slightly greater care in handling needed to prevent breakage. In addition, pyrolytic graphite is highly pure carbon and therefore does not itself add any background radiation to the X-ray spectrum. These properties are all in sharp contrast to those of other possible support materials for electrodeposited filters.
RECEIVED for review April 12,1971. Accepted May 28,1971. This work was supported by Undergraduate Research Participation Grant No. GYO-7516 from the National Science Foundation, D. G . P. was a National Science Foundation Undergraduate Research Participant, 1970, at Seton Hall University, while a student at Montclair State College.
Hydrated Porosijr of Macroreticular Cation Exchange Resins via Nuclear Magnetic Resonance L. S . Frankel Rohm and Haas Company, 5000 Richmond Street, Philadelphia Pa. 19137 CONVENTIONAL GEL ION EXCHANGE resins consist of a continuous network of quasi-homogeneous copolymer (1). Macroreticular resins have two discrete phases, a gel phase as described above and a phase composed of large pores or voids which are occupied in the hydrated state by water molecules (2). One of the most important physical characteristics of a macroreticular ion exchange resin is its porosity, or the fraction of the total volume of the resin occupied by the pores. The porosity is conventionally obtained in the dry state via a mercury porosimeter or a helium densitometer (2). However, of greater interest is the porosity of the hydrated resin and a subsequent comparison with the porosity of the dry resin. There is to our knowledge no simple way of determining the hydrated porosity. The determination of the hydrated porosity requires knowledge of the distribution of the total water between the gel phase and the pores. The only significant contribution to this problem we are aware of utilizes a mercury porosimeter and studies the porosity as a function of hydration. These results have been interpreted to indicate that the hydration of the gel phase is complete prior to any water entering the pores via capillary action (2). Our attention was called to this problem during our study of the nuclear magnetic resonance (NMR) spectra of ion exchange resins (3-16). We wish to (1) W. Riernan 111 and H. F. Walton, “Ion Exchange in Analytical Chemistry,” Pergamon Press, New York, N. Y., 1970, p 13. (2) K. A. Kun and R. Kunin, J . Polym. Sci., Part C , 16, 1457 ( 1967). (3) J. E. Gordon, J. Phys. Chem., 66, llSO(1962). (4) D. Reichenberg and I. J. Lawrenson, Trans. Faraday SOC.,59, 141 (1963). (5) R. H. Dinius, M. T. Emerson, and G. R . Choppin, J. Phys. Chem., 67, 1178 (1963). (6) J. P. devilhers and J. R. Parrish, J . Polym. Sci., Parr A , 2, 1331 (1964). (7) T. E. Gough, H. D. Sharma, and N. Subramanian, Can. J. Chem., 48, 917 (1970). (8) R. W . Creekmore and C. N. Reilley, ANAL. CHEM.,42, 570 (1970). (9) L. S.Frankel, Can. J. Chem., 48,2432 (1970). (10) R. W. Creekmore and C. N. Reilley, ANAL.CHEM.,42, 725 (1970). (11) A. D&covA, D. Dosko&lovB, g. SwvEik, and J. Stamberg, J. Polym. Sci., Part B, 8, 259 (1970). (12) L. S. Frankel, ANAL.CHEM.,42, 1640 (1970). (13) D.G. Howery and M. J. Kittay, J. Macromol. S i . , Part A , A(4), 1003 (1970). (14) D. G. Howery and B. H.Kohn, Anal. &IC., 3, 89 (1970). (15) H. Sternlicht, G. L. Kenyon, E. L. Packer, and J. Sinclair, J. Amer. Chem. Sac., 93, 199 (1971). (16) H. D. Sharma and N. Subrarnanian, Can. J . Chem., 49, 457 (1971).
