Anal. Chem. 1984,
intensity of a particular component is usually much greater than that of other components in the samples being analyzed, the conditions can be chosen to optimize the measurement of the weaker component.
Table IV. Summary of Measurement Conditions for Determinations Shown in Table 111"
determination
hex,
nm
PRFS 1 1 2 3 1 2 3
nm
381 453 417
360 360 360
381 453 417
360 360 360
417 381 453
360 360 360
381 453 417
360 360 360
381 417 453
360 360 360
381 417 429
360 360 360
381 417 453
PRFS 3 1 2 3
Aem
360 360 360
PRFS 2
PRFS 4 1 2 3
PRFS 5 1 2 3 ss 1 1 2 3 ss 2 1 2
3
2199
56,2199-2204
CONCLUSIONS The work described here demonstrates the improved accuracy that can be obtained when wavelength selectivity is combined with fluorescence lifetime selectivity. When PRFS is used, this improvement can be achieved without increasing the number of measurements that must be taken or the complexity of data analysis. The only differences are that phase-resolution equipment is used, detector phase angles must be adjusted in addition to wavelengths, and the molar fluorescence intensities used in the simultaneous equations are phase-resolved rather than steady-state values. In all other respects the PRFS and the steady-state procedures are identical. Although this work has involved the use of three independent measurements for determination of three unknowns, the precision of the determinations could be improved by the use of more measurement condition sets to generate more equations for overdetermination of the components. For components with extensive spectral overlap, the use of selectivity based on fluorescence lifetimes is especially attractive, and the PRFS approach offers a methodologically convenient means for implementing this selectivity. Registry No. POPOP, 3073-87-8; Me2POPOP,1806-34-4; anthracene, 120-12-7. LITERATURE CITED Veselova, T. V.; Cherkasov, A. S.; Shirokov, V. I . Opt. Spectrosc. (Eng/. Trans/.) 1970, 29, 617-618. Lakowicz, J. R.; Cherek, H. J . Blochem. Biophys. Methods 1981, 5 , 19-35. Birks, J. B.; Dyson. D. J. J . Sci. Instrum. 1961, 38, 282-285. Spencer, R. D.; Weber, G. Ann. N. Y . Acad. Sci. 1969, 158, 381-376. Lakowicz, J. R.; Cherek, H. J . Blol. Chem. 1981, 256, 6348-6353. McGown, L. B. Anal. Chlm. Acta 1984, 157, 327-332. Lakowicz, J. R.; Cherek, H.; Baiter, A. J. Blochem. Blophys. Methods 1981, 5 , 131-146. Ware, W. R.; Baidwin, B. A. J . Chem. Phys. 1964, 4 0 , 1703-1705.
Each of the three independent sets of conditions are given for each phase-resolved (PRFS) and steady-state (SS) determination.
(SS l ) , whereas all of the best PRFS results are obtained by using the emission maxima for anthracene and POPOP and the wavelength (453 nm) at which the relative contribution of anthracene is low compared to POPOP and Me2POPOP. For a particular multicomponent system, the optimal combinations of wavelengths and detector phase angles must be determined experimentally and will be dictated by the particular requirements of the analysis. For example, if the
RECEIVED for review April 2, 1984. Accepted June 6, 1984.
Versatile, Efficient Raman Sampling with Fiber Optics S c o t t D. Schwab and Richard L. McCreery*
Department of Chemistry, T h e Ohio State University, Columbus, Ohio 43210 A flber optic Raman probe is described in whlch both the exclting laser ilght and the collected Raman scattering are conducted by optlcal fibers. The technique requires no alignment of sample wlth Input beam or collection optlcs, and the sample may be a great dlstance from the spectrometer or In a hostlle environment If desired. Theoretical caiculatlons demonstrate what factors in probe design determine the collection efficiency and the sampling depth of the probe. Dependlng on configuratlon, the Raman signal from the flber probe was from 1 to 9 tlmes as large as that from a conventional llqukl sampilng system. I n addttlon to hlgh collection efflclency the flber probe does not employ a focused beam, so the lncldent power denslty at the sample Is as low as one four-hundredth that of a conventional focused system. Appllcatlons to liquids, solids, low-temperature samples, and electrochemically generated species are described.
