1202
JOHN S. LEIGH,JR.,AND GEORGEH. REED
Electron Paramagnetic Resonance Studies in Frozen Aqueous Solutions. Elimination of Freezing Artifacts]
by John S. Leigh, Jr.,* and George H. Reed Department of Biophysics and Physical Biochemistry, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 10104 (Received October 12, 1970) Publication costs assisted by the United States Public Service
Methods for eliminating perturbations on the electron paramagnetic resonance (epr) line shapes of frozen aqueous solutions of paramagnetic species are examined. Solvent-solute segregation which occurs in slowly fronen samples results in high “local” concentrations of paramagnetic species, and the epr spectra exhibit strong dipolar and exchange interactions. These intermolecular effects are only partially removed by rapid freezing techniques. Imbedding the aqueous samples in a polydextran gel prior to freezing results in magnetically dilute samples whose epr line shapes show superior resolution suitable for extracting spin-Hamiltonian parameters. Spectra for aquo complexes of transition metal ions, a metal complex, and a neutral free radical are shown to illustrate the dramatic improvements in spectral resolution for the gel-frozen samples. Introduction Solid state electron paramagnetic resonance (epr) spectra of transition metal complexes contain useful information about structures and electronic configurations of the complexes.2 For complexes which are formed in aqueous media, preparation of solid samples by freezing the solutions typically produces “magnetically concentrated” specimens which exhibit strong intermolecular magnetic interaction^.^ The “slow” freezing of an aqueous salt solution usually promotes ice crystal formation with consequent solute-solvent segregation. The segregated solutes do not freeze until an approximate eutectic composition is reached. This means that “local” concentrations of paramagnetic ions in the frozen samples may be of the order of 1 to 10 Furthermore, freezing rates which are sufficiently rapid to overcome the solute segregation effects are difficult to a ~ h i e v e . ~This , ~ paper describes the use of a polydextran gel to achieve magnetic dilution in frozen aqueous solutions. Epr spectra for aquo complexes of paramagnetic ions and an aqueous solution of a nitroxide free radical are presented to illustrate the method. Experimental Section Epr spectra were recorded on a Varian E-3 spectrometer operating a t 9.2 GHz. Temperature for all measurements was 77°K. Three techniques were used for obtaining frozen samples. (1) Aqueous samples were placed in 3 mm i.d. quartz tubes and frozen by dipping the tube into liquid nitrogen. ( 2 ) A special quartz tube as described by Bray and Pettersson’ was filled with methylcyclohexane and chilled in a bath of Dry Ice and acetone. The sample was taken up in a syringe fitted with a polyethylene nozzle (drawn to a fine point).8 The sample was injected into the cold The Journal of Physical Chemistry, Vol. 76,Z o . 9 , 1971
methylcyclohexane, and the resulting snow was packed into the bottom (3 mm i d . ) part of the tube. (3) Sephadex G-25-80 (Pharmacia), which had been allowed to swell in distilled water, was poured into a small Buchner funnel (15 mm i d . , 6 mm depth) and excess water was removed by suction. The aqueous sample (-0.5 ml) was poured over the bed of Sephadex and allowed to move into the gel for a minute without suction. Excess moisture was removed by gentle suction, and the moist gels were packed into 3 mm i.d. quartz tubes and frozen in liquid nitrogen. Results M ) which Epr spectra of llInClz solutions (5 X were frozen by the three different methods are compared in Figure 1. I n Figure 1A the sample was frozen by siniply dipping the quartz sample tube in liquid nitrogen. The absence of hyperfine structure is a consequence of strong intermolecular magnetic interact i o n ~ . The ~ spectrum obtained by rapid freezing9 is (1) This work was supported in part by Public Health Service Grant GM 12446 and by Public Health Service Fellowships GM 32,263 (J. S. L.) and GM 34,539 (G. H. R.). (2) J. 5 . Griffith, “The Theory of Transition-Metal Ions,” Cambridge University Press, London, 1961. (3) R.T . Ross, J . Chem. Phys., 42, 3919 (1965). (4) “International Critical Tables,” Vol. IV, E. W. Washburn, Ed., McGraw-Hill, New York, N. Y., 1928, p 254. (5) C. S. Lindenmeyer, G. T . Onok, K. A . Jackson, and B. Chalmero, J . Chem. Phys., 27, 822 (1957). (6) W. B. Hillig in “Growth and Perfection of Crystals,” R. H. Doremies, B.W. Roberts, and D. Turnbull, Ed., Wiley, New York, N. Y., 1958, p 350, (7) R. C . Bray and R.Pettersson, Biochem. J., 81, 194 (1961). (8) R.C. Bray, ibid., 81, 189 (1961). (9) Bray* has estimated that freezing times of -10 msec are feasible by this technique. Spectra with markedly better resolution than in Figure 1B have been obtained for us by Dr. Helmut Beinert using a “ram” freezing apparatus.
1203
EPRSTUDIES IN FROZEN AQUEOUS SOLUTIONS
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Figure 3. Epr spectra for 1 X 10-8 F solution of Cr (NOa)a: A, slowly frozen; B, frozen in Sephadex gel.
