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1969

The Determination of Selenium.Robert Lewis OsburnLouisiana State University and Agricultural & Mechanical College

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This dissertation has been microfilmed exactly as received 70-9081

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

byRobert 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

PAGECHAPTER

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 theOxidation-Diazotization Reaction ........... 35

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

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

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

V. Time Study for the Coupling Reaction at60 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; HydroxylamineHydrochloride.......................... . 59

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

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

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

viii

LIST OF FIGURES

FIGURE PAGE

1. Absorption Spectra of VariousConcentrations 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 Duringthe Oxidation-Diazotization Reaction ....... 39

4. Variation of Absorbance with Temperature During the Oxidation-DiazotizationReaction................................. 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 at60 Degrees Centigrade...................... 45

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

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

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

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

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

12. The Standard Curve for the AdoptedMethod................................... 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 .0 1 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 (8 3). 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<i 0.37 PPm

respectively.

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 .0 1 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 6 8). This nuclide has two convenient gamma-

rays for counting purposes at 0.27 an<i 0.14 MeV, and its half-life

of 120 days is more than adequate to allow complete chemical se­

paration from other activities. The main disadvantage of using the

selenium- 75 radionuclide for analytical purposes is the long acti­

vation time needed. Usually samples are activated for 7 to 14 days

which gives only 5 to 10 percent of the specific activity that is

obtainable when an activation of one half-life is used. This

results in an equivalent loss of sensitivity, which is undesirable

for trace analysis.

16

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, 6 1), air (107), ores (35, 66, 130,95)* water

(77* 9 6), lead (73)* sulfur (117)* dietary cystine (128), human

teeth and hair (53* HO), meteorites (3 3)* 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.

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

e. Miscellaneous

S.awicki (98) has reported two new sensitive rea-

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 (2 0).

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 3j3 l_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) h2no2S'+sN +

h2no2s 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* 7percent (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

I. Reagent Blank II. 10 Micrograms Se(lV)

5 5 --III. 25 Micrograms Se(lV) IV. 125 Micrograms Se(lV)

IV

70--

III

II

100

Wavelength in Millimicrons

Figure 1

Absorption Spectra of Various Concentrations of the Coupled Product

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- Correctedtion of of HC1 bance Absor-of Selenium during at bance

Selenium (l-tg) reaction 5Mfm|J.(mg/1)

0 .0 0 .0 0 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 .9 0 0.075 0.060

5-0 25.00 5.15 0.090 0.0755-0 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

0.10 -j,

0 .0 9

0.08 - -

0 .0 7 . .

0.06 - -

0 .05

0.0it- --

O.O3 --

0.02 • -

0.01 - -

2 3 h 5Concentration of Hydrochloric Acid (Normality)

Figure 2

Variation of Absorbance with Concentration of Hydrochloric Acid During the Oxidation-Diazotization Reaction V_Mo\

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- Correctedtion 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 .0 1 8

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

0.08 - -

0.06 - -

0.0*+ --

0.02 - -

+ +15 30 1+5 60 75 90

Time (minutes) Figure 3

105 120 135 150 165 180

Variation of Absorbance with Time During the Oxidation-Diazotization Reaction

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<l 120 minutes. Therefore, a reaction

time of 90 minutes was used for all future studies.

TABLE III

TEMPERATURE STUDY FOR THE OXIDATION-DIAZOTIZATION REACTION

Concentration of Selenium (mg/1)

Amount of Selenium

(Hg) 5°c 25 °C

Blank Corrected Absorbance at 51*4 mi

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

0.3

0.2

0.1

1 PPM

Temperature (Degrees Centigrade)

Figure 4

Variation of Absorbance with Temperature During the Oxidation-Diazotization Reaction

Corrected

Absorbance

0.60

O.5O ..

0 .1*0 ■■

0,30 -■

0.20 - ■

0.10

5 cX—*25 C

"c0—-050 c P— 0 60 c P—>70 c

b 3 6 i 'X

Milligrams Se(IV) per liter

Figure 5

The Effect of Temperature During the Oxidation Diazotization Reaction on Standard Curves for the System

■p"Vji

44

TABLE IV

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

Concentra- Amount Reaction Absor- Correctedtion of Time bance Absorbanceof 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

0.350 T

O.JOO -•

0.250 -■

0.200 - -

0.150 -» 1 1 1 1—45 60 75 90 105

Time (minutes)15 30 120 135 150 I65 180

Figure 6

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

■p-\J1

k6

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.

47

TABLE V

TIME STUDY FOR THE COUPLING REACTION AT 60 DEGREES CENTIGRADE

Concentra­tionof

Selenium(mg/1)

Amount of Selenium

(M-g)

Reactionlime

(minutes)

Absor­bance

at-544my,

CorrectedAbsorbance

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 .1*0

O.35 -•

0.30 --

0.25 -■

0.20tT "fe"15 50

Time (minutes)

Figure 7

Variation of Absorbance with Time During the Coupling Reaction at 60 Degrees Centigrade -p-00

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, 3 0 , 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- Correctedtion 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.5290.0 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 .6- ■

Volume in Milliliters

Figure 8Variation of Absorbance with Volume

During the Coupling Reaction

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 .0 5, 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)

Concentra­tionof

Selenium(mg/1)

Amountof

Selenium(/ig))

PercentageSulfanil­amide(w/v)

