380
Anal. Chem. 1981. 53. 369-374 A
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samples are recorded in the T mode. Since analogue noise varies in proportion to the square root of the signal, the Z signal analogue noise may be considerably smaller than the digital resolution at all slit widths. This situation is illustrated in Figure 4. A portion of the spectrum of a blue glass filter is shown under three instrumental conditions. The ordinate has
been expanded lOOOX and the digital resolution steps are clearly evident. The wavelength interval within which noise causes the signal to dither between discrete counts is much smaller than the apparently noiseless intervals. Consequently averaging does little to improve accuracy. Neither the analogue noise nor digital resolution of the Io signal can be discerned in Figure 4 since these correspond to less than 0.1% of the T value. However, the Io signal changes with wavelength; hence the ADC counts of Io increase with decreasing wavelength. Transmittance then appears to change within the noise-free intervals. Now consider this same spectral region recorded in the A mode as shown in Figure 5. Some noise is detectable, but ADC steps cannot be identified. Averaging can be reliably used to improve readability. From eq 1the digital resolution is expected to be more than 200X better in Figure 5 than in Figure 4. LITERATURE CITED (1) Ingle, J. D., Jr.; Crouch, S. R. Anal. Chem. 1972, 44, 1375. (2) Kaye. W.; Barber, D.; Marasco, R. Anal. Chem. 1880, 52, 437A.
RECEIVED for review May 1,1980. Accepted October 10,1980.
Low Absorbance Spectrophotometry Wilbur Kaye Beckman Instruments, Inc., Imine, Callfornia 927 13
Recently there has been a resurgence of interest in lowabsorbance UV-VIS spectrophotometry stimulated largely by developments in optoacoustics (I). Any improvement in low-absorbance capability can be utilized in lowering analytical detection limits, improving analytical accuracy, reducing sample volume, and permitting the study of weak transitions. For many years the generally accepted lower limit of reliable absorbance measurements by conventional spectrophotometry has been 0.002 A (2). This absorbance level is considerably higher than the noise level specified for many high-quality spectrophotometers and warrants reassessment. The instrument used here was a modified Beckman DU-8capable of being operated with a noise of 9 X lo4 A RMS at 0 A (3). This rivals noise in laser lensing, optoacoustics, and fluorescence excitation techniques. However, a number of problems have had to be resolved before this excellent S/N could be converted into useful absorbance data. EXPERIMENTAL SECTION Apparatus. The spectrophotometerused here was a prototype Beckman DU-8 modified by replacing the side-windowR928HA photomultiplier and diffuser with an R375 end-window photomultiplier mounted within a Halon (Diano Corp., Woburn, MA) lined enclosure. The argument for use of such an optical integrator has been given elsewhere (4). The source for the spectra shown below was a 50-W tungsten-halogen lamp. It was totally enclosed in such a way as to minimize Schlieren (5). The monochromator consisted of a conventional Littrow mounted holographic system with fixed slits having spectral slit widths of 0.1,0.2,0.4, 1,2, and 4 nm. The measured half-bandwidth with the narrowest slit was 0.18 nm. The novel portion of the optical system allowing the accurate measurement of low-absorbance spectra is identified in Figure 1. The exit slit, S, is imaged 1 in. from the right-hand side of the sample compartment by transfer mirrors M1 and M2. The sample compartment is isolated from the remainder of the 0003-2700/81/0353-0369$01.00/0
Table I. Sources of Error at Low Absorbance 1. S/Nand drift 2. sample defocusing/displacement 3. internal reflections 4. cell scatter 5. sample scatter/Schlieren 6. hysteresis 7. tracking error purgeable optical train by windows W1 and W2. A large concave mirror M3 reimages the beam on the entry port of the optical integrator and detector D. Either an aperature at A may block all but the specularly transmitted rays or a flag at A may block these rays and permit 30' of the forward scattered rays to be detected. Alternately both transmitted and scattered rays may be detected. The size of the entry port of the optical integrator is dictated by the size of the beam with the widest slit after being defocused by a 10-cm cell filled with CSz, a material of high refractive index. It should be apparent that rays scattered from points nonconjugate to the integrator port will not be efficiently collected by mirror M3. A quantitative estimate of the collector efficiency is given in Fi@;ure2. This graph was obtained by placing a thin transluent Mylar film in the beam and measuring the detected signal as a function of the distance of the film from the W2 toward W1. The signal has been arbitrarily normalized on its maximum value. RESULTS AND DISCUSSION Many studies have addressed the numerous sources of error in spectrophotometry (6). Those factors which have a special significance for low-absorbance measurements are identified in Table I. These will be analyzed briefly and their solution will be illustrated with some demanding low-absorbance spectra. Noise and Drift. Noise in this instrument is a function of analogue (shot and Schlieren) noise, read average, and 0 1981 American Chemical Soclety
370
ANALYTICAL CHEMISTRY, VOL. 53,NO. 2, FEBRUARY 1981
Flgure 1. 100
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Simplified diagram of the DU-8 optics. I
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Detector response vs. position for a scattering film.
