Time-Resolved FT-IR Absorption Spectroscopy Using a Step-Scan Interferometer WOLFGANG UHMANN, ANDREAS BECKER, CHRISTOPH TARAN, and F R I E D R I C H SIEBERT* Institut [Er Biophysik und Strahlenbiologie der Albert-Ludwigs-Universit~t, Albertstrasse 23, D-7800 Freiburg, F.R.G. (W.U., A.B., Ch.T., F.S.); and Max-Planck-Institut [Er Biophysik, Kennedyallee 70, D-6000 Frankfurt a.M. 70, F.R.G. (F.S.) The implementation of time-resolved step-scan FT-IR spectroscopy with a commercial interferometer is described. With the use of the photoreaction of the biological system bacteriorhodopsin as an example which exhibits infrared spectral changes smaller than 10 -2 absorbance units, the quality of the method is demonstrated. A comparison with conventional flash-photolysis experiments with a monochromatic infrared monitoring beam clearly demonstrates the multiplex advantage. The advantage of covering the total time course of the reaction allows for a variety of data analysis, such as forming difference spectra between intermediates of the reaction and the deduction of time courses of absorbance changes at selected wavenumbers. The mirror stability is better than _+1.5 nm, which is sufficient for the reliable measurement of small absorbance changes. Index Headings: Step-scan Fourier transform Spectroscopy; Biochemical systems; Bacteriorhodopsin. INTRODUCTION Time-resolved vibrational spectroscopy can provide detailed information on molecular relaxation processes and on mechanisms of chemical reactions. 1-3 Resonance Raman spectroscopy is the predominant technique, but increasing interest is now being shown in time-resolved infrared spectroscopy. There are two main advantages of this method: (1) the monitoring beam does not disturb the system being investigated, and (2) nonchromophoric systems can be investigated. The most widely employed method uses a monochromatic monitoring beam and the infrared transients are sampled point by point over the spectral region of interest. The time resolution extends from microseconds up to the femtosecond range? -12 Recently, broad-band methods, using a monochromator and multichannel detection, have also become available. ~3,~4 Although FT-IR spectroscopy has revolutionized vibrational spectroscopy, there are only few reports on timeresolved FT-IR spectroscopy. But the advantages of F T - I R spectroscopy should, in principle, also prevail for time-resolved studies. The fast-continuous-scan technique, allowing time resolution of a few milliseconds, is at the slow end. Here, the advantage of modern FT-IR spectrometers is exploited, in that the time needed to acquire an interferogram is of the order of only several milliseconds? 5,~s The stroboscopic technique was proposed many years ago. With this technique, commercial continuous-scan instruments can be employed as well? 7'~s A repetitive process is triggered many times during the acquisition of a large number of interferograms, until, Received 9 July 1990; revision received 27 October 1990. * Author to whom correspondence should be sent. 390 Volume 45, Number 3, 1 9 9 1 by reshuffling of the collected data, a complete interferogram can be reconstructed for each time of interest. Apparently, this method is prone to artifacts, is In a modified technique, the process and the digitization of the transient are triggered at each zero-crossing of the HeNe laser interferogram. 19This requires that the transient of the process be shorter than the time between two sampling points of a static interferogram measured under the same conditions. In principle, a combination with the stroboscopic technique is also possible, which is, however, susceptible to the same artifacts. One of the problems with these techniques is the correlation of the not very precise clock provided by the laser interferogram with the data acquisition, on the one hand, and the correlation of the data acquisition time with the requested knowledge of the precise mirror position, on the other hand. The difficulties are especially severe if only small spectral changes occur during the process being studied, such as during the reaction process of biochemical systemsY °,21 In cases where one is interested in the fast part of a reaction, which has, however, a slow decay, the method is inefficient. The slow part is sampled with the same time resolution as the fast part, and the data of the slow