4806
J . Phys. Chem. 1993,97, 4806-4814
Increasing Contact Ion Pairing in the Supercooled and Glassy States of “Dilute” Aqueous Magnesium, Calcium, and Strontium Nitrate Solution. Implications for Biomolecules Gerhard Fleissner, Andreas Hallbrucker, and Erwin Mayer’ Institut fur Anorganische und Analytische Chemie, Universitat Innsbruck, A-6020 Innsbruck, Austria Received: December 29, 1992
FTIR spectra of aqueous DzO solutions of magnesium ( O S and 0.05 M), calcium (0.5,0.2,0.05, and 0.02 M) and strontium ( O S M) nitrate in their glassy states at 78 K and of 0.5 M calcium nitrate in supercooled solution from 300 to 235 K are reported and compared with those recorded at 300 K. All four fundamental vibrational regions of the nitrate ion and a combination band could be evaluated by careful subtraction of the D20 solvent. Evidence for increasing contact-ion pairing in going from ambient temperature to the glassy state is based on (i) development of a new band in the v4 band region at 727 cm-* (Mg) and increasing intensity of the band at ~ 7 4 cm-I 0 (Ca and Sr), (ii) doubling of bands in the V I and v 2 band regions, and (iii) fourth-derivative curves of the v j band region and its curve fitting (Ca). The latter also indicated, in addition to “free” nitrate and the contact-ion pair, the presence of a third species which is tentatively assigned to ion triplets. Relative intensities of bands due to “free” nitrate and contact-ion pair were determined by curve fitting the v4, v2, and v 1 band region. For the glassy 0.5 M nitrate solutions, the relative area of the band assigned to the contact-ion pair is Sr > C a z M g for the v4 band region and Sr > C a > Mg for the V I band region. Evidence for increasing ion pairing is seen already in supercooled solution at 1260 K. We suggest that water’s structural changes toward a more open, fully hydrogen-bonded tetrahedral network in its supercooled and glassy state are at the bottom of increasing contact-ion pairing with decreasing temperature. Implications for biomolecules stabilized by contact-ion pairs are discussed with respect to cold denaturation of proteins, cryoenzymology, and cryofixation. Introduction We have recently reported that contact-ion pairing and aggregation increases in dilute aqueous solutions of alkali-metal thiocyanates when going from ambient temperature to the supercooled and glassy state.’ The evidence was based on FTIR spectra of the CN stretching band region. Increasing contaction pairing with decreasing temperature came as a surprisebecause the opposite trend is generally observed for the ambient to subcritical temperature region2“ We had attributed it to water’s density maximum and the anomalies of supercooled water and aqueous solutions7which are caused by structural changes toward a more open, fully hydrogen-bonded tetrahedral network in its supercooled and glassy state.8-’2 By “dilute” we understand in this and the earlier study’ electrolyte concentrations which still show the anomalies of supercooled water,7 although to a lesser extent. We now extend our FTIR spectroscopic study of contact-ion pairing in the glassy state obtainable by “hyperquenching”’3-17 to aqueous solutionsof magnesium, calcium,and strontium nitrate. Calcium nitrate was chosen because for this electrolyte Spohn and Brill6 recently have shown by Raman spectroscopy increasing contact-ion pairing in going from 299 to 723 K. Magnesium and strontium nitrate were included to study the effect of ionic radius and to enable a comparison with the work of Irish and co-workers18-2° at mainly ambient temperature. The three electrolytes investigated in this study show similar behavior as the alkali-metal thiocyanates,’ i.e., increasing contaction pairing in going from ambient temperatureto the supercooled and glassy temperature region. For calcium nitrate in particular we can show for the first time, from a comparison with Spohn and Brill’s work6, that for the same solute contact-ion pairing increases both in going from ambient temperature either to the supercooled and glassy state or to subcritical temperature. Increasing contact-ion pairing with decreasing temperature in going to the supercooled and glassy state can have implications for the structure and function of biomolecules in supercooled and glassy dilute aqueous solution because contact-ion pairs can 0022-365419312097-4806%04.00/0
contribute significantly to their thermal ~tability.~I-~’ We will consider in particular its possible importance for cold denaturation of for c r y o e n z y m ~ l o g yand , ~ ~for ~ ~~ryofixation.~~35 ~
Experimental Section The glassy, or vitrified, solutions were prepared by so-called “hyperquenching” of aerosols on a CsI window held at 78 K. Briefly, droplets of =3-pm size made by means of an ultrasonic nebulizer were suspended in N2gas and allowed to enter a highvacuum cryostat through a 400-pm aperture. Once inside, the droplets moved toward the CsI window at supersonic speeds and deposited on it. Estimated cooling rate is 106-107 K s-l.33 For further experimental details see refs 1 and 13-17. Salts of p.a. quality were from Merck. The hydrates were dried over P2O5, and solutions were made with DzO(99.7%). The concentrations given in the figures are those of the standard solutions used for generating the aerosols. There are changes in concentration during the various stages of the vitrification procedure, as pointed out in refs 13 and 15. For investigation of the supercooled region, 0.5 M Ca(N03)2 solutions were emulsified in an oil phase containing 75 wt % liquid paraffin and 25 wt % lan0lin3~9~~ and investigated between AgCl plates. The influence of the emulsifier on spectral quality is discussed in detail in refs 1 and 37. The FTIR spectra were recorded in transmission on Biorad’s FTS 45 at 2-cm-’ resolution (UDRl), by coadding 1000 scans. Peak maxima are given in the figures to 0.1 cm-I and were found to be reproducibleto a few tenths of a wavenumber. Deconvolution parameters and pass bands for second and fourth derivatives are given in the figure legends. Note that the second derivatives are shown inverted. Distortion of the infrared spectra by the Christiansen effect, as reported for the OH (OD) stretching band region of vitrified water,I4 seems to be less pronounced for the spectra of dissolved electrolytes shown in this and the earlier study.’ The amount of crystalline ice in the vitrified samples was determined via the decoupled OH stretching transition from 0 1993 American Chemical Society
Increasing Contact Ion Pairing residual H20 in the deuterated solutions. For this the sample was heated to 160 K, held at 160 K for 15 min for complete crystallization, and cooled to 78 K, and its spectrum recorded at 78 K. The amount of crystalline ice in a hyperquenched solution could be determined to f2% by comparison of its spectrum with that of the crystallized sample but recorded at the same temperature of 78 K. For 0.5 and 0.2 M solutions, no ice could be detected this way. The 0.05 and 0.02 M solutions contained 15% ice, respectively. Spectra of the supercooled 0.5 M Ca(N03)2 solution in D 2 0 were recorded in transmission, with the sample between AgCl plates, from 298 to 235 K in emulsified solution. The absence of crystalline ice and also of crystalline hydrate was ascertained by followingthe phase transition via the decoupled OH stretching transition from residual H20. For the comparatively uneven AgCl plates optimal sample thicknesswas obtained without spacer, in contrast to the earlier study.’ The Raman spectrum was recorded on a computerized Coderg PHO instrument at 4-cm-’ resolution by using 488-nm excitation. For curve fitting, either Biorad’s bandfit software was used with the Nelder-Mead algorithm or Spectra-Calc’s Curvefit. The quality of the fits was controlled by comparing the deconvoluted original band with the deconvoluted sum of the curve-fitted component bands, as suggested in ref 38. For curve fitting, the spectrum of the solvent was subtracted either with a glassy electrolyte solution of closely similar band shape (e.g., 0.5 M CaC12solution) or with the multipoint spline function routine.
