On the photophysical properties of new luminol derivatives and their synthetic phthalimide precursors

Article abstract of DOI:10.1007/s10895-010-0598-0

The photophysical properties of a series of structurally related 4-aminophthalimides and the corresponding 5-aminophthalic hydrazides (luminols) are reported. Absorption, steady-state, and time-resolved fluorescence spectra of luminols exhibited substitution, solvent, and pH dependence. Singlet lifetimes have been determined by time-resolved laser flash spectroscopy. UV spectra in gas phase and DMSO solution were calculated by TD-DFT which revealed the existence of two low-energy excited singlet states with strong pH-sensitivity. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s10895-010-0598-0

J Fluoresc (2010) 20:657–664
DOI 10.1007/s10895-010-0598-0
ORIGINAL PAPER
On the Photophysical Properties of New Luminol Derivatives
and their Synthetic Phthalimide Precursors
Raúl Pérez-Ruiz & Robert Fichtler & Yrene Diaz Miara & Matthieu Nicoul &
Dominik Schaniel & Helfried Neumann & Matthias Beller & Dirk Blunk &
Axel G. Griesbeck & Axel Jacobi von Wangelin
Received: 23 November 2009 /Accepted: 4 January 2010 /Published online: 29 January 2010
#
Springer Science+Business Media, LLC 2010
Abstract The photophysical properties of a series of struc-
Introduction
turally related 4-aminophthalimides and the corresponding
5
-aminophthalic hydrazides (luminols) are reported. Absorp-
Aromatic molecules with both electron-donating and
electron-accepting substituents (I) have been intensively
investigated for their special photophysical properties
which distinguish them from simple donor or acceptor-
substituted arenes (Scheme 1) [1]. Among them, phthali-
mide derivatives (II) play an important role with diverse
applications as dyes, brighteners, bioactive drugs, bio-
markers, or photocatalysts [2]. Aminophthalic hydrazides
tion, steady-state, and time-resolved fluorescence spectra of
luminols exhibited substitution, solvent, and pH dependence.
Singlet lifetimes have been determined by time-resolved laser
flash spectroscopy. UV spectra in gas phase and DMSO
solution were calculated by TD-DFT which revealed the
existence of two low-energy excited singlet states with strong
pH-sensitivity.
(
III) belong to a related class of compounds that display
.
.
.
Keywords Fluorescence Luminols Phthalimides
unique photophysical and photochemical properties but are
still on an infant stage with regard to applications. The
parent molecule luminol, 5-amino-2,3-dihydrophthalazine-
.
Singlet lifetime pH dependence
1,4-dione, has been known for decades for its robust
chemiluminescence (CL) in the presence of oxidants, which
led to practical applications as analytical probe for metal
ions, oxidants, or anions [3]. Spectroscopic properties of
luminol, particularly its fluorescence behavior, have been
the subject of numerous investigations due to its intense
solvent-dependent emission and pronounced Stokes shifts.
Mukherjee and co-workers [4] studied the lowest excited
singlet state of luminol which exhibits a red-shifted
emission in protic solvents. Recently, Vasilescu and co-
workers [5] performed a spectroscopic study of luminol in
dimethyl sulfoxide (DMSO), DMSO-water and alkaline
DMSO-water showing a reversible acid-base equilibrium
with distinct fluorescence behaviour.
While numerous studies have been performed with
luminol and simple mono-substituted luminol derivatives,
only a handful of higher substituted compounds have been
investigated. Structural variations were carefully studied by
Brundrett and White [6], who investigated the influence of
the luminol constitution on its CL behavior. Subsequently,
White and Bursey [7] observed a beneficial effect on the
Electronic supplementary material The online version of this article
doi:10.1007/s10895-010-0598-0) contains supplementary material,
which is available to authorized users.
