Bruno Lunelli lstituto Chimico "G. Ciamician" University of Bologna
1
Lyophilization is the operation by which water or another solvent is removed by sublimation from a frozen phase or system. Since lyophilization takes place a t low temperature and pressure ( r w m to about -80°C; 10-210-6 mm Hg, respectively) and in closed apparatus, thermal denaturations, reactions with atmospheric gases, loss of dangerous or expensive substances, as well as frothing and splashing, are completely avoided. The residue is an extremely subdivided, porous mass which rapidly re-absorbs the solvent to give the initial solution and is very active physically and chemically. Therefore, lyophilization is widely applicable for the handling of both natural and synthetic substances: its main drawback is the high cost of the apparatus needed to produce and maintain the low pressure required. Here we propose a method based on an easily constructed apparatus which allows laboratory-scale lyophilizations to he carried out easily and inexpensively, requiring only the intermittent use of an ordinary mechanic oil pump or even of a water aspirator. The apparatus, sketched in the figure, consists of a kind of a demountable Pyrex vacuum trap, D, provided with a high vacuum stopcock, F, and a tangential inlet tube, C. Tube C ends with a socket, B, if special vessels are used or with a cone to fit standard flasks. The high vacuum needed for lyophilization is produced by substituting the usual gas phase consisting of air, nitrogen, or argon, with the vapors of a solvent which a t room temperature has a vapor pressure larger than the ultimate vacuum of the available pump hut an extremely low vapor pressure a t liquid nitrogen temperature.
Lyophilization Apparatus vapors from the solvent in D to diffuse in all the available space. Evacuation through F and waiting for diffusion of the vapors are repeated two or three times, thus completely replacing the initial gas phase.2 The lowest half of the trap is immersed into the liquid nitrogen contained in a Dewar and allowed to stand until the lyophilization is finished. Overnight operation is quite convenient and safe because there are no moving parts nor water nor electricity to be supplied. The snow which forms after a while on A provides a sufficient insulation to prevent undue rise of temperature. When the snow melts the operation is ended. If the material to be lyophilized is contained into a number of vials, these are partially introduced into a layer of fine lead shot previously put in A to ensure a proper supply of heat from the exterior; then one proceeds as outlined above for a solution. The quantity of liquid nitrogen used up in the lyophilization can be estimated on the basis of the following data: substance/boiling point a t 760 mm Hg (unless otherwise stated), "C/AH0 of boiling, cal mol-'/melting point a t 760 mmHg, "CIAH" of melting/other data: nitrogen / -195.8/1360/-210.0/112/ density at -195.8 is 0.808 gram ml-1; water /100.0/9717/0.0/1436; benzene /80.1/7350/ 5.512350; formic acid /100.7/4755/8.4/3030; acetic acid/ 117.8/11650/16.6/5600; dimethyl sulfoxide 125 a t 0.6
Operation The material or the solution to be lyophilized is placed into vessel A and cooled until it begins to freeze. Then F is opened, vacuum is applied for a few seconds, and the sample is degassed by repeated freezing and thawing, swirling the contents while it is a liquid and applying the vacuum for a few seconds while it is frozen. Finally the sample is rapidly frozen to avoid segregations, taking due care, in the case of an aqueous solution, to swirl the freezing liquid to avoid cracking the flask. Air or an inert gas is admitted through F, then E is rapidly opened' and about 1 ml of solvent introduced into D. This solvent may be either the same or different from that present in the sample-it should only have a t room temperature a vapor pressure higher than the ultimate vacuum of the pump used. Then E is closed and F is opened for a few seconds, removing most of the gases present in the system. Now one waits for a few minutes to allow the 'In the interior of a plastic hag filled with an inert gas if air is harmful. 21f the available pump produces an ultimate pressure lower than the vapor pressure of the solvent over the solution (with an efficient oil pump this is the case for most solutions) the operations listed in this paragraph are omitted.. *Only in this way could we prepare biphenylene-8-dn of satisfactory isotopic purity, Pecille, C. and Lunelli, B., to be published. Peeile, C., and Lunelli, B., J. Chem. Soe., A1910, 690; Lunelli, B., and Peeile, C., Gozr. Chim. Ital., 99,496 (1969). T o r instance in the study of the reduction of phthalimide or isatine in dimethylsulfoxide, Farnia, G., private communication.
Lyophilization apparatus. The sizes given are intended for handling up to about30 rnl of solvent.
Volume 50, Number& August 1973 / 577
mmHg/12640 a t 0.6 mmHg/18.4; ammonia /-33.3/5560/ -77.8/1380. Typical cases in which lyophilization gives results o t h erwise difficult or impossible to obtain include the preparation of instantaneously soluble samples for kinetic pur-
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poses,3 the separation of the exchange medium in the preparation of deuterated compounds by hydrogen-deuterium exchange,4 the removal of high boiling solvents often used in electrochemistry under exclusion of moisture and oxygen.5