Figure 1. Epr spectra for 5 X 10-4 F solution of MnC12: A, slowly frozen; B, rapidly frozen; C , frozen in Sephadex gel.
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Figure 2. Epr spectra for 5 X lo-' P solution of Cu(NO& A, slowly frozen; B, frozen in Sephadex gel.
shown in Figure 1B. An improvement in the extent of magnetic dilution is evidenced by the appearance of 6KMnhyperfine structure. Figure 1C illustrates the dramatic improvement in spectral resolution achieved when the sample is imbedded in the polydextran gel prior to freezing. The hyperfine structure including "forbidden" hyperfine transitions is partially resolved, and line widths of less than 20 G are observed. Proton relaxation rate measurements for water in the unfrozen gels gave no evidence for Mn(I1) complexation to the dextran matrix.
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Figure 4. Elpr spectra for 1 x 10-3 M solution of 2,2,6,6-tetra1nethylpiperidinol-4-oxyl-l: A, slowly frozen; B, frozen in L3ephadex gel.
Some of the spin-Hamiltonian parameters for the aquo-cupric ion can be readily obtained from the gelfrozen spectrum for a Cu(NO& solution shown in Figure 2B. The resolution in the spectrum 2B should be contrasted to the structureless spectrum (Figure 2A) obtained by the slow freezing method. From the The Journal of Physical Chemistry, Vol. 76, N o . 9,1971
1204 hyperfine structure in the axially symmetric, powder line shape one calculates A l l = 117 G. Values for g, and gil are estimated to be 2.10 and 2.38, respectively. Analogous results for the aquo-chromic ion are shown in Figure 3. The solution was made up from the hydrated nitrate salt. Slow exchange of the six hydration waters from the chromic ionlo precludes any complex formation with the gel. The peak to peak line width of the gel-frozen spectrum (Figure 3B) is only 40 G compared with the 200-G line width of the slowly frozen sample (Figure 3A). Spectra for frozen aqueous solutions (1 mM) of an uncharged molecule (2,2,6,6-tetrame t hylpiperidinol-4oxyl-1) which contains a nitroxide free radical are given in Figure 4. The spectrum for the slowly frozen sample shows the intermolecular interactions brought about by solvent-solute segregation. From the gelfrozen spectrum one can immediately measure A , = 33.3 G. Other components of the A tensor and the g tensor components can, in principle, be estimated by spectral simulations. The epr spectra for the slowly frozen samples in Figures 1 4 illustrate the effects of dipolar and/or exchange broadening. If during the freezing process the unpaired spins of neighboring molecules are brought into sufficiently close proximity, electron exchange interactions become strong enough to narrow the resonance signal. The slowly frozen spectrum for the M n (11)-adenosine triphosphate complex (Figure 5A) shows this exchange narrowing phenomenon. Spectra for rapidly frozen samples were broader than that in Figure 5A but exhibited no hyperfine structure. The line width of 260 G in Figure 5A is to be contrasted to the -500-G hyperfine envelope in the gel-frozen spectrum (Figure 5B).
Discussion The results presented here indicate that imbedding aqueous solutions in polydextran gels prior to freezing effectively eliminates solvent-solute segregation, and the resulting samples are magnetically dilute. The gel method is faster, more convenient, and gives better results than the rapid freezing method. Ross3 has demonstrated the use of high concentrations of salts or miscible organic liquids to induce glass formation in the frozen aqueous samples. However, for solutions of transition metal complexes the high concentrations of "inert" solutes may directly or indirectly perturb the chemical equilibria and thereby alter the chemistry of
The Journal of Physical Chemistry, Vola76,No. 9,1971
JOHN S. LEIGH,JR.,AND GEORGE H. REED
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Figure 5. ISpr spectra for Mn(I1)-adenosine triphosphate, MnClz = 1 X 10-8 F , ATP = 1.1 X F , pH = 8.0: A, slowly frozen; B, frozen in Sephadex gel.
the sample. Our preliminary experiments have indicated no complex formation between the gel and transition metal ions. Thus the gel appears to function as a truly inert matrix and chemically distinct samples are obtained by the gel-freezing method. Epr studies of transition metal complexes with biological macromolecules are often carried out in frozen aqueous solutions. l1 While the macromolecules themselves provide for effective magnetic dilution, upon freezing the "local" buffer or salt concentrations may reach levels detrimental to the integrity of the tertiary or quaternary structure of the macromolecule. Polydextran gels with pore sizes sufficient to accommodate the macromolecule could provide protection against high salt concentrations which might otherwise result during freezing process. Freezing aqueous solutions of paramagnetic species in a polydextran gel provides samples whose epr spectral line shapes are suitable for deriving spin-Hamiltonian parameters, I n addition quantitative analyses of paramagnetic components of aqueous samples could be performed through use of an appropriate internal standard signal. (10) J. P. Hunt and R. A . Plane, J . Amer. Chem. SOC.,76, 5960 (1954). (11) H.Beinert and G. Palmer, Advan. Enzymol., 27, 105 (1965).
