Anal. Chem. 1985, 57,2253-2256 (15) Nechaev, E. A.; Volglna, V. A. Russ. J. Phys. Chem. (Eng. Trans/.) 1974, 48, 1364-1367. (16) Snyder, L. R. “Princlples of Adsorptlon Chromatography”; Marcel Dekker: New York, 1968; p 163. (17) Flscher, W.; Kulling, A. 2.Elekfrochem. 1958, 60,680-688. (18) Umland, F. Z . Elekfrochem. 1956, 60,711-721. 1191 Shiao. S. Y.:. Mever. ~. R. E. J. Inoro. Nucl. Chem. 1981. 43, 3301-3307. (20) Cornelius, E. B.; Mulliken, T. H.; Mills, G. A.; Oblad, A. G. J . Phys. Chem. 1955, 59, 809-813. (21) Perl, J. B. J . Phys. Chem. 1985, 69, 220-230. (22) Churms, S. C. J . S.A h . Chem. Inst. 1986, 79, 98-114. (23) Parks, G. A. Chem. Rev. 1965, 65, 177-196. (24) Schmitt, G. L.; Edeimuth, S. H., unpublished results.
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(25) Helfferlch, F. “Ion Exchange”; McGrall-HIII: New York, 1962. (26) Sillen, L. 0. “Stability Constants of Metal-Ion Complexes”; The Chemicai Society: London, 1971; Supplement 1, Publication 25. (27) Small, H.; Miller, T. E., Jr. Anal. Chem. 1982, 54, 462-469.
RECEIVED for review Ami1 9. 1985. AcceDted June 10, 1985. Part of this work was sipported by Grant AM28077 awarded by the National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases and was presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, New Orleans, Feb 1985, paper 1104.
Detection Method for Ion Chromatography Based on Double-Beam Laser-Excited Indirect Fluorometry Sun-I1Mho and Edward 5.Yeung* Ames Laboratory-USDOE
and Department of Chemistry, Iowa State University, Ames, Iowa 50011
The concept of Indirect fluorometry for detectlon In Ion chromatography Is presented. A fluorescing eluting Ion Is used to malntaln a constant background slgnal. When the analyte Ions elute, electroneutrallty forces the displacement of an equlvalent amount of the elutlng Ions at the detector. A decrease In slgnal Is then observed. A mass detectablllty of 6.7 ng of chloride Ion Is obtained ( S I N = 3), which corresponds to a concentration detectablllty of 7.5 X IO-’ N. Thls Is posslble due to a novel double-beam arrangement for laserexclted fluorescence, substantlally reduclng the flicker nolse.
Ion chromatography (IC) has become a mature analytical method as a result of research efforts in the last few years (1-3). Significant improvements have been made in the de: velopment of a variety of stationary phases and in new detection methods. Much attention has been given to nonsuppressed ion chromatography, because of the potential for minimizing band broadening. In addition to the standard conductivity detector, several optical methods, including indirect photometric ( 4 ) ,refractive index (5),and direct photometric (6) methods, have been tried to allow the use of standard liquid chromatographic instrumentation. Most of these detectors have very similar limits of detection (LOD), which are in the nanogram range for typical ions. Since these detectors have already been utilized to their full potential, additional improvements in LOD can only come if new detection principles are introduced. In conventional liquid chromatography (LC), the lowest LOD’s have been reported for the fluorescence detector (7). For ions that fluoresce, one can thus expect improved LOD in IC as well. However, the number of ions that do fluoresce is very small, particularly those that are of interest to “real-world”problems. The exceptions are certain rare-earth ions, which phosphoresce in aqueous solutions. The combination of low absorption coefficients and poor emission efficiency leaves the LOD’s for these ions still quite unimpressive. Recently, several indirect detection methods for LC have been demonstrated (4,8). Briefly, the detector responds to some physical property of the chromatographic eluent. So, there is a constant background signal generated at the detector when no analytes are present. When the analyte elutes, it
