The photophysics of some UV-MALDI matrices studied by using spectroscopic, photoacoustic and luminescence techniques

Chemical Physics Letters 426 (2006) 334–340 www.elsevier.com/locate/cplett

The photophysics of some UV-MALDI matrices studied by using spectroscopic, photoacoustic and luminescence techniques Mariana Mesaros a, Olga I. Tarzi b, Rosa Erra-Balsells a

b,* ,

Gabriel M. Bilmes

*,a

Centro de Investigaciones Opticas-CIOp (CONICET-CIC) and Universidad Nacional de La Plata, Casilla de Correo 124, (1900) La Plata, Argentina b CIHIDECAR-CONICET, Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabello´n II, 3 Ciudad Universitaria, (1428) Buenos Aires, Argentina Received 10 January 2006; in final form 7 June 2006 Available online 16 June 2006

Abstract The photophysical behaviour of classical UV-MALDI matrices 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THAP), trans-3,5-dimethoxy-4-hydroxycinnamic acid (SA), trans-4-hydroxy-a-ciano-4-hydroxycinnamic acid (CHC), 9Hpirido[3,4-b]indole (nor-harmane; norHo) and 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho) in acetonitrile was studied by using spectroscopic, luminescence and photoacoustic techniques.  2006 Elsevier B.V. All rights reserved.

1. Introduction Since its introduction, matrix-assisted UV laser desorption/ionization mass spectrometry (UV-MALDI MS) [1,2] has rapidly become a vital tool in the study of macromolecules. UV-MALDI is the method of dispersing a macromolecule (analyte) in a large excess of a matrix. The solid mixture is irradiated with a laser pulse and vaporizes. Analyte gas ions are formed and then detected by MS. The mechanism of ionization during MALDI is still poorly understood and no adequate quantitative model for the complete process exists. Whatever the mechanism may be, one can certainly say UV-MALDI has proven to be of great utility in the MS analysis of otherwise intractable bio- and synthetic polymers. The UV-MALDI technique involves both laser ablation and ionization of the matrix/ analyte mixture after electronic excitation of the matrix. Spectra are dominated by matrix photoproducts, protonated molecules (analyte and matrix), matrix molecular radical cations, and adduct ions. Several groups have developed models for the ablation process [3–6]. However, *

Corresponding author. Fax: +54 11 4576 3346. E-mail address: [email protected] (R. Erra-Balsells).

0009-2614/$ - see front matter  2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2006.06.030

full models of UV-MALDI wait a more detailed knowledge of ionization mechanisms, kinetics and thermodynamics of the species involved [7,8]. Evidences exist that analyte ionization occurs both in the surface [6,9] and in the expanding plume by a collisional mechanism in the gas phase (matrix-analite collisional mechanism; matrix as [M]+, and/or [M + H]+, and/or [M + alkali metal cation]+) [7,10]. Both processes prompt ionization and ionization in the plume, might involve the ground state and electronic excited states of the matrix. Ehring and Sundqvist [11] studied the 2,5-dihydroxybenzoic acid (gentisic acid; GA), finding that its luminescence quantum yield was very low (<0.2). The authors suggested that most of the energy absorbed by the molecule relaxes via non-radiative pathways and is therefore available for the desorption/ionization process. In spite of this suggestion, and the models of ablation and ionization previously mentioned, the thermal deactivation process from the electronic excited state of the currently used UVMALDI matrices has not been described yet. A knowledge of the thermal deactivation process of common UV-MALDI matrices should play a critical role in understanding why some matrices are ‘hotter’ than others, leading to more abundant post-source decay as well as prompt decay of the analyte (i.e., fragmentation pro-

