Anal. Chem. 1987, 5 9 , 1177-1179
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Electron Spin Resonance Study of Chromatographic Surfaces R. K. Gilpin* and A. Kasturi Department of Chemistry, Kent State University, Kent, Ohio 44242 E. Gelerinter
Department of Physics, Kent State University, Kent, Ohio 44242
Chromatographlc grade slllca has been modlfled wlth Immobillzed TEMPOL molecules. These materials have been studled In several polar and nonpolar solvents by uslng electron spln resonance spectroscopy. Large dlfferences In the shape of the hyperflne spectra as well as a reversal In hyperflne coupling constants have been observed as a function of solvent polarlty. These data are explainable In terms of a major conformational change in the immobilized molecules. I n hydrocarbon solvents the nltroxlde group k In the plane of the surface and associated with surface sllanols. I n alcohols the immobilized groups are displaced from the surface and associated wlth solvent molecules.
A large variety of methods have been used to study chromatographic surfaces. Nuclear magnetic resonance, infrared, and luminescence measurements have been especially important in probing the orientation and dynamics of chemically modified sorbents ( I ) . Another potentially useful technique for studying immobilized layers is electron spin resonance (ESR) spectroscopy. Robb and Smith ( 2 ) have used ESR to examine the conformation of copolymers of vinylpyrrolidone and allylamine adsorbed on silica as a function of surface coverage, molecular weight, and coating solvent. With increasing surface coverage the polymer changes from a flat to a loop configuration. Similarly, Hommel and co-workers ( 3 )have studied the conformation of spin-labeled mono-, di-, tri-, and tetraethylene glycol as well as poly(ethy1ene oxide) grafted onto silica. The population ratio of end segment groups free in solution to those adsorbed to the surface changes with temperature and chain length. These same investigators also have studied the influence of grafting ratio on the polymer chains’ conformation (4). The observed ESR line shapes are the superposition of two types of spectra arising from motionally different states. At low grafting ratios the chains are restricted as the result of a flat conformation along the surface. However, as the grafting ratio increases the chains are extended into solution and are more solvated ( 4 ) . In all of the above studies (2-4) as well as numerous other work the nitroxide radical has served as an important spin labeling group. The spin Hamiltonian for the nitroxide radical is
7f = gPH$
+ AIS + gNPNHd
(1)
where I refers to the 14Nnuclear spin, @, the Bohr magneton, g, the Lande factor, H,,the magnetic field strength, and S , the electron spin value. The first term in eq 1 is the electron Zeeman term. A in the second term arises from the hyperfine interaction between the magnetic moments of the nitrogen nucleus and the free electron. Both g and A are axial and have nearly the same principal axes. The third term is the nuclear Zeeman term. For isotropic systems where the spin labels tumble rapidly enough to completely average the hyperfine
and Zeeman interactions, Aisois ll3(Ax,+ A , + Az,) and giso is l/3(9XX + gyy + g z z h A,, is the component of the 14N hyperfine coupling tensor along the direction perpendicular to the N-0 plane ( 5 ) and experimentally is half the difference between the low- and high-field extremes in the rigid-limit spectrum. Since the polarity of the local environment modifies the localization of the unpaired electron on the nitroxide group, the isotropic nitrogen hyperfine coupling constant, Aiso, is sensitive to solvent (6). Sistovaros and co-workers ( 5 ) have studied the effect of various contact liquids on the 4,4-dimethyloxazolidine-N-oxyl ring chemically anchored to silica and found changes in the hyperfine coupling constant with solvent polarity. Likewise, Hammerstedt and associates (6) have utilized hyperfine coupling as a means of examining the influence of various solvents on membranes of bovine serum. The influence of solvent on biological reproduction also has been studied by ESR (7, 8). In general A,, increases with increases in the solvent’s dielectric constant and is especially sensitive to the hydrogen bonding of the N-0 group (9, IO). Although ESR has been used to examine a number of bulk polymers, biological membranes, and thicker grafted surface films (2-IO), it has not been specifically applied to the study of high-performance liquid chromatographic surfaces nor to the study of related interfacial phenomena. In the current investigation electron spin resonance studies have been carried out on chromatographic silica chemically modified with TEMPOL. Changes in the hyperfine line shapes and A,, coupling have been measured as a function of solvent type and polarity. The data obtained can be rationalized in terms of a major conformational change in the immobilized nitroxide probe as the result of the solvent’s ability to strongly interact with unreacted silanol groups on the surface.
