. H., F. M. Graber, V. P. Guinn, H. R. Lukens, and D. M. Settle. 196 7. Photonuclear activation analysis of biological rna· terials for various elements, including fluorine. Proceedings of the Symposium on Nuclear Activation Technology Life Sciences (IAEA), Amsterdam. pp. 99-113. Bardin. V. V., and V. N. Tolatousov. 1970. Potentiometric determi nation of trace amounts of iodide in aqueous solutions. lzv. Vyssh. Ucheb. Zavod. Khim. Teknol. 13(2):165-167. Birkeland, J. M. 1970. 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