(
:
:
:
:
R. Pérez-Ruiz R. Fichtler Y. Diaz Miara D. Blunk
:
A. G. Griesbeck (*) A. Jacobi von Wangelin (*)
Department of Chemistry, University of Cologne,
Greinstr. 4,
5
0939 Köln, Germany
e-mail: griesbeck@uni-koeln.de
e-mail: axel.jacobi@uni-koeln.de
D. Blunk
e-mail: d.blunk@uni-koeln.de
:
M. Nicoul D. Schaniel
Department of Physics I, University of Cologne,
Zülpicher Str. 77,
5
0937 Köln, Germany
:
H. Neumann M. Beller
Leibniz-Institute of Catalysis,
Albert-Einstein-Str. 29a,
1
8059 Rostock, Germany

6
58
J Fluoresc (2010) 20:657–664
Scheme 1 Push-pull arenes as
model systems for photophysi-
cal studies
NH2
O
O
D
R
NH
NH
R
NR'
R
A
O
O
I
II
Phthalimides
III
Donor-Acceptor-Arenes
Phthalic Hydrazides (= Luminols)
CL quantum yield by methoxy substitution. Brundrett and
White [8] and Gundermann and Drawert [9] showed that
luminols bearing 6-alkyl substituents display enhanced
excited state formation and thus increased CL. Detailed
investigations of structure-activity (CL) relationships have
been limited by the lack of reliable synthetic procedures
toward new luminol derivatives. To address this bottleneck
of synthetic access to new derivatives especially with
multiple substituents, we developed a practical three-step
synthesis of luminol derivatives based upon commercially
available starting materials [10], which is part of a wider
research program directed at practical one-pot procedures
for the efficient synthesis of diversified carbocyclic and
heterocyclic building blocks [11]. Herein, we wish to report
on the detailed investigation of the photophysical properties
of a series of phthalimides and the corresponding luminol
derivatives. The study involves absorption and fluorescence
spectra in DMSO and aqueous buffer solutions at different
pH, the determination of singlet state lifetimes, DFT
calculations of UV spectra in gas phase and solution, and
a discussion of electronic and steric substitution effects.
were placed into quartz cells of 1 cm path length.
Concentrations were fixed as indicated in the text.
Time-resolved fluorescence
Singlet lifetime measurements were performed after excita-
tion with laser pulses of 120 fs length. The pump laser was
an “Integra-C” from Quantronix providing pulses of 2 mJ
with 160 fs duration at 796 nm and 500 Hz repetition rate.
Pulses of 1.4 mJ were sent to an Optical Parametric
Amplifier “TOPAS” from Light Conversion. The OPA
was set to deliver pulses of 355 nm wavelength. The laser
repetition rate was reduced to 50 Hz with the use of a
chopper. The UV pulses were then focused with a 10 cm
focal lens onto the solution under investigation. The sample
was placed slightly before the focus to avoid bubble
formation, the concentration of the probe was adjusted to
exhibit complete absorption of the UV pulse and give light
emission in a small region behind the quartz window. The
emitted light was collected with a lens and sent onto an
ultra-fast photodiode (2 GHz bandwidth). A color filter was
placed before the diode to ensure that no reflected or
scattered light from the pump pulse impinged on the
detector. The spectral traces of the ultra-fast diode were
recorded on a WavePro 960XL oscilloscope from LeCroy
(2 GHz bandwidth). Each trace was the average of 1000
single traces.
Experimental section
Materials
The synthesis of phthalimides 9–16 was realized from
commercial starting materials via sequential thermal cyclo-
addition and palladium-catalyzed transfer hydrogenation on
DFT calculations
3
0 mmol scales [10]. Simple hydrazinolysis of phthalimides
All density functional theory (DFT) calculations were
performed with the Becke three parameter hybrid functional
[12] with the correlation functional of Lee, Yang, and Parr
(B3LYP) [13] and applying the 6-311++G(d,p) [14] basis
set as implemented in the Gaussian 03 program suite [15]
applying an ultrafine integration grid (99,590). Each
geometry optimization was performed under tight optimi-
zation convergence criteria. For each of the optimized
structures the analytical Hessian was calculated (vibrational
analysis) to ensure the absence of any negative Eigen
values and thus to ascertain the minimum character of the
stationary state. Bulk solvent effects were taken into
account by applying a polarizable continuum model
(PCM) [16] with dimethyl sulfoxide parameters. For the
cavity generation, the radii from the UFF force field with
gave the corresponding phthalic hydrazides 17–24 as
colourless to off-white solids [10]. All products were
isolated and purified by silica gel flash chromatography.
Absorption spectroscopy
Absorption spectra were recorded on a Beckman Coulter
UV-DU800. Samples were placed into quartz cells of 1 cm
path length. All concentrations were 10 M.