displaces an equal amount of the eluent at the detector. Even though the detector does not respond to the analyte, the lower eluent concentration at the detector causes a decrease in signal. The analyte can then be monitored as a negative signal, Le., indirectly. So, it should be possible to devise a detection method based on indirect fluorometry. It may then be possible to extend the advantages for fluorescence detection to species that do not themselves fluoresce. To appreciate the potential of indirect measurements, it is necessary to define a figure-of-merit known as the dynamic reserve (DR). This is the ability of a detector to recognize a small change on top of a large background signal. This is quite different from the “dynamic range” concept often used in measurements, which is simply the ratio between the smallest and the largest signal that can be measured, independent of the background level. For example, the absorbance detector for LC can measure a change of AA = 2 X AU when the background absorbance is unity (4,9).The dynamic reserve is simply the ratio of the two, so that DR = 5 X lo3. In other words, the analyte must be at a fractional concentration of at least 1part in 5 X lo3 (of that of the eluent) at the detector before a noticeable decrease in the signal is observed. We note that DR for the absorbance detector cannot be increased by using a larger background absorbance, because noise increases as well (in fact more rapidly) to degrade the minimum detectable AA. So, indirect photometry (4) can only be used at analyte (fractional) concentrations of 1 part in 5 X lo3 or higher. In normal LC situations, this is too high a concentration to be useful. The reason indirect photometry works in IC (4) is because the eluting ion is typically present at a low concentration, e.g., M. So, the analyte need only be at 2 x M to produce a fractional concentration of 1 part in 5 X lo3. The refractive index (RI) detector is also an indirect detector because the solvent provides the major contribution to the signal. One can measure ARI = regardless of the background RI. So, for a solvent-solute RI difference of 0.1, the DR is lo6. This is why RI detectors have been useful in many LC situations. The best DR is obtained with polarimetry (8). When an optically active eluent is used, there will be a background rotation as large at looo (10). By mechanical adjustment to the polarization analyzer, one can suppress this background signal to a level similar to that with an optically inactive eluent. A change of 4 X lo4 deg ( S I N = 3) can still be detected. The DR is then 2.5 X lo7. In ion
0003-2700/85/0357-2253$01.50/00 1985 American Chemical Soclety
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chromatography, the conductivity detector also functions as an indirect detector (11). In the most favorable case, the background conductance can be 50 bmho without affecting the base line noise (12),and changes of 0.01 bmho can be measured. The DR is thus 5 X lo3. Again, this DR becomes useful only because of the low initial concentration of the eluting ions. In fluorometry, the DR depends on the stability of the background fluorescence. This in turn depends on the stability of the light source (flicker noise) and the intensity of the fluorescence (shot noise). Shot noise can be neglected if laser excitation is used to generate the background fluorescence. However, lasers are inherently very noisy sources (compared to, e.g., tungsten lamps), with stabilities in the 1% (DR = lo2) to 0.1% (DR = lo3) range. To increase the DR, intensity stabilization via a doublebeam arrangement with modulation is a possibility. This should be superior to using a reference photodetector for normalization because of difficulties in matching detectors and amplifiers. No double-beam arrangement for fluorescence has been reported previously. The reason is that the experimental conditions are usually deliberately chosen to provide a low fluorescence background. We report here a double-beam laser-excited fluorometric detector for LC, making it possible to demonstrate the concept of indirect fluorescence for detection in IC. The detection of analytes in LC effluents by a decrease in a large fluorescencebackground has been reported earlier (13). However, that report is quite different from the indirect fluorometric scheme described here. Judging from the magnitudes of the decrease for the concentrations used there, a displacement of the eluent by the analyte at the detector cell is not the main mechanism. Also, the anomalies reported in ref 13 cannot be explained by a displacement process. Most likely, there is a combination of fluorescence quenching by the analyte, equilibrium effects (14), and changes in the chemical form of aniline (e.g., protonation and hydrogen bonding) in the presence of the analytes. In this work, we will only be concerned with a one-to-one displacement process between the eluting ions and the analyte ions.
