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1969 The etD ermination of Selenium. Robert Lewis Osburn Louisiana State University and Agricultural & Mechanical College

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OSBURN, Robert Lewis, 1936- THE DETERMINATION OF SELENIUM. The Louisiana State University and Agricultural and Mechanical College, PhJD., 1969

Chemistry, analytical

University Microfilms, Inc., Ann Arbor, M ichigan THE DETERMINATION OF SELENIUM

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Chemistry

by Robert Lewis Osburn B.S., George Peabody College, 1962 August, I969 TO LINDA

who understands. ACKNOWLEDGMENTS

The author wishes to express his gratitude to Professor

Philip W. West, who, through his penetrating insight, calm for­ bearance, and timely encouragement, contributed so much to the

attainment of this goal.

He also wishes to thank the United States Public Health

Service for providing the funds during the entirety of this work

through U. S. Public Health Research Grant No. AP 0072^, National

Center for Air Pollution Control, Bureau of Disease Prevention and Environmental Control. The author gratefully acknowledges the financial assistance received from the "Dr. Charles E. Coates

Memorial Fund of the L. S. U. Foundation donated by George H.

Coates" for the preparation of this dissertation.

iii Patience is bitter, but its fruit is sweet.

Jean Jacques Rousseau

iv TABLE OF CONTENTS

PAGE

DEDICATION...... ii

ACKNOWLEDGMENTS...... iii

QUOTATION...... iv

LIST OF TABLES ...... viii

ABSTRACT ...... x

CHAPTER

I. INTRODUCTION ...... 1

II. CONVENTIONAL METHODS FOR THE

DETERMINATION OF SELENIUM...... 12

A. Titrimetric Methods...... 12

B. Gravimetric Methods...... 13

C. Neutron-Activation Analysis...... 15

D. Polarographic Methods...... 16

E. Photometric Methods...... 17

1. Spectrophotometry...... 17

a. Particles in Suspension...... 17

b. Equivalent in Liberated

Iodine...... 17

c. Pyrrole Color Complex...... 18

d. Diaminobenzidine Complex ...... 18

e. Miscellaneous...... 19 PAGE

CHAPTER

2. Fluor imetry...... 20

5. Emission Spectroscopy...... 21

X-Ray Fluorescence

Spectroscopy ...... 21

F. Ring Oven Method ...... 21

G. Catalytic Methods...... 22

III. THE DETERMINATION OF SELENIUM BY

THE HYDROXYLAMINE OXIDATION METHOD ...... 2k

A. Introduction...... 2k

B. Preparation of Reagents and

Solutions...... 27

1. Standard Selenium Solution ...... 27

2. Hydrochloric Acid...... 27

3. Hydroxylamine Hydrochloride

Solution...... 27

I4-. Sulfanilamide Solution...... 27

5. N-(1-Naphthyl)ethylenediamine

Dihydrochloride Solution ...... 28

6. Interference Solutions ...... 28

C. Apparatus...... 28

IV. RESULTS AND DISCUSSION...... 30

A. Preliminary Experiments...... 30

B. The Oxidation-Diazotization

Reaction...... 37

vi PAGE CHAPTER

C. The Coupling Reaction...... k6

D. Concentration Studies......

1. Sulfanilamide (p-aminobenzene-

sulfonamide) ...... 52

2. N-(1-Naphthyl)ethylenediamine

Dihydrochloride...... 55

5. Hydroxylamine Hydrochloride...... 58

E. The Standard Recommended

Procedure...... 58

F. Interference Studies ...... 61

G. Statistical Evaluation ...... 71

H. Conclusion...... 71

SELECTED BIBLIOGRAPHY...... ik

VITA ...... 92

vii LIST OF TABLES

TABLE PAGE

I. Effect of Acid Concentration During the Oxidation-Diazotization Reaction ...... 35

II. Time Study for the Oxidation-Diazotization Reaction at Room Temperature...... 38

III. Temperature Study for the Oxidation- Diazotization Reaction...... 4l

IV. Time Study for the Oxidation-Diazotization Reaction at 60 Degrees Centigrade...... 44

V. Time Study for the Coupling Reaction at 60 Degrees Centigrade...... 47

VI. Volume Study for the Coupling Reaction ...... $0

VII. Concentration Study; Sulfanilamide, (p-amino- benzenesulfonamide)...... 53

VIII. Concentration Study; N-(1-Naphthyl)ethyl- enediamine Dihydrochloride ...... 56

IX. Concentration Study; Hydroxylamine Hydrochloride...... 59

X. Variation of Absorbance with Concentration of Selenium(lV) for the Adopted Method .... 62

XI. Interference Studies of Diverse Ions ...... 64

XII. Statistical Analysis of Absorbance at Various Levels of Selenium(lV) ...... 72

viii LIST OF FIGURES

FIGURE PAGE

1. Absorption Spectra of Various Concentrations of the Coupled Product...... 33

2. Variation of Absorbance with Concentration of Hydrochloric Acid During the Oxidation- Diazotization Reaction...... 3 6

3. Variation of Absorbance with Time During the Oxidation-Diazotization Reaction ...... 39

4. Variation of Absorbance with Temperature During the Oxidation-Diazotization Reaction...... 42

5. The Effect of Temperature During the Oxidation-Diazotization Reaction on Standard Curves for the System...... 43

6. Variation of Absorbance with Time During the Oxidation-Diazotization Reaction at 60 Degrees Centigrade...... 45

7. Variation of Absorbance with Time During the Coupling Reaction at 60 Degrees Centigrade...... 48

8. Variation of Absorbance with Time During the Coupling Reaction at 60 Degrees Centigrade...... $1

9 . Variation of Absorbance with Concentration of Sulfanilamide (p-aminobenzenesulfonamide) . 5^

10. Variation of Absorbance with Concentration of N-(1-Naphthyl)ethylenediamine Dihydrochloride...... 57

11. Variation of Absorbance with Concentration of Hydroxylamine Hydrochloride ...... 60

12. The Standard Curve for the Adopted Method...... 63

ix ABSTRACT

Selenium dioxide has been used extensively as an oxidizing agent in organic chemistry, particularly for the oxidation of alde­ hydes, ketones and unsaturated hydrocarbons. It is well known that selenium dioxide can be used to oxidize hydroxylamine hydrochloride to nitrous acid under acidic conditions. A new spectrophotometric method for the determination of submicrogram quantities of selenium dioxide has been developed based upon this reaction.

In a strongly acidic medium, (6.8N hydrochloric acid), even trace amounts of selenium dioxide (as selenious acid) oxidize hydro­ xylamine hydrochloride to nitrous acid when heated at 60 degrees centigrade for a period of 90 minutes. When sulfanilamide is present in the system, diazotization of the amine takes place as the oxida­ tion reaction proceeds. The diazonium salt thus formed is stable under the reaction conditions employed. Subsequent to the oxida- tion-diazotization step N-(l-naphthyl)ethylenediamine dihydrochlo­ ride is added and heating at 60 degrees centigrade is continued for

30 minutes, resulting in the formation of a highly-colored coupling product which has a maximum absorbance at 5^ millimicrons.

This approach to the determination of selenium takes advan­ tage of the extreme sensitivity which is inherently present in anal­ ogous procedures for the determination of nitrites. The method therefore provides a means for the spectrophotometric determination of selenium in the 0.01 to 0 .2 parts per million range. The method is sensitive, reproducible and accurate and should be especially useful in air pollution surveys where a knowledge of the absolute concentration of selenium oxides in the atmosphere is most important. CHAPTER I

INTRODUCTION

Selenium was identified as an element in 1817 by the

Swedish chemist, Berzelius, in a residue from the production of sulfuric acid. It was named from the Greek word, selene, meaning the moon, because of its resemblance to tellurium, which had been discovered earlier and was named from the Latin word, tellus, meaning the earth.

The chemistry of sulfur, selenium, and tellurium are similar, but their abundance in the earth's crust varies. Sulfur occurs freely in abundant quantities, while selenium and tellurium are rare with an estimated percentage of 10”4 and 10"9, respective­ ly. Intensive explorations in the United States have not been suc­ cessful in finding large deposits of selenium. At the present time, selenium is chiefly obtained commercially as a by-product from the electrolytic refining of copper.

The element selenium can be traced in an orderly way from its origin in the earth's crust to specific geological formations, to its distribution in specific genera and groups of plants which require the element for their growth, to its accumulation in veg­ etation, and to its subsequent toxicity to birds or mammals that consume the seleniferous foods.

The disease syndrome produced by selenium is an age-old disease and has been observed in numerous areas throughout the world. The type of chronic selenium poisoning known as alkali disease is characterized by loss of hair and deformation and

sloughing of the hoofs in cattle, hogs, and horses that have con­

sumed moderately seleniferous grains and forage grasses over a

period of several weeks or months. The term alkali disease came

into usage among the early settlers of the Western United States, who erroneously believed the ailment to be due to alkali or mineral

salts in water or soil. Around 1295 Marco Polo was, perhaps, re­

ferring to alkali disease when he described a poisonous plant grow­

ing in Western China which, when eaten by his beasts of burden, had

the effect of causing the hoofs of the animal to drop off (91)*

In the Western hemisphere, chronic selenosis resembling

alkali disease in livestock, causing malformations in chicks and

children and loss of hair and nails of the people, was first re­

corded by Father Pedro Simon of Colombia in I56O (104).

The earliest record of the poisoning known as alkali

disease in the United States was reported in 1857 by Dr. T. C.

Madison, an Army surgeon, who described the symptoms of a fatal

disease which afflicted the cavalry horses at Fort Randall in the

territory of Nebraska (76).

In fifteen of the western states, acute and chronic poison­

ing of unknown origin, often attributed to poisonous forage plants,

have been fatal to large numbers of cattle and sheep on the grazing

ranges. More than 15,000 sheep died in a region north of Medicine

Bow, Wyoming during the summers of I907 and 1908. The deaths were

attributed to grazing in an area where woody aster and Gray's vetch

abundantly grew (126). 3

In 1932> some native species of Astragalus were used for studies of the chemical properties and physiological effects of these plants (lj). Animals that survived the extended feeding period showed lesions which suggested the action of an irritant poison, presumed to be of mineral origin. It became evident that certain native milk vetches growing on restricted soils could have been responsible for many acute and chronic poisonings of live­ stock. Later studies by Beath, Eppson, and Gilbert (8) showed that a number of species of plants have the ability to convert selenium from soils to an available form. These plants are called selenium indicator plants or "Beath's selenium indicators," be­ cause of their ability to accumulate several thousand parts per million of selenium.

The administration of selenium compounds to livestock or laboratory animals causes toxic symptoms in the animals, but typical alkali disease or blind staggers is not produced. The syndrome alkali disease is produced by seleniferous indicator plants. The selenium compounds in the plants that can cause vari­ ous disease syndromes in livestock are not known. Only a limited amount of data are available on the compounds which are present in indicator plants and those present in grains and grasses.

In 1938; experimental evidence was presented (113) showing that selenium is required for the growth and development of sele­ nium indicator plants. It has been suggested that selenium may be an essential element for these groups of plants. Evidence is 4 now accumulating that selenium is not only an essential element

for indicator plants but has a significant dietary function in mammals as well as birds (101, 100, 89, 82).

The elements selenium and sulfur are closely related both crystallochemically and in certain aspects of their distri­ bution in minerals and rocks. Some sulfide ores or impure sulfurs are enriched with selenium. Selenium forms selenides and sulfo-

selenides of silver, copper, lead, and mercury, and selenites with copper, cobalt, and lead. Selenium frequently occurs as impure

selenides and as an impurity in most sulfides. The sulfides of copper, iron, mercury, and silver often contain high concentrations of selenium. Tiemannite from Utah contains about 24 percent se­

lenium (83). A sample of naumannite from Silver City, Idaho, con­ tained 23 percent selenium (102). Davidson (25) has reported that common epithermal silver-gold and antimony deposits are highly se- leniferous but that less common gold-silver and mercury deposits rarely contain high concentrations of selenium. Analysis of 98 samples of pyrite and sulfide ores from Utah, Nevada, Idaho and

Oregon has been reported by Lakin and Byers (69). One sample of ore from Park City, Utah, contained 540 ppm selenium. The sub­ stitution of selenium for sulfur in numerous sulfide minerals has been suggested by a number of workers (17, 18, 24, 122). This substitution has been proposed on the basis of the similarity of the chemical and physical properties of selenium and sulfur. The radii of S2“ (1.74A) and of Se2- (I.92A) are so close that selenium can and does substitute in the lattices of sulfides. Ill spite of the fact that selenium and sulfur are closely

related crystallochemically and geochemically, there is a vast

difference in their actions physiologically. Sulfur can be pre­

sent in living cells in comparatively high concentrations while

selenium is toxic in low levels. The toxicity of selenium is com­

parable to that of arsenic. Selenium is one of the few elements

known to be absorbed by food and forage plants in sufficient amounts

to create a toxicity hazard to animals. Acute selenium poisoning

results from the ingestion, usually in a single feeding, of a

sufficient quantity of highly seleniferous plants which produces

very severe symptoms. In many cases death follows within a few hours. Selenium is carried by the circulatory system to all

organs of the body. In the case of acute poisoning the highest

concentrations are found in the liver, blood, kidney, spleen, and brain. The muscles, hide, hair, and bones usually contain only

trace amounts. Dudley (31) reported 4-25 PPm in the liver, kidney,

and spleen, and 7-27 ppm in blood. On the average, the blood con­

tains 5_ 15 PPny liver 22 ppm; kidney 10 ppm; spleen 8 ppm; and heart 4 ppm. In horses and mules the selenium content of the

stomach, kidney, and liver was higher than that of the other tis­

sues analyzed (81).

