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ABSTRACT

A SPECTROFLUOROMETRIC AND SPECTROPHOTOMETRIC
STUDY OF THE COMPLEXES OF MORIN WITH
GADOLINIUM(III) AND LUTETIUM(III)

by Jack Edward Weiler

Gadolinium (III) and lutetium (III) both form fluorescent complexes
with morin. The various factors which affect the fluorescence of these
complexes in a dioxane-water solvent system were investigated. The
nature of these complexes was also investigated.

Gadolinium and lutetium form 1:2 complexes with morin, which
show maximum fluorescence intensity at pH 4. 25 and 3. 75, respectively.
The equilibrium constants for these complexes are 14 .8 and 32.9,.
respectively.

The fluorescence intensity peaks for both complexes are between
505 and 510 mu and their absorption peaks are at 410 mu. The morin
absorption peak is at 355 mu and it is nonfluorescing.- Excitation radiation
of 436 mu produces somewhat more. fluorescence intensity than does 365
mu. Increasing dioxane content in the solution increases the fluorescence
intensity. The fluorescence intensity and absorbance increase linearly
with increasing gadolinium 01' lutetium concentration over a range of 0 to
100 'y in the presence of 400 'y morin per 25 ml. of 50—50 dioxane-water
solution. Higher concentrations of morin result in concentration quenching.

The addition of europium, holmium, and ytterbium, which form
nonfluorescing complexes with morin, produce changes in the fluorescence
intensity of the complexes of gadolinium and lutetium. Initially the

fluorescence produced by 365 mu radiation increases due to the reduction

Jack Edward Weiler

of free morin, while that produced by 436 mp. radiation remains relatively
unchanged. After approximately 60 7 of nonfluorescing lanthanide ion is
added, in the presence of 400 y morin and 35 'y gadolinium or lutetium per
25 ml. the fluorescence produced by the 365 mu radiation is relatively
unaffected by further addition while that produced by the 436 mp. radiation
decreases due to absorption by the additional complex formed.

Tartrate, oxalate, sulfate, and phOSphate anions destroy the com-
plexes. Chloride, bromide, iodide, carbonate, acetate, and tetraborate
anions show little effect in 1. 0 mg per 2.5 m1 concentration.

Instrumental variations can produce a satisfactory linear concen-

tration fluorescence intensity relationship in the 0-10 7 region.

A SPECTROFLUOROMETRIC AND SPECTROPHOTOMETRIC
STUDY OF THE COMPLEXES OF MORIN WITH
GADOLINIUM(III) AND LUTETIUM(III)

BY

Jack Edward Weiler

A THESIS

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

MAST ER OF SCIENCE

Department of Chemistry

1961

ACKNOWLEDGMENTS

The author wishes to express his appreciation to Dr. Andrew
Timnick for instruction, guidance, and encouragement during this
investigation and course of study. '

The author also wishes to thank Dr. L. Fleck for advice, aid
and for laying such excellent groundwork in this project.

For financial aid and generous educational leave-of-absence
time, the author is indebted to the Dow Corning Corporation, Midland,
Michigan.

Without the encouragement and support of the author's wife,
Lucille, this project could not have been completed.

**************

ii

VITA

Name: Jack Edward Weiler
Born: August 22, 1928, Chicago Heights, Illinois.

Academic Career:
Bloom High School, Chicago Heights, Illinois (1946)
University of Chattanooga,Tennessee (1946-1948)
University of Illinois, Urbana, Illinois (1948-1950)

Degree Held:
B. S. Chemistry, University of Illinois (1950)

Industrial Career:
Victor Chemical Works, Chicago Heights, Illinois (1950-1953)
United States Army-Chemical Corps, Army Chemical Center,
Edgewood, Maryland (1954-1955)
Dow Corning Corporation, Midland, Michigan (1956-)

iii

TABLE OF CONTENTS

INTRODUCTION ..........................
HISTORICAL ........................
EXPERIMENTAL .........................
INSTRUMENTATION ......................
REAGENTS .......................
PREPARATION OF REAGENT SOLUTIONS ..........
EXPERIMENTAL PROCEDURES ...............
Method of Sample Preparation ..............
Instrumental Measurements ................
Parameters Affecting Fluorescence ............
Instrumental Variations to Obtain Greater Response
Per Unit Concentration ..................
Nature of the Complex ....................
Equilibrium Constant of the Complex ............
DISCUSSION OF RESULTS ...............
General . . ..........................
Parameters Affecting Fluorescence .............
Instrumental Variations to Obtain Greater Response Per
Unit Concentration . . . .................
Nature of the Complex . ...................
Equilibrium Constant of the Complex ..........
SUMMARY ..........................
CONCLUSIONS ...........................

LIT ERATURE CIT ED .......................

iv

Page

11
14
14
15
16
18
19
21
24

25
25

43
46
57
58
62
64

67

LIST OF FIGURES

FIGURE

10.

11.

12.

. Block Diagram of Spectrofluorometer ..........

. Hydrolysis Curve of Lutetium Perchlorate-Perchloric

Acid When Titrated with Standard Sodium Hydroxide .

Absorption Spectra of Morin and the Complexes of
Morin with Lutetium(III) and Gadolinium(III) in 50-50DW
atpH4.25 .............

. Fluorescence Spectra of Morin and the Complexes of

Morin with Lutetium(III) and Gadolinium(III) in 50-50DW

. Effect of pH on the Fluorescence and Absorbance of

Gadolinium(III)-Morin Complex in 50-50DW .......

Effect of pH on the Fluorescence and Absorbance of
Lutetium(III)-Morin Complex in 50-5ODW ........

. Effect of Morin on the Fluorescence and Absorbance of

87 ‘y Gadolinium(III) per 25 ml in 50—50DW at pH 4. 25 .

. Effect of Morin on the Fluorescence and Absorbance of

84 7 Lutetium(III) per 25 ml. in 50-50 DW at pH 4. 25

. Effect of Gadolinium(III) on the Fluorescence and

Absorbance of 400 y Morin per 25 ml. in 50-50DW at
pH 4. 25 ......... . . . . . ........

Effect of Lutetium(III) on the Fluorescence and Absorb-
ance of 400 'y Morin per 25 ml. in 50-50DW at pH 4. 25.

Effect of Anions on the Fluorescence of the Morin
Complexes of Lanthanum(III), Gadolinium(III), and
Lutetium(III) at pH 4. 25 .................

Effect of Dioxane Content on the Fluorescence and Ab-
sorbance of the Gadolinium(III)-Morin Complex at pH
4.25. ........... ...... .

Page

8

13

26

27

28

29

31

32

3‘3

34

36

37

LIST OF FIGURES - Continued

FIGURE

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Effect of Dioxane Content on the Fluorescence and
Absorbance of the Lutetium(III)-Morin Complex at pH
4. 25 ...... . ....... . . ............

