Anal. Chem. 1985, 57,538-545
538
Enhancement of Luminescence and Raman Spectroscopy by Phase-Resolved Background Suppression J. N. Demas*' and R. A. Keller*
University of California, Los Alamos National Laboratory, Chemistry Division, Los Alamos, N e w Mexico 87545
Phase-resolved spectroscopy (PRS) Is shown to reduce lumlnescence backgrounds In Raman spectroscopy, and Raman or other scattering backgrounds in luminescence spectroscopy. I n PRS a two-component sample Is excited wlth a modulated source; the emission associated wlth the dlfferent component llfetlmes exhlblts modulated dgnals that are phase shlfted wlth respect to each other. These two signals are separated by a phase-sensltlve detector that permlts complete suppression of one component while only partially attenuatlng the other. PRS can resolve signals that are more than a hundred times smaller than the backgrounds. PRS Is effectlve In reducing nonstatlstical nolse but has no effect on statlstlcal nolse. Examples that are glven Include the faithful resolution of the Raman spectrum of water from the 3 4 s fluorescence of a water solution of rhodamlne 66. Error sources are discussed. Instrumentatlon made from commercially available components is described.
Photoluminescence and Raman spectroscopy are pervasive tools in fundamental scientific studies and in numerous areas of analysis including process control, waste monitoring, immunoassay, and biochemistry ( I ) . The sample blanks frequently set the ultimate limits on the selectivity and sensitivity of these measurements. In fluorimetric analyses the blank is frequently dominated by the noise in scattered exciting radiation that leaks through the detection system or by the noise in solvent Raman scattering. Raman scattering is especially troublesome since it is shifted in wavelength from the excitation and often overlaps the sample luminescence. In photoexcited flame emission experiments, exciting light scattered from flame particulates frequently obscures the signal. Raman signals are generally very weak. They are, thus, easily obscured by sample luminescences that arise from intrinsic sample or solvent emissions, or from impurities ( 2 ) . These problems are especially severe in complex biological media. Detection limits in analytical chemistry are ultimately determined by noise in background signals. The most fundamental noise source in sensitive fluorescence measurements is statistical noise ( r ~ ' / ~in) the number of photoelectrons (n). Other important noise sources include amplitude and frequency instabilities in the laser output and concentration fluctuations of the species responsible for the background signal. In this paper we describe an adaptation of phase nulling that permits enhanced visualization of underlying signals by background reduction and improves detection limits by reducing nonstatistical background emission noise. We first discuss existing background reduction methods including frequency modulation (3),simple blank subtraction, and time discrimination ( I , 3-16). We then discuss briefly the fundamentals of phase-resolved spectroscopy (PRS) and On sabbatical leave from the Chemistry Department, University of Virginia, Charlottesville, VA 22901.
its extension to background suppression. We present extensive data showing that PRS is a valuable tool in luminescence and Raman spectroscopy, even when the lifetimes of the excited species are quite short. The success of frequency modulation in reducing background from Raman, Mie, and Rayleigh scattering in fluorescence analysis results from the fact that the excitation of scatter is essentially wavelength independent while the excitation of fluorescence can be a sharp function of the wavelength. Obviously, this technique works best for species with sharp excitation spectra. Time discrimination capitalizes on lifetime differences between components. Since there is a difference in lifetime between scattered radiation (Rayleigh, Raman, or Mie) and sample luminescence, time discrimination can separate the overlapping components. Time resolution can be pulsed or modulated. In pulsed time resolution, the components are separated by using a fast excitation pulse and selectively gating the detection system to maximize the amount of the desired component relative to the undesired one (3-6). Flash lamps are used to discriminate against fluorescences in phosphorimetry ( 6 ) . By use of picosecond pulses from a mode locked CW laser, significant enhancements of Raman spectra have been achieved even in the presence of intense short-lived fluorescence interferents ( 4 , 5 ) . Modulated time resolution is the subject of this paper and is discussed below. Our current work involving fluorimetric analyses has been plagued with Raman interferences (17). Elimination of Raman lines from a fluorescence can be considered a matter of time resolution, and we set out to determine the advantages and