A SPECTROFLUOROMETRIC STUDY OF
MORIN AND 5,1-DlCHLORO-8-QUINOLINOL
COMPLEXES OF LANTHANUM (Ill)

Thesis {or flu Degree 0‘ pk. D.
MICHIGAN STATE UNIVERSITY

Lawrence LaRoy Fleck

1961

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ABSTRACT

A SPECTROFLUOROMETRIC STUDY OF MORIN AND
5, 7-DICHLORO-8—QUINOLINOL COMPLEXES
OF LANTHANUM (III)

by Lawrenc e ‘LaRoy Fleck

Lanthanum (111) forms fluorescent complexes with morin or 5, 7-
dichloro-8-quinolinol. The various factors which affect the fluorescence
of these complexes in 50 percent dioxane-50 percent water and the nature
of these complexes were investigated.

At pH 5. 5 lanthanum (III) forms a 1:2 complex with morin (lanthanum
(III): reagent). The value of the equilibrium "constant" for this complex
is approximately 0. 6.

Solutions of lanthanum (III)-morin at the Optimum pH of 5. 5 are
excited to nearly equal fluorescence intensities by the 365, 405 and 436 mu
mercury radiations yielding a fluorescence band whose peak is between
505 and 510 mu. Under these conditions morin is relatively non-
fluorescing. Fluorescence intensity of the complex increased with increas-
ing dioxane content. The fluorescence intensity varies linearly when the
concentration of lanthanum (III) is varied between 0-80 7 with the morin
concentration fixed at 400 7 per 25 ml. Morin concentrations greater
than 400 7 decreased the fluorescence intensity of the complex by concen-
tration quenching. The addition of up to 130 'y of samarium (III), which
forms a non—fluorescing complex with morin, to 25 ml. solutions con-
taining 40 y lanthanum (III) and 400 'y morin does not significantly change
the fluorescence intensities originating from these solutions when excited
by 365 mu radiation. The rate of change in the fluorescence intensity of
the complex corresponds to l. 8 fluorescence intensity units decrease per

. . . 0
degree centigrade increase over the temperature 1nterval 9—50 .

Abstract Lawrence LaRoy Fleck

The absorption band peaks of the complex and morin are at 410 and
356 mu, respectively, and the absorptivities of the complex and morin
at 410 mu are 4. 00 x 104 and Z. 28 x 103 liters per mole-cm., respectively.
The pKa of morin is 6. 7.

The species, which is responsible for the fluorescence yield of 50
percent dioxane-50 percent water solutions containing lanthanum (III)
and 5, 7-dichloro-8-quinolinol at pH 9, is probably the 1:3 complex
(lanthanum (III):reagent). The complex is excited by the 365 mu mercury
radiation to yield a fluorescence band peak between 526 and 530 mp,
while the absorption maximum is at 388 mu. For solutions containing
1000 7 of reagent, the lanthanum (III) concentration range in which the
fluorescence intensity changes linearly with concentration is approxi-
mately 0-70 7 of lanthanum (111) per 25 ml. Maximum fluorescence
intensity readings for solutions containing 80 7 of lanthanum (III) are
obtained when the reagent concentration is varied between 800-1500 7
per 25 ml. Above 1500 7 of reagent, the fluorescence intensity decreases
by concentration quenching. When 0-35 7 of samarium (III) are added to
solutions containing 40 7 lanthanum (III) and 1500 7 of reagent in 25 ml. ,
no appreciable change in intensity is noted. Maximum fluorescence
readings are obtained in solutions containing between 50 and 55 percent
dioxane. Addition of sodium carbonate to solutions of the complex reduces
the fluorescence intensity, therefore, carbonate free base must be used
in the development of the complex at pH 9. An increase in temperature
decreases the fluorescence intensity 3. 0 fluorescence intensity units per
degree centigrade over the temperature range 14-450.

The logarithms of the consecutive formal stability constants, K1
and K2, for the lanthanum (111)-5, 7-dichloro-8-quinolinol complexes are
7. 3 and 6. 5, respectively. The value of K3 can not be determined by a

titrimetric method since lanthanum (III) tends to hydrolyze at higher pH

Abstract Lawrence LaRoy Fleck

values. The pK and pK of 5, 7-dichloro-8-quinolinol at 25° are

NH OH
estimated to be 1. 6 and 9. 11, respectively.

Both morin and 5, 7-dichloro-8—quinolinol have potential analytical
applications as fluorometric reagents for the determination of microgram

quantities of lanthanum (III) in pure solutions or in solutions containing

trace amounts of samarium (III).

A SPECTROFLUOROMETRIC STUDY OF MORIN AND
5, 7-DICHLORO-8-QUINOLINOL COMPLEXES
OF LANTHANUM (III)

BY

Lawrence LaRoy Fleck

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1961

ACKNOWLEDGMENTS

The author wishes to express his appreciation to
Dr. Andrew Timnick for counsel and guidance given through-
out the investigation and preparation of this thesis.

Acknowledgment is also extended to the Socony Mobil
Oil Company, and the Dow Chemical Company, for financial
aid.

A special acknowledgment is extended to the author's
wife, Helen, and family for encouragement and assistance
rendered throughout the course of his graduate studies.

3:: >:< >’,< 3:: * 3:: >1: >§< >:< >:< >:< >:< :1: >5:

ii

VITA

Name: Lawrence LaRoy Fleck

Born: February 24, 1931 in Kalamazoo, Michigan

Academic Career: St. Augustine High School, Kalamazoo,

Michigan (1945- 1949).

Western Michigan College, Kalamazoo,
Michigan (1951-1955)

Michigan State University, East Lansing,
Michigan (1955-1961)

Degrees Held: B. S. Western Michigan College (1955)

M. S. Michigan State University (1958)

iii

TABLE OF CONTENTS

Page
INTRODUCTION .......................... 1
HISTORICAL ............................ 5
EXPERIMENTAL ......................... 10
INSTRUMENTATION .................... 11
REAGENTS ......................... 15
Preparation of Reagent Solutions ........... 16
EXPERIMENTAL PROCEDURES . . . . .......... 17
Introduction ................... . . . 17
The Effect of Temperature ............ . 18
Lanthanum (III)-Morin System ........... . 19
Solution Preparation Procedure ....... . l9
Lanthanum (III)- 5, 7—Dichloro- 8- quinolinol System . 20
Solution Preparation Procedure ...... . 20
Evaluation of the Complex Stability Constants. 21
Titration Procedure .......... . 21
DISCUSSION OF RESULTS ..................... 23
LANTHANUM (UH-MORIN SYSTEM ........... . 24
Effect of Experimental Variables on the Absorbance
and Fluorescence of the Complex ...... . 24
Nature of the Reaction . . . . . . 37
Composition of the Complex and Estimation of the
Equilibrium Ratio, K'eq ........... . 39
LANTHANUM (III)- 5, 7-DICHLORO- 8-QUINOLINOL
SYSTEM ........................ 44
Effect of Experimental Variables on the Fluorescence
of the Complexes ................ 47
Evaluation of the Lanthanum (III)-5, 7-Dichloro-8-
Quinolinol Stability Constants ......... 56
SUMMARY AND CONCLUSIONS .................. 65
INTRODUCTION ....................... 66
LANTHANUM (III)-MORIN SYSTEM . s . ......... .. 66
LANTHANUM (111)-5, 7-DICHLORO-8-QUINOLINOL
SYSTEM ........................ 69
LITERATURE CITED ...................... . 72

iv

TABLE

II.

III .

IV.

LIST OF TABLES

Estimated Equilibrium Ratio, K'eq, Value of the
Lanthanum (III)-Morin Complex in 50 Percent
Dioxane—Water at 25 .................

Effect of Sodium Carbonate on the Fluorescence
Intensity of Lanthanum (III)—5, 7-Dichloro-8-
quinolinol in 50 Percent Dioxane-Water at pH 9. 0 . .

Fluorescence Stability Data on 50 Percent Dioxane-
Water Solutions of Lanthanum (III)-r5, 7-Dichloro-8—
quinolinol at pH 9. 0 ..................

pR- and '5 Values for Titration of Lanthanum (III)
Perchlorate-5, 7-Dichloro-8-quinolinol in 50 Per-
cent Eioxane-Water with 0. 0862 N Sodium Hydroxide
at 25 .........................

Page

45

55

57

63

LIST OF FIGURES

FIGURE Page

1. Block diagram and radiation paths of spectro-
fluorometer .................... . . 12

2. Absorption and fluorescence spectra of lanthanum
(III)-morin and the absorption spectrum of morin in
50-50 DW at pH 5. 5 ................. . 25

3. Effect of dioxane content on the fluorescence and
absorbance of the lanthanum (III)-morin complex at
pH 5. 5. . . . ..................... 26

4. Effect of pH on the absorbance of lanthanum (III)-
morin and morin in 50-50 DW at 410 mu ....... 28

5. Effect of pH the fluorescence lanthanum (III)-morin
and morin in 50-50 DW ............... . 29

6. Effect of lanthanum (III) on the fluorescence and
absorbance of 400 7 morin per 25 m1.in 50-50 DW at
pH 5. 5 ......................... 31

7. Effect of lanthanum (III) on the fluorescence of 1000 7
morin per 25 ml. in 50-50 DW at pH 5. 5 ....... 32

8.‘ Effect of morin on the fluorescence and absorbance
of 80 7 lanthanum (III) per 25 ml. in 50-50 DW at
pH 5. 5 ......................... 33

9. Effect of samarium (III) on the fluorescence of
lanthanum (III)—morin in 50-50 DW at pH 5. 5 . . . . 35

10. Effect of samarium (III) on the fluorescence of
lanthanum (III)-morin in 50-50 DW at pH 5. 6. . . . . 36

11. Spectrophotometric curves showing isoabsorptive
point .......................... 38

12. Determination of composition of the lanthanum (III)-
morin complex in 50-50 DW at pH 5. 5 by method of

continuous variations ................ .. 40

vi

LIST OF FIGURES - Continued

FIGURE Page
13. Determination of composition of the lanthanum (III)—
morin complex in 50-50 DW at pH 5. 5 by slope
ratio method ....................... 42

14.

15.

16.

17.

18.

19.

20.

21

22.

Absorption spectra of 5, 7-dichloro-8-quinolinol (QClz)
and lanthanum (III)-QC12 in 25 m1. of 50-50 DW . . . .

Fluorescence spectra of lanthanum (111)-5, 7-dichloro-
8-quinolinol in 50-50 DW ................

Effect of dioxane content on the fluorescence of
lanthanum (III)-5, 7-dichloro-8-quinolinol at pH 9. 0
in 50-50 DW .......................

Effect of pH on the fluorescence of 5, 7-dichloro-8-
quinolinol and lanthanum (III)-5, 7-dichloro—8-
quinolinol in 25 ml. of 50-50 DW ............