Effect of Holmium(III) on the Absorbance of the Gadolin-
ium(III)-Morin Complex per 25 m1. of 50—50DW at pH
4.25 .............. . . .....

Effect of Holmium(III) on the Fluorescence of the Com-
plexes of Morin with Gadolinium(III) and Lutetium(III) in
SO-SODW at pH 4.25. . .........

Effect of Ytterbium(III) on the Fluorescence of the
Complexes of Morin with Gadolinium(III) and Lutetium
(111) in 50-50DW at pH 4.25 . . .....

Effect of Europium(III) on the Fluorescence of the Com-
plexes of Morin with Gadolinium(III) and Lutetium(III)
in SO-SODW at pH 4.25. . . . . ........

Instrumental Variation--Effect of Increased Mono-
chromator Slit Settings on Fluorescence Readings . .

Instrumental Variation-~Effect of Increasing Instru-
mental Sensitivity on Fluorescence Readings . .

Spectrophotometric Isoabsorptive Point for the
Gadolinium(III)-Morin Complex in 50-50DW at pH 4. 25

Spectrophotometric Isoabsorptive Point for the Lutet-
ium(III)-Morin Complex in 50-50DW at pH 4. 25 . . .

Spectrophotometric Isoabsorptive Point for the Gadolin-
ium(III)-Morin Complex in 50-50DW at pH 5. 25 .

Spectrophotometric Isoabsorptive Point for the Lutetium
(III)-Morin Complex in 50-50DW at pH 5.4 .......

Determination of the Composition of the Gadolinium(III)-
Morin Complex in 50-50DW at pH 4. 25 by the Method of
Continuous Variations . . . . . . . . ..........

Page

38

39

4O

41

42

44

45

47

48

49

50

51

LIST OF FIGURES - Continued

FIGURE

25.

26..

27.

28.

29.

Determination of the Composition of the Lutetium(III)-
Morin Complex in 50-50DW at pH 4. 25 by the Method of
Continuous Variations ...................

Determination of the Composition of the Gadolinium(III)-
Morin Complex in 50-50DW at pH 7.05 by the Method of
Continuous Variations ...................

Determination of the Composition of the Lutetium(III)-
Morin Complex in 50-50DW at pH 7. 25 by the Method of
Continuous Variations ...................

Determination of the Composition of the Gadolinium(III)-
Morin Complex in 50-50DW at pH 4. 25 by the Slope Ratio
Method ...........................

Determination of the Composition of the Lutetium(III)-
Morin Complex in 50-50DW at pH 4. 25 by the Slope
Ratio Method ........................

vii

Page

52

53

54

55

56

INT R ODU CT ION

Many compounds are photoluminescent. Photoluminescence is the
general term which describes the emission of radiant energy by a com-
pound after having first absorbed radiant energy. If the time delay
between the absorption and emission is short, that is to say of the order
10"8 seconds, the effect is more specifically termed fluorescence.

The emitted energy is usually of longer wavelength than the excita-
tion wavelength. The excitation radiation excites the molecule to one of
the many available vibrational energy levels of an excited electronic
singlet state. In the excited molecule any vibrational energy above the
lowest vibrational energy level of the excited electronic state is quickly
lost by collisions or changes in mode of vibration leaving the molecule
in the lowest vibrational energy level of the first excited electronic state.
The molecule then returns to some vibrational level of the ground state
with the loss of energy which results in fluorescent radiation of longer
wavelength than the exciting radiation .(14, 15, 35).

-abc) relates the fluorescence intensity,

The equation If = Q 10(1-10
If, to the incident radiation intensity, Io, the quantum efficiency of the
absorbed radiation in producing fluorescence, Q, the absorptivity, a,
path length, b, and the concentration, c. For low concentrations, it 'may
be shown that the equation simplifies to If=QIoabc (5, 35) and the fluores-
cence intensity is proportional to the concentration.

Many of the tripositive lanthanide ions in their salts fluoresce.
Gadolinium is in this group. Some, however, yield no fluorescence as
pure salts and lanthanum and lutetium are members of this group (21).

Only three tripositive lanthanide ions form fluorescent complexes
with morin (2',4', 3, 5, 7 pentahydroxyflavone). They are lanthanum,
gadolinium, and lutetium (19). The electronic structure of the other
lanthanide ions prevents fluorescence by the complexes formed through
intramolecular energy transfer (7, 16, 26, 36) which results in internal
quenching. The three ions which yield fluorescent complexes have an

empty, half-full, and full 4f shell.

It was the purpose of this investigation to'extend the systematic
study of the fluorescent morin complexes of the lanthanide series begun
by Fleck (7). . Factors effecting fluorescence such as pH of solution,
solvent composition, reagent concentration, excitation conditions, and
instrumental conditions were to be investigated. In addition, the nature
of the complex was to be investigated through Spectrophotometric

measurements.

HISTORICAL

Many books, chapters of books, and articles have been written
which are devoted to fluorescence and cover the field rather completely.

A few books which are readily available are: "Fluorescence and Phos-
phorescence" by P. Prigsheim (21), "Fluorescence of Solutions" by

E. Bowen (3), and "The Chemical Aspects of Light" by E. Bowen (2) give
a fairly complete coverage of the subject. In addition review articles by
White appear biyearly in Analytical Chemistry covering the field of organic
and inorganic fluorescence and instrumental development (28, 29, 30, 31,

32, 33, 34). This series is an excellent source of references.

Fleck (7) in the initial phase of this work compiled an excellent
bibliography on the fluorescence of cation complexes of morin. Morin has
been tested as a reagent for the fluorometric detection of a large number
of cations (1, 4,10, 20, 22, 23, 27). Included in this group are Be(II),

Y(III), Sc(III), La(III), Li(I), Th(IV.), Zr(IV), Al(III), Ga(III), Ge(IV), Sn(IV),
Sb(IV), Zn(II), Gd(III), and Lu(III).

Milkey and Fletcher (17, 18) conducted an investigation of the
thorium(IV)-morin complex in slightly acidic ethanol-water mixtures.

This investigation and the work of Fleck (7) were used as a pattern for this

work in hopes that comparative data could be obtained.

EXPERIMENTAL

INSTRUMENTATION

Spectrofluorometer

 

Fleck (7), of this laboratory, modified a Spectrofluorometer con-
structed in this laboratory by Thommes (24). This instrument was used
throughout this investigation. A block diagram of this instrument is shown

in Figure 1. The components are listed here:

Ultraviolet Source

 

A Hanovia S-H high pressure mercury arc with a Hanovia 110/120
volt constant voltage transformer was used as an ultraviolet source. This
source provides seven mercury lines (436, 405, 365, 313, 303, 265, and

254 mp.) which are intense enough for use in fluorescence work.

Ultraviolet Monochromator

 

A Bausch and Lomb Model 33-86-40 grating monochromator was

connected to the cell compartment with a light-tight 0-ring gasket.