part, which comprise the largest fraction, are discarded. Another disadvantage of these methods is the necessity to digitize the data with high resolution, i.e., with at least 16 bits. This limits the time resolution, based on currently available technology, to above 1 ~s. Another approach to time-resolved F T - I R spectroscopy is the implementation of the so-called step-scan technique; 22,23here, the interferometer mirror is held at a fixed position during the time course of the process and the transient is digitized. In this way, by moving the mirror step-wise, one obtains the time courses of the change of the interferogram at each sampling point, and from the data set the spectral changes can be obtained by the Fourier transform at each time of interest. Obviously, in this method, the time resolution is only limited by the detector rise-time, by the electronics, especially by the AD convertor, and by the signal strength. In timeresolved absorbance spectroscopy, since only differences have to be measured, the resolution of the AD convertor can be reduced to 12 or even 8 bits. In principle, with modern AD convertors, this would improve the time resolution up to a few nanoseconds. Recently, three papers have appeared that describe time-resolved infrared measurements with the step-scan method. In two of them, a commercial instrument was also used. 24,25 In the third publication, a special interferometer was developed. 2s In Fig. 1, the principle of the data acquisition for the 0003-7028/91/4503-039052.00/0 © 1991 Society for Applied Spectroscopy APPLIED SPECTROSCOPY I, .,,/t TERMINAL \/ ~CONTE , LECT. CPU RAM //, DMA ' ; / ;Itl FIG. 1. Principle of time-resolved FT-IR spectroscopy. Axis g represents the optical path difference of the interferometer, axis t represents time evolution of the process, and axis I represents the intensity of the interferogram. Only a small part of the interferogram is shown. The sampling points along the g axis are shown. three methods of time-resolved FT-IR spectroscopy (continuous-scan, stroboscopic, step-scan) is depicted. The central part of an interferogram is shown, which is a function of the mirror path g, which itself is normally a function of time t. In addition, a simple process has been assumed that results in changes of the interferogram which decay exponentially in time t. In the continuous-scan method, the time constant of the process has to be long in comparison to the time needed to collect a total interferogram. Thus, several interferograms are collected after the start of the process, i.e., several cross sections parallel to the I-g plane are made for each time of interest. With the stroboscopic method, the time for acquisition of the interferogram can be comparable to the time constant of the process. This means that, to cover the total time course, a large number of cross sections must be made, which are parallel to the I axis and an axis lying in between the t and g axes. In the stepscan technique, cross-sections parallel to the t - I plane are made. Whereas, in previous implementations of the step-scan technique for time-resolved FT-IR spectroscopy, special homebuilt interferometers were developed, we report here on the realization of this technique using a commercially available interferometer equipped with both continuousscan and step-scan facilities. 2~ As a performance test, time-resolved difference spectra of a photobiological system, i.e., of bacteriorhodopsin, 2s'29 are presented. Here, only very small absorbance changes occur since only a few groups of the total system (chromophore and protein) undergo molecular changes. 2°,21 The largest absorbance change is less than 0.01 at an average background absorbance of the sample between 0.5 and 1. This imposes special conditions on the stability of the instrument. D E S C R I P T I O N OF T H E I N S T R U M E N T Figure 2 shows the principle of the instrument. The optical bench is a homebuilt spectrometer with two sam- i.