Applications of picosecond spectroscopy to analytical chemistry

Applications of picosecond spectroscopy to analytical chemistry

trenaYsWin analyticalchemistry,vol. 8, no. I, 1989 Chromatogr., 391(1987) 296. 14 R. M. Caprioli and T. Fan, Biochem. Biophys. Res. Commun., 141 (198...

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trenaYsWin analyticalchemistry,vol. 8, no. I, 1989

Chromatogr., 391(1987) 296. 14 R. M. Caprioli and T. Fan, Biochem. Biophys. Res. Commun., 141 (1986) 1058. 15 R. M. Caprioli, B. DaGue, T. Fan and W. T. Moore, Biothem. Biophys. Res. Commun., 146 (1987) 291. 16 A. E. Ashrott, J. R. Chapman and J. S. Cottrell, J. Chromatogr., 394 (1987) 15. 17 T. Takeuchi, S. Watanabe, N. Kondo, M. Goto and D. Ishii, Chromatographia, 25 (1988) 523.

29 Dr. Daido Zshii is a professor of chemistry at Nagoya University. His recent interests center on the combination of micro-LC with MS and the development of a chromatographic system for liquid, supercritical fluid and gas chromatography using a single system. Dr. Toyohide Takeuchi is a research associate of chemistry at Nagoya University. He is currently working in the field of microcolumn chromatography. Their address is Department of Applied Chemistry, Faculty of Engineering, Nagoya University, Chikusaku, Nagoya 464, Japan.

Applications of picosecond spectroscopy to analytical chemistry G. J. Blanchard Red Bank, NJ, U.S.A. Analytical spectroscopies usually employ frequency resolution to obtain selectivity. Another degree of selectivity inherent in spectroscopic measurements is time. Picosecond time resolved spectroscopy is useful for analytical applications ranging from rejection of fluorescence from Raman signals to quantitation of mixtures offluorophores based on their individual lifetimes andlor dynamical properties, where frequency resolution will not provide the required selectivity.

Introduction Traditional analytical spectroscopic techniques rely on frequency resolution to obtain selectivity. The validity of this approach is based on the premise that each analyte possesses a unique combination of spectral properties related to its molecular identity. This approach has proven to be quite useful for a variety of techniques, such as atomic absorption/emission and liquid phase NMR spectroscopies, because of the characteristically narrow resonances observed. Other techniques, such as molecular fluorescence and absorption, do not always offer this same selectivity due to the typically broad spectral features being examined. Ultraviolet and visible absorption and emission measurements are used extensively for characterization and/or quantitation of organics due to their high sensitivity. Using these techniques it is not always possible to detect individually multiple components in a sample because of spectral overlap interference. Thus sensitive absorption and emission measurements would benefit from an added degree of selectivity. Time domain measurements offer the possibility of additional selectivity since different spectrally overlapped fluorophores are likely to have different fluorescence lifetimes. This principle has been demonstrated using time-correlated single photon count-

ing’ and more recently by quantitating the ratios of four different fluorophores in a single solution using phase resolved techniques2. In addition to lifetime selectivity, time resolved spectroscopy has been used to separate analyte fluorescence from interferent fluorescence and scattering3T4. The traditional strength of fluorescence lifetime measurements, however, has been in biomolecular identification and characterization. Commercial instrumentation is now available for making such fluorescence lifetime measurements. In addition to the selective detection of different chemical species in solution, time resolved measurements are capable of selecting one spectral process over another. For example, Raman spectroscopy using visible excitation is known to suffer from fluorescence background interference. Non-resonant Raman spectroscopy differs from fluorescence in that there is a fixed phase relationship between the incident and emitted light with Raman ([email protected] = 0), where& with fluorescence there is no similar constraint. Because of this phase relationship, non-resonant Raman scattering is essentially an ‘instantaneous’ process, i.e. Raman scattering can occur only during excitation. Fluorescence, because there are no restrictions on A$ and the intermediate state is real, can be significantly longer lived for some organics, on the order of picoseconds to several nanoseconds. By detecting only during excitation, Raman scattering will comprise a larger relative fraction of the signal than if continuous detection is used. Thus time resolution gives significant spectral process selectivity even for small signals against a large background. This principle has been demonstrated by Harris et a1.5 in the time domain and more recently by Wirth and Chou6 using phase resolved techniques to obtain time resolution. Picosecond time resolved spectroscopy can also be used to measure rotational motion in liquids. Since

