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
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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£
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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)
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High-pass filler
integrations-time
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time- resolved
Ol, O2,O3 controlled by
computer
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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
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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 ~
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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
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310
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140
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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 . W i t h t h e u s e o f a f a s t e r d e t e c t o r ( s u c h as a p h o tovoltaic MCT detector) and faster electronics, the timer e s o l u t i o n c a n e a s i l y b e e x t e n d e d i n t o t h e 10-ns r e g i o n .
S u c h i n v e s t i g a t i o n s a r e in p r o g r e s s .
ACKNOWLEDGMENTS
We are grateful to J. M. Weft, A. Simon, and J. Gast of the Bruker
Company for continuing technical advice. This work was supported by
the Deutsche Forschungsgemeinschaft, SFB 60, G-9, and by the Ministerium fiir Wissenschaft und Kunst Baden-Wiirttemberg.
1. Time-Resolved Spectroscopy, Advances in Spectroscopy, Vol. 18,
R. J. H. Clark and R. E. Hester, Eds. (John Wiley & Sons, Chichester, 1989).
2. Time-Resolved Vibrational Spectroscopy, Springer Proceedings in
Physics 4, A. Laubereau and M. Stockburger, Eds. (Springer-Vetlag, Berlin, 1985).
3. Time-Resolved Vibrational Spectroscopy, G. H. Atkinson, Ed. (John
Wiley & Sons, New York, 1983).
4. F. Siebert, W. M~intele, and W. Kreutz, Can. J. Spectrosc. 26, 119
(1981).
5. K. Gerwert, R. Rodriguez-Gonzalez,and F. Siebert, in Time-Resolved Vibrational Spectroscopy, SpringerProceedingsin Physics
4, A. Laubereauand M. Stockburger,Eds. (Springer-Verlag,Berlin,
1985), p. 263.
6. P. Glyn,M. W. George,P. M. Hodges, and J. J. Turner, J. Chem.
Soc., Chem. Commun.1989, 1655 (1989).
7. P. L. Bogdan and E. Weitz,J. Am. Chem. Soc. III, 3163 (1989).
8. P. A. Bottomley,Radiology170, 1 (1989).
9. H. Hermann,F.-W. Grevels,A. Henne,and K. Schaffner,J. Phys.
Chem. 86, 5151 (1982).
I0. J. D. Berkerle, M. P. Casassa, R. R. Cavanagh,E. J. Heilweil,and
J. C. Stephenson,J. Chem. Phys. 90, 4619 (1989).
11. W. Kaiser, R. J. Baeuerle, T. Elsaesser, H. J. Huebner, and A.
Seilmeier, in Ultrafast Phenomena 6, SpringerSeries in Chemical
Physics 48, T. Yajama,Ed. (Springer-Verlag,Berlin,1988), p. 452.
12. H. Graener, T. Q. Ye, R. Dohlus, and A. Laubereau, in Ultrafast
Phenomena 6, Springer Series in Chemical Physics, T. Yajama,
Ed. (Springer-Verlag, Berlin, 1988), p. 458.
13. E. J. Heilweil, Opt. Lett. 14, 551 (1989).
14. M. A. Young and G. C. Pimentel, Appl. Opt. 28, 4270 (1989).
15. K. Gerwert, Ber. Bunsenges. Phys. Chem. 92, 978 (1988).
16. M. S. Braiman, P. L. Ahl, and K. J. Rothschild, Proc. Natl. Acad.
Sci. USA 84, 5221 (1987).
17. A. W. Mantz, Appl. Opt. 17, 1347 (1978).
18. W.M. Grim III, J. A. Graham, R. M. Hammaker, and W. G. Fateley,
Int. Lab. 1984, 67 (1984).
19. J. J. Sloan, in Proceedings of the 7th International Conference on
Fourier Transform Infrared Spectroscopy, D. G. Cameron, Ed.,
Proc. SPIE 1145, 10 (1989).