Effect of lanthanum (III) on the fluorescence of 1000 7
5, 7-dichloro-8-quinolinol in 50-50 DW at pH 8.8 . . .

Effect of 5, 7-dichloro-8-quinolinol on the fluorescence
of 80 7 lanthanum (III) per 25 ml. in 50-50 DW at pH
8. 8 ............................

pH titrations for lanthanum (III), lanthanum (III)-5, 714
dichloro-8-quinolinol and 5, 7-dichloro-8-quinolinol
in dilute perchloric acid solutions of 50-50 DW. . .

.‘ Effect of pH on the absorbance at 369 mu of 1000 7

5, 7-dichloro-8-qui‘nolinol in 25 m1. of 50-50 DW.

Plot for the evaluation of log K1 and log K2 for the
lanthanum (111)-5, 7-dichloro-8-quinolinol complexes
in 50-50 DW .......................

46

48

49

51

52

53

59

60

62

INTRODUCTION

e

«\u
u

EX

EX

C11.

When many organic and some inorganic substances are excited by a
relatively high- energy source, usually an ultraviolet light source,
fluorescence, a particular type of photoluminescence, is observed. In
this phenomenon there is only a relatively short, finite time delay between
the absorption and emission of radiant energy.

Fluorescence is produced in molecules by the absorption of radiant
energy of a frequency within the normal absorption band of the molecule,
usually in the ultraviolet region of the Spectrum. The absorbed radiation
excites the molecule to one of many vibrational energy levels of an upper
excited electronic singlet state. (A singlet state in an unsaturated mole-
cule is considered to be characterized by a pair of unsaturation (1T)
electrons having paired, antiparallel, spins (23).) Within a period of less
than 10’12 second (26), the energy above the lowest vibrational level of the
first excited electronic singlet state may be dissipated by a non- radiative
transition. The molecule, within a period of about 10'9 second, then
returns to an excited vibrational energy level of the ground electronic
singlet state. This latter transition, which is a singlet -—-)I=singlet transi-
tion, gives rise to a fluorescence band (23, 26). Since the fluorescence
transition usually involves less energy than the absorption transition,
the fluorescence band appears at longer wavelengths than the absorption
band.

The intensity or power of the fluorescence radiation I emitted by

f,
a solution of the fluorescing solute in a non-fluorescing solvent, is related

be), where I0 is

to the concentration by the relationship If = 010 (l-lO'a
the intensity of the incident radiation, Q is the quantum efficiency of the
absorbed radiation in producing fluorescence, _a_,,is the absorptivity, and

b is the cell width. For low concentrations it has been shown, that by
means of an exponential series transformation, the above equation simpli-

fies to If = Qloabc (10). Consequently, at low concentrations the fluor-

escence intensity If is proportional to c., the concentration.

A relatively limited number of inorganic substances fluoresce in
both the crystalline state and in solution with their characteristic line
or band spectra. Among these substances are the tripositive lanthanide
ions of samarium, europium, terbium, and dysprosium (5, 40), and
gadolinium (40). However, the trivalent salts of praseodymium, neodymium,
holmium, erbium, and thulium are reported to be luminescent only in
liquid or solid solution and as activators in crystal phOSphors (40).
Furthermore the tripositive ions of lanthanum, cerium, ytterbium, and
lutetium do not yield line fluorescence spectra as pure salts or in crystal
phosphors (40).

Although a number of the tripositive lanthanide ions fluoresce,
extensive quantitative analytical applications of this phenomenon have not
been exploited. The main reason for the apparent lack of interest in
determination of the lanthanides by measurement of their characteristic
line fluorescence is that exciting radiation wavelengths shorter than 250
mu are necessary to obtain adequate energies for excitation. The
lanthanide ion absorptions are weak and narrow for wavelengths longer
than 250 mg and as a result excitation of moderate intensities require
great intensities of exciting light. For wavelengths shorter than 250
mg, the lanthanide ions are relatively strongly absorbing and have
continuous absorption bands (9, 18). Unfortunately, the uncontrolled nature
of the exciting sources presently available, such as arc or spark dis-
charges for wavelengths shorter than 250 mu, exclude accurate measure-
ments of the fluorescence intensities (ll).

' A few organo-lanthanide complexes can be excited in the near ultra-
violet and visible region of the spectrum to emit intense fluorescence
which is characteristic of the complex. Lanthanum (III), gadolinium (III),
and lutetium (III) are the only tripositive lanthanide ions which contain
sufficiently stable cation electronic configurations to yield an observable
fluorescence which is characteristic of their complexes. The remaining

tripositive lanthanide ions, having other than zero, seven or fourteen 4f

electrons, are considered to diminish the fluorescence yields of the com-
plex due to the interaction between the electronic field of the metal ion
and the optical electrons of the organic entity responsible for the fluor-
escence in the complex. This strong "internal quenching"* effect of the
tripositive lanthanide ions other than lanthanum (III), gadolinium (III),
and lutetium (III) is apparently caused by the deactivation of the fluor-
escence of the complex by an "intramolecular energy transfer" (27, 47, 58)
process which is brought about by spin-orbital interactions (8). In quali-
tative detection tests, lanthanum (III), gadolinium (III), and lutetium (III)
were found to be the only tripositive lanthanide ions which formed
fluorescent complexes with morin (2’, 4’, 3, 5, 7-pentahydroxyflavone (37)
and 8-quinolinol (37, 43).

To the present time, most of the work done on the fluorescence of
lanthanide complexes has been of a qualitative nature in which the various
factors which affect fluorescence have been disregarded. It was the pur-
pose of this study to initiate a systematic study in which, (1) organic
reagents are tested as to whether or not they form fluorescent complexes
with lanthanide ions, (2) the characteristics of the complexes are estab-
lished, and (3) the various factors, such as the affect of solvent, pH of
solution, reagent concentration, temperature, and excitation wavelength,
which affect the fluorescence are tested. Lanthanum (III) was selected as
the representative member of lanthanide ions which form fluorescent

~COInplexes with some organic compounds. The organic compounds

selected were morin, 8-quinolinol, and 5, 7-dichloro-8-quinolinol.

*Quenching is the phenomenonby which substances showing fluor-
escence have their emission reduced or extinguished by substances added
F0 tIhe solutions, changes in temperature, solvent, or physical state, or
Increase in concentration of absorbing species. When it is due to none of
these, however, but represents a real reduction of "quantum yield" due
to Conversion of light energy of the excited Species, it may be called

"tru e quenching " (6).

HISTORICAL

Several books concerning the theoretical and practical aspects
of fluorescence have been written. Pringsheim in a book entitled
"Fluorescence and Phosphorescence"’(40) discussed the theoretical
aspects of fluorescence and phosphorescence and reviewed existing
information on many substances which are known to fluoresce. ‘ In this
book, one complete chapter is devoted to the fluorescence of the
lanthanide ions in crystals, solutions, and in phosphors. ' In a book
"'intended for the use of students and practical workers, 1' entitled
"Fluorescence of Solutions" by Bowen and Wokes (6), the-most signifi-
cant facts pertaining to fluorescence theory and the practical aspects of
fluorescence measurement have been considered. In "The Chemical
Aspects of Light, " Bowen (5) has presented, insofar as possible, a non-
mathematical treatment of the modern concepts of the interaction of
light with matter. The author (5) also discussed the transformation of
absorbed radiation and its relation to fluorescence. Although there are
several other books or chapters within books which are devoted to
fluorescence, the above noted works contain a reasonably complete
treatment of the subject.

In a series of review articles by White (48, 49, 50, 51, 52, 53, 54, 55),
covering the period of 1939 to 1960, organic and inorganic applications of
fluorometric analysis and the fundamental developments in instrumen-
tation are discussed. Approximately 1000 references are cited in these
review articles.

Morin has been used extensively as a reagent for the fluorometric
analysis or fluorometric detection of a relatively large nmnber of metals
(2, 8,19, 42, 46).

Sill and Willis (41) have reported a fluorometric method for the
determination of beryllium (II) by complexation withmorin in a basic
solution which contained EDTA to complex traces of impurities contained

in the solvent. Bismuth, silver, mercury, gold and platinum metals

interfered in this method. Yttrium (III), scandiurn (III), lanthanum (III),
and lithium (I) were reported to yield a green fluorescence which was
similar to beryllium(II), while thorium (IV) and zirconium (IV) produced
a yellowish fluorescence when complexed with morin. The authors
reported that ions such as lanthanum (III) and thorium (IV) formed
fluorescent complexes with morin evenxinr the presence-of EDTA.
Cerium (IV), praseodyrnium (III), neodymium (III), samarium (III) and
uranium (VI) reduced the fluorescence intensities of the beryllium (II)
morin solutions.

Fletcher and Milkey (29) conducted a very thorough fluorometric
study of the thorium (IV)-morin complex formed in slightly acidic
ethanol-water solutions. The effect on the fluorescence of the complex
by such variables as concentration of acid, ethanol content, thorium (IV),
morin and complex concentrations, time, temperature and wavelengths
of exciting radiation were studied to determine experimental conditions
which yield maximum fluorescence. Equations which account for the
contribution to the total fluorescence by three components in this system
when the fluorescence is measured in a transmission-type fluorometer
were derived.

Analytical fluorescence tests employing morin as a reagent for
the detection of small amounts of aluminum (III), beryliurn (H), gallium
(III), germanium (IV), tin (IV), antimony (V), thorium (IV) and zirconium
(IV) have been worked out by Patrovsky (35).

Geiger and Sandell (16) have developed a fluorometric method for
the determination of submicrograrn quantities of zirconium (IV) with
(morin in 2 M hydrochloric acid, ethanol-water solutions. . The various
experimental factors which affect the fluorescence of the complex were
tested. More than 30 elements including lanthanum‘ (III) were individually
tested to determine their effect on the fluorescence of the complex.
Solutions containing one or two mg. of lanthanum (III) in the presence of

300 7 of morin did not yield any measurable fluorescence.

The influence of various anions on the morin fluorescence test
for aluminum (III), gallium (III), beryllium (II) and zinc (II) were evaluated
by Bishop (3). Citrate, fluoride, oxalate, phosphate, tartrate or vanadate
ions diminish the fluorescence intensity of complexes formed between any
of the above listed cations and morin. The presence of any individual
anion which forms a complex or a precipitate with any of the above listed
cations can be detected by its quenching of the metal ion-morin complex
fluorescence.

Pollard, et al. (38) employed paper chromatograms for separation
and organic complexing agents for identification in a study to develop
tests for distinguishing between lanthanon groups and for identifying cer-
tain individual lanthanides. The morin and 8-quinolinol complexes of
lanthanum (III), gadolinium (III), and lutetimn (III) were the only ones
which could be detected visually by their fluorescences.