It is generally believed that dietary, chemical and physical

factors may interfere with reproduction of embryonic development.

The significance of selenium in reproduction, decreased fertility and malformation has been reported by numerous investigators in different species of animals. The economic loss caused by the effects of selenium on reproduction in livestock is not presently known. Toxic and subtoxic amounts of selenium can interfere with fertility and reproduction in experimental animals. The inhibition due to selenium on reproduction can be either primary or secondary.

The primary effect may be due to interference of selenium with the critical oxygen requirements, fertility and reproduction could be reduced even in low-grade chronic selenosis. The secondary effect of chronic selenosis is emaciation. Conception cannot take place in emaciated animals (125, 40).

The selenium problem is especially important because of the possibility of human injury from' the consumption of grains, vegetables, eggs, dairy products, and meats from affected areas.

Extensive investigations of the poisoning of animals have been made, but the research required on human phases of the problem has hardly been touched. Investigations into the sources of selenium to which man is exposed in seleniferous regions have shown wide occurrence of the element in foods of animal origin. It is be­ lieved that a concentration of 5 PPm in most foods or one tenth this concentration in milk or water is potentially dangerous.

Smith and Westfall (105) found that eggs, meat, and milk contain significant amounts of selenium in seleniferous regions. Selenium in wheat and grain is of special interest because of the wide­ spread use of wheat in bread and breakfast foods. The per capita consumption of wheat products is about 160 pounds per year. The selenium content of drinking water is rarely high enough to produce toxic effects in man. However, a recent report

(7) indicates that well water obtained from the Wasatch geological

formation from a depth of about 1^0 feet contained 9 ppm selenium and produced chronic selenosis in humans. This is the first in­ cident where the selenium concentration in water was high enough

to produce chronic symptoms in humans similar to alkali disease in

livestock.

Few attempts have been made to determine the existence, outside the known seleniferous areas, of public health hazards caused by selenium. This problem is not confined to the United

States since grain is exported from the United States to various parts of the world. Although toxic wheat is diluted with nontoxic grain during storing and milling processes, and in well-balanced diets, the consumption of bread and cereals per day is limited, possible harmful effects could occur in individuals who consume more than normal amounts of cereal products or who may have chronic diseases involving the kidney and liver. Selenium, therefore, could play an important role either directly or indirectly in the health of the general public.

A survey of the selenium content of urine samples was made on industrial workers in Rochester, New York (106). There is no selenium in the soil in that area or within 1,000 miles of the region. The workers included in the survey had had no recent exposure to selenium. However, when random urine samples were collected from 60 men, 'JO percent of the samples contained selenium. 8

The selenium content varied from a trace to 0.025 PPm °f selenium.

Another series of studies included repeated daily collection for four consecutive days. The urinary excretion of selenium per day was from 0.02 to 0.1 ppm. The rate of excretion was constant in the subjects included in this study. Further investigation (106) to determine the possible sources of selenium in this survey showed that bleached flour and bread contained from 0.26 to 0.53 PPm selenium; rye bread and cracked wheat bread 0.39 an

The increasing use of selenium in manufacturing processes poses an important industrial health hazard. Two types of indus­ tries are involved: those that extract, mine, treat, or process selenium-bearing minerals and those that utilize selenium in man­ ufacture of pigments, in the chemical industry, in the manufacture of rubber as a vulcanizing agent, and in rectifiers and photocells.

Selenium and its compounds used in industry are an ever present po­ tential health hazard (30, 56).

The extent of this hazard depends primarily upon the types of processes being carried on. Selenium-bearing dusts, fumes, vapors, or liquids can present definite hazards, the scope and degree of which are dependent upon the processes involved and the protective devices used to dissipate the noxious materials. Dust, such as that of elemental selenium, may be of such composition that on inhalation no soluble selenium compound is liberated.

But soluble dusts, such as selenium dioxide, selenium trioxide, and certain halogen compounds, can prove toxic due to the ease 9 with which they are absorbed by the tissues of the lung, the ali­ mentary canal, and perhaps the skin. The most toxic vapors include hydrogen selenide and certain organic compounds such as methyl sel- enide, ethyl selenide, and various aromatic selenides. In industries where selenium dioxide, selenium trioxide, sodium selenate, and so­ dium selenite are used, the atmospheric concentration should be be­ low 1.0 milligram per cubic meter of air (90 ).

When guinea pigs were exposed to hydrogen selenide (0.001-

0.004 milligram per liter) 50 percent of the animals were killed within 30 days. The accidental exposure of men to hydrogen sel­ enide has an immediate and drastic effect. The odor, similar to hydrogen sulfide, produces olfactory fatigue rapidly so that toxic concentrations may not be detected after exposure to the gas for several minutes. The gas usually produces a copious flow of tears and nasal mucus (32). Other exposures to hydrogen selenide have been reported by various investigators (12, 86, 111). The thresh­ old limit given by the American Conference of Government Industrial

Hygienists for hydrogen selenide is 0.05 ppm selenium or 0.2 milli­ gram per cubic meter of air.

Selenium dioxide and sodium selenite readily form seleni- ous acid which is among the more toxic and irritating compounds of selenium. It penetrates the skin readily and produces local irrita­ tion and inflammation. The exposure to Se02 dust produces severe dermatitis (93)> painful inflammation of nail beds due to penetration of the dioxide under the nails (^7)> burning of the eye with intense pain, lacrimation, and congestion of the conjunctiva (80), and 10

allergic reactions in the eyes (U7). The toxic effects of Se02 dust may be due to its rapid conversion to selenious acid when the dust comes in contact with moist surfaces of the tissues. Cutaneous disorders in workers handling Se02 have shown that the selenite ion is responsible for its toxic action. Skin lesions produced by Se02 resembling hematomas in a state of resorption have been described

The possible long-term effect is believed to be renal and liver damage. Fibrosis in the lung may develop due to continued exposure to dust and gases in the air. Disposal of industrial waste containing selenium is a serious problem. Burning cannot be carried out in residential areas because the smoke has an obnoxi­ ous odor as well as toxic fumes. If the material is dumped and allowed to drain into rivers, there is the danger of creating a toxic contamination of streams and surrounding soil. It has been suggested (3) that the selenium be reclaimed for further use, but it is not likely that this approach can be used under industrial conditions.

The seriousness of the selenium problem has been further emphasized by the report of West (15), who found selenium to be present in cigarette papers as well as in other paper. The pres­ ence of selenium in cigarette papers must be considered signifi­ cant because this is the first known toxic material found to be universally present in cigarette papers. Because of the known toxicity of selenium and because of the carcinogenicity of ingest­ ed selenium, the findings were considered significant in terms 11 of lung cancer and emphysema among cigarette smokers. Another far-reaching implication of this report is the hazard of air pollution which arises due to the burning of paper refuse materi­ al of all kinds in areas which have open-air city dumps or in­ adequate incinerator systems for waste disposal.

The most frequently used methods for the determination of selenium are time consuming, use reagent solutions which are generally unstable and must be prepared daily, and require a careful control of pH. The present study was undertaken in order to develop a method for the determination of submicrogram quanti­ ties of selenium with the sensitivity and accuracy needed for trace analysis and at the same time rapid and reliable enough to be of utility in laboratories where selenium analyses are routine­ ly made. CHAPTER II

CONVENTIONAL METHODS FOR THE

DETERMINATION OF SELENIUM

A. TITRIMETRIC METHODS

By far, the most sensitive and accurate of all of the titrimetric methods for the determination of selenium are the iodometric procedures. It is usually necessary, however, that a quantitative separation from the other elements be made if the full potential of these methods is to be realized.

The iodometric method as proposed by Klein (6 7) is based upon the distillation of selenium as SeBr4, precipitation of se­ lenium and titration with thiosulfate. This method is widely used for the determination of quantities of selenium of more than 0.01 milligram in plants, plant extracts, water, animal tissue, urine, soil and rocks.

After digestion of the sample in H2S04 -HN03, selenium is separated from all other elements except arsenic, tin, germanium, small amounts of antimony, and partially from tellurium by dis­ tillation with HBr and Br2 from acid solution:

H2Se03 + IfflBr -» SeBr4 + 3H20

The distillate contains selenium as selenious acid:

SeBr4 + 3H20 -* H2Se03 + ^HBr

12 13

Selenium is precipitated from the distillate by reduction to the

element with sulfur dioxide and hydroxylamine hydrochloride.

H2Se03 + 2H2S03 -* Se + 2H2S04 + H20

H2Se03 + 2NH20H • HC1 -» Se + N20 + 4H20 + 2HC1

After standing overnight, the precipitate is dissolved in dilute HBr-Br2:

Se + 2Br2 + 5H20 -* H2Se03 + taBr

The titration is carried out by the addition of excess

standard thiosulfate and 5 drops of starch. After 30 seconds

the excess thiosulfate is back titrated with standard iodine

solution;

H2Se03 + ll-HBr + *)-Na2S203 -* Na2S4Se06 + Na2S4Os + Ij-NaBr + 3H20

2Na2S203 I2 Na2S406 2NaI

Other less widely used titrimetric methods are permangan­ ate titrations in either acid (U8) or alkaline (60) solution, argentometric titration (55).? a method similar to the Volhard method for the determination of chloride, and the back titration of excess standard thiourea solution with standard eerie sulfate after reduction and precipitation of selenium by thiourea (27)*

B. GRAVIMETRIC METHODS

Sulfur dioxide is most often used for the reduction of selenium metal which is collected by filtration and subsequently Ik weighed. In the procedure of Goto and Kahita (50) the sample is treated with 5 milliliters of perchloric acid, and nitric acid if necessary to dissolve the sample. It is then fumed gently to remove the nitric acid, cooled and diluted to 100 milliliters with

9N hydrochloric acid. Sulfur dioxide is added for about 15 minutes after the precipitation of selenium begins. The solution is filtered through a fritted-glass crucible and washed with 9N hydrochloric acid and hot water.

Boto and Ogawa (51) have reported that selenium may be pre­ cipitated quantitatively from solutions that contain 5 milliliters of concentrated sulfuric acid per 100 milliliters of Ij-N to 9^ hydrochlo­ ric acid.

A combination of sulfur dioxide and hydroxylamine hydrochlo­ ride to reduce selenium, which may be in the form of selenious or selenic acid, has been used by deSalas (26) for the gravimetric determination of selenium. In this procedure, 30 milliliters of concentrated hydrochloric acid saturated with sulfur dioxide is added to 50 milliliters of solution followed by 1 milliliter of

20 percent hydroxylamine hydrochloride. The solution is allowed to stand for several hours to complete the precipitation before filtration. In all of these procedures, the red selenium on the filter should be washed thoroughly first with 9^ hydrochloric acid and then with water at room temperature before being converted to the black form with hot water, since it is not possible to wash the black form free of impurities. The sample is dried at IO5 degrees centigrade for 1 hour, or to constant weight. 15

Sulfur dioxide has the advantage over many of the stronger reducing agents in that it does not reduce copper to the metal. If the concentration of selenite ion is not too low, i.e. greater than

1 milligram of selenium per 100 milliliters of solution, sulfur di­ oxide is very efficient in reducing selenious acid to elemental se­ lenium.

Other less widely used methods for the gravimetric deter­ mination of selenium are the mercuric nitrate precipitation of se­ lenious acid as HgSe03 (28), percipitation as selenium sulfide by hydrogen sulfide (84), and the thiourea reduction of selenious acid (27)•

C. NEUTRON-ACTIVATION ANALYSIS

Neutron-activation analysis is a useful and highly sensitive method for determining trace elements. For the deter­ mination of selenium, most workers have used the selenium-75 radionuclide(99;71^ 68). This nuclide has two convenient gamma- rays for counting purposes at 0.27 an

The selenium-77m radionuclide has also been used for the determination of selenium (85, k-, ^6), in spite of the fact that it is exceedingly short-lived, with a half-life of 17*5 seconds. In order to determine the element, an activation period of a few seconds and a multichannel analyzer focused on the 0.16-MeV gamma-ray peak must be used. This method can be quite satisfactory provided the half-life of the peak if measured, since many other short-lived nuclides have a gamma-ray of approximately the same energy.

Other selenium isotopes which have been used for the determination of selenium by neutron-activation analysis are

3.9-minute selenium-79m (75? 7Mj> 57-minute selenium-8lm (127)> and 18-minute selenium-81 (10). These procedures usually require a combination of rapid chemical separation and gamma-ray spectro­ metry and are not as widely used.