digital resolution. These factors have been addressed before (3,5). All previously described conditions for optimizing S/N and minimizing drift have been employed here. Sample Defocusing. When sample absorptivity is low it is usually best to use as long a sample path as possible. However, anything introduced into the beam alters beam focus on the detector and the longer the cell the worse the problem. Most detectors exhibit pronounced changes in sensitivity across the face of the photocathode; hence any change in the spatial distribution of light on the photocathode will cause a change in anode current. The beam may move to either a more or less sensitive area of the cathode. It is not uncommon for a 10-cm cell filled with a nonabsorbing liquid to appear to transmit over 100% for this reason. Frosted silica diffusers are most often employed to spread the beam over the photocathode and minimize spatial sensitivity. The more diffuse the scatter, the lower the spatial sensitivity, but at the expense of overall sensitivity. A compromise must always be made between scattering and transmitting properties of a diffuser. The efficiency of an optical integrator is determined by the port size and reflectivity of the cavity lining and typically exceeds that of a diffuser at the shortest wavelength. The spatial sensitivity can be measured by scanning the image of a narrow slit across the face of the detector, diffuser, or cavity port by means of the adjustment screws on mirror M3 (Figure 1). The change in sensitivity with the integrator is typically 2% of that with the diffuser. If the photocathode surface is normal to the beam(s), the sample and reference cells are of equal length, exhibit no wedge, and are filled with material of the same refractive index, the defocusing errors compensate. All instrument designs and sample circumstances do not meet these criteria. When measuring the optical properties of pure materials these criteria can seldom be met. One circumstance of particular concern occurs when measuring transmittance of glass photometry standards such as the NBS 930 filters. Cell Window Scatter. Any scatter of the beam out of the cone of detection will attenuate the beam. The cell windows are one source of scatter. It is very difficult to see the scatter from a well-cleaned cell under ordinary illumination; however, when directing an unfocused 1-mW HeNe laser beam through
such a cell the surface scatter is readily visible. The surface scatter must be attributed to residual polish marks that are ubiquitous to all cell windows. Actually it is possible to polish cells so that little scatter is detectable with laser illumination, but, to my knowledge, no such spectrophotometer cells have hitherto been made (7). Sample cells are often sold in “matched” pairs and the “matching” is largely for surface scatter. However, it is increasingly difficult to “match” cells as absorbance sensitivity increases. In any event it is of questionable value if the surfaces are not well cleaned. Most spectroscopists develope their favorite technique for cleaning cells, some good and some poor. A major difficulty is in ascertaining when one has done an adequate job. The scatter accessory for the DU-8 permits a quick test of cell scatter. Rotation of the flag into the beam causes the detector to respond only to the forward scattered rays which largely originate at the cell. A typical well-cleaned cell filled with filtered water will scatter about 0.1% of the incident rays into the 30° cone. Dirty cells may scatter 10-50 times more light. It is also possible in the DU-8 to use the same cell to hold solvent (blank) and solution (sample)and thereby compensate for cell scatter. No attempt is then made to clean the surfaces between the cell fillings. While this is of some inconvenience, it is the method of ultimate accuracy. Measurements of surface scatter must take into consideration the position of the scattering surface relative to the focus of the detector optics. The further the surface from the normal slit image the more defocused the surface image on the port of the optical integrator as seen in Figure 2. Sample Scatter. All liquid samples scatter light sufficiently to attenuate the beam in a DU-8 by a detectable amount, even when the liquid is perfectly clean. This scatter arises from thermally induced inhomogeneities on a molecular scale, It is possible to calculate the approximate scattering coefficient knowing the Raleigh factor, Re. If the scatter is isotropic the coefficient, as,is (8) To a first approximation this coefficient also varies with the fourth power of the wavelength. However, the scattering from foreign particles suspended in the sample cannot be treated by so simple an equation. The angular distribution of rays scattered from particles whose size is larger than the wavelength of light is complicated but is largely in the forward direction. It is not adequately appreciated how much scatter can occur from particles in supposedly clean liquids, particularly aqueous solutions. Even the particles in fresh triply distilled water can scatter considerablymore light than the water molecules themselves. It has been shown that only a momentary exposure of water to the