Spectrofluorometer

 

A Beckman Model DU Spectrophotometer modified for use as a
Spectrofluorometer as indicated in the block diagram was used. The
light-tight cell compartment was equipped with a quartz cylindrical con-
verging lens to produce a parallel beam of ultraviolet light focused on

the center of the sample cell.

Sample C ell

 

Two clear window silica cells 10 x 20 x 50 mm, purchased from
the Farrand Optical Company were used. The cells were reproducibly
positioned in the cell compartment by means of a 10 x 20 mm cell holder
situated so that the ultraviolet beam struck the 10 mm side. These cells

are transparent to the above listed excitation radiation.

 

 

 

 

Beckman
"T / Monochromator
// i
' I \
Fluorescent ‘ \
Bediation ' t
|
l
Bausch and Iomb ‘ Photomultiplier
Grating Monochromator ) Tube 301131118
1;: rL——-—= '

 

 

 

 

 

 

/

Mercury Are ~ /
301131138 AC Power Supply and
' Photomultipller Control Unit

/

Trans former

 

 

 

Figure 1.4 Block Diagram ofl:.Speotrof1uorometer

Spectrophotometer

 

The Beckman Models DU and DK-Z spectrophotometers were used
for the absorption measurements. The Model DU was used for the single
wavelength measurements for the calculation of Keq and the composition
studies. A matched pair of 0. 988 cm silica cells was used with these

instruments .

C onstant T emperature Bath

 

A Sargent constant temperature bath, equipped with a thermo-
regulator, No. 7530, and a heater control unit, No. S-2770, was set to
maintain a temperature of 25 i 0. 20.

pH Meter

A Beckman Model G pH meter equipped with a glass-saturated
calomel microelectrode pair, was standardized with Beckman standard

pH 4 buffer solution.

REAGENTS

Ammonium Hydroxide

 

Reagent grade ammonium hydroxide was distilled and stored in a
polyethylene bottle as an approximately 0. 1 M solution. . It was protected

by an ascarite absorption bulb.

Dichlorofluorescein

 

Eastman Kodak White Label.

Dioxane

A modification of the procedure of Hess and Frahm (12) was used to

purify commercial grade dioxane. A mixture of 4 l. of dioxane, 400 m1.

10

of distilled water, and 54 m1. of concentrated hydrochloric acid was
refluxed for 12 hours. A slow purge of nitrogen was maintained by means
of a bubbler. After cooling, potassium hydroxide pellets were slowly
added, with stirring, until the solution was saturated and separated into
two layers. The dioxane was decanted and treated with additional
potassium hydroxide. The partially dried dioxane was then decanted into
a screw-cap bottle containing anhydrous calcium chloride. After stand-
ing at least 24 hours the dioxane was filtered into a 5 l. round-bottom
flask, refluxed over calcium hydride for 12 hours and distilled. A 50 m1.
forecut and about 300 ml. heel were discarded. The product distilled

at 100 i 0. 50 and was stored in a screw-cap brown bottle in the dark.

Europium S e squioxide

 

Heavy Metals Co. , Chattanooga, Tennessee. Purity unknown.

Gadolinium S e s quioxide

 

Michigan Chemical Corp. , St. Louis, Michigan. Labeled 99. 9%

pure.

Holmium Se squioxide

 

Michigan Chemical Corp., St. Louis, Michigan. Labeled 99.9%

pure.

Hydrochloric Acid

 

Bakers reagent grade.

Lutetium S e s quioxide

 

Michigan Chemical Corp., St. Louis, Michigan. Labeled 99.9%

pure.

11

Morin Dihydrate

 

Doctor Theodor Schuchardt, Munchen, Germany.

Perchloric Acid

 

Bakers analyzed reagent grade, 70-72%.

Sodium Salts

 

The various sodium salts used to determine the effects that different

anions have on the fluorescence of the complex were all reagent grade.

Water

 

All of the water used for the fluorescence work was distilled water
that had been passed through a "Crystalab Demineralizer" ion exchange

column to remove any cations present.

Ytterbium S esquioxide

 

Michigan Chemical Corp., St. Louis, Michigan. Labeled 99. 9%

pure.

PREPARATION OF REAGENT SOLUTIONS

Ammonium Hydroxide

 

The ammonium hydroxide used for pH adjustment was about 0. 03N

and was prepared from the distilled material.

Dichlorofluorescein

 

A 0. 4 7 per ml solution in 4% ethanol was prepared by dilution of
a stock solution prepared by dissolving a weighed quantity of the reagent

in 95 % ethanol.

12

Lanthanide S e s quioxide

 

Portions of the various sesquioxides were ignited to constant weight
in platinum crucibles at 750 i 250. The ignited sesquioxide was dissolved
in a measured amount of perchloric acid so that the final solution had a
0. 2 M perchlorate ion concentration and about 0. 02 M cation concentration.
Due to the purity of the reagents these solutions were not standardized.
However, the hydrolysis curve of lanthanum (III) perchlorate shown by
Fleck (7) and Freiser eta}. (15) indicates a distinctive separation between
the titration of the excess perchloric acid and the hydrolysis of the
lanthanide perchlorate. A typical hydrolysis curve is shown in Figure 2.
The concentrations based on the weight of sesquioxide used and the

amount titrated are shown.

Weight Titration
EurOpium (III) 0. 0197 M 0. 0189 M
Gadolinium (III) 0. 0243 M 0. 0240 M
Holmium (III) 0. 0220 M 0. 0218M
Lutetium (III) 0. 0228 M 0. 0227 M
Ytterbium (III) 0.0213 M 0.0209 M

Aliquots of these solutions were taken for the preparation of the working

solutions of 1.0, 0.1, 0.01 and 0.001 mg. of lanthanide sesquioxide per ml.

Ldorin

 

A weighed portion of the reagent was dissolved in purified dioxane

to produce a solution of 0.00148 moles per liter (0. 5 mg/i‘nl).

Perchloric Acid

 

The perchloric acid used for pH adjustment was about 0. 04 N and

was prepared by dilution of the reagent acid.

13

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EXPERIMENTAL PR OCEDUR ES

To establish the parameters affecting the fluorescence of the com-
plexes of gadolinium and lutetium with morin, a standard approach was
adopted. This approach was based on the results obtained in the investi-
gation of lanthanum and morin by Fleck (7). The variable being investi-

gated was altered while the other possible variables were held constant.

Method of Sample Preparation

 

The lanthanide perchlorate solution1 was measured quantitatively
into a 50 ml beaker and the morin solution quantitatively added to it.
Pure dioxane was added to give a total dioxane volume of 12. 5 ml.
Demineralized water was added to give a total solution volume of approxi-
mately 21-22 ml. The pH of the solution was adjusted with dilute NH4OH
and/or HC104 to within j; 0. 3 pH units of the desired pH and the solution
was transferred to a 25 m1. volumetric flask.7' After one hour in a 250
thermostated water bath the solution was diluted to volume and thoroughly
mixed. The final pH adjustment was made by the addition of small
increments of dilute NH4OH and/or HC104 (less than 0. 1 ml). . These
additions were made by using 1 ml. hypodermic needles and syringes.
The solution was returned to the thermostated bath until needed for

spectrofluorometric or Spectrophotometric measurement.