M. Mesaros et al. / Chemical Physics Letters 426 (2006) 334–340

cesses of carbohydrates [12,13]). Furthermore, the knowledge of this matrix property might be helpful in providing an additional aspect to keep in mind in order to choose the proper matrix to perform better UV-MALDI-MS analysis. As it is well known, the type of fragmentation observed also depends on the type of analyte molecular ion formed, which to some extent depends on the matrix too [7,12,13]. On the other hand, in an early work was demonstrated that laser ablation generates an acoustic signal which is directly proportional to the amount of material removed [14,15]. This principle has been used for quantification in both UV-MALDI [16] and IR-MALDI [17]. From a different point of view, Golovelev [18] described the basic principles and experimental results of laser-induced acoustic desorption (LIAD) of some salts and biomolecules. As part of a comparative study of the photochemistry of the most popular matrices used in UV-MALDI-MS, we decided to examine the photophysical processes occurring after the electronic excitation of acetonitrile solutions of the following compounds: 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THAP), trans-3,5dimethoxy-4-hydroxycinnamic acid (SA), trans-a-ciano-4hydroxycinnamic acid (CHC) together with the recently described 9H-pirido[3,4-b]indole (nor-harmane; norHo) and 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho) [19– 21]. As it is known, they behave in general as efficient matrices in UV-MALDI-MS analysis of proteins and carbohydrates, among other analytes [3,7,8,12,13,19–21]. In this work, we study the absorption and emission properties of those molecules in steady-state conditions; its photostability; its singlet oxygen production by means of time resolved phosphorescence detection, and its calorimetric behaviour and triplet state properties by using photoacoustics measurements. 2. Experimental Spectrograde and HPLC grade acetonitrile was purchased from J. T. Baker and was used without further purification. 2-Hydroxybenzophenone (2-HBP), phenalenone (PH), GA, THAP, SA, CHC, norHo and Ho were purchased from Aldrich and were used without further purification. The absorption measurements were performed with a UV–Visible spectrophotometer Shimadzu UV-1203. All measurements were made with 1 cm stopped quartz cells at 298 K. Steady-state spectra and quantum yields were determined with a spectrofluorimeter PTI QM-1 Quanta Master as described elsewhere [22]. Photoacoustic measurements were performed by using a set-up already described [23]. Basically, a Q-Switched Nd:YAG laser (7 ns FWHM) operating at 355 nm, was used as excitation source (1 mm diameter in the cell). A home-made ceramic PZT (4 · 4 mm) transducer with an appropriate amplifier was used to detect the acoustic sig-

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nals. The resolution time of the experiments was sR = 1 ls. Measurements were performed averaging the acoustic signals generated by 64 laser shots for better signal to noise ratio. The absorbances of the solutions were checked before and after each set of laser shots. 2-HBP was used as calorimetric reference (CR) [24]. For the experiments, sample and reference solution concentrations were matched to absorbance values between 0.1 and 0.2 at the laser wavelength. Signals were also processed mathematically using deconvolution methods, combined with an appropriate kinetic model. Experiments were performed at open air and under controlled atmosphere, bubbling N2 or O2 in the solution, during 15 min. Time resolved phosphorescence detection was used for singlet oxygen detection. The near IR luminescence of O2 (1Dg) was observed at 90 geometry through a 5 mm thick AR coated silicon metal filter with wavelength pass >1.1 lm and an interference filter at 1.27 lm by means of a preamplified (low-impedance) Ge-photodiode (Applied Detector Corporation, time resolution 1 ls). Simple exponential analysis of the emission decay was performed with the exclusion of the initial part of the signal. Phenalenone, with /DPh = 0.95 [25] was used as reference. 3. Results and discussion Absorption and emission data obtained for the studied compounds are shown in Table 1. It is important to note that in UV-MALDI experiments (N2 laser, 337 nm), matrices and analyte are prepared in neutral solutions (i.e. acetonitrile, acetonitrile–water or water for carbohydrates and glycoconjugate compounds) and in acid medium (i.e. H2O–trifluoroacetic acid 0.1% for protein analysis) [7,8,19,20]. Thus, matrix molecules are protonated species in the latter experiments. Therefore, we studied the photophysics of the selected compounds also in acid solution (MeCN + HClO4). The UV-absorption spectra of THAP, GA, SA and CHC are similar in neutral and in acid solution as well as their luminescence deactivation process (results not shown). Ho and norHo show the characteristic b-carboline bathochromic shift described elsewhere [22,26] and its fluorescence quantum yield show different values too (Table 1). No photobleaching was detected after UV longer irradiation time experiments for GA, THAP, SA, norHo and Ho, showing for that compounds good photostability. In the case of CHC an irreversible photobleaching of the irradiated solution was observed: hypsochromic shift of kmax, from 336 to 310 nm. For this compound, also irreversible changes in the amplitude of the measured acoustic signals were observed increasing the number of excitation pulses. Singlet oxygen phosphorescence measurements for THAP and GA did not show any evidence of singlet oxygen formation in our experimental conditions. On the contrary, norHo and Ho in neutral solutions showed clear evidences of singlet oxygen production with efficiencies of 0.30 and 0.20, respectively. These values diminished