atmosphere can result in detectable particle contamination (9). Fortunately, it is possible to filter most samples directly into the cell and remove the particles without affecting solute concentration. Disposable 0.2-1m pore size “Acrodisc” filters (Gelman Sciences, Ann Arbor, MI) were used for the aqueous samples in this study. Nonaqueous solvents usually pose less of a problem from foreign particles and may be filtered through Teflon filters if necessary. The scattering accessory of the DU-8 may be used to evaluate sample scatter but cannot easily descriminate between cell window scatter and sample scatter. Nor should it be assumed that one can predict the magnitude of beam attenuation caused by scatter from the signal obtained with the flag in the beam. The angular distribution of scatter from foreign particles is almost never known. The solid angle of rays detected by the unapertured DU-8 system is 0.84 steradians. This is only 6.7% of the total4r solid angle. However, the scatter from large particles and window polish marks is
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
concentrated in this forward cone. As an example it was found that approximately 30% of the light scattered by an imperfect polish on a 1-cm cell filled with filtered water was detected with the flag in the beam. Internal Reflections. Inspection of Figure 1 reveals a number of surfaces which may influence the detected signal by virtue of internal reflections. The surfaces usually discussed in studies on this subject are those between the sample cell and the windows of the sample compartment. In the DU-8 the windows on the sample compartment are tilted sufficiently so these internally reflected rays are not detected. Another less avoidable reflection problem occurs between the external surfaces of the sample cell (or filter). These two surfaces are almost always plane and normal to the beam. If the material in the cell does not absorb, these reflections increase the signal by about 0.16%. In low-absorbance studies of solutions, both reference and sample signah will be similarly affected and the error will be compensated. It is seldom compensated when studying neat liquids or filters. In high-absorbance measurements the sample partially absorbs the internally reflected rays introducing a small nonlinearity into the Lambert-Beer relationship. In thin samples with well-polished flat and parallel surfaces, this type of internal reflection can be effectively amplified and complicated by interference. A more troublesome internal reflection occurs between the diffuser or integrator and the cell windows. This problem is somewhat worse when using an optical integrator than a diffuser because more light scatters backward from the integrator. In the DU-8, the magnitude of this error is a function of the detector acceptance angle and the distance between the cell surfaces and the slit image in the sample compartment. When a 1-cm cell is located in the normal position and the full 30” detection cone is employed with the optical integrator, the doubly reflected signal amounts to slightly more than 0.5%. It is often possible to move the cell suficiently away from the slit focus to reduce this error to about 0.01%. As with other types of reflections this error is compensated when studying solutions. Hysteresis. Photomultiplier hysteresis has been shown to influence photometric linearity (5). This nonlinearity is negligible over the low-absorbance region of interest here, but hysteresis must be taken into consideration for the best results. This hysteresis is manifested as an overshoot or undershoot of anode current when abruptly changing the light level on the photocathode. Such an abrupt change occurs during a “gain-set” operation in the DU-8 and results in a slow equilibration of the signal. The error is within the specifications for the instrument but is needlessly large by the low-absorbance standards set here. The problem can be surmounted by renormalizing with the “auto-zero” operation after hysteresis has equilibrated (usually within 15 s). Sample Schlieren. Thermally induced refractive index gradients within the sample can displace the beam on the detector and introduce an apparent signal drift. This problem is worse with the diffuser than the integrator. The evaporative cooling accompanying the act of filling a cell with a volatile sample can result in significant Schlieren for periods up to 15 min. Fortunately a bubble within the cell can easily be manipulated to stir and equilibrate the sample. 100% Adjust. This control is common to most doublebeam spectrophotometers, but is absent on this instrument. It is usually intended to compensate for mismatch of beams and cells. If properly set with solvent in both beams, the control has a valid function. However, there is a dangerous temptation to set this control either with no cells in the beams or with solvent and solution in separate cells. The “auto-zero” and “run” controls on the DU-8 do normalize the instrument and a wide range of scale expansions and suppressions are available, but the plotted ordinate always identifies these
371
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Figure 3. Vapor-phase spectrum of iodine.