EXPERIMENTAL SECTION The reagents used in this study are reagent grade. All solutions are made with deionized water. A 2.2 X lo-' M sodium salicylate solution, pH 4.9 without any buffer ions, is used as a fluorescing eluent. The chromatographic peaks of the nonfluorescing inorganic ions are detected as an indirect fluorescence signal. The chromatographic system used is conventional and composed of a syringe pump (ISCO, Lincoln, NB, Model 314), an injection valve (Rheodyne, Berkeley, CA, Model 7010) with a lO-bL sample loop, and an anion chromatographiccolumn (Vydac, Hesperia, CA, 302 IC, 4.6 mm X 25 cm, 15 pm) connected to the sample flow cell. The flow rate used is 2.25 mL/min. The same eluent is introduced by a peristaltic pump (Gilson Medical Electronics, Middleton, WI, Minipuls 2) with a flow rate of 0.36 mL/min into a reference flow cell. The experimental arrangement for the fluorescence detection system is shown in Figure 1. A linearly polarized 325-nm UV beam of a HeCd laser (Liconix, Sunnyvale, CA, Model 4240NB) at about 7 mW is used as an excitation source. The polarization of the beam is modulated at 100 kHz by an electroopticmodulator (Lasermetric, Inc., Teaneck, NJ, Model 3030), which is driven by a high-frequency modulator driver (Conoptics,Inc., Danbury, CT, Model 25). Unipolar positive-goingsquare waves (0 to + L O V) from a wave generator (Wavetek, San Diego, CA, Model 162) are in turn applied to the modulator. A calcite beam displacer (Karl Lambrecht Corp., Chicago, IL, MBDA10) splits the polarization-modulated beam into two separate beams. The perpendicularly polarized beam continues straight through while the horizontally polarized beam goes through the calcite at 6'. The horizontallypolarized beam emerges displaced 3.3 mm apart from the first, and parallel to it. The two beams are directed to the centers of the sample and reference flow cells, respectively. In
g---
Flgure 1. Experimental arrangement for double-beam fluorescence detector: HeCd, 325 nm CW laser; PC, Pockels cell; MD, modulatlon driver; WG, wave form generator; FL, focuslng lens, focal length = 20 cm; BD, calclte beam displacer; OF, optical flat; FC, Now cells; F, filters; PMT, photomultiplier tube; P, power supply; LA, lock-in amplifier; CR, chart recorder.
//
3.3mm
5 Mm
Flgure 2. Flow cells for double-beam fluorometry: two 1.0 mm id. X 2.0 mm 0.d. quartz tubes are mounted on an aluminum block. PMT
is posltloned in front of the paper;
e-,
excitation laser beams.
other words, during one half-cycle of the modulation the laser beam is directed to the sample cell, and during the other half-cycle it is directed to the reference cell. A J z in. thick quartz window is used to balance the intensities of the two beams based on the natural reflectivity of the surfaces, which is dependent on angle and on polarization. A schematic of the sample and reference cells is shown in Figure 2. Two parallel 1mm i.d. X 2 mm 0.d. quartz tubes are held 5 mm apart by an aluminum block. A 20 cm focal length quartz lens is placed before the calcite displacer in order to focus the 1.2 mm diameter laser beam down to 68 pm at the flow cells. The fluorescence is collected at ca. 90' relative to the excitation laser beam by a photomultiplier tube (RCA, Harrison, NJ, Type 1P28) after passing through two filters (Corning Glass, Corning, NY, 0-52 and 5-58). The aluminum holder of the two flow cells is tilted at a 20° angle, so that the scattered light produced by the cell walls is not hitting the cathode of the photomultiplier tube. The photomultiplier tube output is directed to a lock-in amplifier with a 10-stime constant (EG&G, PAR, Princeton, NJ, Model HR-8) through a current-to-voltage converter (lo3V/A). Initially the fluorescence signals from the sample and reference cells are maximized and balanced (during the two half-cycles of the modulation) by adjusting the angle of the quartz flat and the collection angle of the photomultiplier tube while the intensity is being monitored by an oscilloscope (Tektronix, Beaverton, OR, Model 7904). Fine balance of the fluorescence intensities is accomplished by monitoring the output of the lock-in amplifier with 1-s time constant.
