Electric Heater for Microprocedures and Melting Points TONY CIFONELLI, University of Minnesota, Minneapolis, Minn. between the center and the outer regions of the block when the temperature rises above 100" to 125" C. To determine the area of uniform temperature range, a solut,ion of a substance possessing a conveniently low melting point and low volatility a t temperatures near the melting point-e.g., urea-is spread on t,he surface of the plate and evaporated by raising the temperature of the plate somewhat. A thin coating of the substance remains on the surface. Now the temperature is raised rapidly until within a few degrees of the melting point, after which the
Various microprocedures have been worked out for use with the electric heater described. The drying of precipitates, the heating of substances at constant temperatures over long periods, and the like may also be carried out on this hot plate. A wide range of controllable temperatures is obtained.
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THERMALLY controlled electric heater of simple and economical construction is described. It has been used by the author for carrying out microprocedures including boiling points (3, 6 ) , extractions ( I ) , fractionations (4, 6), sublimations (3, 6, ?'), and sealed capillary reactions (3),and also for melting point determination (2,8). .4 wide range of controllable temperatures is obtainable. APPARATUS
Au electric flatiron of 5-amperc mpaci(y and 550-watt rating is used in the preparation of t,he heating block. TYOholes of &mm. diameter are drilled diagonally throu h the base (11 mm. thick), so that they meet at the center of t8e block (Figure 1) One hole is for a 360" thermometer (Figure 2) and the other is fo; a microreaction capillary tube. Several other small-diameter holes which need not terminate in a central point, are drilled through the base for holding tubes to he used for sublimations, sealed capillary reactions, and the like. .1 variable-voltage transformer or a rheostat of 5-ampere capacity may be used for therma,l COntr~Jl,but lamp bank control has given the author very satisfactory results. For finer temperature control, a slide-wire rheostat, map be used in conjunction with the lamp bank. Figure 3 shows the current input. necessary for obtaining a convenient temperature range. By increasing the current input, proportionately higher temperatures will be obtained. If desired, time-temperature and constant radient calibration curves may he obtained by the met'hod descrifed by Dowzard and Russo ( 2 ) .
Figure 2.
Thermometer and Boiling Point Capillaries in Position in Heater
heatirig is adjusted to give a rise of about I" per minute. The areas within which the substance melts for 0.5" or 1.0' intervals are noted, these are the demarcation areas of uniform temperature ran e and give an indication of the best distribution over the surface. This process niay be repeated with ot,her substances of different melting point,s.
For determining boiling points (3, 6 ) the author has found the following procedure satisfactory: A narrow capillary (0.3-mm. interval diameter and 7 to 8 cm. long) is used. KOtapered stem is re uired. Liquid is drawn up to a column of 5 mm. or more, then &e capillary is sealed in the usual way. Care should be taken not to make the sealing bead too large; otherwise, proper contact of the whole length of the
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Figure 1.
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Top V i e w of Heater
h short preliminary working acquaintance suffices to establish familiarity with the arrangements necessary for satisfactory operation under various demands.
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PROCEDURE
The procedure for taking melting points is essentially as described by Shriner and Fuson (8). Small amounts of the substance are deposited on the hot plate throughout small interval rises of temperature. The moment an instantaneous fusing is observed, the temperature is read and taken as the melting point of the substance. Glass wool is used for wiping off the surface of the heater. In taking the melting point, the area over the thermometer bulb is used, because noticeable temperature differences arise
A M!PERES
Figure 3.
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Temperature-Ampere istics of Heater
Character-
February, 1944
Fi ure 4.
Position of Liquid in Capillary at Microboiling Point
ANALYTICAL EDITION
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the solvent has been cooled the tube is resealed and heated until solution is complete. Then the solution is centrifuged to the other bulb and allowed to cool until crystallization is complete. For sublimations, tubes similar t o those used in microfractionations may be used. If only minute amounts of substances are to be sublimed, tubes 1 or 2 mm. in diameter may be heated in the small-diameter holes of the heater. For fractional sublimation (7), a long tube (about 10 cm.) is used. A little powdered glass wool in the tubes holds back unsublimable impurities. Hubacher (6)illuatrates clearly the advantages and limitations of vacuum sublimation and believes this method “is worthy of greater consideration”, a belief which is strongly shared by the anthor of this paper.
