-
bi
0*2[~(Xi) +~(XO)]
(A-6)
Note that since ai is in units of cm in this appendix (to be consistent with the usual units of b ) , the values of bi that are expressed here are for use with particle sizes in cm. The analyst may wish t o divide such values of bi by lo4 in order to use them with particle sizes expressed in pm (as was done for Table I and for Equation 4 in the text). The values of bi tabulated in the paper were not obtained by the approximation A-5, but by the following method. Equation A-1 was used to calculate the ratio Si/Soi for particle sizes ranging from 1 to 500 pm. Then, the value of bi was found that best satisfied the approximation
(A-7) for that set of calculated values of SilSoi vs. ai. A convenient method for performing such a fit is to re-write the approximation as
I t is generally found that this value of bi varies slowly with ai, so that it is not important how one chooses the "best" value of bi from Equation A-8. For generating Table I, the value of bi was chosen that fit best when the value of Si/SoL was in the range 0.25-0.5, which corresponds to a particle correction, (1 biai)', in the range 2-4. I t was decided that errors in the choice of bi would not be very serious when the correction factor was much less than 2, because the effect of such errors would be small (Figure 4). Also, errors in the choice of bi for use when the correction factor was greater than 4 should not be much more disturbing than the errors due to other causes for such large corrections (Figures 3 and 4). Most of the values of bi in Table I are given to only two significant digits; in many cases, there is an uncertainty of 1t o 3 in the second digit, because the empirical correction does not match the detailed calculations exactly. The two x-ray tube spectra used t o generate Table I are probably fair approximations for most commonly-used Crand W-target x-ray tubes operated in the range of 40-50
+
kV (constant potential). Noticeable differences in calculated values of bi for long-wavelength lines could result from tube and experimental characteristics that affected the long-wavelength portion of the x-ray spectrum striking the sample (9, 1 5 ) . Relevant characteristics include the x-ray take-off angle from the tube, the tube-window thickness, the presence of a filter, and the length of any air or helium path from the tube to the sample.
ACKNOWLEDGMENT The author is grateful to L. S. Birks and J. V. Gilfrich, of the Naval Research Laboratory, and to J. Wagman, of the Environmental Protection Agency, Research Triangle Park, N.C., for many useful discussions during the preparation of this paper.
LITERATURE CITED (1) L. S.Birks, J. V. Giifrich, and P. G. Burkhalter, "Development of X-Ray Fluorescence Spectroscopy for Elemental Analysis of Particulate Matter in the Atmosphere and in Source Emissions", Envlron. Prot. Agency (US.) Rep., EPA-R2-72-063, November 1972. (2) J. V. Gilfrich, P. G. Burkhaiter. and L. S.Birks, Anal. Chem., 45, 2002 (1973). (3) P. G. Burkhaiter, "Trace Metal Water Pollutants Determined by X-Ray Fluorescence", Nav. Res. Lab. Rep., 7637, Aug. 1973. (4) J. R. Rhodes and C. B. Hunter, X-Ray Spectrom., 1, 113 (1972). (5) C. B. Hunter and J. R. Rhodes, X-Ray Spectrom., 1, 107 (1972). (6) T. G. Dzubay and R. 0. Nelson, Adv. X-Ray Anal., 18, 619 (1975). (7) J. W. Criss, unpublished work on x-ray absorption and fluorescence in particles of various shapes and orientations, Naval Research Laboratory, Washington, D.C. 20375. (8) L. S. Birks, "X-Ray Spectrochemical Analysis", 2nd ed., Wiiey Interscience, New York, N.Y., 1969. (9) J. V. Gilfrich and L. S.Birks, Anal. Chem., 40, 1077 (1968). (10) J. W. Criss and L. S.Birks, Anal. Chem., 40, 1080 (1968). (11) W. H. McMaster et al., "Compilation of X-Ray Cross Sections", Lawrence Radiation Laboratory, Livermore, Calif., USRL-50174, Sec. II, Rev. 1. 1969. (12) L. S.Birks. "Electron Probe Microanalysis", 2nd ed., Wiley interscience, New York, N.Y., 1971. (13) L. Siiverman, C. E. Billings, and M. W. First, "Particle Size Analysis in industrial Hygiene". Academic Press, New York. N.Y., 1971. (14) J. V. Giifrich et ai., Anal. Chem., 43, 934 (1971). (15) D. B. Brown et ai., J. Appl. Phys., 46, 4537 (1975).