RESULTS AND DISCUSSION To establish a stable base line with a fluorescing eluent, the key was a double-beam arrangement with modulation. We can estimate the number of photons reaching the phototube per second. For a M solution of ion with a molar ab-
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
sorptivity of lo3 L/(mol cm), about 2.3% of the light is absorbed. For 7 mW excitation at 325 nm, this corresponds to 2.6 X 1014photons absorbed. Losses due to quantum efficiency of the phototube, transmission of filters, quantum efficiency of fluorescence, and collection optics can be estimated to be a factor of IO4. So, shot-noise is at the 6 X lo4 level and can be neglected here. The measured flicker noise (on a photodiode) in the laser is With the modulation system (100 kHz operation) in Figure 1, the flicker noise is reduced to 2 x IOd, as measured by combining both beams onto a common photodiode before the fluorescence cells. We note that if the modulated beams are directed to separate photodiodes that are balanced electronically before demodulation,the noise level is or higher. This emphasizes the need for using a single detector for double-beam optics, so that true compensation between the reference/sample signals is possible. To accomplish this in fluorescence, the offset placement of the reference/sample flow cells in Figure 2 allows variable collection efficiencies at a common phototube depending on the placement of the latter. The optical flat in Figure 1 can also be adjusted to change the relative powers of the beams going into the two cells. Also, the alignment of the cells with respect to the two beams can be adjusted so that different optical path lengths are used in the two cells, i.e., shining across different chords in the cylindrical cells. Of the three methods for compensation, the first is preferred because thermal effects and light scattering can be properly accounted for there. In principle, the reference cell should reproduce the flow conditions of the sample cell. In practice, we found that as long as there is moderate flow in the reference cell of a solution identical with the eluent, the base line will be stable to 2 X lo4. At this level of noise, a peristaltic pump, with pulsating flow, is found to be adequate. A static reference cell is not acceptable because the fluorescence intensity is found to decrease as a function of time, presumably due to heating. Balancing the signal for the two half-cycles of modulation produces a zero output on the lock-in amplifier, so that more sensitive scales can be used to monitor the analytes. Useful results will still be obtained as long as the output of the lock-in amplifier stays on-scale for the base line of the chromatogram. However, the base line will drift with changes in the laser power unless the two fluorescencesignals are finely balanced initially. Since ion exchange chromatography is the basis for indirect detection here, the choice of the eluting ion is important. The main factors influencing the choice are fluorescence efficiency, absorption coefficient, freedom from quenching, lack of complex equilibria with the analytes, and good elution properties a t low concentrations. Certain rare-earth ions in aqueous solution show emission and may be suitable for cation exchange chromatography. We found that the combination of weak absorption and weak emission makes shot-noise a serious problem. The long emission lifetimes also make it impossible to modulate at high frequencies to reduce flicker noise in the laser. Many organic dyes in the ionic form fluoresce strongly. However, the large sizes of these molecules sometimes lead to interactions with the column other than via an ion-exchange process. For example, we tested sodium fluorescein as a possible eluting ion for anion exchange chromatography. This dye molecule can be excited with the 488 nm output of an argon ion laser in an arrangement idential with Figure 1. Sodium fluorescein is strongly adsorbed onto our ion exchange column, as indicated by the breakthrough curve. The adsorption equilibrium competes with the ion exchange equilibrium such that poor chromatographic peak shapes are observed at an eluting ion concentration below M. Good results were obtained with sodium salicylate as an eluent for anion exchange chromatography. Absorption a t 325 nm, a
2255
Figure 3. Fluorescence chromatogram for a mixture of IO3-and CImol) of IO3-; (b) 0.16 pg (4.5 X lo-’ mol) ions: (a) 0.88 pg (5 X M sallcylate, pH 4.9; flow rate, 2.25 mL/min. of CI-; eluent, 2.2 X