 

1This investigation covers the fluorescence characteristics of
both the gadolinium and lutetium morin complexes. All of the work was
carried out in exactly the same manner in both cases. For simplicity
and ease of reading the term RZO3--meaning gadolinium or lutetium
sesquioxide and lanthanide ion--meaning Gd(III) and Lu(III) will be used.
In all cases reference to holmium, europium or ytterbium- will be specific.

2All work in this investigation was carried out using 25 m1. volu-
metric flasks unless otherwise specifically stated.

15

Instrumental Measurements

 

Spectrofluorometric Measurements

 

All spectrofluorometric measurements were made with the instru-
ment described in the instrumentation section and shown in Figure 1.

The mercury arc, AC power supply and Beckman DU were turned on and
allowed to warm up for at least thirty minutes to insure instrumental
stability. The photomultiplier sensitivity control knob was set to the
"full" position. The ultraviolet monochromator entrance and exit slits
were set to 1.0 and 0. 5 mm. respectively.

The ultraviolet monochromator was adjusted to pass the 365 mu
mercury line. The Beckman DU sensitivity knob was set three turns from
the clockwise position, the selector switch to the 0. 1 position, and the
Beckman monochromator for 530 mp. The percent T scale was set to 50.
The 0. 4 7 per m1 dichlorofluorescein solution was placed in the cell,
the shutter opened and the instrument balanced by adjustment of the
Beckman entrance slit. Under the conditions used, the normal slit
setting was 1.1 i 0.05 mm.

, After standardization, as indicated above, the Bausch and Lomb,
and the Beckman monochromators were adjusted to the desired wavelength

and measurement of the samples fluorescence recorded as "% T" readings.

Spectrophotometric Measurements

 

All SpectrOphotometric measurements not used for calculations of
the Keq for the complex by the slope-ratio method, were carried out on
the Beckman DK-Z using the standard procedure and silica cells. The data
for the Keq and the slope-ratio method were obtained on a Beckman DU

using standard procedures .

16

pH Measurements

 

All pH measurements were made with a Beckman Model G battery
operated pH meter. The instrument was (allowed to warm up at least one-
half hour. It was adjusted using standard Beckman pH 4. 0 solution.

All readings were made to within 1; 0. 01 pH units. The instrument was
equipped with micro electrodes. The sample was in the closed shielded

compartment during the reading period.

Parameters Affecting Fluorescence

Effect of pH 0f the Solution

 

Solutions containing 0. 1 mg of R203,1 and 0.4 mg of morin were
adjusted to the desired pH readings with dilute NH4OH and/or HCIO4.
Twenty-eight solutions covering the range of 2. 7-10.0 pH units were
examined. The fluorescence of these solutions was measured at 510 mp
under an excitation of 365, 405, and 436 mp.. Several of these solutions
were scanned to obtain the fluorescence Spectra. The absorbance of

some of these solutions was measured.

Morin Conc entration

 

Solutions containing 0. 1 mg of R203 and amounts of morin ranging
from 0 to 2. 0 mg were adjusted to pH 4. 25 i”. 0.05]. . The fluorescence and
absorbance measurements were made. Fluorescence excitation wave-

lengths were 365, 405, and 436 mp.

RZOLConc entration

 

Solutions of 0.4 mg of morin and R203 ranging from 0. 005 mg to

1. 0 mg were made and the pH adjusted to 4. 25 :1; 0. 05. Fluorescence

-—.

A

1This notation is used for simplicity. Standard stock working solu-
tions were made to contain 1.0, 0.1, and 0.01 mg R203 per ml. This
notation avoids the use of other units which would be different for the two
sesquioxides. Data shown in figures, however, are noted as micrograms
of lanthanide ion per 25 ml volume for ease of comparison with data by
Fleck (7) on lanthanum-morin complex.

17

measurements were made under excitation of 365, 405, and 436 mp.

Absorbance measurements were made.

Anion Effect

 

The effect of 1. 0 mg of ten common anions (chloride, bromide,
iodide, sulfate, carbonate, tetraborate, phosphate, acetate, oxalate, and
tartrate), all added as the sodium salts, on the fluorescence of the com-
plex was determined. Solutions of 0.4 mg morin and 0. 1 mg of LazO3,

GdzO3, or 1.41.1203 were prepared and examined under 365 mp excitation.

Composition of the Solvent

 

Solvent mixtures of from 3 to 90% dioxane in water1 were investi-
gated. Solutions were prepared which contained 0. 1 mg of R203 and
0.4 mg of morin. The pH was adjusted as usual to 4. 25 i 0.05; however,
the actual pH of the solutions of greater than 50% dioxane is somewhat
doubtful. Freiser (8) indicated that solutions above 50% dioxane do not
exhibit true pH. Several authors have stated that nearly true pH values
are obtained below 50% dioxane (6, 8, 9). Fluorescence and absorbance

measurements were made.

Concentration of Nonfluorescing Lanthanide Morin Complexes

 

The solutions used in this portion of the investigation contained 0. 04
mg R203, 0. 4 mg morin, and 12. 5 m1 dioxane and were adjusted to a pH
of 4. 25 :1; 0. 05. In addition these solutions contained amounts of either
H0203, szO3, or EuzO3 ranging from 0.01 to 1.00 mg. The fluorescence
measurements were obtained under excitation of 365, 405, and 436 mp.

In the case of the gadolinium solutions the absorbance measurements, as

well, were obtained on the Beckman DK-2.

 

1No attempt was made to correct for the volume contraction on mix-
ing dioxane and water. In all cases the percentage refers to dioxane
content per total volume. Fleck (7) and Freiser (8) indicate the error to
be about 2% at 25°.

18

Time Stability of the Complex

 

The fluorescence of a solution of 0.1 mg of R203 and 0.4 mg morin
was examined closely over a period of six hours and then periodically for

nine days .

Wavelength of Exciting Radiation

 

The fluorescence of a solution of 0. 1 mg of R203 and 0. 5 mg morin
was examined under nine different wavelengths of exciting radiation.
The Bausch and Lomb monochromator was adjusted to irradiate the sample
cell with the following wavelengths; 254, 265, 297, 302, 313, 365, 405,
408 and 436 mp.

Instrumental Variation to Obtain Greater Response
Per Unit Concentration

 

Inc reas ed Illumination

 

The instrument was set up and standardized normally, then the
Bausch and Lomb monochromator entrance and exit slits were increased
from 1.0 and 0. 5 mm respectively, to 2.0 and 1.0 mm, respectively.
Solutions of 0. 4 mg morin and 0. 005 to 0. 04 mg R203 were prepared and
adjusted to pH 4. 25 i 0. 05. Solutions of 0.4 mg morin and 0. 005 to
0. 04 mg LaZO3 were prepared and adjusted to pH 5. 5. The fluorescence

of these solutions under excitation of 365, 405, and 436 mp was measured.