EXPERIMENTAL SECTION Materials. Dimethyldichlorosilane and 2,2,6,6-tetramethylpiperidine oxide (TEMPOL) were purchased respectively from Petrarch Systems (Levittown, PA) and Aldrich Chemical Co. (Milwaukee, WI). The porous chromatographic support was LiChrosorb Si-60 silica (dp = 10 pm, SA = 550 m2/g) from Merck (EM Labs., Elmsford, NY). 2-Propanol and 1-butanolwere HPLC grade from MCB Inc. (Norwood,OH). The hydrocarbon solvents octane and decane were gold label from Aldrich Chemical Co. (Milwaukee, WI). Toluene, tert-butyl alcohol, and HPLC grade hexane were purchased from Fisher Scientific (Pittsburgh, PA). Synthesis of Spin-Labeled Silane. A mixture of 0.5 mmol of TEMPOL and 0.5 mmol of dry triethylamine was prepared in dry benzene. This solution was slowly combined with a cooled solution of 0.5 mmol of dicholorodimethylsilane which also was prepared in dry benzene. The reaction mixture was allowed to warm to ambient conditions and stirred for 3 hours. Following this the mixture was refluxed for an additional 4 h. The resulting solution was cooled and the precipitate, triethylamine hydrochloride, formed was removed by filtration. The filtrate was placed in a rotoevaporator and the benzene removed from the spin-labeled silane and unreacted TEMPOL. Subsequently,hexane was added to the product mixture and the vessel was capped and left
0003-2700/87/0359-1177$01.50/0 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
A
Figure 1. Synthesis of TEMPOL modified silica.
in the freezer overnight. The insoluble TEMPOL was filtered off and the hexene removed from the silane with a rotoevaporator. Preparation of Spin-LabeledSilica.Silica,2.5 g, was slurried with water for 3 h, the water decanted off, and the silica dried at 110 OC for 5 h. Forty milliliters of dry toluene was added to the dried silica followed by addition of 5 mL of toluene which contained (0.1 mmol) spin-labeled silane. The resulting reaction mixture was refluxed for at least 12 h with dry nitrogen bubbling through the solution. Subsequently, the derivatized silica was washed 5 times with 50-mL portions of dry toluene, twice with 50-mL portions of water-saturated toluene, and 4 times with 50-mL, portions of ethyl ether sequentially. To ensure the absence of any remaining unbonded material the spin-labeled silica was packed into a 4.6-mm-i.d. stainless steel tube and 250 mL of a 50/50 mixture of 2-propanol/deionizedwater was pumped through the column. This was followed by 100 mL of 2-propanol and 200 mL of deionized water. The labeled silica was unpacked and dried under vacuum at 80 "C overnight. The overall reaction scheme used in the current work is summarized in Figure 1. Electron Spin Resonance Studies. Spectra were acquired at the X-band by using an IBM Instruments Inc. (Danbury, CT) Model 2OOD-SRC ESR spectrometer interfaced to a CS-9000 computer. Samples were placed in 4-mm-0.d. quartz tubes and deoxygenated by using at least 10 freeze-thaw cycles while applying a vacuum. Following this treatment, the tubes were heat sealed under 0.5 atm of nitrogen. Peak positions in the ESR spectra were selected by the IBM software package. In all cases hyperfine splitting values (i.e., A,) were obtained from the rigid limit spectra (not shown) acquired at 180 K. The amount of TEMPOL bonded to the surface was determined by comparing the integrated area of the immobilized spectra obtained in 1-butanol with known amounts of TEMPOL dissolved in dibutyl phthalate at room temperature. By this procedure the derivatized materials contained approximately0.002 pmol of TEMPOL per square meter area of silica surface.