Table I. Transmission of Ensign-Bickford HC-212-T Fibers as a Function of Laser Wavelength laser A, nm
Y,O dB/m
514.5 496.0 488.0 476.5 457.9
0.018 0.014 0.014 0.020
0.021
y is defined as (10/b) log (Zo/Zt) where I,, is intensity incident on a length b of fiber and Zt is the transmitted intensity. y is dea
termined from a ratio of transmissions through fibers of different length and is independent of coupling losses.
A variety of applications of optical fibers to spectroscopic problems have been described, with particular emphasis on UV-vis absorption and fluorescence techniques ( I ) . Fiber-
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ANALYTICAL CHEMISTRY. VOL. 56. NO. 12. OCTOBER 1984
mgwa I. opacal amngemmt tor fiber optk -man probe. L, is a 5cmwlenglhLnspavldedwimmespexLmhator~,wNch also m s ~ e l p l i c amlector. l Th, sample is carmined h a m vial Into v h k h the probe is inserted. d is the sample depth used In all cakuiatians.
based sampling devices allow the sample to be remote from the spectrometer, perhaps in a hostile environment, and the fiber probe can be made very small for use in vivo (2). While the development of fiber optic probes for Wyis spectrometry has been s u e d , these techniques provide little structural information and are not highly selective. While infrared absorption spectrometry can provide structural information, it is not amenable to fiber optic probes because of poor transmiasion of infrared light hy g h or plastic fibers. Since Raman scattering spectrometry normally uses visible light which is efficiently transmitted by optical fibers, it can provide vibrational information about the sample yet still be easily coupled to a fiber optic probe. Fiber optics have been used to collect spontaneous Raman from a sample (3),and hollow fibers have been used to hold samples for both spontaneous Raman ( 4 ) and coherent anti-Stokes Flaman spectrometry (CARS) (5). CARS generated in a tlame has been transmitted away from the hostile environment to a remote spectrometer using fibers optin (ti), and a fiber optic illumination device was developed for samples which were sensitive to a focused laser beam (7). In an earlier paper, we reported a fiber optic probe for Raman spectrometry in which fibers both carried the laser light to the sample and collected the scattered signal (8). Unlike previous fiber systems for Raman, ours requires no alignment of sample with probe or laser beam, and the sample may he remote from both laser and spectrometer. The present paper describes the theoretical basis of the fiber probe's performance and several improvements in design which lead to greatly increased collection efficiency. Finally, several applications of the fiber probe will be described.
EXPERIMENTAL SECTION The fiber optic probe was designed to mate directly to a Spex 1403 Spectrometerwith a Model 1459 sample illuminator system. The general optical arrangement is shown in Figure 1, with one fiber uurying the laser light to the sample and 18 collecting the Raman light. The 18 collection fibers were arranged in a vertical line at the spectrometer and placed at the position normally occupied by a sample capillary. The elliptical collector of the spectrometer imaged the slit-shaped output of the collection fibrs
mt
Flpv 2. Ratcndmcqaph ot the pobe end. -led by canid by the Rbers. Tim canter fiber is me inpm fiber wmch c a v b s the lase# light lo sample. The entire array ammeter 1s 1.0 mm.