RECEIVEDfor review June 9, 1975. Accepted October 10, 1975. The calculations reported here were supported in part by the Environmental Protection Agency under Interagency Agreement D5-0344.
Vidicon Detection of Resonance Raman Spectra: Cytochrome c William H. Woodruff* and George H. Atkinson* Department of Chemistry, Syracuse University, Syracuse, N. Y. 732 10
The resonance Raman spectrum of the heme protein cytochrome c (6.0 X M) is detected using continuous laser excitation, a silicon intensified-target vidicon tube, and a spectrograph with a holographically ruled diffraction grating (HRDG). Image intensification is required to attain acceptable vidicon sensitivity, and the HRDG is required to eliminate grating artifacts. Vidicon dark current is the overwhelming source of noise in the present study. Noise from this source can be virtually eliminated either by cooling the detector or by using short-pulse laser excitation. In the latter case, time-resolved resonance Raman (TR3) spectros186
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
copy can be contemplated. The results suggest that TR3 experiments can be performed with laser energies (singiepulse or total, muitipie-pulse) of approximately 100 mJ.
There has been much recent interest in the use of multichannel detection systems in spectrometry (1-3 and references therein). At present, the most popular multichannel detector is the silicon vidicon, the analytical applications of which were pioneered by Pardue, Margerum, and coworkers ( 4 - 7 ) . As commercial vidicon detection systems have
DOUBLE MONOCHROMATOR
I
s3 PHOTOMULTIPLIER TUBE
u
1
TUBE
------OMS Is'
S I - s 3 ,S L I T S
GI , G 2
-/
MI - M S . MIRRORS
VIDICON SPECTROGRAPH
CONVENTIONALLY RULED DIFFRACTION GRATINGS ( 1 2 0 0 G R O O V E / m m BAUSCH AND L O M B )
M3
PATH SI
-.-
I I"$
' -
45. SW ING A WAY
x
I I
n
OUT
.-
G l i k - ~
I
, CONVENTIONALLY RULED GRATING
PATH
HOLOQRAPH ICALLY RULED GRATING (PLANE)
Figure 1. Schematic diagram of apparatus used in the present study.
Spex 1401 spectrometer used ( a ) in its normal, double monochromator configuration and (b)as a vidicon spectrograph. In the latter configuration, grating G1 is replaced by a 1800 groove/mm Jobin-Yvon holographically ruled diffraction grating become available, additional applications of these devices have' emerged (8-10). We now report the first use of vidicon techniques to detect resonance-enhanced Raman spectra of dilute species in solution. Our interest in vidicon detection of Raman spectra is a result of the intrinsic ability of a vidicon system to detect transient spectral phenomena while preserving the multiplex advantage of a spectrograph. T h e importance of these features t o Raman spectroscopy was first demonstrated by Delhaye ( I I ) , who, using image converters and a T V camera, obtained spectra of neat liquids and gas mixtures in the millisecond time regime. Delhaye pointed out the potential of this method for time-resolved Raman spectroscopy of reacting chemical systems. However, the feeble nature of normal Raman scattering has generally prevented this potential from being realized. Recently, it has been shown that for systems with accessible electronic transitions, the resonance Raman (RR) effect can enhance t h e intensity of Raman scattering by many orders of magnitude (12) and references therein). Thus, a combination of resonance-enhanced Raman scattering and sensitive vidicon detection techniques can reasonably be expected t o fulfill the experimental requirements for obtaining Raman spectra of dilute, short-lived chemical species. In order t o establish these experimental conditions, we have examined the RR spectrum of cytochrome c, in aqueM concentration range. Cytoous solution in the chrome c is a small heme protein (mol wt = 12400) which functions as an electron-transport entity in the respiratory redox chains of many organisms (13). We have chosen cytochrome c as the test system for vidicon detection of resonance Raman scattering because its RR spectrum is well known (12), because it is commercially available in high purity, and because of the similarity of the chromophore of cytochrome c t o those of more complex members of t h e respiratory redox chain. In the present study, both conventional (double monochromator/photomultiplier) and vidicon (silicon intensified-target tube) detection schemes were used in companion experiments to obtain estimates of 1) t h e laser energy, 2) t h e optical components, and 3) t h e detector sensitivities required t o perform time-resolved resonance Raman (TR3) spectroscopy.