−4
Steady-state fluorescence
Emission and excitation spectra were carried out using a
Perkin-Elmer LS-50B luminescence spectrometer. Samples

J Fluoresc (2010) 20:657–664
659
O
O
O
NH2
NH2
NH2 O
O
O
NH
+
+
O
H , Δ
Pd/C
N H
2
4
NH
NH
R
NMe
R
O
O
R
NMe
Δ
O
O
NMe
O
R
1-8
O
introduction
of diversity
NH2
NH2
O
1
O
R
R
R
R
R
= H
17(ref.)
= Me 18
= Et 19
= i-Pr 20
= Bn 21
R1
R1 = Me 10
1
R1
1
1
1
1
R
R
R
= Et
11
NH
NH
NMe
1
1
= i-Pr 12
= Bn 13
R1
O
R1
O
NH2
NH2
O
O
O
NMe
S
S
NH
NH
14
15
16
22
23
O
NH2
NH2
O
O
O
NH
NH
NMe
O
O
NH2
NH2 O
NH
NH
24
NMe
O
O
Scheme 2 Synthesis of phthalimide and luminol derivatives (parent luminol 17 as reference)
individual spheres at the hydrogens have been applied. The
UV-vis spectra were computed for the optimized structures
by calculating the first 10 excited singlet states with time-
dependent density functional theory (TD-DFT) [17].
component coupling of O-benzyl carbamate with the
corresponding unsaturated aldehyde and N-methyl maleimide
afforded the Cbz-protected aminocyclohexenes 1–8
[10a,10b,11]; 2) thermal treatment in the presence of catalytic
amounts of palladium-on-charcoal resulted in deprotection
of the amine function and concomitant oxidation of the
Results and discussion
1
0
.00
.95
1
0
Synthesis of phthalimide and luminol derivatives
1,0
1
1
1
14
1
1
2
3
The synthesis of phthalic hydrazide derivatives 17–24
involved a three-step sequence: 1) acid-catalyzed three-
0
0
,8
,6
0.90
0.85
0.80
5
16
Table 1 Absorption (abs) and emission (em) properties of phthali-
mides 10–16
3
80
390
400
λ (nm)
410
420
a
a
b
−1 c
0,4
Compound
λabs (nm)
ε
λem (nm)
Δλ (cm )
1
0
397
400
401
401
404
393
390
5712
6306
6124
5520
5909
5302
5170
470
475
472
475
454
466
460
3912
3948
3751
3885
2726
3986
3902
0
0
,2
,0
11
1
1
1
1
1
2
3
4
5
6
400
450
500
λ (nm)
550
600
650
Fig. 1 Normalized fluorescence spectra (λex=400 nm) of phthali-
mides 10–16 in DMSO under aerobic conditions; c=3.3·10 M.
Inset: Normalized absorption spectra of 10–16 in DMSO; c=10
−
6
a
c=10−4 M; c=3.3·10−6 M; Stokes shift
b
c
−4
M

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60
J Fluoresc (2010) 20:657–664
Table 2 Spectral properties of 17–24: absorption (abs) and emission
Table 3 Singlet state and fluorescence data of luminol derivatives in
(
em) maxima and Stokes shifts
DMSO
a
a
b
−1 c
a
b
8
−1 c
Compound
λ
abs (nm)
ε
λ
em (nm)
Δλ (cm
)
Luminol
E
S
(kcal/mol)
τ
S
(ns)
Ф
F
k
F
·10 (s
)
c
1
1
1
2
2
2
7
8
9
0
1
2
297
58
302
67
305
68
304
70
305
72
333
7768
7508
8972
9676
5872
7752
6232
8196
9320
10016
9048
9852
10152
8176
8304
6156
7916
17
18
19
20
21
22
23
24
75
72
72
72
72
70
75
75
1.8
2.0
2.0
2.0
2.2
0.9
2.8
1.8
0.15
0.23
0.21
0.31
0.48
0.21
0.40
0.34
0.8
1.1
1.0
1.5
2.1
2.3
1.4
1.8
3
405
425
429
430
430
3241
3718
3863
3771
3625
2392
3
3
3
a
b
Singlet energy; fluorescence quantum yield (for luminol, Ф
see ref. [5]; fluorescence rate constant, k = ф / τ
F F S
F
=0.15,
3
c
3
3
80
98
418
435
higher ring strain and reduced mesomeric delocalization of
the amide moiety, phthalimides 10–16 exhibit higher
stretching frequencies of the carbonyl bands (1755–
680 cm ) in the characteristic IR region than the six-
membered phthalic hydrazides 17–24 (1675–1589 cm ).