D. P0LAR0GRAFHIC METHODS

A polarographic method for the determination of selenium in water in the 5 to 100 milligrams per liter range has been used

(1^). In this procedure, the selenium is first separated from other elements by reduction to the element and then dissolved in a mixture of hydrogen bromide and bromine. After the bromine lias been swept out with carbon dioxide, the solution is polarographed at 400 to 65O millivolts with a tungsten-saturated calomel elec­ trode. Increased sensitivity can be achieved by using a solution

2N with hydrochloric acid in a range of 300 to 65O millivolts. 17

Dolezal and Cadek reported that the height of the wave is proportional to concentration of selenium in the range of 5 x 10"6 to 2 .5 x 10”3 moles of selenium in the analysis of pyrite samples

(29). After dissolution of the sample, SeBr4 distillation, and reduction to the metal, the selenium is dissolved in nitric acid, which is fumed off with sulfuric acid. Subsequently the solution is neutralized to pH 8 and polarographed. At this pH the half-wave potential of selenium is -l.Ml-V vs. a standard calomel electrode.

A polarographic method has been developed by Hahn and

Kleinworth (5^-) which depends upon trie precipitation of Tl2Se from a standard solution of TINO3 followed by the determination of ex­ cess thallium. In an ammonical solution and under the proper con­ ditions, Tl2Se is precipitated quantitatively by hydrazine sulfate.

E. PHOTOMETRIC METHODS

1. Spectrophotometry

a. Particles in Suspension

The sol methods, which have an analytical range of

50 to 5^000 micrograms of selenium per gram of sample, differ from one another principally according to the reductant used. The two most common reductants are hydrazine and stannous chloride (116,

131). Sols formed by stannous chloride contain substantial amounts of coprecipitated tin oxides while the hydrazine-reduced selenium sols are presumably sols of the pure element.

b . Equivalent in Liberated Iodine

A greater coloration than that achieved with the formation of the selenium hydrosol alone can be obtained by 18 using potassium iodide as a reducing agent for selenious acid due to the liberated iodine (79)* A further gain in sensitivity can be realized by the addition of a soluble starch solution (129.,

These procedures have an analytical range of 0.5 to 20 micrograms per milliliter and have been utilized for the determination of se­ lenium in natural waters (70).

c. Pyrrole Color Complex

Selenious acid may be detected by pyrrole in the presence of selenic, tellurous, and telluric acids (57) • The re­ action depends upon the oxidation of pyrrole to "pyrrole blue" by selenious acid; the selenious acid being reduced to the element.

A quantitative procedure has been developed based on this reaction

(108), but it has not been widely used for the determination of selenious acid.

d. Diaminobenzidine Complex

Since Hoste first proposed 3,3'-diaminobenzidine

(3*3'*^*^,-Tetraaminobiphenyl) as a sensitive reagent for the qualitative determination of selenium in 19^8(59)* numerous spec- trophotometric methods for the quantitative determination of sele­ nium using this reagent have been reported. These are by far the most widely used procedures for determining small amounts of se­ lenium in various kinds of samples including stainless steels and copper (16, kj), biological materials (109, 9, 58, 63, 22, 21), sulfuric acid (23, 61), air (107), ores (35, 66, 130,95)* water

(77* 96), lead (73)* sulfur (117)* dietary cystine (128), human teeth and hair (53* HO), meteorites (33)* tellurium (11^), and telluric acid (115). The yellow diphenylpiazselenol which forms when 3> 3 '-diaminobenzidine is added to a solution containing se­

lenious acid has an absorption maximum at 420 millimicrons.

H2N nh2

h2n- NH2 + 2H2Se03 -»

S e = N N = S e

N ■N + 6h2o

These procedures, in general, have a lower detection limit of

0 .2 5 to 5 micrograms per milliliter depending on the type of sample being analyzed. In spite of the widespread use of this reagent for the spectrophotometric determination of selenium, there are several serious limitations. The control of pH during formation of the complex and subsequent extraction is extremely critical, and all strong oxidizing and reducing agents must be absent.

e. Miscellaneous

S.awicki (98) has reported two new sensitive rea- gents which might be substituted for 3,3'-diaminobenzidine for the determination of selenium. One is 4-dimethylamino-l, 2-phenyl- enediamine, which reacts with selenious acid to produce a bright red color in IN hydrochloric acid. The other is 4-methylthio-

1,2-phenylenediamine, which also reacts with selenious acid to 20 give a purple-blue color in 50 percent sulfuric acid. Both of

these colored products are piazselenols similar to the complex

formed by 3>3 '-diaminobenzidine. Other reagents have been eval­ uated as possible reagents for the determination of selenium in­ cluding o-phenylenediamine (5)? and if—substituted ^-phenylene- diamines (112), but they are not as sensitive as 3>3’-diamino­ benzidine.

2. Fluorimetry

The diphenylpiazselenol which is formed in the reaction of selenious acid with 3>3 ’-diaminobenzidine has been used for the fluorimetric determination of selenium by several workers. This complex is excited at 425 millimicrons and fluoresces with maximum intensity at about 565 millimicrons. The sensitivity of this pro­ cedure is 0.02 micrograms of selenium per gram of sample, and it has been used for the analysis of various biological materials

(19, 121, 34) and arsenic (88). For the fluorimetric work of high sensitivity, this reagent suffers the disadvantage of having a fluorescence sensitivity which is comparatively low.

Another reagent which can determine selenium down to

0.002 micrograms per gram of sample, was introduced by Parker and

Harvey (87) in 1962. The reagent, 2,3-diaminonaphthalene, reacts with selenious acid to form the 4, 5-Benzopiazselenol, which fluo­ resces strongly at ^kO millimicrons, and has been used for the analysis of copper, zinc, aluminum, and sulfuric acid (72), various biological materials (1, 120) and in conjunction with isotopic di­ lution for the determination of selenium in cereal grains (20). 21

3* Emission Spectroscopy

Due to the low spectral sensitivity of selenium,

little work has been done in developing practical methods for its

spectrographic determination. However, using photographic plates which are sensitive to short wave length ultraviolet radiation a

procedure has been developed to determine selenium in the con­

centration range of 0.0015 to 2 percent in pyrites and sulfide

ores of copper (118). Using similar plates, a method also has been reported for detecting selenium in dried salts to 0.001

percent (9*0 *

4. X-Ray Fluorescence Spectroscopy

The intensities of the few x-ray emission lines of

selenium are very low and are of little analytical value except

in samples in which the concentration is greater than 10 ppm.

Selenium has a peak at a wave length of 1.106A. and this emission

peak has been used for the determination of selenium in plant mate­ rial (11, 57)* Matrix effects are extremely important in the analysis

of plant materials due to the differences in background emission obtained from different samples types. For this reason, it is

imperative to apply a correction procedure in order to obtain

consistent results.

F. RING OVEN METHOD

The ring oven is a simple apparatus which has been demon­

strated to be useful for the detection and determination of vari­

ous elements by chemical methods where only very small quantities 22 of test material are available. Not only can the ring oven be used to concentrate the material being tested into a ring of very small area, but it also enables one to apply separation techniques such as solvent extraction and precipitation to quantities of test material in the nanogram to microgram range. West and Cimerman

(125) have developed a method for the identification and quanti­ tative estimation of air borne selenium particulates based upon the reaction between selenious acid and 3j3l_diaminobenzidine.

The average relative error for the procedure is 5 percent when applied in the range of 0 .1 to 0 .5 micrograms of selenium.

G. CATALYTIC METHODS

The use of catalyzed and induced reactions in microchemi­ cal and trace analysis is increasing in importance. These methods can be very sensitive and highly selective or even specific when appropriate conditioning procedures are employed. Since the feasibility of automation of catalytic determinations has recently been demonstrated (52)^ quantitative kinetic methods are becoming more widely appreciated.

Recently, Kawashima and Tanaka (6 2) have proposed a pro­ cedure for the determination of selenium based on the catalytic reduction of 1, K} 6, 11-tetraazanaphthacene. The method is very sensitive but it is subject to interference from several ions in­ cluding tellurium. Feigl and West (39) developed a highly sensi­ tive spot test for selenium based on the catalytic effect of ele­ mental selenium on the reduction of methylene blue by sodium sul­ fide. Goto, Hirayama, and Ikeda (^9 ) have reported a procedure 23 for the determination of selenium based on this reaction. Their method, however, is neither sensitive nor selective. More recently,

West and Ramakrishna (12^) have developed a method for the deter­ mination of traces of selenium in air, water and biological mater­ ial. By using EDTA as a general masking agent, the procedure is quite specific, and by taking advantage of the inducing effect of

Fe(lll), as little as 0.1 microgram of selenium in 10 milliliters, can be measured. CHAPTER III

THE DETERMINATION OF SELENIUM BY

THE HYDROXYLAMINE OXIDATION METHOD

A. INTRODUCTION

Selenium dioxide has been used extensively by organic

chemists as an oxidizing agent for unsaturated hydrocarbons, alde­

hydes, ketones, heterocyclic nitrogen compounds, terpenes, sterols,

fatty oils, and other natural products. This topic has been the

subject of a review by Watkins and Clark (II9 ).

Several analytical methods which are based on the use of

selenium dioxide as an oxidizing agent have been discussed in the

previous chapter. These procedures lack sensitivity and hence can­

not be used for the determination of trace amounts of selenium.

Postowsky, Lugowkin, and Mandryk (92) investigated the

oxidation of arylhydrazines by selenious acid. They found that

the arylhydrazines were oxidized to diazonium salts, which could be coupled with aromatic amines to produce intensely colored azo dyestuffs. Feigl and Demant (3 8) employed this reaction for the detection of small amounts of arylhydrazines.

Yoe and Kirkbright (65) have reported a spectrophotometric method for the determination of selenium based on the spot test developed by Feigl. They used selenious acid to oxidize phenyl- hydrazine p-sulfonic acid to the corresponding diazonium salt in

strongly acidic medium. After the addition of 1-naphthylamine,

the pH of the system must be adjusted to between 1.8 and 2.2 by

2k 25 by the addition of sodium acetate to achieve the maximum yield of the coupled azo dyestuff. The useful analytical range of the procedure is 2 to itO micrograms in a maximum sample volume of 2 milliliters.

Feigl (36) has mentioned that hydroxylamine hydrochloride is oxidized to nitrous acid by selenious acid under strongly acid­ ic conditions. It is commonly known that nitrites react with pri­ mary aromatic amines in acidic solutions with the formation of diazonium salts which will couple with certain compounds to form intensely colored azo dyes. Because of the extreme sensitivity of the diazotization-coupling reaction sequence with which nitrite determinations are routinely made in the parts per billion range., this reaction offers a unique and attractive approach for the determination of submicrogram quantities of selenium.

Various combinations of reagents for the diazotization- coupling reactions have been used by different workers including sulfanilic acid and 1-aminonapht.halene (2, 6, !&), sulfanilic acid and N, N-dimethy1-1-aminonaphthalene (^-5); sulfanilic acid and N-(l-naphthyl)ethylenediamine dihydroc.hlori.de (97); anc* sul­ fanilamide and N-(l-naphthyl)ethylenediamine dihydrochloride (6b}

IO3). Since the last combination of reagents is somewhat more sensitive than the others, it was chosen as the basis for the development of a new spectrophotometric method for the determina­ tion of selenium which can be represented by reactions (1), (2), and (3).

(1) HSe03" + H+ + NHaOH -* Se + HN02 + 2H20 26

(2) HgNO^S—^ ^ — NH2-W02"+2H+ - HgNOgS— ^ ^ — N^+2H20

+ (5) h 2n o 2S' sN +

,+ h 2n o 2s NCH2CH2NH2 + H

The work presented here is a systematic, study of the vari­

ous factors which influence these reactions. The study was carried

out in order to determine the optimum reaction conditions and thus

achieve maximum sensitivity. The method which has been developed

provides a means for the spectrophotometric determination of sele­

nium in the 0.01 to 0.2 parts per million range., that is both

accurate and reproducible. 27

B. PREPARATION OF REAGENTS AND SOLUTIONS

All inorganic chemicals used in this work were analytical

reagent grade. Double distilled water was used for the prepara­

tion of all aqueous solutions.

1. Standard Stock Selenium Solution

A solution containing 0.05 milligram of selenium per milliliter was prepared by dissolving 50 milligrams of pure sele­

nium metal in a few drops (minimum necessary) of concentrated

nitric acid, boiling gently to expel brown fumes and remove

excess nitric acid and making up to 100 milliliters with distilled water. The standard stock solution was diluted as necessary for

preparing standard working solutions.

2. Hydrochloric Acid

Concentrated hydrochloric acid used was approximately

37 percent HC1, sp. gr. 1.18, from Mallinckrodt Chemical Works.

3. Hydroxylamine Hydrochloride Solutions

Solutions of 0.1, 1, 5* 10* 12, 16, 20, JO, 40, and

50 percent (w/v) were prepared by dissolving the required amounts

of hydroxylamine hydrochloride (Mallinckrodt Chemical Works or

J. T. Baker Chemical Co.), in the appropriate volumes of dis­

tilled water.