Results and Assignment An aquated nitrate ion is known to generatevibrational bands at -718 ( ~ 4E’), , 4 3 0 ( ~ 2A”), , =lo49 (v’,AI’), and -1354 and -1410 ( ~ 3 ,E’) c ~ - ~ . ~Lowering J* of the hypothetical D3h symmetry is evident by splitting of u3 into two components, and often by infrared activity of V I . Contact-ion pair formation is seen most clearly by development of a second band at -740 cm-I. Sometimes doubling of the nondegenerate vI and u2 bands, and development of a second and third set of bands in the u3 region has been observed. The above-given notation is strictly valid only for D3h symmetry, but in the absence of detailed knowledge of the symmetry of theaquated or contact-paired ion it is common praxis to use it also for the latter. We will speak in the following of “free” nitrate but understand that it contains varying amounts of solvent-shared and/or solvent-separated ion pairs which are spectroscopically undistinguishable. In H20 solutions only the V I , u2, and v3 band regions are observable by FTIR spectroscopy because the u4 band region between 700and 750 cm-I is covered by water’s intenselibrational band. In D20 solution this band is shifted sufficiently to lower frequency to allow, by careful subtraction of the solvent spectrum, for 0.5 M solutionsevaluation of all four fundamental vibrational regions. Therefore we have studied mainly D20 solutions, and have investigated only several H 2 0 solutions to confirm similar contact-ion pairing for glassy H 2 0 and D20 solution. FTIR spectra of the same stock solutions were recorded at 300 K for comparison and are also shown. Figure 1 depicts for demonstration the original FTIR spectrum (lower curve) of a hyperquenched and glassy 0.5 M Ca(N03)2 solution in D20 from 2000 to 500 cm-l and the bands due to nitrate ion after subtraction of the solvent (upper curves), with ordinate scaling factors as given in the figure. The weak band at -1 780 cm-I is a combination band of v1 and u4; the other bands are in the characteristic fundamental band regions. Proper subtraction of the glassy solvent spectrum is not trivial and most critical for the band at -720 cm-1 because in this region the intensity of the D2O librational band increases steeply. In the original spectrum no band can be observed at -720 cm-I, only a weak peak at -740 cm-I can be seen. Subtraction of a
The Journal of Physical Chemistry, Vol. 97, No. 18, 1993 4807
-I
2m
u I
I
1600
1200
I
800
cm-I
Figure 1. FTIRspectrum of 0.5 M calcium nitrate DzOsolution recorded at 78 K i n its glassy state (bottom) and nitrate’s fundamental modes and combination band after subtraction of the solvent (top). The scaling factors refer to the ordinate.
glassy D 2 0 spectrum is unsuitable because the band shape is altered by the electrolyte. We have generated therefore FTIR spectra of glassy D20 solutions with electrolytes which do not have bands in this critical region. A 0.5 M glassy CaC12solution was finally chosen for subtraction because its band shape in the librational region most closely resembled that of the nitrate solutions. After subtraction of the solvent spectrum a multipoint spline function routine was used to generate a flat base line. Nevertheless, the two bands at -720 and 740 cm-I do show differences in relative intensities even when using spectra of very similar band shape for subtraction of the solvent. We estimate the error in our procedure to be *5% of the relative intensity of the two curve-fitted component bands. Subtraction of the solvent in the other fundamental band regions and in the region of the combination band for both spectra recorded at 78 and at 300 K is straightforward and less subject to error. For the u1 band region, subtraction of the D20 deformation band either with a multipoint spline function routine or with the spectrum of a D20 solution gave, after curve-fitting, relative intensities of the two components within 1%. For D2O solutions recorded at 300 K, subtraction of the solvent spectrum in the u4 band region is also simple because the intensity of the librational band increases more gradually in solution than in the glassy state, and solvent subtraction by either method described above also gave results within 1%. The experimentaldifficultiesin correct subtraction of the glassy solvent spectrum in the u4 band region aresimilar to those reported frequently for subtraction of water’s deformation band from the amide I band region of proteins (see for example ref 39). It requires FTIR spectra of high quality and therefore was problematic or impossible with dispersive IR instruments. In the following we show as so-called “original” bands spectra of the nitrate ion but after subtraction of the solvent spectrum. Only the v3 band region is shownwithout subtractionof the solvent. The number of component bands in composite band profiles was determined by deconvolution and second and fourth derivatives. The curve-fitted component bands are also shown in the figures. v4 Band Region from 700 to 750 cm-1. We start with this region because, as pointed out above, it is most informative for observing contact-ion pairing. In Figure 2 are compared spectra of hyperquenched and glassy 0.5 M magnesium, calcium, and strontium nitrate (a) with those of the solution recorded at 300 K (b). Increasing contact-ion pairing in going from 300 K to the glassy state is seen most clearly from a comparison of the two spectra of magnesium nitrate solution on top: at 300 K, only one band assigned to the aquated nitrate ion, containing solventseparated and for solvent-shared ion pairs, is observed at 719 cm-1. This is consistent with Chang and Irish’s report that at 298 K contact-ion pairing starts only at >3.9 M.I8 In the glassy state, however, the original band profile shows clearly two components, with relative intensity of 71% for the band at high frequency (727 cm-1) and 29% for the band at low frequency (716 cm-I).
4808 The Journal of Physical Chemistry, Vol. 97, No. 18. 1993
Fleissner et al.
I
,
I
Figure 2. v4 band region, after subtraction of the solvent, and the curvefitted component bands of 0.5 M magnesium, calcium and strontium nitrate D20 solution recorded at (a) 78 K in their glassy states and (b) 300 K in solution.
