Femtosecond Spectroscopic Study of Carminic Acid-DNA ...Spectroscopic... · Femtosecond Spectroscopic Study of Carminic Acid-DNA Interactions ... 2.2.2 Time-Resolved Optical Spectroscopy

Femtosecond Spectroscopic Study of

Carminic Acid-DNA Interactions

Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur Erlangung des Doktorgrades

vorgelegt von

Dipl.-Phys. Radu Comanici

Aus

Piatra Neamt

2007

Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der

Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 29.07.2007

Vorsitzender der Promotionskommission: Prof. Dr. Bänsch Eberhard

Erstberichterstatter: Prof. Dr. Carola Kryschi

Zweitberichterstatter: Prof. Dr. Rainer Fink

Contents

1. Introduction........................................................................................................................ 10

2. Materials and Methods ...................................................................................................... 13

2.1 Materials....................................................................................................................... 13

2.1.1.1 DNA ................................................................................................................... 13

2.1.1.2 Binding Mode..................................................................................................... 17

2.1.1.3 Structural, Electronic and Spectroscopic Properties of Carminic Acid ............. 19

2.2.1 Stationary Optical Spectroscopy............................................................................... 21

2.2.1.1 UV/VIS Absorption Spectroscopy..................................................................... 22

2.2.1.2 Fluorescence Spectroscopy ................................................................................ 25

2.2.1.3 Measurement of Fluorescence Quantum Yield and Fluorescence Lifetime...... 27

2.2.2 Time-Resolved Optical Spectroscopy....................................................................... 31

2.2.2.1 Femtosecond Transient Absorption Spectroscopy............................................. 31

2.2.2.2 Femtosecond Fluorescence Up Conversion ....................................................... 41

3. Experimental...................................................................................................................... 48

3.1 Materials: Chemicals and Sample Solutions ............................................................... 48

3.2 Methods........................................................................................................................ 49

3.2.1 UV/VIS Absorption and Fluorescence Spectroscopy........................................... 49

3.2.2 Determination of the Fluorescence Quantum Yield ............................................. 49

3.2.3 Fluorescence Titration Experiments ..................................................................... 50

3.2.4 Femtosecond Transient Absorption Spectroscopy................................................ 50

3.2.5 Femtosecond Fluorescence Up-Conversion Technique........................................ 51

3.2.6 Computations ........................................................................................................ 53

4. Results and Discussion ...................................................................................................... 54

4.1. Stationary Optical Spectroscopy................................................................................. 54

4.2. Femtosecond Spectroscopy......................................................................................... 67

4.2.1 Transient Absorption Spectroscopy ...................................................................... 67

4.2.2 Fluorescence Up-Conversion Spectroscopy ......................................................... 70

5. Conclusions........................................................................................................................ 80

6. References.......................................................................................................................... 82

List of Figures

Fig. 2.1.1 Heterocyclic bases A: pyrimidines 1: uracil, 2: thymine, 3: cytosine; ................ 14

Fig. 2.1.2 Structure components of the common nucleotides. .............................................. 14

Fig. 2.1.3 Tautomers of uracil. .............................................................................................. 15

Fig 2.1.4 Binding of guanine with cytosine .......................................................................... 16

Table 2.1.5 Redox potential of the DNA bases at a pH value of 7. ...................................... 16

Fig. 2.1.5 Structure of a section of DNA............................................................................... 17

Table 2.1.1.2 Thermodynamic binding parameters for the interaction of doxorubicin,

daunorubicin, hydroxyrubicin and the ββββ anomer of doxorubicin with calf thymus DNA; Keq

is the binding constant and n is the number of base pairs per binding site. .......................... 18

Table 2.1.1.3 Free energy of anthracycline antibiotic binding to calf thymus DNA. ........... 19

Fig. 1.3.1: Structure formula of carminic acid. ..................................................................... 20

Fig. 2.2.1 Spectrum of electromagnetic radiation: the spectral range of optical spectroscopy

is depicted in enlarged form. ................................................................................................. 22

Fig. 2.2.1.1 Schematic representation of a UV/VIS absorption spectrometer. ..................... 24

Figure2.2.1.2.1 Jabłoński term scheme. ................................................................................ 25

Fig. 2.2.1.2.2 Setup of a fluorescence spectrometer.............................................................. 27

Fig 2.2.2.1.1 Energy scheme of the electronic states involved in a pump-probe.................. 33

experiment; excited states relaxation dynamics detected by absorption changes of the probe

............................................................................................................................................... 33

pulse are: 1) bleaching; 2) excited state absorption; 3) stimulated emission. ....................... 33

Fig. 2.2.2.1.1 Schematic representation of the fs transient absorption spectroscopy

experiment; BS: Beam Splitter; FM: Flip Mirror; DS: delay stage; VA: variable attenuator;

P: polarizer; L: lens; WLC plate: rotating fused silica plate for white light continuum

generation; PM: parabolic mirror; SHG: BBO crystal for second harmonic generation; BC:

Berek compensator; BD : beam dump; BG: BG38 filter; GG: GG420 filter; ND: neutral

density filter. .......................................................................................................................... 36

Fig. 2.2.2.1.4 a) Intensity as a function of time for a Gaussian laser pulse b) Time

dependence of the frequency for a positive nonlinear index of refraction, n2....................... 39

Fig. 2.2.2.1.5. Representation of the wlc probe pulse as a composition of the temporally

shifted, different spectral sub-pulses having the same pulse width as the pump pulse......... 39

Table 2.2.2.2 Relative quantum efficiencies ηηηηq (normalized relative to KDP), damage

thresholds Ithr, and cut-off wavelengths for fluorescence up-conversion with 800 nm pump

pulses. .................................................................................................................................... 44

Fig. 2.2.2.2.1 Femtosecond up-conversion technique apparatus; DM: dichroic mirror; HW:

half wave plate; CCD video camera for the visual superposition of the beams in the BBO

crystal; Mono: monochromator; PM: photomultiplier. ......................................................... 46

Scheme 1: Dissociation reaction of carminic acid. ................................................................ 54

Fig. 4.1.1: pH dependence of the spectral features of carminic acid in water measured by

UV/VIS absorption spectroscopy. ......................................................................................... 55

Fig. 4.1.2: Dual fluorescence of carminic acid with a blue emission peak at 470 nm (22700

cm-1) and an orange emission peak at 570 nm (15100 cm-1)................................................. 56

Fig. 4.1.3: Orange fluorescence of the tautomer at 15100 cm-1. ........................................... 57

Fig 4.1.4: Normalized fluorescence spectra of 5µM carminic acid in BPES (dashed line) and

5µM carminic acid with 5µM DNA in BPES (solid line) ..................................................... 58

Fig. 4.1.5: Absorption of carminic acid in BPES; the band structure (thin solid line) was

analyzed by fitting with a superposition of four Gaussian function (dashed line)................ 59

Fig. 4.1.6: Absorption of carminic acid in DMSO; the band structure (thin solid line) was

analyzed by fitting with a superposition of four Gaussian function (dashed line)................ 60

Scheme 2: Molecular structure of the normal form of carminic acid (CAH) and the three

tautomers (CAH T1, CAH T2, CAH T3) .............................................................................. 61

Table 4.1.1: Calculated values of the total energy (ET), the binding energy (EB), the absolute

energy (Eabs), the energy of the S0-Sm transition (∆∆∆∆(S0-Sm)) and S0-Sn transition (∆∆∆∆(S0-Sm))

transition and of the oscillator strength (f). .......................................................................... 62

Fig. 4.1.7: The spectra of the orange fluorescence of 5 µM carminic acid in BPES. The band

structure of the spectra (thin solid line) was analyzed by fitting with a superposition of

Gaussian functions (dashed line). .......................................................................................... 64

Fig. 4.1.8: The spectra of the orange fluorescence of 5 µM carminic acid in DMSO; the

band structure of the spectra (thin solid line) was analyzed by fitting with a superposition of

Gaussian functions (dashed line). .......................................................................................... 65

Fig. 4.1.9: Concentration dependence of bound carminic acid, cB, on the DNA

concentration; the experimental data (dots) were obtained from fluorescence titration of 6

µM carminic acid with DNA in BPES and were fitted (solid line) employing the

relationship cB= cT⋅⋅⋅⋅ cDNA/(KB-1+cDNA) with KB= 5.0××××10

5 (M nucleotide)-1. .......................... 67

Fig. 4.2.1.1 3-D plot of the temporal evolution of the transient absorption spectra obtained

for carminic acid in BPES. .................................................................................................... 68

Fig. 4.2.1.2 Transient absorption spectrum (thin solid line) recorded at the delay time ττττ= 1

ps; the fit (thick short dashed line) arises from the superposition of 8 Gaussian functions

assigned to four different time constants (thick solid, dashed, dashed dotted and dotted

lines). ..................................................................................................................................... 69

Fig. 4.2.2.1 Fluorescence-up conversion spectrum (dots) fitted by a superposition (thin solid

line) of two Gaussians (dashed line)...................................................................................... 70

Fig. 4.2.2.2 3-D plot of the temporal evolution of the fluorescence up-conversion spectra

obtained for 0.6 mM carminic acid in BPES......................................................................... 71

Fig.4.2.2.3: 3-D plot of the temporal evolution of the fluorescence up-conversion spectra

obtained for 0.6 mM carminic acid and 3.9 mM DNA in BPES........................................... 72

Fig. 4.2.2.4: Fluorescence up-conversion decays of 0.6 mM carminic acid in BPES (thin

solid line) detected at 14910 cm-1 in the parallel-polarization geometry (I(t)par) and in the

perpendicular-polarization geometry (I(t)perp); both fits were obtained using the parameters:

a = 0.37, b= 0.91, ττττ1= 1.7 ps, ττττ2= 33 ps and r(0)= 0.137. ...................................................... 73



perpendicular-polarization geometry (I(t)perp); both fits were obtained using the parameter:

a= 0.50, b= 0.92, ττττ1= 1.5 ps, ττττ2= 47 ps and r(0)= 0.202. ...................................................... 74

Fig. 4.2.2.6: Fluorescence up-conversion decays of 0.6 mM carminic acid and 3.9 mM

DNA in BPES (thin solid line) detected at 14910 cm-1 in the parallel-polarization geometry

(I(t)par) and in the perpendicular-polarization geometry (I(t)perp); both fits were obtained with

the parameter: a= 0.50, 0.97, ττττ1= 1.9 ps, ττττ2= 48 ps and r(0)= 0.182. .................................... 75


DNA in BPES (thin solid line) detected at 16490 cm-1 in the parallel-polarization geometry

(I(t)par) and in the perpendicular-polarization geometry (I(t)perp); both fits were obtained with

the parameter: a= 0.50, b= 0.97, ττττ1= 1.1 ps, ττττ2= 61 ps and r(0)= 0.243. ............................... 76

Table 4.2.2.1: The amplitudes, a and b, the time constants, ττττ1 and ττττ2, and the anisotropy,

r(0), were obtained from the best fit of the fluorescence up-conversion decay curves that are

detected at 14910 cm-1 and 16490 cm-1. ................................................................................ 78

Introduction

10

1. Introduction

Drugs for treatment of cancer on basis of anthracyclines have been extensively

studied for decades in an effort to optimize their therapeutic function. These compounds are

believed to develop their cytotoxic effect by penetrating into the tumor cell nucleus and

interacting there with DNA [1-4]. The formation of drug-DNA complexes is determined by

the structural features of the anthracyclines composed of a dihydroxy-anthraquinone

chromophore with one or two glycosyl side chains. The formation of intercalation

complexes has been observed to inhibit the DNA replication and the RNA transcription that

blocks the gene expression [3]. Irradiation with light enhances the cytotoxicity of

anthracyclines (e.g. daunomycin) by several orders of magnitudes [5-8]. This photo-

activation effect is understood to originate from an ultrafast electron transfer reaction from a

G base of the DNA to the intercalated chromophore, which is associated with the oxidation

of the G base and the reduction of the dihydroxyanthraquinone [7, 8]. This hypothesis is

based on femtosecond spectroscopy studies of daunomycin-DNA and adriamycin-DNA

complexes, yielding a decrease of the S1 state lifetime of the drug by three orders of

magnitude when DNA is present [7]. The lifetime of the daunomycin-DNA complex with

τ= 290 fs was ascribed to the occurrence of the photo-induced electron transfer from the G

base to the 1,4-dihydroxyanthraquinone chromophore. On the other hand, other

radiationless decay processes such as intersystem crossing, internal conversion and excited-

state intramolecular proton transfer (ESIPT) may also be enhanced by conformational

reorganization that daunomycin experiences in the hydrophobic environment of the DNA

base pair stacking. Despite extensive research activities on the examination of photo-

activated anthracycline-DNA complexes [7-11] to date there exists no unambiguous

evidence for photo-induced oxidation of the DNA and moreover, the excited-state relaxation

dynamics as well as the structural mechanism at the molecular level are more hypothetical

than really understood.

Introduction

11

The proton transfer is a fundamental process and stay for instance at the basis of

various biochemical reactions such as occurring in enzymes [12-17]. In technical

applications proton transfer reactions are employed to build up optical memories or as

fundamental process for proton transfer LASER.

Excited state intramolecular proton transfer (ESIPT) is induced by optical excitation

that changes the electronic configuration of the molecule and thereupon, promotes the

tautomerization reaction in the electronically excited state. Proton transfer may occur

between two functional groups, for instance between (–OH, -NH2) and (-C=O, -N=),

resulting either in a tautomerization reaction or in an incomplete transfer of a hydrogen

atom. Both are associated with the rearrangement of the molecular structure. This structural

rearrangement results into a neutral tautomer having an energetically distinct molecular

configuration in the excited state which is responsible for the Stokes shift of the tautomer

fluorescence relatively to the fluorescence of the normal form.