[iRS23£ F d ~ I TR,RECBRD, IINTERFACEI FIO. 2. Schematics of the instrument. The optical part is represented by the globar (GL), the interferometer with movable (MM) and fixed (FM) mirrors, the sample (S), and the MCT detector (D). The laser, triggered by the transient recorder, excites the photoreaction of the sample. The signal from the detector is amplified by the dc-coupled pre-amplifier (PAMP) and further processed by the main amplifier (MAMP), providing the ac- and dc-coupled channels. ple chambers, equipped with the interferometer of the Bruker IFS 88 spectrometer together with the control electronics. For better stability of the mirror in the stepscan mode, the optical bench can be evacuated to about 5 Torr. In addition, it is mounted on a vibrationally decoupled table. A standard MCT-detector (cutoff 700 cm -1) from Judson is used. The control electronics of the interferometer are connected to the host computer (68020-type from Eltec Elektronik, 2 Mbyte RAM) via a serial interface. In this way, the host computer can send the necessary commands to the control electronics for continuous and step-scan modes. The signal from the detector is amplified by a dccoupled preamplifier. Basically, it represents a currentto-voltage convertor and uses a constant bias-voltage across the detector element3 ° A compensating current is provided, which is set manually, in order to subtract the dc-part of the detector current. In this way, essentially the "ac" part of the static interferogram can be recorded. The signal from preamplifier is fed to a special main amplifier, the principles of which are shown in Fig. 3. Since for absorbance difference spectroscopy not only the spectral change of the intensity but also the background spectral intensity (i.e., the single-beam spectrum of the sample) has to be measured, this main amplifier provides two outputs: output dc, corresponding to the value of the static interferogram, and output ac, corresponding to the time-resolved change of the interferogram. This is realized by an integrating feedback loop. If the loop is closed, the dc output reproduces just the input (i.e., the value of the static interferogram), whereas at the ac output, the static interferogram level is blocked. The dc output is directly digitized by a 16-bit AD converter. The ac output is further amplified, increasing the dynamic range, and the corresponding transient is digitized by a 12-bit, 100-kHz transient recorder, from which it is transferred to the host computer. To allow for a fast settling of the electronics (an order of 10 ms) and, at the same time, for a low high-pass frequency for the ac part (0.1 Hz), a quasi sample-hold mechanism is provided by the switch "AC/DC," controlled by the computer. If the APPLIED SPECTROSCOPY 391 switch is off, the ac output is now quasi dc-coupled but is compensated by the voltage over the condensor C, corresponding to the interferogram level before the switch was in the off position. The lower limit of the high-pass cutoff frequency is limited only by the droop of the condensor. With this main amplifier, the static interferogram and the time-resolved change of the interferogram can be measured during the same scan. This feature ensures that the same phase can be used for phase correction of both the static and time-resolved spectra. The evaluation of the data is shown in Scheme I. Usually, to save measuring time, single-sided interferograms are recorded. The static interferogram, Io(g), is used to calculate the single-beam spectrum So(v) of the sample. The corresponding phase ~o(v) used for phase correction is stored. For the calculation of the phase, triangular apodization was employed. The data from the time-resolved signals AIg(t) are rearranged so that for a specified time t the change of the interferogram is obtained, A/t (g). From the Fourier transform of AP(g), together with the stored phase ~o(v), the spectral change of the intensity at the specified time t, ASs(v), is obtained. Again, triangular apodization is used. Since the spectral change of the intensity contains both positive and negative values, the usual procedures for phase correction31,32 cannot be directly applied, but the stored phase from the single-beam spectrum has to be used. From this, together with the single-beam spectrum So(v), the spectral absorbance change at the specified time t, AAt(v), is obtained. Here, the different amplifications and resolutions of the digitization for the static and time-resolved interferograms, respectively, have to be corrected for. To(g) zxlg(t) /\ So(t,) 1 ~(u) zxIt(g) L~Str(U) L~Sti(v) Z~S (u) = zXS r(V)cos(~) + ~S i(~)sin(p) • G1 G2 AC/D CR"~~ - ~ ' ~ l - ~ High-pass filler integrations-time ~_ 1 ~ R"~- -~_ fost-scon G3 time- resolved Ol, O2,O3 controlled by computer ~RC FIG. 3, Schematics of the main-amplifier. G1 is a special home-built, low-noise amplifier which is dc-coupled. G2 is an AD 507; G3 is composed of an AD 507 and a 50J. The feed-back OPAM is also an AD 507, whereas the integrating OPAM is a high-impedance AD 515. The OPAM of G2 and G3 is used in the noninverting mode. Switch AC/DC and the gains of G1-G3 are controlled by the computer. 