2020
ANALYTICAL CHEMISTRY
The operating cycle of this apparatus consists of five steps: equilibrating the two phases in tubes C, decanting upper phase into tubes B, shifting tubes C one position down the series relative to tubes B, emptying tubes B into tubes C, and returning tubes C to the original position relative to B. Equilibrating. Clamp T is loosened and tubes C are equilibrated by oscillation about axis T V . Tubes C are then held in a horizontal position until the phases separate. Decanting. With handle R in position 1, tubes B are raised to the side arms of tubes C by rotation about axis D. Minor adjustment a t axis T V permits each side arm to enter the mouth of the corresponding tube. I n this position tube C-1 is in relation with tube B-1. Both series of tubes are rotated forward together until I rests on H , bringing tubes C to the vertical position. In this operation the upper phase flows into B leaving the lox-er phase in C. Shifting. Tubes C are raised slightly so that b will clear the rims of B. Handle M is shifted to position 2. With minor adjustments a t T V , the pouring spouts of series B enter the mouths of b. In this position tube B-1 is in relation with tube C-2. Both series of tubes are rotated backward together until tubes C are horizontal. The upper phases flow back into tubes C displaced one position along the series. Returning. Tubes B are returned to the rest position. Another volume of upper phase is placed in C-1. The cycle of operation is now repeated from the equilibrating step. DISCUSSION
The above apparatus proved very satisfactory in the purification of the antifungal antibiotic, bacillomycin R ( 1 ) . Troublesome emulsions developed in some tubes but these n ere immediately centrifuged, so that there was very little delay. In order to avoid holdup due to surface tension effects, the various tubes (including centrifuge tubes) were coated on their inner surfaces with a liquid silicone lubricant. At any stage in the procedure the contents of any tube C can be removed by rotating C backa-aid past the horizontal position until the entire contents of the tube are contained in cap a. a can then be removed for centrifuging or other operation. This is particularly useful when the phases do not separate readily after shaking. The emulsion is removed and centrifuged a t 500 times gravity to cause it to separate. The separated phases can then be replaced in the apparatus. The addition of reagents to break any emulsions formed is unnecessary because contents of any tube may be centrifuged without difficulty. The choice of solvents is therefore not limited by the possibility of heavy emulsion formation. The apparatus may be used in the single withdrawal procedure by the addition of a 26th C tube to collect the upper phase from tube B-25. A motor may be attached to F to make the shaking mechanical. The conversion of the apparatus into a completely automatic one is not considered advisable because this would greatly increase the cost of the apparatus. LITERATURE CITED
(1) Badad, J., Pinsky, A., Turner-Graff, R., and Sharon, N., ,Vatwe, 170,618 (1952). (2) Craig, L. C., J.Biol. Chem., 161, 321 (1945). (3) Craig, L.c.,and Post, o., -4h-i~~. CHEM.,21, 500 (1949). (4) Raymond, S., Ibid.,21, 1292 (1949). Paper 62, Agricultural Research Station, Rehovot, Israel, 1953 Seriee.
Assignment of Wave lengths on Spectra Recorded with Beckman Model DK-1 Spectrophotometer S. Z. Lewin and R. H. Fairbanks, Department of Chemistry, N e w York University, N e w York, N. Y.
Model DK-1 recording spectrophotometer, TwhichBeckman employs the same monochromator as the Beckman HE
Model DU, has a nonlinear wave-length scale. The accurate assignment of wave lengths on spectra recorded by this instrument is therefore not a simple matter. The manufacturer supplies pressure-sensitive tapes on which are printed the wavelength scales appropriate to each of the scanning speeds of the instrument, but these tapes are intended only for the approximate location of wave lengths. They are somewhat elastic, and care should be taken when applying them to the chart paper not to stretch them to different extents in different regions. The tape provides wave-length markers only along a narrow strip of the chart, and the markers have to be extended to the absorption peaks either by eye or by the use of drafting equipment. Finally, they are a continuing source of expense, and a quantity of tapes must be kept on hand. For more accurate work, the manufacturer recommends that the wave-length scale of the monochromator be used directly. A very rapid and convenient technique, as accurate as the direct use of the monochromator scale, consists in transferring the wave-length scroll markings of the monochromator to a sheet of transparent plastic, which thereafter serves as the wavelength standard, and the wave lengths of absorption peaks are read diiectly by placing it on top of the chart to be measured. The technique used for accurately inscribing the plastic sheet is as follows.
A spectrum is run with no specimen in either beam, so that a flat 100% line is traced out. The wave-length scroll of the mono-
chromator is viewed with the aid of a magnifying glass, and as each graduation on the scroll passes underneath the hairline of the rider which sits on the scroll, the sample compartment shutter is quickly closed and opened. This places a sharp vertical line on the chart a t a position corresponding to the wave length involved. (The term “horizontal” refers to the direction of feed of the strip chart, “vertical,” to the direction of travel of the recorder pen.) With care, a t scanning speeds of 10 or slower and 0.1-second time constant, these vertical lines can be placed on the chart paper in exact correspondence to every single marking on the monochromator wave-length scroll. This procedure should be repeated on several separate charts to establish the reproducibility of the marking technique. The correct markers are then transferred to a 1-inch strip of paper which is pasted along the center, parallel to the long axis, of a 12 X 28 inch right rectangle of 0.25-inch Plexiglas (these dimensions are appropriate for the region 800 to 2850 mfi a t speed 10). By means of a T-square and steel scriber, lines are scratched on the Plexiglas from top to bottom along the 12-inch dimension, leaving a gap corresponding to the width of the paper strip for the tens, and no gap for the hundreds of units. Wavelength designations are inscribed on the reverse side of the plastic sheet. In order to ensure that the plastic sheet is placed on the chart with its ruled lines parallel to the axis along which the recorder pen travels, it is convenient to inscribe a line perpendicular to the wave-length markers in the center of the plastic sheet. In using the sheet, this line is lined up with a grid line of the chart paper. When running a spectrum, several wave-length markers are placed on the record by manipulating the sample compartment shutter as described above. The plastic sheet is then placed on the chart with the ruled side facing the paper, and is carefully lined up with the vertical wave-length markers and with the appropriate horizontal chart grid line. Absorption peak wave lengths are then read directly off the plastic scale, with visual interpolation between scale markings. If the plastic sheet is illuminated from one edge, the light scattered into the ruled lines makes these lines stand out sharply. It has been suggested (by Lee Cahn, Beckman Instrument Corp., Pasadena, Calif., private communication) that a convenient procedure for comparisons of different spectral charts would be to inscribe the wave-length grid on a transparent sheet that is illuminated from below by means of fluorescent lamps, with the charts placed on top of this viewing fixture. The use of the plastic wave-length scale described makes possible the greatest accuracy in comparisons of wave lengths of the absorption peaks of different specimens run on the same instrument. It is recommended that the markings on the plastic chart be calibrated by comparison with standard spectra, such as the emission of mercury, or absorption of water or chloroform.