factors. This removes the temptation to insert unknown “correction factors” into the spectra. Vapor-Phase Spectra. From a photometric viewpoint vapor-phase spectra are the simplest to obtain. Reference and sample scans may use the same cell, and window scatter, defocusing,and internal reflections are compensated. Gases do not usually contain significant levels of scattering particles. Of course the bandwidths of many absorption bands in gases are narrower than the instrument bandwidth (resolution); hence there is a premium on the use of the narrowest possible slits resulting in less than optimum S/N. Figure 3 shows the spectrum of iodine vapor in air at 25 “C. A pathlength of only 1 mm has been selected in order to pose a challenge to the instrument. The transmittance mode was used to minimize digitization error. Analogue noise was high enough to permit averaging, and a “read average” of 7 was employed. A scan speed of 4 nm/min proved adequate for low tracking error. The background (Io)varied slowly with wavelength, hence could be stored at a speed of 10 nm/min. The wavelength interval was 40 nm. Consequently the background scan required 4 min and the sample scans 10 min. Two scans are shown; one with and one without iodine in the cell. Peak-to-peak noise is about 0.025% (0.oOOl A) and drift is negligible. Solute Spectra. The majority of spectra fall in this class. The instrument is usually normalized with a solvent-filled cell in the beam followed by a scan of the solution in the same or a “matched” cell. Surface scatter, sample defocusing,and internal reflections should compensate if absorbance is low. Filtration should eliminate sample scatter. Accuracy then need be limited only by S/N and drift. These limiting factors are usually determined by the wavelength interval to be covered and the required resolution. A particularly difficult situation is illustrated with the interesting spectra of europium solutions. The valence shell of 4f electrons responsible for absorption is not the outermost shell; hence the absorption bands are not broadly perturbed by neighboring atoms. Many of the absorption bands are remarkably narrow. The absorption band arising from transitions from the lowest ground state, 7Fo,to the lowest excited state, 5Do, is only about 0.12-nm half-bandwidth. However, selection rules forbid this transition and it is detectable only when the europium ion exists in a perturbing field produced by neighboring ions and atoms (10).These properties make the Eu3+ion a useful probe of molecular environment. Unfortunately the band is so weak it has been considered “out of the question” to study by conventional absorption techniques (11). Special techniques such as fluorescence excitation (12) and photoacoustic spectroscopy (13)have been employed. Furthermore, fluorescence is strongly quenched by OH vi-
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
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Spectrum of 0.2 M Eu3+ in 3 M NO3-.