2. The interferometer control electronics are set into stepscan mode. Hereby, the SCS and SCL values and the sampling point spacing are used and the mirror moves to the position determined by SCS; switch AC/DC is still on. 3. The fluctuations of the laser signal, caused by residual motions of the mirror, are monitored. If they are smaller than a set value, the controller signals OK. 4. The static interferogram is digitized at this mirror position. 5. AC/DC is switched off. 6. The process is initiated and the time course of the change of the interferogram at this mirror position, AI°(t), is stored in a transient recorder and transferred to the host computer. This is repeated several times for signal averaging. 7. AC/DC is switched on. 8. The mirror is moved to the next point, determined by the sampling point spacing. 9. From 3 onwards, this sequence is repeated, until the mirror has moved over the required distance determined by SCL. RESULTS So(u) + List(u) LXAt(v) = lg So(v) Scheme I. Data evaluation of time-resolved step-scan absorption spectroscopy. The principle of an experiment measuring time-resolved absorbance changes follows this procedure: 1. In continuous-scan mode, the centerburst position, in units of zero-crossings of the laser interferogram, kHeNe/ 2, is obtained. From this and from the resolution the start, SCS, and the total length of the step-scan movement, SCL, are determined. The high-wavenumber limit determines the distance between two successive sampling points in units of ~ S e S J 2 . :392 Volume 45, Number 3, 1991 A useful system for testing the instrument is the cyclic photoreaction of bacteriorhodopsin, which is a chromoprotein located in the so-called purple membrane. 2s The chromophore is all-trans retinal, which is bound to the protein via a protonated Schiff base to a lysine. 2s Upon absorption of light, bacteriorhodopsin undergoes a cyclic photoreaction involving several intermediates which are characterized by their absorption maxima (Fig. 4). BR568 is the initial state, and the L550 and M412, N520 and 0640 intermediates have time constants in the range accessible to the instrument. From our own measurements, using the conventional flash-photolysis setup, time-resolved infrared difference spectra between bacteriorhodopsin and the L550 and M412 intermediates of the photoreaction are available and can be used for comparison. 4,5 In addition, static low-temperature difference spectra for these intermediates have been published. 2°,21 6000 H 3000 0 0540 I ( N $2o ) IJ -3000 -6000 -9000 BRsT° G |! 100 0 K59° I -100 Ls5o -200 0 256 512 768 1 :4 H÷ Fia. 4. The bacteriorhodopsin system. Upper part: the chromophore of bacteriorhodopsin, all-trans-retinal, bound to a lysine of the protein via a protonated Schiff base. Also shown is the site of isomerization of the retinal chromophore during the photoreaetion, i.e., the Cla=C14 double bond. Lower part: the cyclic photoreaetion of bacteriorhodopsin. The numbers behind the intermediates show the respective approximate absorption maxima in the visible region. The numbers by the arrows show the approximate time constants of the respective intermediates at room temperature. 2.0 ~ c O " 1.0 d . ~ £ 0.0 Since bacteriorhodopsin is very stable, the cyclic photoreaction allows the sample to be flashed with many thousands of light pulses. The samples of bacteriorhodopsin were prepared in the form of hydrated films of purple membranes. They contain a small amount of water which renders the sample transparent enough in the mid-infrared spectral range, but which warrants unchanged photocycle kineticsY To be able to resolve the L550 to M412 transition with the 100-kHz transient recorder, we cooled the sample to 0°C. For excitation of the sample, a dye laser pumped by an excimer laser was used. The pulse energy at the sample was about 3 mJ, the area of the sample approx. 