If the original exposure time is extended, development with the fluorescent bulb will not be required. I n this case a real image will immediately appear upon the Linagraph print. When nanow-strip chromatograms or chromatograms containing several limbs are photographed, it is desirable to cut a thin opaque mask to fit between limbs to avoid possible overexposure of the paper. The developed print image of the areas of the chromatogram containing ultraviolet-absorbing materials appears white to light gray in contrast to the darker background of the print. The print is then superimposed upon the original chromatogram and fractions of the latter are cut in subdued room light. Alternatively, the prints may he saved in a lighttight container for future analysis of the chromato-
grams or for reference. Further viewing of the print, however, should he done under subdued room light. Such prints will not serve as permanent records, as continued exposure to light will darken them. To secure permanent records, it is better to employ a high contrast paper to obtain prints of the paper chromatograms. The best print is obtained when the original chromatogram is exposed to Kodak Azo F-5 single weight paper for 1 second. The paper is developed with Versatol developer diluted l to 10 with water, fixed, and dried as usual.
Losses due to even 1-minute exposure of the ultraviolet-absorbing materials to the ultraviolet light are not significant if the paper is thoroughly dry before exposure. In no case is the loss greater than that which results from conven-
tional spot test analysis of representative strips of the paper chromatogram. ACKNOWLEDGMENl
The author would like to acknowledge with appreciation support received from Grant HF-6137 National Institutes of €Iealth, National Heart Institute. Thanks are also due to J. T. Groet, Graphic Reproduction Division, Technical Service Center, Eastman Kodak Go., Rochester, N. Y., for supplying information concerning the use of Kodak Linagraph direct print paper, and to W. F. Swann and W. T. Swanton, Industrial Sales Division, EastmanKodak Co., forgeneronsly supplying the photographic paper for this study.
Rapid Evaporation of Solutions in Test Tubes Charles E. Pierce and Theodore D. Perrine, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, U. S. Department of Health, Education, and Welfare, Bethesda, Md.
purification proceINdures, such asorcolumn chromatogANALYTICAL
raphy, a large series of fractions is often obtained. These fractions, as obtained b y an automatic fraction collector, usually consist of equal volumes of solution stored in individual tubes. The evaporation of the solvent from these fractions, which is often the prerequisite to further work, is tedious. To improve the efficiency of this operation, a number of experiments were carried out. The practice has been to warm the tube in a heated bath and blow a stream of gas into the tube, evaporate the liquid in vacuo, or use a combination of both. With many solvents, practical evaporation rates can be achieved only vhen one of these methods i s employed. With one of the more effective devices (R. R. Williams and T. D. Spies, “Vitamin B, and Its Use in Medicine,” p. 141, Maemillan, New York, 1938) it was possible to evaporate 10 ml. of water in 1 hour (at an unstated bath temperature). This required a fairly large gas stream, and experience shovs that it works best when the gas inlet jet i. continually adjusted to maintain the tip close to the liquid surface. Unless the gas stream strongly riffles the liquid surface, this method of evaporation is not efficient. Difficulty arises becanse the geometry of test tubes is such that the free liquid surface is small and bumping occurs readily. Radin [ANAL.CHEM.28, 542 (1956)l described an evaporator designed to overcome this. Experiments were designed to agitate or whirl the tubes so that the free sur-
face would he greatly increased and humping reduced. The methods included high and low speed rotation of the tube about ita axis, low frequency agitation, and slow rotation of a series of tubes symmetrically placed about an i n c h e d vertical axis. Only high speed rotation gave a film thin enough to prevent undesirable bumping unless the volume of the liquid in the tube was very small, in which case any of the methods was reasonably satisfactory. Unfortunately, the power requirements and heat generation necessary to operate a high speed rotary vacuum seal are such that high ~. meed rotation is not practical. A Eecond objection is that this type of motion, while i t produces a thin film, induces no movement. of the liauid relative to the tube wall, and hence produces a “dead” surface as opposed to the rolling surface found, for example, in a falling-film still. (The rolling surface can he obtained if the speed of rotation is continuously varied.) The Evapo-mix, manufactured by the Lahoratory Glass and Instruments Carp., New York 31, N. Y., is designed to oscillate a series of 10 tubes about an axis perpendicular to the axis of the tubes, at frequencies up to 125 cycles per second. This device can impart a rolling motion to the liquid similar to that obtained in a falling-film still, and has a centrifugal component which should Serve to minimize humping or spraying during rapid distillation. Theoretically, this should provide excellent evaporation rates, and tests indicate that within certain limits this type of agitation is satisfactory. ~
Figure 1. Evopo-mix showing springloaded tube holders and modified manifold and tube covers
The machine, as supplied, requires certain modifications to protect the sample from conta.mination. Other modifications have been made for convenience in use. (Figure 1). An improved model now availiable is much the same as the one described here, except for the stopcocks. Stopcocks are superior to pinch clamps, which are affected by instrument vibration. Also one does not always have 10 tubes to he evaporated a t one time. DESCRIPTION OF APPARATUS
The Evapo-mix machine consi rack to hold a maximum of 10 ti to u) mm. in diameter and 120 VOL. 30, NO. 12, DECEMBER 1958
mm. long. The tubes are cushioned in rubber and clamprd in place by means of individual screw and lock-nut fasteners (Figure 4 4 ) . The rack is oscillated about its long axis by an eccentric and variable speed motor. The whole assembly is mounted on a thermostatically controlled water bath, so that the lower parts of the tubes are immersed. A manifold is mounted above the tubes and connected to them by short lengths of Tygon tubing and inverted conical adapters of polyethylene (Figure 2). This arrangement of the manifold system would lead to contamination of the samples if organic solvents were being evaporated. Operating tests indicated that the screw and lock-nut device had a tendency to shake loose a t high frequencies, and was inconvenient to use. The rheostat speed control on the motor was not satisfactory.