on the entrance slit of the monochromator. The output of a Coherent 90-5 argon ion laser was pagsed through the Spex premonochromator and foeused onto the single input fiber using the spectrometer's illumination optics. Both the input and collection fibers were mounted on a kinematic mount similar to t h e provided by Spex for acceaaories. The entire fiber awemhly muld be replaced hy a conventional sampling system in a matter of seconds. The input fiber was mounted on a small X-Ystage (Newport FP-I) to allow po8itioning of the fiber with respect to the input beam. The collection fibers were terminated at the spectrometer with a &axis mount (Newport FP-2) to allow optimization of the Raman signal. The 18 collection fibers and single input fiber were made from Ensign-Bickford HC-212-T 200 rm core diameter silica fibers which have a numerical apenure of 0.4. The cladding consists of a 15 rm thick layer of a hard propietmy fluorocarbon coating which is chemically more resistant than silicone rubber and allows closer parking of the fibers ink? a bundle. Unlike the prohe reported prenously (8). the cladding remains on the fihers, and the refractive index of materials in contact with the cladding exterior does not affect transmission efficiency. The fibers are sold with an additional 200 r m thick protective coating which was removed from the fiber at both the sample and spectrometer ends. The input fiber wan cut with a tungsten csrhide knife and mounted in the Newport X.Y stageat the spectrometer. The 18 collection fibers were mounted in a 6-mm metal tube with Torr-Seal (Varian). arranged in a slit as described above. The total lenglh of fiber from spectrometer to probe was about 2 m in this work. but could be much longer ifdepired. The probe itself was comtructed by d i n g the 19 fibers,less the protpetive costing but with the cladding, into a 1.1 mm i.d. glass capillary with Ton-Seal. The capillary was then mounted in a larger glass tube for easier handling. but this outer tube could he customized for a particular application. The probe was sanded to a flat surface with 600 grit silicon carbide paper and then polished with 5-um and U.05-ern alumina. A photomicrograph of the end of the finished probe IS shown in Fipure 2. Approximately 75% of the 488-nm laser light incident on the input fiber was transmitted to the sample, with most of the loss occurring during coupling. The light logq in the fibers is about 0.5% per meter. but this value is wavelennh dependent. as will be discussed helow. Torr-Seal was tested for chemical compatibility with several liquids which might be encountered in use of the probe. No harmful effects of the solvents on the probe were observed after
ANALYTICAL CHEMISTRY, VOL. 56, NO. 12, OCTOBER 1984
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a
I
t
0
efficiency. The half angles of the input and collection cones are determined by the appropriate numerical apertures.
several days of immersion in water, acetone, benzene, pentane, acetonitrile, or methanol, but some degradation of the Torr-Seal was observed after 2 days in dimethylformamide. Spectral slit widths were set as noted in figure legends, and spectra were obtained and plotted with the Spex Datamate system. With the exception of the low-temperatureCC1, spectrum, scans consisted of 0.1 or 0.3 s integration periods at fixed wavenumber separated by 1cm-' monochromator steps. Typical average scan rates were in the range of 2-5 cm-l/s. For the high-resolutionCC14spectrum, monochromatorsteps of 0.05 cm-' were used and the integration time was 0.5 s.
0.4
0.2
Flgure 3. Variables involved in the theoretical simulation of collection
0.6
D (mm)
Flgure 4. Theoretical figure of merit, as defined in the text as S for a single collection fiber plotted as a function of the distance between the radii of the Input and collection fibers. R , = R, = 200 pm, n = 0.075, and n , = 0.15.
5P
.30
THEORY A theoretical examination of the fiber probe was undertaken in order to understand the experimental factors affecting collection efficiency and sampling depth. The laser light will define a cone as it enters the sample, with the half angle determined by the coupling optics and the numerical aperture of the fiber. Similarly, there is a cone at each collection fiber from which the scattered light reaches the spectrometer; this cone is determined by the numerical aperture of the collection fiber or by the spectrometer f / number. The overlap between the input and collection cones is the region of solution from which the scattered light is sampled. The geometry of the problem is shown in Figure 3 for a single cQllection fiber. For some point x , y, z in the input cone, the laser irradiance (W/cm2) will be given by eq 1, assuming the light distribution
PO
Io = TR? across the fiber cross section is homogeneous and no absorption occurs in the solution. Po is the laser power at the end of the input fiber and Ri is the radius of the input cone a t the distance z from the fiber end. Ri is a function of z and the numerical aperture of the exit cone from the input fiber. The Raman intensity, lR,in W/sr from a volume element dx dy dz at a point x , y, z is given by (2), where p is the differP,,~Nax dy az IR = (2)
TR?
entia1 Raman scattering cross section in cm2molecule-l sr-l, assumed to be constant over the range of angles monitored, and N is the number density of scatterers. Finally, the Raman power, P,,incident on the collection fiber is given by eq 3, where R2 is the radius of the collection (3)
0 1
2 SAMPLE
3 DEPTH
4
5
(CM)
Flgure 5. Theoretical value of T for the fiber bundle as a function of sample depth and numerical aperture of the collection fibers. Sample depth is the distance d through a liquid sample between the end of the probe and the wall of the cell. n , = 0.075 and R , = R, = 200 pm. Numbers on theoretical curves are collection fiber numerical apertures. Points are experimental data fit to the theoretical curves as described in the text. The experimental points were obtained from the 2944-cm-l band of acetonitrile with an input of 30 mW at the sample.
