machine-prepared phosphors showed better precision than those prepared by hand, and the two runs with the machine are essentially in agreement. ACKNOWLEDGMENT
The authors thank Charles G. Bay and Leonard B. Riley for suggestions in the building of the phosphor-making machine, and A. G. King, Wayne Mountjoy, Irving Frost, and John C. Antweiler for confirmatory experiments establishing its usefulness.
LITERATURE CITED
(1) Centanni, F. A., Ross, A. M., De Sesa, M. A., ANAL.CHEM.28,1651 (1956). (2) Grimaldi, F. S., May, Irving, Fletcher, M. H., U. S. Geol. Survey, Circ. 199 (1952). (3) Michelson, C. E., U. S. Atomic Energy Comm., HW-36831 (1955). (4) Price, G. R., Ferretti, R. J., Schwsrtz, Samuel, ANAL.CHEM.25, 322 (1953). (5) Sadowski, G. S., Gentry, J. R.,
Hemphill, H. L., Clinton National Laboratory, CNL-23 (1947). (6) Thatcher, Leland, U. S. Geological Survey, oral communication.
( 7 ) Zimmerman, J. B., Canada Dept. Mines Tech. Surveys, Mines Branch, Memo. Series 114 (1951). (8) Zimmerman, J. B., Rabbits, F. T., Kornelsen, E. D., Canada Dept. Mines Tech. Surveys, Mines Branch, Radioactivity Div., Topical Rept. TR-122/53 (1953).
PITTSBURGH Conference on Analytical Chemistry and Applied Spectroscopy, February 28 to March 2, 1956. Publication authorized by director, U. S. Geological Survey. Part of investigations undertaken by the Geological Survey on behalf of the Division of Raw Materials, U. S. Atomic Energy Commission.
Protein Determination for large Numbers of Samples Gail Lorenz Miller, The Pioneering Research Division, Quartermaster Research and Engineering Center, Natick, Mass.
Rosebrough, Farr, and RanL dall's procedure for colorimetric determination of protein [J.Biol. Chem.
samples and reagents a t 50" C. accelerates development, reducing the time to minutes.
193, 265 (1951)] is particularly convenient when only a few samples are deter.2 mined at one time. Two modifications in procedure make it possible to determine large numbers of samples. Use of a comparatively smaller volume of more concentrated alkaline copper reagent and a larger volume of more dilute Folin phenol reagent permits introduction of a proportionately larger volume of phenol reagent with sufficient force to ensure adequate preliminary mixing, and final mixing can be postponed until after the reagent has been added to all the samples. Heating the final mixtures of
One-milliliter aliquots of alkaline copper reagent composed of 10 parts of 10% sodium carbonate in 0.5N sodium hydroxide and 1 part of 0.5% copper sulfate in 1% potassium tartrate are added to I-ml. aliquots of protein solution in colorimeter tubes 14 mm. in outside diameter. After the mixtures have stood for 10 minutes, 3-ml. aliquots of a 1 to 11 dilution of Folin phenol reagent are added to the samples as forcibly as practicable. The mixtures of samples and reagents are heated for 10 minutes a t 50" C. in a constant temperature water bath. After the mixtures are cooled to room temperature, absorbance is read at wave lengths of 540 to 750
OWRY,
mp, depending on the sensitivity required. For addition of the alkaline copper reagent and the Folin phenol reagent, hand-operated plunger-type pipets or motor-driven automatic pipets are very useful. With automatic pipets, glass instead of stainless steel valves are required when the phenol reagent is used, because of the problem of corrosion. Entirely analogous results were obtained with bovine serum albumin and with gelatin, although, as expected, the color with gelatin was about half that with bovine serum albumin. The reproducibility of analyses over the range of 0.04 to 0.20 mg. of protein in the modified test is, on the average,to 2%, essentially the same as in the original test.