2
2
3
4
297
58
297
57
3
401
408
2995
3501
−
1
1
−
1
3
a
c=2.5·10−5 M; c=2.5·10−6 M; Stokes shift
b
c
Spectral properties of phthalimides
cyclohexene ring in one operation [10d]; 3) subsequent
hydrazinolysis gave the luminol derivatives (17–24) as air
stable solids in good yields [10e] (Scheme 2).
The absorption spectra of DMSO solutions of phthalimides
10–16 show a single maximum in the 390–404 nm range
(Table 1, Fig. 1 inset). The fluorescence spectra of 10–16
were recorded with a constant excitation wavelength of λex=
400 nm under aerobic conditions (Fig. 1). Emission maxima
appeared between 454 and 475 nm, benzothiophene deriv-
ative 14 exhibited the most blue-shifted fluorescence at
454 nm. The lowest singlet energies were determined from
the absorption/emission spectra overlap as 65–66 kcal/mol
From a practical standpoint it is important to note that all
starting materials and reagents for the synthesis of 17–24
are commercially available. The procedure has been
optimized to allow for the preparation of luminols on
multi-gram scales. The reactions can be performed in
standard glassware without resorting to oxygen/moisture-
free conditions. The overall yields over three synthetic steps
were in the range of 31–62 %. As a consequence of the
1
0
0
,0
,8
,6
NH2
O
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
NH
NH
18
19
2
0
O
21
22
18
2
2
3
4
absorption
excitation
emission
0,4
0
,2
0,0
2
50
300
350
400
λ (nm)
450
500
550
600
0
3
6
9
12
15
τ (ns)
Fig. 2 Normalized absorption, excitation, and emission spectra of 18
Fig. 3 Normalized fluorescence decays (λex=355 nm) of luminols
in DMSO; c=2.5·10−
6
M
18–24 in DMSO under aerobic conditions; c=1.3·10
−3
M

J Fluoresc (2010) 20:657–664
661
Table 4 Spectroscopic data
in aqueous buffer solutions
pH3
pH7
pH12
(
10 vol% DMSO) at
a
a
λ
abs
λ
em
Δλ
λ
abs
λ
em
Δλ
λ
abs
different pH
1
1
1
2
2
7
8
9
0
1
294, 351
301, 360
303, 360
304, 362
–
430
443
446
450
–
5234
5240
5356
5402
–
301, 351
309, 356
311, 357
313, 359
–
–
–
301, 351
312, 354
316, 357
317, 357
317, 362
443
5516
5589
5632
–
446
450
–
All λ values in nm; c=
−
4
22
23
–
–
–
322, 337, 381
303, 347
304, 349
437, 492
427
3294, 5852
5399
5343
322, 337, 381
303, 347
1
2
.5·10 M (λabs); c=
.5·10− M (λem);a λex=350 nm
6
296, 350
294, 351
428
431
5206
5288
(
380 nm for 22); Stokes shift
2
4
429
305, 349
−
1
in cm
.
for all phthalimides. Moderate Stokes shifts were observed
from the difference of absorption and emission maxima
(see for example Fig. 2); two separate fluorescence bands
were observed for benzothiophene compound 22. The
addition of one methyl group in the donor-acceptor system
of parent luminol 17 did not significantly alter the
fluorescence properties (see 23 and 24). Although the para
orientation of electron-donating methyl substituents and an
electron-accepting carbonyl moiety is likely to exert
electronic communication across the arene, steric effects
appear to be more dominant. The introduction of 6,8-
disubstitution in 18, 19, 20 and 21 and anellation with
benzothiophene (22) prompted moderate red-shifts of the
emission band (Δλem=20–25 nm) in comparison with 17.
Although this might be a direct consequence of the
electron-donor abilities of the substituents and facilitate
the formation of an internal charge transfer state after local
(
1
(
Table 1). Comparable values were observed for derivatives
−1
−1
−1
0 (3912 cm ), 11 (3947 cm ), 12 (3751 cm ), 13
−1
−1
−1
3885 cm ), 15 (3939 cm ), and 16 (3901 cm ). A
significantly smaller Stokes shift value was determined for
−1
the sulfur-containing derivative 14 (2677 cm ).