TABLE I: Curve-Fitting Analysis of the u4 Band Region (from 680 to 780 cm-I) of 0.5 M Magnesium, Calcium, and Strontium Nitrate Solution in D20,Recorded Both at 78 K in Their Glassy States and at 300 K in Solution cation Mg Ca Sr
vmlr (cm-l)
fwhh (cm-l)
% Gauss
(A) Recorded at 78 K in Their Glassy States 126.7 15.4 99 716.1 10.1 100 740.0 14.0 99 718.8 12.3 100 736.5 13.9 94 719.1 10.5 100
% relat area 71 29 72 28 80 20
(B) Recorded at 300 K in Solution Ca Sr (Mg
736.6 718.0 73 1.3 717.6 718.8
10.7 16.1 11.2 12.5 16.1
86 100 70 100
51 49 55 45
original band, not fitted!)
For the calcium and strontium salts we also observe in going from 300 to 78 K an increase in the relative intensity of the band a t -740 cm-I assigned to contact-ion pairs.4s18-20The relative intensity of the band assigned to contact-ion pairs increases as Sr > Ca > Mg for spectra recorded at 300 K,4,18-20 but it is Sr > Ca E Mg for the spectra recorded in the glassy state. Peak frequencies (umax), full- widths at half-height (fwhh), percent Gauss, and percent relative band area are listed in Table I. At 300 K the fwhhof the curve-fitted band at high frequency, assigned to contact-ion pairs, is -11 cm-I, but it is 14-15 cm-I for the spectra recorded at 78 K. This is unusual because we expect, and indeed have observed for other band regions, decrease in fwhh with decreasing temperature. It possibly indicates for the spectra of glassy solutions the presence of a second component which cannot be resolved. For magnesium nitrate in glassy solution the peak frequency of the band at high frequency is much lower than those for Ca and Sr (727 cm-l vs 740 and 737 cm-I, Table I) and possibly reflects differences in the polarizing power of the cations. But, its value is still within the range assigned to contact-ion pairs in aqueous solution, e.g., 728 cm-l for Na+N03- or Ag+N03- (see Table 15 in ref 4). We are aware of Irish and Brooker’s discussion (p 286 in ref 4) of the u4 band region of the Raman specttum of aqueous beryllium nitrate solution, with two resolved components a t 717 cm-I for the free ion and at 725 cm-I: the latter band was attributed to either contact-ion pairs or solvent-shared ion pairs, and definite assignment was not possible. We nevertheless prefer to assign the band at 727 cm-1 to Mg2+N03- contact-ion pairs,
$2 780
760
740
720
700
cm-1
Figure 3. v4 band region of the Raman (top) and infrared spectrum (bottom, after subtraction of the solvent) and the curve-fitted component bands of a 2.7 M strontium nitrate DzO solution recorded at 300 K.
despite its similar value and the above-mentioned discussion, for the following reasons: (i) the ut band region of glassy magnesium nitrate solution consists clearly of two components (Figure 4) and (ii), in the u3 band region splitting of the two main components increases in going from the liquid to the glassy state from 46 to 61 cm-1 which is similar to that observed for Ca and Sr (Figure 7), and in the fourth-derivative curves of the glassy state spectra similar patterns of peaks are observable for all three cations (Figure 8). An estimate for the relative concentrations of the two species can be obtained from the relative integrated intensities of the two curve-fitted band components as follows: the molar Raman intensities of the bound and free forms of nitrate are effectively equal for both the doublets in the vI and u4 region,u but the molar infrared intensities are not. Irish and co-worker~~*~O have remarked that for the u4 band region the molar infrared intensity of the bound species is much larger than that of the “free” species. In Figure 3 we now compare the Raman spectrum of a 2.7 M S r ( N 0 3 ) solution ~ in D20 with the FTIR spectrum of the same solution, both recorded at 300 K. This concentration was chosen because Bulmer et aLzohave reported and analyzed its Raman spectrum. The band shape of the Raman spectrum is similar to that shown in Figure 1 of ref 20 except for the better resolution of the two components in our curve. The relative integrated intensity of the curve-fitted Raman spectrum is 42% for the component at 717 cm-l and 58% for the component at 734 cm-I, but for the FTIR spectrum it is 22% for the band at 717 cm-I and 78% for the band at 733 cm-I! Therefore, for equal molar Raman intensities, the molar infrared intensity of the band at ~ 7 4 cm-I 0 due to contact-ion pairs is about 2.6 times larger than the molar infrared intensity of the band a t =720 cm-1 due to aquated nitrate. This factor allows in principle to obtain an estimate for the relative molar concentration of the bound and “free” nitrate ion. It gives for the glassy 0.5 M strontium nitrate solution -60% contact-ion pairs, and for the calcium and magnesium salt 4 0 % . But these values should not be taken too literally because the factor can depend on the cation,4 concentration, and temperature. We have to point out that the relative integrated intensities of our curve-fitted Raman spectrum differ somewhat from those reported by Bulmer et al.,zo despite similar overall band shape of the two spectra. It is conceivable that better resolution in our Raman spectrum gives a better fit of the composite band.
Increasing Contact Ion Pairing
The Journal of Physical Chemistry, Vol. 97, No. 18, 1993 4809 b
,
1
IomJ
I
I
Sr Figure 4. (a) YI band region, after subtraction of the solvent, and the curve-fitted component bandsof 0.5M magnesium, calciumand strontium nitrate D20 solution recorded in their glassy states at 78 K. (b) Corresponding deconvoluted spectra (with fwhh = 14 em-I and K factor of 2.5).
b
860
840
Figure 6. (a)
820
800 cm-1
umax(em-[)
fwhh (em-')
% Gauss
% relat area
1046.7 1036.6 1047.4 1036.2 1045.6 1035.1 1044.8 1034.4 1044.9 1034.6 1045.1 1034.6 1045.8 1034.8
12.5 15.0 10.7 14.6 13.2 13.5 12.5 13.3 11.0 12.4 10.8 12.2 12.9 13.1
100 44 39 74 100 81 100 68 100 71 100 60 100 73
43 57 26 74 69 31 51 43 43 57 41 59 77 23
Ca (0.5M) Ca (0.2MI Ca (0.05M)
- 2
0.02M
Ca (0.02M)
Sr (0.5M) cm-1 YI
840
cation
Mg (0.05M)
Figure 5. (a)
8-40
Me. (0.5M l I .
u, / \
800 cm-1
band region, after subtraction of the solvent, and the curve-fitted component bands of 0.5M magnesium, calcium, and strontium nitrate D20 solution recorded in their glassy states at 78 K. (b) Deconvoluted spectra (with fwhh = 8 em-' and K factor = 2.5).