1*. Sulfanilamide (p-aminobenzenesulfonamide) Solution

Solutions of 0.05;, 0.1, 0.5;, 1^ 2, 3* 5* 6? anc* 7

percent (w/v) were prepared by dissolving the required amounts of

sulfanilamide (Mallinckrodt Chemical Works), in the appropriate

volumes of 1:1 hydrochloric acid-distilled water (v/v). It was 28 necessary to heat the higher concentrations of this reagent on a water bath in order to effect solution. If there is a visible residue after all of the sulfanilamide has dissolved, the solution should be filtered, while hot, through a fine porosity sintered glass funnel. This solution is stable for a month if kept tightly stoppered and in a refrigerator when not in use.

5. N-(1-Naphthyl)ethylenediamine Dihydrochloride

Solutions of O.O5, 0.1, 0.2, 0.3, 0.4, O.5, and 0.6 percent (w/v) were prepared by dissolving the appropriate amount of the reagent (Fisher Scientific Co.), in a 1 percent (v/v) hydrochloric acid solution. This solution should be kept in a lightproof container and tightly stoppered. It is stable for IQ-

14 days if kept in a refrigerator when not in use.

6. Interference Solutions

Various solutions of diverse ions used for the inter­ ference studies were prepared by dissolving the calculated weight of each compound in distilled water in order to give 10 milligrams per milliliter of the respective interfering ions.

C. APPARATUS

A Beckman Model DB Spectrophotometer (Beckman Instruments,

Inc.) equipped with 1 centimeter quartz cells was used for making absorbance measurements.

A Sargent Heater and Circulator for Thermostatic Baths,

115V, 5^-60 cycles, 110 watts (E. H. Sargent and Co.), was used to maintain constant temperatures. All weighings were made with a Top-Loading precision balance (Mettler Instrument Co.), Model P120.

Other equipment used in this study was as follows: test tubes (2.5 x 20 cm); size No. 11 cork stoppers; volumetric flasks; volumetric and graduated pipets; beakers; and other general glassware. CHAPTER IV

RESULTS AND DISCUSSION

A. PRELIMINARY EXPERIMENTS

In the early stages of this study, many preliminary ex­ periments were carried out in order to test the feasibility of the hydroxylamine-oxidation approach for the determination of selenium. From the reactions shown on pages 25 and 26, it can be seen that, by the principle of mass action, the oxidation of hy­ droxylamine hydrochloride to nitrous acid should be more favorable in acidic medium. Secondly, it can be seen that the diazotization of sulfanilamide(p-aminobenzenesulfonamide) is also favored by high acidity whereas the coupling reaction between the diazonium salt intermediate and N-(l-naphthyl)ethylenediamine dihydrochloride should be retarded in a highly acidic medium.

A further factor which had to be considered was the order of addition of the reacting species. There are three reasonable ways in which the reactants can be combined. The first of these is to introduce the hydroxylamine hydrochloride into a solution of selenious acid containing hydrochloric acid and allow the oxidation reaction to proceed for a period of time, followed by the addition of sulfanilamide and a time lapse in order for the diazotization reaction to occur. Finally, the coupling reagent (N-(1-naphthyl)- ethylenediamine dihydrochloride) is added with the subsequent for­ mation of the coupled azo dyestuff. A second approach is to allow the oxidation and diazotization reactions to proceed at the same

30 time by adding both hydroxylamine hydrochloride and sulfanilamide initially to a solution of selenious and hydrochloric acids followed by the addition of the coupling reagent after the oxidation- diazotization reaction sequence has gone to completion. The third choice is to combine all reactants in a common system.

All of these procedures were carried out for comparison purposes and by visual comparison of the intensity of the highly- colored product, it was readily seen that the second approach of oxidation-diazotization followed by the coupling reaction was the most promising approach to pursue.

Further preliminary investigations disclosed the necessity of thorough mixing after the addition of each chemical in order to obtain reliable and consistent results. These studies also revealed that the reaction mixture, especially the diazotization product was photochemically unstable and, therefore, the procedure should be carried out in the dark or at least in subdued light.

In order to obtain the absorption spectra of the mixed rea­ gents and different concentrations of the coupled azo product, the following experiment was carried out:

First, 3 milliliters of standard selenium(lV) solutions with concentrations of 2, 5* 25* and 50 milligrams per liter(10, 25,

125, and 250 |j,g of selenium(lV) respectively) were transferred to lightproof vessels (2 .5 x 20 cm test tubes wrapped in aluminum foil and fitted with size No. 11 cork stoppers) containing 3 milliliters of concentrated hydrochloric acid. This was followed by the addition of 2 milliliters of 50 percent (w/v) hydroxylamine hydrochloride 32 solution and 2 milliliters of 0.5 percent (w/v) sulfanilamide solu­ tion, with particular care taken to insure thorough mixing after the addition of each reagent. The reaction mixture was then allowed to stand at room temperature (about 25 degrees centigrade) for ^5 minutes. After this time had elapsed, one milliliter of 0.1 percent

(w/v) N-(l-naphthyl)ethylenediamine dihydrochloride solution was added and the volume was adjusted to 50 milliliters, followed by a reaction time of 30 minutes at room temperature for the coupling reaction. A reagent blank, which contained 5 milliliters of dis­ tilled water instead of selenium, was treated in the same manner.

The absorption spectra were taken with reference to distilled water using 1 centimeter quartz cells with a Beckman Model DB spectro­ photometer between 660 and MK) millimicrons. As shown in Figure 1, the coupled product has a maximum absorbance at millimicrons.

At this wavelength the reagents show little absorbance. However, a reagent blank should be used in all determinations to compensate for any change in color of the reagents due to prolonged storage.

Since the oxidation and diazotization reactions are both influenced by the acidity of the reaction medium, it was necessary to establish the conditions of optimum acidity for the oxidation- diazotization reaction sequence as evidenced by the color intensity of the coupled product. To accomplish this, 5 milliliters of a standard selenium solution containing 5 micrograms of selenium(lV) per milliliter was introduced into the lightproof reaction tubes followed by the addition of 2 milliliters of 50 percent (w/v) hydroxylamine hydrochloride solution. Varying amounts of $ $ Transmittance 100 5 5 70-- --III. 25 Micrograms Se(lV) 25Micrograms --III. IV. 125 Micrograms Se(lV) 125 Micrograms IV. II. 10 Micrograms Se(lV) 10 Micrograms II. I. Reagent Blank Blank Reagent I. Absorption Spectra of Various Concentrations Concentrations Variousof Spectra Absorption Wavelength in Millimicrons Wavelength of the Coupled Product theCoupled of Figure 1 Figure III II IV concentrated hydrochloric acid were then added and the volume was adjusted with distilled water in order to obtain the desired norm­ ality, taking into account the amount of hydrochloric acid added in the sulfanilamide solution. Two milliliters of 0.5 percent (w/v) sulfanilamide solution was added and the reaction mixtures were allowed to stand at room temperature for minutes. The coupling reagent was then added, and after adjusting the volume to 50 milli­ liters, the reaction was continued for an additional minutes.

Absorbance measurements were made against distilled water, at

5^4 millimicrons in a Beckman Model DB spectrophotometer using

1 centimeter quartz cells. These results are presented in TABLE I, and Figure 2 shows a plot of blank corrected absorbance against norm- ality of hydrochloric acid during the oxidation and diazotization reactions. The absorbance due to the azo product is a maximum at concentrations of hydrochloric acid above 6.1 normal. Therefore, for all future experiments, 10 milliliters of concentrated hydro­ chloric acid was added resulting in a reaction mixture which is

6.8 normal with respect to hydrochloric acid during the oxidation- diazotization reaction.

In order to determine the most favorable reaction time for the oxidation-diazotization reaction, 5 milliliter aliquots of a standard selenium(lV) solution which contained 1 microgram of selenium per milliliter were treated in the same manner as in the preceeding experiment with the exceptions that 10 milliliters of concentrated hydrochloric acid was added to each reaction tube and the oxidation-diazotization reaction sequence proceeded for a 35

TABLE I

EFFECT OF ACID CONCENTRATION DURING THE OXIDATION-DIAZOTIZATION REACTION

Concentra- Amount Normality Absor- Corrected tion of of HC1 bance Absor- of Selenium during at bance Selenium (l-tg) reaction 5Mfm|J. (mg/1)

0 .0 0.00 0 .9 8 0.015 Blank

5-0 25.00 0 .9 8 0.050 0.015

5.0 25.00 1.95 O .057 0.022

5-0 25.00 2.93 O .057 0.042

5-0 25.00 3.90 0.075 0.060

5-0 25.00 5.15 0.090 0.075

5-0 25.00 5.61 0.094 O.O79

5-0 25.00 6 .1 c 0.096 0.081

5-0 25.00 6 .6 8 0.096 0.081

0 .0 0 .0 0 7.00 0.015 Blank Corrected Absorbance -j, 0 1 . 0 0.08 9 0.0 0.05 0.05 0.06 . . 7 .0 0 0.0it- -- O.O 0.02 0.01 3 ------•- --

Hydrochloric Acid During the Oxidation-Diazotization theOxidation-Diazotization Reaction During Acid Hydrochloric ocnrto fHdohoi cd (Normality)of HydrochloricConcentration Acid Variation of Absorbance with Concentration of Concentration of Variation Absorbance with 2 3 3 2 Figure2 h 5 V_M o\ 37 different length of time for each tube. After completion of the coupling reaction, the absorbances were measured at 5 ^ millimicrons as previously. The results are shown in TABLE II and Figure

These data show that the oxidation-diazotization reaction is not completed even when the reaction time is 3 hours at room temperature.

B. THE OXIDATION-DIAZOTIZATION REACTION

Since the time required for a determination to be completed is one of the practical factors which must be considered in any procedure to be used for routine analyses, it was considered nec­ essary to carry out a detailed study of the oxidation-diazotization reaction in order to ascertain the effect of temperature on the rate of reaction for the system.

Investigations were made in which the temperature during the oxidation-diazotization reaction sequence was 5, 25, ^0, 50> 60, and 70 degrees centigrade. A refrigerator was used for the study carried out at 5 degrees and in the others a thermostatically cont­ rolled water bath which maintained the temperatures at -1 degree centigrade was used. For each of these experiments, the following procedure was used:

Five milliliter portions of standard selenium(iv) solutions containing O.ij-, 1.0, 5*0 an(i 10.0 micrograms per milliliter respect­ ively were transferred to four lightproof reaction tubes and

2 milliliters of 50 percent (w/v) hydroxylamine hydrochloride solu­ tion was added followed by 10 milliliters of concentrated hydrochloric acid and 2 milliliters of 0 .5 percent (w/v) sulfanil­ amide solution. The oxidation-diazotization reaction was then 38

TABLE II

TIME STUDY FOR OXIDATION-DIAZOTIZATION REACTION AT ROOM TEMPERATURE

Concentra- Amount Reaction Absor- Corrected tion of Time bance Absor- of Selenium (minutes) at bance Selenium (|j,g) (mg/1)

0 .0 0 .0 15 0.014 Blank

1.0 5.0 15 0.032 0.018

1.0 5.0 30 0.035 0.021

1.0 5.0 ^5 O.O39 0.025

1.0 5.0 6o 0.045 0.031

1.0 5.0 90 0.054 0.040

1.0 5.0 120 O.O67 O.O53

1.0 5.0 180 0.084 0.070

0.0 0.0 180 0.015 Blank Corrected Absorbance 0.06 0.08 0 0.02 . 0 *+ ------5 0 + 6 7 90 75 60 1+5 30 15 Variation ofTime During Variation Absorbance with theOxidation-Diazotization Reaction + ie (minutes)Time Figure 3 Figure + 105 120 135 5 165 150 180 vo carried out at the appropriate temperature for 60 minutes. At the

end of this time, 1 milliliter of 0.1 percent (w/v) N-(l-naphthyl)-

ethylenediamine dihydrochloride was added plus 30 milliliters of

distilled water followed by a 30-minute coupling reaction time at

room temperature. A reagent blank was also run simultaneously for

each of these experiments using 5 milliliters of distilled water.

The absorbance measurements were made at 5^- millimicrons in a

1 centimeter cell against distilled water. The data obtained are

presented in TABLE III. Figures Ij- and 5 show plots of blank cor­

rected absorbance versus reaction temperature and concentration of

selenium(lV) in milligrams per liter, respectively. These data show

that the coupled product shows a maximum absorbance when the

oxidation-diazotization reaction is carried out at 60 degrees

centigrade.

To establish the optimal time at 60 degrees centigrade for

the oxidation-diazotization reaction, the following study was made:

Five milliliter portions of a standard selenium(lV) solution containing 5 micrograms per milliliter were treated as in the

previous experiment except that the oxidation-diazotization reaction was run at 60 degrees centigrade for 15, 30, U5, 60, 75, 90, 120,

and 180 minute periods of time. In this experiment, the coupling reaction was also carried out 60 degrees centigrade for 30 minutes.