Absorption and steady-state fluorescence of luminol
derivatives 17–24
Previous to this study, detailed photophysical data of the
parent luminol 17 were determined in DMSO [4, 5] and a
series of other solvents [18]. In order to use luminol as
reference system and evaluate comparable spectroscopic
data, we performed all photophysical measurements in
DMSO. Table 2 summarizes the relevant spectral data that
were collected for luminol derivatives 18–24. Absorption
spectra of 18–21, 23, and 24 in DMSO exhibited two
distinct bands which were comparable to the parent
compound 17, whereas three red-shifted maxima were
found for benzothiophene 22. Concerning peak positions,
the excitation spectra completely matched the absorption
spectra. The band in the 355–380 nm region was in all
cases stronger than the 295–320 nm band. The presence of
these two bands in the absorption as well as excitation
spectra indicate the presence of two distinct electronically
excited states S and S . It is thus obvious that excited
excitation to S , we postulate a pivotal steric effect induced
1
by two substituents in ortho-positions to the amino and
carbonyl groups of the parent luminol system. This could
directly affect the geometry of the excited states and thus
influence their energy and lifetime [18]. Moderate Stokes
1
1
0
.2
.0
.8
1
2
luminol derivatives relax from the S (π,π*)-state to the S
2
1
0.6
0.4
(
π,π*)-state and, after a possible complex formation with
DMSO, emit from the S (π,π*)-state. For compound 22,
1
three bands were detected in both, absorption and
excitation spectra, all slightly red-shifted in comparison
to 17. The anellated benzothiophene in 22 serves as a
strong electron-donating group which, in addition to the
two local excited singlet states, might stabilize an internal
charge transfer state.
0
0
.2
.0
2
50
300
350
λ (nm)
400
450
The emission spectra of the luminol derivatives 17–21,
3 and 24 showed broad bands between 405 and 430 nm
2
Fig. 4 Normalized absorption spectra of 19 in H O/DMSO (9/1 v/v)
at pH3 (▬), pH7 ( ) and pH12 ( ); c=1.3·10 M
−
3
2

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62
J Fluoresc (2010) 20:657–664
pH
Scheme 3 pH-dependent
fluorescence of luminol
derivatives
pH
NH3
O
O
NH2
O
O
NH2
O
NH
NH
H
NH
N
N
N
R
R
R
-
H
O
fluorescent
fluorescent
non-fluorescent
−
1
shifts (≥ 3000 cm ) were determined for all compounds
except the sulfur-containing compound 22 (Table 2).
The excited singlet state energies were calculated from
the intersection of excitation and emission bands and
exhibited no significant dependence on the substitution
pattern (Table 3). In contrast to the excited state energies,
the fluorescence quantum yields showed significant
changes in comparison to luminol 17. Dibenzyl derivative
of the new luminol derivatives 18–24 under identical
conditions. Absorption and steady-state fluorescence meas-
urements of luminols 17–24 were performed in water at
different pH (commercial buffer solutions of pH 3, 7 and 12
with 10 vol% DMSO). As can be seen from Table 4, at least
two absorption maxima were found. Benzothiophene
derivative 22 exhibited an additional red-shifted absorption
band at 381 nm. Poor solubility of 22 (pH 3) and dibenzyl
derivative 21 (pH 3 and 7) accounted for the lack of reliable
data under these conditions. When increasing the pH, the
low-energy transition of diethyl-substituted luminol 19 is
subtly blue-shifted and accompanied by a moderate
increase of the extinction coefficient (Fig. 4); a much more
pronounced effect becomes apparent from the high energy
transition that shows a batho- and hyperchromic behavior
with increasing pH.