3 capillary with tlir surface of lie plate \vi11 not be eff’ected. The capillary is so DISCUSSION placed that the sealed end is approximately a t the center of the block and the D o m a r d and Russo (8) emphasize the open end projects about, 2 cm. off the necessity for maintaining a 0.5” per minute 3ide (Figure 2). temperature rise in melting point determina\Then apprombing the boiling point, ,the liquid starts moving rest,lessly back tions, when within 3” or 4” of the supposed :tnd forth in the capillary. When the melting point. A glance a t Table I shows ,boilingpoint is reached, the liquid remains that the heating characteristic of this heater Figure 5. Microextracting Tube -;t:itionary, and the lower meniscus coinis such that a slow temperature rise is imperacides with t,he edge of the hlock (Figure 4) , (‘ooline of the block causes the liauid to tive for the most accurate results. The rehe drai-n into the c~ipill~try, and a Fepetition of the boiling point sults in Table I indicate that, if the rate of heating is greater .tletermina,tion is pnssible. than about 1’ per minute, the heating of the thermometer lags For carrying out iiiic~~oextr;tctions,a n extraction tube having significantly behind that of the heater’s surface. :L small bulb at each end is fashioned (Figure 5 ) . The m a t e d to be ext,racted is inti,oduced into the ca illary, which is t a ped A comparison was made of the melting points obtained by slightlv to shake all the material into t i e lower bulb. $any this block and of an oil bath (Table 11). A uniform agreement is materikl adheres to the walls of the capillary, a small wad of noted for the melting points obtained by the two methods. glass wool is used to pus11 i t down into the bulb. Next, an It is suggested that the experimenter carry out some pi-(%“iIit,eriixl” filter of powdered glass wool or asbestos is prepared. When the cn,pillary has cooled, the solvent is introduced into liminary boiling point determinations, using capillaries of the tube and centrifuged to the lower bulb. Enough solvent various sizes (0.1 to. say, 0.7 mm.) to acquaint himsdf with the is used to fill t,he bulb about; LWO thirds, and is cooled by submerging the lower bulb in an ice bath. When the solvent has cwoled sufficiently, the upper end of the capillary is sealed. For carrying out, tjhe extraction, the heater is maintained a t a Table 1. Effect of Heating Rate on Lag of Thermometer constant temperature, which should be about equal to that of Temperature the boiling oint, of the solvent. When the heater has reached (Using m.p. of urea a8 piirfnce temperature indicator) the requiref temperature, the capillary tube is laid horizontally on the surface of the heater and allowed to heat for several Melting Point Temperature Rise (Urea) minutes, so that maximum solution of the substance is effected. * C./nin. c. Then the hot solut,ion is centrifuged to the other bulb. A small quantity of liquid is allowed to remain in the stem on the side 132.7 0.2 132.4 0.9 with the empty bulb, so that a vapor e uilibrium will be ef131.5 2.5 fected, t,hus preventing t>hetendency of t%e solvent to go over 130 5.0 to the empty bulb during heating. The bulb containing the 128 13 solution is submerged in the ice bath and kept there until maximum precipitation has occurred. Then the liquid is recentrifuged t o t,he otJherhnlb, arid the operations are repea,ted until tlie Table 11. Comparison of Melting Points Obtained b y Electric est,ract.ion is co~nplete. Heater and Oil Bath Melting Point The completeness of extraction is conveniently deterniincd Substanre Heater Oil bath t y use of the polarizing microscope (1). To prepare t,he extra(.c. c. t.ion tube so that it, may be examined under the microscope, it is 132.5 132.5-133 Urea 147 147-8 -4nhydracetone b e n d necessary to flatten a portion of the tube on the same side as tlic’ Benzimidazole “precipit,ation” bulb. During the extraction proredure, som(’ 168 183.5-184 168-9 p-Aminobenzoio acid 183.5 2-Acetylamino-1. I - n a ~ h t t ,I , ~~ ~ ~ i o ~ m 201 201-2 soliition should be allowed t o remain in the flattened area, R O Fluorescein 320 t.hat crystals Kill deposit. in this area. These crystals may then be examined for homogeneity and other characteriativ!: Table 111. Boiling Point Values Obtained with 0.3-Mm. BOIC t,hat>may be required to establish their identity. Capillaries For microsyntheses, tubes similar to the one described for Observed Boilin Point Liquid ( t 0 . 2 0 c.3 microextraction may be used. The methods used in introducing c. the reactants and in preparing t,he internal filter are the same 5 0 .0 tert-Butyl chloride as for microextractions. After the reaction hae been coni04.5 Methanol 78.0 Ethanol pleted, the empt>yend of the tube is opened, and solvent (cha7183.0 Aniline coal is also added if decolorization is necessary) is int.roduced and 207.0 Tetralin wntrifuged to thc hnlb cont>jiniiigthe resrt,ion mixillre. Aftvi.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
proper size of tube required for most accurate results. I t has been the author’s experience that, when capillaries of less than 0.3-mm. internal diameter are used, the boiling point is too low; with larger capillaries, the boiling point is t,oo high (Table III). LITERATURE CITED
(1) Benedict, H.C . , IND. ENO.CHEM.,ANAL.ED., 2,91 (1930). (2) Dowzard, E., and Rusao. M.. Ibid., 15,219 (1943).