The results given in Figure 6 and TABLE IV show that the optimum reaction time for the oxidation-diazotization reaction at 60 degrees

centigrade is between 75 an

time of 90 minutes was used for all future studies. TABLE III

TEMPERATURE STUDY FOR THE OXIDATION-DIAZOTIZATION REACTION

Concentration Amount of Blank Corrected Absorbance of Selenium Selenium at 51*4 mi (mg/1) (Hg) 5°c 25°C 4o°c 50 °c 60 °c 70 °c

0.00 Blank 0.011 0.00 0.009 0.005 0.009 0.015

0.1*0 2.00 0.005 0.016 0.016 0.015 0.016 0.013

1.00 5.00 0.012 0.031 0.037 0.049 0.049 0.042

5.00 25.00 0.023 0.151 0.180 0.243 0.263 0.197

10.00 50.00 0.035 0.325 0.351 0.463 0.532 o .4oo Corrected Absorbance 0.6 0.4 0.2 0.3 0.1 During the Oxidation-Diazotization Reaction theOxidation-Diazotization During Variation of Absorbance with Temperature of Absorbancewith Variation eprtr (DegreesCentigrade) Temperature 1 PPM Figure 4 Corrected Absorbance 0.10 0.20 ,0 -■ 0,30 0 .O .. O.5O 0.60 . 1*0 -■ ■■ —7 c P—>70 c 60 0 — P 0 5 0 - — 0 X xdto iztzto ecino tnadCre o theforSystem StandardonCurves Reaction Oxidation Diazotization —*25 5 C c c "c

The Effect of Temperature During theofTemperatureDuring Effect The ilgasS(V pr liter perSe(IV) Milligrams 3 i 'X i 6 3 b Figure 5 Figure Vji ■p" 4 4

TABLE IV

TIME STUDY FOR THE OXIDATION-DIAZOTIZATION REACTION AT 60 DEGREES CENTIGRADE

Concentra- Amount Reaction Absor- Corrected tion of Time bance Absorbance of Selenium (minutes) at Selenium (p.g) (mg/1)

0.0 0.00 15 0.014 Blank

5.0 25.00 15 0.200 0.186

0.0 0.00 50 0.011 Blank

5.0 25.00 50 0.281 0.270

0.0 0.00 45 0.014 Blank

5.0 25.00 45 0.510 0.296

0.0 0.00 60 0.012 Blank

5.0 25.00 60 0.528 0.516

0.0 0.00 75 0.015 Blank

5.0 25.00 75 0.558 0.525 0.0 0.00 90 0.011 Blank

5-0 25.00 90 0.540 0.529

0.0 0.00 120 0.014 Blank

5.0 25.00 120 0.549 0.555 0.0 0.00 180 0.016 Blank

5.0 25.00 180 0.524 O.508 Corrected Absorbance .O -• O.JOO T 0.350 .5 -■ 0.250 0.200 0.150 -- 15 30 Oxidation-Diazotization at Oxidation-Diazotization Reaction -» 5 0 5 0 105 90 75 60 45 Variation of Absorbance with Time During the Duringof Time Absorbance with Variation 1

1

ie (minutes)Time Figure 1

6 1 — 60 Degrees Centigrade Degrees 120 135 5 I65 150 180 \J ■p- 1 k 6

C. THE COUPLING REACTION

A comparison of the data given in line four, column seven of TABLE III and line eight, column four of TABLE IV shows that more of the highly-colored coupled product is formed when the coupling reaction is carried out at 60 degrees centigrade than when it is run at room temperature for the same JO-minute time period. There­ fore, a time study at 60 degrees was performed for the coupling reaction. Using 5 milliliters of a selenium(IV) standard solution which contained 5 micrograms per milliliter this study was made under the optimum conditions which had been established up to this point. Coupling reaction times of 5, 15, $0, and 60 minutes were used and from the results shown in TABLE V and Figure 7> it can be seen that the most favorable coupling reaction time at

60 degrees centigrade is 30 minutes.

As previously mentioned, the rate of reaction for the coupling process should be higher as the acidity of the reaction medium is decreased. This can readily be noted from reaction (3) on page 26, where the hydrogen ions are seen to appear on the right side of the equation. For this reason, most methods used for nitrite determinations decrease the acidity of the medium to about pH 2 by the addition of sodium acetate solution prior to the coupling reaction. However, when this was attempted for the present system, the result was extremely high blanks. Thus, it is more advantageous to raise the temperature of the system during the coupling process in order to obtain a reasonable reaction time rather than to lower its acidity. 4 7

TABLE V

TIME STUDY FOR THE COUPLING REACTION AT 60 DEGREES CENTIGRADE

Concentra­ Amount of Reaction Absor­ Corrected tion Selenium lime bance Absorbance of (M-g) (minutes) at- Selenium 544my, (mg/1)

0.0 0.00 5 0.006 Blank

5.0 25.00 5 0.295 0.289

0.0 0.00 15 0.009 Blank

5.0 25.00 15 0.332 0.323

0.0 0.00 50 0.007 Blank

5.0 25.00 50 O.538 0.331

0.0 0.00 45 0.008 Blank

5.0 25.00 45 0.320 0.312

0.0 0.00 60 0.011 Blank

5.0 25.00 60 0.322 0.311 Corrected Absorbance 0 0.30 --0.30 -•O.35 0.25 -■0.25 0.20 . 1*0 15 Variation of Absorbance with Time During theof TimeDuring Variation Absorbancewith Coupling Reaction at Reaction Coupling ie (minutes)Time Figure 50 60 Degrees Centigrade Degrees 7 t T "fe" 00 -p- Kershaw and Chamberlin (64) have reported that for the

same amount of nitrite nitrogen a more intense color is developed when the reaction mixture is diluted to a final volume of 50 milli­

liters than when the final volume is less than 50 milliliters. In view of the similarities between the hydroxylamine oxidation pro­ cedure being developed and their method for the determination of nitrites, it was considered necessary to investigate the effect

of volume of the reaction system during the coupling reaction on

the color intensity of the final product. Five milliliter portions of a standard selenium(iv) solution containing a total of 25 micro­

grams of selenium were examined under the same conditions as the preceding study except immediately prior to the coupling reaction,

sufficient distilled water was added in order to bring the final volumes to 20, 25, 30, 35 > 40 , 45, and 50 milliliters. The coupl­

ing reaction was then carried out at 60 degrees centigrade and

absorbance measurements were made. TABLE VI presents the data

and Figure 8 shows the variation of blank corrected absorbance with volume of the system during the coupling reaction. Clearly,

a final volume of 25 to 30 milliliters is the optimum final volume

for the hydroxylamine oxidation procedure and not. 50 milliliters as in the Kershaw and Chamberlin method for nitrite determination.

D. REAGENT CONCENTRATION STUDIES

In order to determine the most suitable concentration of

each of the reagents used in this procedure, investigations were made to establish the manner in which the blank corrected absor­ bance of the coupled product varied as the concentrations of 50

TABLE VI

VOLUME STUDY FOR THE COUPLING REACTION

Concentra- Amount Final Absor- Corrected tion of Volume bance Absor- of Selenium (ml) at bance Selenium (^g) 5^*mji (mg/)-)

0.0 0.00 20 0.000 Blank

5.0 25.00 20 0.1+20 0.1+20

0.0 0.00 25 0.001+ Blank

5.0 25.00 25 0.550 0.526

0.0 0.00 50 0.006 Blank

5.0 25.00 30 0.555 O.529

0.0 0.00 55 0.006 Blank

5.0 25.00 55 0 .1+75 0 .1+69 0.0 0.00 1+0 0.005 Blank

5.0 25.00 1)0 0.1+25 0.1+20

0.0 0.00 ^5 0.007 Blank

5.0 25.00 ^5 0.380 0-575

0.0 0.00 50 0.006 Blank

5.0 25.00 50 0.352 O.326 Corrected Absorbance o o .6- ■ Variation of Absorbance with Volumeofwith Variation Absorbance During theCouplingReaction During Volume inVolume Milliliters Figure 8 sulfanilamide, N-(l-naphthyl)ethylenediamine dihydrochloride, and hydroxylamine hydrochloride were varied.

1. Sulfanilamide (p-aminobenzenesulfonamlde)

Five milliliter aliquots of a standard selenium(iv) solution containing 1 microgram per milliliter of selenium were transferred to the lightproof reaction tubes followed by the addition of 2 milliliters of 50 percent (w/v) hydroxylamine hydro­ chloride solution and 10 milliliters of concentrated hydrochloric acid. Two milliliter portions of sulfanilamide solutions of vary­ ing concentrations were then added. More specifically, the concen­ trations studied were 0 .05, 0 .1, O.5, 1.0 , 2.0 , J.0 , 4.0 , 5*0 j

6.0 , and 7*0 percent (w/v). The reaction mixtures were then tightly stoppered and heated on a thermostatically controlled water bath set at 6ol"l degrees centigrade for 90 minutes. At the end of this time, 1 milliliter of 0.1 percent (w/v) N-(1-naphthyl)- ethylenediamine dihydrochloride was added plus 5 milliliters of distilled water and heating was continued using the same water bath as above set at 6oi"l degrees centigrade for an additional

30 minutes. Reagent blanks were run at 0.5, 5*0 > anc^ 7-0 percent sulfanilamide respectively and care was taken to insure thorough mixing after the addition of each reagent. Absorbance measure­ ments were made at 544 millimicrons in a Beckman Model DB Spectro­ photometer using a 1 centimeter quartz cell and these data are presented in Figure 9 and TABLE VII. Since the absorbances of the blanks were essentially independent of sulfanilamide concentration, an average value was used in making corrections for the color due 53

TABLE VII

CONCENTRATION STUDY; SULFANILAMIDE (p-AMINOBENZENESULFONAMIDE)

Concentra­ Amount Percentage Absor­ Corrected tion of Sulfanil­ bance Absor­ of Selenium amide at bance Selenium (/ig)) (w/v) 541w)m (mg/1)

0.0 0.00 0.5 0.014 Blank

0.0 0.00 5.0 0.015 Blank

0.0 0.00 7.0 0.012 Blank

1.0 5.00 0.05 0.025 0.011*

1.0 5.00 0.1 0.035 0.021

1.0 5.00 0.5 0.104 0.090

1.0 5.00 1.0 0.166 O.I52

1.0 5.00 2.0 0.244 0.230

1.0 5.00 3-0 0.279 0.265

1.0 5.00 4.0 0.306 0.292

1.0 5.00 5.0 0.321 0.307

1.0 5.00 6.0 0.339 O.325

1.0 5.00 7.0 0.341 0.327

*An average blank value of 0.014 was used. Corrected Absorbance 0.0 0 . 2 - ■ l 2 Concentration of SulfanilamideofConcentration Variation of with Variation Absorbance ecnaeSlaiaie (w/v)SulfanilamidePercentage Figure 9 b 6 7 VJl 4 =" to the reagents themselves. From these results, it can be seen that for the hydroxylamine oxidation procedure, the amount of the azo product which is produced by the coupling reaction increases drastically as the concentration of the sulfanilamide solution in­ creases until a maximum absorbance is reached at 6 percent (w/v) sulfanilamide. In order to dissolve this amount of sulfanilamide in 1:1 hydrochloric adic-distilled water (v/v), it is necessary to heat the mixture on a water bath. Once dissolution has taken place, the solution should be left in the 60 degree thermostati­ cally controlled water bath until after it has been used in order to prevent the sulfanilamide from crystallizing out of solution.

2. N-(1-naphthyl)ethylenediamine Dihydrochloride

In the reagent concentration study for the coupling reagent, 5 milliliter portions of standard selenium(iv) of 1 micro­ gram per milliliter concentration were treated in the same way as in the previous experiment except that a 6 percent (w/v) sulfanil­ amide solution was used throughout and 1 milliliter portions of the coupling reagent which had concentrations of 0.05, 0 *1, 0 -2,

0 .3, 0 .1+, 0 .5, and 0.6 percent (w/v)-were used. TABLE VIII pre- I sents the data and Figure 10 shows a plot of the variation of blank corrected absorbance as the concentration of the coupling reagent varies. A maximum in absorbance is reached at concentra­ tions of N-(l-naphthyl)ethylenediamine dihydrochloride of O.h per­ cent (w/v) or greater. Therefore, a coupling reagent concentration of 0.5 percent (w/v) was incorporated into the standard procedure being developed. 56

TABLE VIII

CONCENTRATION STUDY; N-(l-NAPHTHYL)- ETHYLENEDIAMINE DIHYDROCHLORIDE

(Coupling Reagent)

Concentra­ Amount Percentage Absor­ Corrected tion of Coupling bance Absor­ of Selenium Reagent at bance Selenium (^g) (w/v) 54imyi (mg/ml)

0.0 0.00 0.1 0.014 Blank

0.0 0.00 0.5 0.016 Blank

0.0 0.00 0.6 0.017 Blank

1.0 5.00 0.05 0.227 0.211*

1.0 5.00 0.1 0.335 0.319

1.0 5.00 0.2 0.411 0.395

1.0 5.00 0.3 0.417 0.401

1.0 5.00 0.4 0.427 0.411

1.0 5.00 0.5 0.424 0.408

1.0 5.00 0.6 0.427 0.411

*An average blank value of 0.016 was used. Corrected Absorbance t -- .to o 0 . 20 ' 2 0. 4 0. .6 0 .5 0 .4 0 .3 0 .2 0 i . o of N-(l-naphthyl)fethylenediamineDihydrochloride Variation of Absorbance with Concentration of Variation Absorbance with ecnae (w/v)Percentage Figure 10 + 58

3. Hydroxylamine Hydrochloride

Using 2 milliliters of each solution in a series of

hydroxylamine hydrochloride solution which had concentrations of

0 .1, 1.0 , 5.0 , 10.0 , 12.0 , 16.0 , 20.0 , 30.0 , I40.0, and 5O.O per­

cent (w/v), 5 milliliter aliquots of a standard selenium(iv) so­

lution containing one microgram per milliliter were analyzed under

the optimum conditions which had thus far been established. The

results of this study are given in TABLE IX and Figure 11. These

data show that the intensity of the color developed in the system

is highly dependent upon the concentration of the hydroxylamine

hydrochloride solution used and that a maximum absorbance is obtain­

ed when the concentration of this solution is 10 to 12 percent

(w/v).