Lederer (24, 25) studied the separation of lanthanides by paper
chromatography. The location of the individual lanthanides on the chromato-
grams was accomplished by illuminating the chromatogram, which had
been treated with a solution of 8-quinolinol, with ultraviolet light and
noting where fluorescence appeared. Lanthanum (III), erbium (III), and
lutetium (III) complexes emitted a green fluorescence, while all other
lanthanide» complexes appeared only as brown or black spots.

White, _et 31. (56) have determined the Optimum pH of solutions,
absorption band peaks, maximum excitation wavelengths, and the fluor-
excence band peaks for a number of metal chelates including aluminum
(III)-morin, beryllium (ID-morin and lithium (1)-8-quinolinol.

Several investigators have studied the fluorescence of metal ion-
8-quinolinol complexes (12, 31, 32, 57). Ohnesorge and Rogers obtained
spectrofluorometric data on a number of group III A metal-8-quinolinol
complexes. The effects of acidity (33) and different solvents (34) on the

fluorescence of these complexes were studied.

Popovyelt and Rogers (39) obtained the fluorescence spectra,
intensities, and "efficiencies" of zinc (II) chelates of 8-quinolinol,
8-quinolinol-5-sulfonic acid, 2-methy1-8-quinolinol, and 5, 7-dichloro-8-
quinolinol as a function of solvent and substituents. The fluorescence
spectra of. 2-methy1-8—quinolinol chelates with gallium (III), indium (III),
magnesium (II) and cadmium (II) were also obtained. The solvents tested
were chloroform, ethanol, carbon tetrachloride, dimethylformamide,
ether, water, and tetrahydrofuran. The fluorescence intensities were
found to be highest in ”inert" solvents.

~ Nishikawa (30, 31, 32) in a series of papers on fluorometric analysis
discussed the fluorescence behavior of chloroform solutions or suspensions
of metal chelates of the metals in groups I, II, III, and IV in the periodic
table. The organic reagents which were employed in these studies
included 8-quinolinol, 5, 7-dichloro-8-quinolinol, 5, 7-dibromo-8-quinolinol,
and 5, 7-diiodo-8-quinolinol.

Nishikawa (31) also discussed the relationship between fluorescence
of metal ion-8-quinolinol complexes and the position of the metal in the
periodic table. The author stated that the following relationships exist
between the position of metallic elements in the periodic table and the
ability of their ions to form fluorescent complexes:

1). Group I, II, III and IV metal ions form fluorescent complexes.

2). Group IIa, 111a and 11b metal ions form the most intensely

fluorescing complexes.

3). Within a group, the lower the atomic number of the metal,

\ the stronger will its complex fluoresce.

4). Metal ions which have a high magnetic susceptibility do not

yield fluorescent complexes.

Fassel and Heidel (11) have develOped a method for the determin-
ation of 0. 005 to 100 per cent terbium in complex lanthanide mixtures
by measurement of fluorescence- intensity at 545 mg, the maximum
peak of the strongest fluorescence band emitted when aqueous solutions

containing terbium (III) are irradiated with ultraviolet light.

EXPERIMENTAL

10

11

INSTRUMENTATION

The spectrofluorometer used in this work is a modification of one
constructed in this laboratory by Thommes (44). The only change in the
instrument was the replacement of the Farrand grating monochromator
and associated light source with a high resolution Bausch and Lomb
Model 33-86-40 grating monochromator, and a Hanovia S-H high pressure
mercury arc and the required Hanovia 110/120 volt constant voltage
transformer. Figure 1 shows a block diagram of the modified instrument
and the excitation and fluorescent emission radiation light paths.

The Bausch and Lomb monochromator was connected by a light
tight seal to the cell compartment by means of a rubber O-ring gasket.
The monochromator and a quartz cylindrical converging lens which was
mounted in the cell compartment to replace the factory equipped collective
lens, were positioned so that the narrow, nearly parallel beam of light
from the source was focused on the center of the cell. This arrangement
was found to give satisfactory Operation.

The S-H mercury arc served as the source for excitation in all
the experimental work. This lamp emits seven mercury lines which are
sufficiently intense for fluorescence work. These are the 436, 405, 365,
313, 303, 265, and 254 mu lines.

Clear window silica cells, 10 x 20 x 50 mm. , purchased from
the Farrand Optical Company, were employed for all fluorescence
measurements made in this study. The cells could be reproducibly
positioned in the instrument cell compartment by means Of a specially
constructed cell holder. These cells are quite transparent to all the
principle mercury lines listed above.

The procedure employed in obtaining fluorescence intensity measure-

ments was as follows:

12

 

Beckman Monochromator

 
 
   
   

Fluorescent /
Radiation '

Bausch and Lomb

' Monochromator
H

Photomultiplier
ube Housing

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

./
Grating

Mercury Arc

Housing “7l—'
AC Powe Supply and Photomultiplier

Control Unit

 

 

 

Transgrmer

'Figure 1. . Block Diagram. and Radiation‘Paths Of
Spectrofluorometer

l3

1) The mercury arc lamp, AC Power Supply, and Beckman DU
instrument were allowed to warm up for at least thirty minutes prior to
use in order to insure maximum instrument stability.

2) The photomultiplier sensitivity control knob was set to the "full"
position and the Beckman DU sensitivity control knob set three turns
from its clockwise limit.

3) The Bausch and Lomb monochromator entrance and exit slits
. were set to 1. 0 and 0. 5 mm. , respectively, and the Beckrnan DU slit
adjusted to approximately 1. 0 mm.

4) The BeckInan DU instrument was zeroed with the "dark current"
control knob before each series of measurements. The instrument was
calibrated with a 0. 4 7 per ml. dichlorofluorescein standard solution by
the following procedure. The cell containing the standard solution was
placed in the cell holder, the phototube shutter was opened and with the
selector switch in the 0. 1 position, slight adjustments were made in the
Beckman DU slit width so that a reading of 50 on the o/OT scale was
obtained. For this calibration procedure the Beckrnan DU and Bausch
and Lomb monochromators were set at 530 and 365 mu, respectively.

5) After the instrumental Operational variables were established
through the calibration procedure, the fluorescence intensities of the
solutions investigated were obtained at the desired excitation and emission
wavelengths. No corrections for the variations of photomultiplier tube
sensitivity for various wavelengths were applied to fluorescence Spectral
data.

A Beckman DU or a DK-2 spectr0photometer was used for all
absorption measurements. The Bechnan DU, however, was employed
exclusively for the single wavelength measurements required for the
stability and complex composition studies. Matched one cm silica cells
were employed in all these measurements.

A Sargent constant temperature bath, equipped with a number 7530

thermo-regulator and associated Sargent number S- 2770 heater control

14

unit, was maintained at 250 i 0. 2 for the preparation and storage in
flasks of all solutions employed in this study. After thermal equilibrium
of the flasks and contents was attained, aliquots were withdrawn from
the flasks and the single wavelength fluorescence measurements were
made within one minute after sample withdrawal.

A Beckrnan Model G pH meter with a glass-saturated calomel
electrode pair was used for all measurements except those employed in
the chelate stability studies. The pH meter which was standardized at
pH 4 or 7 with Beckrnan standard buffer solutions, gave satisfactory
performance for all solutions containing less than 70 percent dioxane.
Erratic electrode behavior was noted for solutions of very high dioxane
content.

The apparatus employed in the alkalimetric titrations, which were
performed for the determination of the chelate stabilities of the lanthanum
(111)-5, 7-dichloro-8-quinolinol system, consisted of a Beckrnan Model G
'pH meter equipped with a macro saturated calomel and glass electrode
pair and a titration vessel which was maintained at 250 in the constant
temperature bath. The electrical lead of the glass electrode was shielded
with grounded aluminum foil to eliminate any stray electrical pickup.
The electrode pair was mounted in a polyethylene disk which was one-
quarter inch thick and four inches in diameter and was equipped with
ports for a nitrogen inlet tube, two semi-micro burets tips, and an
electric motor driven glass stirring paddle. The electrode assembly
was positioned on top of either a 250 or 400 ml. beaker and the cell unit
rigidly mounted in the 250 water bath. The Beckrnan pH meter was
calibrated as previously indicated at pH 4 and 7 and was found to give

satisfactory operation for this system.

15
REAGENTS

The purification of a number of chemicals employed in this investi-
gation was not deemed necessary due to their initial high degree of

purity. The chemicals, labeled purity and source are:

Dichlorofluorescein Eastman Kodak, white label

Lanthanum sesquioxide Optical Grade,» Heavy Minerals CO. ,
Chattanooga, Tennessee

Morin dihydrate Doctor' Theodor Schuchardt,
Miinchen, Germany

Perchloric acid 70-72 percent Bakers Analyzed Reagent

Potassium acid phthalate Primary Standard, Bakers Analyzed
Reagent, oven-dried for 2 hours at 105

Samarium sesquioxide Labeled purity 99. 9 per cent, Michigan
Chemical Corporation, Saint Louis,
Michigan

Sodium acetate trihydrate A. C. S. , Fisher Certified Reagent

Sodium carbonate A. C. S. , Fisher Certified Reagent

Matheson Coleman and Bell 8-quinolinol was purified by sublima-
tion under the reduced pressure of a water aSpirator in a large sub-
limation apparatus maintained in a water bath at approximately 70-720.
The melting point of the pure white needles of the sublimed 8-quinolinol
was found to be 74-74. 50 (u. c. ); American Chemical Society specifi-
cations, 72. 5-73. 50.

The 5, 7-dichloro-8-quinolinol was prepared by chlorination of
8-quinolinol dissolved in ethanol (17). The reagent was recrystallized
variously from glacial acetic acid, acetone, and absolute alcohol and
dried in an oven at 1050 for approximately two hours.

Commercial grade dioxane was purified by a modification Of the
procedure of Hess and Frahrn (21). A mixture of 54 ml. of concentrated
hydrochloric acid, 400 ml. of distilled water, and 4 l. of dioxane was

refluxed for 12 hours, during which time a slow stream of nitrogen was

l6

bubbled through the solution to sweep out aldehydes. The solution was
cooled and potassium hydroxide pellets added slowly with mixing until
the solution became saturated and a second layer formed. The dioxane
was decanted, treated with fresh potassium hydroxide pellets to remove
adhering aqueous liquor and transferred to a large screw capped bottle
containing anhydrous calcium chloride. The following day the dioxane
was filtered into a clean flask, refluxed for 12 hours over calcium
hydride, and distilled. A 100 to 100. 50 fraction was collected and the
dioxane stored in an amber colored screw capped bottle.

The distilled water employed throughout this investigation was
allowed to pass through a "Crystalab Deeminizer" ion exchange column

in order to remove possible metal ion impurities contained in the water.

Preparation of Reagent Solutions

 

A 0. 1 M stock solution of ammonium hydroxide was prepared from
distilled reagent grade ammonia and stored in a polyethylene bottle
equipped with an ascarite absorption bulb and glass siphon assembly.