brations, and deuterated solvents had to be used. No difficulty was encountered in studying these bands with the DU-8. Figure 4 shows the spectrum of a 0.2 M E u ( N O ~solution )~ in a 1-cm cell. For preparation of the solution, the oxide was dissolved in excess nitric acid and the solution was filtered into the cell. A solution of equivalent (3 M) NO3- concentration was placed in a “matched” cell and used as the blank. Because of the narrow bandwidth of the 578-nm (7Fo- 5Do) band, the narrowest slit width was used along with a read average of 7. A scan speed of 2 nm/min was required to achieve an acceptable tracking error. The background spectrum varied slowly with wavelength; hence it was possible to scan it a t a speed of 10 nm/min. The three scans shown (background normalization scans are never plotted) required a total of 26.4 min, yet there is little evidence of drift. The absorbance is 0.00353 and the molar absorptivity is 0.0177 cm-’ M-1. Resolution of this instrument at a spectral slit width of 0.1 nm was measured with a low-pressure mercury lamp and found to be 0.18 nm (half-bandwidth). The observed bandwidth of the 578-nm band is seen to be about 0.30 nm; hence the sample bandwidth must be about 0.12 nm. This implies that the true peak intensity must be higher than that shown. The ratio of the peak intensity of the narrow 578-nm band to the broader 591-nm band should be a sensitive measure of instrument resolution. In fact this spectrum provides a good test of instrument performance for S/N, resolution, tracking error, and wavelength accuracy. Solutions of Eu3+in other anions and solvents have been studied. For example, perchlorate solutions have been studied without finding any evidence of the 578nm band (11). Figure 5 shows the spectrum of 0.2 M Eu(C104), in 10% excess perchlorate. A 10-cm cell was used easing the S/N problem. There is now no difficulty in using the absorbance mode. Splitting of the broad 591-nm band is quite evident. There is only a slight trace of the 578nm band. To study this band better, the transmittance mode was used with 200X scale expansion and the same cell was used for reference and sample scans. The results are seen in Figure 6. Refilling the cell increased the time between scans increasing the possibility of drift. Two solvent scans were run along with the solution scan. Drift appears to be about equal to noise and corresponds to about 0.00005 A . There appears to be an absorption or scatter continuum amounting to 0.0008 A under the 589-nm band and the peak band absorbance is O.OO0 86 A above this. Band absorptivity is 0.00043 cm-l M-I or 16 times lower than the apparent limit of the previously published work (12).
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Spectrum of 0.2 M EIJ(CIO,)~ 0.2 M Eu (ClO,)3 PATH lOcm SLIT 0.2 nm
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Expanded scale spectrum of 0.2 M Eu(CIO,),.
The possibility always exists that the cell windows might have been contaminated between solvent and solution fillings or that the filter did not adequately remove scattering particles. The flag was inserted into the beam and absorbance readings were taken to monitor this possibility. On conversion to transmittance, the scatter (above that with no cell in the beam) was 0.069% when filled with the blank and 0.014% with the Eu3+solution. It then seems unlikely that the continuum absorption can be attributed to foreign particles or window contamination. Spectrum of Neat Materials. The final and most difficult type of absorption spectrum to be discussed here is that of a neat material. In this case it is not always possible to completely compensate for cell scatter, defocusing, or internal reflections. The best that can be done is use two cells of widely different pathlength. This compensates for the large reflection losses at the two air-silica and two sample-silica interfaces. The best way to minimize cell scatter effects is to “super” polish the cell windows. Defocusing is likely to introduce serious errors unless a good optical integrator is used. Internal reflection problems can be minimized in this instrument by mounting cells far from the slit image in the sample compartment and inserting the detector aperture in the beam. Unfortunatelythere is insufficient room to place both windows of a 10-cm cell far enough from the slit image to render internal reflections negligible. Recently there has been a flurry of interest in the higher harmonics of the CH stretching vibration of benzene by long path absorption (14, 15), thermal lensing (16-18), laser wavelength modulation (19), and photoacoustic (20-24) spectroscopy. The sixth and higher harmonics occur in the
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
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Table 11. Band Centers and Peak Absorption Coefficients of the Sixth, Seventh, and Eighth Harmonics of the C-H Stretching Vibrations in Benzene h (6), nm LY (6), cm-I h (7), nm LY (7), cm" h ( 8 ) , nm CY (\8), cm-I ref 605 607 608.1 606.7 609.8 607.3 607.0
17 x 10-4 21 6.0 7.2 20 22 22.3
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531.6 531.0
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15 16 17 20 475.3 475
19
9 . 2 x 10-5 9.8
24
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99.2
99.45
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REFERENCE 1 mm C&
SLIT 0.5 nm
REFERENCE 1 mm C6H6
1 0
0.0088
0.0044
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Fbure 7. Spectrum of the sixth ham-tonic of the CH stretching vibratbn in benzene.