E. THE STANDARD RECOMMENDED PROCEDURE

On the basis of the results which have been presented and

described in this chapter, the following procedure is recommended

for the determination of selenium(iv) by the hydroxylamine oxidation

method that has been developed. Five milliliters of sample which

contains between 0.01 and 0.20 micrograms per milliliter of se-

lenium(iv) are introduced into a lightproof reaction vessel and

2 milliliters of 10 percent (w/v) hydroxylamine solution is added.

After the addition of 10 milliliters of concentrated hydrochloric

acid, 2 milliliters of 6 percent (w/v) sulfanilamide solution in

1:1 hydrochloric acid-distilled water (v/v) is added, the reaction

tubes are tightly stoppered, and the reaction mixture is heated

in a thermostatically controlled water bath set at 6ol"l degrees

i 59

TABLE IX

CONCENTRATION STUDY; HYDROXYLAMINE HYDROCHLORIDE

Concentra­ Amount Percentage Absor­ Corrected tion of NHo0H * HC1 bance Absor­ of .... Selenium ... . (w/v) at bance Selenium (^s) (mg/ml)

0.0 0.00 0.1 0.009 Blank

0.0 0.00 10.0 0.012 Blank

0.0 0.00 50.0 0.014 Blank

1.0 5.00 0.1 0.331 O.3I9*

1.0 5.00 1.0 0.384 0.372

1.0 5.00 5.0 0.518 O.506

1.0 5.00 10.0 0.539 0.527

1.0 5.00 12.0 0.537 0.525

1.0 5.00 16.0 0.499 0.487

1.0 5.00 20.0 0.454 0.442

1.0 5.00 30.0 o .4 to 0.428

1.0 5.00 lo .o 0,437 0.425

1.0 5.00 50.0 0.427 0.415

*An average blank value of 0.012 was used. Corrected Absorbance 0.2 0.6 0 5 Variation of Absorbance with Concentration of with Variation Absorbance of Hydroxylamine Hydrochloride ofHydroxylamine 10 ecnae (w/v)Percentage Figure 11 20 25 30 61 centigrade for 90 minutes. This is followed by the addition of

5 milliliters of 0.1 percent (w/v) N-(l-naphthyl)ethylenediamine dihydrochloride in 1 percent (v/v) hydrochloric acid plus 1 milli­ liter of distilled water and a further heating period of 30 minutes in a constant temperature water bath set at 6ol"l degrees centigrade, again taking care to insure that the reaction tubes are tightly stoppered. Absorbance measurements are made at 5 ^ millimicrons and the amount of selenium(iv) present in the sample is determined from a standard curve such as the one shown in Figure 12. It is impera­ tive that the reaction medium be mixed thoroughly after the addi­ tion of each reagent in order to obtain consistent and reliable results.

F. INTERFERENCE STUDIES

To determine the effect of diverse ions on the determination of selenium(iv) by the hydroxylamine oxidation method which has been developed, 0.1 milliliter of varying concentrations of each ion to be tested was added to 5 milliliters of a standard selenium(iv) so­ lution which contained a total of 1 microgram of selenium i.e.,

0.2 parts per million. The selenium was then determined by the re­ commended procedure, and the results of this study are presented in

TABLE XI. Those ions which interfere when present in a thousand­ fold excess are: BrO^ , TeO^2 , La^+ , Br , V0 ^ , Fe^+ , CrO^2 , CN ,

CIO^", MoO^2", i", SCN", Cu2^, Bi5+, UO^2" , B ^ 2-, 10^", SeO^2" ,

NO, , and V0 _ . However, when the concentrations of BrO, and 5 5 5 2- TeO, were reduced to a hundred-fold excess, the concentrations 5 of La 3+ , Br - , and V0 ^ 3- reduced to a fifty-fold excess; the con-

2- 2- - centrations of Fe , CrO^ , Cr^O^. , CN , and C10^ reduced to 62

TABLE X

VARIATION OF ABSORBANCE WITH CONCENTRATION OF S ELENIUM(IV) FOR THE ADOPTED METHOD

Concentra­ Amount Absor­ Corrected tion of bance Absorbance of Selenium at Selenium 0*8 ) (mg/1)

0.00 0.00 0.009 Blank

0.01 0.05 O.OI5 0.006

0.02 0.10 0.020 0.011

0.0^ 0.20 0.027 0.018

0.05 0.25 O.O55 0.021+

0.10 0.50 0.060 0.051

0.20 1.00 0 .111). 0.105 Corrected Absorbance 0 0.000 -- 5 2 .0 0 . 100 -- 00 0.05 The Standard Curve for theforCurveStandardMethod Adopted The Milligrams of Selenium(iv) per Liter perSelenium(iv)of Milligrams Figure 10 0.15 0 .1 c 12 0.20 TABLE XI

INTERFERENCE STUDIES OF DIVERSE IONS

Se (IV) Diverse ion Amount Added Corrected Added (jig) Added Added (jig) As Absorbance

0(Blank) - - - 0.010

1.0 - 0 - 0.105

1.0 Ba2+ 1000 BaCl2 0.10k

1.0 Ca21" 1000 CaCl2 0.106

1.0 Li+ 1000 LiCl 0.105

1.0 M g ^ 1000 MgCl2 0.108

1.0 Ni2* 1000 n i c i 2'6h 2o 0.109

1.0 K+ 1000 KC1 0.105

1.0 Na+ 1000 NaCl 0.105

1.0 Sr2 *" 1000 SrCl2 0.102 TABLE XI (continued)

1.0 so2" 1000 Na2S04 0.109

1.0 1000 Na2S03 0.106 S032' 1.0 Cr?+ 1000 CrCl^ 0.106

1.0 F- 1000 NaF 0.102 2- 1.0 Si0_, 1000 Na2Si03-9H20 0.110 3 2- 1.0 uok 1000 Na2S0 j^-2H20 0.107

1.0 C d ^ 1000 CdCl2 0.107

1.0 Sn2^ 1000 SnCl2 0.103

1.0 Co2+ 1000 Cocl2 0.114

1.0 Zn2* 1000 ZnCl2 0.102

1.0 M n ^ 1000 MnCl2 0.103

1.0 Sb5+ 1000 SbCl^ 0.101

Zr^+ 1000 ZrCl, 0.112 1.0 4 1.0 B e ^ 1000 BeCl2*4H20 0.106

1.0 Malonate 1000 Malonic Acid 0.102 TABLE XI (continued)

1.0 Tartrate 1000 Tartaric Acid 0.105

1.0 Oxalate 1000 Oxalic Acid 0.103

1.0 GeO^2" 100 NaoGe0 ^ 0.100 ^ 3 1.0 1000 0.101

1.0 H2P02_ 100 NaH2P02 0.103 1.0 1000 0.100

1.0 bo2" 100 NaB02 0.104

1.0 1000 o . i o4

NaJHPO, 0.101 1.0 hpo42" 100 2 4 1.0 1000 0.103

1.0 La*’+ 1000 LaCl, 0.184 3 1.0 100 0.108

1.0 Br“ 100 NaBr 0.118

1.0 50 0.105

CT\ TABLE XI (continued)

1 . 0 vo^5" 100

1.0 50

1.0 MoO^2" 100

1.0 25

1.0 10

1.0 Fe^+ 100

1.0 50

1.0 25

1.0 10

1.0 i" 1000

1.0 10°

1.0 10

1.0 s o n " 1000 NaSCN

1.0 w o

1.0 10 TABLE XI (continued)

1.0 Cu2* 1000 CuCl2 1.800

1.0 10 0 . 11*8 BiCl, 2.000 1.0 Bi3+ 100 5 1.0 10 1.900 Na.UO, 2.000 1.0 uo^2- 1000 2 4

1.0 10 0.160

1.0 CN~ 50 NaCN 0.121 0.102 1 .0 25

1.0 Citrate 1000 Citric Acid 0.100

1.0 BrO “ 1000 NaBrO.. 0.141 5 5 1.0 100 0.108 NaCIO, 1.700 1.0 CIO " 1000 3 1.0 100 0.225

1.0 25 0.101 ON 00 TABLE XI (continued)

1.0 10^“ 100 NalO, 0.151 3 1.0 10 0.126

1.0 TeO^2- 1000 Na TeO, 0.126 2 3 1.0 100 0.106

1.0 Hg21" 100 HgCl2 0.107

1.0 1000 0.104

1.0 Al5+ 100 AlCl 0.100 3 1.0 1000 0.102

1.0 HAsO^2" 100 Na2HAs0^'7H20 0.101

1.0 1000 0.106

Na B, 0 • 10H 0 2.000 1.0 1000 2 4 7 2

1.0 10 0.348

1.0 S e ° ^ 2 “ 100 Na2Se0^-10H20 1-900

1.0 10 0.343 o\ TABLE XI (continued)

1.0 NO, 100 Mn(N03 )2 2 . 0 0 0 3 1.0 50 1 .5 0 0

1.0 25 0.458

1.0 10 0.142

1.0 VO," 1000 NaVO, 0.1T2 3 3 1.0 100 0.159

1.0 10 0.146

1.0 Cr(\2” 100 Na2Cr0^‘4H20 0.159

1.0 50 0.139

1 .0 25 0 . 1 0 8

1.0 Cro0 100 0 . 1 3 7 ^ i 1.0 50 0 . 1 1 5

1 .0 25 0.110 71 2- a twentyfive-fold excess and that of MoO^ reduced to a ten-fold excess, no interference was observed.

Of the remaining ions which interfere when present at a 2_l_ 2— ten-fold excess, only Cu and SeOj,, are likely to be significant 2~ in air pollution studies. Se0^ is easily reduced to Se(lV) by boiling for ten minutes in a solution which is kN with respect to hydrochloric acid. The interference due to Cu can be readily obviated using an ion exchange procedure or by extraction from a solution which has been acidified with hydrochloric acid into chloroform as its cupferron complex.

G. STATISTICAL EVALUATION

TABLE XII presents the results of a statistical evaluation of selenium(iv) determinations at the respective concentrations using the hydroxylamine-oxidation procedure which has been developed.

H. CONCLUSION

A new spectrophotometric method for the determination of submicrogram quantities of selenious acid has been developed.

The procedure is based upon the oxidation of hydroxylamine hydro­ chloride to nitrous acid by selenious acid followed by the diazo- tization of sulfanilamide by the nitrite produced and subsequent coupling of the diazonium salt with N-(l-naphithyl)ethylenediamine dihydrochloride.

Reaction parameters such as temperature, time and reagent concentrations have been studied in detail and optimum conditions for the system have been established. The range of determination 72

TABLE XII

STATISTICAL ANALYSIS OF ABSORBANCE AT VARIOUS CONCENTRATIONS OF SELENIUM(IV)

Number of Concentra- Precision, $ Determina- tion of Standard Relative tions Selenium Deviation Error (mg/1) (mg/1)

10 0.01 0.0016 12.0 10 0.02 0.0021 8.0

10 o.ob- 0.0025 b.5 10 0.05 0.0027 b.8

10 0.10 0.00l<6 k.k 10 0.20 O.OO57 2.2 extends from 0.01 to 0.20 milligrams of selenium(lV) per liter.

The method is simple, sensitive, and reproducible. There are no common interferences which cannot be easily obviated. 74

SELECTED BIBLIOGRAPHY

1. Allaway, W.H., and Cary, E.E., "Determination of Submicrogram

Amounts of Selenium in Biological Materials," Anal. Chem., 36,

1 3 5 9 (1 9 6 4).

2. American Public Health Association, Standard Methods of Water

Analysis, p . k6 (1936)•

3« Amor, A.J., and Pringle, P., "A Review of Selenium as an

Industrial Hazard," Bull. Hyg., 20, 239 (1949)*

4. Anders, O.U., "Use of Very-Short-Lived Isotopes in Activation

Analysis," Anal. Chem., 33, 1706 (I9 6 I).