Femtosecond (fs) resolved transient absorption spectroscopy experiments of ESIPT

processes may give information of the structural relaxation dynamics following the proton

transfer. The drawback of transition absorption spectroscopy is that this technique does not

allow for the specific detection of only one photophysical deactivation process: the

fluorescence, the internal conversion or the intersystem crossing. To obtain an indepth

insight in the scenario of structural, radiationless and radiative relaxation the ESIPT

reactions of the compounds under study have been studied using complementary fs-

resolving techniques, as the fs transient absorption spectroscopy and the fluorescence up

conversion spectroscopy.

Our research work presents stationary and time resolved optical spectroscopic studies

of carminic acid-DNA complexes using standard techniques of stationary spectroscopy as

well as femtosecond-resolved transient absorption spectroscopy and fluorescence up-

conversion technique. We have focused onto carminic acid (7-α-D-glycopyranosyl)-

9,10,dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracenecarboxylic acid), since

this natural dye consists of a tetrahydoxy-anthraquinone chromophore, with a pendant

Introduction

12

glycosyl moiety and thus constitutes the essential structural features of the anthracyclines.

The main impetus for us is to understand the complex mechanism and dynamics of the

excited-state relaxation of carminic acid in the presence of DNA. Carminic acid as a

carboxylic acid is expected to dissociate in water, so that two forms should coexist at pH =

7, the fully protonated acid (CAH) and the deprotonated anion (CA-).

Although the UV/VIS absorption spectra and the fluorescence spectra of carminic

acid and their dependence on the pH value had been still investigated in detail [18-20], no

contribution by tautomerization reactions, that may occur in the ground state (S0) as well as

in the excited state (S1), to the spectroscopic properties were taken into account. This is

really surprising, since excited-state intramolecular proton transfer (ESIPT) has been shown

to accelerate the rate of internal conversion by reducing the energy gap between the S0 and

S1 states [20]. In order to get a comprehensive understanding of the ESIPT kinetics of

carminic acid and its sensitivity to the microenvironment of the DNA base stacks, there are

performed ZINDO/S calculations of the electronic spectra of the four carminic acid

tautomers in both its non-dissociated and dissociated forms. In addition, have been studied

the binding of carminic acid to DNA upon performing fluorescence titration experiments.

Materials and Methods

13

2. Materials and Methods

2.1 Materials

2.1.1.1 DNA

Deoxyribonucleic acid (DNA) is a nucleic acid in the form of a double helix that

contains the genetic instructions specifying the biological development of all cellular forms

of life and many viruses. DNA is a long polymer of nucleotides that encodes the sequence

of the amino acid residues in proteins using the genetic code, a triplet code of nucleotides.

DNA consists of two complementary strands linked by hydrogen bonds. Each strand of

DNA is a chain of nucleotides consisting each of a heterocyclic base, a sugar and one or

more phosphate groups. In the most common nucleotides the base is a derivative of purine

or pyrimidine as being adenine (A), cytosine (C), guanine (G) or thymine (T) (see Figure

2.1.1), and the sugar is the pentose deoxyribose or ribose (see Figure 2.1.2) [21, 22].

Between the two strands, each base can only mate with one distinct base: resulting into one

of the following combinations: A+T, T+A, C+G and G+C. For instance, A on one strand of

double-stranded DNA will match properly only with a T on the other complementary strand.

A pair of two nucleotides is a so-called base pair [21].

The oxidation of DNA occurs most likely at guanine residues due to the lowest

reduction potential of this base in comparison with cytosine, thymine, and adenine [23, 24]

(see Table 2.1.5).


14

1 2 3

1 2

Fig. 2.1.1 Heterocyclic bases A: pyrimidines 1: uracil, 2: thymine, 3: cytosine;

B: purines 1: adenine, 2: guanine.

OBase

OP

OO-

OP

O

O-O

PO-

O-

O

HO OH -(Ribose) H - (Deoxiribose)

Fig. 2.1.2 Structure components of the common nucleotides.

The uracil base (A1) is a pyrimidine and have been found in RNA making a pair with

the adenine. In DNA it is replaced by thymine. Theoretically the uracil base can be

A

B

Nucleotide

diphosphate

Nucleotide

monophosphate

Nucleotide

triphosphate


15

combined with any base, although it only mates with adenosine via a hydrogen bonding. As

hydrogen bond acceptor the uracil makes up to three hydrogen bonds. Uracil occurs as two

tautomers that are at pH=7 the lactam (keto) and the lactim (enol).

Fig. 2.1.3 Tautomers of uracil.

Another pyrimidine base is thymine of DNA (A2). Thymine can be formed by a

methylation reaction of the uracil base.

The third base in the class of pyrimidines is cytosine (A3). It forms with guanine a

pair with three hydrogen bonds. The methylation reaction of cytosine to 5-methylcytosin is

conducted by an enzyme. However, uracil will be produced in a side reaction.

The adenine (B1) base belongs to the purines. Originally it was called vitamin B4.

Adenine forms several tautomers that can be rapidly interconverted and are often considered

as being equivalent. In DNA adenine binds through two hydrogen bonds with thymine.

The second purine base is guanine that forms pairs with adenine in DNA and with

cytosine in RNA. Guanine occurs as two tautomers, a keto form and an enol form. It binds

with cytosine via three hydrogen bonds. The carbon atom 6 in Figure 2.1.4 (left) of guanine

acts as the hydrogen acceptor, while the nitrogen at position 1 and the carbon at position 2

act as hydrogen donors. In comparison with guanine the hydrogen donor of cytosine is the

amino group and the hydrogen acceptors are the carbon atom 2 and the nitrogen 3 (right).


16

N

NH

NH2

O

HN

N NH

N

H2N

O

Fig 2.1.4 Binding of guanine with cytosine

The one-electron oxidation potential can be further lowered when G is contained as GG or

GGG in DNA sequences.

Base redox potential

Adenine 1.42 V

Guanine 1.29 V

Thymine 1.7 V

Cytosine 1.6 V

Table 2.1.5 Redox potential of the DNA bases at a pH value of 7.

Electron transfer measurements in solution indicate that the redox potential of

guanine depends also on the substituent at the nitrogen (9). For example the redox potential

of guanosine (Gs) is higher for 0.1 V than that of guanine (G).

The double-stranded structure of DNA provides a simple mechanism for DNA

replication: the DNA double strand is first separated down the middle, and the other half of

each new single strand is recreated by an enzyme by finding the correct base in the mixture

1 1 1 1 12

1 1 1 1 1

1

1 1 1 1 13

2

4 1 16

1 15 7

1 1 1 1 18

1 1 1 1 1

9

1 1 1 1 12 1 1 1 1 1

1

1 1 1 1 13

4 1 15

1 16

Cytosine Guanine


17

of the four bases and pairing it with the original strand. This replication mechanism is

sensitive to the nearest environment. For instance, any chemical compound attached at the

DNA double strand may perturb the separation into single strands.

Fig. 2.1.5 Structure of a section of DNA.

2.1.1.2 Binding Mode

Numerous compounds, with varying sizes and diverse structurally groups, bind to the

duplex DNA molecule. The cation-substituted quinone derivative binds to the DNA with

different affinities depending on the structure of the quinones and the sequence of the DNA.

In the case of anticancer antibiotics the binding mode of anthracycline to DNA is

crucial for their efficiency in the cancer therapy. Daunomycin is one of the most known


18

anthracycline antibiotics and exhibits a unique DNA binding sequence specificity by

preferential binding to the triplet sequences 5’TACG and 5’T

AGC [25].

Compound Keq/105 (M-1) n(bp) δ logK/δlog [M+]

Doxorubicin

Daunorubicin

Hydroxyrubicin

β anomer of doxorubicin

29.0±2.0 3.4±0.1 -0.96 ± 0.1

6.9±0.2 3.4±0.1 -1.25 ± 0.1

1.9±0.1 3.4±0.1 -0.18 ± 0.1

0.15 4.3±0.5 -0.91 ± 0.1

Table 2.1.1.2 Thermodynamic binding parameters for the interaction of doxorubicin,

daunorubicin, hydroxyrubicin and the β anomer of doxorubicin with calf thymus DNA; Keq is the

binding constant and n is the number of base pairs per binding site.

The polyelectrolyte theory quantitatively describes the thermodynamic linkage

between cation and charged ligand binding to DNA. The cations are condensed around the

DNA in order to neutralize the highly negatively charged backbone. The cations are

nonspecifically bound and are mobile. Binding of positively charged ligand to the DNA

results in the release of bound cations, since the positive charge on the ligand may serve to

neutralize the DNA in the place of cation.

The doxorubicin and hydroxorubicin binding data are summarized in Table 2.1.1.2.

Both of these compounds possess the protonated daunosamine group and are still positively

charged at a pH value of 7. The β anomer of doxorubicin is a stereoisomer of the parent

compound with an altered orientation of the C-1’ adriamycine. The table also contains the

values of the slope (δ log K/δ log [M+]) ranging from -0.91 to -1.25 for anthracycline

antibiotic binding to DNA. The exception is hydroxyrubicin with a value of 0.18. For the

binding of a monovalent intercalator to DNA the quantity (δ log K/δ log [M+]) is predicted

to be -0.88 by Record and coauthors [26, 27] and -1.24 by Friedman and Manning [28].


19

Friedman and Manning calculated a value of (δ log K/δ log [M+]) = -0.24 for the binding of

an uncharged intercalator to DNA.

The observed free energy of antibiotic binding is defined as ∆Gobs° = - RT ln Kobs.

Values calculated for the binding constant are shown in the Table 2.1.1.3. The observed free

energy may be partitioned into two contributions:

∆Gobs° = ∆G° + ∆Gel II.1.1

where ∆G° is the nonelectrostatic contribution to ∆Gobs° and ∆Gel is the polyelectrolyte

contribution to ∆Gobs°.[11]

Compound R1 R2 ∆Gobs° ∆G° ∆Gel° δ ∆G

Doxorubicin

Daunorubicin

Hydroxyrubicin

β anomer of doxorubicin

OH NH2 -8.8 -7.8 -1.0 0

H NH2 -7.9 -6.9 -1.0 0.9 OH OH -7.2 -7.0 -0.2 0.8 OH NH2 -5.7 -4.7 -1.0 3.1

Table 2.1.1.3 Free energy of anthracycline antibiotic binding to calf thymus DNA.

2.1.1.3 Structural, Electronic and Spectroscopic Properties of Carminic Acid

Carminic acid (7-α-DGlucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-

9,10-dioxo-2-anthracene-carboxylic acid) is an antraquinone dye with a wide application

spectrum in the alimentary and textile industry. For instance it is used as dye in food and

drinks. The molecular structure (Figure1.3.1) consists of an anthraquinone chromophore, a


20

sugar residue, and a carboxyl group. Thus the carminic acid has a good solubility in water.

As being a carboxylic acid it is a weak acid so that its dissociation equilibrium crucially

depends on the pH value of the aqueous solution. The differences in electronic structures of

the dissociated and undissociated carminic acid are reflected by their absorption spectra

measured at different pH values between 2 and 9. At a pH value lower than 5, the carminic

acid is fully protonated (undissociated) and exhibits maximum absorption at 490 nm. With

rising pH value the absorption maximum is gradually shifted to 550 nm. The isosbestic

point appears at 515 nm [20].

Fig. 1.3.1: Structure formula of carminic acid in 2D and 3D.

O

OH

O

OH

CH3O

HO

OH

OH

HO

OH

OOH

HO


21

Cyclovoltammetric studies of the interaction of carminic acid with human serum

albumin (HSA) show that carminic acid has a sensitive linear sweep voltammetric reductive

peak at -0,54 V (vs. SCE). After the addition of HSE in the carminic acid solution the peak

decreased markedly [29].

2.2 Methods

2.2.1 Stationary Optical Spectroscopy

The electronic structure and the optical properties of organic molecules are examined

using stationary and time-resolved techniques of optical spectroscopy. Interactions between

photons and molecular states may lead to an indepth insight into the molecular electron

configuration and excited-state relaxation processes.

The fundamental methods for the study of molecular optical properties are the

stationary techniques of UV/VIS absorption and fluorescence spectroscopy. The wavelength

range of these techniques conventionally reaches from the UV(180 nm) to the near infrared

at 900 nm.

Spectroscopy is the study of matter by investigating light, sound, or particles that is

emitted, absorbed or scattered by the matter under investigation. The energy of a light

quantum is hυ = k · 1,24 · 10-4eV cm. The spectrum of electromagnetic radiation can be

situated between the 50 Hz, the normally AC frequency, and secondary high radiation at

1020 Hz shows in the Fig. 2.2.1.


22

Fig. 2.2.1 Spectrum of electromagnetic radiation: the spectral range of optical spectroscopy

is depicted in enlarged form.