Increased Illumination and Changed Standardization

 

The Bausch and Lomb monochromator entrance and exit slits were
set at 2. 0 and 1. 0 mm. respectively, and the wavelength at 365 mp.
The normal 0. 4 7 per ml dichlorofluorescein solution was used. On the
Beckman DU, the sensitivity knob was adjusted to three-quarters of a
turn from the counter clockwise position, the selector switch set to the

1. 0 position and the percent T set on 35. The wavelength was set at 530

19

mp and with the slit open the instrument balanced by slight adjustments
of the Beckman entrance slit between 1.7 and 1. 8 mm.

Samples containing 0. 25 mg morin and a range of 0. 0002 to 0. 010
mg R203 were prepared and examined under excitation radiation of 365
and 436 mp.

The instrument was adjusted to "normal" conditions and the solu-
tions containing 0. 005 and 0. 010 were examined to give comparative

measurements.

Nature of the Complex

 

Isoabs ogtive Point

 

To determine whether more than one complex was being formed,
fourteen solutions were prepared which contained 0.4 mg morin and from
0.01 to 1.00 mg of R203. These solutions were adjusted to pH 4. 25 i 0.05,
and the absorbance measured from 350 to 450 mp using the BeckmanDK-Z.

Some of these solutions were then readjusted to pH of 5. 30 i". 0. 1

and some to pH 7. 0 i 0. 1 and the absorbance again measured.

Method of Continuous Variations

 

The method of continuous variations suggested by Job (13) and
Vosburgh and Cooper (25) was employed to prove the nature of the com-
plexes formed by the reaction of morin and either gadolinium or lutetium.
Equal molar solutions of morin, gadolinium and lutetium were prepared
by dilution of the master stock solutions. These solutions were 2.70 x 10"
M. Twelve solutions of constant total molar concentration of the two
reactants were prepared. The mole ratio (R3/R3+morin) was varied from
0.1 to 0. 9. The pH of these solutions was adjusted to 4. 25 i 0. 05 and
the absorbance measured using the Beckman DU at 354 and 410 mp.

The "Y" values were calculated and plotted against the mole ratio.

20

Since, at higher pH values, the complexes no longer fluoresce,
the above solutions were adjusted to a pH of 7.05 i 0. 05 by the addition
of small amounts of dilute NH4OH. The small error introduced by the
increased volume was neglected. . The absorbance was again measured.
Since there is a shift in the absorption peak with increase in pH these
solutions were measured at 395 and 435 mp. . The "Y" values were again
calculated and plotted against the mole ratio.

Solutions containing one part lanthanide ion and 1, 2, or 3 parts
morin were prepared and absorbance measured on the Beckman, DK-2

from 500 to 340 mp.

Slope Ratio Method

 

The slope ratio method of Harvey and Manning (11) was also used
to confirm the nature of the complex. To accomplish this, the following

investigations were conducted.

. Lanthanide Concentration Variation in the Presence of Large Excess

 

Morin. --Sixteen solutions containing gadolinium or lutetium in the concen-
tration range of approximately 0.6 x 10’6 M/l to 10. 5 x 10‘6 M/l in the
presence of a large excess morin (1.157 x 10" M) were prepared by the
standard technique. The absorbance of these solutions was determined
with the Beckman DU at a wavelength of 410 mp. The data were plotted

against concentration and the slope of the line determined.

. Morin Concentration Variation in the Presence of Large Excess

 

Lanthanide. --Twelve solutions containing morin in the concentration range

 

of 5.9 x 10"6 to 6.5 x10’5 M/l in the presence of a large excess of
gadolinium (4.84 x 10"3 M/l) or lutetium (4. 56 x 10"3 M/l) were prepared
by the standard technique. The absorbance of these solutions was measured
at 410 mp with the Beckman DU. The data were plotted against the concen-

tration and the lepe of the line determined.

21

Equilibrium Constant of the Complex

An estimation of the equilibrium constant may be obtained by employ-
ing the technique used by Milkey and Fletcher (17) in their investigation
of the thorium-morin complex. The following information was obtained in
order to perform the calculations of the equilibrium constant of the com-

plex under the conditions of the investigation.

Abs orptivity of Morin

 

The absorptivity of morin under the following conditions was
determined. Solutions were prepared which contained no lanthanide ions,
12. 5 m1 dioxane and had a final pH of 4. 25 i”. 0. 05. These solutions con-
tained morin covering the range of 1.177 x 10-5 M to 2. 364 x 10" M.
The absorptivity, (a) was calculated from the measurements made on the
Beckman DU using the formula a = A/bc where:

a - absorptivity of morin(expressed in 1/mole-cm)
A - absorbance of the solution

b - cell length

c - concentration of morin in moles/liter.

Absorptivity of the Lanthanide Morin Complex

 

The absorptivity of the complex was calculated from the slope ratio
data obtained in the presence of a large excess of the lanthanide ion using
the above formula. This large excess insured the complete reaction of
all of the morin added to form the complex and therefore one—half of the
morin concentration could be related directly to the concentration of the
complex.

Concentration of the Components in Solution After
Complex Formation

 

 

The calculation employed by Milkey and Fletcher (17) to determine
the concentration of the components of a solution of the Complex was

carried out on the data obtained in the slope ratio investigation.

22

The concentration of the complex was calculated as follows:

A-aMH(MH)b
X = .
b(aRMz+" ZaMH)

 

where,
X = moles of complex per liter
A = absorbance of the solution at 410 mp
MH = total concentration of morin in moles/liter

aMH = molar absorptivity for the morin at 410 mp
aRMI: molar absorptivity of the complex at 410 mp
b = cell path length in cm (0. 988 cm)

and, in addition, the concentration of the uncombined morin and lanthanide

ion remaining in solution may be calculated as,

Y = (MH) - um.
and
z = (R?) - (X),
where,
Y 2 moles of uncombined morin/liter
Z = moles of uncombined lanthanide ion per liter
RT+3 = total moles of lanthanide ion (Gd or Lu)/liter.

Calculation of the Equilibrium Constant

 

The information gained through the calculation of the absorptivities
and the subsequent calculation of the concentrations of the components
present in solution may be utilized in the calculation of the equilibrium
constant of the complex under the conditions of this investigation; namely,
a solution of 12. 5 ml dioxane per 25 ml total volume at a pH 4. 25 i 0.05.

The calculations were made considering the reaction to be:

23

+
R+3 + 2M = = = R(M)z + 2H+ and the equilibrium constant to be:
. .1. ' 2
eq R+ ] MH
where,
+

[R(M)z ] = X as calculated above

[133] ' = 2 as calculated above

[MH] = Y as calculated above

[H+] = The hydrogen ion concentration calculated from the pH

measurement of the solution

The solutions used for this calculation were those used to determine
the nature of the complex by the method of continuous variations. . Calcu-
lations were also carried out for the solutions used in the slope ratio

method.

