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
Figure 1. Schematic of thermoelectric freeze-drying unit 0.
b. C.
d. e.
d. g.
Thenocouple leads Cold stage 112 CP 2-31-10 thennoelectric module Intermediate h a d sink 2 CP 2-31-10 thermoclectrlc modules He01 exchanger Cooling lines for heat eishonger
obtained. (These modules may he ohtained from Materials Electronic Products Corp., Trenton, N. J.) The unit is composed of alternately stacked heatsinks and thermoelectric modules. The first stage of the unit is a heat exchanger (f), consisting of a copper block (76 X 38 X 18.5 mm.) with water passing through it. This water is obtained by tapping in on the diffusion pump cooling system and is fed through the base plate by adapting two existing feed-throughs. Two CP 2-31-10 modules (e), are soldered t o the upper side of this stage. When soldering the thermoelectric modules, care must he taken not to exceed the melting point of the solder used in the construction of the module. The manufacturer includes with the modules sufficient solder for the construction of the unit. The solder supplied has a melting point slightly below that used in the module, so hy careful heat control this problem can he avoided. The intermediate heat sink (d), is a 63- X 38- X 6-mm. piece of copper soldered on t o of the two C P 231-10 modules. Soliered to this is a CP 2-31-10 (c), and soldered to this is a piece of copper (31.5 X 19 X 1.25 mm.) which is used as the cold stage. The current needed for the operation of the unit (maximum 8 amp.) is supplied by a variable d.c. power pack (the one used in this laboratory has a
evaporator by means of an existing octal header. The electrical leads from the modules are attached hy the manufacturer, and using these leads, the three modules are connected in series. The unit must he checked as to the direction of current flow before increasiug t h e current to the useful range. If t,he current flow is reversed, the heat exchanger will be of no use and the unit will generate enough heat to melt the solder joints. This will also occur if the unit is operated without the heat exchanger operating efficiently-e.g., insufficient flow of water. A thermocouple (a) is attached to the upper surface of the cold stage for monitoring the temperature at that point. After an initial calibration of the d.c. power pack, the temperature may be read directly from the ammeter as is shown hy the graph, Figure 2. A typical sequence for sample preparation would be as follows: The specimen is frozen at approximately -65" C. and the temperature then raised to just helow its freezing point. After sufficient time for sublimation of the ice, the temperature is again lowered to a minimum. This is to ensure against deformation by the heat developed in shadowing or coating. The metal deposition is then completed. This coating may serve two purposes. It will show the shape of the specimen when in the frozen state and give the
Figure 3. Freeze-dried, soft emulsion shadowed with germanium
specimen support so as not to allow it to collapse upon warming. This operation also allows the freeze-drying to he done on the grid to he used in the microscope. Therefore, after warming the specimen to room temperature and breaking vacuum, it can he transferred to the microscope with no additional operations. RESULTS AND DISCUSSION
-mt
The freeze-drying unit has been used in this laboratory on soft emulsions with some success. Figure 3 is of a soft emulsion and shows little deformation taking place when the above procedure is followed. The particle has lain over on its side showing the amount of deformation that has occurred. Although a soft emulsion has been used to demonstrate one use of the unit, it is by no means the most important application of this type of apparatus. Any operation requiring a carefully controlled low temperature, whether in or out of a vacuum system, could
NO. 13, DECEMBER 1 965
1785