2.2.1.1 UV/VIS Absorption Spectroscopy

The total energy of a molecule in its electronic ground state with exception of the translation

and internal nuclear energy is the sum of the electronic energy (Ee), the vibrational energy

(Ev) and the rotational energy (Er):

Et = Ee + Ev + Er II.2.2.1

The total energy for the excited state (Et’) is the sum of the electronic vibrational and

rotational energy from the excited state (Ee’, Ev’, Er’). The absorption transition can be

defined as:

102 105 108 1011 1014 1017 1020 1023

800 20000

700 300 600 500 400

Near

infrared

Red

750 680 622 597 370 315 280 492 455 575 Crim

son

Orange

Yellow

Green

Blue

Violet

UV A

UV B

UV C

105 104 103 100 10 1 100 10 1 100 10 1 100 10 1 100 10 1 100 10 1 100 1 100 10

km m mm µm nm pm

Röntgen radiation

Gamma radiation

IR Microwave Radio wave Longest wave Cosmic radiation

λ

Hz

eV 10-12 10-9 10-6 10-3 100 103 106 109

10-6 10-3 100 103 106 109 1012 cm-1


23

∆Ex = Ex’- Ex , II.2.2.2

where x represents t, e, v, or r. The ∆Er is in the order of magnitude of 10 cm-1, typical

values of ∆Ev are about 103 cm-1 and those of ∆Ee are around 3 • 10

4 cm-1. While excited

electronic states may be investigated using the UV/VIS absorption spectroscopy, the

rotational absorption transitions and vibrational transitions occur in far infrared and near

infrared spectrum, respectively.

The absorption of visible and UV light by organic molecules with a closed shell is

associated with electronic transitions between the electronic singlet ground state (S0) and

higher excited singlet states (Sn, with n=1,2,3,…). Transitions of σ electrons occur via

excitation at photon energies equivalent with wavelengths between 120 and 200 nm. At

wavelengths longer than 200 nm electrons in p-, d-orbitals and π molecular orbitals may be

excited.

To quantitatively characterize absorption transitions of molecular electronic states in

solution samples Lambert-Beer’s law defines the molar extinction coefficient ε. If a

monochromatic light beam with the intensity I0 is incident on a rectangular cell (i.e. cuvette)

with the path length d (in cm), containing the concentration c of the compound under study,

the intensity I of the transmitted light beam decay mono-exponentially over the path length

of the cuvette:

I = I0 e- ε c d , II.2.2.3

The prior conditions of Lambert-Beer’s law are that, first, the fraction of the incident

light absorbed by the sample is independent of the total intensity of the light source, second,

the concentration of the absorbing molecules is infinitely small and third, the incident light

beam falls orthogonally of the plan-parallel quartz windows of the sample cuvette. The so-


24

called absorption or absorbance, d, of the cuvette, is defined as the decadic logarithm of the

ratio I0/I:

A= cdI

I⋅⋅= ε0lg II.2.2.6

where I0 is the intensity of the incident light, I is the intensity of the transmitted light, ε the

molar decadic extinction coefficient (in l cm-1 mol-1), c is the concentration (in mol/l) and d

is the path length of the cuvette (in cm).

The experimental absorption spectrum is plotted absorption, A, versus the

wavelength, λ.

Fig. 2.2.1.1 Schematic representation of a UV/VIS absorption spectrometer.

In practice, a chromophore giving rise to light absorption by a totally (dipole) allowed

optical transition has values of ε larger than 10 000 l/(mol cm). For the excitation of absorption

transitions in the UV regime a deuterium high pressure arc is employed, whereas a tungsten-

halogen lamp provides for the excitation in the visible. The excitation light is dispersed using a

grating monochromator. The intensity of the monochromatic excitation light, I(λ), after the sample

cuvette, and that of the excitation light after the reference cuvette, I0(λ), are detected using exactly

Grating Beam Splitter

Sample

Reference

Detectors

PMT 2

PMT 1 UV VIS lamps


25

the same type of photomultiplier tube (PMT1 and PMT2). The absorption spectrum for the

wavelength λ is obtained by subtracting the intensities of the transmitted light beams (A(λ1) = lg

I0(λ1) – lg I(λ1)).

2.2.1.2 Fluorescence Spectroscopy

Fluorescence is a dipole-allowed spontaneous emission of light that occurs from an

electronically excited singlet state. In the first excited singlet state the electron in the lowest

unoccupied molecular orbital (LUMO) is of opposite spin orientation relatively to the other

electron in the highest occupied molecular orbital (HOMO). Consequently, the return to the

ground state is spin allowed and occurs rapidly by emission of a photon. The fluorescence

lifetime of organic compounds in solution ranges from ps to hundreds of ns. Photo-physical

processes following an optical absorption transition of molecular systems are described in

terms of the Jabłoński term scheme (Figure 2.2.1.2.1). The ground state, the first and

second singlet states, are denoted by S0, S1 and S2, respectively, whereas T1 is the first

excited triplet state and the sublevels of each electronic state arise from the fundamental and

the overtones of one representative vibration with the vibrational quantum number 0, 1 and

2. The electronic absorption transition between the ground states S0 and the S1 or S2 occur

within 10-15 s.

Fig. 2.2.1.2.1 Jabłoński term scheme.

2 1 0

hυA hυF hυA

Internal conversion Intersystem

Crossing S1

S2

S0

T1

Absorption

Fluorescence

Phosphorescence

hυP

Internal conversion


26

In general, the electronic excitation of molecules involves the occupation of higher

vibrational levels of the S1 or S2 states. With a few rare exceptions, molecules in condensed

phases rapidly relax to the vibration less first excited electronic state (S1). This nonradiative

relaxation process is the so-called vibrational relaxation that occurs in 10-12 seconds or less.

Nonradiative deactivation of higher excited singlet states to the S1 state takes place by

internal conversion with time constants between ps and ns that are given by the energy gap

between the involved states (Fermi’s Golden rule). Since the lifetime of the excited S1 state

is about 10-8 s, vibrational relaxation has to occur prior to fluorescence, internal conversion

and intersystem crossing. The radiationless deactivation processes of the S1 state as being

internal conversion and intersystem crossing occur in competition with the fluorescence.

While the internal conversion promotes the electronically excited molecules to their ground

state by dissipation of vibrational energy, the intersystem crossing occurs via spin flip as an

isoenergetic transition to the vibrational manifold of the triplet system. Its probability is

ruled by spin-orbit coupling and thereupon, by the nuclear configuration of the molecule.

The time constant of the intersystem crossing may vary between ps and ms.

Fluorescence relaxation to the ground state occurs to a higher exited vibrational

ground state. Deactivation of the triplet state (T1) takes place either by phosphorescence or

by internal conversion. Since both processes, the radiative and the radiationless one, are spin

forbidden, their time constants may reach values up to several seconds. In general the

fluorescence spectrum is red shifted in comparison to the absorption spectrum. This

phenomenon is called Stokes shift and may be explained by the Strickler-Berg relationship

[30].

Figure 2.2.1.2.2 shows a setup for measurements of fluorescence spectra. A

fluorescence spectrometer consists of an excitation part and a detection part. The excitation

light may be either generated by a xenon high-pressure arc, or by a laser. For the xenon

high-pressure arc the excitation wavelength is selected with a grating monochromator (i.e.

excitation grating). A beam splitter reflects a part of the excitation light to a photon counter

that monitors light intensity instabilities and enables therefore their compensation for the


27

final spectrum. The emitted fluorescence light is isotropic and has to be collected by a

converging lens. An ideal detection configuration is the collection of fluorescence emission

under an angle smaller than 90° relatively to the excitation beam. For anisotropy

measurements two polarizers are used, one in front of the sample and the other behind the

sample.

Fig. 2.2.1.2.2 Setup of a fluorescence spectrometer.

2.2.1.3 Measurement of Fluorescence Quantum Yield and fluorescence lifetime

A quantitative measure for the fluorescence probability of an excited molecule is

acquainted by the determination of the fluorescence lifetime and the fluorescence quantum

yield. The fluorescence quantum yield is given by the number of emitted photons relative to

the number of the absorbed photons. In the case of compounds with a large fluorescence

quantum yield, approaching unity, the displayed emission is very intensive.

PMT 2

PMT 1

Slit 1

Slit 2

Source Counter

Slit 3

Slit 4

Excitation grating

Emission grating


28

The fluorescence quantum yield is the ratio of the number of photons emitted from

the compound under study and the number of photons absorbed by the substance. The

processes governed the Γ and knr with both depopulating the excited state. The fraction of

fluorophores which decays by emission, and hence the quantum yield is given by:

nrk

Q+ΓΓ

= II.1.3

where Г is the radiative decay rate.

The quantum yield can be close to one, if the radiationless decay rate is much smaller

than the rate of Stock’s losses. For simplicity, all nonradiative decay processes of the first

excited singlet state (S1) are accounted for with the rate constant knr. The lifetime of the S1

state is defined by the mean time the molecule spends in the excited state prior to their

return into the electronic ground state. The fluorescence lifetime is given by:

nrk

1

+Γ=τ II.1.4

The fluorescence emission is a random (spontaneous) process: a few molecules emit

their photons at precisely t = τ. More than 60% of the fluorescing molecules decay earlier

than t = τ, while the other 40% will emit at t > τ.

τ/0)( teItI −= II.1.5

The natural radiative lifetime or intrinsic lifetime and is given by:

Γ

=1

nτ II.1.6


29

The natural radiative lifetime can be calculated from the absorption spectrum (ε) and the

fluorescence spectrum (F) of the fluorophore. The radiative decay rate Γ can be calculated

using:

∫∫∫ =⋅=Γ − ν

ννε

ννν

ννd

dF

dFn

)(

/)(

)(1088.2

3

219

∫−−− ><⋅νννε

νd

n)(

1088.2 13219 II.1.7

F(ν ) is the fluorescence spectrum as function of the wavenumber (in cm-1), ε(ν ) is

the absorption spectrum, and n is the refractive index of the medium. The integrals are

calculated for the S0 – S1 absorption transition and by integrating over the total fluorescence

spectrum, respectively.

The natural lifetime can be obtained from the measured fluorescence lifetime (τ) and

quantum yield:

Q

n

ττ = II.1.8

This equation results from Eq. II.1.3, II.1.4 and II.1.6.. Many biochemical

fluorophores do not behave as predicted for aromatic compounds. There is often poor

agreement between the lifetime calculated using Eq. II.1.7. and II.1.8. that may be explained

by protein residues acting as fluorescence quenchers.

Scintillators are generally used because of their high fluorescence quantum yield.

High fluorescence quantum yields are a result of large Γ values. Hence, their fluorescence

lifetimes are generally short, around 1 nanoseconds. The quantum yield of phosphorescence

in condensed matter is extremely small at room temperature. The triplet to singlet transition

is spin forbidden, and the rate of spontaneous emission is about 103 s-1 or smaller.


30

Fluorescence anisotropy measurements are commonly used for biochemical studies

of macromolecules (i.e. proteins, DNA). This type of measurement technique provides

information of the size and shape of the molecules or the rigidity of various molecular

environments.

Dipole-allowed optical transitions are associated with the absorption or emission of

light the electric field vector is aligned in parallel to the transition moment. The transition

moment has a definite orientation with regard to the molecular axes. The random orientation

of fluorescent molecules in solution generates an isotropic environment. Upon excitation

with polarized light, one selectively excites those fluorescent molecules whose absorption

transition dipole momentum is parallel to the electric field vector of the excitation light.

This photo-selective excitation results in a partially oriented population of fluorophores and

in a partially polarized fluorescence emission. Emission also occurs with the light polarized

along the fixed axis in the fluorophore. The relative angle between these moments

determines the maximum measured anisotropy. The fluorescence anisotropy (r) and

polarization (P) can be calculated with help of the equations:

II.1.9

II.1.10

where III and ⊥I are the fluorescence intensities of the vertically (||) and horizontally (┴)

polarized emission, when the sample is excited with vertically polarized light.

Decrease of fluorescence anisotropy can be caused by several phenomena. The most

common case is rotational diffusion. This type of diffusion occurs during the lifetime of the

excited state and displaces the emission dipole momentum of the fluorophore. Measurement

⊥

⊥

⊥

⊥

+−

=

+−

=

II

IIP

II

IIr

II

II

II

II

2


31

of this parameter provides information about the relative angular displacement of the

fluorophore between the time of absorption and the time of emission. The rotational

relaxation time of most fluorophores in solution ranges between 50 and 100 ps. This implies

that the molecule may rotate several times during its lifetime of the excited state (1-10 ns).

2.2.2 Time-Resolved Optical Spectroscopy

2.2.2.1 Femtosecond Transient Absorption Spectroscopy

In order to investigate interaction between solute and solvent, high time resolution is

an essential aspect. One of the spectroscopic experimental techniques used in this thesis is

the femtosecond-resolved transient absorption pump-probe spectroscopy. In such an

experiment a strong femtosecond (fs) pump pulse is used to initiate a photoreaction, and the

reaction dynamics is followed by recording the absorbance of an week monitoring pulse, as

a function of the time delay between the pump pulse and the monitoring pulse. In order to

eliminate the possible noises introduced by possible intensity fluctuation of the laser the

monitoring pulse is divided in a probe and a reference pulse. The probe pulse in spatially

overlapped with the pump pulse in the sample, while the reference pulse passes through a

region of the sample which is unaffected by pump pulse. The change of the optical density

(∆OD) is obtained as a negative logarithm of the ratio of the intensity of the probe and

reference pulse as follows:

ref

probe

I

IOD log−=∆ II.2.1

Liquid phase absorption and emission spectra generally contain broad bands, which

makes it essential to examine the temporal behavior of the photoinduced reaction over a


32

wide range of probe wavelengths. In the experiment the probe pulse has a white light

continuum (wlc) spectrum. The wlc is generated by the χ(3) process of self-phase modulation

of an intense femtosecond laser pulse propagating through a dense but transparent medium

[31, 32], here a fused silica disc. Under proper experimental condition a wlc can be made to

extend from the near ultraviolet to near infrared. After the sample, the wlc is dispersed by a

spectrograph and detected by a charged coupled device (CCD) camera which allows for

simultaneous measurement of the intensities of the probe and reference pulse for a wide

range of wavelengths present in the wlc. In this sense the pump-probe signal, ∆OD (λ, ∆t),

is obtained as a function of the probe wavelength and the delay time ∆t between the pump

and the probe pulses.

