Fluorescence spectroscopic behaviour of folic acid

Article abstract of DOI:10.1016/j.chemphys.2009.10.026

The fluorescence spectroscopic behaviour of folic acid (FA) in 4 M HCl (dominant bi-cationic form), 0.1 M HCl (bi-cationic and cationic form), citric acid-NaOH pH 6 buffer (neutral form), 0.1 M and 4 M KOH (anionic form), and trifluoroacetic acid is studi

Full text of DOI:10.1016/j.chemphys.2009.10.026

Chemical Physics 367 (2010) 83–92
Contents lists available at ScienceDirect
Chemical Physics
journal homepage: www.elsevier.com/locate/chemphys
Fluorescence spectroscopic behaviour of folic acid
*
A. Tyagi, A. Penzkofer
Institut II – Experimentelle und Angewandte Physik, Universität Regensburg, Universitätstrasse 31, D-93053 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The fluorescence spectroscopic behaviour of folic acid (FA) in 4 M HCl (dominant bi-cationic form), 0.1 M
HCl (bi-cationic and cationic form), citric acid–NaOH pH 6 buffer (neutral form), 0.1 M and 4 M KOH
(anionic form), and trifluoroacetic acid is studied. The thermal stability is investigated. Absolute absorp-
tion cross-section spectra are determined and compared with fluorescence excitation spectra. Intrinsic
fluorescence quantum distributions and fluorescence quantum yields are extracted from fluorescence
spectra measurements. The temporal fluorescence decay after picosecond pulse excitation is studied.
The fluorescence quenching mechanisms for the different ionic forms of FA are discussed: excited-state
proton release for bi-cationic FA, photo-physical non-radiative relaxation for cationic FA, and photo-
induced intra-molecular electron transfer for neutral and anionic FA. Aerobic FA in 4 M KOH at elevated
temperature dehydrated to 9,10-dehydro-folic acid. Its photo-dynamics was governed by twisted intra-
molecular charge transfer and photo-isomerisation.
Received 7 September 2009
In final form 28 October 2009
Available online 1 November 2009
Keywords:
Folic acid
Absorption cross-section spectra
Fluorescence excitation spectra
Fluorescence quantum distributions
Fluorescence lifetimes
Photo-induced proton release
Intra-molecular electron transfer
9,10-Dehydro-folic acid
Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction
glutamic acid part have not yet been determined [9]. They are ex-
pected to be similar to the pK values of free glutamic acid in aque-
Folic acid (pteroylglutamic acid, abbreviated as FA) is a widely
distributed vitamin (called vitamin BC, vitamin M, or vitamin B9).
It is involved in single carbon transfer reactions in metabolism,
and it is the precursor of the active tetrahydrofolic acid coenzyme
[1]. It was first discovered in spinach [2]. Folic acid deficiency
causes growth weakness in mammals and different kinds of anae-
mia; it causes a failure to make the purines and thymine required
for DNA synthesis [3]. FA is a potential agent for cancer prevention
[4,5] by free radical scavenging and antioxidant activity [6]. FA is
made up of the building blocks pterin, p-amino-benzoic acid, and
glutamic acid. Folate is the preferred name of folic acid and folic
acid derivatives.
The chemistry and biochemistry of folates and pterins is re-
viewed in [1,7,8]. Depending on the pH of aqueous solutions, folic
acid exists in different ionic states. The structural formulae of FA in
neutral, anionic, cationic, and bi-cationic form are shown in Fig. 1
[9]. The change from the neutral form to the anionic form occurs
with a dissociation constant of pKn,a = 8.38 [9], the change from
neutral form to cationic form has a dissociation constant of
pKn,c = 2.35, and the pK value for the transformation from cationic
form to bi-cationic form was reported to be pKc,bc = 0.20 [9]. The pK
ous solution (pK = 2.1 and pK = 4.07 [10]). Absorption spectra of
a c
FA in its different ionization states are found in [1,7–9,11,12]. Fluo-
rescence studies on folic acid were carried out in [13–19]. No fluo-
rescence was found in [13,14]. pH dependent fluorescence
behaviour was reported in [15] where fluorescence spectra are
shown for pH 2, 5, 6, 7, 8, and 9. Fluorescence quantum yields were
found to be less than 0.005 in [16,17] for acid and basic forms of FA.
Fluorescence quenching processes are discussed in [16,18–20]. The
photo-stability of FA was studied in [1,8,16,19,21–26].
In this paper a fluorescence spectroscopic characterization of FA
in 4 M aqueous HCl (pH ꢀ ꢁ0.6, dominant bi-cationic folate), 0.1 M
aqueous HCl (pH ꢀ 1, mixture of cationic and bi-cationic folate),
aqueous citric acid-NaOH pH 6 buffer (neutral folate), 0.1 M aque-
ous KOH (pH ꢀ 13, anionic folate), 4 M KOH (pH ꢀ 14.6, anionic fo-
late), and trifluoroacetic acid (TFA, pK = 0.5 [27]) is undertaken. The
thermal stability of FA in these solvents is studied. Fluorescence
emission spectra, fluorescence excitation spectra, and temporal
fluorescence decay traces are measured. The fluorescence quantum
distributions, EF(k), and the fluorescence quantum yields, /F, are
calculated by calibration to standard reference dyes. Fluorescence
lifetimes are extracted from temporal fluorescence traces obtained
by picosecond laser pulse excitation and streak-camera or micro-
channel-plate photomultiplier signal detection. Schemes of the
photo-dynamics are derived from the experimental results. The
fluorescence behaviour of bi-cationic folate is interpreted in terms
values of the a-carboxyl group and of the c-carboxyl group of the
* Corresponding author. Tel.: +49 941 943 2107; fax: +49 941 943 2754.
E-mail address: alfons.penzkofer@physik.uni-regensburg.de (A. Penzkofer).
0301-0104/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.chemphys.2009.10.026

84
A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
and in TFA. Additional measurements were carried with FA in
Tris–HCl pH 8 buffer (25 mM Tris + 150 mM NaCl + 5 vol.% glycerol,
pH adjusted with HCl, Tris = (HOCH2)3CNH2).
O
COO-
H α
1'
2'
5'
O
N
H
3'
4'
9
Folic acid is poorly soluble in aqueous solution at room temper-
ature, but the solubility rises with temperature [1]. Therefore, we
dissolved folic acid in the aqueous solvents of different pH at
70 °C and then let the samples cool down to room temperature.
The so prepared samples remained dissolved without agglomerisa-
tion for a few days. FA is good soluble in TFA. The temporal stability
of the samples at room temperature was tested by measuring the
absorption spectra after sample preparation in certain time inter-
vals over a period of a few days. No remarkable absorption changes
were observed. The dye aggregation was checked by 90° static light
scattering experiments [31] using a HeNe laser (kL = 632.8 nm) for
excitation and a photomultiplier for detection. Centrifuged sam-
ples showed approximately the same light scattering as the corre-
sponding solvents. Heating of FA in 4 M HCl reversibly shifted the
bi-cationic – cationic equilibrium to the cationic side (rise of long-
wavelength absorption, see below). Prolonged heating of FA in TFA
caused absorption spectral changes due to thermal degradation
(see below). Prolonged heating of FA in 4 M KOH oxidized FA to
9,10-dehydro-folic acid (DHFA, see below).