TABLE Ik Curve-FittingAnalysis of the V I Band Region (from 1000 to 1100 cm-l) of Magnesium (0.5 and 0.05 M), Calcium (0.5, 0.2, 0.05 and 0.02 M), and Strontium (0.5 M) Nitrate DzOSolution Recorded in Their Classy States at 78 K
a
1i
820 v2
band region and the curve-fitted component bands of
0.5,0.2, and 0.02M calcium nitrate DzO solution recorded in their glassy states at 78 K. (b) Deconvoluted spectra (with fwhh = 14 em-' and K factor = 2.5 for 0.5and 0.2M solution, but 1.5 for 0.02 M solution). Note in both sets of curves the increasing intensity of the band assigned to "free" nitrate at low frequency with increasing dilution. V I BandRegion from 1000 to 1100 cm-I. In Figure 4 we show R I R spectra for glassy 0.5 M magnesium,calcium,and strontium nitrate D20 solution. The original bands (a) show clearly asymmetry at low frequency. Their deconvoluted spectra (b) show resolution into two bands, with peak frequencies given on the bands. The curve-fittedband components (a) show increasing intensity of the high-frequency component in going from Mg to Sr. Spectra at ambient temperature are not shown because only one band at =lo45 cm-I could be detected. Figure 5 shows for glassy Ca(NO& solution in D20 the influence of concentration on the relative intensities of the two component bands. In the original curves (a) the intensity of the component at high frequency clearly decreases in going from 0.5 to0.2 and 0.02 M. Similar behavior is found for the deconvoluted spectra (b). The relative integrated intensities of the two component bands are quantified by curve-fitting (a). The spectra shown in Figures 4 and 5 are consistent with the assignmentof thecomponent band at -1035 cm-1 to "free" nitrate ion, and of the band at -1045 cm-' to contact-ion pairs. The band parameters are listed in Table 11. For further support of the assignment we have also studied for the magnesium salt the influence of concentration, by recording
the FTIR spectrum of a 0.05 M hyperquenched and glassy solution. The result of the curve-fitting analysis of the v l band region is also included in Table 11. It is in line with the abovegiven assignment because the relative band area of the component at high frequency decreases upon dilution. v2 Band Region from 800 to 860 cm-I. Figure 6 shows original spectra (a) for glassy 0.5 M magnesium, calcium, and strontium nitrate solution and their deconvoluted spectra (b). Band asymmetry at high frequency is seen for both Ca and Sr in both the original curves and the deconvoluted spectra (marked by arrow), but it is only weakly observable in the deconvoluted spectrumof Mg. For Ca and Sr the band asymmetry was resolved into two component bands (a). The band at 824 cm-I is assigned to the contact-ion pair, the band at 831 cm-I to the "free" nitrate ion. Relative areas of the component bands are similar to those assigned in the v4 and V I band region to the two nitrate species. We have to point out, however, that this curve-fitting analysis is the least reliable because of severe band overlap. Band overlap is probably also the reason why we cannot observe two components for the Mg salt. The band parameters are summarized in Table 111. v3 Band Region from 1200 to 1600 cm-'. Figure 7 compares original spectra (without solvent subtraction) of glassy 0.5 M magnesium, calcium, and strontium nitrate solution in DzO recorded at 78 K (a) with those recorded in solution at 300 K (b). The most obvious difference between the two sets of curves is the much better separation of the two main componentsin the spectra of the hyperquenched glasses. In Figure 8 we show the fourth-
4810
I
i
I
Fleissner et al.
The Journal of Physical Chemistry, Vol. 97, No. 18, 1993
l
1600
A
I
l
1500
1400
1300 cm-1
1600
,
,
1500
1400
'
1300 cm-1
Figure 7. v3 band region of 0.5 M magnesium, calcium, and strontium nitrate D20 solution recorded at (a) 78 K in their glassy states and (b) 300 K.
a 1600
Figure 9. Top:
1500
1400
1300
cm-1
band region, after subtraction of the solvent, of 0.5 M calcium nitrate D2O solution recorded in the glassy state at 78 K, and the six curve-fitted component bands. Bottom: comparison of the deconvoluted spectra obtained from the original band and from the sum of the curve-fitted component bands (with Spectracalc/Curvefit: y = 12.3 and K = 0.097).
I
1600
#
,
1500
I
,
1400
,
,
,
l
1300 cm-1
1 , , 1600 1500
I
I
1400
,
,
; ,
1300 cm-1
Figure 8. Fourth-derivativecurves of the spectra shown in Figure 7: (a) spectra recorded at 78 K in their glassy states; (b) recorded at 300 K. The labeling of the peaks for glassy calcium nitrate solution in (a) is the same as that used in Figure 9 for the curve-fitted component bands (passband = 0.10).
TABLE IIk Curve-Fitting Analysis of the v2 Band Region
(from 800 to 860 cm-1) of 0.5 M Calcium and Strontium Nitrate D20 Solution Recorded in Their Glassy States at 78 K cation Ca Sr
(Mg
umax (cm-1)
fwhh (cm-I)
830.7 824.3 830.5 824.6 828.9
9.1 9.6 7.7 8.3 10.4
% Gauss
7% relat area
100 22 84 78 100 19 85 81 original band, not fitted!)
