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Synthesis and Chemiluminescence Studies of Luminol and Derivatives

Filipe M. C. Menezes1, Carlos A. M. Afonso

1, Carlos M. B. Baleizão

1, Luís F. Veiros

2, Mário N. Berberan-Santos

1

1CQFM-Centro de Química-Física Molecular and IN-Institute of Nanoscience and Nanotechnology, Instituto Superior

Técnico, 1049-001 Lisboa, Portugal. E-mail: Fax: +351 218464455; Tel: +351 2184192212

Centro de Química Estrutural, Departamento de Engenharia Química e Biológica, Instituto Superior Técnico, Av.

Rovisco Pais, 1049-001 Lisboa, Portugal

Luminol is a well-known chemiluminescent compound with a strong blue emission. Owing to its

visible emission, chemiluminescence mechanism and high quantum yield, it has been used in biosensors,

sensitive metal ion detection and in several in vivo applications.1

In this work, we report the synthesis and properties of some new derivatives of luminol in our search

for green chemiluminescence as well as some photophysical and computational studies on luminol and the

respective diphthalate anion.

1. Introduction

Since its synthesis in 1928 by Albrecht,2

luminol (Figure 1, species A) has been subject to

several mechanistic and synthetic studies, not

only to fully understand the processes underlying

its chemiluminescence but also to obtain more

chemiluminescent compounds with emission in

other colours.

It is believed that the reaction itself can

follow several pathways. In water, the

chemiluminescence quantum yield is just 1%.

Nevertheless, it is in this medium where most of

luminol’s interesting applications are found.1,3

Figure 1: Generic Scheme for luminol’s (A)

chemiluminescence. B is the related aminodiphthalate.

Here we report the effect of N-acylation of

luminol (aniline moiety) in its chemiluminescence

and in the photophysical properties of the

chromophore. The acyl groups chosen were

trifluoroacetyl, benzoyl, ethyl carbamate and two

still unknown structures obtained with reaction

with dimethyl carbamic chloride. Computational

studies on luminol’s tautomerism and acidity as

well as spectroscopic studies on luminol and the

aminodiphthalate (B) were performed. The

absorption spectra are compared with theoretical

results.

2. Computational Details

All theoretical calculations were

performed with Gaussian 03 program package,4

using the restricted approximation of the

wavefunctions.

Geometry optimizations were performed

with Möller-Plesset second order perturbation

theory with 6-31G** basis set. MP2 calculations

with Dunning’s aug-cc-pVTZ basis set were

performed for single point energy calculations.

Charges are analysed using natural population

analysis for the most refined basis sets.

Solvent effects were considered using the

polarisable continuum model (PCM)5

in single

point energy calculations using the optimized

MP2/6-31G** geometry. The augmented version

2

of Dunning’s triple zeta basis set was once again

used in these calculations.

The Hessian matrix was calculated

analytically for the optimized structures to prove

the exact location of correct minima in potential

energy surfaces (only positive frequencies) and to

estimate thermodynamic parameters at 298 K and

1 atm.

TDDFT calculations using

PBE1PBE/aug-cc-pVTZ were used to predict the

absorption spectra of luminol. The solvent

dielectric was used in the calculations. Optimized

MP2/6-31G** geometries were also used in these

calculations.

3. Experimental

3.1. Reagents and Solvents

All the solvents used in synthetic

experimental procedures were dried and/or

distilled according to standard procedures.6

Reagents for synthetic procedures were

all reagent grade and samples for spectroscopic

studies (and solvents) were all with spectroscopic

purity, being also commercially available.

3.2. Instrumentation

NMR spectra were recorded in an

Ultrashield Bruker Avance III 300 or Bruker

Avance III 400. The solvent and the standard are

in brackets in the synthetic procedure description.

All shifts are reported in ppm and coupling

constants in Hz.

For fluorescence, excitation spectra and

chemiluminescence studies a Spex Fluorolog

F112A fluorimeter was used.

UV/Vis spectra were all recorded in a

Shimadzu UV-3101PC UV-Vis-NIR spectro-

photometer.

Aqueous solutions pH was measured in a

Crison micro pH 2001.