1 cm 2. The laser is blocked both from the detector and from the interferometer by Ar-coated Ge windows. The 100kHz, 12-bit transient recorder is equipped with a dual dwell-time facility. Although the minimum dwell-time is 10/~s, the electronic bandwidth of the total system determining the noise is 5 MHz. Thus, the noise corresponds to a sampling rate of about 10 MHz. To increase the signal-to-noise ratio, we averaged 16 signals at each interferogram sampling point. Spectra are taken with a resolution corresponding to one spectral data point per approximately 3.8 cm -1, without zero-filling. Since almost no important spectral information about the system is contained in the spectral range above 1800 cm -1, the free spectral range is limited to 1950 cm -~ by a long-pass filter (OCLI). This reduces the number of interferogram sampling points for a onesided interferogram to 512 (sampling point spacing 8XneSe/ 2). For phase-correction, data at 128 additional sampling points on the other side of the centerburst were collected, • -1.0 - 2 . 0 --------~--------~-----~--~-----~--------~------2000 1600 1600 14-00 1200 1000 800 wovenurnbere(crn -1) Fro. 5. Principle data of a step-scan measurement with a resolution of approx. 4 cm -I. (a) Static interferogram; (b) complete difference interferogram at 500 #s after the flash. Due to the different amplification and digitization,ordinate units are differentfrom those of 5a. (c) Single-beamspectrum derivedfromthe interferogramshownin 5a. (d) Phase-spectrum derived from the interferogramshown in 5a; ordinate units are in radians. increasing the total number of interferogram sampling points to 640. Figure 5a shows the static interferogram with left-sided zero-filling, and Fig. 5c the corresponding single-beam spectrum of the sample. The strong bands at 1660 and 1550 cm -1 represent the amide I and amide II bands of bacteriorhodopsin. The cutoffs below 900 and above 1950 cm-1 are due to the CaF2 window and the long-pass filter, respectively. The small offset at the beginning of the interferogram is due to the nonperfect manual compensation of the preamplifier, whereas the slow drift is mainly due to the drift of the detector, but also, to some degree, to a small monotone intensity change along the mirror path. Since triangular apodization was used, both for the phase determination and for the calculation of the spectra, these discontinuities do not cause oscillations in the phase or intensity spectra. In Fig. 5d the phase spectrum is shown. Since the static interferogram was sampled by a dc method, the spectrum exhibits only APPLIED SPECTROSCOPY 393 loo 65O I (3 48O 310 -o 140 0 o_ -31 ~ 0 E 250 500 750 1000 150 300 450 600 750 900 channel channel Fro. 6. Signals representing the change of the interferogram at a fixed mirror position (a), and at fixed wavenumber, i.e., 1528 cm -1 (b). From channel 0 to 511, dwell-time is 10 tts, and from channel 512 to 1023 dwell-time is 100 gs. The laser flash was triggered at channel 150. a linear dependence, characteristic for the phase shift caused by the beamsplitter. The distortions of the spectrum in the cutoff regions are probably due to the nonlinearity of the detector, and, therefore, using the phase in these regions would lead to erroneous results. Figure 6a represents the flash-induced change of the interferogram at a mirror position far from the centerburst. At channel 512 the dual dwell-time was switched from 10 to 100 #s. In some cases, near the centerburst position, small distortions of the signal are observed. They are due to residual fluctuations of the mirror and can be seen only at the immediate slopes of the centerburst. At that position, residual movements of the mirror will exert their largest effect. To quantify the mirror fluctuations, we measured the He-Ne-laser signal with the mirror held at a fixed position (Fig. 7). The signal amplitude is approximately ± 75 mV. The amplitude of the laser interferogram is ± 7 V. From these two values and the laser wavelength of 632.8 nm, the geometrical fluctuations of the mirror can be calculated to ± 1.1 nm. In Fig. 5b, the complete flash-induced difference interferogram is shown at 500 us after flash. This corresponds to the maximum of the signal in Fig. 6a. The same zero-filling as in Fig. 5a is applied. To obtain the interferogram, we subtracted the signal at the last sampling point from the signals at all the other interferogram sampling points; i.e., the "dc" part of the difference interferogram