V O L U M E 28, NO. 2, F E B R U A R Y 1 9 5 6 purification of a solid compound mixed with impurities common to its origin. PROCEDURE
By combination of evacuation and inflow of nitrogen through stopcocks Z, N, and B , flush air out of the apparatus; dissolve the compound in a suitable volatile solvent and draw the solution into flask E . Close P with a stopper. Chill E1 to induce crystallization in the well. Switch st'opcock Z to the vacuum posit'ion and H and B to the points where suction applies only to flask 9 0 . Ilotate the apparatus (suspended on the shaft R ) YO" counterclockwise, thus throwing the solution onto filter plate C. At the same time open cock A- to admit nitrogen into top of condenser J , thus forcing fluid through C. Chill well A1 in a refrigerant mixture to induce the filtrate t.0 deposit additional solid. Rot'ate apparat'us back to the vertical position, holding filtrate solids in A I , while the clear solvent returns to Ao. Close -VI turn B so that flask A communicates only with E, and turn H so that vacuum applies only to the top of condenser J . Again chill E1 and apply gentle heat to '40,so that the solvent will distill under reduced pressure through B back into E,. (If some solid has accident,ally plugged filter disk C, this may be cleared easily by momentarily closing stopcock B so that vapors from A are forced t,hrough C during redistillation.) Restore apparatus to normal pressure with nitrogen, close the passage from E through B to A, and warm Eo until refluxing solvent redissolves the precipitated solid. Chill to induce crystallization of the compound, rotate, filter again by suction, distill back the solvent, etc., and crystallize a third or fourth time if desired. Finally, introduce fresh cold solvent through P so that the compound may be washed and freed of solution bl- suct,ion. Det,ach flask A containing filtrate solids and attach a closure cap or empty flask in it8splace. Turn Z to the vacuum position, close S, and open B and H to the three-way positions. Bring up an infrared lamp or suitable heater and dry the compound under reduced pressure. (Because the unit centering around E is compact, it, ma? be detached from A and J and put into a vacuum drying oven if desired.) hfter drying, admit nitrogen, detach the condenser and closure cap, and weigh to determine the amount of pure, dry compound deposited in E (the unit having previously been weighed empty). Modifications. T o use E as a reactor vessel it is necessary only to detach A , put a cap over the joint, and introduce a Tefloncoated magnetic stirrer into well E t,o promote good mixing of the contents. Again, all operations can be conducted under the appropriate gaseous atmosphere. Reagents may be added through a dropping funnel attached to condenser joint P. If a substance is to be carbonated as in a Grignard process, chlorinated, or oxidized, the desired gas can be introduced by way of the cocks Z, H , and B and bubbled into the medium through the filter disk while the unit is in the horizontal position. With stopcock S closed, excess gas passes out through P and may be metered, absorbed, or otherwise treated according to requirements. One problem is the control of gas pressures when the apparatus under vacuum is restored to normal pressure (stopcock LV closed, Z in vacuum position, Z is then switched to nitrogen inflow). A simple device that has proved satisfactory in this laboratory is shown in Figure 2. The long reservoir tube provides a supply of gas under pressure greater than atmospheric as regulated by the height of liquid in the graduated cylinder, while excess gas bubbles out through C. By use of a viscous mineral oil in the cylinder, the surge of liquid when stopcock Z (Figure 1) is switched from vacuum to nitrogen occurs a t a moderate rate, and Figure 2. Gas none can be forced out through the reservoir for prestop because of the Kjeldahl trap. sure control The operator, by slightly increasing the nitrogen flow from t,he tank valve, can bring the system quickly to equilibrium, for which purpose the rise or fall of liquid levels provides a convenient indicator. An advantageous alteration of the unit in Figure 1 is attachment of a suction tube containing a filter disk like C to the side of flask A .
283 Upon crystallization of filtrate solids in A (easily induced by partial evaporation of solvent through cock B or by chilling), a secondary purification can be accomplished by drawing out the remaining solution through the suction tube. Thereafter, it is necessary only to introduce fresh solvent into flask E and repeat the crystallization, gathering pure components in both flasks E and A. The proposed design lends itself to compactness of construction around the central unit, E, so that a model could be constructed on a small scale to permit manipulation of quantities approaching micro dimensions. (For miniature scale operation a test tube could replace flask E and filter disk C be correspondingly reduced in size.) On the other hand, increase in size to handle large batches is no problem. RESULTS
The apparatus described has proved valuable in the purification of sensitive compounds-e.g., drying-oil acids like eleostearic and licanic acids which must be handled under inert gas-and in the purification of precious substances xhere losses must be kept low-e.g., the resolution of d l mixtures by preparation of diastereo isomers where repeated crystallization is necessary. ACKNOWLEDGMENT
The author is grateful to the Research Corp. of New York for support of a project during which this apparatus was perfected.