In a heterodyne detection scheme, the electric field of the strong pump pulse,

Epump(z,t), and the electric field of the weaker probe pulse, Eprobe(z,t), generate a non-linear

polarization, PNL, in the sample, where z is the sample thickness. The non-linear

polarization acts as a source term in the Maxwell’s equation to generate a signal field, ES.

The probe field is called the local oscillator field [33], and in these terms the total measured

intensity is proportional to the square of the total field. In order to obtain the change in

optical density, a reference field is needed, Eref , which is the probe field unaffected by the

pump, Eprobe(0,t). The total detected intensity can then be written as [33]:

)),(),0(*Im(2),(

|),0(||),0(|),(

|),(),0(||),(|),(

22

22

tzEtEItzI

tEtEtzI

tzEtEtzEtzI

Sproberefprobe

refproberef

Sprobeprobeprobe

−∝−

=∝

+=∝

II.2.2

The signal field ES is usually weak so that the detected intensity is approximately

proportional to the sum of the cross term, and the reference term, |ES (z,t)|2 , in Eq. II.2.2 is

neglected. In order to normalize the measured intensity the probe is divided by the reference


33

intensity, and to achieve the change in the optical density, the negative logarithm is taken of

this ratio as:

ref

refprobe

ref

probe

I

II

I

IOD

−⋅

−≈

−=∆

)10ln(

1log II.2.3

The approximation in Eq. II.2.3 is valid only under the condition, when (Iprobe – Iref)/ Iref <<1

[34].

Fig 2.2.2.1.1 Energy scheme of the electronic states involved in a pump-probe

experiment; excited states relaxation dynamics detected by absorption changes of the probe

pulse are: 1) bleaching; 2) excited state absorption; 3) stimulated emission.

In a pump-probe experiment there will be three contributions to the transient

absorption pump-probe signal (see Figure 2.2.2.1.1). First, the strong pump pulse excites

the molecule from the ground state S0 to a higher lying state SN. The probe pulse may also

excite the molecules from the ground state to the same excited state as the pump pulse,

which will result in a bleaching of the ground state, 1) in Figure 2.2.2.1.1. This may be

observed as an increase in the detected probe intensity since less probe photons is absorbed

relative the reference. An increase in probe pulse intensity is the same as negative ∆OD. For

the situation of the probe pulse (see 2) in Figure 2.2.2.1.1) the molecules in the excited state

undergo so-called transient absorption transitions. This is seen as an increase in ∆OD.

Pump Probe 1)

Probe 3)

Probe 2)

SN+M

SN

S0

S1


34

Finally there may be stimulated emission as the case 3) in the same figure and this is will be

detected as a decrease in the ∆OD.

However most of the samples are transparent in the pump wavelength region, both to

one- and two-photon absorption. In this case there is no excited state in the sample, and

consequently neither bleaching, excited-state absorption nor stimulate emission when the

probe pulse is present in the signal. But even in those cases there will be detectable signals,

when the probe is dispersed after the sample, due to the change in non-linear polarization of

the sample.

Positive values of ∆OD may be ascribed to either bleaching of the ground-state (S0)

population density or stimulated emission, while negative values of ∆OD are generally

assigned to absorption transitions from the first excited singlet state, S1, to higher singlet

states (i.e. transient absorption). Both phenomena, S0-state bleaching and transient

absorption from the S1 state are induced by the pump pulse. Both, recovery of the S0-state

population density and decay of the transient absorption exhibit mono-exponential behavior

with a time constant that is the lifetime of the S1 state, if the latter is determined by pure

monomolecular photo-physical processes.

The experimental setup for the fs transient absorption pump-probe spectroscopy is

depicted in Figure 2.2.2.1.1. The central wavelength of the pump pulse is adjusted to the

energy gap between the electronic ground state (S0) and the first excited singlet state (S1) of

the molecules under study, while a white-light continuum is used for the probe pulse in

order to enable a large variety of absorption transitions from the S1 state to higher lying Sn

states. A delay stage is used to vary the path length of the probe pulse in small steps in order

to obtain a variable time delay between the pump and the probe pulses. The delay stage may

vary the path of the probe pulse down to steps of 1µm, corresponding to 3.3 femtoseconds.

The maximum delay time what can be applied between pump and probe pulses is about 1

nanosecond. A part of 5% from the 800 nm laser beam (fundamental) is reflected by a beam

splitter and sent to the diagnostics part where the time and frequency profile of the laser

pulse can be measured by an autocorrelator and a spectrometer. The 95% transmitted laser


35

beam is raised to a convenient level by a mirror periscope and a λ/2 plate rotates the

polarization of the beam from horizontal to vertical. This gives s-polarized light with respect

to reflection in the horizontal plane which is what the dielectric mirrors, frequently used in

set up, are optimized for. The two time magnifier telescope reduces the beam diameter to a

half and consists of a concave mirror focusing the beam and a convex mirror restoring the

beam collimation. The chopper triggers the detector and synchronizes it with the chopped

light. A second beam splitter divides the beam in a pump beam (90%) and a probe beam

(10%). The pump pulses are frequency doubled in a non linear crystal (BBO). The probe

pulses can be shifted in time by the delay stage. The wlc probe beam is split in a reference

beam and a probe beam. The probe pulses pass the sample cell to spatially overlap there

with the pump pulses, whereas the reference beam crosses the sample unaffected by the

pump beam.


36

Fig. 2.2.2.1.1 Schematic representation of the fs transient absorption spectroscopy

experiment; BS: Beam Splitter; FM: Flip Mirror; DS: delay stage; VA: variable attenuator; P:

polarizer; L: lens; WLC plate: rotating fused silica plate for white light continuum generation; PM:

parabolic mirror; SHG: BBO crystal for second harmonic generation; BC: Berek compensator; BD :

beam dump; BG: BG38 filter; GG: GG420 filter; ND: neutral density filter.

The sample solution is through a flow cell with a thickness of 2 millimeters. A

spectrograph disperses the probe and the reference beams, which are detected by a CCD

camera. A computer controls the experiment and plots the ∆OD as a function of delay time

and frequency.

┴ ||

5%

Ti:Sapphire laser λ/2

DM

pulse characterization

SHG

DS

P λ/2

10% BS 90%

λ/2 L WLC plate PM

probe reference

FS

sample

spectrograph CCD

spectrograph CCD

autocorrelator

chopper

X2 telescope

periscope VA

DM

BD

95% 800nm

400nm

ND GG BG

BC

pump


37

The transient absorption spectroscopy is based on a Ti: Sapphire laser system that

consists of a mode-locked oscillator (Coherent, Mira) pumped by an argon ion laser

(Coherent, Innova 310) and a Ti: Sapphire regenerative amplifier (Alpha 1000 US, BM

Industries). This femtosecond laser system produces 40 fs output pulses at 800 nm with a

repetition rate of 1 kHz and energies of about 700 µJ. The pump pulses at 400 nm are

obtained by frequency doubling the output pulses in a 0.2 mm BBO crystal, whereas the wlc

probe pulses between 400 and 800 nm are generated by focussing a small fraction of the

fundamental pulses (5%) into a thin rotating fused silica disc. By applying the pump pulses

at 400 nm to the sample solution, carminic acid is non-resonantly excited into the S1 state,

while the excited-state decay dynamics are probed with the temporally delayed white-light

continuum pulses.

Wlc probe pulses are generated by self phase modulation (SPM) occurring as a

parametric χ(3) process in a Kerr medium. SPM involves the modulation of the refractive

index of the medium in response to ultrashort laser pulses. The modulated refraction index

modulates the pulse phases and thereupon the frequencies of the electromagnetic waves

contained in the wave packet. Self phase modulation is based on the Kerr effect accounting

for the nonlinear dependence of the refractive index n(t) on the intensity I(t) of the pulse

propagating along the space coordinate:

220 |)(|)( tEnntn ⋅+= , II.2.1

where n0 denotes the linear refractive index, n2 the nonlinear refractive index and E(t) is the

electric field of the laser beam.

Combining the expression for nonlinear refracting index (II.2.1) with a linear wave

equation [35]:

2

2

2

22

t

E

c

nE

∂

∂−∇ II.2.2

derived from the Maxwell’s equation, where c is the velocity of light and n is the refractive

index, gives rise to the nonlinear term on the right side of the nonlinear wave equation:


38

2

2

02

2

2

202

t

P

t

E

c

nE NL

∂

∂=

∂

∂−∇ µ , II.2.3

with:

EEc

nnEEEPPNL

22

20)3(00

)3(00 ||

2≈== χεµµµ , II.2.4

where the n22|E| term has been neglected and n2 and χ

(3) are assumed to be instantaneous.

Expressing E as a plane wave with the angular frequency ω0 and the wave vector k

propagating in the z- direction with E = A(t)ei(kz- ω0t) + A*(t)e-i(kz- ω0

t) and inserting this into

Eq. II.2.3 give the following equation for the amplitude A(t)[36]:

AAnc

it

A

z

A 22

0

g

||2v

1 ω=

∂∂

+∂∂

, II.2.5

where vg is the group velocity. Both, the group velocity dispersion and linear absorption,

are neglected.

Solving Eq. II.2.5 with respect to the phase reveals a time dependence of the phase

Φ(t). The instantaneous frequency is the time derivation of the phase and can be written as

[37]:

zt

tn

ct

tt

∂∂

−=∂∂

=)(

)()( 00

ωωφω II.2.6

The frequency shift is:

t

tIz

nc

nt

∂∂

=−=)(

)(00

220

0 εω

ωωδω , II.2.7

where I(t) is the intensity of the pulse and is written as:

20 |)(|2)( tAcntI ε= II.2.8


39

Fig. 2.2.2.1.4 a) Intensity as a function of time for a Gaussian laser pulse b) Time

dependence of the frequency for a positive nonlinear index of refraction, n2.

The wlc probe pulse in the propagation direction is a repetition of monotone pulses with

continually increasing central wavelength. These spectral pulse components of the wlc pulse

have the same temporal pulse width as the wlc generating pulse which is the pump pulse.

Fig. 2.2.2.1.5. Representation of the wlc probe pulse as a composition of the temporally

shifted, different spectral sub-pulses having the same pulse width as the pump pulse.

λ


40

The fundamental nonlinear optical phenomena arising from strong light-matter

interactions are parametric three-wave mixing and four-wave mixing that give rise to second

harmonic generation (SHG), sum frequency generation (SFG), difference frequency

generation (DFG) and third harmonic generation (THG). While SHG, DFG and SFG are

parametric χ(2) processes and are derived from the second order term of macroscopic

polarization P(2)=ε0 χ(2) E2, the THG is a χ(3) and is due to P(3)=ε0 χ

(3) E3.

SHG, SFG and DFG directly result from P(2)=ε0 χ(2) E2 for electromagnetic waves the

with angular frequencies ω1 and ω2:

)cos()cos( 222111 zktEzktEE −+−= ωω II.2.9

That are incident on a non-centrosymmetric medium with a non zero χ(2). E1 and E2 are the

amplitudes of the two beams and k1 and k2 are the wave vectors in the direction z with ki

=2πn(λi)/λi. The second order polarization becomes [39]:

[ ]ttEEtEtEEE

ttEEtEtEzEP

)cos()cos(2cos2

12cos

2

1)(

2

1

)coscos2coscos()0(

212121222

2122

122

21

)2(0

2121222

2122

1)2(

02)2(

0)2(

ωωωωωωχε

ωωωωχεχε

−++++ ++=

=++===

P(2) contains as components a static electric field with ½ε0χ(2)(E1

2+E22), the SHG of both

fundamental waves with ω1 and ω2 (i.e. 2ω1, 2ω2), the SFG with ω1+ω2 and the DFG with

ω1-ω2. For efficient SFG and DFG the incoming and generated waves must fulfil the phase

matching condition:

2121 ) ( kkk ±=±ωω II.2.11

Because of ki =2πn(λi)/λi the phase matching condition can be fulfilled by changing the

refractive index n(λ) of one wave with respect to the others. In practice, this can be provided

II.2.10


41

by uniaxial birefringent crystals that have two different refractive index n0 and ne(θ) for the

ordinary and the extraordinary beams, where θ is the angle between optical axis and the

wave vector. The ordinary beam is polarized perpendicular to and the extraordinary beam

parallel to the plane defined by the propagation direction and the optical axis.

2.2.2.2 Femtosecond Fluorescence Up-Conversion

The fluorescence spectrum, quantum yield and lifetime of a chemical compound

depend on its environment and of the solute-solvent interaction. In particular, the

fluorescence lifetime may reflect the kinetics of a chemical reaction. Time-resolved

fluorescence spectroscopy measurements give detailed information about intra- and

intermolecular photophysical and photochemical processes. In the last 30 years, the laser

technology and especially, the ultrafast laser techniques enable precise measurements of

molecular excited-state relaxation dynamics in the nano-, pico- and femtosecond time range.

When analyzing the rise and the decay, often multiexponential of their fluorescence

intensity as a function of time, the pre-exponential factor and the fluorescence time constant

have to be determined accurately after a careful deconvolution using a computer-based

algorithm. Several techniques are used: phase modulation spectroscopy with a modulated

laser pump source on a time scale comparable to the decay times of interest, time-correlated

single photon counting under picoseconds laser excitation, ultra-fast streak cameras

designed for the conversion of time dependent light emission into distance dependent

electron impact on a phosphor screen. Because a sufficient resolution is not possible with

this technique for measurements of fast decay in the range 50 fs – 100 ps, the fluorescence

up-conversion in nonlinear crystals is the best choice.