Vol. 16, No. 2
(3) Emich-Schneider, “Microchemical Laboratory Manual”, pp. 118-35, New York, John Wiley & Sona, 1932. (4) Gettler, A. C.,and Fine, J., IND.ENG Cmm., ANAL.ED., 11, 469 (1939). (6) Hubacher, M. H., Ibid.. 15,448 (1943). (6) Morton, A. A., and Mahoney, J. F., Zbid., 13,494 (1941). (7) Morton, A . A., Mahoney, J. F., and Richardson, Graham, Ibid., 11, 460 (1939). (8) Shriner and Fuson, “Identification of Organic Compoundd’, 1st ed., p. 51, New York, John Wiley & Sone,’1935.
A DIALYSIS CELL For Rapid Quantitative Analytical Determination of Diffusible Components in Blood Plasma PAUL B. HAMILTON AND REGINALD M. ARCHIBALD P i , N. Y.
Hospital of Rockefeller Institute for Medical Research, N e w York
A simple dialysis apparatus is described which in P to 3 hours provides quantitative equilibrium of diffusible constituents in the system and a dialyzate convenient for analyses.
Tit HE
dialysis cell here described has been of use in a variety of procedures. The technique is especially applicable when is desired to prepare a protein-free solution of dialyzable components with any one or a combination of the following conditions: minimal dilution, absence of foreign ions, or exposure to acid or alkaline reagents or to reagents capable of denaturing protein. The apparatus has proved so simple and effective that it can be recommended for general use in analytical dialysis.
cates that the diaphragm is intact and secure. Sausage casing (27/32) has proved satisfactory (supplied by the Visking Corp., 6722 West 65th St., Chicago, Ill.). Some cellophane products are, however, impermeable to water and of course are useless for this purpose. A simple test of permeability is to introduce a measured volume of 0.1 N hydrochloric acid into the central tube and dialyze against 2 volumes of distilled water. If the intact membrane is permeable, at equilibrium the concentration of acid throughout will be 0.0333 N .
APPARATUS
The dialysis cell consists of a small wide-mouthed bottle of about 120-cc. capacity, closed by a So. 8 stop er, through which passes a straight glass tube of 28-mm. out& diameter and 11 cm. long. The central tube is closed a t its lower end by a cellophane diaphragm held securely in place by many turns of an elastic band, and a t the top by a S o . 5 rubber stopper. Both rubber stoppers have intravenous needles (No. 18) passing throu h to allow equilibration of pressure with the atmosphere wikout loss of liquid by evaporation. The apparatus is assembled as shown in Figure 1. The technique is like that employed by Hamilton and Van Slyke (1). Two cubic centimeters of plasma were pipetted into the central tube, a glass marble was introduced to give mechanical stirring as described by Northrup and Kunitz (S), and the central stopper was set in place. Eleven cubic centimeters of distilled water were pipetted into the bottle, and the central tube was pushed into place with glycerol as lubricant, and pushed down till the diaphragm was within 1 or 2 mm. of the bottom of t,he bottle. The bottle was then gently rocked for 2.5 hours, by which time all diffusible amino acids had become u n i f o r m l y d i s t r i b u t e d throughout the total 13 cc. of liquid in the system. A convenient rocking device is depicted in Figure 2. Each assembled cell is secured to a narrow board by a stout elastic band. The board is rocked back and forth by B windshield wiper motor, as described by Kunitz and Simms ( 2 ) . There are several points to note in the operation of the cell. The diaphragm is tested for leaks by immersing it in water and blowing down the open end of the tube; Figurel. DialysisCell the absence of any bubbles indi-
Figure
2. Rocking Device for Dialysis Cell
If the cell is used to prepare dialyzates suitable for ultraviolet spectrographic analysis it is essential to wash the membrane free of substances that absorb light in the ultraviolet. The blanks are easily reduced to low constant amounts by cleansing the membranes in four washes of 100 cc. each of distilled water: each wash is continued for 3 hours with gentle rocking, For exact quantitative York it is necessary to avoid isolation of droplets on the glass walls. This may be accomplished by having the glass parts, especially the central tube, scrupulously clean and free from grease, or the central tube may be coated within and without by a layer of paraffin. In this latter case the glass marble provides the wettable Furface on which to drain the pipet. In this apparatus a shift of fluid across the membrane in either direction does not influence the final concentration of a freely diffusible constituent (one independent of a Donnan equilibrium), since its concentration will be equal on both sides of the membrane. The length of time a cell has to be rocked, in order to have equilibrium of diffusible amino acids throughout the total fluid volume, was established by dialyzing 2 cc. of plasma against 11 cc. of water. Cells were rocked for 15, 30, 45, 60, 90, 120, 150, 180, and 310 minutes and the dialyzate mis analyzed for free alpha-amino acids by the ninhydrin-carbon dioxide method (1). All analyses were in duplicate. Figure 3 shows that equilibrium across the membrane with respect to diffusible free alpha-amino acids was achieved within 2 hours of rocking,