limitations of PRS in the separation of luminescence and Raman spectra. We describe here simple instrumentation for phase resolved, Raman, or luminescence spectroscopy, and present extensive results showing the utility of PRS for enhancement of Raman and luminescence spectroscopy. PRS with a modulated source was originally developed to separate fluorescence components (7). It has subsequently been used to separate phosphorescences and fluorescences from overlapping phosphorescences (8). PRS has proved exceptionally useful to the biochemists for resolution of overlapping fluorescence spectra in complex systems (12-15). Recently, there has been renewed interest in the analytical applications of PRS. PRS has been used to suppress a fluorescent interferent in a fluoroimmunoassay (9) and an intrinsic sample fluorescence in blood plasma bilirubin analyses ( 1I ) . From the work described in the references above it is well documented that fluorescence or scattering components of similar magnitude that differ in lifetime by more than 0.2 ns can be resolved. The analytically interesting question, not addressed previously, is the following: At what level can a weak signal be resolved in the presence of a strong background? Two cases are considered in detail: (1)resolution of scattering and fluorescence when one component is very weak relative to the other and the fluorescence is short lived (3 ns), and (2) resolution of weak long-lived phosphorescence from large scatter or fluorescenceusing inexpensive electronics. Case 1 is demonstrated by the resolution of weak rhodamine
0003-2700/85/0357-0538$01.50/0G 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985
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nents. The fundamental and each harmonic are considered separately using equations analogous to eq 1. To phase resolve two components, the frequency must be high enough for at least one of the components to have appreciable phase shift. For typical nanosecond fluorescence lifetimes, this implies the need for modulation frequencies in the megahertz range. PRS exploits the phase shift between the scattered radiation and the sample luminescence to discriminate or resolve the two components. To perform this resolution the superimposed sine waves are separated with a phase-sensitive detector (e.g., a commercial lock-in amplifier). If I ( t )for the two-component system above is applied to an RMS reading lock-in amplifier, the output, I, of the lock-in amplifier is given by
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i
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0 0
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Figure 1. Frequency effects on phase related signals: (a) phase shift (---) and amplitude attenuation (-) of sample and emission as a function of frequency; (b) attenuation of luminescence (- -) on qua-
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draturing out of scattering and resultant luminescence signal as a function of frequency.
6G fluorescence from strong water Raman scatter and weak water Raman scatter from strong rhodamine 6G fluorescence. Case 2 is demonstrated by the resolution of weak 350-11s phosphorescence from strong 3-ns rhodamine 6G fluorescence. THEORY OF PHASE-RESOLVED SPECTROSCOPY We describe briefly the principles of PRS. Further details and applications are given elsewhere (14, 15). When a multicomponent luminescent or scattering sample is excited by a sinusoidal modulation of frequency, f , the scattered radiation and luminescence are also sinusoidally modulated a t f . Consider a mixture of two components, a and b, excited by a sinusoidal wave form given by 0.5[1 sin ( 2 7 ~ f t ) l . The emission intensity as a function of time is given by (8,9, 14,
+
16)
I(t)= a
+ KAEA sin (ut - 0,) + KBEB sin (ut - OB)
where Oli is the phase angle setting on the lock-in phase shifter. If OA # eB, Oli can be adjusted to suppress completely or "quadrature out" the undesired component. For example, component a can be suppressed if Oli is adjusted so that O A Ob equals 90'. As long as 8A and OB are unequal, the unwanted component can be suppressed completely while the desired component will only be attenuated by the factor sin (OB - OA). For example, consider two components with lifetimes of 2 and 4 ns. A modulation frequency of 40 MHz results in a difference in the phase shifts ( 0 , - OB) of 18.5'. If component a is quadratured out, component b is attenuated by a factor of 3. Optimum Modulation Frequency. Simple considerations indicate that there is an optimum frequency for the resolution of luminescence and sample scatter spectra. The optimum frequency depends on whether one is resolving a luminescence from scatter or scatter from luminescence. First, consider the resolution of a luminescence from scatter. The larger phase shift attainable by chopping at higher frequencies is counterbalanced by the loss of luminescence intensity as the chopping frequency is increased; see eq I b and IC. If component b is the luminescent species, the lock-in signal I with the scattering component quadratured out is given by