fiber and a is the distance from x , y, z to the collection fiber face. Note that r R Z 2 / a 2is the solid angle of the fiber in steradians. We will define the figure of merit, S, as shown in (4), in which the scattered light for all points in the overlap
region of the two cones is integrated. S is independent of scattering cross section or concentration and incorporates the collection efficiency and the volume of the sample region. The integral of (4) was approximated by a summation carried out with an Apple I1 for various combinations of R1,R2, n l , and n2,the numerical apertures of the fibers, and D, the distance between fiber centers. nl and n2 are the sines of the half angles of the respective cones. The total figure of merit for a probe with 18 collection fibers, T , is the sum of 6s for the inner ring of fibers (a= 230 pm) plus 1 2 s for the outer ring (D= 450 pm). Figure 4 is a plot of S as a function of D, all else being
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 12, OCTOBER 1984
W
'
1
2 SOLUTION
4
3 DEPTH
1
5
(cml
Figure 6. Theoretical signal as a function of input and collection numerical apertures. n = n for all curves.
,
equal, and demonstrates the importance of thin cladding on the fibers. Figure 5 demonstrates the dependence of T on sample depth (d in Figure 1) for various values of collection numerical aperture. Clearly the best collection fiber is one with large n2 and thin cladding. As will be shown below, the n2 value is usually limited by the spectrometer rather than the collection fiber. Figure 5 also shows that the effective sampling depth of the probe into the solution is on the order of several millimeters. For example, for nl = 0.075 and n2 = 0.15,90% of the possible signal is collected from a region of solution within 15 mm of the probe end. The signal increased very slightly beyond 5 cm, with a further increase of 3.5% from 5 to 25 cm. Figure 6 shows the effect of increasing both nl and n2,indicating that large numerical apertures (wider cones) yield larger signal and shallower sampling depth. For equal nland n2,90% of the signal is achieved at 22,15,9, and 7 mm for nl = 0.1, 0.2, 0.3, and 0.4, respectively. For the purpose of comparison, the corresponding figure of merit for a conventional sampling system, defined in the same way as S or T above, but with Poand P, equal to the input and collected powers, is equal to dF, where d is the length of the sampled region along the beam axis and F is the solid angle of the collection optics in steradians. For typical values (d = 0.1 cm, F = 0.25 sr), the figure of merit of a conventional sampling system in 0.025. Since the polarization of the laser light diminishes during transmission over even short distances in a multimode fiber, both the illumination and collection steps will be done with essentially unpolarized light. Furthermore, the fiber probe employs a backscatter geometry, differing from the more common 90" arrangement. By examination of the polarizability tensor, it can be shown that for symmetric vibrations, the backscattered signal from unpolarized light is equal to that for polarized light using 90" geometry and pardel polarization. For asymmetric vibrations, the same result is true if the polarizability tensor is symmetric about its diagonal, as is usually the case. In more physical terms, the backscattered spectrum from unpolarized light is the same as a 90" arrangement with parallel observation because backscattered observation is not sensitive to rotation of the induced dipole relative to the input polarization. Therefore one predicts that the spectrum obtained with the fiber probe (backscattered mode with unpolarized light) will have the same relative intensities as a conventional (90" observation) spectrum obtained using parallel polarization from polarized input light.