visible region and are quite weak. Not surprisingly the reported absorption coefficients differ appreciably as summarized in Table 11. It should be noted that the absorption coefficient a, used here is given by the equation
I = Ioe-auL and differs from absorptivity by the factor 2.3026. It is not clear that this factor has been fully recognized in all the above papers. Also the reported values refer only to the absorption not the scattering coefficient. The thermal lensing and optoacoustic methods ostensibly ignore the scattering component. The absorption methods assume the absorption coefficient to be given by the attenuation above a base line drawn between shoulders of descrete bands. In this case the defocusing, cell scatter, drift, and internal reflection effects influence the scattering and not the absorption coefficient. The sample cell chosen for the benzene spectrum was a 10-cm cell with specially polished windows. Examination of the filled cell with the HeNe laser revealed little scatter from the windows but a uniform scatter from the benzene. Very few scattering particles could be seen in the benzene. The reference cell used here was of 1-mm pathlength and was filled with benzene. Thus, the net sample path was 9.9 cm. Unfortunately the windows for this cell were of standard polish. This resulted in a small overcompensation in the transmittance spectrum (i.e., T could be expected to read slightly high). Figure 7 shows the spectrum of the sixth harmonic band of benzene. The bandwidth is sufficiently wide not to require narrow slits and S/N is very good. The uncertainty of the absorption coefficient arises almost entirely in the estimation of the base-line scatter. Measurement of the seventh and eighth harmonics of the CH vibration in benzene presents more of a challenge. Figure 8 shows this portion of the spectrum. The scale expansion is large (200X) and there is an offset. Otherwise the same conditions of Figure 7 prevail. There is an apparent rapid
I
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increase in the scattering coefficient with decreasing wavelength. It is not known how much of this continuum attenuation arises from scattering, shoulders of the electronic a b sorption in benzene, or absorption from impurities. It has been observed that the slope of the continuum attenuation varies considerably with sample purity. Large differences occur between reagent, HPLC, and Ultrex (multipass fractional freezing by J. T. Baker, Chemical Co., Phillipsburg, NJ)grades of benzene. The Ultrex grade is shown here. An estimation of the scattering coefficient can be obtained from eq 1 and the Rayleigh factor for benzene (25). The scattering coefficient at 632.8 nm is found to be 2.12 x cm-*. Transmittance through a 10-cm cell at this wavelength should then be at most 99.788%. That it appears greater than this in Figure 7 can be attributed to the aforementioned errors (although some error in the use of eq 1 should be acknowledged since benzene does not scatter isotropically). If one uses the fourth power of wavelength relationship, a scattering coefficient of 6.68 X lo4 could be expected at the wavelength of the eighth harmonic. The minimum transmittance through a 10-cm cell at this wavelength should be 99.334%. The extrapolated value from Figure 8 is 99.46%. Repeated measurements of the eighth harmonic indicated an uncertainty of less than 20% of the observed coefficient or 2 X lod cm-'. In terms of absorptivity this is 7 x lo+ cm-', considerably below the previously cited state of the art. LITERATURE CITED (1) (2) (3) (4)
Hunter, T. F. Nature(London) 1979, 280, 357. Cetorelll, J. J.; Wlnefordner. J. D. Talanta 1967, 74, 705. Kaye, W.; Barber, D. Anal. Chem., preceding paper In this Issue. Eckerle, K. L.; Venable, W. H.. Jr.; WeMner, V. R. Appl. Opt. 1976,
(5) (6) (7) (8)
Kaye, W.; Barber. D.; Marasco, R. Anal. Chem. 1980, 52, 437A. Reule. A. G. J. Res. NaN. Bur. Stand., Sect. A 1976, 80A, 809. Kaye, W. Anal. Chem. 1973, 45, 221A. Fabellnskli, I. L. "Molecular Scattering of Light"; Pknum Press: New
75, 703.