derivative curves of the original spectra recorded in the glassy state at 78 K (a) and at 300 K (b). The quality of these curves and the high signal-to-noiseratio can be judged by the nearly flat line from -1 500 to =1600 cm-I. The features below 1300 cm-I are due to interference from D20's deformation band. The fourth-derivative curves reveal a larger number of bands in the spectra of the glassy solutions (a) than in those recorded at 300 K (b). The two most intense bands at ambient temperature are also observable in the glassy state at similar frequencies, but the latter contains a number of additional bands. We have attempted curve-fitting of v3 composite bands, by entering frequencies either from fourth-derivatives and/or from the deconvoluted spectra, but had limited success because of the large number of components. For the same composite band we could obtain fits with widely differing relative intensities of the components, but similar "quality" in terms of root-mean-square
u3
(RMS) values. Similar problems in curve-fitting the v3 band region into three pairs were encountered by others.4' Only for the v3 band region of glassy 0.5 M Ca(N03)2solution we could obtain a curve fit which we consider reliable. Figure 9 (top) shows theoriginal band, but after subtractionof thesolvent and correction of the base line and six curve-fitted component bands. Below are compared the deconvoluted spectrum of the original curve with that of the sum of the curve-fitted component bands. Similarity between the two deconvolutedcurves was taken as indicator by Friesen and Michaelian3*for the quality of a fit and was achieved here by altering in small steps peak positions of the component bands. The same procedure is possible in principlefor v3 band regions of other solutionsbut it is very tedious and time consuming. This fit requires six component bands (labeled 1-6) and is therefore indicative for the nitrate ion in three distinct environments. It also indicates that for solutions in the glassy state ion pairing is more complex than suggested by doubling of the bands in the v4, V I and v 2 band region. A tentative assignment is based on a comparison with our spectra recorded at ambient temperature and with Raman spectroscopic studies of aqueous nitrate solutions. From Irish and Walrafen's'g Raman study of aqueouscalcium nitrate solution we can deduce for a 0.5 M Ca(N03)2 solution a concentration of =20%bound and 4 0 % "free" nitrate (read from their Figure 5 and assuming equal molar intensities). Therefore, the two most intense bands in the fourth-derivative curves of spectra recorded at 300 K (Figure 8b) can be assigned to "free" nitrate. In the curve-fit shown in Figure 9, component bands are at similar frequency and are therefore also assigned to "free" nitrate (labeled 3 and 5 ) . Component bands at 1365 and 1438 cm-I for 1 M aqueous Sr(N03)2 solution recorded at 298 K were assigned by Bulmer et al.zoto contact-ion pairs. The curve fit shown in Figure 9 shows bands at similar frequency, namely at 1367 and at 1442 cm-1 (labeled 4 and 2) which are also assigned to contact-ion pairs. This leaves two bands in the curve-fit, with peak maxima at 1311 and 1483 cm-' (labelled 6 and 1) for assignment. The separation of the two peak maxima, i.e., Av3, is much larger than those of the two other sets. We assign tentatively this pair of bands to an ion triplet. Bands with similar frequency and Av3
Increasing Contact Ion Pairing
The Journal of Physical Chemistry, Vol. 97, No. 18, 1993 4811
e
lb
P
dl
u - i ,A\*, Sr
1860 1820 1780 1740
.,
1700cm -1 1700cm
,
~
E A
i8ko
J S
1860 1820 1780 1740 1700cm-1
Figure 10. Combination band region ( V I + u4, from 1700 to 1860 cm-I) and the curve-fitted component bands of 0.5 M magnesium, calcium and strontium nitrate D20 solution recorded at (a) 78 K in their glassy states and (b) 300 K.
TABLE I V Curve-Fitting Analysis of the v3 Band Region (from 1200 to 1600 cm-l) of 0.5 M Calcium Nitrate DtO Solution Recorded in Its Classy State at 78 K
1
1 ~ o1780 1fho 1
I
i+wC~-l IS60
1
1820
1780
1 2 3 4 5 6
vmax (cm-I)
1483.2. 1442.0 1408.5 1367.0 1343.3 1311.1
fwhh (cm-I)
% Gauss
% relat area
44.8 49.2 45.9 32.1 37.4 33.3
48 97 85 100
8 20 25 12 26 9
100 0
splittings were reported for concentrated aqueous Zn(NO& solution recorded at ambient temperature40and at 573 K.5 The Av3 splittings for the three distinct nitrate species of 65,75, and 172 cm-' are consistent with increasingperturbation of the anion in going from the "free" ion to the contact-ion pair and the ion triplet. The band parameters are summarized in Table IV. Ion triplets can also be recognized by a band at 2750 to 760 cm-I in the v4 band regiona4 We did not observe a band in this region in resolution-enhancedcurves, possibly because of its low intensity. But it is conceivable that in spectra of glassy solutions it is at similar frequency as the band due to contact-ion pairs. This would explain the unexpected increase in fwhh of this band in going from 300 to 78 K (Table I). The fourth-derivativecurve of the spectrum of glassy calcium nitrate solution (Figure Sa) shows peaks at similar frequency as the curve-fit and the deconvoluted spectrum (see Figure 9 and Table I) and is thereforeindicative for its usefulness for estimating peak positions in a strongly overlapping composite band. These peaks are also labeled 1-6 and have frequencieswhich are shifted at most by a few wavenumbers, but they are better resolved than in the deconvoluted spectrum. It is important to note that in the fourth-derivativecurves of the spectra of glassy magnesium and strontium nitrate solution (Figure Sa) similar patterns of peak frequencies are apparent. We take this as a first indication that these two glassy solutionsalso contain three distinct nitrate species. Combination Band Region, from 1700 to 1860 cm-1. In Figure 10 are compared spectra of hyperquenched and glassy 0.5 M magnesium, calcium, and strontium nitrate solution (a) with those of the solution recorded at 300 K (b). Figure 11 depicts their deconvoluted spectra in the same order. For the calcium and strontium salt deconvolution separates the original asymmetrical bands into two components. For the magnesium salt only one band was observed even in deconvoluted spectra. However, its increase in fwhh in going from 300 (20 cm-I) to 78 K (30 cm-1) is indicative of two species which cannot be resolved. The
1700cm-1
TABLE V Curve-Fitting Analysis of the Combination Band Region ( V I wh from 1700 to 1860 cm-') of Calcium (0.5 and 0.2 M) and Strontium (0.5 M) Nitrate D20Solution, Recorded Both at 78 K in Their Classy States and at 300 K in Solution W relat cation Y ,, (cm-I) fwhh (cm-I) W Gauss area
+
~
component
,
1740
Figure 11. Deconvoluted spectra of the spectra shown in Figure 10: (a) recorded at 78 Kin their glassy states and (b) at 300 K. (fwhh = 25 cm-I and K factor = 1.8).
~~
(A) Recorded at 78 K in Their Glassy States Ca (0.5 M) Ca (0.2 M)
Sr (0.5 M) (Mg (0.5 M)
1785.4 1764.3 1784.2 1762.2 1783.9 1765.6 1767.0
20.1 25.4 20.3 28.0 20.7 23.2 30.3
89 98 85 98 92 100
29 71 25 75 31 69
original band, not fitted!)
(B) Recorded a t 300 K i n Solution Ca (0.5 M) S r (0.5 M) (Mg (0.5 M)
1783.2 1762.4 1777.7 1762.3 1762.7
14.9 24.4 15.6 18.9 19.7
100 64 96 94
9 91 14 86
original band, not fitted!)
component bands obtained by curve-fitting are also shown in Figure 10and the band parameterssummarizedin Table V.Curvefitting parameters of a 0.2 M glassy calcium nitrate solution are also included in this table. For the calcium and strontium nitrate spectra recorded at 300 K, we assign the weak band at high frequency to the contact-ion pair obtained by combination of their fundamental bands in the V I and v4 band region, and the band at low frequency to yfree" nitrate. The same assignment of the two band components in glassy solution (Figure loa) gives good agreement between calculated and observed peak values for the component at high frequency but a difference of -10 cm-1 for the component at low frequency. For glassy 0.5 M calcium nitrate solution the relative band area due to contact-ion pairs is -29%, but for the 0.2 M solution it is -25%. This decrease in band area with increasing dilution is further support for our assignment. Relative band areas of the curve-fitted component bands of the combination band differ from those of the fundamental bands. We attribute this to a change in molar infrared intensity. We note that a band at 1768 cm-1 in saturated aqueous sodium nitrate solution has been assigned by Irish and Davisa to the combination v1 (A]') at 1049 cm-1 and v4 (E') at 719 cm-I. w 2 and v3 Band Regionof Supercooled Calcium Nitrate Solution. Supercooling of a dilute aqueous solution close to the temperature of homogeneous nucleation requires emulsification to avoid heterogeneous n~cleation.~We have previouslylJ7 and in this study investigatedspectra in detail for possible artifactsoriginating from emulsification,by comparingspectra of emulsified solutions with those of unemulsified solutions over the accessible temper-
4812 The Journal of Physical Chemistry, Vol. 97, No. 18, 1993
i
Fleissner et al.