Pyrex, 8 m p
U
Figure 2. Original manifold and tube covers
/
4
Heovy Springs 4vZ turns 0064 g " p i t c h wire
Light Springs 43/4" turns 0,052' I 3/i6'I pitch wire
MODIFICATION OF APPARATUS
The motor speed mas controlled by a Variac. The manifold system was entirely rebuilt (Figure 3). Tests indicated that for optimum agitation, the connections to the manifold had to be very flexible, which precluded the use of vacuum tubing. The requirement was met by a very flexible grade of rubber tubing, in size 1/4-inch bore x inch wall, supported by a ll4-inch pitch spiral of 22-gage Nichrome wire which had been wound on a 6/32-inchmandrel. Each vacuum tube was fitted with a 3mm. stopcock. In use, the stopcocks are turned on one after the other so that a partial vacuum is maintained in the manifold. This facilitates sealing the tubes, locates leaks, and prevents bumping. The polyethylene adapters ryere altered from conical to a part conical, part cylindrical shape by warming in a steam bath, and then forcing a forming tool into the cone for a distance of about 10 mm. This tool should have a maximum diameter of 20 mm. with a fairly sharp bead a t its forward end. A female B 14/15 joint is a suitable tool. This treatment causes about a 10% mortality among the polyethylene adapters, due t o a split which may appear as much as 3 days after forming, and which appears to be associated with a mold mark. Adapters formed in this .ivayare more convenient t o attach to the tubes, easier to keep on, and 1%-iththem it is easier t o establish and maintain a vacuum seal. The tube support screws were replaced by spring-loaded plungers as illustrated in Figure 4,B. The arrangement shown in Figure 4,C, having the lock nut outside the
frame, was more convenient than the arrangement shown in Figure 4,A, and could be used to advantage if it should be desired to maintain the screw-lock system. RESULTS
It is possible t o get evaporation rates 2070
ANALYTICAL CHEMISTRY
A
B
Figure 4. A.
B. C.
Figure 3. Modified manifold, with '/l-inch bore X 'i6-inch wall rubber tubing supported b y a wire spiral. Curved glass tube with 1O/ 30 joint and glass bulb adapter
Tube holder
Original Modified With lock nut outside
for water of 1.0 ml. per minute; ethanol, 4.2 ml.; methanol, 4.5 ml.; ethyl acetate, 7.2 ml.; and acdone, 10.0 ml. These rates were obtained by concentrating in vacuo in single 20 x 150 mm. culture tubes, using an ordinary glass water pump (25-mm. vacuum, measured a t the pump) and a 44" C. bath temperature. Then the machine is filled to its capacity, the vacuum is decreased somewhat (32 mm.), and the evaporation rate is considerably reduced, although a condenser in the manifold should overcome this. Kater, for example, may be evaporated under these conditions a t a rate of 0.3 ml. per minute per tube. When a stream of gas is used instead of a partial vacuum, the rate of evaporation is slower. For example, blowing air into the top of 20 x 150 mm. culture tubes while shaking in the machine, results in a water evaporation rate of about 0.07 ml. per minute per tube. The bath temperature was 44" C. and the air flow in this experiment was 0.1 cubic foot per minute per tube. The air jets were not close to the liquid surface. The amplitude of vibration of the tubes was such that it was not feasible to insert the tubes deeply into them. Flexible (polyethvlene) jets could be inserted deeper in the tubes, but they required cleaning after each run. At this rate, a 200-cu. foot cylinder of nitrogen would last 3112 hours for concentrating the contents of 10 tubes (140 ml. of water removed per cycle of nitro-
gen). With other solvents under the same conditions, the rates were: ethanol, 0.4 ml. per minute; methanol, 0.7; ethyl acetate, 1.1; and acetone, 1.9. Vacuum evaporation is some 5 to 10 timee as fast as air stream evaporation under these conditions. SPEED OF AGITATION
Frequencies were measured with a stroboscope. The optimum frequcncy of vibration is a subjective matter and is also extremely sensitive to operating variables. In general, the following frequencies (cycles per second) are satisfactory in these machines under the operating conditions as described. 20 x 150 mm. culture tubes, air blast 58 C.P.S.