I =KAFA(EA/~"~)
(14
KA = 1/[1 + KB
1/[1
(u7A)2]1/2
+ (u7B)2]1/2
(IC)
(UTA)
(14
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(WQ)
(le)
= 27Tf
FA = sin (arctan
(1b)
OA = arctan w
539
(If)
a is the average dc amplitude, which we do not have to consider further for ac detection schemes. The subscripts A and B refer to components a and b, respectively. The E's are emission intensities and are related to the concentrations, emission yields, and excitation/detection efficiencies for a and b. 7's are the emission lifetimes. 0's are the phase shifts of the components relative to the excitation. ICs are attenuation factors that account for the inability of the excited state to follow the excitation as the excitation frequency becomes too high. We assume negligible high frequency rolloff in the modulator, PMT, and the system electronics. Inspection of eq 1 shows that if T* # T ~ the , 8's and K s depend differently on frequency. Plots of 0 and K as a function of the dimensionless quantity 2 7 ~ fare ~ shown in Figure 1. If the excitation source is more complex than a sine wave, the wave form may be decomposed into the Fourier compo-
(3a)
- OB)
(3b)
= sin (arctan ( 2 ~ f ~ ~OB) = ) ;0
(3c)
accounts for the signal attenuation caused by quadraturing out the undesired component. K A F A describes the total signal attenuation caused by signal loss a t high frequency and attenuation by quadraturing. F A and KAFA are plotted in Figure 1. The optimum frequency maximizes I and occurs at 2 r f ~ = 1.000 or 0 = 45'. As shown in Figure 1,however, there is a relatively small penalty for working at frequencies somewhat removed from this optimum, especially at higher frequencies where the curve falls off slowly. We now consider resolution of a scatter component from a luminescence where the optimum frequency is different. While the luminescence intensity falls monotonically with increasing frequency, the scatter signal remains unchanged because the scattering behaves as a zero lifetime component. Therefore, to phase resolve scattering from luminescence, the optimum frequency is the highest one possible. The limit is frequently set by the modulator, the lock-in amplifier, or detector roll-off characteristics.
FA
EXPERIMENTAL SECTION Systems. To demonstrate the utility of the PRS for background suppression, we selected several systems that spanned a
540
ANALYTICAL CHEMISTRY, VOL. 57,
=.. 525
550
575
600
NO.
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2, FEBRUARY 1985
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650
675
WAVELENGTH (nm)
Figure 2. Luminescence spectrum of aqueous 400 nM rhodamine 6G (---)and of 130 p M [R~(bpy)~]*+ (- -). Raman spectrum of pure water ( e ) .
S
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slg Ref wide range of properties and fully tested the methodology. These MARKER I systems included the resolution of the rhodamine 6G fluorescence DMM 4MUX 4 L O C K - IN 4 from water Raman, rhodamine 6G fluorescence from [ R ~ ( b p y ) ~ ] ~ + Q luminescence, and the [ R u ( b p ~ ) ~luminescence ]~+ from water Raman. [Ru(bp~),]~+ is the tris(2,2'-bipyridine)ruthenium(II) ion. The luminescence spectra of rhodamine and [Ru(bpy),lZ+as well as the water Raman spectrum are shown in Figure 2. The strong spectral overlap presents challenging separation problems. Figure 3. Schematic representation of a phase-resolved spectrometer: LASER, ionized argon laser; P M T l , signal phototube: PMT2, reference The 350-ns lifetime of [Ru(bpy),]*+permitted resolution on readily phototube; L1, 10 cm focusing lens for A 0 modulator; L2, defocusing available low-frequency lock-in amplifiers and probably represents lens or neutral density filters: DRIVER, modulator oscillator/driver: M, the shortest lifetime that would be encountered in analytically A 0 or EO modulator: HWP, half-wave plate for EO modulator; MON, useful room-temperature phosphorescences ( I , 18,19). The 3-11s monochromator; DR, Compuscan monochromator wavelength drive; rhodamine fluorescence is representative of analytically useful LOCK-IN, lock-in amplifier: MR. mirror; SP, microscope slide beam fluorophores and of fluorescent interferents, and the short lifetime splitter; MUX, analog multiplexer; S,sample: DMM, digital multimeter. presented a substantial challenge in resolution. Finally, the systems are intrinsically important. Rhodamine modulator. The EO modulator suffered from severe EM1 a t is an important taggent used in flow cytometry (In,and the Ru(I1) frequencies above 0.5 MHz. complex is the model system for numerous solar energy conversion For frequencies >0.5 MHz we used an Inrad EFL-200 acousschemes (20). tooptic (AO) modulator equipped with an EFL-C200Pll driver. Chemicals. The rhodamine 