Nuclear Magnetic Resonance Technique for Determining Hydration Numbers R. J. Kula, D. L. Robenstein, and G. H. Reed, Department of Chemistry, University of Wisconsin, Madison, Wis.
w Ivarious several of
of investigations of metal-chelate systems, the crystalline materials were synthesized for purification purposes and for elemental analyses. With water soluble chelates of the (ethylene(EDTA) dinitrilo) tetraacetic acid variety, the crystalline material usually retains several waters of hydration in more or less stoichiometric quantities even after drying. I n fact, it is often nearly impossible to remove all of the mater without decomposing the samples, indicating that the method of complete dehydration is not suitable for determining the hydration number. Although several methods exist for the quantitative determination of hydration numbers, none were found to be particularly convenient for our purposes (5) We feel that we have found a relatively simple and accurate method for deteymining the hydration numbers of a wide variety of materials from measurements of the total hydrogen content of the materials in deuterium oxide (DzO) solutions wing nuclear magnetic resonance (NMR). ;2bout the only experimental requirements with regard to the sample are that part of the material present possess nonexchangeable protons (this condition is normally satisfied for carbon-bonded protons), and that there are no paramagnetic ions present in the sample (rvhich could broaden the proton resonances so extensively that they are not detectable). I n crystalline metalligand complexes of definite stoichiometry the number of carbon-bonded protons is known and can be compared with the number of exchangeable protons by examining the NMR spectrum of the substance in a DzO solution. In EDTA, for example, there are twelve carbon-bonded protons, 4 ethylenic and 8 acetate, which give two proton resonances, either or both of which may be further split by spin-spin interactions. In addition there is a single resonance line, the intensity of which is determined by the contributions from all the exchangeable protons, including those from residual H 2 0 in the DzO solvent, from acidic and basic groups within the substance-e.g., -NH, -COOH, -OH etc.-and from hydration water. If corrections are made for the first two contributions, the hydration number can be determined. With appropriate metal ions and with slight modifications of the technique it is also possible to determine hydration numbers of various metal salts. This procedure is exemplified by the study of hydrated zinc nitrate. THE COURSE
For a comprehensive discussion of quantitative determinations using NMR the reader is referred to the papers by Paulsen and Cooke (6, 7 ) . EXPERIMENTAL
The crystalline materials to be studied are dissolved in 99.87, DzO (obtained from Bio-Rad Laboratories), the solutions are placed in 5-mm. 0.d. precisionbore NMR tubes, and the NMR spectra are recorded on a Varian A-60 spectrometer. Integrals of the resonances are obtained using the integrating unit of the spectrometer. The sample is then removed and, with all control settings maintained constant, an integral is obtained for a pure sample of the 99.8% DzO which is kept in a separate XMR tube. At about 0.2M solute concentrations the residual water in DzO contributes less than 15% to the total exchangeable proton resonance. For calculating the hydration number the following procedure has been adopted. The total integrated signal intensities (measured by the vertical pen deflection) for the ligand proton resonances is divided by the number of protons contributing to the signals giving, in effect, the intensity per proton. Dividing this value into the intensity determined for all exchangeable protons, which has been corrected for residual HzO in the DzO solvent, gives the total number of exchangeable protons per molecule of solute. Subtracting the number of protons known to be present in acidic or basic groups of the complex gives the number of hydration water protons from which the hydration number is obtained. One particular advantage of this method is that virtually no control of the solution pH or temperature (35' i 2' C. in the NMR probe) is required. Another advantage is the small sample size, less than 50 mg. of material normally being used. Studies have been made to determine the effect of concentration, of sweep rates, and of power levels of the radiofrequency field on the integrated intensities. Six different substances were used to study these factors and to provide examples of the applicability of the method. Disodium(ethplenedinitri1o)tetraacetate - dihydrate, NazH2EDTA. 2H20 (Compound A), was obtained from J. T. Baker Chemical Co. (Assay 99.7%) and was used as received. In addition, an ultrapure sample (Compound B) was prepared according to the recommended procedure (1). The molybdenum(V1) complex of N-methyliminodiacetic acid was prepared recently (3) and was shown to have the composition IYa.