5 . Ariyoshi, H., Kiniwa, M., and Toei, K., "UV Spectrophotometric

Determination of Trace Amounts of Selenium with o-Phenylene-

diamine, 11 Talanta, jj, 122 (i9 6 0 ).

6 . Assoc. Official Agr. Chem., Official and Tentative Methods of

Analysis, pp. 222, 527 (1940).

7. Beath, O.A., "Selenium Poisons Indians," Sci. Newsletter, 81

25k (1 9 6 2).

8. Beath, O.A., Eppson, H.F., and Gilbert, C.S., "Selenium and

Other Toxic Minerals in Soils and Vegetation," Wyoming Agr.

Expt. Sta. Bull. No. 206, 1 (1935). 9- Bonhorst, C.W., and Mattice, J.J., "Colorimetric Determination

of Selenium in Biological Materials (with Diaminobenzidine),"

Anal. Chem., ^1, 2106 (1959)*

10. Bowen, H.J.M., and Cawse, P.A., "The Determination of Selenium

in Biological Material by Radioactivation," Analyst, 88, 721

(1963).

11. Brandt, C.S., and Lazar, V.A., "Analysis of Dried Plant Mate­

rial by X-Ray Emission Spectrograph," J. Agr. Food Chem., 6,

306 (1958).

12. Buchan, R.F., "Industrial Selenosis," Occup. Med. 439

( I 9 V 7 ) .

13 . Byers, H.G., "Selenium Occurence in Certain Soils in the United

States, with a discussion of Related Topics," U.J3. Dept. Agr.

Tech. Bull. No. 482, 1 (1935).

14. Cervenka, R., and Korbora, M., "Polarographic Determination

of Selenium in Water," Chem. Listy, 49, H 5 8 (1955)*

15. Chem. Eng. News, 4^(23), 12 (1 9 6 7).

16. Cheng, K.L., "Spectrophotometric Determination of Selenium in

Stainless Steels and in Copper by 3, 3-Diaminobenzidine,"

Chemist-Analyst 45, 6 7 (1956). 76

17- Coleman, R. G., "The Occurrence of Selenium in Sulfides from

Sedimentary Rocks of the Western United States," (Abstr.)

Econ. Geol. 5 1, 112 (1956).

18. Coleman, R.G., and Delevaux, M., "Occurrence of Selenium in

Sulfides from Some Sedimentary Rocks of the Western United

States," Econ. Geol. 5 2, 499 (1957)*

1 9 . Cousins, R.B., "A Fluorometric Microdetermination of Selenium

in Biological Material," Australian J. Exptl. Biol. Med. Sci.

$ 8, 11 (I9 6 O).

20. Cukor, P., Walzcyk, J., and Lott, P.F., "The Application of

Isotopic Dilution Analysis to the Fluorlmetric Determination

of Selenium in Plant Material," Anal. Chim. Acta, 30> 473

(1964).

21. Cummins, L.M., Martin, J.L., and Maag, D.D., "An Improved

Method for Determination of Selenium in Biological Material,"

Anal. Chem., 430 (1 9 6 5)*

22. Cummins, L.M., Martin, J.L., Maag, G.W., and Maag, D.D.,

"A Rapid Method for the Determination of Selenium in Biologi­

cal Material," Anal. Chem., 3 6, 362 (1964).

23* Danzuke, T., and Ueno, K., "Determination of Trace Amounts of

Selenium in Sulfuric Acid. Colorimetric Method Using 3j 3*“

Diaminobenzidine," Anal. Chem., 30. 1370 (1958)• 77

24. Davidson, D.F., "Selenium in Some Epithermal Deposits of

Antimony, Mercury, Silver, and Gold," U.£k Geol. Survey Bull.

No. 1112-A. 1 (I960).

2 5. Davidson, D.F., and Lakin, H.W., "Metal Content of Some Black

Shales of the Western United States," U.S. Geol. Survey Res.,

Profess. Paper No. 424-C, 3 2 9 (1961).

26. de Salas, S.M., "Selenium in Water. II. Methods of Determina­

tion," Rev. Obras. Sanit. Nacion (Buenos Aires), 20, 264

(1947).

27* Deshmukh, G.S., and Sankaranarayanan, K.M., "Reduction of

Selenious Acid by Thiourea," J. Sci. Research Banaras Hindu

Univ., 2, 5 (1952-3).

28. . "Estimation of Selenium by Mercuric Nitrate," J.

Indian Chem. Soc., 29» 527 (1952).

29. Dolezal, J., and Cadek, J., "Determination of Small Amounts

of Selenium in Pyrites and Similar Material," Chem. Listy, 49,

1 1 5 2 (1955).

30. Dudley, H.C., "Selenium as a Potential Industrial Hazard,"

Public Health Repts. (U.S.) £2, 281 (I9 3 8).

31. • "Toxicology of Selenium, I, A Study_of the Distribu­

tion_of Selenium in Acute and Chronic Cases of Selenium

Poisoning," Am. J. Hyg. 23, I6 9 (1936). 32. Dudley, H.C., and Miller, J.W., "Toxicology of Selenium, VI,

Effect of Subacute Exposure to Hydrogen Selenide," J. Ind.

Hyg. Toxicol., 2£, VfO (I9 U).

33* DuFresne, A., "Selenium (Photometric Determination with

Diaminobenzidine) and Tellurium in Meteorites," Geochim. et

Cosmochim. Acta 20, l4l (i9 6 0 ).

3k. Dye, W.B., Bretthauer, E., Seim, H.J., and Blincoe, C.,

"Fluorometric Determination of Selenium in Plants and Animals

with 3>3 '-Diaminobenzidine, " Anal. Chem., 35, 1 6 8 7 (19^3)'

35* Eiss, M.I., and Gieseck, P., "How to Determine Selenium in

Ores and Cyanide Solutions (Photometrically with Diaminoben­

zidine)," Eng. Mining J. 160, No. 12, 102 (1959)*

3 6. Feigl, F., Spot Tests in Organic Analysis, 7th ed., p. 231,

Elsevier, New York (I9 6 6).

37-______• Spot Tests in Inorganic Analysis, 5th ed., pp. 3^>~

^7, Elsevier, New York (1 9 5 8).

3 8. Feigl, F., and Demant, V., "Mikrochemische Nac.hweise Organis-

cher Verbindungen mit Hilfe von Tupfelreaktionen-Nachweis von

Arylhydrazinen Sowie von Arylhydrazonen and Osazonen,"

Mikrochim. Acta 1, 1 3^ (1937)*

39- and West, P.W., "Test for Selenium Based on

Catalytic Effect," Anal. Chem., 19, 351 (19^7)* 79

1+0. Franke, K.W., Moxon, A.L., and Tully, W.C., "A New Toxicant

Occurring Naturally in Certain Samples of Plant Foodstuffs,

XIII, The Ability of Rats to Discriminate between Diets of

Varying Degrees of Toxicity," Science 8 3, 330 (1 9 3 6).

41. Franke, K.W., and Potter, V.R., "A New Toxicant Occurring

Naturally in Certain Samples of Plant Foodstuffs, IX, Toxic

Effects of Orally Ingested Selenium," J. Nutrition, 10, 213

(1935).

42. Frant, R., "Schadelijke Werking van Seleendioxyde op de

Menselijke Huid," Ned. Tijdschr. Geneesk., 93? 874 (1949).

4 3. Gebauhr, W., and Spang, A.Z., "(Photometric) Determination of

Low Selenium Contents in Copper as Piazselenole (From Diamino­

benzidine) After Dry Separation," Anal. Chem., 175, 175 (i9 6 0 ).

44. Geiersberger, K., and Durst, A., "Determination of Tellurium

and Selenium. II. Detection of Selenium in Tellurium and of

Tellurium in Selenium," Z. Anal. Chem., 135, 11 (1952).

4 5. Germuth, F.G., "Dimethyl-Alpha-Naphthylamine for Determination

of Nitrite Ion," Ind. Eng. Chem., Anal. Ed., L, 28 (1 9 2 9)*

46. Gibbons, D., and Simpson, H., "Short-Lived Radioactive

Isotopes in an Activation Analysis Service Program," Paper

SM 32/3^ presented at the Conference on Short-Lived Radio­

isotopes, International Atomic Energy Agency, Vienna, (I9 6 2). 80

47• Glover, J. R., "Some Medical Problems Concerning Selenium in

Industry," Trans. Assoc. Ind. Med. Officers 4, yk (195*0•

48. Gooch, F.A., and Clemons, D.F., "The Volumetric Determination

of Selenite Using Permanganate," Am. J. Sci., ^0, ^1 (1895)*

4 9 . Goto, H., Hirayama, T., and Ikeda, S., "Catalytic Analysis

(XX) Determination of Selenium," J. Chem. Soc. Japan, Pure

Chem. Sect., ]]>, 6 5 2 (1952).

50. Goto, H., and Kakita, Y., "Determination of Tellurium and

Selenium. I. Determination of Tellurium and Selenium in the

Presence of Perchloric Acid with Sulfur Dioxide," Sci. Repts.

Research Insts. Tohoku Univ., Ser. A, 4, 28 (1952).

51. Goto, H., and Ogawa, T., "Determination of Tellurium and

Selenium. II.," Sci. Repts. Research Insts. Tohoku Univ. Ser.

A, 4, 121 (1952).

52. Hadjiioannou, T.P., "Catalytic Microdetermination of Mercury,"

Anal. Chim. Acta 35, 351 (1 9 6 6).

5 3. Hadjiimarkos, D.M., and Bonhorst, C.W., "Photometric Determi­

nation of Selenium in Human Teeth with Diaminobenzidine," Oral

Surg., Oral Med., and Oral Pathol., 25, 113 (1959)*

5 4. Hahn, H., and Kleinworth, W., "An Indirect Polarographic Meth­

od for the Determination of Microquantities of Selenium," Z.

Anal. Chem., 151, 9 8 (1956)» 81

55. Hahn., H., and Biohl, R., "Argentometric Determination of

Selenium," Z. Anal. Chem., 149, 40 (1 9 5 6).

5 6. Hamilton, A., and Hardy, H.L., Industrial Toxicology. 2nd ed.,

Paul B. Hoeber, Inc., New York (19^9).

5 7. Handley, R., "Fluorescent X-Ray Determination of Selenium in

Plant Material," Anal. Chem., 52, 1719 (I960).

5 8.*- Handley, R., and Johnson, C.M., "Microdetermination of Sele­

nium in Biological Materials (Photometrically with Diamino­

benzidine)," Anal. Chem., 3l» 2105 (1959)*

59* Hoste, J., "Diaminobenzidine as a Reagent for Vanadium and

Selenium," Anal. Chim. Acta 2 , k02 (1948).

60. Issa, I.M., and Issa, R.M., "Volumetric Estimation of Selenite

and Tellurite with Alkaline Permanganate," Chemist Analyst,

4 5, 16 (1956).

61. Kaneko, H., "Colorimetric Determination of Selenium in

Sulfuric Acid with 3>3 '-Diaminobenzidine," Ryusan 13, 2 6 9

(i9 6 0 ).

62. Kawashima, T., and Tanaka, M., "Determination of Submicrogram

Amounts of Selenium(lV) by Means of the Catalytic Reduction

of 1, 4, 6, 11-Tetraazanaphthacene," Anal. Chim. Acta, 40,

137 (1 9 6 8). 6 3. Kelleher, W.J., and Johnson, M.J., "Determination of Traces

of Selenium in Organic Matter. Combined Spectrophotometric

(Method with Diaminobenzidine) and Isotope Dilution Method,"

Anal. Chem. , 1429 (1961).

64. Kershaw, N.F., and Chamberlin, N.S., "Determination of Nitrites,"

Ind. Eng. Chem., Anal. Ed., _14, ^>12 (1942).

6 5. Kirkbright, G.F., and Yoe, J.H., "A New Spectrophotometric

Method for the Determination of Microgram Amounts of Selenium,"

Anal. Chem., 55, 8 0 8 (1965).

6 6. Kitazato, T., and Saeki, Y . , "Determination of a Small Amount

of Selenium in Ores (Photometrically with Diaminobenzidine),"

Bunseki Kagaku 8 , 422 (1959)*

6 7. Klein, A.K., "Report on Selenium," J. Assoc. Offic. Agr. Chem. ,

2 6, 546 (1943).

68. Koch, R.C., and Roesmer, J., "Application of Activation

Analysis to the Determination of Trace Elements in Meat,"

Food Sci., 2X, 309 (1962).

6 9 . Lakin, H.W., and Byers, H.G., "Selenium Occurrence in Certain

Soils in the United States with a Discussion of Related Topics:

7th. Report," U.S. Dept. Agr. Tech. Bull. No. 950, 1 (1948).

' , ) -V 83

70. Lamberts J.L., Arthura P., and Moore, T.E., "Determination

of Trace Amounts of Selenium in Water," Anal. Chem., 23, 1101

(1951).

71. Leddicotte, G.W., "The Radiochemistry of Selenium," National

Academy of Sciences Report NAS-NS 3^30 (1961).