trends in analytical chemistry, vol. 8, no. <, 1989


the rotational dynamics of an analyte are related to its size and shape, measurement of this property offers the possibility of obtaining greater selectivity than that available with fluorescence lifetime measurements. In fact, both lifetime and dynamical information is present in most time resolved data. Applications of dynamical information will be discussed in detail here. It is but one application, however, of time resolved spectroscopy to analytical chemistry. Time resolved spectroscopy offers selective detection capabilities based on fluorescence lifetime, spectroscopic process (RamanHluorescence) and characteristic molecular motion. It is most applicable where frequency domain spectral overlap renders traditional analytical spectroscopies incapable of providing an answer. Time resolved measurement schemes There are a variety of ways to time resolve spectroscopic processes. The choice of a specific scheme depends mostly on the intended application of the measurement. Time resolution is usually accomplished through the use of short light pulses. Measurements can be performed using nanosecond or picosecond pulses, and the mechanism of pulse formation differs significantly between these two time regimes. A complete discussion of picosecond pulse formation has been presented before7 and is beyond the scope of this review. The discussion here will be confined to measurements using mode-locked lasers, which are capable of several picosecond time resolution. Using passively mode-locked lasers it is possible to obtain pulses of several femtoseconds (lo-l5 s) duration’, but such short pulses have, as yet, found limited application in analytical chemistry. Early time resolved measuremems were made by detecting the decay of sample fluorescence following pulsed excitation. If polarized excitation is used, dynamical information can be obtained. A schematic representation of such an experiment is presented in Fig. 1. The temporal resolution of this type of measurement is limited by the speed of the photomultiplier (PMT) detector and/or the detection electronics and is, at best, greater than 100 ps with standard ‘fast’ PMTs. Deconvolution of the (large) instrument response from the signal is usually necessary to obtain chemically useful information. Nonetheless, lifetime measurements have been used widely for sample characterization and depolarization measurements have found use in characterizing motions of large (excited state) molecules of biological significance’ as well as of small molecules in liquids of moderate viscosityi’. Recent advances in microchannel plate technology and detection elec-

polarized pulse



Fig. 1. Schematic of a typical fluorescence depolarization experiment. A portion of the input pulse is used to synchronize data acquisition, the color filter eliminates scatter interference from the source and the Polaroid is used as the polarization analyzer. For certain applications a monochromator is used in place of the color filter. The fzuorescence intensity is usually attenuated before entering the photomultiplier detector.

tronics speed have improved the time resolution of these techniques to the -100 ps range. The detection sensitivity of fluorescence is high, and the equipment required is relatively simple, but the large instrumental response function and the ability to detect only fluorescent molecules limit the selectivity and versatility of these techniques. There are several instrumental schemes which offer improved time resolution (-10 ps full width at half maximum response) over fluorescence depolarization. All of these are pump/probe techniques and are more complex experimentally than fluorescence depolarization. The chief use of these has been in the study of molecular motion, and they fall into three major categories: (i) transient dichroism, (ii) transient grating, (iii) stimulated emission/transient depletion. The transient dichroism technique was devised by Shank and IppFn11,12 and treated exhaustively by Waldeck et al. . This measurement scheme detects the anisotropy induced in the absorption of the probe pulse arising from the absorption of the pump pulse. It offers time resolution limited by the crosscorrelation of the mode-locked laser pulses. This technique is, however, fraught with potential artifacts and the interpretation of the results is not straightforward13. Other pump/probe techniques, described below, are less prone to artifacts and produce signals which are less dependent on the peculiarities of the spectrometer.