6'
10
N
H
β
N
CH2
4
5
HN
3
6
7
γ
CH2
COO-
2
8
1
H2N
N
N
Folic acid (neutral)
O
COO-
H
O- K+
N
H
N
CH2
CH2
COO-
N
N
H
H2N
N
N
Folic acid (anionic)
O
COOH
O
N
H
H
Absorption cross-section spectra of the samples were deter-
mined with a Cary 50 spectrophotometer (from Varian, Darmstadt,
Germany). The absorption in the long-wavelength absorption tails
was measured using cells of 10 cm length; otherwise cells of 1 cm
length were used.
N
CH2
HN
N
CH2
H
H2N
N
N
COOH
H+ Cl-
The fluorescence spectra, SF(k), of the samples were measured
with a Cary Eclipse spectrofluorimeter (from Varian). For absolute
intrinsic fluorescence quantum distribution, EF(k), calibration of
the samples, the dyes 2-amino-pyridine in 0.1 normal aqueous
H2SO4 (absorption below 330 nm, fluorescence quantum yield
/F = 0.60 [32,33]), POPOP (1,4-di-(5-phenyloxazolyl)benzene) in
ethanol (absorption below 380 nm, /F = 0.85 [34]), and coumarin
314T in ethanol (absorption below 460 nm, /F = 0.87% according
to data sheet of Kodak) were used as fluorescence standards. The
fluorescence was collected perpendicular to the excitation path.
The samples were excited with vertical polarized light, and the
fluorescence polarized to the magic angle (54.7° to the vertical)
with a polarizer was detected. The fluorescence quantum yield,
Folic acid (cation)
O
COOH
H
O
N
H
N
CH2
HN
N
H2
CH2
+
H2N
N
N
COOH
2Cl-
H+
Folic acid (bi-cation)
Fig. 1. Structural formulae of folic acid in neutral (sum formula: C19H19N7O6, molar
mass Mm = 441.4 g molꢁ1), anionic, cationic, and bi-cationic form.
/F, is obtained by integration over the fluorescence quantum distri-
R
bution, i.e. /F ¼ EFðkÞdk.
Fluorescence excitation spectra, Eex;k ðkÞ, were also determined
det
with our Cary Eclipse spectrofluorimeter by collecting the fluores-
of photo-induced proton release and subsequent thermal activated
internal conversion. For cationic folate a temperature independent
non-radiative decay is found. For neutral folate and anionic folate
the fluorescence is thought to be quenched by intra-molecular
electron transfer. Higher excited-state deactivation towards the
ground-state via conical intersection are made responsible for
excitation wavelength dependent fluorescence quenching (viola-
tion of Vavilov’s rule of excitation wavelength independent fluo-
rescence quantum yield [28,29] and of Kasha–Vavilov’s rule of
exclusive fluorescence emission from S1 state with efficiency inde-
pendent of the excitation wavelength [30]).
cence at a fixed emission wavelength of kdet and scanning the exci-
tation wavelength k through the absorption region [35]. Eex;k ðkÞ is
det
defined as Eex;k ðkÞ ¼ SF;mðkdetÞ=PinðkÞ ¼ ½1 ꢁ TðkÞꢂ/FðkÞ where
j
det
SF,m(kdet) is the magic-angle fluorescence signal at the detection
wavelength, Pin(k) is the excitation power, T(k) = exp[ꢁ
the transmission ( : absorption coefficient, ‘: sample length),
/F(k) is the fluorescence quantum yield belonging to the excitation
a(k)‘] is
a
wavelength, and
j is a proportionality constant. In the case of high
transmission (T > 0.8) it is Eex;k ðkÞ ꢀ j0
a
ðkÞ/FðkÞ ¼
00raðkÞ/FðkÞ. If
j
det
the fluorescence quantum yield, /F, is independent of the excita-
tion wavelength, then the shape of the fluorescence excitation
spectrum is equal to the shape of the absorption spectrum
A(k) = 1 ꢁ T(k), and for high transmissions it is approximately equal
2. Experimental
to the shape of the absorption cross-section spectrum, ra(k).
Folic acid (purity P0.985, water content 7.9%) was purchased
from Schircks Laboratories, 8645 Jona, Switzerland. The other chem-
icals were bought from Sigma–Aldrich, Germany, in highest purity
quality. The chemicals were used without further purification.
The samples were studied under aerobic conditions (solvents
used as delivered). FA was dissolved in 4 M aqueous HCl, 0.1 M
aqueous HCl, aqueous citric acid–NAOH pH 6 buffer (commercial
solution from Aldrich), 0.1 M aqueous KOH, 4 M aqueous KOH,
Fluorescence decays of the samples were studied by picosecond
laser pulse excitation and time-resolved fluorescence detection.
Measurements at room temperature and at liquid nitrogen tem-
perature (77.2 K) were carried out with excitation at 351.3 nm. A
mode-locked Nd:phosphate glass laser system [36] with second
harmonic generation (in a non-critical phase-matched CDA crystal
[37]) and third harmonic generation (frequency mixing of funda-
mental and second harmonic light in an angle phase-matched

A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
85
BBO crystal [37]) was used for excitation pulse generation (pulse
duration DtL ꢀ 6 ps, wavelength kL = 351.3 nm). The fluorescence
signal was detected with an ultrafast streak-camera (type C1587
temporal disperser with M1952 high speed streak unit from
Hamamatsu) [38]. The streak-camera has a time resolution of
about 3 ps in the fastest streak speed of 300 ps/15 mm (time
window 165 ps), and a time resolution of about 100 ps in the slow-
est streak speed of 10 ns/15 mm (time window 5 ns). For measure-
ments at 77.2 K the sample cells were purged into an optical glass
dewar filled with liquid nitrogen.
Further fluorescence lifetime measurements at room tempera-
ture were carried out by sample excitation at 400 nm (pulse dura-
tion ca. 4.5 ps) using second harmonic pulses of a Ti:sapphire
femtosecond laser oscillator-amplifier system (laser system Hurri-
cane from Spectra Physics) and signal detection with a micro-chan-
nel-plate photomultiplier (Hamamatsu type R1564-U01) in
combination with a fast digital oscilloscope (LeCroy type DSO
9362).
absorption spectrum of FA in TFA is similar to the absorption spec-
trum of FA in 4 M HCl. In 0.1 M HCl, FA is present in single cationic
and bi-cationic form (see below). The absorption strength,
R
½
raðkÞ n kꢂdk, of the first absorption band is moderate. The absorp-
tion cross-section spectrum of FA in citric acid–NaOH pH 6 buffer
(neutral folate form) has a weak absorption band around 440 nm
and a medium absorption band peaking at 345 nm. The long-wave-
length absorption band is attributed to charge-transfer state
absorption [41,42]. For FA in 0.1 M KOH and 4 M KOH (anionic fo-
late form) the absorption behaviour is similar to the neutral situa-
tion. The first absorption band has its maximum at 365 nm. A long-
wavelength absorption tail is present.