336

M. Mesaros et al. / Chemical Physics Letters 426 (2006) 334–340

Table 1 Photophysical data of compounds used in UV-MALDI as matrices Matrix

Absorption dataa kmax (nm)

log e

kf (nm)

/fO2

/fN2

/D

aO2

aN2

sTO2 ðnsÞ

sTN2 (ls)

/T

kP (nm)

THAP

281 322

4.1 3.4

399

<103

<103

<103

1

1

<200

<0.2

1

–

GA

336

3.5

427

0.05

0.05

<103

0.96

0.96

<200

<0.2

1

–

norHo

281(sh) 288 339 350

4.1 4.2 3.6 3.6

355

0.20

0.35

0.30

0.75

0.15

<200

0.75

0.60

410d

norHo + H+(c)

304 376

4.2 3.7

443

0.35

0.75

<0.1

0.70

0.35

<200

>5

0.05

458d

Ho

278 (sh) 287 336 348

3.5 3.7 3.2 3.3

355

0.30

0.40

0.20

0.70

0.01

<200

1.5

0.70

404d

Ho + H+(c)

289 (sh) 302 370

3.7 3.8 3.3

428

0.50

0.60

<0.1

0.55

0.25

<200

>5

0.30

460d

a b c d

Emission datab

Singlet oxygen data

Calorimetric data

Triplet state data

A = elc; A, absorbance; c, 1 · 105 mol dm3; e in M1 cm1; solvent: acetonitrile; T: 298 K. c, 1 · 106 mol dm3; solvent: acetonitrile; T: 298 K. Addition of 1% perchloric acid (0.5 mol dm3). Ref. [33].

dramatically to 0.1 when experiments were conducted with the compounds in acid solutions (MeCN + HClO4). Fig. 1a shows typical photoacoustic signals detected by the piezoelectric transducer of the sample (THAP) and the calorimetric reference (2-HBP), in acetonitrile at 298 K, when both solutions have the same absorbance. As can be seen, almost the same signal was obtained for sample and reference. The peak to peak amplitude of the first acoustic pulse (H) of this signals were used to measure the prompt heat released to the medium by the sample, after excitation.

Fig. 1b shows the behaviour of H for same absorbance solutions of THAP and 2-HBP, as a function of the excitation fluence (F), measured under three different atmospheres: air (Air), nitrogen (N2) and oxygen (O2). As can be seen, coincident linear plots of H versus fluence (F) were obtained for sample and reference. A similar behaviour was obtained for other absorbances, showing linear correlations and good reproducibility not affected by atmosphere changes, at fluences F < 18 J/m2. These results can be interpreted in terms of Eq. (1) [27]: H =F ¼ Kað1  10A Þ

ð1Þ

800

0.06

Air N2 O2

600

H (mV)

PA signal amplitude (a.u.)

0.12

0.00

2-H BP 400

200

-0.06

5

6

7

8

0

0

4

8

12

16

2

Fluence (J/m ) Fig. 1a. Photoacoustic signals of 2-HBP (atmosphere: Air, solid line) and THAP (atmosphere: N2, dotted line; O2, dashed line) in acetonitrile solutions.

Fig. 1b. Amplitude of the photoacoustic signals as a function of laser fluence for acetonitrile solutions of THAP (atmosphere: Air, N2, O2) and 2-HBP (atmosphere: Air).