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The second measurement scheme, transient grating generation, was developed by the Fayer group14 and has found widespread application to a variety of studies characterizing and understanding molecular motion and excitation transport15,16. This technique is based on the generation of an interference grating at the common focus of two non-colinear picosecond pulses which are coincident in time. Their overlap in the sample causes a spatial variation in the incident electric field, and consequently, in some physical property of the sample. The probe beam is Bragg diffracted by the induced grating and its diffracted intensity monitored as a function of delay time. The physical or chemical phenomenon being examined depends on the pump and probe pulse wavelengths and the particular sample. The transient grating technique is thus very versatile, but requires relatively high peak power pump pulses (-lo5 W)14 to form the transient grating. Low repetition rate systems like the frequency doubled, Q-switched and mode-locked Nd:YAG laser are common light sources for these experiments. The detection sensitivity of this technique is limited ultimately by the audio frequency noise present on the source. The third pump/probe measurement scheme offers most of the wavelength versatility of the transient grating technique, but tends to generate much smaller signals. The type of laser used in most of these experiments, however, has unique low noise properties. When measurements are made using synchronously pumped dye lasers and multiple modulation detection17”*, shot-noise limited detection sensitivity is attainable. A one-monolayer detection limit has been obtained for stimulated Raman measurements’9’28, and an absorption sensitivity of -2.10e6 has been achieved21. This technique (schematized in Fig. 2) measures either small gains or losses on the transmitted probe beam and allows examination of both ground state and excited state phenomena by setting the pump and probe wavelengths appropriately. As with the other methods, most chemical applications have been to studies of molecular motion, where the signals are obtained by probing parallel and perpendicular to the pump polarization. Fluorescence lifetimes can be measured easily and directly with this technique by probing at a polarization of 54.7” with respect to the pump. All of the picosecond resolved schemes discussed above use both pump and probe laser pulse trains. Time resolution is accomplished by varying the total pathlength (between the source and sample) of one pulse train with respect to the other. Because a 1-ps delay change corresponds to a pathlength change of 0.3 mm, controlling the probe pathlength is typically done with a translation stage. The use of mechanical





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Fig. 2. Schematic of a pumpiprobe transmission measuremer The boxes labeled ‘8 MHz’ and ‘12 MHz’ are modulators to e code a sine wave modulation on the pulse trains. The lock-in ar plifiers detect the encoded signal at 20 MHz (8 + 12), the prod power at 12 MHz. The (small) signal A T is doubly demodulate The computer acquires the signal and reference channel data at controls the probe delay time.

timing control limits the scan speed and introducc the possibility of delay time dependent alignment a tifacts. Recently, both of these limitations have bee overcome rather elegantly with the development ( the as nchronous optical sampling (ASOPS) ted nique 21 . With this scheme the repetition rates of th pump and probe lasers differ by a frequency Y. Th delay time between the pump and probe pulses consequently ‘strobed’ at this difference frequent: In the original report v = 10 kHz, demonstrating i rapid signal averaging capability. Detection of th small signals generated by these experiments wi benefit greatly from this advance. ASOPS can be al plied to combustion diagnostics monitoring23 an shows promise as an extremely versatile and eff cient detection scheme. Characterizing molecular motion As noted in the above descriptions of picosecon resolved detection schemes, most of the work in th area has been aimed toward the study and characte ization of solute molecular motion in the liqui phase. These studies offer information about the siI and shape of the probe molecule, which is useful 1 analytical chemists in providing additional selectiv ty for characterizing chemical systems. The first-level description of rotational diffusion given by the Debye-Stokes-Einstein (DSE) mode124 7,



Or 60






trends in analyticalchemistry, vol. 8, no. I,1989

where rOr is the orientational relaxation time of a hard sphere in a continuum fluid, T,Iis the fluid viscosity, V is the hydrodynamic volume of the sphere, k is the Boltzmann constant and T is the temperature. D is the rotational diffusion constant of the probe molecule. This simple relation is valid only for “hard sphere” solutes and contains information about the probe volume but not shape. Perrin later derived the equations for a general ellipsoid, so that a deviation from spherical probe shape could be accounted for25. A modification of eqn. 1 to account for probe molecular shape and solvent-solute interactions can be written as: z




Or kTS

where r,,, q,V, k and T have the same meanings as in eqn. 1, F is a frictional term to account for the solvent-solute boundary condition, equal to 1 in the stick limit and less than 1 in the slip limit, its exact value depending on the shape of the probe molecule26 and S is the probe molecule shape factor25. In practice, it is usually not possible to determine unambiguously the terms ‘F’ and ‘S’ from experimental data because both of these terms are model dependent and the entire premise of the modified DSE model is not physically realistic. Most chemical systems of interest do not follow either stick or slip behavior precisely, and different solutes can behave differently even in the same solvent. The DSE model still serves, however, as a good and simple model for the approximation of rotational diffusion in low viscosity solvent systems. In addition to the above limitations, the term r,, is not measured directly. In a typical rotational diffusion experiment the quantities q,(t) and I’(t) are measured independent of one another and are combined to form the induced orientational anisotropy function, R(t), according to

(3) The time constant of the decay of R(t) is taken as an approximation of r,,. Chuang and Eisentha127 have related the experimental term R(t) to the probe molecule diffusion constant in a model independent manner. A general expression for R(t) is: R(t) = t,FkqiqjYiYjexP l-3(0,