RESULTS AND DISCUSSION For liquid samples, the probe end is merely immersed in the liquid, with no regard to positioning except the distance
from the probe end to the bottom of the sample cell. Except for the capillary arrangement discussed below, the sides of the sample vial did not interfere with the expansion of the input beam. Once the fibers are interfaced to the spectrometer by tuning for maximum signal on a Raman line from a known sample, no further adjustment is necessary, and use of the probe is trivial. For short lengths (1-3 m), of the fibers used here, the numerical aperture of the beam existing the probe, nl, will be determined by the coupling optics of the laser beam into the fiber. With the standard Spex optics, nl is 0.1, a value which is reduced when the beam enters a sample with refractive index greater than 1.0. In acetonitrile (n = 1.342), for example, the exit numerical aperture becomes 0.1/1.342 = 0.075. With the probe immersed in acetonitrile, the Raman intensity was measured as a function of the distance between the probe end and a glass wall (d in Figure I),the result shown in Figure 5. Since /3 plus several instrumental variables (e.g., monochromator throughput, PMT sensitivity, etc.) are not known, an absolute comparison of the magnitudes of theoretical and experimental results cannot be made, but their depth profiles can be compared. By variation of the input parameters for the theory described earlier, a good fit between theory and experiment was obtained for n2 = 0.15. The fit was obtained by normalizing the experimental data to match theory at a sample depth of 5 cm and then adjusting n2 to obtain the best fit of the entire curve. This value is the collection fiber numerical aperture at the sample, so it corresponds to a value of 0.15 X 1.34 or 0.20 in air at the spectrometer. Note that this value is less than the 0.4 numerical aperture of the fiber, indicating that the spectrometer rather than the fiber is limiting collection efficiency. An independent estimate of the numerical aperture of the spectrometer was made by placing masks between the fiber output and the collection optics; this approximate value was 0.20-0.22. If nl is varied by changing the coupling optics, the depth profile has the shape predicted by theory with the total experimental Raman signal for n, = 0.3 being 49% that for n, = 0.075. The corresponding value from theory is 47% (n2= 0.15 in all cases). A comparison of spectra from the fiber probe and the conventional Spex capillary arrangement is shown in Figure 7 , with the laser powers at the sample being equal for the two cases (30 mW). The Spex system uses a focused beam with a 10-pm beam waist and a power density of 4 X lo4 W/cm2, whereas the fiber optic power density is always less than 100 W/cm2. The fiber probe has approximately (f20%) the same signal magnitude as the usual Spex system with no base line shift or distortion of the spectrum. A factor of 9 or more improvement in signal may be achieved by directing the probe output down the bore of a 1.1 mm i.d. capillary containing the sample liquid. Internal reflection constrains the input beam within the capillary and directs the Raman back to the collection fibers. For example, the 2944-cm-' band for acetonitrile had a peak count rate of 1.75 X lo5s-l for 4 mW input power (at the sample) into a 70-pL sample, an improvement of a factor of 9 over the spectrum in Figure 7 after a correction is made for the input power difference. Once the viability of the fiber probe was established with liquid samples, a variety of spectra were obtained to assess its versatility. The resonance Raman spectrum of electrogenated chlorpromazine cation radical shown in Figure 8 was obtained by inserting the probe into a tubular electrode (1.8 i.d.). A normal Raman spectrum of reduced chlorpromazine precursor obtained at higher concentration is shown for comparison. Spectra of the radical generated inside the electrode were easily obtained, with detectable Raman intensities being observed within 200 ms after electrolysis began. While this time response is slower than that reported for
ANALYTICAL CHEMISTRY, VOL. 56, NO. 12, OCTOBER 1984
i
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20 KHZ
T
100 AV
3300
1700 cm-l
,
Flgure 7. Raman spectra for neat acetonitrlle obtained with the fiber probe (upper)and Spex caplllary system with elliptical mirror collector and focused input beam (lower). Thirty milliwatts of 488-nm laser light reached the sample in both cases. Except for sampling mode, all of the conditions for both spectra, including the 3 cm-' band-pass, were identical.
0
$ X Y
A V . cm-'
Flgure 9. Spectrum of frozen acetonitrile at 77 K. The probe was immersed in liquid as usual and then the entire probe and sample vial were immersed in liquid nitrogen. There was 40 mW of 488-nm light at the sample, band-pass = 1 cm-'. helium or liquid nitrogen, the Raman line width was 0.8 cm-l, and the five isotope peaks for the 459-cm-l band were base line resolved with an instrumental band-pass of 0.4 cm-l. The upper limit for temperature will be determined by the epoxy used to bind the fiber probe together. With a different epoxy or binding compound, it is likely that temperatures well above those used here could be achieved. Since the transmission of the fibers varies with wavelength, there is a possibility of distortion of the relative intensities of peaks, particularly for long fibers. To assess the severity of this error, the transmission of the fibers was measured for the argon ion laser lines by comparing the light transmission of a 1-m fiber with that of one 25 m long. The results are shown in Table I, expressed as y in db/m such that the transmitted intensity will decrease by 1-104~1~ per meter of fiber. The distortion introduced in the relative intensities of Raman bands by variations in transmission can be predicted from the data in Table I to be about 10% for a 70-m collection fiber and 457.9-nm excitation. The loss in Raman light over this length would be 20-28%. For the 1-3 m lengths normally used in a laboratory, the wavelength dependence of transmission is negligible.