M. Mesaros et al. / Chemical Physics Letters 426 (2006) 334–340

H/F (mV.m2/J)

60

40

20 2-HBP THA 0 0.00 0.06 0.12 0.18 0.24 0.30 0.36

1-10(-A) Fig. 1c. Amplitude of the fluence-normalized photoacoustic signals as a function of the fraction of absorbed energy for acetonitrile solutions of THAP and 2-HBP (atmosphere: Air).

800 N2 O2

600

H (mV)

were K is an experimental constant containing the thermoelastic properties of the solution and instrumental factors, A is the sample absorbance value and a is the fraction of energy released to the medium as prompt heat, within the time resolution of the experiment. As can be observed in Fig. 1c for THAP and 2-HBP, a linear plot of H/F versus the fraction of absorbed energy by the sample, for the A range of 0.102–0.170 was obtained, independently of the atmosphere (data of Fig. 1c corresponds to air atmosphere). Taking into account that for the calorimetric reference 2-HBP a = 1 and that the measurements for sample and reference were performed in the same experimental conditions, the ratio of the H/F values obtained for sample and reference yields the sample a value. Thus, for THAP a = 1 ± 0.03 was obtained. As it was discussed by several authors [28,29] prompt heat means the heat integrated by the transducer in processes faster than roughly sR/5 with sR the resolution time of the experiment. When a value of a = 1 is obtained in this conditions, it is accepted that there are no energy-storing species present. In this situation the lifetime of the involved states must be shorter than sR/ 5. Then, we assume that THAP releases to the medium all the absorbed energy as prompt heat and the lifetime of the triplet state is shorter than s = 200 ns (considering sR = 1 ls). No evidence was found for the formation of photoproducts or intermediate species with lifetimes higher than this time (i.e. triplet state; phototautomers). When THAP acid solutions were analyzed no changes were observed for the a values with respect to neutral solutions. For GA, experiments conducted in neutral and acid acetonitrile solutions under different atmospheres (N2, O2 and Air) and at the same absorbance, showed in all the cases the same acoustic signal (results not shown). Fig. 2 is a typical plot of H versus F for same absorbance samples of GA (in N2, O2 and Air) and 2-HBP (air). From this plots, the slopes H/F of sample and CR, at several absorbances

337

Air 2-HBP

400

200

0

0

4

8

12 2

Fluence (J / m ) Fig. 2. Amplitude of photoacoustic signals as a function of laser fluence for same absorbance acetonitrile solutions of GA (atmosphere: N2, Air, O2) and 2-HBP (atmosphere: Air).

and under the same experimental conditions were obtained. In all the cases the change of the atmosphere does not produce any modification in the slopes, within experimental error. From these plots a = 0.96 ± 0.03 was determined for GA in N2, O2 and Air solutions (see Scheme. 1). In this case, a simple energy balance equation can be used: Ea ¼ /F Ef þ aEa

ð2Þ

where /F is the fluorescence quantum yield and ka = hc/Ea, kf = hc/Ef are the absorption and fluorescence maximum wavelengths measured experimentally (see Table 1). Then, as in the case of THAP, we can assume that no energy-storing species are present and the lifetime of the triplet state is again shorter than s = 200 ns. In agreement with this result, in a previous work [30] for GA was found the evidence of the formation of a transient species with lifetime lower than 100 ns (i.e. keto-enol phototautomer). This species is not affected by changes in the atmosphere and reverse to GA by means of reversible intramolecular rearrangement. For nor-Ho and Ho a different photoacoustic behaviour, respect to THAP and GA, was found under different atmospheres. As is shown in Figs. 3 and 4 the plots of H versus F for these substances were different under N2 and Air and were drastically modified in the presence of a triplet quencher as O2 [31]. NorHo and Ho plots of the acoustic signal as a function of time in N2 atmosphere show also a clear phase shift between the waveforms of samples and the calorimetric reference (2-HBP). In O2 and air atmospheres no shift was observed. These results can be interpreted by proposing the production of an energy storing species, i.e. the triplet state with a life time between 200 ns 6 s 6 5 ls (sR/5 6 s 6 5sR with sR = 1 ls) [30,31], efficiently quenched by O2 via T1 ! So induced intersystem crossing. In order to determine the value of a for norHo an Ho and an estimation of the lifetime of the transient storing