+ $3 + a)exp[-(6D +$Ga)exp[-(6&2A>t]



+ 2A)t] t


Where i,j,k are cyclic permutations of x,y,z. The terms q and y are projections of the pumped and probed transition dipoles on the molecular Cartesian axes, D = 12

3 X,Y,Z

Di, A is a term relating to the an-

isotropy of the probe molecule shape and the a and /? terms are permutations of the q, y, D and d terms. The significance of eqn. 4 is that it relates the experimental quantity R(t) directly to the molecular quantities D,, D,, D, and the pumped and probed transition dipoles. Depending on the functional form of the R(t), it may or may not be possible to determine uniquely the Cartesian components of D. In most cases, however, different molecules of similar volume will possess different values of D due to their different chemical identities. Analytical applications There have been a variety of analytical applications of time-resolved spectroscopy. Most of them are based on resolving multiple phenomena or components which contribute to a steady-state Raman or fluorescence signal. These interferents can range from Rayleigh and/or Mie scattering, depending on the sample, to background fluorescence. Hieftje’s group has demonstrated the value of time resolution to biomedical assays with the elimination of bilirubin interference in the measurement of fluorescein emissior?. The Hieftje group has also developed novel optical fiber sensors to measure fluorescence lifetimes in ‘real-world’ environments2s. These sensors have already been used in the determination of iodide in solution2’. Fluorescence lifetime measurement and lifetime based discrimination techniques have established themselves as analytical methods. Measurement of dynamical processes allows, however, for greater selectivity than lifetime alone. For a given solvent it is known that similar molecules reorient according to different values of D3’ (see Fig. 3). The uniqueness of the value of D for a particular solute in a solvent is exactly analogous to the uniqueness of the fluorescence lifetime of a given fluorophore2. Dynamical information allows for greater selectivity because of the greater number of possible decays in R(t). There are up to five exponential decays observable in R(t) in addition to the transition polarization information; the decay of fluorescence is represented typically by a single exponential. Time domain rotational diffusion measurements have been used before for the determination of individual species in a two-component mixture31’32. In that work, a fluorophore was bound to two macromolecules of different size and shape. Identification of each macromolecule was made by measurement of the rotational diffusion

[email protected] in analyticalchemistry, vol. 8, no. I, 1989

“$JIn;” Oxozin: 1 18





-r (PSI

Resorufin 7- (PSI


AZ 16



&I 48


?I 22



L- 35





+ 24



f. 98




Fig. 3. The structures and measured reorientation times for the isoelectric molecules oxazine 118 and resorufin in the series of butanols. Solution concentrations for all of the measurements were -20 uM. The errors are 95% confidence intervals. Note the difference in measured times for the two molecules in the same solvents.


a R(0) = 0.40




z C




-2.0 5


times of the fluorophore-macromolecule complexes32. In addition to selectivity based on molecular shape and volume, the number of decays in R(t) can provide valuable information on the environment surrounding the probe molecule33. Changing the local environment of the probe molecule can cause a change in the number of decays present in R(t), as is shown in Fig. 4. This change in local environment, while manifested strongly in the rotational diffusion data, is ver difficult to detect with other analytical techniques 2 . Thus, though little explored as a routine analytical technique, picosecond rotational diffusion measurements offer the promise of greater selectivity than the more widely used fluorescence lifetime measurements and better time resolution than the more traditional nanosecond fluorescence depolarization technique’. Summary Picosecond time resolved spectroscopy has the potential to become a widely used and selective technique for the characterization of analytes in the liquid phase. In the past, the equipment required to perform picosecond pump/probe measurements has been both expensive and relatively complex. As mode-locked laser technology continues to progress, the applicability of picosecond techniques to analytical chemistry will increase. Picosecond resolved techniques are useful for fluorescence rejection from Raman signals5T6, interferent discrimination3Y28 and to the resolution of several spectrally overlapped components in solution2. In the future, when picosecond pump/probe spectrometer systems become more common, the characterization of molecules based on their dynamical behaviors1’32 promises to rival fluorescence lifetime measurements because of


400 Delay Time






b b1+b2-OX5



,s L

-c -1.5 -

-2.0 5










Fig. 4. The probe molecule cresyl violet. (a) The decay of R(t) for cresyl violet in ethylene glycol at 26°C (v = 17.3 cP). (6) The decay of R(t) for cresyl violet in I-dodecanol at26”C (v = 16.7 cP). Note the single decay in (a) and the two-component decay in (b). I-dodecanol exhibits small-domain structure between its freezing point of 24°C and -30°C. Confinement of the cresyl violet by these structured domains causes its motion to be restricted, resulting in a two-component decay. These data are adapted from ref. 30.

the added selectivity attainable diffusion experiments.

with the rotational

Acknowledgment The author is grateful to Professor D. V. Naik for several stimulating discussions and a critical reading of the manuscript.