Automatic Multistage Semimicro Zone Melting Apparatus A. P. Ronald, Chemistry Section, Fisheries Research Board of Canada, Technological Station, Vancouver, 8. C.
technique of zone melting, the mathematics (1, 9) and principles (8) of which have been adequately described, is not entirely new in metallurgical purification (8, 9, I S ) , b u t is relatively new in organic chemistry. Rock (11) has purified benzene; Hesse and Schildknecht (6),fatty alcohols; and Handley and Herington (S), benzoic acid, pyrene, anthracene, and crysene. The limited success of chromatographic techniques in the sterol field suggested that a suitable apparatus might make zone melting applicable to problems in this laboratory. This method for purifying organic compounds requires that a narrow molten band travel slowly down a vertical column of the solid substance in a glass tube; impurities are separated and normally concentrated a t the bottom of the column. A horizontal THE
964
ANALYTICAL CHEMISTRY
tube may be used in metallurgy (b), but in organic separations when the molten zone passes and the material crystallizes the solid drains away from the side of the tube and seeps back. No available apparatus (3-7, 11, 1.2) was entirely suitable for the purpose. An automatic macromodel (6) was investigated, b u t the disengagement of the drive motor by the system of pawl and ratchet was not clearly understood, and i t did not appear suitable for adaptation to a micro scale. A zone melting apparatus could have been modified by changing the heater carriage to a sample carriage and building a bank of heaters through which the sample tubes could pass, b u t the apparatus described here was completed before the advertisement appeared (10). An apparatus ( 7 ) apparently similar to the commercial one has been described, but
the mechanical operations are different. The apparatus described here is cheaper, smaller, and self-contained. PRINCIPLE
OF
OPERATION
The apparatus (Figure 1) consists of a drive motor which raises a platform that supports four tubes containing the material to be purified. As the platform rises, the tubes pass through a heater which creates a well defined molten zone of material. The molten zone passes slowly down the column to the bottom. The platform reverses, travels quickly to its starting position, and again commences its slow ascent. The slow drive speed for raising the platform is attained by a synchronous motor with a drive shaft speed of 120 revolutions per hour. This is reduced by external gears to 20 revolutions per hour. The last reduction is by a bevel gear with a n internally threaded hub
in " " " , I- I
Figure 1 . General layout of mechanical components Drive motor, Bodine Type NSY-12R Reverse motor, Bodine Type CL-2 Clutch solenoid, GE magnetic switch solenoid 1 6 2 5 2 Bevel gears, two-1 6-tooth, one-64400th Spur gears, two-24-tooth, one-36-tooth, one-52-tooth
ni 9 11
Table 1.
Values of Component Parts of Electrical Circuit Resistors Rl 100-ohm 50-watt R2, R3, R4, %ohm 50-watt rheostats R5 R6, R7, R8, Loop of No. 26 bare nickelR9 chromium wire
c1 Y'
~ U I D EEAR
platform, in 6 seconds, to its original position, where on striking another microswitch it is stopped. After about 15 seconds the clutch engages and the drive motor starts. The delay before commencement of the recycle is effected by incorporation of a delay mechanism in the circuit, installed to avoid damage to the gear system.
Vl, v2
Relays P & B Relay, KA 11D, 110volt. d.c.. D.P.D.T. Guardian R e l a i Series 200, 115-vo1tJ ax., D.P.D.T.
1 2
Switches M1, M 2 , M3 Unimax. Type 2 HBW-1,
P i
I
Figure 3. Circuit diagram of apparatus
-ih-g P+--
RI
I
R5
L Figure circuit
4.
Heater
(Figure 2), through which passes a s/sinch-diameter rod with 16 threads to the inch, fixed by a guide bar attached at right angles to the rod and sliding in a slot so that it cannot rotate. The bevel gear on completing 16 revolutions raises the platform 1 inch. The gear turns 20 times in 1 hour; thus the platform travels 1.25 inches per hour. AUTOMATIC RECYCLE. On reaching a predetermined height the guide bar attached to the bottom of the threaded rod lifts a lever which operates a microswitch. The power is cut off from the drive motor, which is then disengaged from the system through the action of a solenoid-operated dog clutch. The reverse motor cuts in, returning the
microswitch
.