3.3. Chemiluminescence

Chemiluminescence assays were carried

out as described by Rauhut and co-workers:25

K2S2O8 6.010-2

M, H2O2 3.010-2

M, and luminol

or one of its derivatives 5.010-5

M.

3.4 Synthesis

N-trifluoroacetyl Luminol (TFALum)

To 100 mg of Luminol (0.564 mmol) were

added 2 mL of DMF (complete dissolution). 90 µL

of triethylamine (TEA; 0.646 mmol) were added.

88 µL (0.628 mmol) of trifluoroacetic anhydride

were added directly and slowly. After one hour, a

white dispersion substituted the yellow colour

indicating the end of the reaction. 50 mL of H2O

were rapidly added and the isolated solid

crystallized in EtOH-H2O mixture (20 and 5 mL

respectively). The yield was 70%. Alternatively,

H2O-acetone mixtures can be used. The isolated

crystals were white and cotton like and melted at

335 ºC. They showed the following NMR data:

1H NMR (400 MHz, DMSO D6, TMS): 14.5 (1H, s);

12.1 (2H, large s); 8.7 (1H, d, 8.1); 7.9 (1H, t, 8.1);

7.7 (1H, d, 8.0).

13C NMR (100 MHz, DMSO D6, TMS): 161.3;

154.5; 152.0; 138.2; 135.2; 126.3; 121.7; 120.6;

120.2; 115.9.

19F NMR (376 MHz, DMSO D6, CFCl3): 75.5 ppm.

N-benzoyl Luminol (BnLum)


added 4 mL of DMF (complete dissolution) 150 µL

of TEA (1.08 mmol) were added. 200 µL of BnCl

(1.72 mmol) were directly and slowly added. The

solution acquired a yellow colour and a white

dispersion appeared. The reaction progress was

3

followed by TLC. After completion, 50 mL of H2O

were rapidly added and the precipitate was

purified by crystallization in EtOH-H2O mixture (20

and 5 mL respectively). The yield was 34%.

Crystallization just in EtOH can also be

performed. yielding product with similar

composition. The isolated product was a white

powder that decomposed at 206 ºC and showed

the following NMR data:


7.3 and 8.3 ppm (nH, m); 7.0 (1H, d, 8.2); 6.6 (1H,

d, 7.6).

13C NMR (75 MHz, DMSO D6, TMS): 164.6 ;

162.9; 162.3; 151.5; 146.3; 136.1; 135.3; 130.7;

130.5; 129.7; 129.5; 127.8; 127.6; 126.7; 117.5;

110.4; 108.5.

Ethyl Carbamate Luminol (ECLum)


added 4 mL of DMF (complete dissolution). 150

µL of TEA (1.08 mmol) were added. Then, 200 µL

of ethyl chloroformate (2.10 mmol) were directly

and slowly added. The solution achieved a yellow

colour and within 30-60 min a white dispersion

appeared, ending the reaction. 50 mL of H2O were

added. EtOAc was used to recover more product

from the aqueous phase and the product

crystallized in EtOH. The yield after crystallization

was 28%. The product obtained was pale yellow

with rod form, melted at 150 ºC and exhibited the

following NMR data:


7.5 (1H, t, 6.6 and 7.4); 7.4 (2H, s); 7.0 (1H, d,

7.4); 6.7 (1H, d, 6.6); 4.3 (2H); 1.3 (3H).

13C NMR (100 MHz, DMSO-D6, TMS): 162.8;

152.5; 151.6; 145.7; 135.4; 126.4; 117.6; 110.4;

108.2; 66.1; 14.3.

Figure 2: Derivatives with assigned structure.

DMU1Luminol


added 4 mL of DMF (complete dissolution). 200

µL of dimethyl carbamic chloride (2.17 mmol)

were directly and slowly added. The solution got

yellow and the system heated and kept for one

day at 60 ºC. A white dispersion appeared

indicating the end of the reaction. 50 mL of H2O

were added, the precipitate dissolved and the

solvent was evaporated. The solid was

crystallized in EtOH (10-15 mL). The amount of

product obtained was 104.3 mg. Alternatively

MeOH can be used as crystallization solvent. The

isolated product was a white powder with the

following NMR data:


9.0 (1H, d, 6.7); 7.9 and 7.8 (2H, [t, 8.1] and [d,

6.8]); 7.6 (1H, d, 7.8); 7.5 and 7.4 (2.5H, m); 2.5

(0.5H, s); 1.1 (2.5H, t, 7.0).