20 x 150 mm. culture tubes, vacuum 67 C.P.S. 12-ml. centrifuge tubes, air blast 63 C.P.S. 12-ml. centrifuge tubes, vacuum 54 C.P.S.
No difference was found in the evaporation rate of Tl-ater in tubes held by screws or springs, The latter are more convenient to operate, and offer more reliable support. A t very high speeds of vibration the tube3 tend t o move in the holders. This can be overcome by wrapping the tubes at the support point with adhesive tape. PATTERN OF AGITATION
The type of agitation attained in this
ever, for type l agitation is more liable to incur bumping than type 2, where the centrifugal force acting on the liquid tends to prevent spray-over. In the case of very rapid, ebullient distillation, it is often impossible to maintain type 2 agitation. The strength of the plunger springs is an important factor in determining the speed a t which a given type of agitation will occur. Two types of springs were investigated (Figure 4,B). The lighter springs produce a given type of agitation a t a lower speed than do the heavier. With vacuum adapters the heavier springs are advantageous. Lower operating speeds are desirable to reduce the noise level and the splashing of water from the heating bath.
machine is complex, and depends on the size and shape of the tube, the type of support, the speed of the vibration, and the nature and amount of liquid in the tube. Two distinct general patterns of agitation were observed. Type 1, surface splashing, in its more vigorous manifestations violently agitates the entire contents of the tube, and may cause cavitation. Type 2, rotary agitation, results in the formation of a vortex which usually reaches to the bottom of the tube. I n general, type 1 agitation appears a t low speeds, increases in vigor, then transforms into type 2, as the speed increases. At the highest speeds, type 2 agitation usually reverts to type 1. An attempt vias made to define the parameters of these types of agitation until it was discovered that there was no difference in evaporation rates. This is not the only consideration, how-
TYPE OF TUBE
In general, the round-bottomed tubes
are superior to conical tubes in evaporation rate, although the diameter appears to be relatively unimportant. For example, the evaporation rate in 16-mm. tubes was about the same as that in 20mm. tubes.
VOLUME OF SOLUTION
In a given tube, small volumes evaporate more rapidly than large volumes. The maximum safe loads are (approximately): 20 x 150 mm. culture tubes, 10 ml.; 12-ml. conical centrifuge tubes, 3 ml. Type 2 agitation cannot always be induced in 20 X 150 mm. tubes when the volume is less than 7 ml. When methanol is being evaporated, the volumes stated may need to be reduced because of the tendency of methanol t o bump.
Method for Elimination of Interference Fringes in Spectra of Thin Films Charlotte Lutinski, The Perkin-Elmer Corp., Norwalk, Conn. HE
presence of interference fringes
Tin the infrared transmittance spectra of thin organic coatings and films affects the accuracy with which these spectra can be analyzed. Interference fringes normally arise from multiple reflections between plane parallel surfaces. This effect can be observed in Figures 1,A, and 2 , A . Fringes may occur throughout the entire infrared region. However, the sample thickness and absorption bands will determine where the fringes \Till be seen. The observed interference fringe pattern is superimposed on the absorption spectrum of the sample of interest and distorts band shape, thus affecting the
accuracy of qualitative and quantitative analysis. A method has been developed which eliminates the interference fringe pattern from the absorption spectrum. When the film is sufficiently thick, the separation of the fringes is less than the resolution of the infrared spectrometer. Films cast directly onto salt plates do not exhibit fringes. Because of the intimate contact, there is no abrupt reflective index change between the second film surface and the first salt surface. By coating a thin film with Nujol and pressing it against a thick rock salt nindow, an effective thick sample can be obtained which has
minimum surface reflection a t the interface, and, therefore, no observable interference fringe pattern. Absorption bands introduced by the Nujol are weak, while the rock salt window does not introduce any additional absorption over the region of interest. If the C-H region is of interest, some nonhydrocarbon liquid such as perfluorokerosine may be used in place of Nujol. Other low index of refraction crystalline windons can also be used. Figures 1 and 2 illustrate this technique, utilizing, in the first case, reflectance to measure a coating, and, in the second case, transmittance directly through a film of polystyrene.
FREQUENCY (CM-')
1
2
3
Figure 1.
4
5
6
7
8 9 WAVELENGTH (MCRONS)
10
11
12
13
14
15
Reflectance spectra showing absorption of this organic coating on metal A.
Interference fringes overlap absorption bands VOL. 30, NO. 12, DECEMBER 1958
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