On-off
10-amper e D .P.S.T toggle
T1
Transformers Hammond, Type 1129R60CY, 6-volt, lo-ampere center tapped
OPERATION OF APPARATUS
When the electronic delay circuit (Figure 3) is switched on, 15 seconds elapse before the relay coil, 1, is energized, cutting off, through the action of relay contact lA, the filament voltage to tube V1 and making the plate voltage to tube V 2 . The relay is held on until the circuit to the coil is broken, a t which point even immediate application of current to the circuit would not energize the coil until 15 seconds had elapsed -the time required for the filament of tube V l to heat up and allow sufficient current to flow to energize the coil. Relay contact 1B controls the action of the clutch solenoid and the drive motor. When coil 1 is not energized, the clutch solenoid is in operation. This disengages the drive motor from the system and closes microswitch M 3 , which is so positioned that the circuit to the reverse motor is not complete unless the clutch is fully disengaged, Even with the clutch disengaged the reverse motor will not act until its circuit has been completed. This can occur only in the following manner. MicrosFvitch A41 is closed either by action of the bar attached to the threaded rod or by manual manipulation. Coil 2 is then energized and relay contacts 2A and 2B close and break the circuit to the delay unit through relay contact 2A. When the circuit to the delay unit is broken, 2A makes a circuit to its own coil; this is necessary, as microswitch M I is opened as soon as the reverse motor comes into operation. Relay contact 2B makes a circuit to microswitch M 3 . When the filament of tube 112 has cooled sufficiently to cut off the power supply to coil 1, relay contact 1 opens, the drive motor stops, and the clutch disengages and completes the circuit through M 3 to the reverse motor. The rod then travels down until the bar strikes microswitch M2, opening the self-holding circuit to coil 2. 2A and 2B open, the reverse motor stops, the circuit to the delay unit is made, and after 15 seconds coil 1 is again ener-
Condensers 50-mfd. electrolytic 117Z6GT, tubes converted to ordinary triode, doubling current-carrying capacity
switch
Fuses Two 2-ampere1 Type 3-AG N1, 11-2
Indicator Lights Keon bulb, Type GE-NE 51, resistor-type mountings
gized. The clutch engages, the drive motor starts, and a new cycle commences. CONSTRUCTION OF APPARATUS
The mechanical components were assembled on an aluminum plate 10 X 24 X 3/8 inches. The bevel gear assembly was first manufactured and assembled and then mounted in the center of the plate. The reverse motor gear shaft was assembled, and the 16-tooth bevel gear was fastened to one end of a 2-inch length of '/(-inch stainless steel shaft, by means of a pin driven through the hub of the gear and through the shaft. The 52-tooth spur gear was fastened to the other end by drilling a '/(-inch hole in the gear and fitting it to the shaft, and a small hole was drilled parallel to the shaft so that it cut a keyway from the shaft and the gear. The hole was then tapped and a screw inserted which held the shaft and the gear firmly together. The gear assembly was mounted on 1/4-inch brass pillow blocks, fastened to an aluminum mounting block of such thickness that the bevel gears meshed correctly. The slow-speed drive motor shaft was mounted by a similar procedure. A hub was brazed to each spur gear which was to be mounted on a motor shaft, a hole drilled and tapped on each hub, and a setscrew inserted. The gear was held in position on the drive shaft of the motor by the setscrew. VOL. 31, NO. 5, MAY 1 9 5 9
965
*
This allows easy mounting, simple adjustment, quick removal, and disengagement of any motor while tests are being carried out on the correct positioning of the electrical components of the system. If the gear system is not mounted properly and a n y tightness occurs, difficulty will be experienced in disengaging the clutch; careful alignment is necessary and the clutch teeth should be angled as much as possible. Microswitches A l l and ill2 can be mounted in many ways. One convenient mount is on the side of a rightangled bracket, mounted on the outside of the guide bar support. A slot is cut in the side of the bracket to allow easy positioning of the microswitches. The mechanism of the microswitch is actuated through coming in contact with that part of the guide bar which protrudes through the guide bar support. Each heater unit is constructed from a single loop of No. 26 bare nickelchromium wire attached to copper rods approximately 5 inches long and inch in diameter, and held in place by a Bakelite strip which provides both insulation and a means of adjusting the height of the heater. Each heater is controlled by a rheostat, wired in the manner of a voltage divider (Figure 4). The type of circuit used gave the best
individual control for each heater. K i t h all the heaters working at maximum temperature the load is about 13 amperes. However, the 6-volt centertapped 10-ampere Hammond transformer worked satisfactorily and there was no indication of heating due to overloading. To determine the effectiveness of the apparatus a trial run was carried out with mixtures of 1 and 0.1% 2,4-dinitrophenylhydrazine in naphthalene. Approximately 500 mg. of the mixture was heated and drawn into a preheated tube 25 em. long (outside diameter 4 mm., inside diameter 2 mm.), to a height of about 15 cm. Five passes completely removed the 2,4-dinitrophenylhydrazine from approximately 90% of the O.lyOmixture and concentrated it in a 1-em. zone above the point which the heater reached a t the end of each run. Fifteen passes were required to remove the impurity completely from approximately 80% of the 1% mixture, concentration taking place in the bottom 3 cm. of the tube.