EXPERIMENTAL The concepts involved in vidicon spectrometry have been dis-
cussed in detail previously (1-4, 6-10) and will not be repeated here except for the technical problems which are unique to Raman spectroscopy. Likewise, the experimental considerations which apply to conventional (i,e., photomultiplier-detected) Raman spectroscopy are well established (14). Apparatus. The spectrometer used in this study was a Spex 1401 double monochromator (Czerny-Turner, 0.85m, f/7.8)with straight, bilateral slits (Spex Industries, Metuchen, N.J.). The fore-optics were of the Nestor design ( 1 5 ) , Suprasil throughout, with a f / l objective lens having 2.5-inch focal length. The photomultiplier tube used for the conventionally scanned Raman spectra was a Centronix Q4283S-25 having s-20 response (Bailey Instruments, Saddle Brook, N.J.). The photomultiplier was maintained at -30 OC using a thermoelectrically cooled housing (Pacific Photometric Instruments, Emeryville, Calif.). The standard gratings in the spectrometer were 1200 groove/mm, 5000-8, blaze, conventionally ruled diffraction gratings (CRDG's) manufactured by Bausch and Lomb and supplied by Spex. For the vidicon experiments, grating G1 (See Figure la) was replaced by a 1800 groove/ mm holographically ruled diffraction grating (HRDG) manufactured by Jobin-Yvon and also supplied by Spex. The vidicon detection system was a Princeton Applied Research "Optical Multichannel Analyzer" (OMA) console (Model 1205A, Princeton Applied Research Corporation, Princeton, N.J.) with a Model 1205D silicon intensified-target (SIT) vidicon detector head having S-20 photocathode response. The OMA head was mounted, using a fabricated adapter, in the focal plane of the single-spectrometer slit exit of the Spex 1401 (See Figure l b ) . Spectra were recorded on a Heath stripchart recorder for the conventionallydetected spectra or on a Hewlett-Packard X-Y plotter for the vidicon-detected spectra. The excitation source for these experiments was a Spectra-Physics model 166-03 argon ion laser (SpectraPhysics Corporation, Mountain View, Calif.). Reagents. Cytochrome c was'from horse heart, Sigma Type VI (Sigma Chemical Company, St. Louis, Mo.). Solutions of cytochrome c were made up 6.0 X M by direct weighing of the protein, and were reduced to ferrocytochrome c with a small excess of solid sodium dithionite. Procedure. Raman spectra were obtained by transverse excitation with the sample contained in 1-mm i.d. Kimax melting-point capillary tubes. Continuous-wave excitation at 5145 8, (19436 cm-') was used, with a laser power at the sample of 100 mW. The special procedures which must be followed in obtaining resonance Raman spectra of heme proteins are discussed elsewhere (16, 17). The differences between the spectrometer configurations necessary for conventional and vidicon-detected Raman spectroscopy are shown in Figure 1. T o change from the double monochromator configuration (Figure la) to the vidicon spectrograph (Figure lb), it is necessary to rotate the 45O folding mirror out of the light path and to replace the normal 1200 groove/mm CRDG a t G 1 with the 1800 groove/mm HRDG (the vidicon head may remain in place in either configuration). Both types of gratings are supplied on the same type of kinematic mount, and are therefore interchangeable. ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
187
CYTOCHROME EXCITATION
2. ( C O N V E N T I O N A L
RESULTS AND DISCUSSION
SPECTRUM)
C
19436 CM-1, 5 0 J (100mW. 5 O O S E C I
F