Although femtosecond mode-locked Ti: Sapphire lasers which offer tunability and

pulse duration similar to those of dye laser but higher stability and ease of use are also

available, ultrafast kinetics in the range of femtosecond cannot be resolved in the


42

nanosecond or picoseconds technique since the limiting factor of their response function is

the detector. The time-gated up-conversion technique is by far the best choice for the

femtosecond time range.

The phenomenon of fluorescence up-conversion is based on the parametric three-

wave mixing process of SFG. The fluorescence emitted after an fs laser pulse excitation is

mixed with the other half of the laser pulse in a nonlinear optical crystal to generate sum

frequency radiation. The crystal acts as an ultra-fast optical “light gate” that is “open” when

the pump pulse is “on”. This method is particularly suited for laser pulses of moderate peak

powers but very high repetition rate. Common nonlinear crystals are cut from β-barium

borate (BBO), potassium dihydrogen phosphate (KDP), lithium iodide (LIO) or urea. In

general BBO crystals are used because of their optical transparency in the UV range.

The fluorescence light with the energy EF and the pump laser beam with the

energy EP are focused into the BBO crystal under phase matching conditions so that the sum

frequency light with the energy ES is produced:

SS hE ν= II.3.1

PP hE ν= II.3.2

FF hE ν= II.3.3

The phase matching conditions are:

PFS ννν += II.3.4

PFS kkkrrr

+= , II.3.5

where Skr, Fkr and Pk

r are the wave vectors of the sum frequency, fluorescence and pump

pulses, respectively. In the case of collinear configuration the phase matching Eq. (II.3.5)

becomes:

S

S

P

P

F

F nnn

λλλ=+ II.3.6


43

where n is the crystal refractive index at the wavelength λ.

In uniaxial crystals with nx = ny = n0 (ordinary index) and nz =ne (extraordinary

index), the refractive indices vary with the wavelength according to Sell Meier’s equation:

22

2 λλ

DC

BAn +

−= II.3.7

For type I crystals (O + O → E), the fluorescence and the pump are polarized parallel to

each others. In type II crystals (E + O → E) or (O + E →E), the pump and fluorescence

beams are polarized perpendicularly. For type I crystals the phase matching angle can be

calculated using the following equation:

2

S,0

2

S,e

2

S,0m

2

S

m

2

n

1

n

1

)n

1

)(n

1(

sin

−

−

=θ

θ II.3.8

where nS(θm) satisfies the following equation:

P

SPO

F

SFOmS nnn

λλ

λλ

θ ,,)( += II.3.9

The phase mismatch is given by

)(1

PPFFSSpPS nnnc

kkkk ϖϖϖ −−=−−=∆ , II.3.10

where c is the velocity of light and ωi is the angular frequency (i

in2

c2

λπ

ϖ = ). Since

fluorescence is polychromatic, isotropic radiation, the fluorescence light has to be collected

and to be focused onto the crystal and the phase matching angle has to be adjusted in

dependence on the wavelength of interest. The acceptance angle increases inversely with the

crystal length.


44

Only a narrow band of the fluorescence spectrum is up-converted for an appropriate

phase matching angle θm. The full spectrum is obtained by varying the angular position of

the crystal.

The quantum efficiency for up-conversion varies as the ratio of the power PP of the

pump of the beam to its area A in the BBO crystal, the square of both the thickness L and

the effective nonlinear coefficient of the crystal deff according to:

)(nnce

A/PLd2n

mP0F.0FF

3

0

P

22

eff

2

q θλλ

π= , II.3.11

where ε0 is the permittivity in vacuum. The quantum efficiency, ηq, the damage threshold,

Ithr(GW/cm2), and the cut-off for the up-conversion fluorescence calculated for a pump

pulse wavelength at 800 nm, λf,min(nm) are summarized in Table 2.2.2.2.

Crystal type ηq Ithr(GW/cm2) λf,min(nm)

KDP (ooe)

LiIO3 (ooe)

BBO (eoe)

BBO (ooe)

urea (eeo)

1.0

44

4.1

6.5

6

30 (ps regime)

15 (ps regime)

50 (ps regime)

50 (ps regime)

5 (ps regime)

344

480

264

249

313

Table 2.2.2.2 Relative quantum efficiencies ηq (normalized relative to KDP), damage

thresholds Ithr, and cut-off wavelengths for fluorescence up-conversion with 800 nm pump pulses.

A typical fluorescence up-conversion experiment is schematically described in

Figure 2.2.2.2.1. The femtosecond laser source is a Ti: Sapphire oscillator (Coherent

MIRA) pumped by continuous wave Coherent VERDI V10 laser with a 10 W output power.

Typical performances of the Ti: Sapphire oscillator is 1.8 W output power in the mode-

locked regime at 800 nm and 76 MHz repetition rate. The autocorrelation trace shows pulse

duration of 125 femtoseconds. The second harmonic at 400 nm is generated in a 1 mm thick


45

BBO crystal and separated from the fundamental 800 nm beam by a dichroic beam splitter.

After passage though a delay line, the residual fundamental is focused by a 100 mm lens

onto a 0.2 mm-thick type II BBO crystal producing the SFG signal. The 400 nm pulse beam

used for excitation is focused by a 1.5-inch concave mirror into the 1 mm thick flow quartz

cuvette cell what contains the solution sample. The fluorescence is collected by another

concave mirror with a 4 inch off axis. After the mirror the fluorescence passes through a 1

mm optical filter (Schott GG420) and is focused into the up-conversion crystal by a similar

concave mirror (4 inch off axis). The up-converted light beam is collected by a 150 mm UV

transparent lens, passes through another filter (Schott UG11), and is focused onto the

entrance slit of a 0.25 µm monochromator. The slit width corresponds to a 10 nm band-pass

filter. The spectrally selected UV light is detected by a photomultiplier connected to a lock–

in photon counter. All the experiments are performed at the magic angle between the

polarization of the excitation and the detection beams. The cross correlation trace between

the fundamental laser beam and the second harmonic give a full width at half maximum

(FWHM) value of 210 femtoseconds, confirmed by recording the Raman line (2900 cm-1 for

the C-H and 3500 cm-1 for the O-H stretching mode, respectively) of pure methanol with the

fluorescence up-conversion apparatus [40].


46

Fig. 2.2.2.2.1 Femtosecond up-conversion technique apparatus; DM: dichroic mirror; HW: half

wave plate; CCD video camera for the visual superposition of the beams in the BBO crystal; Mono:

monochromator; PM: photomultiplier.

Time resolved fluorescence spectra are usually obtained by recording kinetics at

different wavelengths and using the spectra reconstruction method. An alternative method is

to measure directly time resolved spectra to avoid the too long acquisition of the kinetic

data. In principle the acquisition of one complete spectrum at a particular delay time after

excitation does not suffer from long term variation of the experimental conditions. There are

two points of view to consider. First, different spectral components of the fluorescence

propagate with different group velocities in the various traversed media between the sample

cell and the non linear crystal. The second point is the limited half bandwidth of sum

frequency generation, typically 20 nm at 450 nm in a 0.2 mm thick BBO crystal. The

temporal and spectral response has thus to be corrected for. For the temporal correction,

while scanning the monochromator, the group velocity dispersion (GVD) is calculated as a

function of wavelength and the difference in propagation time is compensated by adjusting

Verdi 10 W

Ti: Saphire 2W

Lock in

PM

CCD

Mono.

1 mm flow cell

θ BBO 0.2 mm

BBO 1 mm

HW

DM

800 nm

800 nm

400 nm

GG420


47

the delay line. For the spectral correction, the optimal phase matching angle is calculated as

a function of wavelength and the crystal is rotated to the correct angular position using a

step motor and a PC computer. Moreover, the spectra are corrected for the spectral response

of the up-conversion apparatus. This correction is based on the comparison of the

fluorescence spectrum at a long time after excitation (typically 500 ps, when the molecular

system is spectrally relaxed) to the steady-state fluorescence spectrum corrected itself for

the response function of the conventional spectrofluorometer [40].

Experimental

48

3. Experimental

3.1 Materials: Chemicals and Sample Solutions

Carminic acid was purchased from Aldrich (99% purity) and was used without

further purification. Salmon sperm DNA (low molecular weight) was obtained from Fluka

Biochemika. DNA concentrations were determined upon performing UV/VIS absorption

spectroscopy measurements at 260 nm using the extinction coefficient ε(260nm) = 6600 (M

nucleotide)-1 cm-1. Ultrapure water was obtained from a Millipore system. The buffer

designated as BPES [11] consists of Millipore water with 6 mM Na2HPO4, 2 mM NaH2PO4,

1 mM Na2EDTA, and 185 mM NaCl, all from Fluka, and has a pH value of 7.0.

UV/VIS absorption spectroscopy and fluorescence spectroscopy measurements of

carminic acid in BPES, Millipore water or dimethylsulfoxide (DMSO) (Merck, pro

analysis) were performed at carminic acid concentrations between 5 µM and 10 µM,

Sample solutions (carminic acid in BPES) used for the fs resolved spectroscopic

investigation were prepared to attain an optical density of about 0.5 at 400 nm for an optical

path of 0.4 mm or 2 mm. For the determination of the pKa value of carminic acid we have

titrated carminic acid in Millipore water at a concentration of 5 µM or 1 mM with an

aqueous 5µM NaOH solution (e.g. 1 mM NaOH). The pH value was potentiometrically

measured using a pH meter (pH 522, WTW) equipped with a glass electrode (pH Electrode

Blue Line 23pH, Schott). The results are presented in the next chapter.

Experimental

49

3.2 Methods

3.2.1 UV/VIS absorption and fluorescence spectroscopy

UV/VIS absorption spectra of carminic acid in BPES, in Millipore water or in

DMSO were recorded on a Perkin-Elmer Lambda 2 spectrometer. The fluorescence spectra

were taken with a Jobin-Yvon FluoroMax-3 spectrofluorometer in the magic-angle

polarization configuration. All the stationary spectroscopy experiments were performed at

room temperature for carminic acid concentrations between 5 µm and 10 µM, employing a

quartz cell with an optical path length of 10 mm. For the determination of the pKa value of

carminic acid the different pH values were set by titration either with 1 M NaOH or with 1

M HCl. All experiments on DNA were performed using the BPES buffer solution (pH= 7).

3.2.2 Determination of the Fluorescence Quantum Yield

The fluorescence quantum yield Φ of carminic acid in Millipore water was

determined with reference to rhodamine 6G in ethanol (Φ0= 95 % [15]) using the

relationship:

2

00

2

0)101(

)101( 0

nS

nSA

A

⋅−

⋅−Φ=Φ

−

−

IV.2.1

where S and S0 are the integral intensities of the fluorescence spectra measured in the

magic-angle polarization configuration in the range of 500 - 800 nm, A and A0 are the

absorbances at the excitation wavelength of 480 nm of carminic acid and rhodamine 6G,

respectively, n denotes the refractive index of water and n0 that of ethanol.

Experimental

50

3.2.3 Fluorescence Titration Experiments

The fluorescence spectra were taken with a Jobin-Yvon FluoroMax-3

spectrofluorometer in the magic-angle polarization configuration. For the fluorescence

titration experiments an excitation wavelength of λexc = 320 nm with a slit width of 10 nm

was typically used, and the spectrum was recorded from 360 nm to 800 nm with a detection

slit width of 10 nm. The relative fluorescence intensities were determined by integrating

over the whole spectrum.

3.2.4 Femtosecond Transient Absorption Spectroscopy

The setup used for the femtosecond transient absorption spectroscopy [41] is based

on a femtosecond Ti: Sapphire laser system that consists of a mode-locked oscillator

(Coherent, Mira) pumped by an argon ion laser (Coherent, Innova 310) and a Ti: Sapphire

regenerative amplifier (Alpha 1000 US, BM Industries). This femtosecond laser system

produces 40 fs output pulses at 800 nm with a repetition rate of 1 kHz and energies of about

700 µJ. The pump pulses at 400 nm were obtained by frequency doubling the output pulses

in a 0.2 mm BBO crystal, whereas white-light continuum (wlc) pulses between 400 and 800

nm were generated by focussing a small fraction of the fundamental pulses into a thin

rotating fused silica disc. By applying the pump pulses at 400 nm to the sample solution,

carminic acid was non-resonantly excited into the S1 state, while the excited-state decay

dynamics were probed with the temporally delayed wlc pulses.

Transient absorption spectra were obtained by determining the differential absorption

in the sample versus the delay time τ between the pump and the white-light continuum

pulses. To compensate for eventual intensity fluctuations, the wlc was split into two parts,

one overlapping with the pump beam in the sample and thus serving as probe pulse and the

Experimental

51

other one, non-overlapping with the pump beam, serving as a reference. After passage

through the sample, both probe and reference beams were dispersed in a spectrograph (Spex

270 M) and detected using a 1024 x 512 pixels CCD camera (Princeton Instruments). The

readout rate of the camera was 30 Hz achieved by a chopper that modulates both beams.