338

M. Mesaros et al. / Chemical Physics Letters 426 (2006) 334–340 OH COCH3

COOH

HO

OH

OH

HO

Gentisic acid (GA)

2,4,6-trihydroxyacetophenone (THAP)

N

N

N H

N H

CH3

nor-Harmane (norHo)

Harmane (Ho)

N

N

N H

H

N H

CH3

Protonated norHo (norHo + H+)

H

Protonated Ho (Ho + H+)

COOH C

CH 3O

H

C

H H

C

COOH

C

CN HO HO

OCH3

trans-3,5-dimethoxy-4-hydroxycinnamic acid (SA)

trans- α -cyano-4-hydroxycinnamic acid (CHC)

Scheme 1.

600

800

2-HBP N2

O2

O2

600

Air

H (mV)

H (mV)

400

Air N2

2-HBP

400

200

200

0 0

4

8

12

16

0 0

20

Fluence ( J / m ) Fig. 3. Amplitude of photoacoustic signals as a function of laser fluence for acetonitrile solutions of norHo (atmosphere: N2, Air, O2) and 2-HBP (atmosphere: Air).

species in N2 atmosphere, we used a deconvolution method. The deconvolution method is the best for treating cases like this, where a clear phase shift between the observed waveform for sample and reference exists. In this situations the observed energy normalized acoustic wave S(t) is a convolution of the system response R(t) and the rate of heat evolution q(t) as is shown in Eq. (3) [32]:

4

8

12

16

20

Fluence (J/m2)

2

Fig. 4. Amplitude of photoacoustic signals as a function of laser fluence for acetonitrile solutions of Ho (atmosphere: N2, Air, O2) and 2-HBP (atmosphere: Air).

SðtÞ ¼ H ðtÞ=Eð1  10A Þ ¼ RðtÞ  qðtÞ Z t ¼ RðuÞqðt  uÞ du

ð3Þ

0

where H(t) is the observed waveform of the sample and R(t) is the system response which can be determined experimentally by using the CR. Assuming a kinetic decay model with a fast decay and one storing species, and performing

M. Mesaros et al. / Chemical Physics Letters 426 (2006) 334–340

the deconvolution by an iterative deconvolution program based on the Levenberg–Marquardt v2 minimization procedure [32] the values of s and a can be calculated as: CðtÞ ¼ RðtÞ  ðða=sÞ et=s Þ

ð4Þ

Resolution of Eq. (4) by deconvolution yielded, for norHo very good fits with s1 = 10 ns, a1 = 0.15, and s2 = 730 ns, a2 = 0.33. For Ho s1 = 10 ns, a1 = 0.01, and s2 = 1.5 ls, a2 = 0.34. Then, triplet state lifetimes values around 750 ns and 1.5 ls can be assumed for norHo, and Ho, respectively. The range of our calculated value for norHo agrees with the sT = 600 ls determined by flash photolysis experiments [33]. Thus, for norHo and Ho under N2, a simple energy balance equations can be used: Ea ¼ /F Ef þ aN2 Ea þ /T ET

ð5Þ

where /F, Ea, Ef were previously defined, kP = hc/ET is the phosphorescence maximum wavelength measured experimentally, and aN 2 is the fraction of energy released as heat obtained by the deconvolution method with data obtained under N2 atmosphere. Eq. (5) allowed to estimate the triplet quantum yields /T. Thus, /T = 0.60 and 0.70 were obtained for norHo and Ho, respectively (Table 1). In O2 atmosphere norHo and Ho triplet states are efficiently quenched and singlet oxygen formation takes place by energy transfer. Taking into account that for O2 (1Dg) in acetonitrile s = 80 ls the /D values determined for norHo and Ho in acetonitrile are /DnorHo = 0.30 and /DHo = 0.20. To determine if the triplet state is total or partially quenched in O2 atmosphere Eq. (6) can be used: Ea ¼ /F Ef þ aO2 Ea þ /D ED