York, 1966; p 38. (9) Kaye. W. J. ColbM Interface Scl. 1974, 46, 543. (IO) Sayre, E. V.; Mlller, D. 0.;Freed, S. J. Chem. phys. 1957. 26, 109,
374
Anal. Chem. 1901, 53, 374-377
(11) Honocks, W. D., Jr.; Sudnlck, D. R. Sclence 1979, 206, 1194. (12) Haw, Y.; Stein, G. J . Phys. Chem. 1971, 75, 3868. (13) Sawada, T.; Oda, S.; Shlmlzu, H.;Kamada, H. Anel. Chern. 1979, 57, 888. (14) Stone, J. Appl. Phys. Lett. 1975, 26, 183. (15) Stone, J. Appl. Opt. 1978, 77, 2876. (18) Long, M. E.;Swofford, R. L.; Albrecht. A. C. Sclencs 1976, 797. 183. (17) Swofford, R. L.; Long, M. E.; Albrecht, A. C. J. Chem. Phys. 1976, 65. 179. BU~~M~ M.Y s.; , w e l l , J. A.; Albrecht, A. C.; Swofford, R. L. J. Chem. Phys. 1979, 70, 5522.
Moses, E. I.; Tang, C. L. Opt. Lett. 1979, 7, 115. Tam, A. C.; Patel, C. K. N.; Kerl, R. J. Opt. Lett. 1979, 4 , 81. Patel, C. K. N.; Tam, A. C. Appl. phys. Lett. 1979, 34, 467. Patel, C. K. Cn.; Tam, A. C. Chem. Phys. Lett. 1979. 62, 511. Patel, C. K. N.; Tam, A. C.; Kerl, R. J. J. Chem. Phys. 1979, 77, 1470. (24) Tam, A. C.; Patel. C. K. N. Opt. Lett. 1980, 5 , 27. (25) Kaye, W.; McDaniel, J. B. Appl. Opt. 1974, 13, 1934. (19) (20) (21) (22) (23)
Received for review May 1,1980. Accepted October 10,1980.
Gel Chromatography for the Isolation of Phenolic Acids from Tobacco Leaf M. E. Snook,' P. J. Fortson, and 0. T. Chortyk Tobacco Safety Research Unit, Sclence and Education Administratbn/Agricuitural Research, United States Department of Agriculture, P.O. Box 5677, Athens, Georgia 30613
Phenolic acids are known to occur widely in plants. Generally, paper or thin-layer chromatography is used for its isolation and identification in plant extracts (1-3). However, individual acids are difficult to quantitate by these methods, and only the major components can be determined. Our recent Sephadex LH-20 gel chromatographic work on the isolation of dihydroxybenzenes of cigarette smoke ( 4 ) suggested to us the applicability of this procedure to the isolation of leaf phenolic acids. Our interest in leaf phenolic acids arises from their possible role as precursors of the tumorigenic tobacco smoke catechols. Since phenolic acids are similar in polarity to dihydroxybenzenes (catechols, resorcinols, hydroquinones), they should be adsorbed and separated by the gel in a similar manner. To our knowledge, the application of gel chromatography to the isolation of plant phenolic acids has not been previously reported. EXPERIMENTAL SECTION Extraction of Tobacco Leaf. NC 2326 flue-cured tobacco was ground to pass a 32-mesh screen. The tobacco (200 g dry wt) was ground with 2.5 L of 1.0 N NaOH for 5 min in a Waring blendor. The resulting slurry was filtered through no. 2 Whatman filter paper by vacuum, and the residue was washed with 500 mL of HzO. The total filtrate was filtered again and extracted with 600 mL of ethyl acetate (EtOAc). The aqueous solution was acidified to pH 1.0 (6 N HCl), saturated with NaCl, and extracted with EtOAc (3 X 600 mL). The EtOAc solution was dried over anhydrous MgSO,, filtered, and evaporated to yield 6.9 g of a leaf acids extract (3.4% yield). The acids extract was dissolved in 25 mL of MeOH/CHC13 (l:l,v/v) in preparation for gel chromatography. Gel Chromatography, The gel column was a 1.25-cm i.d. X 55cm LC-type column (LaboratoryData Control, Riviera Beach, FL), packed with Sephadex LH-20 in CHC13. Solvent flow was 2 mL/min and 5-mL fractions were collected. One-milliliter aliquots of acids extract were introduced on the column with a loop injection valve. For the standard phenolic acids, the initial CHC13 solvent was programmed from CHC13 to 10% MeOH/ CHC13from gel fractions 40 to 55 and subsequently held at 10% MeOH/CHC13. The solvent program for the tobacco acids extract