Chromatographic Chamber for Simultaneous Preparation of M a n y Paper Chromatograms Irving R. Hunter, David F. Houston, and Harry 5. Owens, Western Utilization Research Branch, U. S. Department of Agriculture, Albany 10, Calif.
an advantage in preparing paper chromatoI grams,frequently especially in quantitative work, to run a number of T IS
papers simultaneously under identical conditions. This operation requires a great deal of space and equipment if 18 X 22 inch papers are used. -4trend to smaller papers has resulted, and several investigators (1-3) have found that amino acids can bc nicely separated on smaller papers. I n this laboratory, a convenient chromatographic tank has been devised by modification of commercial photographic developing tanks. It will accommodate as many as 60 papers 9 X 9 inches or smaller. APPARATUS
The complete assembly, shown in Figure 1, consists of a large outer tank, into which one to three smaller ones can be inserted. These are the actual developing units. All construction material is stainless steel except the neoprene gaskets. The outer tank
Figure 1.
Stainless steel chromatographic tank
284
ANALYTICAL CHEMISTRY
serves as an air or water bath to maintain constant temperature, which has been shown necessary by Rockland ( 4 ) for maximum reproducibility of Rr values. If the assembly is located in a constant-temperature room, three developing tanks can be inserted into the outer jacket. If a constant-temperature room is not available, the outer jacket can be used as a water bath. The center tank can be replaced by a thermostatically controlled heating and stirring unit. The developing tanks are held in the outer jacket by stainless steel clips ( A , Figure 1). These are shaped to engage the flange of the inner tank as shown, and are then screwed to the outer tank. Two clips are used, one on each end of the developing tanh. When the outer jacket is filled with water, the buoyant effect can be utilized to level the inner tanks. Their height and position above the outer jacket are regulated simply by adjusting the screws holding the clips to the outer container. Where an air bath is used, the leveling adjustment can be made by inserting shims between the clip and the outer jacket. The covers of the inner tanks are secured tightly by stainless steel bolts, washers, and wing nuts. The bolts are welded to the flange of the inner tank and are not removed when the cover is taken off. When frequent inspection of the papers is desired, a plate-glass cover can be used instead of a metal cover. Here a modified outer jacket is employed, whereby the glass cover is held against the gasket by clamps arranged on swivels attached to the outer tank (Figure 2).
1
Figure 3.
Stainless steel rack
T316 stainless steel; spot-welded construction
with a second solvent in the usual way. Colors of the resulting spots are subsequently developed by a suitable indicator, such as ninhydrin for amino acids or aniline-trichloroacetic acid for reducing sugars.
Ae many as 40 papers have been run simultaneously with this apparatus. This capacity is a decided advantage in quantitative paper chromatography, where several replicates of each determination are desired. Figure 2.
Clamp arrangement ACKNOWLEDGMENT
The tanks are designed to accommodate 9 x 9 inch chrornatographic papers, upon which adequate resolution of 18 amino acids or several sugars has been found practicable. Papers of this size were obtained ready cut (Schleicher and Schull) in 500-sheet packages. They are fastened to the racks shown in Figure 3 with stainless steel spring clips. Each rack will hold five papers, and four racks can be inserted in each developing chamber. Hence the total capacity of the apparatus is 40 to 60 papers. Xormally there is sufficient space in the tank so that the end papers do not come in contact with the walls of the tank. Certain solvent systems cause excessive curling and bowing of the papers. In these solvents only four papers are placed on a rack. In this way contact between the papers or with the tank walls is prevented.
The authors wish to thank Earl L. Muller and John G. Gill of the plumbing shop for their aid and advice and B. C. Lovett and 1.Floy Bracelin for preparing the illustrations. LITERATURE CITED
(1) Datta, S. P., Dent, C. E., and Harris, H., Science 112, 621 (1950). (2) Redfield, R. R., Bioehem. et Biophys. Acta 10, 344 (1953). (3) Rockland, L. B., Blatt, J. L., and Dunn, M. S., ANAL. CHEM.23, 1142 (1951). (4) Rockland, L. B., and Underwood, J. C., Ibid., 26, 1557 (1954). ~ I E X T I OofN specific products or equipment does not constitute endorsement by the Department of Agriculture over others of a similar nature not men-
tioned.
OPERATION
The mixture to be analyzed is applied from a micropipet as a spot in one corner 1 inch from each edge of the paper. The papers are then clipped to the frame with care that the spotted corners are all at the same lower corner of the frame, and that the papers are level and do not touch the sides of the frame. Wearing surgical gloves during manipulation of the papers avoids finger marks on the final chromatogram. The papers rest against the crossbars, A (Figure 3), which prevent contact between adjacent papers. This does not interfere with migration of the spots in any way. The paper-filled racks are placed in the chromatographic tank containing about 3500 ml. of the appropriate solvent and left until the solvent front has reached the top of the paper. The racks are removed and the papers allowed to air-dry. In twodimensional work, the papers are then rotated 90" and treated
Convenient Starch Electrophoresis Apparatus Kenneth Paigen', Virus laboratory, University of California, Berkeley, Calif.
'
O X E electrophoresis on a preparative scale has come into in-
creasing use, and starch, first introduced by Kunkel and Slater (b), has proved a versatile supporting medium. Electrophoresis in starch has been used in this laboratory for some time, and a simple and convenient apparatus has been developed which possesses a number of advantages, particularly in construction 1
Present address, RosweIl Park Memorial Institute, Buffalo, N. Y .