13C NMR (100 MHz, DMSO D6, TMS): 168.5;

160.9; 152.0; 145.1; 135.6; 134.8; 129.7; 129.6;

127.9; 127.6; 126.4; 121.8; 118.3; 115.3.

DMU2Luminol

The reaction and workup procedures

were exactly the same as the ones for DMU1Lum

but without heating during the reaction. The

product was crystallized in H2O-EtOH mixture (15

and 5 mL respectively). 212.7 mg of pale yellow

granulate contaminated with luminol were

obtained. The product exhibited the following

4

NMR data (peaks matching luminol were marked

with an L inside brackets).

1H NMR (400 MHz, DMSO D6, TMS): 13.8 (1H, d,

10.8); 11.3 (3H, large s); 9.3 (1H, d, 10.8); 8.0

(1H, d, 8.1); 7.8 (1H, t, 7.6 and 8.1); 7.6 (1H, d,

7.6); 7.3 (L); 6.8 (L); 6.7 (L); 3.1 (s).

13C NMR (100 MHz, DMSO D6, TMS): 161.7 (L);

161.1; 152.9; 151.9 (L); 151.1 (L); 138.9; 135.3;

134.3 (L); 126.9 (L); 126.7; 120.8; 118.0; 116.9

(L); 115.3; 110.8 (L).

4. Results and Discussion

4.1. Computational

Figure 3: Luminol’s tautomeric forms computationally

studied.

Table 1: Relative Gibbs energy for luminol’s tautomers

(tautomer B as reference). All calculations with

MP2/aug-cc-pVTZ level of theory. Energies in kcal/mol.

Gas DMSO H2O

A 7.24 5.25 5.16

B 0.00 0.00 0.00

C 3.85 2.04 1.86

D 9.58 8.58 8.38

E 49.6 45.5 44.8

F 41.3 38.7 38.2

The conformations for luminol tautomers

presented in Figure 3 were the computationally

most stable ones. With respect to their relative

energy, as Table 1 shows, tautomers without

imine character in ring 1 (6 member ring always

on left in Figure 3) are the most stable. Of those,

the less stable is the tautomer with aromaticity

extension through the two 6 member rings

(tautomer D), being the tautomer with the carboxyl

farthest from the aniline moiety enolized the most

stable one (B).

To account for the relative stability order

we have verified that not only the strength of the

hydrogen bonding would be relevant but also that

electronic density (charge) analysis proved quite

useful. From the former parameter, the stronger

the hydrogen bond between the aniline and closer

carboxyl group the most stabilized is the structure

(supplementary material S1). But because A has

stronger hydrogen bond than C and due to the

small difference of the hydrogen bond distance

between A and B (A-1.93 Å; B-1.92 Å; C-1.981

Å), the charge distribution must be evoked. The

latter parameter (charge distribution) shows that

the most stable tautomers have higher electronic

density in the most electronegative atoms, ring 2

(cf. supplementary material S2).

Regarding the aromaticity, its complete

loss in ring 1 appears to greatly destabilize the

whole structure yielding species at least 38

kcal/mol less stable (E and F). As for the

aromaticity extension, it should not be important

enough to account for the complete loss of

carboxyl groups (that also weakens the hydrogen

bond strength). Therefore, the main parameters

affecting luminol’s tautomers relative stability were

proposed to be (by increasing order of

importance) (i) aromaticity and electronic

delocalization (increase in the number of

molecular orbitals with character), (ii) hydrogen

bonding, and (iii) charge distribution. Regarding

other studies, the energetic distribution of

tautomers that we propose here is slightly

different from a previous DFT study.8

The main

5

difference is in the relative stability of tautomers A

and C. While in our theoretical calculations

tautomer C was the second most stable tautomer,

the other calculations predicted that instead of C it

is A the second most stable species. According to

our initial method and basis set scan, that may

have to do with the description of luminol’s aniline

functionality.

