Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX
pubs.acs.org/jchemeduc
Introduction to Time-Resolved Spectroscopy: Nanosecond Transient Absorption and Time-Resolved Fluorescence of Eosin B Erik P. Farr,* Jason C. Quintana, Vanessa Reynoso, Josiah D. Ruberry, Wook R. Shin, and Kevin R. Swartz Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States S Supporting Information *
ABSTRACT: Here we present a new undergraduate laboratory that will introduce the concepts of time-resolved spectroscopy and provide insight into the natural time scales on which chemical dynamics occur through direct measurement. A quantitative treatment of the acquired data will provide a deeper understanding of the role of quantum mechanics and various phenomenological expressions in predicting kinetic rates for fluorescence, phosphorescence, and nonradiative decay mechanisms. This laboratory framework focuses specifically on spectroscopy in the nanosecond regimeassisted by various steady-state spectroscopic techniquesin order to fully characterize the electronic structure and the picosecond-to-microsecond dynamics of the dye eosin B. There is great flexibility in both the recommended lab duration (1 week to several months) and course level (upper-division to graduate) due to the numerous additional experiments that may be performed at varying levels of difficulty. The necessary components include pump and probe light sources, photodiode detectors, a programmable signal delay generator, and an oscilloscope for measurements with requisite resolution. The cost of building this experiment from scratch is less than $20,000 at the time of publication, but costs are expected to decrease over time and alternate excitation sources are available. Although this lab requires some expertise with optical spectroscopy to initially build and troubleshoot, use by students has been a straightforward and valuable experience. KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Physical Chemistry, Hands-On Learning/Manipulatives, Spectroscopy, Fluorescence Spectroscopy, Molecular Mechanics/Dynamics, Kinetics, Instrumental Methods
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INTRODUCTION The physical sciences have long endeavored to push the boundaries of detection, and the capability of atomically precise imaging via scanning tunneling or electron microscopy is generally introduced to undergraduates. Spatial resolution, however, is only one such frontier being actively researched equally important have been advances in temporal resolution, which are presently in the femtosecond and attosecond regimes in the field of ultrafast spectroscopy. Unlike advances in spatial resolution, dynamics on ultrashort time scales can be complex to visualize and interpret but are nevertheless equally critical to the fundamental nature of chemistry. It is this natural time scale of chemistryof making and breaking bonds, diffusion, solvation, energy/electron transfer, etc.that we believe is under-represented in upper-division undergraduate chemical education and warrants novel laboratory approaches. A summary of the time scales of these processes in liquids is provided in Figure 1. In this laboratory exercise, the time scales and photophysics of fluorescence, phosphorescence, intersystem crossing, and nonradiative decay mechanisms were explored by direct measurement with transient absorption and time-resolved fluorescence in multiple curriculum settings. We contrast the direct measurement of lifetimes in this laboratory to the variety of historical experiments that indirectly measure these rates, such as Stern−Volmer procedures.1 The primary benefit of measuring such lifetimes directly is that any data obtained for fluorescence and phosphorescence decays are unambiguousduring administration of this experiment, students made a strong intuitive connection to these time © XXXX American Chemical Society and Division of Chemical Education, Inc.
scales when observing the dynamics in real time through an oscilloscope and when performing subsequent analyses through least-squares fitting of the exponential decays. These exponential decay rates may be directly influenced through concentration,2 solvent environment,3,4 quenching,3 nearby heavy nuclei via the heavy-atom effect,5 aggregation interactions,6−8 or comparisons to different dyes,9 some of which were explored as extensions or advanced topics as described below. Most importantly, the nanosecond regime in this lab is used to conceptually bridge macroscopic and ultrashort time scales; at this time femtosecond and attosecond techniques are still prohibitively expensive and technical for undergraduate use, but concepts from the nanosecond regime are transferable to shorter time scales more commonly found in research. In this laboratory exercise, a home-built nanosecond transient absorption spectrometer was employed. It also doubles as a time-resolved fluorimeter with moderate sensitivity when the probe pulses are disabled. Briefly, pump−probe spectroscopy consists of multiple short pulses of light: the early pulses prepare a nonequilibrium state that subsequent pulses may probe as a function of the time delay between them (more information can be found in the Supporting Information, which also doubles as the student laboratory manual). We are among only several using time-resolved spectroscopy in an undergraduate setting,10−20 allowing us to expand upon the existing Received: December 7, 2017 Revised: March 5, 2018
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DOI: 10.1021/acs.jchemed.7b00941 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 1. Overview of time scales of molecular motions in liquids. Time resolution, unfortunately, limits this particular advanced undergraduate laboratory to time scales longer than 500 ps. The introduction of the provided student manual makes clear that these time scales contain critically important chemical dynamics phenomena.