&IoO&fIDA 4H20 (Compound C). The molybdenum(V1) complex of EDTA was prepared according to the published method (8) in which the composition was found to be
Sa4(~\1003)2EDTA. 8H20 (Compound D). As an additional example of a system which contains exchangeable protons as well as carbon-bonded protons, HzHg(II)-EDTA 2l/2HZO (Compound E) was prepared ( 2 ) . Sodium acetate trihydrate (Compound F) was obtained as the reagent grade product from Mallinckrodt Chemical Works and was used as received. Zinc nitrate-hexahydrate (Compound G) was from J. T. Baker and was also used as received. RESULTS AND DISCUSSION
Because the line width of the exchangeable-proton resonance is considerably less than most of the ligand proton resonances (often by a factor of two or three), the sveep time has a marked effect on the integrated intensities. In particular it mas noted that a t faster sweep rates low results were obtained for the amount of water present. To circumvent this problem it mas useful to make the total sweep width 100 c.p.s. on the recorder, and to employ sweep rates of 1.0 c.p.s./.econd or less. Most of the tabulated data were obtained using a sweep rate of 0.4 c.p.s./ second. The small line width of the exchangeable-proton resonance corresponds to a long relaxation time for these protons, which means that saturation can occur at relatively small radiofrequency fields, this effect also causing low results. Studies of the effect of radiofrequency field intensites indicated that saturation effects are first observed a t about 0.08 milligauss on our instrument, and therefore, all data were obtained a t intensities of 0.06 milligauss or less. Investigations of the effect of solute concentration verified the suspected conclusion that the results are independent of concentration. However, to obtain reasonable signal-to-noise ratios, the concentrations generally had to be greater than 0.05.11. Most of the results reported here were obtained using solutions with concentrations between 0.1 and 0.5M. Besides the low concentration limit the only other limiting condition is that saturated solutions should not be employed-i.e., samples containing undissolved solid in the S M R tube. The results from such solutions consistently gave high results compared to unsaturated solutions, suggesting that perhaps some water (or protons) were exchanged between the solvent and the solid. That the results are not concentration-dependent means that the technique is even further simplified because no weighings are necessary. The material can merely be VOL 37, NO. 13, DECEMBER 1965
1783
Table I.
Compound A B C D E F G ~
Determination of Hydration Number
Waters of hydration Literature Experimental 2 2
4 8 2.5 3 6
dissolved in high purity D20 and the spectrum integration performed. I n Table I the results of the determinations have been summarized. It can be seen that in general this method gives results which are precise to about 0.05 water of hydration. The data for the disodium-EDTA compounds, A and B, illustrate that one must exercise caution in working with hydrated materials because previous treatment of the sample may have affected its hydration number. This is particularly important for a substance like EDTA\ which is often used as a primary standard. The difference of 0.1 waters of hydration in -4between the experimental and the assumed values might not seem particularly significant until one considers that this difference would cause an error of nearly 0.5% in preparing a standard solution of EDTA. Spontaneous loss of hydration water may occur with certain substances For example, for Compound C the freshly prepared material has a hydration number of almost exactly four. The same material stored under atmospheric conditions for several months, h o w r c r , is found to lose water, giving a value of 2.8 a t the time these determinations
Std. dev.