Heating of FA in 4 M HCl from 22 °C to 85 °C increased the
absorption strength of the long-wavelength absorption band
(k > 360 nm) approximately a factor 1.8. Cooling down the sample
brought the absorption back to the original situation. Due to this
behaviour we interpret the long-wavelength absorption band of
FA in 4 M HCl as originating from cationic FA (heating shifts bi-cat-
ionic – cationic equilibrium towards the cationic side, see below).
Heating of FA in TFA irreversibly raised the absorption strength
of the long-wavelength absorption band (k > 360 nm, no decrease
in absorption by cooling down) likely by increasing the fraction
of single-cationic FA (FAH+). The absorption spectrum changes of
FA in TFA due to heating up to 65 °C and keeping the sample at this
temperature are shown in Fig. 3a. The long-time heating (>8 h) de-
creased the absorption band centred at 380 nm and decreased
more strongly the dominant absorption band centred at 315 nm.
The chemical reactions causing thermal degradation are not iden-
tified here.
For picosecond fluorescence excitation at 449 nm our Ti:sap-
phire laser system was operated at 397 nm and the radiation was
red-shifted by stimulated Raman scattering in ethanol (Stokes shift
2928 cmꢁ1) [39] using a Raman generator – amplifier system [40]
with two 5 cm cells in series (distance between cells ca. 30 cm for
divergence reduction).
3. Results
The absorption cross-section spectra of the samples at room
temperature are shown by the thick curves in Fig. 2. The dye con-
centrations were around 4 ꢃ 10ꢁ5 mol dmꢁ3. In all cases no vibron-
ic structure is seen indicating an inhomogeneous broadening of the
transitions. The shapes of the spectra depend on the pH of the
aqueous solutions (ionicity of the folates). The main absorption
band of FA in 4 M HCl peaks at 320 nm. A weak absorption plateau
is present in the 360–430 nm region. FA is dominantly present in
bi-cationic form. The long-wavelength absorption plateau is attrib-
uted to the presence of some FA in single-cationic form. The
Prolonged heating of FA in 4 M KOH irreversibly converted FA
into a new species. The temporal absorption spectrum develop-
ment at 85 °C is shown in Fig. 3b. The spectra have isobestic points
at 381 nm and 298 nm indicating that only one new species was
formed. The new species has its absorption maximum at 425 nm
and its absorption cross-section there is 5.1 ꢃ 10ꢁ17 cm2.
We think that a double bond is formed between C9 and N10 (for
numbering see Fig. 1) by the oxidative action of the dissolved
a
a
b
b
Fig. 2. Absorption cross-section spectra (thick curves) and fluorescence excitation
spectra (thin curves, detection wavelength 480 nm, normalized to absorption cross-
sections at 370 nm) of folic acid in various solvents. The used solvents are indicated
in the figure.
Fig. 3. Development of absorption coefficient spectra due to sample heating.
Storage times, theat, of samples at temperatures # are indicated. (a) FA in TFA at
# = 65 °C. (b) FA in 4 M KOH at # = 85 °C. Inset shows absorption coefficient at
425 nm versus heating time.

86
A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
oxygen under the catalytic action of KOH. The following reaction is
thought to take place:
460 nm. Excitation into the long-wavelength absorption tail results
in an enhanced red-shifted emission similar to the pH 6 situation.
In the case of short wavelength excitation a weak additional emis-
sion around 350 nm could be resolved. This weak emission may be
due to the presence of some p-amino-benzoic acid fragments [44]
and some higher excited state emission.
Fluorescence quantum distributions of FA in TFA which was
thermostated at 65 °C for 3 h are displayed in Fig. 5a for different
excitation wavelengths. The spectra have their maxima at around
480 nm. The fluorescence quantum yield rises with excitation
wavelength.
The fluorescence quantum distributions of FA in 4 M KOH and of
9,10-dehydro-FA (DHFA) in 4 M KOH (aerobe sample kept at 85 °C
for 10 h) are displayed in Fig. 5b. The FA spectra have their maxima
at around 490 nm, the DHFA spectrum peaks at around 580 nm.
The fluorescence quantum yield of FA rises with excitation wave-
length (/F(320 nm) ꢀ 0.00036, /F(338 nm) ꢀ 0.00063). The fluo-
rescence quantum yield of DHFA is /F = 0.021 0.002.
The excitation wavelength dependence of the fluorescence
quantum yield, /F, of FA in the studied solvents at room tempera-
ture is summarized in Fig. 6. The total fluorescence quantum yield,
KOH;85 ꢄ
!
FA þ O2
C 9; 10-dehydro-FA þ H2O2:
The inset in Fig. 3b shows the build-up of absorption at 425 nm at
85 °C with time t. The absorption dependence is well fitted by
aðt ꢁ t0Þ ¼
aðt0Þ½1 ꢁ expðꢁk1tÞꢂ;
ð1Þ
with a quasi-unimolecular rate constant of k1 = 9.4 ꢃ 10ꢁ5 sꢁ1 (dis-
solved O2 concentration is much higher than FA concentration). The
consumption of FA is approximately given by
d½FAꢂ
¼ ꢁk2½O2ꢂ½FAꢂ ¼ ꢁk1½FAꢂ;
ð2Þ
dt
with k1 ¼ k2½O2ꢂ. Heating an anaerobe sample of FA in 4 KOH under
the same conditions as the aerobe sample resulted in no absorption
spectra changes (no dehydration occurred). In passing, it is noted
that a similar dehydration is taking place in the biogenesis of green
fluorescent protein [43].
In Fig. 4a–e fluorescence quantum distributions, EF(k), of FA in
the different aqueous solvents and in TFA at room temperature
are shown. In each sub-figure EF(k) curves are shown for several
excitation wavelengths. All emission bands are rather broad. They
show no vibronic structure. The fluorescence efficiency depends on
the excitation wavelength. Vavilov’s rule of excitation wavelength
independent fluorescence emission efficiency [28,29] is violated
for the studied folate samples.
For FA in 4 M HCl and in TFA the main emission band is centred
at 485 nm. A short-wavelength emission band centred at 370 nm is
additionally resolved in the case of short wavelength excitation.
The main emission is attributed to S1–S0 emission from single-
cationic FA (excited-state proton release, see below), and the
short-wavelength emission is attributed to S1–S0 emission from
bi-cationic FA (see below). For FA in 0.1 M HCl the emission max-
imum occurs at around 470 nm. No short-wavelength-emission
band was resolved. For FA in pH 6 buffer the fluorescence emission
peak emission occurs at 445 nm. In the case of long-wavelength-
excitation (kexc P 400 nm) the emission maximum shifts to longer
wavelength. For FA in 0.1 M KOH the main emission band peaks at
/F,tot (thick curves), and the quantum yield of the short wavelength
emitted fluorescence, /F,short (thin curves), are shown. In all sol-
vents the fluorescence quantum yield varies with excitation wave-
length. The highest fluorescence quantum yield in the few percent
range was obtained for FA in TFA.