DISCUSSION OF RESULTS

24

25

General

Morin (2', 4',3,5,7 pentahydroxy flavone) forms complexes with
gadolinium and lutetium which fluoresce when exposed to ultraviolet
radiation. The absorption spectra of morin and the two complexes are
shown in Figure 3, and the corresponding fluorescence spectra are shown
in Figure 4. These spectra conform to those given by Fleck (7) and
Milkey and Fletcher (17, 18). Figure 3 shows that there is an~ absorption
peak at 355 mp for the reagent and 410 mp for both of the complexes.
Figure 4 indicates a maximum fluorescence intensity for the complex
at 505-510 mp and essentially no fluorescence exhibited by the reagent
under these conditions. Since both gadolinium and lutetium were treated
identically, both will be discussed and compared under the same headings

used in the experimental section.

Parameters Effecting the Fluorescence and Absorbance

Effect of pH of the Solution

 

Figures 5 and 6 show the effect that change in pH has upon the
absorbance and fluorescence of the complex. The dip in the absorption
spectra may be accounted for by considering two factors: 1) the absorption
maxima of both the reagent and the complex tend to shift to higher wave-
length with increase in pH, and 2) the change in pH above 6 effects much
less shift so the curve tends to level off.

The increase in fluorescence noted under excitation of 365 mp radi-
ation at the higher pH values is due to the increased fluorescence of the
reagent in this region.

It is of interest to note that the maximum fluorescence intensity of
the gadolinium complex is found in the pH range of 4. 0-4.75 while that
of the lutetium complex is in the range of 3. 50-4. 25. This is considerably

lower than that found by Fleck (7) for the lanthanum complex.

26

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Effect of Morin Coéncentration

 

The effect of increasing the morin concentration, while holding the
lanthanide ion concentration constant, is shown in Figures 7 and 8.
The fluorescence intensity increases until the morin concentration is in
the region of twice that of the lanthanide ion. After this point reagent
concentration quenching occurs. This effect is most pronounced when
the excitation wavelength is 365 mp. as this is nearest to the wavelength
of maximum absorption for the reagent. The absorption of the complex
itself and equilibrium considerations cause the curvature noted before the
maximum is reached. The absorption spectra are shown for reference.

The maximum for the lutetium complex occurs at a lower morin
concentration than that for the gadolinium. . This is explained by consider-

ing the effect of pH on the equilibrium of the reaction involved, that is:

+
R”3 + ZMH === R(M)2 + ZH+.

Due to the sharpness of the pH effect on the fluorescence of the lutetium
complex and also for ease of handling, all of the investigations were
carried out at pH 4. 25, despite the fact that this was a little above the

optimum for lutetium.

Effect of the Lanthanide Concentration

 

Figures 9 and 10 show that both complexes exhibit a linear increase
in fluorescence and absorbance over the range of 0-100 7 per 25 m1.
Figures 18 and 19 confirm that the lower range (0-40 7) is linear and could

be used for analytical purposes.

Effect of Anions

 

The anions investigated fell into two classes: 1) those which destroyed
the fluorescence of the complex completely (sulfate, oxalate, tartrate, and

phosphate), and 2) those which had little or no effect (chloride, bromide,

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Fluorescence Intensity, 1510

33

 

 

 

 

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Figure 9. Effect of Gadolinium( III) on the Fluorescence
and Absorbance of 4005' Morin per 25 ml of
50-50. DW at pH 4.25

 

 

 

 

 

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Figure 10. Effect of Lutetium(III) on the

Fluorescence and Absorbance of 4008

Morin per 25 ml 012 50-50 DW at pH 1+.25.

35

iodide, carbonate, acetate, and tetraborate). The absorption spectra,
not shown, indicate that this is not a matter of quenching but rather
complete supression of the complex formation. These data are shown

graphically in Figure 11.

Effect of the Composition of the Solvent

 

The fluorescence and absorbance of a given amount of complex
increases with increasing dioxane concentration, as shown in Figures 12.
and 13, and then falls off above 70 percent dioxane -30 percent water.
However, at the higher dioxane levels the pH values are doubtful. The
absorbtion spectra of these solutions, not shown, tend to confirm this
as they show a shift of maxima to higher wavelength which is an indication
that the solutions were really at higher pH values than-the measurements

indicated.

Effect of*Nonf1uorescing Lanthanide Complexes

 

To establish the formation of a complex between morin and holmium,
europium or ytterbium, the absorbance of various solutions was measured.
Figure 14 is a typical curve which indicates that the complex is being
formed. Figures 15-17 show the effect of the above three cations on the
fluorescence of the gadolinium and lutetium complexes.

. Initially fluorescence produced by 365 mp excitation increases while
that produced by the 436 mp. excitation remains constant. This is due to
the removal of free morin from solution through the formation of additional
complex and as a result reduces the absorption of the 365 mu excitating
radiation. The fluorescence produced by the 436 mu excitation falls off
while that produced by the 365 mu excitation remains constant. This is
a result of two factors: 1) the absorption of the 436 mp radiation by the
complex formed, and Z) eventually by competition between the ions for the
morin, since these complexes are relatively weak and the equilibrium

constants for the complexes would be of the same order of magnitude.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Morin Complexes of Lanthanum( III) Gadolinium(III),
and Lutetium(III) at pH 4. 25 1n.56-5o ow.

§

365

Fluorescence Intensity, 1510

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Morin Complexes of lanthanum III)

Gadolinium(III),
and. Lutetium( III) at pH 4. 25 in 5

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43

Time Stability of the Complex

 

The fluorescence of the complex remains relatively constant for a
period of at least two days. After this period the fluorescence began to
fall off. This drop might be partially accounted for by the fact that this
investigation was carried out by making all of the measurements on the
same sample and contamination during storage and handling could result

in lower fluorescence.

Wavelength of Exc itiniR adiation

 

Excitation wavelengths of 436, 365, and 405 mp. produced fluorescence
of sufficient intensity (in decreasing order) to be of value in the detection
of the gadolinium or lutetium complex. Below 365 mu the five lines in-

vestigated produced little or no fluorescence.

Instrumental Variation to Obtain Greater Response per
Unit Concentration

 

 

Figure 18 indicates that by doubling the slit width of the ultraviolet
monochromator, the fluorescence intensity recorded was almost doubled
for a given amount of complex. Under these conditions readings satis-
factory for analytical use were obtained over a range of 0-40 7 per 25 ml
of the lanthanide ion.

By using the increased illumination in conjunction with the changes.
in standardization of the Beckman DU, the intensity of the fluorescence
is increased six fold. Figure 19 illustrates that the 0-10 7 per 25 ml
range would yield very satisfactory results if used for the determination
of small amounts of gadolinium or lutetium.

The fluorescence of the lanthanum complex is shown in Figure 18

for comparison.