10.02
300 K 260 K 250 K 240 K 235 K 78 K
78K
kaarGGz
860
840
si0
SOocm-~'
Figure 13. u2 band region of a supercooled 0.5 M emulsified calcium nitrate solution in D20 cooled in steps from 300 to 235 K: (a) original spectra, (b) and (c) their secondderivatives and thedeconvoluted spectra. The spectrum of the hyperquenched and glassy solution recorded at 78 K is shown for comparison. (Pass-band for b was 0.50,and fwhh and K factor for c was 14 cm-I and 2.5.)
cm- 1 Figure 12. u3 band region of a supercooled 0.5 M emulsified calcium nitrate solution in D20 cooled in steps from 300 to 235 K. The spectrum of the hyperquenched and glassy solution recorded at 78 K is shown for comparison. The deepening of the trough between the two main peaks, which increases with decreasing temperature, can be used to associate a spectrum with its temperature of recording. Imperfect subtraction of an emulsifier band caused the artefact at ==1470cm-'in the curves recorded from 300 to 235 K. ature range 1260 K. We found that emulsification leads to only minute changes of fwhh and peak maxima of bands as long as thesample thickness is kept sufficientlysmall. The v4 band region is unfortunately not accessible because of intense bands of the emulsifier, and the v I band region does not show temperaturedependent effects, but the v3 and v 2 band region does and is shown in the following. Figure 12 shows the original v 3 band region of a 0.5 M calcium nitrate solution recorded at selected temperatures from 300 to 235 K and for comparison the spectrum of a hyperquenched and glassy solution recorded at 78 K which was taken from the same stock. This set of curves shows a regular trend: the peak maximum of the main component at high frequency shifts with decreasing temperature from 1399 cm-I at 300 K to 1413 cm-I at 235 K, whereas the peak maximum of the main component at low frequency shifts very little. Therefore the separation of the two main components increases and the trough between the two main bands deepens with decreasing temperature. It is important to note that both trends are continued in going from 235 to 78 K by hyperquenching where the peak maximum has shifted further to 1419cm-I. At 235 K,whichis thelowesttemperatureaccessible by slow supercooling, the change in peak maximum at high frequency and separation of the two main components is about half of that observable in going from 300 to 78 K. We do not want to imply that this also means half of the increase in ion pairing because the composite band at I8 K consists of six component bands (Figure 9). But it indicates extensive changes in ion pairing already in supercooled solution, in a temperature region accessibleby slow cooling, toward that found in the glassy state. In Figure 13 are shown spectra of a supercooled and emulsified 0.5 M calcium nitrate solution from 780 to 860 cm-I at selected temperatures and are compared with those recorded in the glassy state at 78 K: (a) the original bands, (b) and (c) their secondderivativecurves and the deconvoluted spectra. In the resolutionenhanced curves a second component at low frequency is indicated already at 300 K by asymmetry of the band. Its intensityincreases with decreasing temperature, and it is clearly separated at -240 K from the main peak. This growing component has the same
peak frequency as the peak at 78 K assigned above to contact-ion pairs, and therefore indicates more clearly than Figure 12 increasing contact-ion pairing with decreasing temperature already in this temperature region accessibleby slow supercooling. We are aware that increasing intensity of the band at low frequency with decreasing temperature could also be interpreted by two unresolved component bands present already in the spectrum at 300 K which have different temperature dependence of their peak maxima, namely with a component at -829 cm-I whose peak maximum does not shift and a second component whose maximum shifts to lower frequency with decreasing temperature. This situation would also generate increasing separation of the two components but is basically different in that for this case the relative band areas of the two components, and therefore the concentrationsof the two distinct nitrate species, do not have to change with decreasing temperature. However, we consider it unlikely because, as Figure 13c shows, the peak maximum of the low-frequency component does not change with temperature from ~ 2 4 K, 0 where it becomes clearly separated from the main peak, to 78 K. V I / V ~Infrared Band Intensity Ratios. These ratios have been used as an indicator for perturbation of the nitrate ion by a cation, and to differentiatebetween electrostaticand increasinglycovalent i n t e r a ~ t i o n . ~For ~ , ~the ~ , ~0.5 ~ M solutions recorded at 300 K ratios of the total band intensity in the V I band region divided by that of the v 2 band region are 0.75 (Mg), 0.56 (Ca), and 0.63 (Sr). These values are lower than that reported for aqueous calcium nitrate and other alkaline-earth metal nitrate solution (1.45 f 0.12 for CaI9). For the 0.5 M hyperquenched and glassy solutions the ratios are 1.1 (Mg), 0.99 (Ca), and 0.78 (Sr), and for dilute glassy calcium nitrate solution the values are 0.99 (0.2 M) and 0.83 (0.05 M). There is clearly an increase in intensity ratio in going from solution at 300 K to the glassy state at 78 K, indicating increasing perturbation of the anion by the cation. The ratio is highest for Mg both at 300 K and in the glassy state. Similar behavior was reported for pure alkali metal nitrate melts.42 The ratiosobtained for the glassy state solutionsare clearly below the value of >2 which supposedly indicates increasing covalent interaction,I9 and therefore we conclude that the additional spectral features observable in the spectra of glassy solutions are due to electrostatic interaction. Discussion Vitrification of pure liquid water and dilute aqueous solutions by "hyperquenching" at estimated cooling rates of -106-107 K s-l (ref 33) has been confirmed by diffraction,l'-I3 differential scanning calorimetry,16J7 and infrared spectral study of the decoupled OH (OD) stretching mode.14 But, for dilute aqueous solutions, this does not exclude the possibility of liquid-liquid immiscibility by spinodal decomposition during quenching into
Increasing Contact Ion Pairing a water-rich and solute-rich phase. This is an important point because our interpretation of the spectral features in glassy electrolyte solutions presupposes homogeneous distribution of the solute. Thermodynamically, the Occurrence of liquid-liquid immiscibility is conditional upon a positive free energy of mixing of the components, and, for an immiscibility gap with an upper critical point, upon a positive enthalpy of m i ~ i n g . ~ 3For . ~ the ~ binary solutions investigated in this study the enthalpy of mixing is negative at ambient t e m p e r a t ~ r eTherefore, .~~ phase separation during hyperquenching is possible only when these values become positive. However, this seems unlikely. In addition, the gradual change in spectral features of glassy electrolyte solutions seen upon dilution in this and the earlier study’ are not easily explained by phase separation of solutions into a water-rich and solute-rich phase. We have also to point out here that earlier conjectures for liquid-liquid immiscibility in concentrated aqueous lithium chloride s o l ~ t i o n s during ~6~~~ slow cooling into their glassy states were later disproven by careful study by small angle neutron s ~ a t t e r i n g Therefore, .