convenient wavelength for laser excitation, is strong and emission around 420 nm is efficient. The salicylate ion is structurally similar to phthalate or benzoate ions, which are well-behaved eluting ions for IC (2). With the sodium salt dissolved to 2.2 X M, the pH was measured to be 4.9. So, most of the salicylate is in the ionized form. A pH buffer was not needed to control the equilibrium and was avoided to ensure a well-defined ion-exchange pathway with only the eluting ions. A chromatogram of iodate and chloride ions eluted with salicylate and detected by indirect fluorescence is shown in Figure 3. . The magnitudes of the signals can be compared to those predicted from the displacement of the eluting ions by the analyte ions on a per equivalent basis. The background fluorescence signal, as determined by irradiating only the sample flow cell, was 45 mV measured by the lock-in amplifier. The peak heights of iodate and chloride in Figure 3 corresponded to 0.80 mV and 0.72 mV, respectively. So, the signals at the peaks corresponded to decreases of roughly 1/56 and 1/63 of the total fluorescence, or a displacement of 3.9 X lo4 N and 3.5 X N of salicylate ions, respectively. Now, 5 X mol and 4.5 X mol of the two ions, respectively, were injected, and these were diluted to peak volumes (fwhm) of 1.2 mL and 1.3 mL. The actual concentrations of the analyte ions at the peaks are then 4.2 X lo4 N and 3.5 X lo4 N. This is in good agreement with the predicted decrease in salicylate concentrations considering that a triangular peak shape is used to approximate the Gaussian peak. So, an ion exchange mechanism for separation and a displacement mechanism at the detector (due to electroneutrality) are responsible for the observed chromatogram. The noise level in Figure 3 corresponds to a flicker noise of 2 X (0.01mV). The LOD for chloride is then 6.7 ng injected, at SIN = 3. The fact that the signal magnitude is predictable indicates that this fluorometric scheme can also be used for quantitation without standards (9, 12),by eluting the analytes first with salicylate and then with a nonfluorescing ion of comparable strength, e.g., phthalate. The LOD of 6.7 ng is comparable to the mass LOD for indirect photometric (9) and conductvity (12) detection. We note however that the chloride peak here has a k’ = 19 compared to the nitrate peak in ref 9, which has a k’ = 3.5. The number of theoretical plates here is 1600, which is comparable to that in ref 9 and to the manufacturer’s specifications. So the concentrationLOD (SIN = 3) at the fluorescence detector is 1.5 X lo-’ N and is somewhat better than the concentration
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LOD at the absorption detector (9),which is 3 X N. The reason why k'is so large is because of the low concentration of eluting ion used. It is well-known that the logarithm of the adjusted retention time is roughly inversely proportional to the logarithm of the concentration of the eluting ion (15). We confirmed that diluting the eluting ion by a factor of 2 (same pH) causes the retention time of chloride ions to increase roughly by a factor of 2. The base line fluctuations can be maintained to the same (2 X 10"') level. For the same injected amount, the chromatographic peak showed the same S I N ratio, so that the mass LOD was still 6.7 ng. However, the peak concentration was only half of that in Figure 3 because of the larger k'. The concentration LOD was thus improved to 7.5 X N at the detector. Another way to rationalize the above is that the concentration LOD is always equal to the eluting ion concentration multiplied by three times the fractional base line noise in indirect fluorometry. We can compare the ultimate potentials of the various common detection modes in IC. For absorbing ions, if a suitable wavelength can be chosen, the molar absorptivity can be as high as lo4 L/(mol cm). A realistic absorbance LOD ( S I N = 3) is 3 X AU for standard absorption detectors. So, for a 1cm cell, the concentration LOD is 3 X lo4 N. For nonabsorbing ions, one can use indirect photometry to achieve the same LOD, provided that a highly absorbing eluting ion can be found. Conductivity detection is limited by the inability of the transducer to detect small changes much below pmho, which is equivalent to a concentration LOD of N for most ions. The LOD for fluorescing ions, such as salicylate, should be comparable to other fluorometric schemes for LC (16),i.e., in the 5 X N range. For indirect fluorometric detection of nonfluorescing ions, the base line stability and the eluent concentrationare the determining factors. The intensity of fluorescence can further decrease by a factor of 1000 before the shot noise (fundamental limit) becomes comparable to the present flicker noise of 2 X 