was subtracted. The ordinate scale is the same as that in Fig. 6a. This shows that the modulation of the interferogram is about 50%. Since the difference spectrum consists of several positive and negative bands, the corresponding interferogram has no clear centerburst and resembles somewhat the free induction decay in FTN M R spectroscopy. Figure 8a-d shows the difference spectra for 20, 100, 600, and 5000 #s after the flash, respectively. The first and third spectra correspond to the BR568-L550 and BR568-M412 difference spectra, respectively. Negative bands reflect the initial state, (i.e., BR568), positive bands the photoproducts. For comparison, we show in Fig. 9a and 9b the corresponding two spectra measured with the flash photolysis instrument with infrared monitoring beam2 ,s4 Within the limits imposed by the noise of the flash photolysis spectra, the spectra of both methods are in excellent agreement. (The noise in the step-scan spectra, which can be estimated from the spectral range above 394 Volume 45, Number 3, 1991 - 100 0 10 20 30 time (ms~ 40 50 FIG. 7. Laser-signal of the interferometer at a fixed mirror position. There are small 50-Hz distortions caused by interference from the line; ordinate units: mV. Maximum signal amplitude is approx. ±75 mV. 1750 cm -1 in the 20-us spectrum, is much smaller.) In the flash photolysis measurements, the range above 1700 cm -1 was omitted, but the comparison of the step-scan data with later measurements 21,35 shows that bands in this range are also in very good correspondence. Since the L550 and M412 difference spectra are obtained from the same experiment, the difference between the two difference spectra directly provides the M412-L550 difference spectrum, without any need for normalization. This is shown in Fig. 10. It is beyond the scope of this b c d 1800 1630 14t60 12'90 11'20 wevenumbers (cm -1) 950 FIG. 8. Flash-induced difference spectra at 20 (a), 100 (b), 600 (c), and 5000 (d) its after the flash. Resolution 4 cm-1; temperature 0°C. The spectra are scaled to each other. The size of the largest band at 1528 cm -1 in spectrum c corresponds to an absorbance change of 5 * 10-3. l o b o o ~ u3 ~ I o L550 o O4 ¢,4 M412 C,4 o 1800 16'30 14f60 12'90 11'20 wavenumbers (cm -1) 950 Fro. 9. Flash-photolysis difference spectra of the BR568-L550 (a) and of the BR568-M412 (b) transitions24 article to discuss the spectra at greater detail. Only a few points will be emphasized. The strong bands at 1538 and 1562 cm -~ correspond to the ethylenic modes of the retinal chromophore in the L550 and M412 intermediates, respectively. It should be noted that, as in resonance Raman spectra, as the L550 mode is split. However, whereas in the latter spectra the splitting causes an additional band around 1550 cm -~, here a down-shifted band is observed at 1527 cm-L Since both the 1538- and the 1527-cm -~ bands shift down upon deuteration of the retinal chromophore at C-15, they must be attributed to the ethylenic modes. Since no fingerprint bands (between 1300 and 1100 cm -~) of the initial state can be seen, we can rule out the suggestion that the BR568-M412 difference spectrum contains already back-reacted BR568, which would cause the band at 1527 cm-L Thus, both bands at 1538 and 1527 cm -~ must be attributed to the L550 species. Due to the overlap with the large ethylenic band of BR568 (negative band at 1528 cm -~ in Fig. 8a) and with amide II bands, it was hitherto impossible to identify these bands directly in the BR568-L550 or BR568-M412 difference spectra. The same splitting, although less resolved, is also observed when the two spectra of Fig. 9 are subtracted. The negative band at 1622 cm -x is also shifted down by deuteration of the retinal chromophore at C-15. Therefore, this band can be assigned to the C=N stretching mode of the Schiff base of the M412 intermediate. It is interesting to note that the corresponding band of the L550 species cannot be identified. The negative band at 1276 cm -~, which can also be seen as a negative band in the 20-gs spectrum (Fig. 8a), deserves some further discussion. This band of the BR568 state has been attributed to a tyrosine which has already undergone molecular changes in the K610 intermediate. 