With respect to luminol’s acidity, we

verified that in solvent’s dielectric, the most acidic

protons of luminol were the hydrazide ones. Both

are practically equally labile, being the energetic

discrepancy less than 1 kcal/mol (Figure 4). As for

the second most acidic proton of luminol, we

verified that it should be an anilinic one instead of

the other hydrazide proton (Figure 5). The energy

difference between those two proton abstractions

is at least 10 kcal.mol-1

. The most stable species

are once again the ones retaining the hydrogen

bonding where the aniline’s nitrogen acts as

acceptor. Supplementary material S3 shows the

same energetic relations but for gas phase

calculations.

Figure 4: Relative stability of luminol’s monoanionic

base conjugates according to MP2/aug-cc-pVTZ/SCRF

calculations on optimized MP2/6-31G** geometries.

Figure 5: Relative stability of luminol’s dianionic base

conjugates according to MP2/aug-cc-pVTZ/SCRF

calculations on optimized MP2/6-31G** geometries.

4.2. Absorption and Fluorescence

In this work we have performed

absorption and fluorescence studies on luminol

and its derivatives in water at high pH, in DMF

and in DMSO.

Regarding absorption spectra in aprotic

media (Figure 6, left) we observed that the

generated electronic states come all from *

transitions (the same conclusion for the other

media studied). While luminol and ECLum (EC)

have both two electronic transitions (that may

come from several tautomeric forms), in the other

derivatives the relation between the energy of the

excited states is greatly affected, meaning that the

absorption spectra pattern is changed. With

respect to the fluorescence, we have observed

that all species relax to a similar structure (both in

geometry and in energy), accounting for the

emission resemblance of all those species. Of all,

the most curious case is TFALum (TFA) that has

two emission bands in DMSO. This may be an

artefact from tautomerism. When changing to

DMF (Supplementary material S4), the absorption

pattern is changed (more resolution) but the

fluorescence spectra are equal to DMSO’s. The

only exception is TFALum that only exhibits one

emission band in DMF.

G(k

cal/

mo

l)

DMSO

Alpha

Delta

Eta

Nu

Omikron

G(k

cal/

mo

l)

DMSO

Beta

Epsilon

Zeta

Xi

6

In water at pH in the range 11.5-11.8

(S5), we have verified that TFA hydrolizes.

Regarding the other spectra, BnLum (Bn) and

DMU1Lum (DMU) show just one very broad

absorption band. ECLum retains the resemblance

of luminol’s spectrum in water. As for fluorescence

in aprotic media, DMU1Lum shows a quite

complex emission. The other species exhibit a

maximum and a shoulder that we have verified to

come from acid-base reaction in the excited state.

4.3 Theoretical Spectra

With respect to theoretical absorption

spectra, we observed that using MP2/6-31G**

optimized geometries on TDDFT calculations with

PBE1PBE/aug-cc-pVTZ level of theory allowed us

to predict with some accuracy the absorption

spectrum of luminol. In Figure 7 we can observe

that the highest error is in the maximum

absorption wavelength of the first electronic

transition. The same type of error occurs in

DMSO’s theoretical absorption spectrum

(presented in supplementary material S6 and S7).

Regarding the oscillator strengths, the theoretical

predictions were more accurate in water than in

DMSO. We can also verify from Figure 7 that the

two tautomeric species predicted by us to be the

most stable in both DMSO and water are the ones

that best fit to the experimental absorption

spectrum (assuming that a fit to maximum

absorption wavelength is performed). Because in

water we only observe two transitions, and

because the transitions only show two maxima,

the best fit of the spectra excludes luminol’s

tautomer A as a significant contributor to the

ground state systems of luminol.

With respect to the error in the predicted

maximum absorption wavelength, we have

verified that it may be related to the level of

contamination in the transitions (*). Another

possibility is the contribution of the aniline moiety

to the molecular orbitals involved in the transition.

That proposal comes directly from the fact that

MP2 calculations described the aniline

functionality in a different way than DFT methods.