2
1 displayed a more than tripled fluorescence quantum
yield (0.48 vs. 0.15 for 17 [5]). The singlet state decay
traces of 18–24 showed mono-exponential behavior
(
Fig. 3). As can expected from the significantly smaller
Stokes shift, 22 has a shorter singlet state lifetime. This is in
full accord with its largest fluorescence rate constant among
the luminols studied here.
pH-dependence of absorption and steady-state fluorescence
Based on the high acidity of the phthalic hydrazide moiety
(
pK 12.7 in DMSO [19], cf. pK of acetic acid in DMSO=
a
a
A recent spectrophotometric study of luminol (17) in
DMSO, DMSO-water and alkaline DMSO-water showed
a pH dependence of its absorption and fluorescence
properties [5]. We investigated the photophysical properties
12.6), it can be assumed that the hydrazide is fully
deprotonated (dianion stage) at high pH and non-protonated
at pH 3. The 5-amino group (aniline) is not subject to
deprotonation even at pH 12, however, protonation occurs
Fig. 5 Amino-protonated
(
upper structure and spectrum)
and non-protonated (lower
structure and spectrum) phthalic
hydrazide 18 calculated by
B3LYP/6-311++G(d,p)

J Fluoresc (2010) 20:657–664
663
under acidic conditions. Thus, a complex equilibrium
Summary
+
between ArNH , ArNH , hydrazide mono- and dianionic
2
3
species is operative under various pH conditions (Scheme 3).
To make the story even more complicated, theoretical
calculations [20] revealed the existence of different tautomers
The photophysical properties of a series of substituted
phthalimides and their corresponding luminol products
were investigated and compared with that of the parent
luminol. All compounds were available by facile multi-
component reactions from simple starting materials. De-
tailed spectroscopic data of luminol derivatives 17–24 were
collected with respect to absorption and fluorescence
behaviour. Singlet lifetimes (0.9–2.8 ns, cf. 1.8 ns for
luminol 17) and quantum yields of the fluorescent excited
singlet states (0.21–0.48, cf. 0.15 for 17) were determined
in DMSO. Significant differences were detected depending
on the substituents in particular for the presence of
heterocyclic ring anellation (see benzothiophene 22).
Additionally, the photophysical behavior of luminol deriv-
atives 17–24 were studied under different pH conditions in
aqueous buffer solutions. The absorption spectra revealed
several species with a major presence of the dianionic
luminol derivative at pH 12 which exhibited no significant
fluorescence. Theoretical calculations support the assump-
tion of a stronger absorbance of the low-energy transitions
under acidic and neutral pH conditions.
(
which is especially important for the neutral and the
monoanionic species) that make a detailed analysis of the
absorption behavior challenging. The most pronounced
changes in absorption are the increased absorption in the
long-wavelength region of the UV-vis spectra (between 300
and 350 nm) at increasing pH values coupled with slight
absorption shifts. When raising the pH from 3 to neutral, this
trend can be correlated with the increasing absorption of the
neutral hydrazide portion of the molecule. Identical effects (of
different magnitudes) were also detected for luminol deriv-
atives 17, 18, 20, and 21 as well as for the mono-alkylated
compounds 23 and 24, respectively. As expected, strong
fluorescence was observed at pH 3 for all derivatives whereas
their intensities decreased to ∼50% at pH 7. In consistency
with literature results [5], no emission of any luminol
derivative was detected at pH 12: This is in full agreement
with the formation of the dianionic phthalhydrazide species
and the delocalization of the negative charges and, conse-
quently, the fluorescence quenching by internal charge
transfer. Finally, the increase in Stokes shifts at higher pH
can be interpreted as an increased charge shift stabilization of
the excited state relative to the ground state.
Acknowledgments Financial support from the Alexander von
Humboldt Foundation (postdoctoral fellowship to R. P.-R.) and the
DAAD (fellowship to Y. D-M.) is gratefully acknowledged. A. J.v.W.
is an Emmy-Noether fellow of the Deutsche Forschungsgemeinschaft
(DFG). This work has been partially funded by the 7th framework
program of the European Union (R. F. and A. J.v.W).
Theoretical considerations
In order to investigate the background of the pH-dependence
of the absorption spectra of luminol derivatives 17–21, the
electronic absorption spectra (UV-vis) of the 6,8-dimethyl
derivative 18 were calculated in the gas phase and in DMSO
environment in the protonated and non-protonated form
mimicking the situations at pH=3 and pH=7, respectively.
As can be seen in Fig. 5, the absorption spectrum of
protonated 18 (resembling low pH) is dominated by a
benzene-type transition at 219 nm and several transitions
with lower extinctions between 267 and 304 nm. In the non-
protonated state (resembling neutral pH), a strong band at
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