Figure 2. Chemical structure of eosin B. As a xanthene/fluorescein dye derivative, it exhibits fluorescence with relatively high quantum yields but is induced to phosphoresce by the heavy-atom effect through the bromine substituents.
Eosin B is commonly used in tissue staining for microscopy in conjunction with hematoxylin as one of the primary methods for medical diagnosis and biological research,22 but it has more recently been used as a reporter of the local hydrogen-bonding environment23 and for surface-selective second-harmonic generation.24 It is a strong absorber and emitter and relatively stable as a xanthene/fluorescein derivative. The bromine atoms in the eosin structure significantly reduce the near-unity fluorescence quantum yields of typical fluorescein dyes9,25,26 by increasing the triplet yield through their internal heavy-atom effect,27,28 while also matching the energies of the first excited singlet state and lowest triplet state,29 allowing intersystem
repertoire of experiments and extend the literature to include faster dynamics. Eosin B, shown in Figure 2, was chosen to be the molecule of study because of the experimental constraints of the pump (355 nm) and probe (532 nm) wavelengths, the appreciable quantum yields of both fluorescence and phosphorescence,9,21 and the fact that fast intersystem crossing allows the assumption of a negligible nonradiative contribution. Although this instrument was specifically designed with eosin B in mind, the instrument is easily adaptable to other pump wavelengths or expansion to include broadband multichannel detection17,18 for use with other systems. B
DOI: 10.1021/acs.jchemed.7b00941 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 3. Representative Jablonski diagram of the relevant processes for this laboratory exercise.
the case in research settings. There is substantial ambiguity in the literature for the lifetimes and yields of eosin B, as summarized in Table 1, but it was found that the nature of the unsolved problem was appropriate for a research-based curriculum36 in this laboratorybecause there is no single answer, the students were forced to trust their data and make a claim based on their analysis. Other dyes that are bettercharacterized with longer lifetimes, such as eosin Y, erythrosine, or rose bengal, may be substituted for a more straightforward laboratory experience. The core concepts of this lab involve photophysics for real molecular systems. The experimentally measured fluorescence and phosphorescence decays for most molecules in dilute solutions are unimolecular processes and therefore decay exponentially. A critical clarification, however, is that empirical measurement of fluorescence or triplet decays yields the sum of all competing processes involving the electronic state of interest:
crossing. Another important advantage of eosin B is the roughly proportional singlet and triplet yields, lending itself well to measurement of both processes30−33this is a critical point for eosin B, as it provides acceptable signal for both photophysical processes (and the others shown in Figure 3), subsequently allowing characterization of the intermediate process, intersystem crossing. There are very large ranges of reported singlet lifetimes (0.1−5 ns)21,31,32,34,35 and triplet lifetimes (nanosecond to microsecond)31 at room temperature in ethanol. With the experimental apparatus for this laboratory, we measure the fluorescence and phosphorescence lifetimes (τF and τP) to be approximately 800 ps and 1.6 μs respectively; these mechanisms may be visualized in the Jablonski diagram shown in Figure 3, in which the rough order of events occurs left to right. These lifetimes made eosin B a good candidate for pushing the boundaries of our home-built system and provided students a window into the shortest feasible time scales and, as a secondary learning objective, the analyses required to understand data that is nearly instrument-limited, as is often
kF,exp = kF,true + k S,nr + kISC + [SQ]k SQ C
(1)
DOI: 10.1021/acs.jchemed.7b00941 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 4. Basic schematic including the optics and arrangement used in our instrument. The reference diode detects only a small portion of the 355 nm pump that leaks through the dielectric mirror.
kP,exp = kP,true + k T,nr + [TQ]k TQ
(2)
where the experimental and true fluorescence and phosphorescence rates are labeled, kS,nr and kT,nr are the nonradiative relaxation rates out of the singlet and triplet states, respectively, kISC is the intersystem crossing rate from S1 to T1, and the final term in each expression is a placeholder for any applicable quencher of either the singlet or triplet state (oxygen being the primary offender in most cases). Several of these may be neglected to a first approximation: the quantum yields for fluorescence and triplet state formation are sufficiently high that kS,nr may be negligiblethis is further motivated by the choice of the short fluorescence lifetime of eosin Band if samples are purged with inert gas prior to measurement, the quenching term for atmospheric oxygen may be neglected. The advantage of making these assumptions is that each of the remaining rates may be measured directly or obtained from solving the kinetic equations with the help of quantum yields, which may be estimated from the equations shown below or found in the literature.9,21,32,34,35,37 ΦF =
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EXPERIMENTAL APPARATUS AND PROCEDURES The core of our instrument consisted of a Crylas GmbH FTSS355-Q3 355 nm pump laser with