The wavelength dependence of the fluorescence quantum yield
is also seen in the fluorescence excitation spectra which are in-
cluded in Fig. 1 for FA in 4 M HCl, 0.1 M HCl, and 0.1 M KOH (thin
curves). For wavelength independent fluorescence quantum yields
the presented normalized fluorescence excitation spectra,
Eex;nðkÞ ¼ Eex;480 nmðkÞrað370nmÞ=Eex;480 nmð370nmÞ, should coincide
with the absorption cross-section spectra, which is not the case.
Over the presented wavelength range the sample transmission
was kept higher than 70%.
Picosecond-pulse-excited temporal fluorescence traces mea-
sured with a streak-camera are shown in Fig. 7 (excitation
a
a
b
c
b
d
e
Fig. 5. (a) Fluorescence quantum distributions at room temperature for FA in TFA
after 3 h at 65 °C. Curves at different excitation wavelengths are shown. (b)
Fluorescence quantum distributions at room temperature for FA in 4 M KOH at
fluorescence excitation wavelengths kexc = 320 nm and 332 nm, and for 9,10-
dehydro-FA (DHFA) at kexc = 420 nm (conversion of FA to DHFA by sample storage
for 10 h at 85 °C).
Fig. 4. Fluorescence quantum distributions, EF(k), of folic acid in studied solvents.
Distributions are shown for several excitation wavelengths, kexc
.

A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
87
room temperature results are also listed in Table 1. Fluorescence
lifetimes at room temperature were also measured by picosecond
pulses excitation at 400 nm and 449 nm where the signal detection
occurred with a micro-channel-plate photomultiplier (full half-
width of response function ꢀ 650 ps). Short fluorescence lifetimes
(range of 200 ps to 1 ns) were determined by signal curve de-con-
volution (the procedure is described in [45]). The results are given
in Table 1 using the presentation
SFðtÞ=SF;max ¼ x1 expðꢁt=sF;1Þ þ x2 expðꢁt=sF;2Þ;
ð3Þ
where x1 and x2 are the fractions of components with fluorescence
lifetimes sF,1 and sF,2
.
The fluorescence of FA in 4 M HCl, in TFA, and in 0.1 M HCl de-
cays single-exponentially. For FA in 4 M HCl (bi-cationic FA) the
fluorescence lifetime increased from 50 ps at room temperature
to 800 ps at 77.2 K. For FA in TFA the fluorescence lifetime in-
creased from ꢀ1.75 ns at room temperature to ꢀ2.6 ns at 77.2 K.
For FA in 0.1 M HCl the fluorescence lifetime was found to be
sF ꢀ 875 ps independent of the temperature.
For FA in pH 6 buffer (neutral FA) the fluorescence decayed bi-
exponentially with a very short component and a slow component
in the case of excitation at 351.2 nm. The very short component
will be attributed to direct locally-excited-state emission and the
slow component will be attributed to delayed locally-excited-state
emission and charge-separated-state emission [38]. The time con-
stant of the very short fluorescence component could not be re-
solved directly from the fluorescence trace in Fig. 7d because of
limited time-resolution. Its value will be extracted below by com-
bining the information of fluorescence trace and fluorescence
quantum yield measurements. The time constant of the short fluo-
rescence component was longer at 77.2 K than at room tempera-
ture indicating a potential energy surface barrier crossing in the
excited-state deactivation. In the case of excitation at 400 nm
and 449 nm (micro-channel-plate signal detection) the very short
Fig. 6. Fluorescence quantum yields of folic acid in studied solvents versus
excitation wavelength. Experimental data points are indicated by symbols. Thick
line connected points: total fluorescence quantum yields. Thin line connected
points: short-wavelength fluorescence quantum yield contributions.
a
b
c
Table 1
Fluorescence lifetimes of folic acid in various solvents at room temperature.
Solvent
4 M HCl
kexc (nm)
x1
sF,1 (ns)
x2
sF,2 (ns)
d
e
351.3
400
449
1
1
1
0.05
ꢀ0.05
ꢀ0.05
0.1 M HCl
pH 6 buffer
0.1 M KOH
4 M KOH
351.3
400
449
1
1
1
0.88
ꢀ0.9
ꢀ0.9
f
351.3
400a
449a
ꢀ0.998
ꢀ0.9
0.00023
1.9
0.5
ꢀ0.002
ꢀ0.1
ꢀ3
ꢀ7.6
ꢀ2.5
60.06
P0.94
351.3
400a
449a
ꢀ0.65
0.021
1.3
1.6
ꢀ0.35
0.62
1
1
Fig. 7. Temporal fluorescence traces belonging to folic acid in studied solvents.
351.3
400a
449a
400b
1
1
1
1
0.013
0.38
0.43
ꢀ0.6
Fluorescence excitation at kL = 351.2 nm with laser pulses of D L
t
ꢀ 6 ps duration.
Fluorescence light is collected for kF P 435 nm (Schott filters KV435 in path) in the
cases of FA in 4 M HCl, TFA, 0.1 M HCl, 0.1 M KOH, and for kF P 418 nm (Schott
filters KV418 in path) in the case of FA in pH 6 buffer. Thin solid lines belong to
measurements at room temperature; thick solid lines belong to measurements at
77.2 K; and dash-dotted curves are exponential regression fit curves. Fitted
fluorescence lifetime parameters are listed.
TFA
351.3
400
1
1
1
0.85
1.75
1.8
449
ꢀ2.6
400c
0.4
0.15
4.9
wavelength 351.2 nm, excitation pulse duration ꢀ 6 ps). The thin
solid curves were measured at room temperature, and the thick so-
lid curves were measured at 77.2 K. The dash-dotted curves are
single-exponential or bi-exponential regression fits. The extracted
fluorescence decay times are indicated in the part figures. The
Covered fluorescence spectral region in the case of kexc = 400 nm and 449 nm was
kdet = 501–566 nm (FWHM range).
a
Shortest time constant could not be resolved (see text).
theat(85 °C) = 10 h.
b
c
theat(65 °C) = 24 h.

88
A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
time constant could not be observed because of too low time res-
olution (its presence is documented by the small fluorescence
quantum yield).
The absorption and emission behaviour may be understood by
(i) bi-cationic FAH22þ excitation followed by photo-induced proton
release [46–48] to cationic FAH+ and subsequent thermal activated
internal conversion from S1 to S0 in the case of excitation at wave-
length kexc < 350 nm, and (ii) by cationic FAH+ excitation with sub-
sequent thermal activated internal conversion from S1 to S0 in the
case of excitation at wavelength kexc > 350 nm. The photo-dynam-
ics is illustrated by the potential energy scheme presented in
Fig. 8a and by the reaction scheme shown in Scheme 1.
The fluorescence decay of FA in 0.1 M KOH and in 4 M KOH (an-
ionic FA) behaved similar as in the case of FA in pH 6 buffer. The
short fluorescence component could be time resolved in the
streak-camera measurements to be in the 10–25 ps range.