was similar to that for standards, but to hasten elution of the dihydroxy aromatic acids, the solvent was programmed from 10 to 40% MeOH/CHC13 from gel fractions 110 to 115. Eluted materials were monitored at 280 nm, and gel fractions (GF) were pooled to yield fraction A (GF 65-84) and fraction B (GF 85-122). Gas Chromatography (GC). The pooled fractions A and B were concentrated under house vacuum on a rotary evaporator to a small volume (about 250 pL), Aliquota of fractions A and B were added to an equal volume of N,O-bis(trimethylsily1)trifluoroacetamide (BSTFA) in microreaction vials fitted with Teflon-lined caps. The trimethylsilyl (Me3Si)derivatives were prepared by heating the mixtures at 86 "C for 15 min. The derivatives were analyzed with a Hewlett-Packard 5830 gas
chromatograph,equipped with a 183-cm x 2-mm i.d. g h column packed with 6% OV-17 on 100/120 mesh Chromosorb G-HP (temperature program, 90-250 OC at 2 "C/min; He flow, 20 mL/min; injector, 290 "C; flame detector, 300 "C). Compounds were identified by comparison of GC retention times to those of standards, by coinjection with standards, and by GC-mass spectral (MS) data. MS data on the Me3Si derivatives were obtained on a 5930A Hewlett-Packard mass spectrometer, interfaced to an HP 5710 gas chromatograph equipped with an identical OV-17 column. W-Recovery Studies. Sali~ylic-~~C acid (ICN Pharmaceuacid (California ticals, Inc., Irvine, CA) and p-hydroxyben~oic-~~C Bionuclear Corp., Sun Valley, CA) were purified by silicic acid column chromatography(100-gcolumn of Mallinckrodt,100-mesh silicic acid). The columns were eluted successively with 1L each of 4% diethyl ether (E)/petroleum ether (PE), 10% E/PE, and 50% E/PE. The 50% E/PE fractions were evaporated and dissolved in 1 L of MeOH. Aliquota of the purified labeled compounds were diluted 1:l with CHC13and chromatographed on the gel column. Recovery of the 14C acids was measured by standard liquid scintillationcounting techniques. Aliquota (5 mL, 100OOO dpm) of each standard were added to additional NaOH extracts of tobacco. The mixture was acidified and extracted with EtOAc. The extract was subjected to gel chromatography, and the GF were counted. RESULTS AND DISCUSSION Sephadex LH-20 gel with chloroform as solvent will separate compounds on the basis of hydrogen bonding effects. Phenolic materials are strongly adsorbed by the gel and require the addition of MeOH in order to be eluted in a reasonable time. In contrast, hydrocarbons, ketones, aldehydes, alcohols, and amines are not retained and are eluted with the void volume of the column or shortly thereafter. This property of LH-20 is ideally suited to the separation and purification of phenolic materials from complex mixtures. The elution characteristics of several phenolic acids from a Sephadex LH-20 column are shown in Figure 1. Acids with internal hydrogen bonding (such as sinapic, ferulic, salicylic, and vanillic) were eluted earlier than hydroxyphenylacetic, coumaric, and monohydroxybenzoic acids. Dihydroxybenzoic acids were eluted much later. Interestingly, cinnamic acids were eluted before the benzoic acids. Other compounds, containing two or more hydroxyl groups, were also found to elute in the same GF as the phenolic acids. These included dihydroxybenzaldehydes and both dicarboxylic and tricarboxylic aliphatic acids. The gel elution characteristics of the dihydroxybenzoicacids were investigated further and are shown in Figure 2. The observed elution order of the dihydroxybenzoic acids appears to depend strongly on steric, electronic, and internal hydrogen bonding factors. I t was also noted that, except for the 2,6-
This artlcle not subject to U S . Copyrlght. Publlshed 1981 by the Amerlcan Chemlcal Society