Approximately 0. l M carbonate free sodium hydroxide was pre-
pared from a saturated solution of the reagent grade pellets and standard-
ized with potassium acid phthalate by titration to the phenolphthalein
endpoint. The sodium hydroxide solution was also stored in a polyethylene
bottle equipped with an ascarite absorption bulb and a glass siphon
assembly.

A 0. 4 7 per ml. solution of dichlorofluorescein in four percent
ethanol, which was employed as the fluorescent standard, was prepared
by diluting an aliquot of a stock solution made by dissolving a weighed
quantity of the reagent in 95 percent ethanol.

Individual stock solutions of lanthanum (III) or samarium (III)
perchlorate in dilute perchloric acid were prepared by dissolving the

required amounts of freshly ignited oxides in l M perchloric acid.

17

Aliquots of these stock solutions were taken for the preparation of the
working solutions. The lanthanum stock solution was analyzed by
precipitation of the lanthanum (III) with 8-quinolinol according to the
procedure of Pirtea (36). The analyzed solution contained 0. 0938 moles
of lanthanum (III) perchlorate per liter. Since the samarium was used
only in interference tests, it was not analyzed quantitatively.

Solutions of morin, 8-quinolinol, and 5, 7-dichloro-8-quinolinol
Of the desired concentrations were prepared by dissolving a weighed
quantity of the solid material in dioxane. The reagents adopted for use
in the analytical studies contained respectively, 0. 2, 1. 0, l. 0 mg. of
morin, 8-quinolinol, and 5, 7-dichlorO-8-quinolinol per ml. Of dioxane.
Even though there was no evidence that these reagent solutions were
unstable, fresh reagent solutions were prepared every two or three

weeks .

EXPERIMENTAL PROCEDURES

Introduction

 

The Spectrofluorometric study Of the complexes which form between
lanthanum (III) and the organic reagents, morin, 8-quinolinol, and 5, 7-
dichlorO-8-quinolinol, was made with dioxane-water solutions. In estab-
lishing the Optimum conditions for the reactions between lanthanum (III)
and the organic reagents, the individual effects of the more important
experimental variables were established. In studying these effects, the
reagents were added in the order described in the solution preparation
procedures.

Preliminary investigations of the effect of pH and dioxane content
on the fluorescence of a lanthanum (III)-organo complex were carried
out on the lanthanum (III)-8-quinolinol system. In the pH region of
maximum fluorescence, this complex is not sufficiently soluble in solu-

tions containing less than 60-70 percent dioxane for satisfactory study.

18

When sufficient lanthanum (III) and 8-quinolinol was introduced into a
50-50 DW* medium and the pH adjusted to attain maximum fluorescence,
a suspension rather than a solution was obtained. Since the fluorescence
intensities of the complex are highly pH dependent and, as previously
indicated, the pH meter readings are erratic and non-reproducible for
solutions containing greater than 60 percent dioxane, no further studies

on the lanthanum (111)-8-quinolinol system were made.

The Effect of Temperature

 

The effect of temperature on the fluorescence of morin and 5, 7-
dichlorO-8-quinolinol complexes of lanthanum (III) was estimated by
heating the solutions contained in 25 ml. glass stoppered bottles in a
water bath or cooling in an ice bath to approximate desired temperature
for recording the fluorescence intensities. The temperature was
measured by a mercury thermometer which was immersed in the
fluorescence cell immediately before and immediately after obtaining
the fluorescence readings. A plot was then constructed of the average
of the two temperature readings against the fluorescence intensities.
For both complexes, a nearly linear decrease in fluorescence intensi-
ties with increasing temperature was Observed. From the respective
plots, a relative temperature coefficient expressed as fluorescence
intensity units decrease per degree centigrade increase was evaluated
for each of the complexes over the temperature range in which the

fluorescence decreased linearly.

 

*Hereafter a 50 percent dioxane-50 percent water solvent will be
designated as 50-50 DW solvent. During this investigation no attempt
was made to correct for the volume contraction on mixing dioxane and
water. When equal volumes of dioxane and water were mixed, the
error was found to be 1. 5 percent at 25°. Freiser stated that the error
was less than 2 percent (7).

19

Lanthanum (III)-morin System

 

In preliminary investigations, dilute ethanol-water and dioxane-
water solutions Of lanthanum (III)-morin or morin were tested to
determine which solvent system would be most satisfactory for the
spectrofluorometric and Spectrophotometric study of the complex.

The results of these studies indicate that higher fluorescence intensities
are Obtained when some definite amounts of lanthanum (III) and morin

are dissolved in a dioxane-water medium than when the same quantities
of lanthanum (III) and morin are dissolved in an equivalent volume Of an
ethanol-water solvent. Furthermore, morin dissolved in ethanol-water
fluoresced noticeably throughout the pH range in which the complex
fluoresced, whereas the morin dissolved in dioxane-water did not.

For these reasons a dioxane-water solvent system was employed through-

out this entire investigation.

Solution Preparation Proc edure

 

The 50-50 DW solutions of lanthanum (III)-morin or morin were
prepared in either 25 or 50 ml. volmnetric flasks. Unless otherwise
noted the concentrations were expressed in units of micrograms, 7,
per 25 m1. of solution. 6

A measured volume of the 0. 2 mg. per ml. of dioxane stock
solution of morin was introduced into a 25 m1. volumetric flask and
sufficient dioxane added so that the total volume in the flask was 12. 5
ml. The required volume of a lanthanum (111) stock solution was added
and the total perchloric acid content adjusted to 0. 1 meq. by adding
the necessary amount of 0. 1 M perchloric acid. Then 0. 65 ml. of
0. 2 M sodium acetate and sufficient water were added to bring the
liquid level nearly to the encircling mark on the neck Of the flask, the
flask was tightly stoppered, inverted several times, and placed in the

250 water bath for at least one-half hour. < Finally, water was added to

20

the flask to bring the liquid level to the mark, the flask stoppered and
the contents thoroughly mixed. The flask with its contents was again
placed in the bath until needed for the measurements.

The above procedure was employed in the preparation of all
solutions with the exception of those prepared for the effect of pH and
the nature of the complex studies. For these solutions the sodium
acetate and the perchloric acid were not added and the pH was adjusted

with dilute perchloric acid or ammonia only.

Lanthanum (111)-5, 7-dichlorO-8-quinolinol System

 

Preliminary investigations had shown that the lanthanum (III)—
5, 7-dichloro-8-quinolinol complexes are sufficiently soluble in dioxane-
water solutions for a Spectrofluorometric study in this medium. NO

other solvents were tested for studies on the complex.

Solution Preparation Proc edure

 

The 50-50 DW solutions Of lanthanum (111)-5, 7Qdichloro-8-
quinolinol were prepared in a similar manner employed in the morin
study. A measured volume of the l. 0 mg. per ml. stock solution Of
5, 7-dichlorO-8-quinolinol in dioxane was added to each 25 ml. volu-
metric flask. The total dioxane content in the flask was adjusted to
12. 5 ml. and a measured volume of a lanthanum (III) perchlorate stock
solution was added. Then, 0. 5 ml. of 0. l M perchloric acid was added
and the solution made basic by the dropwise addition of 1. 25 ml. of
0. l M ammonia solution while carefully swirling the flask to prevent
the formation of a high local concentration of the hydroxide. Water
was then added slowly while swirling the flask until the liquid level
was nearly to the encircling mark on the neck of the flask. The flask
was tightly stOppered, inverted several times, and placed in the 250

water bath for nearly one-half hour. Finally, water was added to the

21

flask to bring the liquid level to the mark, the flask stOppered and

the contents thoroughly mixed. The flask and its contents was again
placed in the bath until needed for the measurments. The single wave-
length fluorescence measurements were obtained one—half hour after
addition of the ammonia.

The above procedure was employed for the preparation Of all
solutions except those for the effect of pH and the evaluation of the
stability constants studies. The effect Of pH study was carried out
in the same manner as that employed for the lanthanum (III)-morin

system.

Evaluation of the Complex Stability Constants

 

The technique Of Freiser and co-workers (15), which is an
adaption of the method of Calvin and Wilson (7) and Bjerrum (4) for the
determination of stabilities for chelating agents possessing more than
one acidic or basic group, was employed for the evaluation of the
stabilities Of the lanthanum (III)—5, 7-dichlorO-8-quinolinol complexes.
Information required to evaluate the consecutive stability constants
and to establish the composition of the complexes, was obtained from
alkalimetric titrations of solutions containing the metal ion and the

chelating agent.

Titration Procedure: A weighed quantity of 5, 7-dichloro-8-

 

quinolinol was transferred to the titration vessel and 105 ml. .of ‘
dioxane added. To this was added a measured volume of the 0. 00938

M lanthanum (III) perchlorate in dilute perchloric acid stock solution
and water so that the total volume of water was 105 ml. The titration
vessel and contents were purged with nitrogen for about ten minutes and
the titration begun. A nitrogen atmosphere was maintained above the
solution during titration. Sodium hydroxide was added in small

increments by means of a semi-micro buret which was equipped with

22

a small ascarite absorption bulb. The volume of the titrant was
decreased to 0. 05 m1. increments in regions of rapidly changing pH
values. With each addition of base an equal volume of dioxane was
added to maintain the desired dioxane-water ratio. The solution was

stirred continuously except when pH readings were taken.

DISCUSSION OF RESULT S

23

24

LANTHANUM (III) - MORIN SY ST EM

In Figure 2, curves A and B Show the absorption Spectra of morin
and the lanthanum (III)-morin complex, respectively, while curve C
shows the fluorescence spectrum of the complex when exposed to 365 mp
radiation. In order to force the formation of the complex a large excess
of lanthanum (III) (16 mg.) was employed.

The complex which forms at a pH of 5. 5 in 50-50 DW can be excited
to nearly equal fluorescence intensities by the 365, 405 or 436 mp
mercury radiations. No appreciable fluorescence radiation can be
detected when the complex is exposed to ultraviolet radiation Shorter
in wavelength than 365 mu.

The absorption peaks of the complex and Of the reagent are at 410
and 356 mu, resPectively, while the fluorescence maximum occurs
between 505 and 510 mu. Single wavelength absorbance and fluorescence

measurements of the complex were taken at their respective peaks.

Effect of Experimental Variables on the Absorbance
and Fluorescence of the Complex

 

 

Although the addition of sodium acetate and perchloric acid to solu-
tions containing lanthanum III and .morin resulted in approximately a 30
percent decrease in the fluorescence readings, the acetate buffer was
used in order to facilitate the preparation of the solutions at pH 5. 5.
The addition of sodium acetate did not Shift the absorption peaks of the
reagent or of the complex.