The transmitted white light intensity of the probe (Tsample) and the reference (Tref)

beams were simultaneously measured on the CCD for delay times τ ≥ 0 ps. This delay time

was varied between 0 and 200 ps by 30 fs steps. The differential absorption is defined by the

equation:

<

<−

=

)0(

)0(log

)(

)(log

τ

τ

τ

τ

ref

sample

ref

sample

T

T

T

Tabsorption

IV.3.1

In this equation, Tref(τ) does not imply any direct dependence of the reference beam on the

time delay τ, only that it was the reference signal recorded simultaneously with the probe

signal that was used thus compensating for any intensity fluctuations. Thus defined, the

transient absorption is negative and the bleaching of the ground-state population density is

positive. The sample solutions were pumped through a flow cell with a thickness of 0.4 mm

or 2 mm. The 0.4 mm-thick flow cell was used for carminic acid concentrations between 1

and 6×10-4 M, whereas sample solutions at a concentration above 1×10-3 M were

investigated in a 2 mm cell. The FWHM of the instrument response function is about 60 fs

in the wlc spectral range of 400 – 800 nm.

3.2.5 Femtosecond Fluorescence Up-Conversion Technique

The femtoseconds resolved fluorescence up-conversion experiments were performed

using a setup that has already been described in detail in the chapter 2.2.2.2. A femtosecond

laser system, consisting of a Ti: Sapphire laser (Coherent MIRA 900), pumped by a 10 W

Experimental

52

CW solid state laser (Coherent VERDI V10), produces 125 fs pulses at 800 nm with a

repetition rate of 76 MHz and a 1.8 W average output power. The second harmonic (SH) is

generated in a 0.5 mm thick BBO crystal and is separated from the fundamental beam by a

dichroic beam splitter. The SH is used as pump pulse for excitation of the fluorescence of

the sample, whereas the fundamental pulse serves as gating pulse for the sum frequency

(“up-conversion”) generation. The fluorescence light is focused into the up-conversion

crystal, and the up-converted light is spectrally analyzed using a 220 mm focal length

double grating monochromator (SPEX 1680). The detection and amplification of the

dispersed up-conversion light is achieved by using a photomultiplier (Hamamatsu R1527P)

in combination with a photon counter (Stanford SR400). The fluorescence up-conversion

experiments were performed at 0° and 90° angle between the polarization axes of excitation

and detection. The cross correlation trace between the laser fundamental (800 nm) and the

SH (400 nm) gives a FWHM value of 175 fs for the apparatus function.

The fluorescence up-conversion spectra were corrected first by subtracting the

background from the fluorescence up-conversion spectra. The background was recorded by

positioning the delay at “negative” time, so that the gating pulse arrives to the up-

conversion crystal well before the fluorescence signal. The current spectral correction curve,

R(λ), was determined experimentally by comparing the normalized fluorescence up-

conversion spectrum at long times (100 ps: I100ps(λ)) with the normalized steady state

spectrum, ISS(λ) recorded for the same sample solution, so that the relationship

R(λ)=ISS(λ)/I100ps(λ) 2.5.1

is obtained. All fluorescence up-conversion spectra were subsequently multiplied with R(λ).

Experimental

53

3.2.6 Computations

Semi-empirical quantum chemical computations of the molecular geometries,

orbitals and electronic energy states of the four carminic acid tautomers in the fully

protonated state (CAH) as well as in the singly deprotonated state (CA-) were performed

using the HYPERCHEM (release 7.1) package program. The geometry optimizations of all

structures were achieved employing the ZINDO/S self-consistent fields molecular orbital

(SCF MO) method at the restricted Hartree-Fock (RHF) level [42]. For the optimizations we

have applied the steepest-descent method in combination with the consecutively performed

conjugate gradient methods, Fletcher-Reeves and Polak-Ribiere (convergence limit of

4.18×10-4 kJ/mol and RMS gradient of 4.18 ×107 kJ/m mol). RHF open-shell and closed-

shell calculations of the electronic energy states and of the electronic transitions were

performed for single configuration interaction (CI) involving 160 total orbitals.

Results and Discussion

54

4. Results and Discussion

4.1. Stationary Optical Spectroscopy

Carminic acid possesses a carboxylic acid function and is thus expected to dissociate

in water:

O

OH

O

O-

CH3

O

HO

OH

OH

HO

OH

OOHHOO

OH

O

OH

CH3

O

HO

OH

OH

HO

OH

OOHHO

+ H+

Scheme 1: Dissociation reaction of carminic acid.

The protonated carminic acid (CAH) and its anion (CA-) differ in their electronic

density distribution and thereupon, exhibit distinct dipole momentum and solvation shell

structure. Their different electronic configurations are reflected in the pH dependence of the

UV/VIS absorption spectrum of carminic acid dissolved in water (see Figure 4.1.1). As it is

obvious from Figure 4.1.1 there are in main two different dissociation equilibria, one exists

at a pH value of 5 and is shifted to CAH, while the other occurs at pH values above 5.


55

Fig. 4.1.1: pH dependence of the spectral features of carminic acid in water measured by

UV/VIS absorption spectroscopy.

For carminic acid in Millipore water a pKa value of 5.55 was determined upon

titration with either an aqueous NaOH solution or an aqueous HCl solution. The pKa value

of 5.55 implies that at pH = 7 the undissociated carminic acid, CAH, and its anion, CA-,

coexist at nearly the same concentration. Photo-exciting carminic acid in BPES (at pH= 7)

with 340 nm light generates two fluorescence emissions, one peaks at 15100 cm-1 (570 nm:

orange emission) and the other at 22700 cm-1 (470 nm: blue emission) ( see Figure 4.1.2).

30000 25000 20000 15000

0.0

0.1

0.2

0.3

0.4

pH= 3

pH= 4

pH= 5

pH= 6

pH= 7

pH= 8

pH= 9

ab

so

rpti

on

wavenumber / cm-1


56

12000 15000 18000 21000 24000 27000

0.0

0.2

0.4

0.6

0.8

1.0

no

rmalized

in

ten

sit

y

wavenumber / cm-1

Fig. 4.1.2: Dual fluorescence of carminic acid with a blue emission peak at 470 nm (22700

cm-1) and an orange emission peak at 570 nm (15100 cm-1)

The intensity of the orange fluorescence is half of the blue fluorescence intensity.

The observation of the dual fluorescence is taken as strong indication to an excited state

intramolecular proton transfer (ESIPT) [13, 43-46]. In general, the excited-state (S1)

tautomer accessed by ESIPT has an energy value comparable to or slightly less than that of

the normal form, while the ground state (S0) of the tautomer is of considerably higher

energy. As a consequence, the fluorescence of the tautomer appears spectrally red shifted

with respect of that of the normal form. That is why we ascribe the blue fluorescence to the

normal form, whereas we expect the tautomer to emit the orange fluorescence. In contrast,

carminic acid that was dissolved in the aprotic solvent DMSO and was analogously photo-

excited at 340 nm, exhibited the orange fluorescence only (see Figure 4.1.3).


57

12000 15000 18000 21000 24000 27000

0.0

0.2

0.4

0.6

0.8

1.0

no

rmalized

in

ten

sit

y

wavenumber / cm-1

Fig. 4.1.3: Orange fluorescence of the tautomer at 15100 cm-1.

The lack of the blue fluorescence implies that the ESIPT occurs in this aprotic

solvent with a rather high probability. This is explained by the fact that DMSO does not

form external H bonds which stabilize the normal form of carminic acid so that ESIPT

becomes improbably in water. Another stabilization effect may arise from efficient

interactions of DNA with carminic acid. Photo-excited BPES sample solutions containing

the same concentrations of carminic acid and DNA, each at 5 µM, were observed to emit

the blue fluorescence with a significantly higher intensity than that of the orange

fluorescence (see Figure 4.1.4). For carminic acid in BPES the ratio of orange fluorescence

intensity to blue fluorescence intensity amounts to 2:1, whereas that one in the presence of

DNA is 0.8:1. The increase of the blue fluorescence intensity at the expense of the orange

fluorescence intensity does not only indicate to strong interactions between DNA and

carminic acid, but also manifests that the intercalated carminic acids experience a further

structural stabilization due to its rigid environment in the base pair stacks of DNA.


58

12000 15000 18000 21000 24000 270000.0

0.2

0.4

0.6

0.8

1.0

carminic acid

carminic acid + DNAn

orm

alized

in

ten

sit

y

wavenumber / cm-1

Fig 4.1.4: Normalized fluorescence spectra of 5µM carminic acid in BPES (dashed line) and

5µM carminic acid with 5µM DNA in BPES (solid line)

In order to obtain information of the dissociation equilibrium in the S0 state, we have

recorded the UV/VIS absorption spectra of carminic acid in BPES (see Figure 4.1.5) and

carminic acid in DMSO (see Figure 4.1.6). The band structure of both spectra (thin solid

line) was analyzed by calculating them as a superposition of four Gaussian functions

(dashed line). As expected the peak areas and the central positions of the four calculated

peaks in the BPES spectrum are different from those in the DMSO spectrum. The large red

shifts of the peak positions in the carminic acid-BPES spectrum in comparison with that of

DMSO are explained by the more efficient solvation of carminic acid by the water

molecules forming strong H bonds to the oxo and hydroxy groups of carminic acid. On the

other hand, the peak areas are determined by the concentration of the respective species and

their extinction coefficients (i.e. oscillator strength). However, the appearance of these four


59

peaks in the absorption spectrum indicates to the coexistence of four different geometric

states of carminic acid in the S0 state.

15000 17500 20000 22500 250000

2000

4000

6000

8000

10000 experiment

Gauss Fit

17890 cm-1

19060 cm-1

19760 cm-1

22230 cm-1

εε εε / M

-1 c

m-1

wavenumber / cm-1

Fig. 4.1.5: Absorption of carminic acid in BPES; the band structure (thin solid line) was

analyzed by fitting with a superposition of four Gaussian function (dashed line)


60

15000 17500 20000 22500 250000

2000

4000

6000

8000

10000 experiment

Gauss Fit

18560 cm-1

19710 cm-1

20340 cm-1

22544 cm-1

εε εε / M

-1 c

m-1

wavenumber / cm-1

Fig. 4.1.6: Absorption of carminic acid in DMSO; the band structure (thin solid line) was

analyzed by fitting with a superposition of four Gaussian function (dashed line)

Two of the gauss fitting peaks are ascribed to the undissociated carminic acid and its

deprotonated anion. Both, the undissociated acid as well as the deprotonated anion may

undergo a tautomerisation reaction in the ground state, and thus both may occur in an

equilibrium distribution as the normal form and as one tautomer. These four species may

also be formed in DMSO, as naturally containing water up to 2 % in vol. In terms of this

rationale the spectrum of carminic acid in BPES was analyzed as follows: the relatively

large peaks at 22230 cm-1 and 19760 cm-1 were assigned to the normal and tautomer form of

the undissociated carminic acid (CAH and CAH T3, see Scheme 2), respectively, whereas

the small peak at 19060 cm-1 and the very large one at 17890 cm-1 represent the lowest-

energy absorption transitions of the deprotonated CA- and its tautomer CA- T3 (see Scheme

2). In the case of carminic acid in DMSO as containing water at ca. 2 % vol. the two small

peaks (18560 cm-1 and 19710 cm-1) at the low-energy edge of the absorption spectrum were


61

analogously ascribed to the absorption transitions of CA-T3 and CA-, respectively. The very

large peak at 20340 cm-1 displays the absorption of CAH T3 and the smaller one at 22544

cm-1 that of CAH.

CAH:

O

OH

O

OH

CH3O

HO

OH

OH

HO

OH

OOH

HO

CAH T1:

O

OH

O

OH

CH3

O

HO

OH

OH

HO

O

OH

OHHO

CAH T2:

O

O

OH

O

OH

CH3O

OH

HO

OH

OH

HO

OHHO

CAH T3:

O

O

O

OH

O

OH

CH3OH

OH

HO

OH

OH

HOHO

Scheme 2: Molecular structure of the normal form of carminic acid (CAH) and the three tautomers

(CAH T1, CAH T2, CAH T3)

Complementary information of the different geometric states of carminic acid in

BPES was obtained upon performing semiempirical ZINDO/S computations on respectively

four tautomers of the undissociated carminic acid and its deprotonated anion in vacuum. We

have calculated the optimized geometries, electronic states and electronic transitions of all

the eight carminic acid structures. The results are summarized in Table 4.1.1 The

undissociated normal form of carminic acid (CAH) has the lowest absolute energy, Eabs= -


62

8041.5 eV and the largest S0-S1 difference (22716 cm-1) with a rather large oscillator

strength of f= 0.269, which supports our assignment of the highest-energy peaks in the

absorption spectra. The similarly low absolute energy of the tautomer CAH T3 (Eabs= -

8041.3 eV), its largely red-shifted absorption band (18900 cm-1) and the even higher

oscillator strength of f= 0.393 also coincide nicely with the spectroscopy data and moreover,

indicates to a S0-state tautomerization equilibrium between CAH and CAH T3. Because of a

different solvation effect the calculated absolute energy values of CA- and CA- T3, with

Eabs= -8014.2 eV and Eabs= -8014.4 eV, respectively, cannot be correlated with those of the

undissociated carminic acid. Nevertheless the nearly equal absolute energy values

significantly reflect the coexistence of CA- and CA- T3 in the ground state. We have

calculated the optimized geometries, electronic states and electronic transitions of all these

eight carminic acid structures. The results are summarized in Table 4.1.1:

ET/kcal/mol EB/kcal/mol Eabs/eV ∆(S0-Sm)/cm-1 f ∆(S0-Sn)/cm

-1 f

CAH -185444 -25700 -8041.5 22716 0.269 23492 0.037

CA- -184815 -25378 -8014.2 20563 0.071 23496 0.148

CAH T1 -185427 -25693 -8040.7 19351 0.606 21801.1 0.056

CA- T1 -184851 -25421.3 -8015.8 18281.1 0.632 23061.6 0.031

CAH T2 -185431 -25698.3 -8040.9 18919 0.555 25395 0.070

CA- T2 -184811 -25579 -8014 19087.7 0.683 25944.7 0.041

CAH T3 -185440 -25707 -8041.3 18900 0.393 28473.5 0.029

CA- T3 -184820 -25397.7 -8014.4 15491.4 0.414 21758.3 0.051

Table 4.1.1: Calculated values of the total energy (ET), the binding energy (EB), the absolute

energy (Eabs), the energy of the S0-Sm transition (∆(S0-Sm)) and S0-Sn transition (∆(S0-Sm)) transition

and of the oscillator strength (f).