The prolonged heating of FA in 4 M KOH converted it to DHFA.
Fluorescence decay traces were measured for excitation at 400 nm.
Signal de-convolution allowed the extraction of a fluorescence
time constant of sF ꢀ 600 ps. The S0-S1 absorption strength
(Fig. 3b) gives a radiative lifetime of srad ꢀ 24 ns (lower wavelength
limit ku = 380 nm used, see Eq. (8) below) leading to a fluorescence
lifetime of sF ꢀ 500 ps (/F = 0.021, see Eq. (7) below).
In the ground-state there exists a reversible thermal equilib-
rium between the bi-cationic and the cationic form for FA in 4 M
HCl. The fraction of molecules in the cationic form is
ꢀ
ꢁ
Eg
kB#
qcat ¼ exp ꢁ
;
ð4Þ
Heating of FA in TFA to 65 °C for 24 h caused bi-exponential
fluorescence decay with time constants of ꢀ400 ps and 4.9 ns
(picosecond excitation at 400 nm).
where Eg is the energy difference between molecules in the cationic
and bi-cationic form, kB is the Boltzmann constant, and # is the tem-
perature. In the excited state the equilibrium is strongly shifted to
the single-cationic form ðpKꢅc;bc < pKc;bcÞ. After photo-excitation of
4. Discussion
the bi-cationic form (frequency
m1) there occurs a transfer to the
cationic form with a rate of [49,50]
The spectroscopic behaviour of FA in the different solvents may
be put into three groups:
ꢂ
ꢃ
Eb1
kB#
kPR ¼ k0 exp
ꢁ
;
ð5Þ
(i) FA in 4 M HCl where FA is dominantly in the bi-cationic form
ðFAH22þÞ; FA in TFA behaves similar to FA in 4 M HCl. The
dynamics involves excited-state proton release.
where k0 ꢀ kB#=h is the attempt frequency of barrier crossing
[51,52] and Eb1 is the barrier height in the excited state path from
the bi-cationic to the cationic form. h is the Planck constant. Low-
(ii) FA in 0.1 M HCl where FA is partly in the cationic form (FAH+)
and partly in the bi-cationic form. The excited-state deactiva-
tion follows photo-physical non-radiative relaxation.
frequency excitation (
from their S0 state to their S1 state. From there occurs radiative
emission (radiative lifetime rad,cat), non-radiative intersystem
m2) directly excites the cationic molecules
s
(iii) FA in pH 6 buffer (neutral form) and FA in 0.1 M KOH and
4 M KOH (anionic form, FAꢁ). The dynamics is dominated
by photo-induced intra-molecular electron transfer.
crossing, and dominantly non-radiative internal conversion from
the S1 potential energy surface to the S0 energy surface via an en-
ergy barrier Eb2. The rate of thermal activated internal conversion
is approximately given by [49,50]
The dehydrated folic acid DHFA in 4 M KOH has a conjugated
p
ꢂ
ꢃ
electron system extending over the pterin and the imine-benzoic
part. The molecules are thought to be present in trans and cis iso-
meric form. The photo-excitation dynamics is thought to be deter-
mined by trans and cis state excitation and photo-induced twisted
intra-molecular charge transfer.
Eb2
kB#
kIC ¼ k0 exp
ꢁ
:
ð6Þ
From the experimental results some parameters are determined
for FA in 4 M HCl (TFA). The fluorescence lifetime of FAH+* was
measured to be sF,s ꢀ 50 ps (1.75 ns) at room temperature. The
Quantum chemical semi-empirical PM3 calculation for FA
fluorescence lifetime is limited by internal conversion. The transfer
determine permanent dipole moments of
l
indicate the polar nature of FA in ground state and the enhanced
l(S0) = 3.06D and
leading to kIC ꢀ 2 ꢃ 1010 sꢁ1 (5.7 ꢃ
ꢁ1
(S1) = 6.67D (1D = 1 Debye = 3.33564 ꢃ 10ꢁ30 C m). These values
rate is given by kIC
ꢀ
s
F;s
108 sꢁ1). The barrier height for deactivation is obtained from Eq.
(6) to be Eb2 ¼hc0mb2 ꢀ2:31ꢃ10ꢁ20 J or mb2 ¼Eb2=hc0 ꢀ1160cmꢁ1
~
~
polar nature in excited state. The calculated dipole moments for
DHFA in trans form are of l(S0) = 6.87D and l(S1) = 9.30D. The lar-
ger dipole moments result from the stretched shape of DHFA in
trans form.
The experimental behaviour of FA and DHFA is discussed in the
following.
(1890 cmꢁ1).
The radiative lifetime, srad, is generally given by
sF
srad
¼
;
ð7Þ
/
F
where /F is the fluorescence quantum yield. This relation leads to a
radiative lifetime of FAH+* of srad,cat ꢀ 36 ns for FA in 4 M HCl
(/F ꢀ 0.0014, sF ꢀ 50 ps) and to srad,cat ꢀ 50 ns for FA in TFA
(/F ꢀ 0.035, sF ꢀ 1.75 ns). Knowing the radiative lifetime of the
ꢀ
4.1. Folic acid in 4 M HCl and in trifluoroacetic acid
FAH+* ? FAH+ S1–S0 transition, the S0–S1 absorption strength, ra
,
The absorption spectrum of FA in 4 M HCl and in TFA (Fig. 2a)
shows a weak absorption pedestal in the 360–430 nm region.
The absorption pedestal increases with increasing sample temper-
ature (curves not shown for 4 M HCl, curves for TFA shown in
Fig. 3a). The rise of absorption with temperature is reversible for
4 M HCl. It is irreversible for TFA. The fluorescence spectra
(Fig. 4a and b) show two emission bands in the case of excitation
below 350 nm: a weaker band centred around 370 nm and a stron-
ger band centred around 485 nm. The fluorescence efficiency de-
creases with decreasing excitation wavelength. The fluorescence
signal decay is single exponential (kexc = 351.3 nm, 400 nm,
449 nm), and the fluorescence lifetime rises with decreasing
temperature.
of this transition may be determined by application of the Stric-
kler–Berg formula [54–56], which reads in rewritten form:
R
Z
EFðkÞk3dk
EFðkÞdk
raðkÞ
nA
c0n3F srad
S1ꢁS0
ꢀ
R
ra
¼
dk ¼
;
ð8Þ
k
8p
S0ꢁS1
S1ꢁS0
where nA and nF are the average refractive indices in the S0–S1
absorption region and the S1–S0 emission region, respectively, and
c0 is the speed of light in vacuum. The obtained results are
ra;2 ꢀ 2:83 ꢃ 10ꢁ18 cm2 for FA in 4 M HCl (nA ꢀ nF ꢀ 1.34) and
ꢀ
ra;2 ꢀ 2:22 ꢃ 10ꢁ18 cm2 for FA in TFA (nA ꢀ nF ꢀ 1.29). The
ꢀ
R
raðkÞdk
ꢀ
experimental absorption strengths, ra;2;exp
ꢀ
of the
S0ꢁS1
k
ꢀ
long-wavelength absorption band in Fig 2a are only ra;2;exp (4 M

A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
89
a
b
c
d
.