A conventionally-scanned, photomultiplier-detected resonance Raman spectrum of a 6.0 X M aqueous solution of iron(I1)-cytochrome c (ferrocytochrome c ) is shown in Figure 2. The peaks observed in this spectrum are due exclusively to resonance-enhanced vibrations of the heme chromophore, since the normal (nonresonance-enhanced) Raman scattering due to the polypeptide protein backbone is far too weak to be observed a t this concentration. This spectrum was taken using the apparatus shown in Figure l a . Figure 3 shows a vidicon-detected spectrum of the same solution using identical excitation conditions, except that the spectrometer configuration is that shown in Figure I b . The spectrum in Figure 3 is a composite of two separate vidicon spectra. This is necessary because the dispersion of the 1800 groove/mm HRDG is such that only a 275 cm-’ segment of the Raman spectrum will fit on the 12.5-mm vidicon screen. I t can be seen from a comparison of Figures 2 and 3 that all of the features of the cytochrome c spectrum observed using photomultiplier detection are also seen in the vidicon-detected spectrum and that the spatial and relative intensity relationships of these features are preserved. The primary experimental challenge in Raman spectroscopy is the detection of relatively few photons a t the Raman-shifted frequencies in the presence of very high light levels a t the excitation laser frequency. In conventional (photomultiplier-detected) Raman spectroscopy, the stray light a t the laser frequency is generally attenuated by using multiple dispersing elements (e.g., double or triple monochromators), but this is clearly impossible in the case of a spectrograph, which can have only a single dispersing element. With a grating spectrograph, however, grating artifacts (“ghosts”) can frustrate attempts to detect Raman scattering. These artifacts are a consequence of systematic errors in the mechanical ruling of conventional diffraction gratings (CRDG), and of high light levels a t the laser frequency. We find that when one attempts to detect weak Raman signals with the vidicon spectrograph (Figure l b ) , the spectra are dominated by grating ghosts if a CRDG is used. T o the limits of our ability to observe, this problem is entirely eliminated if the CRDG is replaced by a holographically ruled diffraction grating (HRDG). (The difference in groove density between the CRDG and the HRDG in our experiments is due to the ready availability of the 1800 groove/mm HRDG, and also to serious polarization anomalies in the currently available 1200 groove/mm HRDG.) The basic silicon vidicon tube is not sensitive enough to
S P E C T R O M E T E R S P E X 1401 U S E 0 4 5 DOUBLE MONOCHROMATOR DETECTOR PHOTOMULTIPLIER TUBE I C E N T R O N I X 042835-251, DC A M P L I F I C A T I O N G R A T I N G S > T W O 1200 G R O O V E l r n m CRDG S ( 0 8 L l
J U 1200
I
1
I
I
I300
1400
I so0
1600
A”,CM”
Flgure 2. Resonance Raman spectrum of 6.0 X M ferrocytochrome c obtained using conventional scanning and photomultiplier detection with the spectrometer configuration shown in Figure l a
Scan rate 1 cm-’/sec, slit width 6 cm-’. dc amplification of PMT signal, senA full scale, PMT voltage 1200 V The details of the operation of the PAR OMA system have been adequately described by previous workers (10). We have obtained Raman spectra of neat solvents (viz., benzene and carbon tetrachloride) in the “real-time” mode of the OMA (32.8 msec vidicon screen scan time), but the Raman scattering of cytochrome c is too weak to be observed in real time with the laser energy used in the present study (approximately 3.3 mJ per vidicon scan). Therefore, the OMA was operated in an accumulation mode in which the total signal obtained over a large number of 32.8-msec scans was accumulated and stored in one memory buffer of the OMA. Accumulation continued until either a preset number of scans was reached or the signal in any one of the 500 channels of the OMA reached its overflow limit of IO5 counts. The overflow limit was normally reached in 1600 to 1700 scans. The accumulation mode has the effect of increasing signal-to-noise ratio (S/N)in propbrtion to the square root of the number of scans accumulated (10) (assuming a constant signal level). The dark current background of the