3506
show how the hydrated porosity may be obtained from the NMR spectra and the resin moisture holding capacity. Data for a gel resin and a macroreticular resin, both of which contains 5 % DVB cross-linking, are reported. EXPERIMENTAL
All measurements were made on a Varian H.R. 60 spectrometer operating at 56.4 MHz. The true density or skeletal density of the dry macroreticular copolymer was obtained on a helium densitometer. The apparent density or the density of the entire mass of dry macroreticular resin was determined in a modified mercury porosimeter (2). Experimental bead copolymers of both the gel and macroreticular type were prepared using commercial grade monomers. The divinylbenzene contained 58.2% DVB as a mixture of ca. 70% m- and 30% p-isomer. The major impurities in the DVB were isomers of ethylvinylbenzene. A stock monomer solution containing 5 % by weight of DVB and 95% by weight of styrene and ethylvinylbenzenes was used for the synthesis of all polymers. The copolymers and resins were prepared by standard techniques (17) DISCUSSION AND RESULTS
The NMR spectra of ion exchange resins will generally show sets of peaks. One peak originates from solvent or counter ions inside the ion exchange resin (interior peak); the other (exterior peak) is from the molecules in the volume of the NMR tube not occupied by the resin beads (void volume of column). The solid resin backbone does not contribute any peaks to the spectrum since its molecular motion is highly restricted. In several previous reports on gel resins, the chemical shift of the hydrogen and sodium form have been shown to be linear functions of the internal molality, f%, of the resin, fir = (QW) (100 - % H20)/% H20. QW is the dry weight capacity of the resin and H?O is the weight per cent water or moisture holding capacity. Molal chemical shifts in the hydrogen form of 0.287 ppm (6), 0.321 ppm (7), and 0.337 pprn (8) have been reported. These values are slightly less than that reported for the effect of hydrogen ion on water, 0.344pprn (18). The molal chemical shift for the 5% DVB gel resin was 0.339 ppm and for the 5 % DVB macroreticular resin, 0.345 ppm. A similar analysis is applicable to the sodium form although the analytical sensitivity is much less. (17) J. A. Marinsky, “Ion Exchange,” Marcel Dekker, New York, N. Y., 1969, Chapter 6. (18) J. C. Hindman, J. Chem. Phys., 36, lo00 (1962).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
Macrareticular resins do not show separate water resonances for pore and gel phase water (19). The intensity of the interior water peak has been shown to account for all of the internal water (19). This implies that the water in the two internal phases undergoes rapid exchange. Under rapid exchange conditions the observed chemical shift from the exterior water, 6, is given by (?O) 6 =
xos,
+ xpsp
(1)
where X , is the mole fraction of the total water present in the resin in the gel phase of the macroreticular resin, 60 is the chemical shift of the water in the gel phase, Xp and 8 p are similarly defined for the pore phase. If one assumes that the chemical shift between water in the pore phase and water in the void volume of the column is zero, the ratio 6/80 gives Xo from which the hydrated porosity may he calculated. = 0 is reasonable since the We believe the assumption water in the pore phase is not influenced by any ionic groups which are all in the gel phase and the fact that the average pore diameter is large compared to the size of a water molecule. It is pertinent to note that the macroreticular copolymer does not show resolvable interior and exterior water peaks. All previous studies suggest that dispersion interactions with the copolymer do not greatly affect the chemical shift (3). The chemical shift between the gel water and the exterior water must now be considered. We will attempt to show that 6, is the same as that of the gel resin of the same crosslinking density. This is equivalent to saying that the internal molality of the gel phase of the macroreticular resin is identical to that of a conventional gel resin having the same crosslinking density. The values of QW (Table I) are virtually identical and are typical for complete monosulfonation of the copolymer. These values are, however, not distinctive of any particular cross-linking density of up to approximately 20 DVB. What is needed is an independent physical measurement which is sensitive to the gel phase cross-linking density of the macroreticular resin but yet totally insensitive to the other unique properties of macroreticular ion exchange resins such as surface area and pore size distribution (2). Associated with a certain cross-linking density is a certain water content (21). Therefore it is sufficient to show that the cross-linking density of the gel phase of the macroreticular resin is identical to that of the conventional gel resin. It has previously been reported that the line width at half height of the counter ion NMR resonance of the N(CH3)4+ionic form of cation gel resins is strongly dependent on the cross-linking density (3). As the cross-linking density is increased, the counter ion rotational freedom decreases and the transverse relaxation rate, and hence the line width, increases. Since all the counter ions are in the gel phase, the counter ion line width should depend on the gel phase cross-linking density and be independent of other physical properties. The counter ion line widths at half height for both 5 DVB resins were identical, 4.5 Hz, and we therefore conclude that the cross-linking density and hence the moisture holding capacities are identical, (The counter ion line width of the N(CHJ4+ ionic form of a 4.75z DVB and an 8.0% DVB gel resin was 3.5 and 14.0 Hz.) The synthetic method for preparing these cation resins would support the fact that the cross-linking densities are (19) L. S . Frankel, J. Phys. Chem., 75,1211 (1971). (20) J. A. Pople, W. G . Schneider, and H. J. Bernstein, “HighResolution Nuclear Magnetic Resonance,” McGraw-Hill, New York, N. Y.,1959. Chapter 10. (21) T. R. E. Kress.man-and J. R. Millar, Chem. Ind. (London), 1961, 1833.