10"'. So, salicylate concentrations down to 1 x lo-? N can be used, and changes due to displacement of 5 X N should be detectable. Essentially, indirect fluorometry potentially allows the same LOD to be achieved for nonfluorescing ions as for fluorescing ions. The reason we are not able to demonstrate such LOD's is because available columns have too high a capacity and will not function at such low eluent concentrations. While IC based on concentrations of eluting ions at the N range will not improve LOD's in absorption or conductivity detectors, it should extend the LOD's for indirect fluorometry to the picogram range. Efforts in developing such low-capacity IC stationary phases, admittedly not an easy task, should be valuable. But, there will be a 10-fold gain in LOD over existing detectors for every 10-fold reduction in eluting ion concentration below 2 X N. At some point, one will be able to justify the added cost and complexity of this new detection scheme. Several other variations on the basic scheme have been tried. The reference and the sample flow cells can be connected in series with a delay loop small compared to the peak volume (17). This way, a separate flow system is not needed, and pressure and temperature fluctuations are readily compensated for. We found that using a 190-pL delay loop here
gives a proper derivative-shapedsignal, as expected (17). The modification did not allow flicker-noise stabilization better than 2 X lo-*, while the signal (peak to peak) decreased by a factor of 2. So, a series arrangement sacrifices some sensitivity for convenience. The excitation laser was also tested without focusing to illuminate a larger volume. In principle, heating effects will be reduced, but in practice, more of the quartz cell walls were illuminated and scattering causes larger base line fluctuations than before. A higher laser power was also tried to increase the fluorescence signal. Since we were not shot-noise limited here, no advantage was found. In fact, laser powers larger than 10 mW for this eluent concentration cause substantial heating and turbulence and increased base line fluctuations. At lower eluent concentrations, one can expect to gain from using higher laser powers, to the limit of solvent (water) absorption. A simpler modulation system is possible with a Bragg cell instead of a Pockels cell. There, the beams are usually not 100% modulated and the beams are not as well separated spatially. So, the studies here are based on a Pockels cell. In summary, we have demonstrated the concept of indirect fluorometric detection in IC by a double-beam optical arrangement. As presented, the concentration LOD is already better than those for indirect photometry or conductivity. Realization of the full potential will have to wait until IC columns with substantially lower capacities can be developed. Other immediate applications of the double-beam arrangement include fluorescence visualization in chromatography (13)and fluorometric versions of replacement IC (18),where a fluorescence background must be maintained at a stable level. Registry No. C1-, 16887-00-6;IO3-, 15454-31-6;salicylate, 54-21-7.
LITERATURE CITED (1) Small, H. I n "Trace Analysis"; Lawrence, J. F., Ed.; Academic Press: New York, 1982; Vol. I, pp 267-322. (2) Fritz, J. S.; Gjerde, D. T.; Pohlandt, C. "Ion Chromatography"; Huthig: New York, 1982. (3) Small, H. Anal. Chem. 1983, 55, 235A-242A. (4) Small, H; Miller, T. E. Anal. Chem. 1982, 54, 462-469. (5) Haddad, P. R.; Heckenberg, A. L. J . Chromatogr. 1982, 252, 177-184. (6) Reeve, R. N. J . Chromatogr. 1979, 177, 393-397. (7) Yeung, E. S.; Sepaniak, M. J. Anal. Chem. 1980, 52, 1465A-1481A. (8) Bobbitt, D. R.; Yeung, E. S. Anal. Chem. 1984, 56, 1577-1581. (9) Wilson, S. A.; Yeung, E. S. Anal. Chim. Acta 1984, 757, 53-83. (10) Bobbitt, D. R.; Yeung, E. S. Anal. Chem. 1985, 5 7 , 271-274. (11) Fritz, J. S.; Gjerde, D. T.; Becker, R. M. Anal. Chem. 1980, 52, 15 19- 1522. (12) Wilson, S. A.; Yeung, E. S.; Bobbitt, D. R. Anal. Chem. 1984, 56, 1457- 1460. (13) su, S. Y.; Juraensen, A.; Bolton, D.; Winefordner, J . D. Anal. Lett. 1981, 14 (A1),-1-6. (14) Vigh, G.; Leitold, A. J . Chromatogr. 1984, 372, 345-356. (15) Gierde, D. T.; Schmuckler, G.; Fritz, J. S. J . Chromatogr. 1980, 187, 35-45. (16) Sepaniak, M. J.; Yeung, E . S. J . Chromatogr. 1980, 790, 377-383. (17) Woodruff, S. D.; Yeung, e. S. J . Chromatogr. 1983, 260, 363-369. (18) Downey, S. W.; Hieftje, G M. Anal. Chim. Acta 1983, 153, 1-13. '
RECEIVED for review May 2, 1985. Accepted June 10, 1985. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. W-7405-eng-82. This work was supported by the Office of Basic Energy Sciences.