.
.
.
.
.
Fig. 8. Potential energy curves and photo-dynamics schemes. (a) Bi-cationic FA relaxation dynamics. (b) Single-cationic FA relaxation dynamics. (c) Neutral and anionic FA
relaxation dynamics. (d) Relaxation dynamics of DHFA in 4 M KOH. The scheme is shown for twisted intra-molecular charge transfer and trans–cis photo-isomerisation.
HCl) ꢀ 4.7 ꢃ 10ꢁ20 cm2 (upper limit of S0–S1 absorption set to
ku = 355 nm) and ra;2;exp (TFA) ꢀ 6.5 ꢃ 10ꢁ20 cm2 (ku = 355 nm) be-
ꢀ
cause of the limited ground-state population. The fraction of mole-
ꢀ
ꢀ
cules in the cationic state is given by qcat
¼
ra;2;exp
=
ra;2 giving
q
cat(4 M HCl) ꢀ 0.023 and cat(TFA) ꢀ 0.029. The energy difference
q
between the cationic and bi-cationic form is obtained from Eq. (4)
to be Eg ¼ hc0mg ꢀ 1.53 ꢃ 10ꢁ20 J (mg ꢀ 770cmꢁ1) for FA in 4 M HCl
~
~
and mg ꢀ 719cmꢁ1 for FA in TFA.
~
The determined fraction, qcat, of molecules in the cationic state
may be used to estimate the pKc,bc value by application of the
Henderson–Hasselblach equation [57] which reads for our
situation:
Scheme 1.

90
A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
!
½FAHþꢂ
singlet band (around 250 nm) results in an increase of the fluores-
cence efficiency. It is thought that some S3–S1 internal conversion
via a conical intersection takes place.
pH ¼ pKc;bc þ log
:
ð9aÞ
½FAH22þꢂ
In 0.1 M HCl (pH ꢀ 0.1) FA exists in cationic and bi-cationic
Rewriting gives
form. The S0–S1 absorption strength of the cationic form is ob-
½FAHþꢂ
tained by application of Eq. (8) to be ra;cat ꢀ 1:36 ꢃ 10ꢁ18 cm2
pHꢁpK
ð
c;bcÞ
;
ꢀ
¼ 10
ð9bÞ
ð9cÞ
½FAH22þꢂ
(
srad ꢀ 460 ns). The experimental S0–S1 absorption strength of
R
raðkÞdk
ꢀ
Fig. 2a (ku = 355 nm) gives ra;cat; exp
¼
ꢀ 6:9 ꢃ 10ꢁ19
S0ꢁS1
k
½FAHþꢂ
1
cm2. The fraction of FA in cationic state is found to be
qcat
¼
¼
;
½FAHþꢂ þ ½FAH22þꢂ 1 þ 10ðpK
c;cbꢁpHÞ
ꢀ
ꢀ
qcat
¼
ra;cat; exp
=
ra;cat ꢀ 0.51. Insertion of this value into Eq. (9d)
gives pKc,bc = 0.99 in good agreement with the data analysis for
and
FA in 4 M HCl.
ꢂ
ꢃ
Since for FA in 0.1 M HCl only about 50% of the molecules are
present in single-cationic form, the true S0–S1 absorption cross-
section spectrum of FAH+ in the wavelength region P360 nm is
approximately a factor of two larger than shown in Fig. 2a.
In the case of excitation below 350 nm both cationic and bi-cat-
ionic FA are excited. Again photo-induced proton transfer in the
excited state is expected to occur as described above for FA in
4 M HCl.
1
qcat
pKc;bc ¼ pH þ log
ꢁ 1 :
ð9dÞ
Insertion of qcat ꢀ 0.023 for FA in 4 M HCl (pH ꢀ ꢁ0.6) gives
pKc,bc ꢀ 1.0. This value is somewhat larger than the value of 0.2 re-
ported in [9].
The radiative lifetime of FAH22þꢅ is obtained from the S0–S1
absorption cross-section spectrum of the bi-cationic FA according
to the Strickler–Berg relation (Eq. (8)). The result is
HCl) ꢀ 12.3 ns (used absorption range from 310 nm to 366 nm)
and
srad,bicat(4 M
No photo-induced electron transfer is observed for FA in 4 M
HCl (cationic pterin and cationic p-amino-benzoic group) and for
FA in 0.1 M HCl (cationic pterin group). The positive charged pterin
group avoids an electron take-up in the excited state.
s
rad,bicat(TFA) ꢀ 15.2 ns (used absorption range from 305 nm
to 370 nm). The bi-cationic fluorescence quantum yield is deter-
mined from the bi-cationic fluorescence quantum distribution in
the short-wavelength range of Fig. 4 (wavelength from 320 nm
4.3. Folic acid in neutral and anionic form
to 420kexc = 310 nm) giving
/
ionic form are obtained by use of Eq. (7) to be
/
F,short(4 M HCl) ꢀ 1.1 ꢃ 10ꢁ4 and
F,short(TFA) ꢀ 3.5 ꢃ 10ꢁ4. The fluorescence lifetimes of the bi-cat-
Folic acid in aqueous solution at pH 6 is present in neutral form
(FA). Appropriate application of Eq. (9c) gives mole fractions of
qneutral ꢀ 0.996 and qanionic ꢀ 0.004 (pKn,a = 8.38). For FA in 0.1
KOH (pH ꢀ 13) and 4 M KOH (pH ꢀ 14.6) the anionic FA content
sF,f(4 M
HCl) ꢀ 1.35 ps and F,f(TFA) ꢀ 5.3 ps. This fluorescence lifetime is
s
determined by the rate of photo-induced proton release which is
kPR
ꢀ
s
giving kPR(4 M HCl) ꢀ 7.4 ꢃ 1011 sꢁ1, and kPR(TFA) ꢀ 1.9
ꢁ1
is
q
anionic(0.1 M KOH) ꢀ 1 ꢁ 2.4 ꢃ 10ꢁ5 and
qanionic(4 M KOH) ꢀ
F;f
ꢃ 1011 sꢁ1. For thermal activated excited-state proton release acti-
1 ꢁ 6 ꢃ 10ꢁ7. The absorption spectra (Fig. 2b) reveal weak long-
wavelength absorption tails which are thought to be due to
charge-transfer state absorption [41,42]. The same overall photo-
dynamics is thought to be going on for FA in neutral and anionic
form in the studied solutions.
vation barriers (Eq. (5)) of mb1 (4 M HCl) ꢀ 520 cmꢁ1 and mb1
~
~
(TFA) ꢀ 710 cmꢁ1 are estimated.
4.2. Folic acid in 0.1 M HCl
For FA in pH 6 solution excitation at room temperature at
kexc = 347 nm (S0–S1 transition) resulted in a fluorescence spec-
trum with emission maximum at 445 nm and a fluorescence quan-
tum yield of ꢀ4.5 ꢃ 10ꢁ4. The temporal fluorescence trace (Fig. 7d,
kexc = 351.2 nm) revealed a fast component (lifetime <100 ps) and a
slow component (lifetime ꢀ3 ns). It is assumed that the fast fluo-
rescence component is due to direct locally-excited-state emission
and that the slow component is due to delayed locally-excited-
state emission and charge-separated-state emission [38].