vidicon detector was accumulated for the same number of scans as used for the signal and stored in the second memory buffer of the OMA. The two buffers were then subtracted to yield the Raman spectrum. The noise in this difference spectrum is due primarily to statistical fluctuation in the cancellation of the two large background signals. An important comparison concerning the signal vs. dark current should be noted. The dark current can be accumulated either for a preset number of scans (normally chosen to be the same number of scans used to obtain the signal) or until the overflow limit is reached. If the latter mode is used, the dark current accumulation proceeds for approximately ten scan cycles more than obtained when signal plus dark current is accumulated. This demonstrates that, for the present study (1600-1700 accumulation cycles full scale), the signal due to real light in the spectrometer is only about of the dark current. This has important consequences for potential S/N improvements in these experiments (vide infra). sitivity 3 X
CYTOCHROME
C
(VIDICON SPECTRUM)
EXCITATION
19436 C M - ’ , 4 8 J 1100 mW. 48 S E C ) SPECTROMETER S P E X 1401 U S E D A S V m SPECTROGRAPH DETECTOR S I T VlDlCONlPAR O Y A l 3ZrnSEC S C I N S GR4TlHG
1800 G R O O V E l m m HRDG I J - Y l
I\ C
E
A
0
I
1
1200
Flgure 3. Resonance Raman spectrum of 6.0 X
1300
I 1.00 A~,CM-I
I I500
M ferrocytochrome c obtained using the vidicon spectrograph shown in Figure Ib
The spectrum is a composite of two 275 cm-’ segments (see text).Details of the experiment are discussed in t h e text 188
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
detect Raman scattering, and a t least one stage of image intensification is necessary to attain the required sensitivity. This finding is in accord with the earlier results of Delhaye (11). In the present study, the commercially available silicon intensified-target OMA detector was found satisfactory. Our objective is to apply vidicon detection techniques to time-resolved resonance Raman (TR3) experiments. We wish to use the results of the present study t o predict the S/N which may be expected in TR3 experiments. This is possible because our S/N arguments are based upon total laser energy considerations, and are independent of whether the actual experiment employs CW laser excitation (as in this report), or is time-resolved using either single-pulse or repetitive-pulse laser excitation. The S/N of the spectrum in Figure 3 is low when compared with that in Figure 2. However, as was pointed out above, the overwhelming source of the vidicon noise is undoubtedly statistical fluctuation in the dark current component of the total vidicon signal. The dark current in the present case is 150 or more times greater than the vidicon signal due to real light, and the statistical noise due to dark current is proportional to the square root of the number of scans accumulated (IO). Thus elimination of vidicon dark current would automatically increase the S/N of the spectrum in Figure 3 by a factor of 39, assuming that the signal remained the same. Effective elimination of vidicon dark current can be accomplished either by cooling the detector substantially (to -40 OC or below), or by drastically reducing the vidicon exposure time required by increasing the intensity of the Raman scattering. For the case of a cooled detector, an experiment otherwise identical to the one which yielded the spectrum in Figure 3 would produce a spectrum with S/N comparable to that of Figure 2. Such an experiment offers a substantial advantage over photomultiplier detection in terms of acquisition time for comparable results (48 sec vs. 500 sec in the present case). The latter (reduced accumulation time) approach requires decreasing the number of vidicon scans accumulated while increasing the per-scan laser excitation energy in proportion. In the single-scan limit, 4.8 