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46

Nature of the Complex

 

Is oabs o rptive Point

 

Figures 20-23 show spectrOphotometric isoabsorptive points for both
lanthanide complexes as determined at a pH of 4. 25 and 5. 2-5.4. The
formation of an isoabsorptive point or a point of common intersection
produced by varying the concentrations of two reacting components is an
indication of the occurrence of a single reaction (17) between the lanthanide
ion and morin at both pH values. The loss of fluorescence with increasing
pH must then result from a change in the complex itself rather than the
formation of a different complex.

In the higher pH range the actual pH value is apparently more critical
and since reproduction of a given pH from sample to sample was very

difficult, no resemblance of order could be found in the absorption spectra.

Method of Continuous Variations

 

Figures 24-27 indicate the formation of a single complex both at pH
4. 25 and 7. 0. The maximum or minimum in the curves occur at the
position which indicates the complex formed has the composition of one
lanthanide ion to two morin ions. . This agrees with the findings for

thorium-morin complex (17, 18) and the lanthanum-morin complex (7).

Slope Ratio Method

 

The data are plotted in Figures 28 and 29. The large excess of the
one component insures complete reaction, to form the complex, of the
measured second component. From this the concentration of the complex
is known and is related to the absorbance measurements. The following

information was obtained from the plots.

47

 

”r l l I I
400 8 Morin per 25 m1 of
50-50 Dw at pH 4.25

O.9--
0.8'-
l
0.7-- 2
3

5’

9‘

l
:-

 

 

Absorbance,IA“10
.°
u\
30 \

 

7 /
Oou— .10
14
cu... «13.x Curve 6.13,;
I___ 2 .7 9 39.
0'2 3 17.4 10 174.
4 34.8; 11 218. - 3
5 52.2 12 261.
6 69.6 13 348.
7 84.0 14 867. 2
o.1~— - -I

Figure 20. Spectrophotometric Isoabsorptive
Point for Gadolinium(III) Morin. 1

Comp ex.
:0 I I I I I I I I

3&0 350 369 Wavelength mu ,390 -400 #25

 

 

 

 

 

‘1' l | | I l | I 1 I

13

bOOXMox-in per 25 m1 of
50-50 DW at pH 4.25

0.9--
0.8--

1
0.7... 3

 

 

 

 

1#
Curve LL13 , K curve L113 , X

1 g 4 8 . 103. . -
o— 2 ' 9 130 —"
0 2 3 . 16.8 10 168.
4 33.6 11 * 210. 3
5 50.2 12 252.
6 4'67.2 13 335.
7 84.0 18 800. 2

0.1— Figure 21. Spectrophotometrm Isoabsorptive
Point for Lutetium(III) Morin 1
Complex. f

.0 I I III II I

3h0 ‘ 356 ' 369 wavelength.mu. 390 #00 #25

 

 

 

Absorbance, A410

“’9

 

4005' Morin per 25 ml of
50-50 Dw at pH 5.25

0.7—
1
0.6-—
2
0.5——
0.1+—
3
MI—
5
Cuvre‘
1
' 2
0.2—- . 3
i 5

0.1L-

 

o | |

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O

807'

162.6

7 .

348. 1

Figure 22. Spectrophotometric Isoabsorptive Point
for Gadolinium(III} Marni Comp ex.

 

 

also 150 160 Wavelength mu 390 1900

425

 

 

 

 

 

51.0 j | I I I I I I
4002‘ Morin per 25 ml of
50-50 m: at pH 5.1I
9997‘
0.8—
097—
1
0.6"—
3
a .
<1:
8‘6-‘5— “ - \
§
3 /
3
40.n—
0.3—
Curve Lu3,b’
l O
2 8.4
, a 16.3
. —— 33.
° 2 5 67.2
0.1— .
Figure 23. Spectmphotometrie Isoabsorptive Point
. for Lutetium(III) Morin Complex
0 I I I I * I I I I

350

360 Wavelength mu. 39°

nob

425

 

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Condition Slope of the Plotted Line
Gadolinium Lutetium
Excess lanthanide ion 2.16 x 104 2.06 x 10"t
Excess morin 4.00 x104 4. 30 x104
Slope ratio l/l.85 1/2.06

The data confirm the one to two lanthanide ion to morin ion ratio for the

complexes formed which produce the fluorescence.

Equilibrium Constant of the Complex

 

The absorptivity of morin (8. 25 x 102 L/mole cm) and of the com~
plexes (GdM: -- 4.45 x 104 L/mole cm and LuM2+ -- 4.17 x 104 L/mole cm)
calculated using the formula a = A/bc, as indicated in the experimental
section, were used in the calculations suggested by Milkey and Fletcher
(17) to determine the concentration of the components at pH 4. 25 i O. 05.
These data are shown in the Appendix. This information was used to _
calculate the equilibrium constants for the complexes which are included
in the Appendix. The average values are 32. 9 for the lutetium complex
and 14. 8 for the gadolinium complex. These are of the same order of
magnitude but slightly higher than the equilibrium constant for the
lanthanum complex reported by Fleck (7).

The calculation was also carried out using the data obtained in the
Slope Ratio Method. These data gave very erratic results because the
reaction was forced to completion with a large excess of one component.
The resulting small concentration of uncombined second component was
used in the calculation of the equilibrium constant. Errors in this small
concentration lead to large variations in the calculated equilibrium con-

stant values. The magnitude of the variation was of the order of fifty fold.

SUMMARY

58

59

Since the complexes formed by gadolinium and lutetium with morin
were both investigated, all general statements made will refer to both of
the complexes. Specific references will be made where differences were
noted.

Absorption peaks for morin and the complexes were recorded at
355 and 410 mp, respectively. , The fluorescence spectra of the complexes
maximized in the 505-510 mp. region. . Morin exhibits little if any fluorescence
except at high pH values.

The pH of the solution has drastic effects on both the fluorescence
and absorbance of the complexes. I Maximum fluorescence intensity of the
gadolinium complex 13%,? occurs at pH 4. 5 while that of the lutetiumcom—
plex occurs at pH 4. 0. It is of interest to note that in both cases the 122
maximum fluorescence intensity occurs approximately a half of a pH unit
lower. The absorption maximum occurs at pH 4. 8 and 4. 2 for the gado-
linium and lutetium complexes, respectively. This maximum is partially
due to a shift in absorption to longer wavelengths withincreasing pH.

The absorbance changes nearly linearly with increasingmorin
concentration over a range of 0 to 500 y in the presence of approximately
85 7 of the lanthanide ion per 25 m1 of solution. . Maximum fluorescence
intensity was noted at approximately 300 'y morin for gadolinium and 200 'y
morin for the lutetium. ~Above these values the fluorescence intensity
falls off due to concentration quenching by the morin. .,1§},2 falls very
rapidly due to the strong absorption of the exciting radiation by free morin
in this region.