20. F. Siebert and W. M~intele, Eur. J. Biochem. 130, 565 (1983).
21. M. Engelhard, K. Gerwert, B. Hess, W. Kreutz, and F. Siebert,
Biochemistry 24, 400 (1985).
22. H. Sakai and R. E. Murphy, Appl. Opt. 17, 1342 (1978).
23. W. Barowy and H. Sakai, Infrared Phys. 24, 251 (1984).
24. R. A. Palmer, C. J. Manning, J. A. Rzepiela, J. M. Widder, and J.
L. Chao, Appl. Spectrosc. 43, 193 (1989).
25. R. A. Palmer, C. J. Manning, J. A. Rzepiela, J. M. Widder, P. J.
Thomas, J. L. Chao, C. Marcott, and I. Noda, in Proceedings of
the 7th International Conference on Fourier Transform Infrared
Spectroscopy, D. G. Cameron, Ed., Proc. SPIE 1145, 277 (1989).
26. G. Hancock and D. E. Heard, Chem. Phys. Lett. 158, 167 (1989).
27. R. Rubinovitz, J. Seebode, and A. Simon, in Proceedings of the
7th International Conference on Fourier Transform Spectroscopy, D. G. Cameron, Ed., Proc. SPIE 1145, 528 (1989).
28. W. Stoeckenius and R. A. Bogomolni, Ann. Rev. Biochem. 51, 587
(1982).
29. D. Oesterhelt and J. Tittor, Trends Biochem. Sciences 14, 57 (1989).
30. P. R. Skar, Signal Conditioning Electronics for a Scanning Interferometer, MS Thesis, Massachusetts Institute of Technology,
Cambridge (1979).
31. L. Mertz, Infrared Phys. 7, 17 (1967).
32. J. Gronholz and W. Herres, Comp. Anw. Lab. 6, 418 (1984).
33. W. M~intele, F. Siebert, and W. Kreutz, in Methods in Enzymology,
Vol. 88, Biomembranes, L. Packer, Ed. (Academic Press, New York,
1982), p. 729.
34. F. Siebert, in Spectroscopy of Biological Molecules: Theory and
Applications--Chemistry, Physics, Biology, and Medicine, C.
Sandorfy and T. Theophanides, Eds. (D. Reidel Publishing Company, Dordrecht, 1984), p. 347.
35. F. Siebert, W. M/intele, and W. Kreutz, FEBS Lett. 141, 82 (1982).
36. M. Stockburger, W. Klusmann, H. Gattermann, G. Massig, and R.
Peters, Biochemistry 18, 4886 (1979).
37. K. J. Rothschild, P. Roepe, P. L. Ahl, T. N. Earnest, R. A. Bogomolni, S. K. Das Gupta, C. M. Mulliken, and J. Herzfeld, Proc.
Natl. Acad. Sci. USA 83, 347 (1986).
38. P. Roepe, P. L. Ahl, S. K. Das Gupta, J. Herzfeld, and K. J. Rothschild, Biochemistry 26, 6696 (1987).
39. S. P. A. Fodor, J. B. Ames, R. Gebhard, E. M. M. Van den Berg,
W. Stoeckenius, J. Lugtenburg, and R. A. Mathies, Biochemistry
27, 7097 {1988).
40. A. J. Dixon, P. Glyn, M. A. Healy, P. M. Hodges, T. Jenkins, M.
Poliakoff, and J. J. Turner, Spectrochim. Acta 44A, 1309 (1988).
41. A. Ansari, J. Berendzen, D. Braunstein, B. R. Cowen, H. Frauenfelder, M. K. Hong, I. E. T. Iben, J. B. Johnson, P. Ormos, T. B.
Sauke, R. Scholl, A. Schulte, P. J. Steinbach, J. Vittitow, and R.
D. Young, Bioph. Chem. 26, 337 (1987).
APPLIED SPECTROSCOPY
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