~~ there is no evidence for phase separation of aqueous electrolyte solution during quenching at normal pressure into their glassy state (see, however, ref 49 for the postulated phase separation in LiCl-H2O glasses a t high pressures). The spectral features described in this and the earlier study’ observable in going from ambient temperature to the glassy state are on the whole assignable within the framework of assignments developed at ambient and high temperatures. There are exceptions, namely, Spohn and Brill6 attributed in their Raman spectroscopic study of aqueous calcium nitrate solution a band, whose intensity increases with increasing temperatureon the lowfrequency side of the V I main band, to contact-ion pairs whereas we have assigned a component band at high frequency. However, the same order as ours was found by Bulmer et aLzofor aqueous strontium nitrate solution. There are advantages in investigating spectra of dilute aqueous solution in their glassy states, in addition to information obtained on increasing contact-ion pairing, in that bands due to different nitrate species can be better resolved in the glassy state than at ambient temperature. This is seen most clearly for the V I and u2 band region of the calcium and strontium salt in Figures 4-6 where the two component bands are clearly observable. At ambient temperature, no such separation of the two components observable in the v4 band region was possible even by resolution enhancement. The increased separability in the glassy-state spectra must be due to differences in the temperature dependence of the peak maxima of the two component bands andf or decreasing fwhh with decreasing temperature. . For the glassy 0.5 M solutions, the relative area of the band assigned to contact-ion pairs is Sr > Ca zz Mg for the v4 band region (Table I) and Sr > Ca > Mg for the u1 band region (Table 11). We note that increase in contact-ion pairing in going from ambient temperature to the glassy state is most pronounced for magnesium nitrate because a t ambient temperature no spectroscopic evidence for contact-ion pairing can be obtained for this salt (see Figure 2b and ref 18). This study confirms our earlier conjecture’ that extrapolation of the temperature dependence of contact-ion pairing into the deeply supercooled and glassy state is not meaningful. For the same electrolyte (calcium nitrate) contact ion pairing increases both in going from ambient temperature to subcritical temperature6 and in going to the glassy state (Figure 2 and Table I). We conclude, as in our earlier study,’ that water’s density maximum and the anomalies of supercooled water and dilute aqueous’ solutions, which are caused by structuralchanges toward a more open, regular tetrahedral network in a comparatively small temperature region8-I2,are at the bottom of the unexpected behavior. Recent X-ray12and neutron scattering”J2 studies of
The Journal of Physical Chemistry, Vol. 97, No. 18, 1993 4813 glassy water confirm that its disordered structureis well described by a fully hydrogen-bonded continuous random network. Contact-ion pairing increases in going to the supercooled and glassy state despite increasing values of the static dielectric permittivity (or constant, BO). We have remarked in our earlier study]that for aqueous solutions in their deeply supercooled state the COT product cannot be used as an indicator for ion association because of water’s structural changes in a comparatively small temperature region. In this context it is interesting to mention a Monte Carlo simulation on ion pairing in two different models for water by Goldman and B a ~ k x One . ~ ~model (ST2 water) was relatively waterlike, the other model, in effect a polar solvent, had the same dipole moment as ST2 water, but it did not have the ST2-model’s point-charge distribution. They found that the waterlike solvent exerted a stronger and qualitatively different dissociating force on the contact-ion pair, relative to the behavior of the polar solvent. It seemed to be the first clear indication that water’s ionizing ability derives, in part, from characteristics other than water’s large to. So, increasing contact-ion pairing is possibly not so surprising, considering water’s structural changes in going from ambient temperature to the glassy state. Finally, we consider the implications of increasing contact-ion pairing with decreasing temperature for the structure and/or function of biomolecules in supercooled solution. Contact-ion pairs can contribute significantly to the thermal stability of proteins.21,22 This contribution is believed to be largely independent of temperature because, as pointed out in ref 22, “at ambient temperature kT is small compared to the electrostatic interaction energy so that ion pairs do not show significant temperature dependence.” This argument does not take into account water’s structural changes upon supercooling.8-12 Our evidence for increasing contact-ion pairing presented in this and the earlier study’ in the supercooled and glassy state is suggestive to consider also for biomolecules the possible effects of increasing contact-ion pairing in this temperature region. We are aware that the effect has been demonstrated so far only for small molecular ions, but it will be much more difficult to prove for large biomolecules. It is important to note that we have found no exception so far. In the following we consider in particular cold denaturation of proteins, cryoenzymology and crofixation. (i) Cold Denaturation of Proteins. The thermodynamics of reversible cold denaturation has been well but its molecular basis is not known. Dill et al.,27in a recent statistical thermodynamic study, find that “cold-denaturation is driven principally by the weakening of the solvophobic interaction”. It is important to know whether or not increasing contact-ion pairing with decreasing temperature also contributes to cold denaturation of proteins by stabilizing the low-temperature conformation to a larger extent than that stable at ambient temperature. (ii) Cryoenzymology. Douzou and ~o-workers2~ have developed cryoenzymology for slowing down and studying enzyme-catalyzed reactions in solution at subzero temperatures. One approach is to use emulsified dilute aqueous solutions to avoid heterogeneous nucleation and allow supercooling close to 233 K.28 Increasing ion pairing is indicated for this temperature region by Figure 13 and Figure 4 of ref 1. It is thus conceivablethat in the supercooled state stabilization of an enzyme by contact-ion pairing differs from that at ambient temperature and therefore, that its structure and function are altered. (iii) Cryofixation. The goal of cryofixation is to immobilize, or “freeze-in”, the native state of dynamic structures and the momentary distribution of all components in a ~ystem.3&3~ This is impossible for small molecules and ions even by “hyperquenching”, as shown in this and the earlier studiesl8l5 by increasing contact-ion pairing with decreasing temperature. It is conceivable that this triggers conformational changes of a biomolecule. For the praxis of cryofixation of biological specimens for, e.g., electron microscopic studies it is important whether or not subsequent
4814
l-.: ..___ -.ai. rieissner et
The Journal of Physical Chemistry, Vol. 97, No. 18, 1993
changes of the biomolecule’s structure, caused by increasing contact-ion pairing, are fast enough to cause detectable structural changes.30-34 This depends on the relative rates of quenching and conformational change and on the spatial resolution of the technique. Evidence for apparently successful cryofixation of carbonyl hemoglobin in aqueous solution on an atomic scale, based on an FTIR spectroscopic analysis of the amide I band region, is given in ref 35.