72. Lott, P.F., Cukor, P., Moriber, G., and Solga, J., "2,3“

Diaminonaphthalene as a Reagent for the Determination of

Milligram to Submicrogram Amounts of Selenium," Anal. Chem.,

25, 1 1 5 9„(1 9 6 3).

73* Luke, C.L., "Photometric Determination of Traces of Selenium

(with Diaminobenzidine) or Tellurium in Lead or Copper," Anal.

Chem., 21, 527 (1959).

74. Maddock, R.S., and Meinke, W.W., University of Michigan

Progress Report, _11 , 68 (1 9 6 2).

75- _• University of Michigan Progress Report, 8, 9k (1959)*

7 6. Madison, T.C., "Sanitary Report-Fort Randall," Written Sept.,

1857j it- Coolidge, R.H. , "Statistical Report on the Sickness

and Mortality in the Army of the United States, Jan. 1855 to

Jan. i860," (U.S.) Congr. 5 6th., 1st. Session, Senate Exch.

Doc. 5 2, 3 7 (i860). 84

77* Magin, G.B., Jr., Thatcher, L.L., Rettig, S., and Levine, H.,

"Suggested Modified Method for Colorimetric Determination of

Selenium in Natural Water (with Diaminobenzidine)," Am. Water

Works Assoc., 52, 1199 ^1960).

78. Mason, W.P., and Bushwell, A.M., Examination of Water, p. 5 0 ,

John Wiley and Sons, New York, (I9 5 I).

79« Meyer, J., and Von Garn, W., "Uber den Nachweis und die

Bestimmung Kleinster Mengen Seleniger Saure II," Z. Anal.

Chem., 55, 29 (1914).

80. Middleton, J.M., "Selenium Burn of the Eye," A.M.A. Arch.

Opthalmol., 5 8, 8 0 6 (1947).

81. Miller, W.T., and Williams, K.T., "Minimum Lethal Dose of Se­

lenium as Sodium Selenite for Horses, Mules, Cattle, and Swine,"

J. Agr. Res., 60, I6 3 (19^0)•

82. Myth, O.H., Oldfield, J.E., Remmert, L.F., and Schubert, J.R.,

"Effects of Selenium and Vitamin E on White Muscle Disease,"

Science, 128, 1090 (1958).

8 3. Newberry, J.S., "The Silver Reef Sandstones," Eng. Min. J., 51,

4 (1881).

84. Noyes, A.A., and Bray, W.C., A System of Qualitative Analysis

for the Rare Elements, p. 330>-Macmillan, New York (I9 2 7). 8 5. Okada, M. , "Non-Destructive Analysis of Selenium by Neutron

Activation Followed by Gamma-Ray Spectrometry," Nature, 187,

59b (i9 6 0 ).

8 6. Painter, E.P., "The Chemistry and Toxicity of Selenium Com­

pounds, with Special Reference to the Selenium Problem," Chem. .

Rev., 28, 179 (19^1).

8 7. Parker, C.A., and Harvey, L.G., "Luminescence of Some Piaz-

selenola - A New Fluorimetric Reagent for Selenium," Analyst,

81, 558 (1 9 6 2).

88. . "Fluorimetric Determination of Sub-microgram Amounts

of Selenium," Analyst, 86, 5b (I9 6 I).

89- Patterson, E.L., Milstrey, R., and Stokstad, E.L.R., "Effect

of Selenium in Preventing Exudative Diathesis in Chicks,"

Pro. Soc. Exptl. Biol. Med., 95, 617 (1957)-

90. Patty, F.H., Industrial Hygeine and Toxicology, Vol. II.,

Wiley (interscience), New York (19^9)*

91. Polo, Marco, The Travels of Marco Polo, Revised from Marsden's

Translation and edited by Manval Kimroff, Chapter kj, p. 81,

Liveright, New York (I9 2 6).

92. Postowsky, J.J., Lugowkin, B.P., and Mandryk, G.T., "Uber

die Reaktion des Selendioxyds mit einigen Aryl-Hydrazinen,"

Ber., 62, 1913 (1936). 86

93* Pringle, P., "Occupational Dermatitis Following Exposure to

Inorganic Selenium Compounds," Brit. ^J. Dermatol. Syphilis,

5 k, 5k (1 9^2).

94. Rockenbauer, W., and Schroll, E., Osterr. Akad. Wiss., Math.

naturw. Kl. Anz., 92, I9 2 (1955)*

95. Saito, M. , "Spectrophotometric Determination of Trace Amounts

of Selenium (in Pyritic Ores with Diaminobenzidine)," Iwate

Daigaku K^gakubu Kenyu Hdkoku. 13, 257 (i9 6 0 ).

9 6 . Sakanoue M . , "Arsenic, Germanium, and Selenium (Photometric

Determination with Diaminobenzidine) in the Thermal Waters of

Misasa Hot Springs, Tottori Prefecture, and Determination of

Phosphate," Nippon Kagaku Zasshi, 81, 2^2 (i9 6 0 ).

97* Saltzman, B.E., "Colorimetric Determination of Nitrogen

Dioxide in the Atmosphere," Anal. Chem. , 2 6, 19^+9 (195^)*

9 8 . Sawicki, W., "New Color Test for Selenium," Anal. Chem., 29,

1376 (1957).

99* Schindewolf, U., "Selenium and Tellurium Content of Stony

Meteorites by Neutron Activation," Geochim. Cosmochim. Acta,

1 2, 1 3b (i9 6 0 ).

100. Schwarz, K., Bieri, J.G., Briggs, G.M., and Scott, M.L.,

"Prevention of Exudative Diathesis in Chicks by Factor 3 and

Selenium," Pro. Soc. Exptl. Biol. Med., 95, 621 (1957), 101. Schwarz, K., and Foltz, C.M., "Selenium as an Integral Part

of Factor 3 against Dietary Necrotic Liver Degeneration," J.

Am. Chem. Soc., £?, 3 2 9 3 (1957).

102. Shannon, W.V., "An Occurrence of Naumannite in Idaho," Am. J.

Sci. , ^0, 3 9O (1920).

103 . Shinn, M.B., "Colorimetric Method for Determination of Nitrite,"

Ind. Eng. Chem., Anal. Ed., 12, 325 (19^0)*

10^. Simon, P.F., "Noticias Historiales de las Conquistas de Tierre

Firme en las Indias Occidentales," Biblioteca Autores Colombianos,

4, 226 Kelly Publ. Co., Bogota (1953).

105. Smith, M.I., and Westfall, B.B., "Further Filed Studies on the

Selenium Problem in Relation to Public Health," Public Health

Repts. (ihSj, ^2, 1375 (1957).

106. Sterner, J.H., and Lidfeldt, V., "The Selenium Content of

'Normal' Urine," J. Pharmacol. Exptl. Therap. , 73 . 205 (19^+1) •

107. Strenge, K., "Determination of Small Quantities of Selenium in

Air and in Biological Materials (Photometrically with Diamino­

benzidine)," Arch. Gewerbepathol. Gewerbehyg., 16, 5 8 8 (1958)*

108. Suzuki, M. , "Colorimetric Method for the Determination of

Selenium in Crude Copper," J^. Chem. Soc. Japan. Ind. Chem.

Sect., 5 6, 3 2 3 (1955). 88

109. Suzuki, Y., Nishiyama, K., Matsuka, Y., Kuwai, S., Oe, M.,

Hujiwaka, H., Wakatsuki, T., Nakanishi, T., and Doi, M.,

"Spectrophotometric Determination of Selenium (in Biological

and Diverse Materials with Diaminobenzidine)," Shikoku Igaku

Zassh, 11, 77 (1957).

110. Suzuki, Y., Nishiyama, K., Takano, Y., Tajiri, T., and

Sakurayama, H., "(Photometric Determination with Diaminoben­

zidine of) Selenium Content of Various Foods, Fertilizers,

and Human Hair," Tokushima J. Exptl. Med., 6 , 2l+3 (1959).

111. Symanski, H., "Ein Fall von Selenwasserstoffvergiftung,"

Deut. Med. Wochschr., JIJJO (1950).

112. Tanaka, M . , and Kawashima, T., "Some 1+-Substituted £-Pheny-

lenediamines as Reagents for Selenium," Talanta, 12, 211 (1 9 6 5).

113 . Trelease, S.F., and Trelease, H.M., "Selenium as a Stimulating

and Possibly Essential Element for Indicator Plants," Science,

81, 70 (1958).

111+. Veale, C.R., "Determination of Traces of Selenium in Tellurium

(Photometrically with Diaminobenzidine)," Analyst, 8 5, 130

(I960).

115- • " M | f Traces of Quadriva-

lent Selenium photometrically with Diamino-

benzidine)," Analyst, 85, 135 (i9 6 0 ). 89

116. Volkov, S.T., "Determination of Selenium in Sulfur and Sulfur

Ores," Zavodskaya Lab., 5 s 1^29 (1936).

117. Wang, Y.C., and T'ung, F.C., "Colorimetric Determination of

Selenium in Minute Quantities in Sulfur (with Diaminobenzi-

dine)." Hua HsuBh Shih Chieh, 3 3U, (1959).

118. Waring, C. L., Worthing, H.W., and Hazel, K.V., "Spectrochemi-

cal Method for the Determination of Selenium," Anal. Chem.,

30, 1504 (1958).

119. Watkins, G.R., and Clark, C.W., "Selenium Dioxide: Preparation,

Properties, and Use as Oxidizing Agent," Chem. Revs., 3 6, 235

(19^5).

120. Watkinson, J.H., "Fluorometric Determination of Selenium in

Biological Material with 2,3**Diaminonaphthalene," Anal. Chem. ,

38, 92 (1966).

121. . "Fluorometric Determination of Traces of Selenium,"

Anal. Chem., 32, 981 (i9 6 0 ).

122. Weeks, A.D., "Mineralogy of Uranium Deposits in Geologic In-

vestigations of Radioactive Deposits," U.S. Geol. Surv. TEI-

620, 123 (1956).

12 3. West, P.W., and Cimerman, C., "Microdetermination of Selenium

with 3 ,3 '-Diaminobenzidine by the Ring Oven Technique and Its

Application to Air Pollution Studies," Anal. Chem., 3 6, 2013

(l 9610. 12k. West, P.W. , and Raraakrishna, T.V. , "A Catalytic Method for

Determining Traces of Selenium," Anal. Chem., to, 9 6 6 (I9 6 8).

125. Westfall, B.B., Stohlman, E.F. , and Smith, M.I., "The Placental

Transmission of Selenium," ^J. Pharmacol. Expt 1. Therap. , 6k,

55 (1956).

126. Wyoming State Board of Sheep Commissioners, 10th. Annual

Report, Cheyenne, Wyoming (I9 0 8 ).

127. Yajima, S., Kamemoto, Y. , Shiba, K., and Onoda, Y., "The

Determination of Impurities in Tellurium and Selenium by

Activation Analysis," J. Chem. Soc. Japan, Pure Chem. Sect.,

82, 3 4 5 (1961).

128. Yang, C.S., Dialameh, G.H., and Olson, E.E., "Photometric

Determination of Selenium Content of Dietary Cystine with

Diaminobenzidine," paper, kjrd. Annual Meeting, Federation

Proc., 18, 555 (1 9 5 9).

129. Yoshino, Y., "Analytical Studies on the Minute Amount of Se­

lenium with Ion-Exchange," _J. Chem. Soc . Japan, Pure Chem. Sect. ,

II, 577 (1950).

13 0 . Zaikoyskii, F.V., "Photometric Determination of Selenium in

Ores with Diaminobenzidine," Byul1. Eauch.-Tekh. Inform.

Minister. Geol. I Okhrany Nedr. S .S.S.R. N o . 6, 72 (1959)• 91

I3I. Zemel, V.K. , "Determination of Selenium and Tellurium in

Sulfidic Ores," Zavodskaya Lab., 1^33 (1936). VITA

Robert Lewis Osburn was born in Franklin, Tennessee, on

March 30, 1936. He attended the public schools of Franklin,

Tennessee, and graduated from high school in 1953- He served in the United States Air Force from 195^ to 1958, as a radio and radar technician.

In September of 1958 he entered Tennessee Polytechnic

Institute at Cookeville, Tennessee. He entered George Peobody

College in September, 1959> an

1962. He married Linda Ruth Mason in December, I9 6 2. In De­ cember, I9 6 3> a daughter, Marla Jean, was born.

From June, I9 6 2, until August, I9 6 6 , he was employed as a research assistant at Vanderbilt University, the Utah-Idaho

Sugar Company, Salt Lake City, Utah, and the University of Cali­ fornia at Davis.

In September, 1 9 6 6, he entered the Graduate School of

Louisiana State University, Baton Rouge, Louisiana. He is pres­ ently a candidate for the degree of Doctor of Philosophy.

92 EXAMINATION AND THESIS REPORT

Candidate: Robert Lewis Osburn

Major Field: Chemis try

Title of Thesis: THE DETERMINATION OF SELENIUM

Approved:

Lfi VJ. Major Professor and Chairman

R f ) Dean of the Graduate School

EXAMINING COMMITTEE:

Date of Examination:

May 27, I969