6G was from Exciton Chemical This system exhibited a 40-MHz optical band width when used Co. or Eastman Kodak. The two samples were equivalent in this with a 70-pm laser beam diameter. A suitable beam waist was work. [R~(bpy),](ClO,)~ was prepared from the chloride salt obtained by placing the modulator at the focus of a 10-cm focal purchased from G. Frederick Smith Chemical Co. by precipitation length lens. The TTL drive signal for the A 0 driver was derived with perchloric acid and recrystallization from water. Water was from a 40.4-MHz bipolar Pierce crystal controlled oscillator (21). deionized, doubly distilled, and filtered. The output was buffered with a 74H00 AND gate and divided Apparatus. A schematic diagram of the complete apparatus by a switch selectable eight-stage binary divider chain to provide is shown in Figure 3. In general features, the system is a conother operating frequencies. When the oscillator was used to ventional luminescence or Raman spectrometer. The excitation trigger the lock-in, the reference signal was derived from the beam is modulated before the sample, and the photomultiplier divider output after buffering with an AND gate, since the TTL (PMT) signal is processed with a lock-in amplifier referenced to gates were incapable of simultaneously driving the 50-Qinputs the modulation frequency. of the lock-in amplifier and the modulator driver. Two modes of triggering the lock-in amplifier were used. At The A 0 modulator, even a t 40 MHz without elaborate EM1 low frequencies we used the TTL output from the oscillator circuit shielding, yielded less severe EM1 than the EO system had at l o MHz) there was severe phase noise For optical triggering of the lock-in, an RCA 1P21 viewed a between the oscillator TTL trigger output and the optical excireflected portion of the sample excitation beam. The full reflected tation beam (22). The external trigger PMT viewing the excitation beam was attenuated and diffused over the photocathode to beam eliminated the phase noise, since the optical sample and reduce PMT overloading and drift. For stable locking, the PMT lock-in trigger signals are phase locked to each other. All high signal was amplified with a X40 gain, 5 0 4 input impedance, frequency measurements reported here were performed by using 150-MHz preamplifier (Comlinear Corp., E-103-N-BNC1, which the external PMT trigger. Under these conditions the phase noise also served as the PMT load resistor. at 40 MHz was less than 0.1'. Excitation System. All samples were excited with the Detection System. The optical system was a Spex 1404 double 514.5-nm line from a light-stabilized Spectra Physics 165 ionized monochromator, a Spex 1459 Raman optical collection system, argon laser. This line gave an excellent match to the absorption and a Compuscan wavelength driver. Typically, all slits were used maximum of the rhodamine. While 514.5 nm was a poor match at the widest setting, 3 mm, which yielded negligible spectral for the 450-nm absorption maximum of [Ru(bpy),I2+,it yielded distortions. The signal PMT was an uncooled Hamamatsu R928 a principal water Raman band that was in almost perfect coinwired with bypass capacitors for high-frequency operation. cidence with the complexes' emission maxima. For low-frequency measurement (200 kHz), we used a the maximum Raman signal in our configuration. A Wavetek 50-MHz PAR 5202 lock-in amplifier. The lock-in simultaneously Model 184 sweep generator supplied the TTL drive for the
ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985
provided outputs for the in-phase and out-of-phase components. As this lock-in had a 5042 input impedance, the PMT signal was preamplified with a XlOO gain PRA 1763 preamplifier or another X40 gain Comlinear preamplifier. Computer Interface. All of the low-frequency measurements were recorded with an analog recorder. For the high-frequency measurements we added a Hewlett-Packard 3052A computerized data acquisition system consisting of a 9825A calculator, a 9862A digital plotter, a 3455A digital multimeter (DMM), and a 3495A eight-channel analog multiplexer. The DMM and multiplexer were controlled over the HP-IB bus. As the acquisition system lacked a real time clock, data acquisition was synchronized to the wavelength scan by monitoring the Compuscan wavelength marker output with one channel of the multiplexer and the DMM. The marker pulse was extended with a passive RC filter to ensure that the DVM always registered a marker pulse. Noise Measurements. The noise levels in the system studied were quantified with the interfaced system. Typically, at a fixed wavelength and regular time intervals, we collected 200-600 data points. A delay of three time constants between each reading prevented data correlation. For experiments in which we simultaneously recorded the in-phase and quadrature lock-in channels, data from both channels were collected essentially simultaneously (