The effect of the dioxane content on the fluorescence and absorbance
of the complex was studied and the results shown in Figure 3. The fluor-

*
escence of the complex in solution at 1222 increases nearly linearly with

 

* . 505 . . .
For notations of the type I , the subscript 1nd1cates the wave-
length of excitation and the superscript denotes the wavelength at which
the fluorescence radiation is recorded.

25

Absorbance - Curves(A and B)

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Absorbance at 410 mu - (Curve B)

 

 

 

 

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27

increasing dioxane content, however, the absorbance at 410 mu reaches
a maximum at about 40 percent and remains essentially constant up to
at least 75 percent dioxane. - A 50 percent dioxane-50 percent water
solution was selected as the Solvent medium for the remaining studies
for the following reasons:

1) Maximum absorbances are attained in 50-50 DW.

2) At high dioxane contents, about 60 percent and above, erratic
and non-reproducible pH readings are obtained.

3) Several investigators have stated that nearly absolute pH
values are obtained in 50-50 DW (13,14,15). Freiser has
stated that corrections must be applied to pH readings for
solutions containing greater than 50 percent dioxane (14).

In Figure 4, curve A Shows the effect of pH on the absorbance Of
lanthanum (III)-morin solutions at 410 mu and curve B shows the effect
on solutions containing pure morin. The absorbances of the lanthanum
(UH-morin solutions reach a maximum at approximately pH 6 and then
decrease. Comparison of the absorption Spectra for these solutions
over the pH range covered shows that the absorption peak shifts to
longer wavelengths at higher pH values.

' An estimation of the ionization constant for the dissociation of
morin can be obtained from curve B, Figure 4. At the pH value where
the absorbance is half its maximum value, morin exists half in the dis-
sociated and half in the associated form. Therefore, the pH at the half
absorbance value should correspond to the pKa of morin. From the plot,
the pH at the half absorbance value yields a pKa of 6. 7. Unfortunately
no pKa value for morin in 50-50 DW could be found in the literature with
which to compare the above results.

The effect of pH on the fluorescence radiation of lanthanum (III)-

morin and pure morin solutions at 505 mu, when the solutions are exposed
505
405
but of slightly lower intensity, hence it was not

to 365 and 436 mu radiation, is shown in Figure 5. . The curve for I

is quite similar to 1:05

36

28

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included. The pure morin solutions, curves C and D, do not fluoresce
at a pH of 5. 5, consequently the excess reagent does not interfere in
fluorescence measurements on the complex.

The bathochromic shift in the absorbance spectra and the decrease
in the fluorescence intensity of the complex as the pH is increased

above 6, suggests that another species, which is non-fluorescing, is
505 and
365

and the Optimum absorbance at 410 mu of the complex is attained

being formed. The maximum fluorescence intensities at I
505
436
at a pH Of about 5. 5; hence the pH was adjusted to this value in the soluw

I

tion preparation procedure.

' In Figure 6, curve A shows the effect of the change of lanthanum
(III) concentration on the absorbance of 400 7 of morin, while curves B
and C Show the same effect on the fluorescence. A nearly linear increase
in the absorbance and fluorescence of lanthanum (III) morin solutions is
attained in the approximate region 0 to 80 7 of lanthanum (III). The
gradual change in lepe of the absorption curve with increasing lanthanum
(III) concentration above approximately 80 7 suggests that the complex
f ormed is relatively weak.

Figure 7 shows the effect of the change in lanthanum (III) concen-
tration on the fluorescence of 1000 7 of morin and clearly indicates the
effect of concentration quenching. As lanthanum (III) is added to the solu-
tion containing the morin, the morin concentration is decreased and the
concentration Of the complex is increased. Thus quenching by morin
absorption of the 365 mp. radiation is decreased and more excitation
radiation is made available to the complex. Consequently, since morin
absorbs more strongly at 365 mu than an equivalent amount of complex
and does not absorb appreciably at 436 mp, 1322
rate with increasing lanthanum (III) concentration, above 50 7 lanthanum
(111), than 1505

436
Figure 8 shows the effect of the variation in morin content on the

increases at a greater

due tO concentration quenching.

fluorescence of 80 7 of lanthanum (III) per 25 ml. ~ Maximum fluorescence

Fluorescence Intensity, I505 - (Curves A and B)

180.

160

140

120

100

80

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31

 

 

 

 

 

 

 

 

Micrograms Lanthanum (III) per 25 ml.

Figure 6. -Effect of Lanthanum (III) on the Fluorescence and
Absorbance of 400 7 Morin per 25 ml. in 50-50 DW

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‘Micrograms Lanthanum (111) per 25 ml.
Figure 7. Effect of Lanthanum (III) on the Fluorescence of 1000 7 Morin
per 25 ml. in 50-50 DW at pH 5. 5

 

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505 505 505 , . .
436’ 1405 and 1365’ were obtained for the three exc1tation

wavelengths at approximately 400 7 of morin, while above 400 7 the

intensities, I

fluorescence intensities decrease due to concentration quenching. For
purposes of comparison, the effect of the morin concentration on the
absorbance of the complex is also shown in this figure.

The effect of change. in temperature on the fluorescence intensity
505
365 0
m1.) at pH 5. 5 was Obtained over the temperature interval 9 to 50 .

at I of the complex (1. 6 mg lanthanum (III) and 400 7 morin per 25

the fluorescence intensities Of 90 and 500 were 228 and 152, reSpectively
and decreased linearly with increasing temperature. From 12 measure-
ments made in the above temperature range, the rate of change corres-
ponds to about a 1. 8 fluorescence intensity units decrease per degree
centigrade increase.

Samarium (III) was employed in order to study the effect that a
lanthanide ion which forms a non-fluorescing complex with morin *
would have on the fluorescence of the lanthanum (III)-morin complex.
Figure 9 shows the results of this study on solutions containing 40 7 of
lanthanum (III) and400 7 of morin and Figure 10 illustrates the same
effect on 80 7 of lanthanum (III) and 1000 7 of morin.

As Figure 9 shows, the fluorescence intensity of the lanthanum

(III) complex remains essentially constant at 1505 and decreases at'

365
2(3): as the samarium (III) content is varied from 0 to 137 7 per 25 ml.

At the lanthanum (III) and morin concentration levels employed, these

I

results indicate that at least a two to threefold excess of samarium (III)
would not cause an appreciable error in the determination of lanthanum
when the complex is exposed to 365 mu radiation.

Figure 10 clearly illustrates the fluorescence quenching effect

due to the formation of a samarium (III) complex in lanthanum (IIl)-morin

 

*
A 50—50 DW solution of samarium (III)-morin, which contained
86 7 samarium (III) and 40903 of morin gave approximately the same
fluorescence intensity at 1436 as a blank solution containing no samarium

(III) .

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solutions. The addition of the samarium (III) to these solutions reduces
the uncombined morin concentration and results in a decrease in
absorbance at 365 mu and an increase at 436 mu. Since, as indicated
in Figure 8, there is appreciable quenching of the lanthanum (III)-
morin fluorescence at 80 7 of lanthanum (III) and 1000 7 of morin per
25 ml, the fluorescence intensity of the lanthanum (III)-morin complex
would be expected to increase at I505 and decrease at 1505

365 436
is added as shown in Figure 10. The slight decrease in fluorescence

when samarium

readings at 1505 when more than 200 7 of samarium (III) is added, is

probably due3t605the competition between samarium (III) and lanthanum
(III) for morin.

A fluorescence intensity-time study was made on a solution of
the complex, which was prepared by the solution preparation procedure
and contained 807 lanthanum (III) and 400 7 morin per 25 ml. , to
determine the stability of the complex. The fluorescence of the solu-
tion was monitored for one week. No significant change in fluorescence

505 505

365 or 1436' The fluorescence
was 64.0

readings at five minutes after solution preparation at I

readings with time was noted at either I

436
and that after one week, 63. 5. Therefore, it would appear that the

complex is formed immediately after preparation and is stable for

periods up to at least one week.

Nature of the Reaction

 

In order to ascertain the number Of complexes which might be
formed and to determine whether the reaction is stoichiometric,
nineteen solutions containing 400 7 of morin and amounts of lanthanum
(III) varying between 0 and l. 6 mg. per 25 ml. as indicated were pre-
pared by the solution preparation procedure. The absorbance spectra

from 340 to 425 mg for these solutions are shown in Figure 11.

Absorbance

38

 

Ullwa~

 

I I
340 350

 

M /
‘2 s.— 7' 400 7 Morin per 25 ml.

169 , ‘81“)
,/

 

  

1 x
(9 (Curves 1-19)
Curve La“, 7 Curve La+3 mg
l O. 0 11 0.16
2 8. 0 12 0 20
7 3 16.0 13 o 24
4 24. 0 l4 0 32
5 32 0 15 0 48
6 48. 0 l6 0. 64
7 64. 0 l7 0. 80
"’ 8 80. 0 18 1. 20
9 96. 0 19 l. 60
10 120. 0
Figure 11. Spectrophotometric Curves Showing Isoabsorptive Point.

I I I I l I 7
360 370 380 390 400 425 450
Wavelength, mp

39

The family of curves in Figure 11 yield an isoabsorptive. point
at 377 mp. which suggest the formation of a single complex and indi-
cates that an equilibrium exists between two colored species (28).
The decrease in the absorbance st 356 mu, the morin peak, and the
increase at 410 mg, the complex peak, as the concentration of

lanthanum (III) is increased, suggests an equilibrium of the type
+3-x
X

La+3 + xMH ——». La(M) + xH+ is involved--where MH = morin.

Composition of the Complex and Estimation
of the Equilibrium Ratio, K'eq

 

 

Job's method of continuous variation (22) was employed to
determine the empirical formula of the complex which forms at a pH
of 5. 5. The total molar concentration of the lanthanum (III) and
morin was maintained constant at 6. 0 x 10"5 moles per liter for a
series of eighteen solutions, while the individual concentrations Of
lanthanum (III) and morin were varied. The molar concentrations
of lanthanum (III) and of morin per 50 ml. were plotted against Y, the
corrected absorbance which is due only to the complex.

Job (22) and Vosburgh and Cooper (45) have shown that the value
of Y at each wavelength is a maximum when the greatest amount of
complex is formed. Figure 12 shows the results obtained when Y for
the complex is plotted at three different wavelengths, 410, 390 and
356 mu. The maximum or minimmn value of Y appears at a ratio of
one mole lanthanm'n (III) to two moles morin and indicates that the
general formula of the complex is LaMz+. The structure of the com-
plex is probably of the same type as that suggested by Fletcher and
Milkey for the thorium (IV)—mor’in complex (28); that is
7' +

c104“,

 

 

 

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41

In order to substantiate the above results, the composition of
the complex was also determined at pH-S. 5 by the slope ratio method
of Harvey and Manning (20). Two series of lanthanum (III)~morin
solutions were prepared and the absorbance at 410 mu recorded.