development of the equipment, and for assistance in preparing the manuscript, and F. C. Freeman for assistance in solving technical problems and helping to construct the prototype. LITERATURE CITED
( 1 ) Burris, L., Jr., Stockman, C. H., Dillon, I. G., J. Metals 7, Trans. 1017 (, -1-9.5.5 ). - - I .
(2) Goodman, C. H. L., Research (London) 7, 168 (1954). (3) Handley, R., Herington, E. F. G., Chem. & Ind. (London) 1956. 304. (4)zbid., 1957, ii84. ( 5 ) Herington, E. F. G., Handley, R., Cook, A. J., Ibid., 1956,252. (6) Hesse, G., Schildknecht, H., Angew. Chem. 68, 641 (1956). (7) Natl. Bur. Standards (V.S.), Tech. News Bull. 39, 81 (1955). ( 8 ) Pfann. W. G.. Chem. Ena. Y e w s 34. .
I
1440 (1956). (9) \ - /
Pfann, W. G., J . Metals
81 i i a m i ) 112) Schumacher. E. E.. J . Metals 5 . ' 1 2 8 (1953). ' (13) Tanenbaum, lI., GOSS, A. J., Pfann, W. G., Ibid., 6 , Trans. 762 (1954). L
ACKNOWLEDGMENT
The author thanks D . R. Idler for suggesting this project, for advice during
4, Trans.
747 (1952). (10) Research Specialties Co., ANAL. CHEX.29, 77A (July 1957). (11) Rock, H., Satu,wzssenschaSten 43, ,LYV",'
A Compact Unit for Concentration of Multiple Solutions at Reduced Temperature A. S. Meyer, J. C. Matthews, and P. Samarco, Schering Corp., Bloomfield, N. J. unit described (Figure l), perT mitting concentration of multiple solutions at reduced temperature, has HE
been used in this laboratory for several years. The tubes containing the solutes are
attached to a holder, K , by means of a stainless steel spring which snugly surrounds the slot of the holder (Figure 2). This holder was designed for accommodating 12 tubes with a maximal diameter of 1 inch. (Smaller tubes are retained equally well by the spring, even if they are located adjacent t o tubes of larger diameter.) If Erlenmeyer flasks
A\
are used frequently, it is better to space the openings farther apart; in the construction described, only alternate openings could be utilized w t h these flasks. Air or nitrogen is introduced from the source, G, through a distributing manifold (D,Figure 3), 16 inches of lightweight rubber tubing 1 ; ~inch in internal diameter, B, and an 8-inch long rigid extension tube 7/32 inch in outside diameter, I , joined to a 3 I 2-inch long 18 gage stainless steel needle IT ith a Yale adapter and square-cut needle end, J. The gas flow is directed to the individual needle by a stainless steel stopcock No. MSO 1, C (items I , J , and C are from Becton, Dickinson and Co.). The end of the needle is placed approximately l/? inch above the surface of the liquid t o p e evaporated. The extension. I , held in
I 7 !I
,D.HoleThru
YD Hole(Threod1for
C H o l e Thru Both Pcs. Fasten Holderto Hub with 3-+"D Screws 120°Aport
Figure 1.
Solvent evaporation unit
Side view drawn to scale 966
ANALYTICAL CHEMISTRY
Figure 2. Test tube holder Cross sections top and side, drawn to scale
4-;"Holes (Thrlad) to Secure Manifold to Lucite Disc
Air Chamber
Figure 3. Manifold Cross section, d r a w to scale