4.4 Excitation Anisotropy

We have studied luminol’s anisotropy as a

function of excitation wavelength using a 9:1

ethanol-methanol glass. The excitation spectra

were obtained following the two emission maxima

observed in the fluorescence at 100 K, 390 nm

and 406 nm. In Figure 8 we can verify that only

one of those excitation anisotropies goes to 0.4 at

the wavelength of the first electronic transition.

The fact that with the emission at 406 nm the

anisotropy has a limiting anisotropy value was

justified by the observed Stokes shift of the

sample.9

We also have verified that the transition

moment for the second electronic transition is

practically orthogonal to the transition moment for

the first transition. Using data from Table 2

(theoretical predictions for the excitation

anisotropies in water) we conclude that the only

two luminol tautomeric forms that are able to

explain the observed anisotropy at 300 nm

(excitation) are B and C but not A.

Even though we have perfectly excluded

luminol’s tautomer A existence, we cannot verify

the existence of the other two tautomeric forms of

luminol separately, i.e., we have not gathered

evidence that tautomers B and C simultaneously

exist.

7

Table 2: Excitation anisotropies theoretically predicted

in water for luminol’s tautomers A, B and C. Sn.Sm

represents the transition moments involved in the

calculated anisotropy. Inside brackets are the

angles between the transition moments.

S1.S2 S1.S3

A0.074

(132.5)-0.11(66.7)

B-0.19(96.9)

0.007(126.0)

C-0.15(73.2)

-0.18(79.6)

4.5. Chemiluminescence

One last study we performed was the

chemiluminescence of luminol’s derivatives we

have synthesized. The chemiluminescence

spectra in Figure 9 show that the maximum

emission wavelength is the same in all species

meaning that the chromophore is somewhat

independent of the aniline functionality. Besides,

we observed that the chemiluminescence

quantum yield of those derivatives is decreased

with respect to luminol’s (cf. Table 3).

Table 3: Chemiluminescence quantum yield of the

derivatives we have synthesized towards luminol’s.

EC Bn DMU

ΦCL 91% 39% 1.5%

We have also verified that the observed

emission is from the aminodiphthalate anion

(species B in Figure 1). One last point worth to be

mentioned is the observation of a shoulder in

luminol’s chemiluminescence that we have

assigned to the acid conjugate of the

aminodiphthalate dianion. This is the first time

such behaviour is observed, meaning that two

species are emitting light in the system we have

studied. We propose that the acid conjugate of

aminodiphthalate comes from an acid-base

reaction in the excited state.

5. Conclusions

We verified that of the 6 tautomeric forms

of luminol studied, the most stable have

aromaticity in ring 1 (trisubstituted benzene ring)

and in the other ring one carboxyl group. The

tautomer with the carboxyl group closer to the

aniline functionality, B, is the most stable one in all

conditions. Regarding acidity, the hydrazide

protons are the most labile, followed by an aniline

one.

From luminol’s derivatives spectra we

verified that the main effect observed is in the

absorption spectra that changes not only between

derivatives but also with the media. The

fluorescence spectra are less affected by

acylation of luminol and the chemiluminescence is

merely decreased its intensity, i.e., the

chemiluminescence quantum yield decreased

upon derivatization.

Besides, we observed that using

MP2/6-31G** geometry optimization followed by

TDDFT calculations (PBE1PBE/aug-cc-pVTZ)

yields good absorption spectra prediction. We

have also performed excitation anisotropy studies

and verified that luminol’s tautomer A (structure by

which luminol is commonly known) cannot

describe the observed results.

6. Acknowledgments

I wish to acknowledge all my laboratory

colleagues.

8

Figure 6: Luminol’s absorption and fluorescence in DMSO. Initials identify the derivatives. Lum stands for luminol, TFA

for its trifluoroacetyl derivative, EC for ECLum, Bn for BnLum and DMU for DMU1Lum.

Figure 7: Luminol’s normalized theoretical absorption (tautomers A, B and C) superposed with the experimental

absorption spectra. Theoretical calculations performed in water’s dielectric. The experimental spectrum was performed in

water at pH 7.15.

Figure 8: Luminol’s excitation anisotropy in 9:1 ethanol-methanol glass at 100 K. Study at two different emission

wavelengths, 390 nm and 406 nm.