The first series contained a large excess of lanthanum (III) (30. 6 mg. ),
which should ensure essentially complete reaction with the morin to
form the complex, and amounts Of morin varying between 0 and 1014 7
per 50 ml. The second series contained a relatively large excess of
morin (2. 0 mg. ), which should be enough to react with essentially all
of the lanthanum (III), and lanthanum (III) varying between 0 and 41. 5 7
per 50 m1.

Figure 13 shows the absorbance of 410 mg, the absorption
maximum for the complex, plotted against the moles of morin per
liter, curve 1, and the moles of lanthanum (111) per liter, curve 2,
for the slope ratio method. The slope of curve 1, which represents
the rate in change in absorbance per mole of morin, is l. 98 x 10“,
while the slope of curve 2, which represents the rate in change in
absorbance per mole of lanthanmn (III), is 3. 96 x 104. On the basis of
the slopes Of the two curves it can be concluded that two moles Of morin
combine with one mole Of lanthanum (HI), Since

Slope curve 1 _ l. 98 x 104 _ A A/mole morin
Slope curve 2 _ 3. 96 x 10; - A A/mole lanthanum (III)

 

 

 

1 mole lanthanum (III)
2 moles morin

 

The molar absorptivities for pure morin and the complex at pH
5. 5 were determined from plots of absorbance against concentration.
Assuming that when morin is added to a large excess of lanthanum (III)
in solution, the morin is completely consumed in complex formation.
For each two moles of morin added, one mole Of complex is formed.
On the basis of this assumption, curve 1 of Figure 13 can be used to

evaluate the molar absorptivity of the complex. Thus the molar

42

Absorbance - (Curve 2)

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43

absorptivity of the complex will be equal to an absorbance value selected
from the curve divided by one-half of the correSponding morin concen-
tration. The data for the molar absorptivity of morin was Obtained from
the absorbance values of a series Of solutions which varied in morin
content from 1. 2 x 10'5 to 7. 0 x 10'5 moles per liter. Both the complex
and morin were found to obey Beer's Law. The absorptivities at 410 me
are:

+ : , 1 4 ' .
aLa(M z for complex 4 00 x O liters/mole cm

. : . 1 3 . o
aM H for morin 2 28 x 0 liters/mole cm

The formal equilibrium constant expression for the lanthanum
4..
(Ill)-morin complex for the reaction, La+3 + 2 M.H “"'La(M)Z+ + 2H' is

_ [Law] m.
‘ [Law [M H12

 

Keq

An estimation of the value Of this constant can be made employing the
equations of Fletcher and Milkey (28).
In the lanthanum (III)-morin system only two components absorb

light: morin, M H and the complex, La(M)z+.

If X = moles Of complex per liter
(M H) = total moles of morin added per liter
(La+3) = total moles of lanthanum (III) added per liter
Y = moles Of uncombined morin per liter
Z = moles of uncombined lanthanum (111) per liter
A410 = absorbance at 410 mp. of solutions containing the complex
b = internal cell length in cm. (1. 00 cm.)
aM H 7 2 molar absorptivity for morin at 410 mu and pH 5. 5
aLaM'; = molar absorptivity for the complex at 410 mu and pH 5. 5
then
A410 = 3M HI (M H) - (ZXII ‘I' aLaMz[X]

and

44

X = A410-3M H(M H)

aLaMz ' 2aM H

»-<:
n

(M H) - 2[X]

Z (La+3) - [X]

Employing the above equations, the equilibrium ratio, K'eq was
calculated from data obtained from the study involving the 18 solutions
which were employed in the determination of the composition of the
complex. The values needed to evaluate the equilibrium ratio and the
values of the equilibrium ratio are tabulated in Table I.

The equilibrium ratio values, K'eq, vary between 0. 02 and 5. 4 with
the majority of the values ranging between 0. 25 and 1. 6. Excluding the
extreme values designated in Table I, the average K'eq is 0. 6 and
indicates that the complex is weak in comparison to the average value
of 1 x 106 found by Fletcher and Milkey (28), for the thorium (III)-morin

complex in a dilute ethanol-water system.

LANTHANUM (III)- 5, 7 - DICHLORO- 8- QUINOLINOL SYSTEM

In Figure 14, curves B, D and F show the absorption spectra
from 260 to 620 mu for 1000 'y of 5, 7-dichloro-8-quinolinol per 25 ml.
in 50-50 DW at several pH values and curves C and E show the effect
of the addition of 80 7 of lanthanum (III) at pH 8. 8 and 6. 8, respectively,
on the same amount of reagent in 50-50 DW. Under various conditions
of acidity, three different species of 5, 7-dichloro-8-quinolinol have

been reported (1, 12).

C1

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47

The absorption band centering around 390 mu for the reagent at pH 8. 8
(curve B) is due to species I, while the peak at 370 mg, the reagent in
l M perchloric acid (curve F), is due to Species [11 . Species 11, the 1
neutral molecule, is represented by curve A, the reagent dissolved in
pure dioxane which has an absorption peak at 326 rm; and does not
absorb in the visible region above 380 mu. The addition of 80 'y of
lanthanum (III) to 1000 7 of 5, 7-dichloro-8-quinolinol (curves C and E)
results in an increase in the absorption in the region of 388 mu. This
increase suggests that lanthanum (III) forms a complex or complexes
with the reagent at both pH 6. 8 and 8. 8 and that the complex absorption
peak is at approximately 388 mp.

The fluorescence spectra of 80 'y lanthanum (III) and 1000 'y of 5, 7-
dichloro-8—quinolinol per 25 ml. at pH 9. 0, (curve A), and at 6. 1,
(curve B), when exposed to 365 mp. radiation are shown in Figure 15.
The fluorescence peak for curve A appears between 526 and 530 mu,
while the peak for curve B is shifted to a slightly longer wavelength
centering about 535 mp” The peak fluorescence intensity at the higher
pH value is more than two and one-half times that obtained at the lower
one.

The optimum excitation wavelength for lanthanum (111)-5, 7-dichloro-

-8-quinolinol is at 365 mp. The fluorescence intensity at I 3 is nearly
505 505 505 365
three to four times that at 1313, 1405, and 1436’ while no apprec1able

fluorescence can be detected when the solutions are exposed to radiation

below 365 mu. On the basis of the above results, single wavelength
530

fluorescence measurements were taken at 1365'

Effect of Experimental Variables on the
Fluorescence of the Complexes

 

 

Figure 16 shows the effect of the variation of dioxane content on
the fluorescence of lanthanum (111)-5, 7-dichloro-8-quinolinol solutions

buffered at pH 9 with ammonium hydroxide-ammoniumperchlorate.

48

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A slight turbidity, which undoubtably accounts for the low fluorescence
readings, is formed in solutions containing less than 40 percent dioxane.
The absorbance readings at 388 mg, the complex maximum, against
dioxane content for the same solutions employed in obtaining the data
for Figure 16 showed a decrease in absorbance values with an increase
in dioxane content. Since maximum fluorescence intensities are
attained in solutions containing between 50 and 55 percent dioxane,
50-50 DW was selected as the solvent for this study.

The effect of pH on the fluorescence of pure 5, 7-dichloro—8-
quinolinol and lanthanum (111)-5, 70dichloro-8—quinolinol at 1:22 is shown
in Figure 17. Lanthanum (III)-5, 7-dichloro-8-quinolinol, curve A,
fluoresces in the approximate pH region 4. 5 to 10 with a maximum and
nearly constant fluorescence intensity between pH 8. 7 to 9. 5, while
the pure reagent, curve B, does not yield appreciable fluorescence
over the entire pH range covered. Since solutions of the reagent or
of lanthanum (III) do not fluoresce, lanthanum (III)-5, 7-dichloro-8-
quinolinol complexes are responsible for the fluorescence yields shown
in curve A.

The effect of the change in lanthanum (III) concentration on the
fluorescence of 10007 of 5, 7-dichloro-8-quinolinol in 50-50 DW at pH
8. 8 is shown in Figure 18. A nearly linear increase in the fluorescence
of the lanthanum (III)-5, 7-dichloro-8-quinolinol solutions is attained
in the approximate region 0 to 70 7 of lanthanum (III). Above approxi=
mately 100 7 of lanthanum (III), the fluorescence intensity tends to
reach a limiting value.

. Figure 19 shows the effect of the variation of 5, 7-dichloro-8-
quinolinol content on the fluorescence of the complex at pH 8. 8.
A maximum and optimum fluorescence reading is attained at] about
800 to 1500 7 of 5, 7-dichloro-8-quinolinol for 80 7 lanthanum (III)
per 25 ml. Above approximately 1500 7 the intensity of fluorescence

gradually decreases due to concentration quenching.

51

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Solutions which were prepared by the solution preparation
procedure and contained 80 7 of lanthanum (III) and 1000 7 of 5, 7—
dichloro-8-quinolinol at a pH of 8. 8 were employed to study the effect
of the variation in temperature on the fluorescence. A plot of
temperature from 14 to 450 against fluorescence intensity readings
(not shown here) indicated a nearly linear decrease in fluorescence
intensity with increasing temperature increase. This decrease
corresponded to approximately 3. 0 fluorescence intensity units per
degree centigrade increase over nearly the entire temperature range
covered. ‘ It would appear on the basis of the above results that
constant temperature conditions should be maintained for any quanti-
tative fluorescence study on the complex.

In preliminary investigations the observation was made that
solutions of the complex at pH 9, which were prepared from ammonia
repeatedly exposed to the atmosphere, gave significantly lower
fluorescence readings than those prepared from ammonia which was
protected from the atmosphere. This suggested that carbon dioxide
absorbed in ammonia, quenches the fluorescence of the complex
and therefore the effect of carbonate was studied.

Since sodium ions would not be expected to produce fluorescence
quenching, high purity sodium carbonate was selected to test the
effect of carbonate on the fluorescence of the complex. The only
modification introduced into the solution preparation procedure was
the addition of varying amounts of sodium carbonate (0-5 mg.) and
O. 1 M ammonia to attain a pH of 9.

Table II shows the results of the effect of carbonate on the
fluorescence of the complex and specifies the amounts of reagents
required per 25 ml. The results of this study clearly indicate that
carbonate has a pronounced quenching effect on the fluorescence
since an increase in carbonate contentl causes'a decrease in the fluor—

escence intensity.

55

Table II. Effect of Sodium Carbonate on the Fluorescence Intensity of
,Lanthanum (III)-5, 7-Dichloro—8-quinolinol in 50 percent
Dioxane-Water at pH 9. 0.

89 7 Lanthanum (III), 1000 7 5,7-Dichloro-8-quinolinol per 25 m1.