Absor­banceat

541w)m

CorrectedAbsor­bance

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 .2- ■

0.06bl 2 7

Percentage Sulfanilamide (w/v)

Figure 9Variation of Absorbance with Concentration of Sulfanilamide VJl4="

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)

Concentra­tionof

Selenium (mg/ml)

Amountof

Selenium( g)

PercentageCouplingReagent(w/v)

Absor­banceat

54imyi

CorrectedAbsor­bance

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

o .to - -

+0.20'o . i 0 .2 0 .3 0 .4 0 .5 0 .6

Percentage (w/v)

Figure 10Variation of Absorbance with Concentration

of N-(l-naphthyl)fethylenediamine Dihydrochloride

583. 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

Concentra­tionof

Selenium(mg/ml)

Amountof

.... Selenium .( s)

Percentage NHo0H * HC1

... (w/v)

Absor­banceat

CorrectedAbsor­bance

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 . 4to 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

0.6

0.210 20 25 300 5

Percentage (w/v)

Figure 11Variation of Absorbance with Concentration

of Hydroxylamine Hydrochloride

61centigrade 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, and5 5 52-TeO, were reduced to a hundred-fold excess, the concentrations

53+ - 3-of La , Br , and V0 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

Concentra­tionof

Selenium(mg/1)

Amountof

Selenium 0*8 )

Absor­banceat

CorrectedAbsorbance

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 . 100- -

0 .0 2 5 --

0.000c .1 0 0 .1 5

Milligrams of Selenium(iv) per Liter00 0.200 .0 5

Figure 12The Standard Curve for the Adopted Method

TABLE XI

INTERFERENCE STUDIES OF DIVERSE IONS

Se (IV) Diverse ion Amount Added CorrectedAdded (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 Mg^ 1000 MgCl2 0.108

1.0 Ni2* 1000 nici2'6h2o 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 S032' 1000 Na2S03 0.106

1.0 Cr?+ 1000 CrCl^ 0.106

1.0 F- 1000 NaF 0.102

1.0 2-Si0_,3

1000 Na2Si03-9H20 0.110

1.0 2-uok 1000 Na2S0j-2H20 0.107

1.0 Cd^ 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 Mn^ 1000 MnCl2 0.103

1.0 Sb5+ 1000 SbCl^ 0.101

1.0 Zr^+ 1000 ZrCl,4 0.112

1.0 Be^ 1000 BeCl2*4H20 0.106

1.0 Malonate 1000 Malonic Acid 0.102

TABLE XI (continued)

1.0 Tartrate 1000

1.0 Oxalate 1000

1.0 GeO^2" 100

1.0 1000

1.0 H2P02_ 1001.0 10001.0 bo2" 100

1.0 1000

1.0 hpo42" 1001.0 10001.0 La*’+ 1000

1.0 1001.0 Br“ 100

1.0 50

Tartaric Acid

Oxalic Acid

NaoGe0^^ 3

NaH2P02

NaB02

NaJHPO,2 4

LaCl,3

NaBr

0.105

0.103

0.1000.101

0.103

0.1000.104

o . i o 4

0.1010.103

0.184

0.1080.1180.105

CT\

TABLE XI (continued)

1 . 0 vo^5" 100

1.0 501.0 MoO^2" 100

1.0 251.0 101.0 Fe^+ 100

1.0 50

1.0 25

1.0 10

1.0 i" 1000

1.0 10°1.0 101.0 son" 1000

1.0 wo

1.0 10

NaSCN

TABLE XI (continued)

1.0 Cu2* 1000

1.0 101.0 Bi3+ 100

1.0 101.0 uo^2- 1000

1.0 10

1.0 CN~ 50

1 . 0 25

1.0 Citrate 1000

1.0 BrO “ 100051.0 100

1.0 CIO " 1000

1.0 1001.0 25

CuCl2 1.800

0 . 11*8

BiCl, 2.00051.900

Na.UO, 2.0002 40.160

NaCN 0.121

0.102

Citric Acid 0.100

NaBrO.. 0.14150.108

NaCIO, 1.70030.2250.101

O N00

TABLE XI (continued)

1.0 10^“ 100

1.0 101.0 TeO^2- 10001.0 1001.0 Hg21" 100

1.0 10001.0 Al5+ 100

1.0 10001.0 HAsO^2" 100

1.0 1000

1.0 10001.0 101.0 Se°^2“ 1001.0 10

NalO, 0.15130.126

Na TeO, 0.1262 30.106

HgCl2 0.107

0.104

AlCl 0.1003 0.102

Na2HAs0^'7H20 0.1010.106

Na B, 0 • 10H 0 2.0002 4 7 20.348

Na2Se0^-10H20 1-9000.343

o\

TABLE XI (continued)

1.0 NO, 10031.0 501.0 251.0 101.0 VO," 100031.0 1001.0 10

1.0 Cr(\ 2” 100

1.0 50

1 . 0 2 5

1.0 Cro0 100 i1.0 50

1 . 0 25

Mn(N03)2

NaVO,3

Na2Cr0^‘4H20

2.000

1 .5 0 0

0.458

0.142

0.1T20.159

0.146

0.159

0.1390 . 1 0 8

0 . 1 3 7

0 . 1 1 5

0.110

712-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 a2_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 Relativetions 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.510 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

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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<i received his B.S. degree in June,

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