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9 J. R. Lakowicz, B. P. Maliwal, H. Cherek and A. Butler, Biochemistry, 22 (1983) 1741.

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16 P. D. Hyde, D. A. Waldow, M. D. Ediger, T. Kitano and K. Ito, Macromolecules, 19 (1986) 2533. 17 P. Bado, S. B. Wilson and K. R. Wilson, Rev. Sci. Instrum., 53 (1982) 706.

18 L. Andor, A. Lorincz, J. Siemion, D. D. Smith and S. A. Rice, Rev. Sci. Instrum., 55 (1984) 64. 19 J. P. Heritage and D. L. Allara, Chem. Phys. Lett., 74 (1980) 507.

20 B. F. Levine and C. G. Bethea, IEEE J. Quantum Electron,, 16 (1980) 85.

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and M. J. Wirth, Anal. Chem., 58 (1986)


22 P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King and N. M. Laurendeau, Appl. Spectrosc., 41(1987) 2. 23 P. A. Elzinga, R. J. Kneisler, F. E. Lytle, Y. Jiang, G. B. King and N. M. Laurendeau, Appl. Opt., 26 (1987) 4303. 24 P. Debye, Polar Molecules, Chemical Catalog Company, New York, 1929, p. 84. 25 F. Perrin, J. Phys. Radium, 7 (1936) 1. 26 C.-M. Hu and R. Zwanzig, J. Chem. Phys., 60 (1974) 4354. 27 T. J. Chuang and K. B. Eisenthal, J. Chem. Phys., 57 (1972) 5094.

28 G. H. Vickers, R. M. Miller and G. M. Hieftje, Anal. Chim. Acta, 192 (1987) 145.

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30 G. J. Blanchard and C. A. Cihal, J. Phys. Chem., 92 (1988) 5950.

31 J. R. Knutson, L. Davenport and L. Brand, Biochemistry, 25 (1986) 1805.

J. R. Knutson and L. Brand, Biochemistry, 25 (1986) 1811. 33 G. J. Blanchard, J. Chem. Phys., 87 (1987) 6802. 34 G. J. Blanchard and M. J. Wirth, J. Phys. Chem., 90 (1986) 2521. 32 L. Davenport,

Gary J. Blanchard has been a member of technical staffsince September, 1985 at Bell Communications Research, Inc., Red Bank, NJ 07701, U.S.A. He received his Ph.D. in analytical chemistry under M. J. Wirth at the University of Wisconsin-Madison in 1985 and his B.S. in Chemistry from Bates College in 1981.

Automatic precipitation-dissolution in continuous flow systems Miguel Valchrcel and Mercedes Gallego Cbrdoba, Spain The principles and use of precipitate formation and dissolution in continuous flow systems are presented and discussed. Automatic analytical methodologies such as indirect determinations of both organic and inorganic anions andpreconcentration-determination of metal traces using an atomic absorption spectrophotometer are compared with their batch (manual) counterparts.


Precipitation is a widely used separation technique in classical analytical chemistry. Despite the extensive developments in automatic methods of analysis, there are few continuous or batch automatic analytical systems based on precipitate formation’. The reasons for this scarcity are: the heteroge0165-9936/89/$03.00.

neous kinetic process, the physical characteristics of the precipitates, their contamination and the need for aging. In addition, weighing is quite a difficult operation to incorporate into automatic systems, except for robotic stations. Our research team has recently approached automatic precipitation-dissolution systems by exploiting the advantages of flow injection analysis (FIA), particularly its simplicity and versatility2’ . Our first efforts in this regard were aimed at the study and application of continuous automatic precipitation-dissolution systems, coupled on-line with a conventional atomic absorption instrument, in implementing different analytical methodologies such as the indirect atomic absorption determination of non-metal species (both organic and inorganic anions) and the determination of traces and sub-traces of metal ions by use of a preconcentration assembly. .CQElsevier Science Publishers B .V.