37 Since this band appears also as a negative band in the M412-L550 difference spectrum, it can be concluded that the corresponding molecular change is at least partially reversed during the L550-M412 transition. This effect was not observed in low-temperature static or steady-state BR570-M412 difference spectra, as Thus, the higher temperature allows the protein to relax earlier. The data analysis allows the deduction of the time course of absorbance changes at any spectral position. A few examples are shown in Fig. 6b and Figs. 11 and 12. tO 1800 16'30 14"60 12~90 11 '20 950 wavenumbers (cm-l~ FIG. 10. M412-L550 difference spectrum, derived from Fig. 8a and 8c. Figure 6b corresponds to the largest band at 1528 cm -1 in Fig. 8a. In a comparison of this time course with the interferogram signal (Fig. 6a), it is obvious that the noise is much smaller. This demonstrates nicely that the multiplex advantage is retained in the step-scan measurements. Some remarks on the signals shown in Fig. 11 are in order: The time course at 1510 cm -1 shows the rise of the ethylenic mode of the 0640 intermediate, for which the amplitude is very small at 0°C. The time course at 1191 cm -1 deviates from that observed at 1528 and 1171 cm-L This is probably due to the rise of the N intermediate. 39This effect becomes even more clear at higher pH, where, after the initial fast absorbance decrease, an absorbance increase to positives values is observed. Signals reflecting very small spectral changes are shown in Fig. 12. (The corresponding relative absorbance changes can be deduced from Fig. 8.) The two signals at 1764 and 1755 cm -1 reflect protonation changes of aspartic acids: Whereas the former signal exhibits a time course similar to the signals observed at 1528 or 1171 cm -1, the signal at 1755 cm -1 exhibits a slower component. This observation was already described previously.2~,a~Similar slow components are also present in the signals observed at I 1504 I I ~l~l..~ I I 1171 I ( ~t~'~'~t I 1191, pH 8.5 1191, pH 7 1 50 400 650 channel 9001 50 400 650 channel 900 FIG. 11. Signals at various wavenumbers, reflecting the various intermediates. With exception of the signal labeled 1191, pH 8.5, dwell times are as in Fig. 6. Due to the slower time course at pH 8.5, the transient recorder was switched from 10 tts to 200 gs at channel 300. APPLIED S P E C T R O S C O P Y 395 C/" L I I I t 50 500 450 600 750 900 channel L i 150 300 450 600 750 900 chennel Fm. 12. S i g n ~ s a t v a r i o u s w a v e n u m b e r s , r e f l e c t i n g p r ~ e i n s t r u ~ u r M changes. 1572 and 1564 cm -1. These are due to changes in the amide I and amide II bands of the protein and, thus, reflect protein structural changes. DISCUSSION The data show that the implemented step-scan technique represents a promising method for time-resolved infrared absorption spectroscopy. Due to the small spectral changes, the example presented imposes special requirements on the performance and stability of the instrument. It is important to note that, for the conventional flash photolysis spectra, 300 to 1000 signals had to be accumulated per point, whereas for the step-scan technique only 16 signals per interferogram point were required. For a one-sided interferogram, the number of sampling points is the same as the number of spectral points. This clearly demonstrates the multiplex-advantage of the FT-IR technique in the case of the step-scan time-resolved measurement as well. The total measurement, requiring 10,240 flashes, lasts about 40 min. Of course, if the spectral changes are larger, the number of flashes and the total measuring time can be reduced. With the present interferometer, the time needed for the mirror to settle at the next position is about 50 ms; however, the time needed to signal to the computer that the position is "OK" is about 0.5 s. This requirement results in a minimum measuring time of 320 s for the same spectral parameters. With the same interferometer, timeresolved emission spectroscopy with a time resolution of 50 ns has recently been described. 