ð6Þ

This equation corresponds to the case in which total quenching take place. Then by using data obtained from independent experiments performed in O2 atmosphere, i.e: /f, kf =hc/Ef, aO2 (determined by using Eq. (1)), ka = hc/Ea, and /D, with ED known from the literature, the energy balance can be checked and the quenching efficiency determined. Results obtained replacing the corresponding data of norHo and Ho in Eq. (5) shows that this equation is correct to describe the photophysical behaviour within 5% error. Then it can be assumed that in O2 atmosphere norHo and Ho triplets states are completely quenched in a time shorter than s = 200 ns. In acid solutions norHo and Ho show important changes in their photophysical behaviour with respect to neutral solutions. Also, in O2 atmosphere, the triplet states are completely quenched very fast, but singlet oxygen efficiency is drastically reduced with respect to the case of neutral solutions (see Table 1). On the other hand, in N2 atmosphere, triplet states lifetime seems to be enlarged, and triplet state efficiency formation is reduced with respect to the neutral solutions as it can be seen in Table 1. SA showed neither significant fluorescence emission nor singlet oxygen generation. Furthermore, photostability studies in solution showed for this compound the well-

339

known trans–cis photoisomerization process. Owing to this fact, the thermal deactivation process of the electronic excited state could not be characterized by the corresponding a value. Complementary flash photolysis and photothermal studies are in progress in our laboratory to characterize in detail the photophysics of this matrix. 4. Conclusions Experiments conducted showed a strong dependence on the matrix structure. The ortho-hydroxyphenyl-carboxylic and ortho-hydroxyphenyl-carbonylic compounds GA and THAP behave as the well known CR 2-HBP under N2, Air and O2, showing a very minor fluorescence and good photostability in solution. The piridoindoles norHo and Ho in neutral solutions show high fluorescence and good photostability with lower a values depending strongly on the atmosphere. The presence of the cinnamic moiety induces different photophysical behavior in the hydroxyphenyl-ethenyl-carboxylic compounds SA and CHC. SA seems to be involved in a trans–cis photoisomerization process. CHC shows to be not stable at all in the conditions of low laser excitations employed in the photoacoustic experiments, even under N2. As UV-MALDI is performed under high vacuum conditions, the results reported for THAP, GA, norHo and Ho under N2 can give an approach at those matrices behaviour. Preliminary results obtained in our laboratory in the UV-MALDI-MS analysis of polysaccharides agree with the fact that THAP and GA, with the highest a, release heat to the medium more efficiently than norHo and Ho. THAP and GA might behave as ‘hotter’ matrices than norHo and Ho. These studies together with the detailed analysis of the photo- and thermal-decomposition of SA and CHC are in progress in our laboratory. Acknowledgements This project was partially financially supported by UBA (X022), CONICET (PIP05/5443) ANPCyT (PICT0212312), and UNLP (11/I083). R.E.-B. is a research member of CONICET and G.M.B. is a research member of CIC and UNLP. References [1] M. Karas, F. Hillenkamp, Anal. Chem. 60 (1987) 2299. [2] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Comunn. Mass Spectrom. 2 (1988) 151. [3] M. Karas, U. Bahr, U. Giessmann, Mass Spectrom. Rev. 10 (1991) 335. [4] R.W. Nelson, M.J. Rainbow, D.E. Lohr, P. Williams, Science 246 (1989) 1585. [5] B. Spengler, J. Cotter, Anal. Chem. 62 (1990) 793. [6] R.E. Johnson, Int. J. Mass Spectrom., Ion Processes 139 (1994) 25. [7] J. Zhang, R. Zenobi, J. Mass Spectrom. 39 (2004) 808. [8] M. Karas, R. Kruger, Chem. Rev. 103 (2003) 427. [9] C.D. Mowry, M.V. Johnson, Rapid Commun. Mass Spectrom. 7 (1993) 569.

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