J of laser energy would be required in 32.8 msec to produce the total Raman signal detected in Figure 3. In principle, this would again result in an improvement in S/N of a factor of 39. In practice, such an experiment is impractical because of the nonexistence of a suitable laser and probable sample decomposition. However, single-scan operation of the vidicon detector can allow substantially lower laser energy to be employed, if a reduction in S/N can be tolerated. Specifically, if the S/N in Figure 3 is considered as acceptable, then the single-scan laser energy required becomes 4.8/39 = 0.12 J. This level of energy is well within the single-pulse capability of commercially available lasers, and can, in fact, be produced a t an appropriate wavelength (5300 A) in 10-20 nsec pulses. Thus the present data
suggest that TR3 experiments on dilute solutions of heme proteins (or other dilute species having comparable Raman scattering intensity) are feasible in the nanosecond time regime, using either single-pulse techniques or repetitive pulses combined with cooled vidicon detection. Based upon the present results, we conclude that the following are requirements for successful single-pulse TR3 spectroscopy of dilute transient species: 1) a laser delivering a t least 100 mJ of energy per pulse a t the appropriate wavelength and time resolution, 2) a spectrometer based upon a HRDG, and 3) a vidicon detector with a t least one stage of image intensification. Experiments to test those conclusions are in progress in this laboratory. NOTE ADDED IN PROOF. Since the acceptance of this manuscript for publication, another report of vidicon detection of a resonance Raman spectrum has appeared (R. Wilbrant, P. Pagsberg, K. B. Hansen, and C. V. Weisberg, Chem. Phys. Lett., 36, 76 (1975)). The authors employed single-pulse laser excitation, and their procedures and results are in accord with our predictions for time-resolved resonance Raman spectroscopy.
ACKNOWLEDGMENT The authors are grateful to Lou Casper of Spex Industries for arranging the loan of the HRDG, and to Richard Pastor and William Davis for their assistance.
LITERATURE CITED K. W. Busch and G. H. Morrison, Anal. Chem., 45, 712A (1973). Y. Talmi, Anal. Chem., 47, 658A (1975). Y. Talmi, Anal. Chem., 47, 697A (1975). R. E. Santini, M. J. Milano, H. L. Pardue, and D. W. Margerum. Anal. Chem., 44, 826 (1972). W. H. Woodruff, D. W. Margerum, M. J. Miiano, H. L. Pardue. and R . E. Santini, Inorg. Chem., 12, 1490 (1973). M. J. Milano, H. L. Pardue, T. E. Cook, R. E. Santini, D. W. Margerum, and J. M. T. Raycheba, Anal. Chem., 46,374 (1974). M. J. Miiano and H. L. Pardue, Anal. Chem., 47, 25 (1975). K. W. Jackson, K. M. Aldous, and D. G. Mitchell, Spectrosc. Lett., 6, 315 (1973). D. 0. Knapp, N. Omenetto, L. P. Hart, F. W. Plankey, and J. D. Winefordner, Anal. Chlm. Acta, 69, 455 (1974). K. W. Busch, N. G. Howell, and G. H. Morrison, Anal. Chem., 46, 575 (1974). M. Delhaye in "Molecular Spectroscopy," The Institute of Petroleum, London, 1968, p 275. T. G. Spiro, Acc. Chem. Res., 7, 339 (1974). L. E. Bennett in "Current Research Topics in Bioinorganic Chemistry", Progress in inorganic Chemistry, Vol. 18, S.J. Lippard, Ed., John Wiley and Sons, New York, 1973, pp 1-176. M. C. Tobin, "Laser Raman Spectroscopy", Chemical Analysis, Vol. 35, P. J. Elving and I. M. Kolthoff, Ed., Wiley-lnterscience, New York, 1971. J. R. Nestor, Princeton University, Princeton, N.J., personal communication. 1974. T. G. Spiro and T. C. Strekas, J. Am. Chem. Soc., 96, 338 (1974). T. C. Strekas, D. H. Adams, A. Packer, and T. G. Spiro, Appl. Specfrosc., 28, 324 (1974).
RECEIVEDfor review September 18, 1975. Accepted October 6, 1975. This research was supported by National Science Foundation Grant MPS75-10156 and by separate Research Corporation Cottrell Grants to WHW and to GHA.
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