The absorption spectrum (Fig. 2a) shows a moderate absorption
band in the 350–450 nm region. The fluorescence spectra show
only one emission band. The fluorescence quantum yield for exci-
tation at 370 nm (S0–S1 transition) is /F ꢀ 0.0019. The fluorescence
efficiency is lower in the case of excitation around 300 nm (S0–S2
transition), and it is higher in the case of excitation around
250 nm (S0–S3 transition). The fluorescence decay is single expo-
nential, and the fluorescence lifetime is approximately the same
at 20 °C and 77.2 K.
The absorption and emission behaviour of the cationic folic acid
(FAH+) in 0.1 M aqueous HCl may be understood by the photo-
dynamics scheme of Fig. 8b. Depending on the excitation
wavelength a transition occurs from the S0 ground-state to a
Franck–Condon level in the first (S1), second (S2), third (S3), or
higher excited singlet state. S0–S1 excitation results in thermally
relaxed S1–S0 emission with a fluorescence lifetime of sF ꢀ 875 ps.
The fluorescence lifetime is thought to be limited by photo-physi-
cal non-radiative relaxation by equipotential internal conversion
and intersystem crossing [58,59]. No barrier crossing is relevant
since the fluorescence lifetimes are the same at room temperature
and at 77.2 K (energy barrier for thermal activated internal conver-
The radiative lifetime of the S1–S0 transition of neutral FA in pH
6 buffer is obtained by application of Eq. (8) (Strickler–Berg for-
mula). The result is srad ꢀ 21 ns (ku = 330 nm). A bi-exponential
fluorescence trace fit according to SFðtÞ=SF;max
¼ Þ
F;1 expðꢁt=s0F;1
j
þj
F;2 expðꢁt=sF;2Þ (see Eq. (3)) gives
j
F,1 = 0.482, s0F;1 ¼ 100ps,
jF,2 = 0.495, sF;2 ¼ 3ns. Accordingly the fluorescence quantum
yield is composed of two components, i.e. /F ¼ /F;1 þ /F;2. The fast
component fluorescence quantum yield, /F,1, is obtained from the
bi-exponential fluorescence trace in Fig. 7d according to
jF;1s0F;1
SF;1
SF;tot
/
¼ /F
ꢀ /F
:
ð10Þ
F;1
jF;1s0F;1
þ
jF;2sF;2
sion via S1–S0 potential energy surface crossing too high). The rate
Thereby SF,1 is the short component fluorescence signal, and SF,tot is
ꢁ1
of non-radiative relaxation is estimated to be knr
ꢀ
sF ꢀ 1.1
the total fluorescence signal. The analysis gives /F,1 ꢀ 1.1 ꢃ 10ꢁ5
.
ꢃ 109 sꢁ1. The S1–S0 radiative lifetime is estimated to be (Eq. (7))
srad ꢀ 460 ns. For excitation into the second excited singlet band
(around 300 nm) the fluorescence efficiency is reduced. This
behaviour may be understood by partial S2–S0 internal conversion
via a conical intersection [53,60,61]. Excitation into the next higher
The short fluorescence lifetime, sF,1, which was experimentally
not resolved, may be obtained from the fluorescence quantum yield,
/
and the radiative lifetime srad (Eq. (7)). The calculated value is
sFF,,11ꢀ 230 fs ðx1
¼
jF;1s0F;1
sF;1 ꢀ 0:998Þ. The slow fluorescence com-
=
ponent with sF,2 ꢀ 3 ns is present with a fraction of x2 ꢀ 0.002.

A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
91
The strong quenching of the fluorescence emission of FA in
aqueous pH 6 buffer is thought to be due to reductive intra-molec-
ular electron transfer from the p-amino-benzoic acid part to the
is obtained by application of Eq. (8) (Strickler–Berg formula,
ku = 330 nm). The result is srad ꢀ 17.2 ns.
FA was also studied in 4 M KOH where FA is also present in anio-
nic form. The fluorescence maximum occurred at 475 nm, and the
temporal fluorescence trace decayed bi-exponentially with
photo-excited pterin part of the molecule. The rate of intra-molec-
ꢁ1
ular electron transfer is given by kIET
ꢀ
s
ꢀ 4:3 ꢃ 1012 sꢁ1
.
F;1
In the fluorescence lifetime measurements at 400 nm and
449 nm with the micro-channel-plate photomultiplier (temporal
half-width of response function is 650 ps) the ultrafast direct lo-
cally-excited-state emission could not be resolved, but its presence
is concluded from the small fluorescence quantum yield. The ob-
served rise in quantum yield with increasing excitation wave-
length indicates some facilitation of excited-state deactivation
with increasing excitation excess energy.
j
F,1 = 0.86 0.06,
50 ps. At 77.2 K the fluorescence decayed bi-exponentially with
F,1 = 0.5 0.1, F,1 = 800 300 ps, F,2 = 0.5 0.1, and F,2 = 6 2 ns.
The anionic folic acid, FAꢁ, follows the same photo-induced in-
sF,1 = 16 4 ps, jF,2 = 0.14 0.06, and sF,2 = 290
j
s
j
s
tra-molecular charge transfer dynamics as neutral folic acid. FA is
locally excited to FAꢁ* (LE). From there occurs direct locally-ex-
cited-state emission (
kIET
sꢁF;11) to FAꢁꢁ+ (CS). The CS state evolves by CS ? LE back
transfer leading to time-delayed locally-excited-state emission
F,2), CS state emission ( F,2), and CS state charge recombination
(kCR
sꢁF;12) back to FAꢁ. The FAꢁ dynamics is indicated in Fig. 8c
in parenthesis.
sF,1) and charge separation (rate constant
ꢀ
At 77.2 K the fluorescence decay of FA in pH 6 buffer remained
bi-exponential with well resolved short fluorescence time con-
(s
s
stant. The parameters are
jF,1 = 0.87 0.07,
sF,1 = 400 150 ps,
ꢀ
j
F,2 = 0.13 0.07, and sF,2 = 5 1 ns. The rate of intra-molecular
electron transfer reduced to kIET ꢀ 2.5 ꢃ 109 sꢁ1 indicating a ther-
mal activated barrier crossing.