RESULTS AND DISCUSSION To prevent d i p o l d i p o l a r broadening, reaction conditions were selected so that only a very small amount of TEMPOL was bonded to the surface. Similarly, oxygen broadening was minimized by deoxygenation. The electron spin resonance spectra of the TEMPOL chemically modified silica in various nonpolar and polar solvents are shown in Figures 2 and 3, respectively. Also included in each figure is a reference spectrum of free TEMPOL dkoved in either hexane or 1-butanol. Although all data in Figures 2 and 3 were obtained from the same reaction batch of silica, during the course of our experiments several different lots of derivatized silica were prepared yielding comparable results. Large differences in the hyperfine line shapes between the bound TEMFOL and that free in solution are clearly apparent for the nonpolar solvents. Referring to Figure 2, the ESR spectra acquired from the immobilized groups in contact with the hydrocarbon solvents are broad, characteristic of severely restricted motion. In the case of the polar solvents, the line shapes are significantly narrower, indicative of increased group mobility. These data can be explained in terms of a conformational change in the immobilized TEMPOL molecules.
---TJTf------
D
W
Flgure 2. Electron spln resonance spectra of spin-labled surface at room temperature in nonpolar solvents: (A) hexane; (B) octane; (C) decane; (D) toluene; (E) TEMPOL in hexane.
B V
V
v
V
Figure 3. Electron spin resonance spectra of the spin-labeled surface at room temperature in polar solvents: (A) 1-butanol; (E)2-propanol; (C) 2-methyl-2-propanol; (D) TEMPOL in 1-butanol.
The bonded TEMPOL ring, which is removed four bonds from the point of attachment (i.e., the surface silicon atom), can rotate into or out of the surface plane. This conformational change results in the nitroxide group being either more rigid and in close proximity to unmodified silanols or isolated from the surface and more motionally dynamic. Similar conformational change have been reported for other immobilized systems. Recently Suffolk and Gilpin (11-13) have investigated cyanoalkyl ligands also chemically anchored to LiChrosorb Si-60 using Fourier-transform infrared techniques. Major changes in the infrared band contour for the nitrile stretch region were observed depending on polarity of the contact solvent. For more highly derivatized materials a portion of the immobilized groups (i.e., those which were not sterically hindered) were found to hydrogen bond with unreacted surface silanols when placed in hexane. These same groups were displaced from the surface by 1-butanol. Likewise, conformation and motional dynamics have been studied for terminally spin-labeled poly(oxyethy1ene) grafted on silica gel as a function of surface coverage ( 4 ) . The spectra could be resolved into a broad component characteristic of slow motion resulting from the end segments adsorbed on the surface and a narrow component characteristic of faster motion of end
Anal. Chern. 1987, 59, 1179-1186
segments free in solution. The ratio of the intensity of the broad to narrow spectral components increased with decreasing coverage. In the current investigation, the proposed two-conformational state displacement model also is supported by hyperfine data. Values of A,, of 36.5 and 37.4 G were obtained for the immobilized nitroxide group in contact with 1-butanol and hexane, respectively. These values are quite high and in reverse order than expected from solvent polarity arguments. For instance, we have observed A,, = 36.5 G for TEMPOL dissolved in water and A,, = 34.2 G for TEMPOL dissolved in silicone oil. The higher A,, value obtained in hexane further suggests that the local environment of the nitroxide group is more polar than that found in 1-butanol. This could occur only via hydrogen bonding of the nitroxide group to a free surface silanol as the result of an in-plane conformation. The high but reversed value of A,, in 1-butanol suggests that the bonded groups are not in the vicinity of silanols but are associated with alcohol molecules. Hammerstedt and co-workers have reported changes in the hyperfine coupling of about 2 G for solvents with large polarity differences (e.g., water vs. hydrocarbons). For example, 3carboxyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl in water has an isotropic hyperfine coupling constant of 16.1 G compared to 14.1 G in octane (6). Further, the results obtained in the current work are similar to the data of Sistovaris and coworkers who have studied the sorption of di-tert-butyl nitroxide on silica and found an A,, value of 38.6 G if the NO group is hydrogen bonded to a surface hydroxyl group (5). When the same spin probe was dissolved in hexane an A,,
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value of 34.1 G was observed.