I
1000
1350
A V ,
1700
cm-1
Flgure 8. Resonance Raman spectrum of chlorpromazine radical cation generated at 0.8 V vs. SCE from a solutlon of 30 mM CPZ in 1 M HCI (upper spectrum). Lower spectrum is normal Raman scattering from 0.2 M CPZ in 1 M HCI. In both cases,514.5-nm excitatlon was used with 5 cm-' band-pass, and the fiber probe was inserted Into a 1.8 mm i.d. tubular electrode. nonfiber experiments (9),the limitation is electronic and not related to the fiber probe. Spectra of powders were obtained by either placing the probe near the powder surface or immersing it in the powder. Pellets were examined by placing the probe near the surface at an angle of 45O. For solid samples, the high degree of scattered laser light caused a silica background from Raman generation within the fibers. The silica background could be corrected by subtraction of a spectrum obtained by reflecting a portion of the laser light back into the collection fibers. Since the fiber probe is remote from the spectrometer, the temperature of the sample could easily be varied over at least a 4 K to 373 K range. Spectra of tRNA in solution were obtained as a function of temperature from 20 OC to 80 O C , using the fiber/capillary cell immersed in a water bath, with the junction between probe end and capillary being sealed by shrinkable Teflon tubing. The spectrum of frozen acetonitrile shown in Figure 9 was obtained a t 77 K by immersing the probe in a vial of acetonitrile and then immersing the entire assembly in liquid nitrogen. Equally good spectra were obtained at 4 K when the assembly was immersed in liquid helium. For a carbon tetrachloride sample in either liquid
CONCLUSIONS The spectra shown demonstrate the applicability of fiber optic Raman probes to a variety of samples. Spectra obtained with the probe have the same appearance and relative intensities as those obtained using 90' geometry with parallel polarization. The high collection efficiency, ease of use, and remote sampling aspects of the probe make it an attractive alternative to conventional Raman sampling arrangements. The Raman signal from the probe presented here is comparable to conventional methods and can be made 9 times more sensitive by coupling a capillary to the fiber probe. In addition, the unfocused beam from the fiber probe has a power density one four-hundredth that of the usual Spex arrangement, great decreasing the likelihood of photolytic damage. Possible drawbacks of the fiber probe include the potential interference from the silica background when examining highly scattering samples and the loss of polarization information within the fibers. For very long fiber lengths, some distortion of the spectrum may occur due to variations in transmission with wavelength. The most likely applications of the fiber optic Raman probe described here will be for routine or remote sampling of liquids or for situations where the sample is in a difficult to access or hostile environment. A valuable aspect of the probe is its simplicity; spectra may be obtained by an unskilled operator
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Anal. Chem. 1904, 56,2204-2210
with no special cells and no attention paid to sample alignment.
ACKNOWLEDGMENT Useful discussions with Prabir Dutta during the course of this work were appreciated. The existence of the hard-clad fibers used here was brought to the authors’ attention by Bolesh Skutnik of Ensign-Bickford.
LITERATURE CITED (1) Chabay, I. Anal. Chem. 1882, 5 4 , 1071A. (2) Peterson, J. I.; Fitzgerald, R. V.; Buckhold, D. K. Anal. Chem. 1884, 56, 62. (3) Trott, G. R.; Furtak, T. E. Rev. Scl. Instrum. 1880, 57, 1493. (4) Walfren, G. E.; Stone, J. Appl. Spectrosc. 1872, 2 6 , 585.
Schaefer, J. C.; Chabay, I . Opt. Lett. 1878, 4 , 227. Eckbreth, A. C. Appl. Opt. 1880, 78, 3215. Yamada, H.; Yamamoto, Y. J . Raman Spectrosc. 1980, 9 , 401. McCreery, R. L.; Fleischmann, M.; Hendra, P. Anal. Chem. 1883, 55, 148. (9) Jeanmaire, D. L.; Van Duyne, R. P. J . Electroanal. Chem. 1875, 66, 235.