In the presence of 400 7 morin both the fluorescence and absorbance
intensity of the complex increases linearly over a range of 9 to 100 7 of the
lanthanide ion per 25 ml solution.

The effect of anions on the fluorescence and absorbance of the com-
plexes falls into two categories: 1) those having little or no effect

(chloride, bromide, iodide, carbonate, acetate and tetraborate), and

60

2) those which inhibit the complex formation completely (sulfate, 'phos-
phate, oxalate, and tartrate).

The fluorescence intensity increases with increasing dioxane concen-
tration up to the point where the adjustment of the pH of the solution is no
longer meaningful. The fact that the pH values of the high dioxane
concentration solutions is in error is borne out by observed shifts in the
absorption peak which indicates a higher pH than that recorded. Similar
effects were noted in the absorption measurements.

The three lanthanide ions, holmium (III), ytterbium (III), and
europium (III) form nonfluorescing complexes‘with morin. A The increase
in absorption-at 410 ms with increasing concentration of these ions in the
presence of gadolinium or lutetium proves this point. The fluorescence
intensity 12;: remains essentially unchanged in the presence of increas-
ing amounts of these three lanthanide ions. . However when the total
lanthanide ion concentration moves above the 100 7 per 25 ml range, the
complexes present in the solution absorb a considerable amount of 436 mu
radiation and reduce the fluorescence intensity. The fluorescence
intensity‘Igég initially increases with increasing concentration of these
three lanthanide ions. This is due to the removal of free morin from
solution through the formation of the additional complex. Free‘morin
absorbs strongly in the 365 mp. range and reduces the excitation radiation.

Both complexes were stable for from two to five days as indicated
. by fluorescence measurements.

. Increasing the sensitivity of the instrument lowers the detection
limit by increasing the instrument response for a given amount of
fluorescence. A very suitable working curve was produced over the range
of 0-10 7 of lanthanide ion per 25 ml. One complex is formed between
the lanthanide ions and morin as indicated by the formationof one iso-
absorptive point (17) in the absorption spectra of a series of solutions

of constant morin concentration and varying lanthanide ion concentration.

61

Job's (13) method of continuous variations and Harvey and Manning's
(ll) slope ratio method indicate this to be a 1:2 lanthanide ion : morin
Complex. This relationship holds true at pH 4. 25 where fluorescence
occurs, as well as at pH 5. 2 and 7.0 where the complex no longer exhibits
the strong fluorescence. This would indicate a change in the structure of
the complex rather than the formation of a different complex is responsible
for the change in fluorescence character of the system.

The equilibrium constants, Keq: 32.9 for the lutetium complex and
14.8 for the gadolinium complex are slightly higher but of the same order

as the value reported for the lanthanum complex (7).

CONCLUSIONS

62

63

The fluorescent complexes formed by gadolinium and lutetium with
morin at pH 4. 25 in a 50-50 mixture of dioxane and water are suitable
for use in the determination of these cations singly over a concentration
range of 1-100 7 per 25 ml of solution.

The three fluorescing complexes formed with morin by lanthanum,
gadolinium, and lutetium are in general affected in the same way by all
the variables investigated. However, they might be separated to some
extent through careful pH control, multiple measurements at different
pH values and excitation wavelengths and the use of simultaneous equations.
The presence of the non-fluorescing lanthanide complexes would not

interfere unless present in relatively large amounts.

LIT ERATU RE CIT ED

64

10.

11.

12.

13.

14.

15.

16.

17.

18.

65

. Bailar, J._ C. Jr. , ed. , "Chemistry of the Coordination Compounds, "

A.C.S._ Monograph Series No. 141, p. 694, Reinhold Publ. Co.

. Bowen, E. J. ,4 "The Chemical Aspects of Light, " 2d ed. , Oxford

Pres s ,. London, 1946 .

. Bowen, E. J. , and Wokes,. F. ,. "Fluorescence of Solutions, "

Longmans, Green, and Co., New York, 1953.

.. Charlot, (3., Anal- Chim. Acta, _1_, 218 (1947).

. Ewing, G. W. , "Instrumental Methods of Chemical Analysis, "

p. 196,. McGraw Hill Co. ,. New York, 1954.

. Fernelius, W. C., Bryant, B. E., and Douglas, B. E.,. J. Am. Chem.

Soc., 1.2, 3784 (1953).

. Fleck, L. L., Ph. D. Thesis, Michigan State University, East Lansing,

Michigan, 1961.

. Freiser, H. , Analyst, 21,, 830 (1952).

Freiser, H., Charles, R. C., and Johnston, W. D., J.Am._. Chem.
Soc., 14, 1383 (1952).

Goto, H. ,. J. Chem. Soc. Japan, 60—, 937 (1939); C. A., E' 4355 (1940).

Harvey, A. E., Jr., and Manning, D- L.,. J. Am. Chem. Soc., _7_2_,
4488 (1950).

Hess, K., and Frahm, H., Ber., 11, 2627 (1938).
Job, P., Ann. Chim., 2, 113 (1928).

Keirs, R. J., Britt, R. D., Jr., and Wentworth, W. E., Anal. Chem.,

.22, 202 (1957).

Lewis, G. N.,. Kasha, M., J. Am. Chem. Soc., 66, 2100 (1944).
Livingston, R. S.,. J. phys. Chem., _6_l_, 860 (1957).
Milkey, R. C., and Fletcher, M. H., Anal- Chem., _2_8_, 1403 (1956).

Milkey, R. C., and Fletcher, M. H., J. Am. Chem..Soc., Z_9_, 5425 I
(1957).

19.

20

21..

66

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

Ibid. , 4730 (1952).

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

. New York, 1949.

22.

23.

24.

25.

26.

27.

28.

29

30.

31.

32

33

34.

35.

36.

Sill, C. W., Willis, C. P., Anal. Chem., _3_l, 598 (1959).
Stevens, H. M., Anal. Chim. Acta, £0, 389 (1959).

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

Vosburgh, W. C., and Cooper, G. R., J. Am. Chem. Soc.', _6__3_,
437 (1941).

Weissman, S. I.,. J. Chem. Phys., 1_0, 214 (1942).

Welford, C., Harvey, J., Am. Ind. Hyg. Assoc., Quart., _1_3, 232
(1952); N.S.A., l, 1069 (1953).

White, C. E., Anal. Chem., 2_1_, 104 (1949).

.- Ibid., 22, 69 (1950).

Ibid., 24, 85 (1952).

 

Ibid., 26, 129 (1954).

 

.- Ibid., _2_8, 621 (1956).

.- Ibid., _§_0_, 729 (1958).

 

Ibid., 32, 47R (1960).

 

Willard, H. H. , Merritt, L., L. , , Jr. , Dean, J.’ A. ,. "Instrumental
Methods of Analysis, " 3d ed. , D. Van Nostrand, New York, 1958.

Yuster, P., Weissman,. S. I., J. Chem- Phys., _11, 1182 (1949).

APPENDIX

67

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