4.4. 9,10-Dehydro-folic acid in 4 M KOH
A reaction coordinate scheme for the photo-excitation dynam-
ics of neutral FA is depicted in Fig. 8c. FA is excited to a locally ex-
cited state FA* (LE) in the first excited singlet band S1. There occurs
charge separation to an intra-molecular zwitterionic state FA+ꢁ
(CS). From there occurs (i) some thermally activated CS ? LE back
Keeping of FA in 4 M KOH at 85 °C for a few hours converted the
first absorption band peaking at 367 nm completely and irrevers-
ibly to a new absorption band peaking at 425 nm and having a
shoulder at 480 nm (see Fig. 3b). The fluorescence shifted to the
red with an emission maximum at 580 nm and with a weak shoul-
der around 490 nm (see Fig. 5b). A fluorescence quantum yield of
0.021 was measured. It is thought that FA is thermally dehydrated
to 9,10-dehydro folic acid (DHFA) under aerobic conditions. Under
anaerobe conditions no dehydration occurred. Trans and cis iso-
mers are thought to be formed by changing from FAꢁ with flexible
9,10 single-bond to DHFA with stiff 9,10 double-bond. The red-
shift of the absorption spectra is caused by the extension of the
transfer causing locally delayed fluorescence (lifetime
some charge-separated-state emission (lifetime F,2), and (iii)
non-radiative charge recombination (rate constant kCR
sꢁF;12). The
sF,2), (ii)
s
ꢀ
involved HOMO and LUMO molecular orbitals of the pterin (P)
and the p-amino-benzoic acid (B) parts are indicated.
FA was also studied in aqueous solution at pH 8 using Tris–HCl
buffer. In this solvent FA is present in neutral and anionic form
(pK = 8.38 [9]). The shape of the absorption spectrum was between
the shapes of the pH 6 and 0.1 M KOH spectra (curve not shown).
The fluorescence spectra followed the spectra of 0.1 M KOH (curves
not shown). The fluorescence quantum yield was measured to be
/F (360 nm) ꢀ 5 ꢃ 10ꢁ4 at room temperature. The fluorescence de-
cay was bi-exponential as in the case of FA in pH 6 and 0.1 M KOH.
The fluorescence lifetimes were the same as for 0.1 M KOH within
our experimental accuracy.
In 0.1 M KOH (pH ꢀ 13) folic acid is present in anionic form
(FAꢁ). The absorption spectrum shows a slowly decaying absorp-
tion tail in the long-wavelength range (>410 nm). The tail absorp-
tion is attributed to charge-transfer state absorption. The
fluorescence spectra show a dominant emission band around
470 nm and a very weak short-wavelength band around 350 nm.
This short-wavelength band may be due to some S2–S0 emission
and some contribution from p-amino-benzoic acid fragment emis-
sion of some degraded folic acid molecules. The fluorescence effi-
ciency is excitation wavelength dependent (Figs. 4e and 6 and
fluorescence excitation spectrum in Fig. 2b). It is smallest around
kexc = 300 nm (S0–S2 absorption band) probably due to non-radia-
tive S2–S0 deactivation via a conical intersection (see situation of
Fig. 8b). The larger fluorescence quantum yield in the case of
excitation at 280 nm and 255 nm indicates some S3–S1 internal
conversion via a conical intersection. The temporal fluorescence
trace (Fig. 7e, excitation wavelength kL = 351.2 nm) shows a bi-
exponential decay behaviour. The parameters at room tempera-
double bond ꢁc1onjugation. The large absorption–emission Stokes
ꢁ1
shift, dmSt ¼ ka;max ꢁ kF;max ꢀ 6300 cmꢁ1 and the still small fluores-
~
cence quantum yield of /F ꢀ 0.021 indicate the occurrence of
photo-induced twisted intra-molecular charge transfer (TICT state
formation) by excited molecular twist between the pterin part and
the imine-benzoyl-glutamate part (bi-radical formation and twist
around C9,N10 bond) [62]. The large Stokes shift is thought to be
due to major conformation (trans or cis) to minor conformation
(cis or trans) photo-isomerisation.
A dynamics potential energy scheme for DHFA in 4 M KOH is
shown in Fig. 8d. Excitation occurs from a planar trans or cis S0
ground-state to a trans or cis excited singlet state. From there oc-
curs locally excited state emission (LE), a molecular twist (TICT
state formation) with excited-state deactivation and some trans–
cis or cis–trans transfer (photo-isomerisation) followed by relaxed
excited-state emission.
5. Conclusions
The fluorescence behaviour of folic acid in aqueous solution and
in trifluoroacetic acid was studied. The bi-cationic, cationic, neu-
tral, and anionic forms could be investigated by varying the pH
of aqueous solutions. FA is only weakly fluorescent in all ionic
forms. The excited-state deactivation mechanisms were found to
depend on the ionic state of the samples.
ture are jF,1 = 0.65 0.1, sF,1 = 21 6 ps, jF,2 = 0.35 0.1, and sF,2 =
620 100 ps. As in the case of neutral FA the short fluorescence
contribution is thought to be due to direct locally-excited-state
emission (longer lifetime indicates a slightly higher barrier for ex-
cited-state charge separation). The slow component is attributed
to delayed locally excited emission and charge-separated-state
emission. At 77.2 K the fluorescence decays bi-exponentially
In 4 M HCl solution FA is dominantly present in bi-cationic
form. Photo-excitation induced a fast excited-state proton release
(FAH22þꢅ ! FAHþꢅ þ HþÞ which was followed by a thermal activated
FAHþꢅ excited state relaxation.
In 0.1 M HCl solution FA is present in bi-cationic ðFAH22þÞ and
single-cationic form (FAH+). The fluorescence lifetime was deter-
mined by photo-physical non-radiative decay (internal conversion
and intersystem crossing). The weak fluorescence emission
with
jF,1 = 0.29 0.06, sF,1 = 600 200 ps, jF,2 = 0.71 0.06, and
s
F,2 = 5 2 ns. The radiative lifetime of the S1–S0 transition of FA

92
A. Tyagi, A. Penzkofer / Chemical Physics 367 (2010) 83–92
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(2003) 261.
resulted from the weak S0–S1 absorption strength (long radiative
lifetime and normal rate of non-radiative decay).
In aqueous solution at pH 6 (neutral form of FA) the fluores-
cence was found to be strongly quenched by photo-induced in-
tra-molecular electron transfer with subsequent non-radiative
charge recombination. The fluorescence behaviour was determined
by direct locally-excited-state emission, delayed locally-excited-
state emission, and charge-separated-state emission.
In aqueous 0.1 M KOH and 4 M KOH solution (anionic form of
FA) the fluorescence quenching behaved similar to the neutral FA
situation. The dynamics was again determined by photo-induced
intra-molecular electron transfer.
The Kasha–Vavilov’s rule of excitation wavelength independent
constant S1–S0 fluorescence emission due to ultrafast higher ex-
cited-state to first excited-state internal conversion was not
obeyed because other relaxation paths from higher excited poten-
tial energy surfaces like conical intersection funnels exist.
FA in 4 M KOH could be dehydrated under aerobe conditions to
9,10-dehydro-FA at elevated temperatures. The photo-dynamics of
DHFA turned out to be determined by twisted intra-molecular
charge transfer and photo-isomerisation.
The low fluorescence quantum yield of FA may have advantages
in its biological vitamin application by avoiding photo-damage due
to the short excited state lifetime. In this respect the situation is
similar to the DNA bases which have very short excited state life-
times due to conical intersection decay paths [63–65].
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The authors thank the Deutsche Forschungsgemeinschaft (DFG)
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