Table I. Summary of Results HCform Na+ form Dry weight capacity of gel resin meq/g dry 4.67 5.22 resin, QW Dry weight capacity of macroreticular 4.65 5.19 resin, meq/g dry resin, QW Chemical shift of gel resin, Hz 55.0 12.0 36.5 Chemical shift of macroreticular resin, Hz 8.0 Mole fraction water present in gel phase of macroreticular resin, XO 0,644 0.67 Per cent water i n gel resin 64.4 57.5 Per cent water in macroreticular resin 73 5 68.0 Hydrated density of macroreticular resin, g/cc hydrated resin 1.10 1.16 Porosity of hydrated macroreticular resin via NMR, cc pores/cc hydrated resin 0.288 0.26 Porosity of hydrated macroreticular resin via per cent water, cc pores/cc hydrated 0.282 resin 0,288 Skeletal density of dry macroreticular 1.437 1.497 resin, g/cc“ Apparent density of dry macroreticular 1.121 resin, g/cc* 1.108 Porosity of dry macroreticular resin, cc 0.260 0.220 pores/cc dry resin Porosity of dry macroreticular resin, cc pores/g dry resin 0. I96 0.239 a The skeletal density is the density of just the solid copolymer backbone. * The apparent density is the density of the entire volume of the macroreticular resin. similar. If the dry weight capacity and the moisture holding capacity of the gel phase are equal, the internal molality of the gel resin and the gel phase of the macroreticular resin are equal. Therefore, 6G is given by the conventional gel resin chemical shift. The distribution of the gel water is then readily calculated from Equation 1. The data are summarized in Table I. The hydrated porosity in cc of pores/cc of hydrated resin, per cent of volume occupied by the pores, is calculated from (% H20)XpDwhere D is the density of the hydrated resin. After establishing that the gel phase of the macroreticular resin and the gel resin are identical, the hydrated porosity may be calculated from the total moisture holding capacity of the two resins. Consider 1 cc of pore water and calculate the number of grams of hydrated gel phase, Z , that must be added to give the observed weight fraction water in the macroreticular resin, WMR, WMR =
(1
+ WCz>/(l + z>
WO is the weight fraction water in the gel phase. The hydrated porosity, P,in per cent by volume, is then calculated from P = D/(l Z )
+
Excellent agreement is obtained uia the above procedure and the NMR chemical shift data (Table I); however, this should not be regarded as independent confirmation. The calculation of Zinvolves W J I-~ W,. Each of the above quantities can generally be obtained to within a few tenths of a per cent. For the hydrogen form of the resin, the chemical shift data are analytically more sensitive; for the sodium form, the moisture capacity data have greater analytical sensitivity.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
0
1507
The porosity of a dry resin is typically expressed on a dry volume or dry weight basis. We wish to compare the porosity of a hydrated resin with that of a dry resin. To make this comparison, it is necessary to express the results from the dry resin on the basis of a hydrated resin. The porosity of the dry resin must be expressed in cc of pores/cc of hydrated resin assuming no swelling occurs; therefore, the volume of pores does not increase upon hydration. This assumption is dearly not valid but is made to allow the above comparison. Assuming no swelling occurs, the porosities of the hydrated macroreticular resins are 0.052 and 0.075 cc pores/cc hydrated
resin for the hydrogen and sodium ionic form. The correct hydrated porosities are summarized in Table I. The correct hydrated porosities are approximately four times that of the dry porosities. ACKNOWLEDGMENT
The author wishes to acknowledge Mr. K. TregeI and Dr. J. Barrett for the synthesis of the ion exchange resins. RECEIVED for review April 6, 1971. Accepted June 7, 1971.