CONCLUSION Because of the high sensitivity of the nitroxide group to the surrounding local microenvironment, immobilized probes which contain this radical can be used to study interfacial properties of the surface. We hope to extend the current immobilization techniques to examine changes in the interfacial region of chromatographic materials from additional chemical modification and from variations in mobile-phase composition and addition of other secondary equilibrium modifiers such as surfactants. LITERATURE CITED (1) Gilpin, R. K. Anal. Chem. 1985, 57, 1465A. (2) Robb, I. D.; Smith, R. Eur. fo/ym. J . 1974, IO, 1005. (3) Hommel, H..; Legrand, A. P.; Balard, H.; Papirer, E. fo/ymer 1983, 2 4 , 959. Hommel, H.; Legrand, A. P.; Tougne, P.; Bahrd, H.; Papirer, E. Macromolecules 1984, 1 7 , 1578. Sistovaris, N.; Riede, W. 0.; Sillescu, H. Ber. Bunsen-Ges. fhys. Chem. 1975, 7 9 , 882. Hammerstedt, R. H.; Amann, R. A.; Rucinsky, T.; Morse, P. D., 11; Lepcock, J.; Snipes, W.; Keith, A. D. Bo/. Reprod. 1978, 14, 381. Cohen, A. H.; Hoffman, B. M. Inorg. Chem. 1974, 13, 1484. Lozos, G. P.; Hoffman, B. M. J . fhys. Chem. 1974, 78, 2110. Chapman, D. A.; Killian, G. J.; Gelerinter, E.; and Jarrett, M. Biol. Reprod. 1985, 32, 884. . . Killian. G. J.: Gelerinter. E.: and ChaDman. D. A. B i d . ReDrod. 1985. 33, 859. (11) Suffolk, B. R.; Gilpin, R. K. Anal. Chem. 1985, 57, 596. (12) Suffolk, B. R.; Gllpin, R. K. Anal. Chim. Acta 1988, 181, 259. (13) Suffolk, B. R.; Gilpin, R. K. J . Chromatogr. Sci. 1988, 2 4 , 423
RECEIVED for review October 23,1986. Accepted December 19, 1986.
Effects of Starting Temperatures and Temperature/Pressure Programming in Optimization of Gas Chromatographic Separation Numbers Louis A. Jones,* Charles D. Burton,l and Thomas A. Dean2 Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204 Thomas M. Gerig and John R. Cook Department of Statistics, North Carolina State University, Raleigh, North Carolina 27695-8203 separation numbers (TZ values) and concurrent carbon numbers, CH, (where TZ = - I ) , were determlned for six homologous n -alkanes under temperature-programmed condltlons using a 15 m X 0.25 mm fused slllca DB-5 column at startlng temperatures of 40, 50, and 60 OC and flow rates ( F ) of ca. 0.33, 0.78, 1.2, and 1.6 mL/mln (lsobarlc or constant head pressure and constant flow) and approprlate temperature program rates (TPR). Values of CH, for lsobarlc flow were unique to the startlng temperature/flow rate and Increased with Increasingflow rates and temperature. Constant flow rates (maintalned by pressure programmlng) produced only one CH, value per startlng temperature, Independent of flow rate, dlfferlng from those obtalned under lsobarlc flow condltlons. For each startlng temperature, nonlinear regression analysls of the data gave TZ = [ A B(TPR) C(log F ) D(TPR)* €(log F ) * F(l0g F)(TPR)](& CH,) -
+
+
+
+
+ -
‘Present address: Northrop Services, Inc., 2 Triangle Dr., Research Triangle P a r k , NC 27709. Present address: D e p a r t m e n t of Chemistry, W a y n e State University, Detroit, MI 48202. 0003-2700/87/0359-1179$01.50/0
1. The maximum efflclency (Le., largest slope value) was determlned by differentiating the slope of this equatlon and settlng TPR to 1 deg/mln and solvlng for F . At a starting temperature of 40 OC, the lsobarlc F was 0.95 mL/mln, constant F was 0.89 mUmln, and values were conflrmed by TZ vs. F graphs. A rapid “two TZ method” for optimizing F and TPR Is presented.
Temperature-programmedgas chromatography (TPGC) has four operational parameters that can be optimized: the column length, the coating thickness, carrier gas flow rate, and the temperature program rate. The latter two control the peak or base width, zob ( I , 2). For width at half peak height, isothermal operation, the efficiency of a capillary column is traditionally described in terms of the height equivalent to a theoretical plate, HETP (3). Two expressions have been used t o describe this property in TPGC, the first being TZ or separation number which was first proposed in 1959 ( 4 ) and subsequently endorsed by Kaiser (5). The TZ value 0 1987 American Chemical Society