63

The undissociated normal form of carminic acid (CAH) has the lowest absolute

energy, Eabs= -8041.5 eV and the largest S0-S1 difference (22716 cm-1) with a rather large

oscillator strength of f= 0.269, which supports our assignment of the highest-energy peaks

in the absorption spectra. The similarly low absolute energy of the tautomer CAH T3 (Eabs=

-8041.3 eV), its largely red-shifted absorption band (18900 cm-1) and the even higher

oscillator strength of f = 0.393 also coincide nicely with the spectroscopy data and

moreover, indicates to a S0 -state tautomerization equilibrium between CAH and CAH T3.

Because of a different solvent effect the calculated absolute energy values of CA- and CA-

T3, with Eabs= -8014.2 eV and Eabs= -8014.4 eV, respectively, cannot be correlated with

those of the undissociated carminic acid. Nevertheless the nearly equal absolute energy

values consistently reflect the coexistence of CA- and CA- T3 in the ground state. The

difference in their oscillator strengths matches perfectly to the observation of a relatively

small absorption peak of CA- in comparison with the four times larger one of CA- T3 (see

Figure 4.1.5).

The absorption spectra and semiempirical computations support the hypothesis that

the tautomers CAH T3 and CA- T3 may be formed in the S0 state. This has been confirmed

by detecting the fluorescence of the tautomers subsequently by selective excitation at 480

nm. The Figures 4.1.7 and 4.1.8 depict the fluorescence spectra recorded from a BPES

solution (see Figure 4.1.7) and from a DMSO solution (see Figure 4.1.8). The Gaussian fit

of the BPES spectrum yielded three peaks positioned at 16487 cm-1, 14906 cm-1 and 14277

cm-1. The 16487 cm-1 peak was assigned to the fluorescence of CAH T3, whereas the peaks

at 14906 cm-1 and 14277 cm-1 display the fluorescence spectrum (electronic and vibronic

transition) of CA- T3.


64

12000 14000 16000 18000 20000

0.0

0.2

0.4

0.6

0.8

1.0 experiment

Gauss Fit

14277 cm-1

14906 cm-1

16487 cm-1

no

rmalized

in

ten

sit

y

wavenumber / cm-1

Fig. 4.1.7: The spectra of the orange fluorescence of 5 µM carminic acid in BPES. The band

structure of the spectra (thin solid line) was analyzed by fitting with a superposition of Gaussian

functions (dashed line).

The analysis of the DMSO spectrum resulted into two peaks (at 17440 cm-1 and

16487 cm-1) only, which were ascribed to fluorescence transitions of CAH T3.


65

12000 14000 16000 18000 20000 22000

0.0

0.2

0.4

0.6

0.8

1.0

experiment

Gauss Fit

16487 cm-1

17440 cm-1

no

rmalized

in

ten

sit

y

wavenumber / cm-1

Fig. 4.1.8: The spectra of the orange fluorescence of 5 µM carminic acid in DMSO; the

band structure of the spectra (thin solid line) was analyzed by fitting with a superposition of

Gaussian functions (dashed line).

The fluorescence quantum yield of the carminic acid tautomers dissolved in

Millipore water was determined as a value of 2.7 %. This rather low fluorescence quantum

yield suggests that the deactivation of the S1 state may occur either through an efficient

intersystem crossing to the T1 state or by an ESIPT followed by internal conversion to the

S0 state. An argument for the ESIPT is the observation of the dependence of the blue

fluorescence intensity on the nature of the solvent shell.

A relative measure for the interactions between carminic acid and DNA was

achieved by performing fluorescence titration measurements. Carminic acid in BPES at the

total concentration of cT = 6 µM was excited at 320 nm. The total fluorescence spectrum

between 360 and 800 nm was recorded in the magic-angle polarization configuration. The

relative fluorescence intensities (I) were determined by integrating the fluorescence spectra.

The fluorescence titrations were performed upon detecting the fluorescence spectrum of

carminic acid in dependence on the DNA concentration cDNA (M in nucleotide) over the

range of 1 nM to 1 mM. Under the simplified approximation that at maximum one carminic


66

acid molecule may bind to one DNA nucleotide, only, the fluorescence titration data may be

described by the equation:

DNAB

DNAT

BcK

ccc

+

⋅=

−1 V.1.1

with KB representing the intrinsic binding constant. The fluorescence intensities of carminic

acid in the absence of DNA (I0), in the course of the titration with DNA (I) and at high DNA

concentrations, i.e. the plateau value (I∞), are used to calculate the concentration of free

carminic acid:

∞

∞

−−

=II

IIcc T

F

0

)( V.1.2

This relationship allows for the determination of the concentration of bound carminic

acid with cB = cT – cf. Figure 4.1.9 shows the fluorescence titration curve depicted as the

concentration of bound carminic acid, cB, versus the DNA concentration, cDNA. An intrinsic

binding constant, KB= 5.0×105 (M nucleotide)-1 provided the best fit (thick solid line) to the

experimental data (dots). The uncertainty in this parameter is about 10 %. This large binding

constant agrees very well with that reported for the doxorubicin-calf thymus DNA complex

with KB= 1.5×105 (M bp)-1 [11] and that of the daunomycin-DNA complexes with KB=

7×105 (M bp)-1[47]. Binding constants of that order of magnitude are typical for strong

intercalative bindings of chromophores to the DNA base pairs [11].


67

0.0 4.0x10-5

8.0x10-5

1.2x10-4

0.0

1.0x10-6

2.0x10-6

3.0x10-6

4.0x10-6

5.0x10-6

6.0x10-6

experimental data

cB= c

T c

DNA/(K

B

-1 + c

DNA)

bo

un

d c

arm

inic

acid

/ M

DNA concentration / M nucleotide-1

Fig. 4.1.9: Concentration dependence of bound carminic acid, cB, on the DNA

concentration; the experimental data (dots) were obtained from fluorescence titration of 6 µM

carminic acid with DNA in BPES and were fitted (solid line) employing the relationship cB= cT⋅

cDNA/(KB-1+cDNA) with KB= 5.0×10

5 (M nucleotide)-1.

4.2. Femtosecond Spectroscopy

4.2.1 Transient Absorption spectroscopy

The excited-state decay dynamics of carminic acid in BPES were investigated utilizing

fs-resolved transient absorption spectroscopy. Figure 4.2.1.1 represents the temporal

evolution of the transient absorption spectra between 0 and 100 ps illustrated as a 3-D plot.

The rather complicated band structure of the spectra obviously originates from a


68

superposition of photo-bleaching of the ground-state population densities (absorption > 0)

and transient absorption of the excited states (absorption < 0). In order to analyze the time

evolution of the involved spectral components, each spectrum was decomposed into 8

Gaussian functions, where photo-bleaching was accounted for by positive amplitude and the

transient absorption by negative amplitude. The Gaussian fits of the transient absorption

spectra for delay times τ from 0 ps to 100 ps in steps of 5 ps were calculated by keeping

constant the peak positions and the widths of the Gaussian functions, whereas the

amplitudes of the Gaussians were varied until the best fit was obtained.

-0,08

-0,06

-0,04

-0,02

0,00

0,02

0,04

1400016000

1800020000

2200024000

2040

6080

100

ab

so

rpti

on

wavenumber / cm

-1tim

e / ps

Fig. 4.2.1.1 3-D plot of the temporal evolution of the transient absorption spectra obtained

for carminic acid in BPES.


69

The temporal evolution of every amplitude, as being a measure for the relative absorption of

the respective spectral component, could be described by a mono-exponential function. The

exponential fits yielded four different time constants for the 8 spectral components, which

are τta= 15±0.5 ps, 20±0.8 ps, 33±1.2 ps and 46±1.4 ps. This result agrees very well with the

observation of four different species of carminic acid (i.e. CAH, CAH T3, CA-, CA- T3) in

the stationary absorption spectrum. Hence each set of components with equal time constants

constitutes a transient absorption spectrum of one species of carminic acid, respectively.

Figure 4.2.1.2 exemplarily displays the transient absorption spectrum (thin solid line)

recorded at the delay time τ= 1 ps. The Gaussian fit (thick short dashed line) results from

the superposition of 8 Gaussian functions that are assigned to the four different time

constants (thick solid, dashed, dashed dotted and dotted lines).

14000 16000 18000 20000 22000 24000

-0.06

-0.04

-0.02

0.00

0.02

ta spectrum

46 ps Gaussian

20 ps Gaussian

15 ps Gaussian

33 ps Gaussian

Gaussian superposition

tran

sie

nt

ab

so

rpti

on

wavenumber / cm-1

Fig. 4.2.1.2 Transient absorption spectrum (thin solid line) recorded at the delay time τ= 1

ps; the fit (thick short dashed line) arises from the superposition of 8 Gaussian functions assigned to

four different time constants (thick solid, dashed, dashed dotted and dotted lines).


70

4.2.2 Fluorescence up-conversion spectroscopy

Complementary information of excited-state decay behaviour were obtained from

fluorescence up-conversion experiments. The rather small fluorescence quantum yield of

carminic acid limits the detection of the up-converted fluorescence emission to the

wavenumber range of 13800 – 17200 cm-1, and thereupon to the observation of two

fluorescence bands, only, one peaked at 14910 cm-1 and assigned CA- T3 and the other one

at 16490 cm-1 ascribed to CAH T3 (see Figure 4.2.2.1).

14000 15000 16000 17000

0.4

0.6

0.8

1.0

spectrum

Gaussians

Gaussian superposion

14910 cm-1

16490 cm-1

flu

ore

scen

ce in

ten

sit

y

wavenumber / cm-1

Fig. 4.2.2.1 Fluorescence-up conversion spectrum (dots) fitted by a superposition (thin solid

line) of two Gaussians (dashed line).


71

Figure 4.2.2.2 shows the 3D-plot of carminic acid in BPES at a concentration of 0.6

mM. Addition of DNA at a surplus concentration of 3.9 mM (in nucleotide) does not change

the spectral features but distorts a little their time evolution (see Figure 4.2.2.3).

0,0

0,2

0,4

0,6

0,8

1,0

1,2

14000

15000

16000

17000

020

4060

80100

inte

ns

ity

wav

enum

ber

/ cm

-1

time / ps

Fig. 4.2.2.2 3-D plot of the temporal evolution of the fluorescence up-conversion spectra obtained

for 0.6 mM carminic acid in BPES.


72

0,0

0,2

0,4

0,6

0,8

1,0

14000

15000

16000

17000

2040

6080

100

inte

nsit

y

wav

enum

ber

/ cm

-1

time / ps

Fig. 4.2.2.3: 3-D plot of the temporal evolution of the fluorescence up-conversion spectra

obtained for 0.6 mM carminic acid and 3.9 mM DNA in BPES.

To achieve indepth insight into the fluorescence decay dynamics and in particular, to

explore the influence of the intercalative interaction with DNA, the up-conversion decay

curves detected at 14910 cm-1 and 16490 cm-1, respectively, both, in the parallel-

polarization and perpendicular-polarization geometry, were recorded (see Figure 4.2.2.4 –

4.2.2.7). Figures 4.2.2.4 and 4.2.2.5 depict the up-conversion decays of carminic acid in

BPES, whereas those of carminic acid in BPES in the presence of DNA are shown in

Figures 4.2.2.6 and 4.2.2.7.


73

-20 0 20 40 60 80 100 120 140

0

200

400

600

800

1000 I(t)

par

fit(t)par

I(t)perp

fit(t)perp

flu

ore

scen

ce in

ten

sit

y

time / ps



perpendicular-polarization geometry (I(t)perp); both fits were obtained using the parameters: a =

0.37, b= 0.91, τ1= 1.7 ps, τ2= 33 ps and r(0)= 0.137.


74

-20 0 20 40 60 80 100 120 140

0

200

400

600 I(t)par

fit(t)par

I(t)perp

fit(t)perp

flu

ore

scen

ce in

ten

sit

y

time / ps



perpendicular-polarization geometry (I(t)perp); both fits were obtained using the parameter: a= 0.50,

b= 0.92, τ1= 1.5 ps, τ2= 47 ps and r(0)= 0.202.


75

-20 0 20 40 60 80 100 120 140

0

100

200

300

400

500

I(t)par

fit(t)par

I(t)perp

fit(t)perp

flu

ore

scen

ce in

ten

sit

y

time / ps

Fig. 4.2.2.6: Fluorescence up-conversion decays of 0.6 mM carminic acid and 3.9 mM DNA

in BPES (thin solid line) detected at 14910 cm-1 in the parallel-polarization geometry (I(t)par) and in

the perpendicular-polarization geometry (I(t)perp); both fits were obtained with the parameter: a=

0.50, 0.97, τ1= 1.9 ps, τ2= 48 ps and r(0)= 0.182.


76

0 20 40 60 80 100 120 140-100

0

100

200

300

400

500

600

700

I(t)par

fit(t)par

I(t)perp

fit(t)perp

flu

ore

scen

ce in

ten

sit

y

time / ps


DNA in BPES (thin solid line) detected at 16490 cm-1 in the parallel-polarization geometry (I(t)par)

and in the perpendicular-polarization geometry (I(t)perp); both fits were obtained with the parameter:

a= 0.50, b= 0.97, τ1= 1.1 ps, τ2= 61 ps and r(0)= 0.243.