0

0.2

0.4

0.6

0.8

1

1.2

250 300 350 400Wavelength (nm)

Norm. Abs. Lum

TFA

EC

Bn

DMU

0

0.2

0.4

0.6

0.8

1

340 440 540Wavelength (nm)

Norm. Fluor.

0

0.2

0.4

0.6

0.8

1

270 290 310 330 350 370 390 410

No

rm.A

bs.

Wavelength (nm)

Exp

A

B

C

-0.2

0

0.2

0.4

250 270 290 310 330 350 370

Wavelength (nm)

r

390

406

Exc

9

Figure 9: Luminol and its derivatives normalized chemiluminescence spectra in water at pH 11.8 using K2S2O8 and H2O2

as oxidants.7

Figure 10: Transitions moments for the first three transitions of luminol’s tautomers A, B and C (from left to right).

0

0.2

0.4

0.6

0.8

1


Norm. Chem. Luminol

ECLum

BnLum

DMULum

10

7. References

1. Ferreira E. C., Rossi A. V., Quim. Nova, 2002,

25, 6, 1003.

2. Albrecht H.O., Z. Phys. Chem., 1928, 10, 70.

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Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J.

Jaramillo, R. Gomperts, R. E. Stratmann, O.

Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W.

Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth,

P. Salvador, J. J. Dannenberg, V. G. Zakrzewski,

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A. G. Baboul, S. Clifford, J. Cioslowski, B. B.

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M. W. Wong, C. Gonzalez, and J. A. Pople,

Gaussian, Inc., Wallingford CT, 2004.

5. Tomasi J., Persico M., Chem. Rev., 1997, 94,

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laboratory chemicals, 4th

ed., Butterworth

Heinemann: Oxford ; Boston, 1996.

7. Rauhut M. M., Semsel A. M., Roberts B. G., J.

Org. Chem., 1966, 31, 8, 2431.

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Wiley-VCH.

11

Supplementary Material

S1: Hydrogen bonding distance in luminol’s tautomeric forms. In E2 and F2, the highest value for hydrogen

bonding is obtained for the interaction where the oxygen atom from the carboxyl moiety acts has acceptor.

Tautomer A B C D E2 F2

d (Å) 1.93 1.92 1.98 1.99 2.14|2.39 2.09|2.38

S2: Charges in luminol’s tautomers in gas and DMSO at highest level of theory (MP2/aug-cc-pVTZ). The atom

labelling is present also below. Charges in atomic units.

A B C

Vacuum DMSO Vacuum DMSO Vacuum DMSO

C1 -0.602 -0.613 -0.617 -0.61 -0.692 -0.678

C2 -0.226 -0.183 -0.241 -0.23 -0.179 -0.165

C3 -0.93 -0.962 -1.045 -1.046 -0.942 -0.954

C4 1.162 1.231 1.639 1.649 1.134 1.168

C5 0.968 0.924 0.832 0.831 1.291 1.28

C6 0.591 0.603 0.729 0.697 0.7 0.67

C7 0.152 0.266 -0.036 -0.019 -0.058 -0.083

C8 -0.064 0.039 -0.241 -0.264 -0.254 -0.232

N1 -0.741 -0.771 -0.762 -0.773 -0.748 -0.755

N2 -0.015 -0.038 0.493 0.48 -0.798 -0.826

N3 -0.04 -0.061 -0.862 -0.889 0.388 0.377

O1 -0.844 -0.995 -0.94 -0.983 -0.507 -0.53

O2 -0.869 -1.017 -0.543 -0.571 -0.968 -1.028

D E2 F2

Vacuum DMSO Vacuum DMSO Vacuum DMSO

C1 -0.674 -0.66 -0.57 -0.573 -0.553 -0.563

C2 -0.244 -0.23 -0.354 -0.35 -0.384 -0.381

C3 -0.839 -0.842 -0.998 -1.008 -0.971 -0.987

C4 1.257 1.268 1.412 1.438 1.53 1.546

C5 1.04 1.031 1.166 1.12 1.15 1.129

C6 0.71 0.68 0.968 0.925 0.963 0.909

C7 -0.044 -0.046 -0.05 -0.04 0.098 0.124

C8 -0.229 -0.225 -0.255 -0.188 -0.545 -0.507

N1 -0.746 -0.754 -1.191 -1.234 -1.199 -1.245

N2 -0.457 -0.51 -0.072 -0.072 0.406 0.392

N3 -0.507 -0.556 0.034 0.033 -0.882 -0.895

O1 -0.513 -0.534 -0.642 -0.647 -0.581 -0.589

O2 -0.552 -0.576 -0.883 -0.94 -0.549 -0.581

12

S3: Energetic relationship between luminol’s base conjugates.