Ultraviolet Refractive Indices of Aqueous Solutions of Urea and Guanidine Hydrochloride J. R. Krivacic and D. W. Urry Dioision of Molecular BiophysicslLaboratory of Molecular Biology, Unicersity of Alabama Medical Center, 1919 Secenth Acenue South, Birmingham, Ala. 35233
INSTUDYING PROTEINS AND POLYPEPTIDES, it is often necessary to use denaturants such as urea and guanidine hydrochloride. In evaluating the nature of the unfolding or dissociation of these macromolecules, optical rotatory dispersion (ORD) is often the tool of choice for following such phenomena. To evaluate the results in a meaningful manner, a parameter is required which is independent of the solvent system used. Since the ORD of molecules depends on the background contributions and the field in which the molecule resides, it becomes necessary to adjust the ORD spectra for these variables. The Lorentz field correction, applied to such spectra, yields a solvent independent molar rotation. Applying such a correction requires knowledge of the refractive index, as a function of wavelength, of the solvent. Furthermore, knowledge of refractive index dispersion is necessary for a wider understanding of light scattering phenomena. In our laboratory, distortions in circular dichroism spectra due to light scattering and self absorption of particulate systems can be corrected by using Mie or Rayleigh-Gans approximations for estimating the scattered light and by employing the absorption flattening considerations of Duysens. To apply such corrections, refractive index dispersion of the solvent system and of the particulate system are necessary (1-3). The construction of particle refractive indices has been demonstrated by Urry et al. (1,2). The refractive indices herein reported have been determined by variable angle single reflection spectrometry. The general principles have been elaborated on by Harrick ( 4 ) and Hansen (5). Specific details are presented in our previous works (6,7). Previously, refractive index data in the ultraviolet have (1) D. W. Urry and J. Krivacic, Proc. Nar. Acad. Sci. U. S . , 65, 845 (1970). (2) D. W. Urry, T. A. Hinners, and J. Krivacic, Anal. Biochern., 37, 85 (1970). (3) D. W. Urry, L. Masotti. and J. R. Krivacic. Arch. Biochem. ‘Biophys., in -press. (4) N. J. Harrick, “Internal Reflection Spectroscopy,” Interscience Publishers, New York, N. Y . , 1967, pp 32, 182-188. ( 5 ) W. N. Hansen, Spectrochim. Acta, 21, 815 (1965). (6) J. R. Krivacic and D. W. Urry, ANAL. CHEM., 42, 596 (1970). (7) J. R. Krivacic and D. W. Urry, Aiial. Biochem., i n press. 1508
been limited with little or no data available at wavelengths shorter than 265 mF. This is particularly true of highly absorbing solutions. It is, in fact, shorter wavelengths that are of the most direct interest to studies on proteins and polypeptides-studies which often employ urea and guanidine hydrochloride solutions. To our knowledge, the method used herein provides the first direct measurements of refractive indices for urea and, guanidine hydrochloride solutions to wavelengths of 2000 A. These direct measurements are compared to fitting the long wavelength data to a Sellmeier-type equation which is commonly extrapolated to shorter wavelengths. Also, the direct measurements are least squares fitted by a general dispersion expression. EXPERIMENTAL
Reagents. Urea was supplied by J. T. Baker Chemical Co. as a “Baker Analyzed” reagent and was recrystallized from aqueous ethanol. Guanidine monohydrochloride was also supplied by J. T. Baker as a “Baker Grade” reagent and was twice recrystallized from aqueous ethanol. Both reagents were stored in the cold and solutions were freshly prepared. Final concentrations were determined by refractive index on a Bausch & Lomb Model 3L AbbC Refractometer and compared to the data of Warren and Gordon (8) for urea and of Kielley and Harrington ( 9 ) for guanidine hydrochloride. Apparatus. Reflection spectra were run on the Cary Model 14 Spectrophotometer using the Harrick Scientific Model RMVA-1 variable angle reflectance attachment with a sapphire hemicylinder (IO). We have previously described the use and calibration of this particular device (7). Procedure. The optical constants are determined from two spectral scans at different angles of incidence. The first angle is above the sapphire-sample critical angle and the second is below the critical angle. Both angles must be above the sapphire-air critical angle. Optical constants are calculated for the samples at 50-A intervals using the equations (8) J. R. Warren and J. A. Gordon, J . Phys. Chem., 70, 297 (1966). (9) W. W. Kielley and W. F. Harrington, Biochim. Biophys. Acra, 41, 414 (1960). (10) N. J. Harrick, ANAL. CHEM., 37, 1445 (1965).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