 

 

Trial Mg. NazCOz MlVHEOlI-IM pH 1:22
1* 0.0 1.25 8.95 78.2
2 0.0 1.25 8.95 78.2
3 1.0 1.25 9.10 70.0
4 2.0 0.87 8.98 44.7
5 3.0 0.68 8.96 35.1
6 4.0 0.49 9.02 28.1
7 5.0 0.31 9.03 22.3

 

a):
Water boiled to remove dissolved carbon dioxide for this trial.

56

The effect of samarium (III) on the fluorescence of solutions
containing 40 7 of lanthanum (III) and 1500' 7 of 5, 7-dichloro-8—quinolinol
per 25 ml. was studied. The readings at 1:22 for O. 0, 17, 34, 52 and
717 of samarium (111) per 25 ml. were 46. 2, 45.4, 45. 9, 43.8 and 41. 5,
respectively, and indicate that samarium (III) in concentrations approxi-
mately equal to lanthanum (III) can be tolerated without causing a sig-
nificant error. However, for concentrations of samarium (III) greater
than this amount a slight decrease in the fluorescence intensities is
noted.

A fluorescence intensity-time study was made on the lanthanum (III)-
5, 7-dichloro-8-quinolinol solutions in order to ascertain its
stability. The results of this study are shown in Table III for solutions
prepared by the solution preparation procedure, trials I and II, and
for one non—buffered solution, trial III, prepared at pH 9 by omitting
perchloric acid and adding only dilute ammonia. The results indicate
that the fluorescent complex develops within at least ten minutes
after addition of ammonium hydroxide and yields essentially constant
fluorescence readings for approximately one to two hours. The fluor-
escence intensity gradually diminishes after one or two hours of
standing and shows a 30 percent decrease after standing about 24 hours.
On the basis of the above results all fluorescence measurements were

made between one-half to one hour after development of the complex.

Evaluation of the Lanthanum-(III)-5,' 7-dichloro-
8-7quinolinol Stability Constants

 

 

To gain information regarding the possible species responsible
for the fluorescence of the lanthanum (III)-5, 7-dichloro-8-quinolinol
system, the formal stepwise stability constants, K1 and K2, were
evaluated by the method of Freiser and co-workers (15). Since
lanthanum (III) tends to hydrolyze above pH 6, the constant K3 could

not be accurately determined by this method.

57

Table III. Fluorescence Stability Data on 50 percent Dioxane-Water
Solutions of Lanthanum (111)-5, 7-Diohloro-8-quinolinol at
pH 9. 0.

80,7 Lanthanum (III), 1000 7 5, 7-Dichloro-8-quinolinol per 25 ml.

 

 

 

Time After Trial’ l Trial 2_ Trial 3
Development I530 I530 I530
of Complex 365 365 365
10 minutes 82.6 78.5 82.1
20 minutes 81.2 80.2 82.2
30 minutes 80.8 80.2 81.0
1 hour 80.2 79.8 81.8
2hours 77.2 77.2 ----
22 hours ---- ---- 59.2

24 hours 63.8 65.5 ----

 

 

1..

58

Figure 20, curve C, shows the pH titration curve for 4. 99 ml.
of a O. 00938M lanthanum (III) perchlorate stock solution. For pur-
poses of comparison the titration curves of 5, 7-dichloro-8-quinolinol
and lanthanum (111)-5, 7-dichloro-8-quinolinol are also shown in this
figure. The first titration break in curve C corresponds to the titration
of perchloric acid and the second one to the precipitation of lanthanum
(III) hydroxide. The acid concentration of the stock solution was
determined from this curve and from samples which were titrated to
the methyl red endpoint with standard sodium hydroxide. The concen-
tration of the acid was found to be 0. 0751 moles per liter. The
hydrolysis curve of lanthanum (III) is almost identical to that obtained
by Freiser gt a} (15).

The acid dissociation constants of the protonated nitrogen KNH

and of the phenol, K which are required for the calculation of the

formal stability consiiints of the 5, 7-dichloro-8-quinolinolate complexes,
were evaluated in 50—50 DW at 250. The pKOH was determined
graphically from curves of pH against ml. of sodium hydroxide which
are required to titrate acidified samples of the reagent. The pH at

the half neutralization point corresponds to the pKOH and yields values
of 9.10 and 9.12 for two trials as shown in Figure 20, curves B and D.
The average KOH was found to be 7. 8 x 10"“).

NH was estimated from a plot of pH against absorbance

at 369 mp for a series of solutions containing 1000 7 of 5, 7-dichloro-

The pK

8-quinolinol per 25 ml. The acidity of the solutions were adjusted
with dilute perchloric acid. The pH at half absorbance value, which
corresponds to the pKNH’ was found to be 1. 6 as shown in Figure 21.

The consecutive stability constants, K1 and K2, for the reaction
of lanthanum (III) with 5, 7-dichloro-8-quinolinol are

LLa<QCI.)ZJ .
[(La(QCIz)fllll(QCIz‘-)]

.

_ [LalQC121+ZJ

K‘ " [1421“] [0612-]

 

 

and K2 :

and can be calculated employing the equations derived by Freiser,et a1. (15).

59

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61

The equations, which were developed for reagents such as 8-
quinolinol from equations expressing material balances of metal
and reagent, charge balance, and the acid dissociation constants

KNH and KOH’ are:

+
— 1
n=——— [T -S(KNH+H )
TM“ HR KNH + .ZI-l’r

 

and

R- 0 KNHXSXKQH
+
H (KNH+ 2H )

. _ +3
average number of reagent spec1es, R , bound to M

:3
ll

11>
11

total moles per liter of the acid added

TM+3 = total moles per liter of the metal ion

THR = total moles per liter of reagent
+ - +
S +— THR+A- Na+OH+H
[Na ] = calculated from amount of base added
.. _ Kw
[OH 1‘ (his

(H+) = from measured pH .
The logarithms of the stepwise formal stability constants K1 and K2
are obtained from a plot of .11 against pR- at values DIET-1: 1/2 and
3/2, respectively.

In Figure 20 curves A and E show the pH titration curve of
lanthanum (III) perchlorate and approximately a fivefold excess of
5, 7-dichloro-8-quinolinol with 0. 0862 N sodium hydroxide. The data
from these titrations were employed to calculate E and pR- for the
complex. Table IV tabulates some calculated values of n, which
are between 0. 07 and 2. 88, and pR- and gives the concentrations
of the reagents which were employed.

Figure 22 shows the results obtained when; is plotted against

pQClz- for the data given in Table IV. The log K, values obtained

62

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64

from this plot are 7.17 and 7. 31 and the log K; values are 6. 46 and
6. 60. The average log K1 and log K; values are 7. 24 and 6. 53,

respectively. Freiser, et al. (15) estimate that the stability constants

determined by this procedure are accurate to within 0. 2 log K units.

SUMMARY AND CONCLUSIONS

(.5

66

INTRODUCTION

A spectrofluorometric investigation of the complexes which form
when lanthanum (III) is added to 50-50 DW solutions of morin or 5, 7-
dichloro-8-quinolinol was undertaken. The various factors which affect
the fluorescence of the active species were investigated and the compo-
sition and stability of the fluorescent complexes were established.
' All of these studies were carried out at 25°. A 50-50 DW solvent ratio

was selected as the solvent medium for these studies.

LANTHANUM (III) - MORIN SY ST EM

The absorption band peaks of morin and of the lanthanum (III)-
morin complex in 50-50 DW at pH 5. 5 are at 410 and 356 mg,
respectively, while the fluorescence maximum of the complex occurs
between 505 and 510 mp. The complex can be excited to nearly equal

fluorescence intensities by the 365, 405, and 436 mp mercury radiations.
505
(1

Single wavelength fluorescence measurements were made at 1365 an

I505
436'
The effect of the dioxane content on the fluorescence of solutions

of lanthanum (III)-morin at pH 5. 5 was established. The fluorescence
intensity of the complex at 1505

365
ing dioxane content, while the absorbance at 410 mp reached a maximum

increased nearly linearly with increas-

value of about 40 percent and remained essentially constant up to at
least 75 percent dioxane.
The optimum pH for the spectrofluorometric study of the lanthanum

(III)-morin solutions was established. The maximum fluorescence
505 and I505
365 436

intensities of morin were obtained for solutions maintained at pH 5. 5.

intensities at I of the complex and minimum fluorescence
This pH also corresponded to the optimum value for absorption measure-
ments on the complex, therefore, the pH of the solutions were adjusted

to 5. 5 for all studies on the complex or morin.

67

The pKa of morin in 50-50 DW was estimated from a plot of
absorbance against pH for a series of solutions containing 400 7 per
25 ml. The estimated pKa value is 6. 7.

The effect of lanthanum (III) concentration on the fluorescence

. . . 505 505
intensities at 1365 and 1436’

1000 7 of morin per 25 ml. was established. A nearly linear increase

and the absorbance at 410 mu on 400.7 or

in the fluorescence intensities and the absorption measurements for
solutions containing 400 7 of morin is attained in the approximate region
0 to 80 7 of lanthanum (III) per 25 ml. For solutions containing 1000 7
of morin, considerable concentration quenching of the fluorescence

was noted.

The effect of the morin content on the fluorescence of 80 7 of
lanthanum (III) per 25 ml. was determined. Maximum fluorescence
intensities were attained at approximately 400 7 of morin. Above 400 7
of morin the fluorescence intensities decreased due to concentration
quenching.

The fluorescence intensity of the lanthanum (III)-morin complex

50
at 1365
rate of change in the fluorescence intensity corresponds to about 1. 8

decreased nearly linearly with increasing temperature. The

fluorescence intensity units decrease per degree centigrade increase
over the temperature interval studied (9 to 500).

The effect of samarium (III), which forms a non-fluorescing
complex with morin, on the fluorescence of solutions containing 40 7
of lanthanum (III) and 400 7 of morin or 80 7 of lanthanum (III) and 1000 7
of morin per 25 ml. was determined. For solutions containing 40 7 of
lanthanum (III) and 400 7 morin, a two to threefold excess of samarium
(III), did not significantly change the fluorescence intensity readings
of the lanthanum ‘(III)-morin complex at 132:. However, at 1:22, the
fluorescence intensity decreased with increasing samarium (III) concen—

tration. The decrease in the fluorescence intensity of the lanthanum

(III)-morin complex when excited with 436 mu radiation is probably due

68

to quenching of the 436 mu excitation radiation by the samarium (III)—
morin complex which absorbs strongly at this wavelength. At 80 7
lanthanum (III) and 1000 7 morin, small changes in samarium (III)
concentration caused a relatively large change in the fluorescence
intensity of the lanthanum (III)-morin complex. On the basis of the
above results and those obtained in the effect of lanthanum (III) concen-
tration on the fluorescence of morin studies, microgram quantities of
lanthanum (III) in pure solutions or in solutions containing low concen-
trations of samarium (III) or possibly other lanthanides which also
form non-fluorescing complexes with morin can be determined fluoro-
metrically.