S4: Luminol’s normalized absorption spectrum in DMF followed by the normalized fluorescence in DMF.

2.50.0

23.8

10.0

20.4

G(k

cal/

mo

l)

GasAlpha

Delta

Eta

Nu

Omikron

0.00.6

3.1

13.7

G(k

cal/

mo

l)

Gas

Beta

Epsilon

Zeta

Xi

0

0.2

0.4

0.6

0.8

1

250 300 350 400 450Wavelength (nm)

Norm.Abs.

Lum

TFA

EC

Bn

DMU

0

0.2

0.4

0.6

0.8

1


Norm. Fluor.

13

S5: Luminol’s normalized absorption spectrum in water followed by the normalized fluorescence in water. pH of 11.8

S6: Luminol’s normalized theoretical absorption spectrum in DMSO superposed with the experimental one. The

excitations of luminol were predicted using tautomer’s A, B and C.

S7: Data collected from TD DFT studies on luminol. On the left superior corner is the identification of the transition. Cont.

stands for contamination on the transition and f is the oscillator strength.

T1 λmax (nm) f Nature Transition Orbitals

AH2O 339.6 0.15 *

44-48 (1.8%);46–47 (43%)

DMSO 340.7 0.16 * (Ring1)44-48 (1.7%);46-47 (44%)

BH2O 329.2 0.17 * Cont. *

43-48 (2.0%);46-47 (42%)

DMSO 330.4 0.19 * (Ring1) Cont.43-48 (1.9%);46-47 (42%)

CH2O 328.9 0.17 * Cont. *

43-48 (2.2%);46-47 (42%)

DMSO 330.1 0.19 * Cont. *43-48 (2.0%);46-47 (43%)

0

0.2

0.4

0.6

0.8

1


Norm. Abs. Lum

EC

Bn

DMU

0

0.2

0.4

0.6

0.8

1

350 450 550Wavelength (nm)

Norm. Fluor.

0

0.2

0.4

0.6

0.8

1

260 310 360 410

No

rm.A

bs.

Wavelength (nm)

Exp

A

B

C

14


A

H2O 286.3 0.02 * Cont. *42-47 (1.3%);45-47 (40%);46-48 (3.4%)

DMSO 286.8 0.02 * Cont. *

42-47 (1.2%);45-47 (30%);46-48 (3.3%)

B

H2O 293.3 0.11 * Cont. *43-47 (1.8%);45-47 (5.6%);46-48 (37%)

DMSO 294.0 0.12 * Cont. *

43-47 (1.7%);45-47 (5.4%);46-48 (37%)

C

H2O 294.6 0.16 * Cont. *43-47 (1.8%);45-47 (1.3%);46-48 (42%)

DMSO 295.4 0.18 * Cont. *43-47 (1.7%);45-47 (1.2%);46-47 (42%)


A

H2O 280.4 0.061 ππ*44-47 (3.8%);45-47 (4.0%);46-48 (37%)

DMSO 280.9 0.067 ππ* (Ring1)44-47 (3.6%);45-47 (3.8%);46-48 (37%)

BH2O 275.9 0.053 ππ*

45-47 (39%);46-48 (5.0%)

DMSO 276.6 0.057 ππ*45-47 (39%);46-48 (4.9%)

CH2O 273.6 0.043 ππ* 45-47

DMSO 274.2 0.046 ππ* 44-47

S8: Molecular orbitals involved in tautomer B first three electronic transitions.

HOMO-3 (43) HOMO-1 (45)

15

HOMO (46) LUMO (47)

LUMO+1 (48)

synthesis and chemiluminescence studies of luminol and ... · chemiluminescence quantum yield is...

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