Acknowledgment. We thank the Forschungsfiirderungsfonds of Austria for financial support (Project P9175-PHY). References and Notes (1) Hage, W.; Hallbrucker, A.; Mayer, E. J. Phys. Chem. 1992, 96, 6488. (2) Petrucci, S. In Ionic Interactions; Petrucci, S., Ed.; Academic Press, New York, London, 1971; Vol. I, Chapter 3. (3) Franck, U. E. Pure Appl. Chem. 1981, 53, 1401. (4) Irish, D. E.; Brooker, M. H. In Advances in Infrared and Raman
Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; Heyden: London, 1976; Vol. 2, Chapter 6. (5) Irish, D. E.; Jaw, T. Appl. Spectrosc. 1983, 37, 50. (6) Spohn, P. D.; Brill, T. B. J. Phys. Chem. 1989, 93,6224. (7) Angell, C. A. In Water, a comprehensive treatise; Franks, F., Ed.; Plenum Press: New York, 1982; Vol. 7, Chapter 1; Annu. Rev. Phys. Chem. 1983, 34, 593. (8) Dore, J. C. In WaterScience Reviews; Franks, F., Ed.; Cambridge University Press: Cambridge, 1985; Vol. 1, Chapter 1. (9) Chen, S. H.; Teixeira, J. Adv. Chem. Phys. 1986, 64, 1. (10) Bellissent-Funel, M.-C.; Teixeira, J.; Bosio, L.; Dore, J. C. J. Phys. 1989. CJ. 7123. (1’1) Hallbrucker, A.; Mayer, E.; O’Mard, L. P.; Dore, J. C.; Chieux, P. Physics Lett. A 1991, 159, 406. (12) Bellissent-Funel, M. C.; Bosio, L.; Hallbrucker, A.; Mayer, E.;SridiDorbez. R. J. Chem. Phvs. 1992. 97. 1282. (13). Mayer, E. J. Appl. Phys. 1985, 58,663. (14) Mayer, E. J. Phys. Chem. 1985,89, 3474. (15) Mayer, E. J. Phys. Chem. 1986, 90,4455. (16) Johari, G. P.; Hallbrucker, A.; Mayer, E. Nature 1987, 330, 552. (17) Hallbrucker, A.; Mayer, E.; Johari, G. P. Philos. Mug. 1989, B60, 179.
- 4
(18) Chang, T. G.; Irish, D. E. J . Phys. Chem. 1973, 77, 52. (19) Irish, D. E.; Walrafen, G.E. J. Chem. Phys. 1967, 46, 378. (20) Bulmer, J. T.; Chang, T. G.; Gleeson, P. J.; Irish, D. E.J. Solution Chem. 1975,4,969. (21) Perutz, M. F. Science 1978, 201, 1187. (22) Jaenicke, R. Philos. Trans. R. SOC.London, Ser. B 1990,326,535. (23) Dill, K. A. Biochemistry 1990, 31, 7133. (24) Brandts, J. F. J. Am. Chem. SOC.1964, 86, 4291. (25) Privalov, P. L.; Griko, Yu. V.; Venyaminov, S. Yu. J. Mol. Biol. 1986, 190, 487. (26) Franks, F.; Hatley, R. H. M.; Friedman, H. L. Biophys. Chem. 1988, 31, 307. (27) Dill, K. A.; Alonso, D. 0.V.; Hutchinson, K. Biochemistry 1989,28, 5439. (28) Douzou, P.; Balny, C.; Franks, F. Biochimie 1978, 60, 151. (29) Douzou, P.; Petsko, G. A. Adv. Protein Chem. 1984, 36, 245. (30) Plattner, H.; Bachmann, L. Int. Rev. Cytolog~1982, 79, 237. (31) Robards,A. W.;Sleytr, U. B. Low TemperatureMethodsinBiologicul Electron Microscopy; Elsevier: Amsterdam, 1985; Chapter 2. (32) Cryotechniques in Biological Electron Microscopy; Steinbrecht, R. A., Zierold, K., Eds.; Springer Verlag: Berlin, 1987, with many sections on
cryofixation. (33) Mayer, E. Cryoletters 1988, 9, 66. (34) Dubochet, J.; Adrian, M.; Chang, J.-J.; Homo, J.-C. Lepault, J.; McDowall, A. W.; Schultz, P. Q.Rev. Biophys. 1988, 21, 129. (35) Mayer, E.; Astl. G. Ultramicroscopy 1992, 45, 185. (36) Broto, F.; Clausse, D. J. Phys. 1976, C9, 4251. (37) Mayer, E. Chem. Phys. Lett. 1987, 139, 370. (38) Friesen, W. I.; Michaelian, K . H. Appl. Spectrosc. 1991, 45, 50. (39) Powell, J. R.; Wasacz, F. M.; Jakobsen, R. J. Appl. Spectrosc. 1986, 40, 339.
Irish, D. E.; Davis, A. R. Can. J. Chem. 1968, 46, 943. Sze, Yu-K.; Irish, D. E. J. Solution Chem. 1978, 7, 395. Williamson, K.; Li, P.; Devlin, P. J. J. Chem. Phys. 1968, 48,3891. Cahn, J. W.; Charles, R. J. Phys. Chem. Glasses 1965, 6, 181. Vogel, W. J. Non-Cryst. Solids 1977, 25, 170. Gmelins Handbuch der Anorganischen Chemie; Verlag Chemie: Berlin, 1939; Vol. 27B, p 87; 1960; Vol. 29, p 178. (46) Angell, C. A.; Sare, E. J. J. Chem. Phys. 1968, 49, 4713. (47) Hsich, S.-Y.; Gammon, R. W.; Macedo, P. B.; Montrose, C. J. J. Chem. Phys. 1972, 56, 1663. (48) Dupuy, J.; Jal, J. F.; Ferradou, C.; Chieux, P.; Wright, A. F.; Calemczuk, R.; Angell, C. A. Nature 1982, 296, 138. (49) Kanno, H. J. Phys. Chem. 1987, 91, 1967. (50) Goldman, S.;Back, P. J . Chem. Phys. 1986,84, 2761. (40) (41) (42) (43) (44) (45)