27 Of course, the spectral changes involved are much larger in this case. Since the spectrum contains only positive values, the usual procedures for phase correction can be directly employed. Static BR568-L550 or BR568-M412 difference spectra can also be obtained at low temperature, stabilizing the respective intermediates. In this case, the usual continuous-scan technique is employed. To obtain a comparable signal-to-noise ratio, one must average at least 100 scans for each single-beam spectrum before and after the illumination. This circumstance appears even more surprising since the electronic bandwidth for the static measurements is only V~0of the bandwidth for the step-scan 396 Volume 45, Number 3, 1991 measurements. Thus, the step-scan technique is more effective in resolving small absorbance differences. This is probably due to the increase in the dynamic range. The flash-induced signal (i.e., the difference of the interferograms before and after the flash) is digitized with at least 8 bits, whereas the interferograms themselves in the continuous-scan measurements are digitized with only 16 bits. An 8-bit change in the interferogram digitized with 16 bits is equivalent to a change amounting to about 0.8 % of the centerburst intensity. A rough estimate shows that the small spectral changes evident from Fig. 8a (AA < 10 -2 absorbance units) will cause much smaller changes of the interferogram. In the continuous-scan measurements, the dynamic range is increased by increasing the number of averaged scans. In the continuous-scan technique, increasing the free spectral range is equivalent to increasing the electronic bandwidth (using the same mirror velocity). This does not increase the noise in the spectrum, since the greater noise in the interferogram, due to the larger bandwidth, is distributed over the larger spectral range. In the stepscan technique described here, the noise in the difference interferogram does not increase if the sampling points are spaced more narrowly (increasing the spectral range). Thus, the same noise amplitude of the interferogram is distributed over a larger spectral range in the difference spectrum, resulting in a reduced noise. Of course, the total measuring time is increased accordingly. If in the additional spectral range no useful spectral information is contained, this increase in sampling points is equivalent to increasing the number of averaged signals at each sampling point. Control measurements without the infrared beam have shown that the flash causes a small sudden rise in the temperature of the sample which decays within several 100/~s. This rise in temperature causes a signal at the detector with an amplitude of about 20 % of the signal shown in Fig. 6a. The emitted radiation is not modulated by the interferometer since the sample is located behind it; i.e., the signal is independent of the mirror position. Therefore, this signal will not cause spectral distortions. However, fluctuations in the laser pulse energy will lead to fluctuations of both the thermal and the interferogram signals. For our laser system, no systematic drift in the pulse energy during the course of the measurement was observed (10,240 flashes). Since the statistical fluctuations amount to about 10 %, we introduced a beamsplitter and detector which measured part of the total light energy integrated over the 16 flashes applied at each sampling point. As expected, the integrated light energy varies only by 2.5%. This fluctuation is less than the noise present in Fig. 6a. Much progress has been made recently in implementing time-resolved infrared spectroscopy with a monitoring beam provided by an infrared laser (CO laser or diode laser), s,v,4°,41 The high intensity of the lasers allows the detection of absorbance changes of 10 -3 at a bandwidth of 10 MHz with a single flash. Unfortunately, the CO laser has no lines below 1700 cm -1. The modern diode laser technology allows the manufacture of lasers tunable over a range of about 400 cm-L Sometimes, intensity fluctuations cause problems, especially if single modes have to be filtered out with a monochromator to reduce the bandwidth and to precisely define the wavelength. In addition, the tuning itself over a broad spectral range c a n b e t i m e c o n s u m i n g . N e v e r t h e l e s s , if s a m p l e s c a n n o t b e t r i g g e r e d v e r y f r e q u e n t l y o r if o n e is i n t e r e s t e d in o n l y a small spectral range, these techniques are very powerful. B u t if t h e s a m p l e a l l o w s m a n y f l a s h e s t o b e a p p l i e d a n d , e s p e c i a l l y , if o n e w a n t s t o c o r r e l a t e d i f f e r e n t p a r t s in t h e s p e c t r u m , t h e F T - I R t e c h n i q u e a p p e a r s t o b e sup e r i o r . 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