When lanthanum (III) is added to 50-50 DW solutions of morin and
the pH adjusted to 5. 5, a stable complex is formed. The complex formed
immediately after addition of the reagents and was stable for at least
one week.

When amounts of lanthanum (III) varying between 0 and l. 6 mg
per 25 ml. were added to solutions containing 400 7 morin and the pH
adjusted to 5. 5, the absorption spectra gave a series of curves yielding
a single isoabsorptive point at 377 mp0 This family of curves with a
single isoabsorptive point suggests that an equilibrium exists between
lanthanum (III) and morin and that a single complex is formed.

Job's method of continuous variations (22) and the slope ratio of
Harvey and Manning (20) was employed to determine the empirical
formula of the fluorescent complex. These studies indicate that
lanthanum (III) forms a 1:2 complex (Lanthanum (III):morin) with morin
at pH 5. 5 and that the complex, La(M):, is the species responsible for
fluorescence in the lanthanum (III)-morin solutions.

The equilibrium ratio, K'eq, for the lanthanum (III)-morin com-
plex was estimated from the absorbance, pH, and reagent concentrations
data compiled in the composition of the complex studies. The average

calculated K'eq ratio was found to be 0. 6.

69

LANTHANUM (III)-5, 7-DICHLORO-8-QUINOLINOL SYSTEM

The absorption spectra from 260 to 620 mg of 50-50 DW solutions
which were adjusted to various pH values and contained 1000 7 5, 7-di-
chloro-8-quinolinol or 80 7 lanthanum (III) and 1000 7 5, 7-dichloro-8-
quinolinol per 25 ml. Were obtained. The absorption band peaks for
solutions of the reagent or the complex at pH 8. 8 centered around 342
and 388 Inn. The addition of lanthanum (III) to solutions of 5, 7-dichloro-8-
quinolinol at pH 8. 8 resulted in an enhancement of the absorption peak
of the reagent at 388 mp. and suggested the formation of a complex or
complexes.

The fluorescence spectra of 80,7 lanthanum (III) and 1000 7 5, 7-
dichloro-8-quinolinol per 25 ml. at pH 9. 0 and 6. l were obtained when
the solutions were exposed to 365 mu radiation. At pH 9. 0 the fluor-
escence peak appeared between 526 and 530 mu, while that at pH 6. l was
shifted to a slightly longer wavelength centering about 535 mu. The
peak fluorescence intensity at the higher pH value was more than two and
one-half times that obtained at the lower one. The optimum excitation-

wavelength for the complex is at 365 mu, therefore single wavelength
530

365'

The effect of dioxane content on the fluorescence of lanthanum (III)-

fluorescence measurements were measured at I

5, 7-dichloro-8-quinolinol solutions buffered at pH 9 with ammonium
hydroxide-ammonium perchlorate was evaluated. Maximum fluorescence
intensities were obtained in solutions containing between 50 and 55 percent
dioxane.

Lanthanum (III)-5, 7-dichloro-8-quinolinol fluoresces in the pH
region 4. 0 to 10 with maximum and nearly constant fluorescence intensi-
ties at pH 6 to 7. 2 and 8. 7 to 9. 5, while the pure reagent shows no signifi-
cant fluorescence over the entire pH range investigated (pH 2. 3 to 9. 7).
Since the reagent is non-fluorescent, the existence of two plateau regions
suggested that more than one lanthanum (III)-5, 7-dichloro-8-quinolinol

fluorescent complex is formed.

70

The effect of the change in lanthanum (III) concentration on the
fluorescence of 1000 7 of reagent per 25 ml. at pH 8. 8 was evaluated.
A nearly linear increase in the fluorescence intensity of the complex
was attained in the approximate region 0 to 70 7 of lanthanum (III).

In a series of solutions adjusted to pH 8. 8 and containing 80 7 of
lanthanum (III) per 25 ml. , maximum fluorescence intensity readings
were obtained for 800 to 1500 7 of 5, 7-dichloro-8-quinolinol. For solu-
tions containing greater than 1500 7 of reagent, the fluorescence intensity
of the complex decreased due to concentration quenching.

The fluorescence intensity of the lanthanum (III)-5, 7-dichloro—8-
quinolinol complex decreased with increasing temperature. This nearly
linear decrease correSponded to about 3. O fluorescence intensity units
per degree centigrade increase over the temperature range 14-450.

On the basis of this result, constant temperature conditions should be
maintained for any fluorescence study on the complex.

The effect of carbonate on the fluorescence of the lanthanum (III)-
5, 7-dichloro-8-quinolinol complex in 50—50 DW at pH 9. 0 was ascertained.
The addition of carbonate to these solutions (0 to 5 mg. sodium carbonate)
resulted in decrease in the fluorescence intensity of the complex.
Approximately a 10 percent reduction in fluorescence intensity was noted
when 1. 0 mg. of sodium carbonate was added to solutions containing
80 7 lanthanum (III) and 1000 7 5, 7-dichloro-8-quinolinol per 25 ml.

On the basis of these results, carbonate-free base should be employed
in the preparation of the complex.

The effect of samarium (III) on the fluorescence of solutions con-
taining 40 7 lanthanum (III) and 1500 7 of 5, 7-dichloro-8-quinolinol per
25 ml. was investigated. Samarium (III) in concentrations approximately
equal to lanthanum (111) can be tolerated without causing a significant
error in the fluorescence intensity reading of the complex.

The lanthanum (III)-5, 7-dichloro-8-quinolinol complex deve10ps

within at least ten minutes after preparation of the complex and yields

71

essentially constant fluorescence readings for about one to two hours.
Therefore, fluorescence measurements on the complex should be
obtained within at least one to two hours after preparation of the complex.

The acid dissociation constants of the protonated nitrogen KNH
and of the phenol in 50-50 DW solutions, KOH' which were required for
the calculation of the stability constants of the 5, 7-dichloro-8-quinolinol
complexes, were evaluated. The average KOH and KNH was found to
be 7. 8 x 10'1_0 and 2. 5 x 10"2 respectively.

The consecutive formal stability constants K1 and K2 for the
lanthanum (III)-5, 7-dichloro-8-quinolinol system were evaluated by the
method of Freiser, El: 11. (15). The stability constant, K3, could not be
accurately determined by this method since lanthanum (III) tends to
hydrolyze at higher pH levels. The average log K, and log K; values
are 7. 24 and 6. 53, respectively.

On the basis of normal valency considerations in which the maximum
coordination number of six is recognized for lanthanum (III), and on the
basis of}; values greater than two attained at pH values greater than
about seven, it is concluded that the species responsible for the fluor-

escence of the lanthanum (III)-5, 7-dichloro-8-quinolinol system at pH 9

is probably a 1:3 complex (lanthanum (III):reagent).

LITERATURE CITED

72

10.

ll.
12.

l3.

14.

15.

l6.

17.

18.

19.

73

. Albert, A., Magrath, 1)., Biochem. J., :11» 534 (1947).

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- Bjerrum, J- - "Metal Ammine Formation in Aqueous Solution, "
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. Calvin, M., Wilson, K. W., J. Am. Chem. Soc., _6_7_, 2003 (1945).
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Ewing, G. W. , "Instrumental Methods of Chemical Analysis, " p. 196
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Fassel, V. A., Heidel, R. H., Anal. Chem., _2_6, 1134 (1954).
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Fernelius, W. 0., Bryant, B. E., Douglas, B. E.. J. Am. Chem.
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Freiser, H., Analyst, _7_7_, 830 (1952).

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Geiger, R. A., Sandell, E. B., Anal. Chim.‘Acta, 16: 346 (1957).

Ghosh, T. N., Lasker, S. 1..., Banerjer, S., J. Ind. Chem. Soc.,
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20.

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22.

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27.

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29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

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40.

74

Harvey, A. E., Jr., Manning, D. L., J. Am. Chem. Soc., Z_Z_,
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Keirs, R. J., Britt, R. D., Jr., Wentworth, W. E., Anal. Chem.,
:22, 202 (1957).

Lederer, M., Nature, _1_7_6, 462 (1955).

Lederer, M., Anal. Chim. Acta, _1_5_, 46 (1956).

Lewis, G. N., Kasha, M., J. Am. Chem. Soc., 66, 2100 (1944).
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Milkey, R. G., Fletcher, M. H., Anal. Chem., _2_8_, 1403 (1956).

Milkey, R. G., Fletcher, M. H., J. Am. Chem. Soc., _7_9, 5425
(1957). <

Nishikawa, Y., J. Chem. Soc., Japan, 22, 631 (1958).
Ibid., p. 1003.
Ibid., p. 1007.

Ohnesorge, W. E., Rogers, L. B., Spectrochim. Acta., _1_5, El
(1959); C. A., 53, 13775 (1959).

Ibid., p. 41; c. A., 53, 13775 (1959).
Patrovsk'y, v., Cheanisty, _47_, 676 (1953); c. A., 48, 3187 (1954).
Pirtea, T. 1., z. anal. Chem., 107, 191 (1936).

Pollard, F. H., McOmie, J. F. W., Stevens, H. M., J. Chem. Soc.,
3435 (1954).

Ibid., 4730 (1952).
Popovych, 0., Rogers, L. B., Spectrochim. Acta., 16, 49 (1960).

Pringsheim, P. , "Fluorescence and Phosphorescence, " Interscience,
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41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

75

Sill, C. W., Willis, C. P., Anal. Chem., _3_1_., 598 (1959).

Snell, F. D., Snell, C. T. , "Colorimetric Methods of Anal. , "
3d ed. , D. Van Nostrand, New York, 1948.

Stevens, H. M., Anal. Chim. Acta, _2_(_)_, 389 (1959).

Thommes, G. A., Unpublished Ph. D. Thesis, Michigan State
University, East Lansing, Michigan, 1956.

Vosburgh, W. C., Cooper, G. R., J. Am. Chem. Soc., 12, 4488
(1950). .

Welford, G., Harley, J., Am. Ind. Hyg. Assoc., Quart., _1_3, 232
(1952); N.S.A., _7_, 1069 (1953).

Weissman, S. I., J. Chem. Phys., 19., 214 (1942).

White, C. E., Ind. Eng. Chem., Anal. Ed., _1_1_, 63 (1939).
White, C. E., Anal. Chem., _2_1, 104 (1949).

121$, _2_2_, 69 (1950).

1213324, 85 (1952).

2.19;» 26, 129 (1954).

11313;, 32?.» 621 (1956).

I_b_i_<_i_._, 30, 729 (1958).

Ibid., 32, 47R (1960).

 

White, C. E., Ho, M., Weimer, E. Q., Spectrochim. Acta, _1_6_,
236 (1960).

White, C. E., Hoffman, D. E., Magee, J. 8., Jr., Ibid., _9_, 105
(1957).

Yuster, P., Weissman, S. I., J. Chem. Phys., 17, 1182 (1949).

I

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