77

The decay curves were evaluated by a merged nonlinear fitting and deconvolution

process directly on the parallel (Ipar) and perpendicular (Iperp) signals, using the model

functions

( ) ( )( ) ( )tftrti par 21 +=

( ) ( )( ) ( )tftrti perp −= 1

These were convoluted by the Gaussian model function, I(t) ∝ i(t) ⊗ G(t). In the fitting we

used

)/texp(b)a()/texp(a.const)t(f 21 1 ττ −⋅⋅−+−⋅+=

and

)/texp(r)t(rtroτ−⋅= 0

where τrot = 196 ps [19]. Ipar and Iperp represent the fluorescence intensity that was detected

in the parallel and perpendicular polarization geometry, respectively. Table 4.2.2.1 contains

the amplitudes, a and b, the time constants, τ1 and τ2, as well as the anisotropy parameter

r(0), which were obtained from the best fit. The short time constants τ1 were ascribed to a

fast structural relaxation process, whereas the long time constants τ2, with 33±0.7 ps and

47±1 ps, coincide with two excited-state lifetimes that resulted from the Gaussian analysis

of the transient absorption spectroscopic data. These lifetimes were assigned to the excited-

states of the CA- T3 and CAH T3, respectively. As obvious from Figure 4.2.2.6 and Figure

4.2.2.7, the presence of DNA is associated with a significant prolongation of these lifetimes:

τ2 changes from 33 ps to 48 ps for CA- T3, whereas CAH T3 exhibits an increase from 47

ps to 61 ps. In addition the anisotropy parameter r(0) becomes significantly larger under the

influence of DNA. The latter effect confirms for a second time the efficient couplings of

carminic acid to DNA. Moreover, the prolongation of the lifetimes arises from the aprotic

rigid environment of the carminic acid incorporated between the DNA base pairs which


78

presumably slows down the nonradiative processes that are the internal conversion and the

intersystem crossing of carminic acid. Reverse ESIPT processes to the excited state of the

corresponding normal form are very unlikely to take place because of their higher excited-

state energy. Since fluorescence up-conversion were generated using a pump pulse at 400

nm, there are two excitation pathways to populate the excited-states of the tautomers, CAH

T3 and CA- T3. One pathway is direct photoexcitation of the tautomers in the ground state

and the other occurs via ESIPT from the excited state of the respective normal form (CAH

and CA- ). The rise of all fluorescence up-conversion decay curves (see Figures 4.2.2.4–

4.2.2.7) takes place within the instrumental response time (i.e. 270 fs).

a b τ1 / ps τ2 / ps r(0)

CA-T3: 14910 cm-1 0.37 0.91 1.7 33 0.137

CAHT3: 16490 cm-1 0.50 0.92 1.5 47 0.202

CA-T3+DNA: 14910 cm-1 0.50 0.97 1.9 48 0.182

CAHT3+DNA: 16490 cm-1 0.50 0.97 1.1 61 0.243

Table 4.2.2.1: The amplitudes, a and b, the time constants, τ1 and τ2, and the anisotropy,

r(0), were obtained from the best fit of the fluorescence up-conversion decay curves that are

detected at 14910 cm-1 and 16490 cm-1.

This may be explained by an ultrafast ESIPT with a proton-transfer time constant

smaller than 270 fs. Just as probable is that no ESIPT takes place and the observed

fluorescence originates from the directly photoexcited tautomers. On the other hand, the

solvent dependence of blue and orange fluorescence intensities (see Figures 4.1.2, 4.1.3 and

4.1.4) unambiguously indicates to the occurrence of ESIPT. This is consistent with the

results obtained from a fs transient absorption spectroscopy study of 1,8-dihydroxy-


79

anthraquinone by Neuwahl and Coworkers [46]. The authors reported the observation of a

practically instantaneous ESIPT with a proton-transfer time constant much smaller 100 fs.

Conclusions

80

5. Conclusions

In a buffer solution at pH= 7 carminic acid was found to occur as undissociated acid

(CAH) and as its deprotonated anion (CA-), both, at nearly the same concentration. The

Gaussian analysis of the UV/VIS absorption spectrum (see Figures 4.1.5 and 4.1.6) gave

indication to four species of carminic acid coexisting in the ground state. On the basis of

semiempirical molecular geometry computations these four species were assigned to the

normal forms of CAH and CA- and to their tautomers, CAH T3 and CA- T3. The existence

of four ground-state species was confirmed by femtosecond transient absorption

spectroscopy experiments. The Gaussian analysis of the spectroscopic data yielded four

different excited-state lifetimes: 15 ps, 20 ps, 33 ps and 46 ps. The solvent dependence of

the blue and orange fluorescence intensity gave indication to ESIPT. Carminic acid

dissolved in the aprotic solvent DMSO does not exhibit blue fluorescence. On the other

hand, in protic environment (BPES) the orange to blue fluorescence intensity ratio is about

2:1, whereas the presence of DNA alters this ratio to 0.8:1. This implies that ESIPT takes

instantaneously place in an aprotic medium that is also partially achieved for carminic acid

by intercalating between the base pairs of DNA. Fluorescence up-conversion experiments

conducted on carminic acid in BPES led to two lifetimes, 33 ps and 47 ps that were

assigned to the tautomers CA- T3 and CAH T3 of no dissociated and deprotonated carminic

acid, respectively. Since the rise time of fluorescence up-conversion transients corresponds

to the instrumental response time constant of 270 fs, only ultrafast ESIPT with a proton-

transfer time constant smaller than 100 fs may be involved in the excitation of the

tautomers. In the presence of DNA the observed lifetimes increase from 33 ps to 48 ps for

CA- T3 and from 47 ps to 61 ps for CAH T3. Their prolongation in the presence of DNA

may be taken as an additional evidence for the formation of intercalation complexes

between DNA and carminic acid. The rigid aprotic environment between the DNA base

pairs are expected to slow down the nonradiative deactivation processes of the

Conclusions

81

electronically excited carminic acid which are internal conversion and intersystem crossing

[13].

References

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Summary

Photo-excited carminic acid and carminic acid-DNA complexes in a buffer solution

at pH= 7 have been examined using a variety of spectroscopy techniques, that are in

particular, the femtosecond resolved fluorescence up-conversion and transient absorption

spectroscopy. The observation of dual fluorescence emission, one peaks at 470 nm and the

other at 570 nm, indicates to an excited-state (S1) intramolecular proton transfer (ESIPT). A

detailed analysis of the transient absorption measurements of an aqueous carminic-acid

solution at pH= 7 yielded four lifetimes for the excited state (S1): 8 ps, 15 ps, 33 ps and 46

ps. On the other hand, only two lifetimes, 34 ps and 47 ps, were observed by fluorescence

up-conversion spectroscopy because of the detection limitation to the long wavelength edge

of the carminic-acid spectrum. The four S1 lifetimes were ascribed to the coexistence of

respectively two tautomer (normal and tautomer) forms of carminic acid, in the

nondissociated state (CAH) and in the deprotonated state (CA-). The fluorescence up-

conversion measurements of carminic acid-DNA complexes exhibited a prolongation of the

fluorescence lifetimes. This effect was taken as evidence for the formation of intercalation

complexes between the carminic acid and the DNA. The intercalative binding of the

carminic acid to DNA was confirmed by the fluorescence titration experiments leading to a

binding constant of KB= 5.0×105 (M nucleotide)-1 that is typical for anthracycline-DNA

complexes.

Zusammenfassung

Die statischen und dynamischen Wechselwirkungen zwischen optisch angeregter

Carminsäure und DNA wurden mittels verschiedener, komplementärer Techniken der

stationären und zeithochaufgelösten optischen Spektroskopie detailliert untersucht. Die

stationäre Fluoreszenz und UV/VIS-Absorptionsspektroskopie die Identifizierung von vier

Tautomeren der Carminsäure im elektronischen Grundzustand ermöglichte, konnte in durch

Messung der UV-Vis-Absorptionsspektren in Abhängigkeit vom pH-Wert nicht nur der

pKa-Wert von Carminsäure bei pH= 7 bestimmt werden, sondern auch noch gleichzeitig die

Koexistenz von nur einfach deprotonierter Carminsäure mit der nicht-dissoziierten Säure für

pH-Werte zwischen pH= 2 und pH= 9 nachgewiesen werden. Die Koexistenz der vier

Tautomeren von Carminsäure im Grundzustand der einfach deprotonierten und

undissozierten Carminsäure wurde mit Ergebnissen aus semiempirischen ZINDO/S

Rechnungen bestätigt. Die statischen Wechselwirkungen und damit die Bindungskonstante

und der Bindungsmodus von Carminsäure-DNA-Assoziaten wurde durch Messung von

Fluoreszenz-Titrationskurven für verschiedene Carminsäure-Konzentrationen untersucht.

Die Carminsäure-DNA-Assoziate haben eine Bindungskonstante von KB= 5.0×105 (M

nucleotide)-1, was auf einen interkalativen Bindungsmodus hinweist. Die duale Fluoreszenz-

emission von Carminsäure mit Maxima bei 470 nm und bei 570 nm zeigt, dass ein

intramolekularer Protonentransfer im elektronisch angeregten S1-Zustand (ESIPT)

stattfindet. Da die Zeitkonstante des ESIPT von der Molekülgeometrie im S1-Zustand

bestimmt wird, wurde der ESIPT als Sonde für die dynamischen Wechselwirkungen

zwischen Carminsäure und DNA genutzt. Die Relaxationsdynamiken von optisch

angeregter Carminsäure und Carminsäure-DNA-Assoziaten wurden mittels Femtosekunden-

auflösender transienter Absorptionsspektroskopie und Fluoreszenz-Aufwärtskonversion

untersucht. Die detaillierte Analyse der gemessenen transienten Absorptionsspektren von

Carminsäure in einer Lösung mit pH= 7 ergab vier Lebensdauern für den S1-angeregten

Zustand: 8 ps, 15 ps, 33 ps und 46 ps, wobei zwei der vier Lebensdauern, mit 34 ps und 47

ps, durch Messungen mittels der Fluoreszenz-Aufwärtskonversion bestätigt wurden. Die

vier Lebensdauern werden zwei Tautomeren von Carminsäure (CAH) und deren jeweils

deprotonierte Formen (CA-) im S1-Zustand zugeordnet. Für die Carminsäure-DNA-

Assoziate wurde eine Zunahme der Lebensdauer im Vergleich zur reinen Carminsäure

nachgewiesen, was als weiterer Hinweis auf eine Interkalation der Carminsäuremoleküle in

die DNA bewertet wird.

Appendix Used Software

Computation: Hyperchem 7.1

Spectral Analyses: Origin 5.0

SigmaPlot 7.0

Peak Fitting Modules

Laser systems

Ti: Sapphire laser: Coherent MIRA 900

Pump laser: solid state laser Coherent VERDI V10

Spectrographs

Absorption: Perkin Elmer Lambda 2

Fluorescence: Jobin-Yvon FluoroMax-3

Lebenslauf

Vorname Radu Constantin Name Comanici Geburtsdatum 09.01.1977 Geburtsort Piatra-Neamt, Rumänien Staatsangehörigkeit Rumänisch Familienstand Verheiratet, keine Kinder

2002 - 2006 wissenschaftlicher Angestellter im Rahmen der Promotion im Institut für Physikalische Chemie I an der Friedrich-Alexander Universität Erlangen- Nürnberg 2001 Masterdiplom unter Betreuung von Prof. Dr. Viorica Simon 1999 - 2001 Master an der Babes-Bolyai Universität Klausenburg in Rumänien 1999 Diplomprüfüng mit Diplomarbeit unter Betreuung von Prof. Dr. Traian Iliescu 1995 - 1999 Student an der Physik-Fakultät der Babes-Bolyai Universität Klausenburg in Rumänien 1995 Abitur am Informatik-Gymnasium Nr. 1 1983- 1995 Schulausbildung mit Berufsabschluss als Software-Analyst und Service für Computertechnik am Informatik-Gymnasium

Acknowledgement

I would like to thank everyone who has contributed in any way to the successful completion

of my PhD thesis.

First of all, I would like to thank Prof. Dr. Carola Kryschi for accepting me in her work

group and giving me the opportunity make my PhD thesis in the Physical Chemistry I

Institute of the Friedrich Alexander University of Erlangen. Also I want to thank all my

colleagues in the work group of Prof. Dr. Kryschi for the good collaboration and for the

friendly atmosphere.

On this way I want to express my acknowledgement to all my colleagues from this institute.

I would like to thank to the entire employees of the mechanic workshop good coordinated

from Mr. Friedhold Wölfel, for the speedy and professional expertise, for the support and

help with electrical and electronics instrumentation to Mr. Dipl. ing. Dirk Harnisch, to Ms

Julianne Roth for her help in administrating the used chemicals.

My special thanks goes to the Dr. Thomas Gustavson and entire collaborators the

Laboratoire Francis Perrin CEA in Gif sur Yvette France, for helping and coordinate the

femtoseconds measurements to realize the time resolved spectroscopy.

I would like to thank my mother and my brother for all their love and support as well as for

the understanding they shows to me in every circumstance.

Last but not least I would like to thank my wife, Karmen, for the great help, support and

understanding she gave to me during my PhD studies.

Documents

Femtosecond Spectroscopic Study of Carminic Acid-DNA ...Spectroscopic... · Femtosecond Spectroscopic Study of Carminic Acid-DNA Interactions ... 2.2.2 Time-Resolved Optical Spectroscopy