RECEIVED for review April 2, 1984. Accepted May 31, 1984. Major support for this work came from the OSU Materials Research Laboratory, with additional support provided by a grant to R.L.M. from the Chemical Analysis division of the National Science Foundation. R.L.M. is a Sloan Fellow for the period 1981-1985 and the support of the of that foundation is acknowledged.
Correlation of Retention Behavior with Quantitative Surface Analysis of Octadecyl Bonded Chromatographic Supports Mark L. Miller, Richard W. Linton,* Stuart G. Bush, and James W. Jorgenson Department of Chemistry, T h e University of North Carolina, Chapel Hill, North Carolina 27514
ESCA and FTIR-PAS are compared to more conventional technlques, transmlsslon near-IR and total carbon analysls, for quantltatlon of octadecylsllyl (ODS) bonded phases for reversed-phase HPLC. All four technlques give responses which Increase llnearly wlth lncreaslng surface coverages on slllca and provide detectlon llmlts of 5 5 % of a monolayer. The chromatographlc behavlor of solutes differing only In functlonal group Is Investigated as a functlon of ODS coverage. The data lndlcate that nonpolar solute retentlon closely agrees wlth solvophoblc theorles of retentlon, while polar solute retentlon Is more Influenced by a partltlon-like mechanism. Thls study supports the postulate that long n-alkyl chains form aggregates on the surface of slllca supports which can Intercalate small solutes. I n addltlon, It Is observed that retentlon on low coverage ODS silica may be dependent on eluent pH for some polar and nonpolar solutes.
The chemical complexity of chromatographic support surfaces is well-known. For example, silica particles contain various surface functionalities including siloxanes, lone silanols, geminal silanols, and vicinal silanols (1). The presence of adsorbed water and bulk (internal) silanols, the process of dehydration/rehydration,the pore-size distribution, and pore structure also effect the physicochemical properties of the silica. A lack of well-characterized, standard chromatographic supports is reflected in a recent study (2). Widely differing capacity factors and selectivities are observed for the same solutes using various commercially available reversed-phase silica packings of the same type. Currently over 60% of the LC work is done in the reversed-phasemode with nearly 80% of that research involving the use of octadecylsilane (ODS) modified columns (3). Therefore, it is important to better understand the nature of support surfaces and retention mechanisms in the reversed-phasemode, particularly on ODs
columns. This is the aim of the analytical research to be described. The most popular model of retention behavior in reversed-phase chromatography is the “hydrophobic”or solvophobic effect, which has been reviewed by Horvath and Melander (4). However, the role of the stationary phase is complicated by the observations that peak shapes and retention times are dependent not only on the type of bonded phase but on related factors such as bonded phase surface coverage and the nature of unreacted sites on the supports (5-10). Previous investigators have examined retention characteristics in reversed-phase chromatography as a function of bonded phase coverage including the effect of bonded phase alkyl chain length (11-13) or the number of alkyl chains per unit surface area (14-16). A linear relationship normally exists between log (capacity factor) and the carbon number of the alkyl group (11-13). The same result holds for plots of log (capacity factor) vs. alkyl group concentration under low coverage conditions (14-16). However, the above bonded phases were prepared from polyfunctional silanes (17). This complicates both the quantitation of surface groups and the relationships between chromatographic behavior and surface activity. With monofunctional silanes used in this study, only monomeric layers can result and the surface reaction unequivocally reduces the original concentration of surface silanols. The traditional method of reporting bonded phase coverage is in units of wmol/m2using total carbon analysis data and the specific surface area of the support, normally determined by BET nitrogen adsorption. The BET determination, however, may not yield a chromatographically relevant surface area, particularly if there are significant numbers of solute and reagent inaccessible micropores. The above approach also is based on an elemental analysis technique and therefore does not furnish direct molecular, surface structural, or silanol information. This study evaluates the utility of the surface analysis techniques, electron spectroscopy for chemical analysis
0003-2700/84/0356-2204$01.50/00 1984 American Chemical Society