No. of measurements
0.06 0.04 0.02 0.11 0.04 0.03 0.03
20 5 4 15 5 5 5
1.89 2.04 4.01 7.79 2.55 3.08 6.19
were made. The low value obtained for Compound D may also be the result of water loss upon standing. Compound G has been included to demonstrate the possibility of extending this method to substances which do not contain nonexchangeable protons. This extension should be applicable to all hydrated metal salts for which the metal ion can coordinate to a ligand containing nonexchangeable protons and for which the exchange of ligands between free and complexed forms is slow on the NMR time-scale (lifetimes normally greater than 0.5 second). The chelate formed between Zn+2 and EDTB is known to exhibit the aforementioned properties ( 4 ) , and therefore a solution containing EDTA in concentrations greater than Zn+2will show separate resonances for free EDTA and for complexed EDTA. The procedure employed has been to dissolve the anhydrous tetrasodium salt of EDTA in 99.8% D20 (approximately 0.5M) and to obtain an integral of the exchangeable-proton resonance. The NMR tube is removed and the metal salt is added to the solution a t a concentration of about 0.2 or 0.3M. With all the spectrometer settings maintained
constant, a new integral of the exchangeable-proton resonance and of the zincEDTA resonances is obtained. The increase in the exchangeable-proton resonance intensity is due entirely to hydration waters of the zinc salt, and from a comparison of the number of protons contributing to this increase and to the zinc-EDTA signals, the hydration number can be determined. The high results are undoubtedly due to the hygroscopic properties of zinc nitrate. Other ions which coordinate with EDTA and which are known to possess the aforementioned exchange properties include Mg(II), Ca(II), Al(III), Y(III), La(III), Zr(IV), W(V1). Pd(II), and Cd(I1). The water content of hydrated salts of these ions might then be analyzed using this technique. LITERATURE CITED
(1) Blaedel, V W. J., Knight, H. T., ANAL. CHEM.726, - 741 (1954). ( 2 ) Kula, R. J., Reed, G. H., Unpublished work, 1965. ( 3 ) Kula, R. J., University of Wisconsin, Madison, unpublished work, 1965. (4) Kula, R . J., ANAL. CHEM., 37, 989 (4) (1965). ( 5 j -Mitchell, J., “Treatise on Analytical
-
Chemistry,” Part 11, Yol. I, I. M. Kolthoff, P. J. Elving, eds., Interscience, Yew York, 196f. ( 6 ) Paulsen, P. J., Cooke, W. D., ANAL. CHEM.36, 1713 (1964).
( 7 ) Ibid.. D . 1721 (8) PecsoG, R. L., Sawyer, D. T., J . Am. Chem. SOC.78, 5496 (1956).
WORKsupported by the National Science Foundation (summer teaching fellowship, G.H.R.) and the Wisconsin Alumni Research Foundation (graduate research assistantship, D.L.R.).
Freeze-Drying Unit for Electron Microscopy Jack D. Hutchison, Summit Research Laboratories, Celanese Corp. of America, Summit, N. J.
at E some time during his career encounters a specimen which must be VERY
ELECTRON
MICROSCOPIST
freeze-dried. The problems involved in freeze-drying for electron microscopy are quite different from those usually encountered in other fields. The sample is usually very small, and after freeze-drying, it may be necessary to vacuum-deposit a metal on it (as in shadowing) to enhance its fine detail or to reinforce its three-dimensional structure. Those samples which must be shadowed are in danger of absorbing moisture in the transfer from the freezedrying unit to the evaporator, or the heat developed in the shadowing operation may be intense enough to melt, and consequently deform, the sample. 1784
0
ANALYTICAL CHEMISTRY
Various methods have been devised
to overcome these obstacles. The most common is that of pumping liquid nitrogen through a cooling cell and doing the freeze-drying in the evaporator. The inconveniences in this operation are the overall complexity of the setup, the equipment needed to pump the liquid nitrogen, and the possibility of leaks developing in the vacuum system (caused by the extreme temperature differentials near the vacuum seals). There has been developed in this laboratory a freeze-drying unit which can be used in the vacuum evaporator without the inconvenience of a coolant such as liquid nitrogen. This is accomplished by using thermoelectric modules using the Peltier effect, which are cur-
rently being manufactured commercially. In this unit it is the cold side of the modules that is of interest. By the proper stacking of these modules a temperature difference sufficient for freeze-drying is attained. This unit is small, it is silent, the heat exchanger uses only tap water a t room temperature, there are no moving parts such as a pump for moving liquid nitrogen, and the temperature is theoretically continuously variable from a minimum of approximately -90’ C. to room temperature. EXPERIMENTAL
The unit designed and built in this laboratory, Figure 1, consists of only three modules stacked so that a minimum temperature of - 65” C. is readily
