recorder, by reducing the input signal, or by reducing pen travel through use of smaller pulleys in the drive mechanism. The last of these methods was chosen to produce a 2-inch transmittance scale because the dead zone of the recorder is then proportionately reduced. I n this way reducedscale transmittance readings are obtained which differ from those of the spectrophotometer record by not over 1%. The zero and calihratiou adjustments of the auxiliary recorder are used to adjust its transmittance scale to exact agreement with that of the spectrophotometer. Where the auxiliary recorder must also he used intermittently for other purposes, reduction of the input signal may he preferred to produce an auxiliary transmittance scale shorter than 5 inches despite the relatively increased dead zone. For this purpose a voltage divider of 50,000-ohm total resistance across the input to the spectrophotometer recorder was found to he sufficiently high to eliminate any effect on the original record. The chart drive shaft of the auxiliary recorder is connected hy a flexible shaft (automobile speedometer cable) and sear train to the chart drive motor shaft of the spectrophotometer. For this purpose 'a pinion gear inserted in the motor shaft drives the flexible shaft through a spur gear. The flexible shaft is connected to the auxiliary chart drive shaft through a Brown recorder chart drive gear train and simple friction clutch which are mounted on a base plate along with the auxiliary recorder (see figure). A choice of auxiliary chart wave length scales is provided by the change gears available for the gear train. Ample flexibility for swinging out the recorder chassis of the spectrophotometer without disconnecting the flexihle shaft is nrovided bv a IOODin the cable and holes'suitabiy Ideated $or passage of the cahle to the auxiliary recorder. A wave length scale of 0.5 inch per micron aud a 2-inch transmittance scale have been adopted for the reducedscale recordings. This yields a spectrum for the 2- to 15.micron region suitable in size for recording on IBM cards. The width of the pen trace limits the wave Y
~~~~~~~
~
~~~~~
length accuracy of these records to about 0.02 micron and results in merging of peaks separated by less than 0.03 micron. The cards, containing a printed transmittance-wave length grid, are held by spring clamps against an alignment bar on a metalplate 5inches wide, one edge of which has regularly spaced slots cut to mesh with the teeth of one of the auxiliary recorder chart drive sprockets. This platen is pressed against an alignment bar fastened to the recorder by a spring-loaded idler wheel as indicated in the figure. The spring clamps simplify exact positioning of the wave length scale of the card on the platen. As the chart drive shaft of the auxiliary recorder is free to be turned forward but not backward, it is essential that the clutch he disengaged when the spectrophotometer chart drive is reversed in direction. The platen of the auxiliary recorder is easily positioned by hand, the platen being moved sidewise to disengage the edge drive slots when it is desired to move it backwards,
engage the clutch and flexihle shaft, r e move the platen and its alignment bar, and replae one of the two retaining plates which prevent the regular chart from becoming disengaged from the chart drive sprockets. Replacement of the pulleys of the pen drive mechanism may also he required if these have been changed. An anxiliary signal controlled hy coupling a potentiometer to the pen drive of the spectrophotometer recorder can be used as input to the auxiliary recorder for spectrophotometers in which an input signal suitable for direct auxiliary recording is not available ( 1 , $1.
I n addition to employing readily available components, requiring comparatively little machine work, and providing flexihility in choice of transmittance and wave length scales, this anxiliary recording system permits the auxiliary recorder to be readily removed and used for other purposes when desirable. To do this it is necessary to dis-
11) Olsen A. L., Johnson, D. J., Pierson, ,R. H.,J . Opt. SOC.Arne?. 46, 354 (1956). 1 2) Strauss, F. B., Thompson, A. E., Chemistry & Industw 1955, 1402.
ACKNOWLEDGMENl
The authors wish to acknowledge the assistance of John Correia who did the machine work involved. LITERATURE CITED
~
MENTION of manufacturers and commercia1 products does not imply they are endorsedorrecommended by theDepartment of Agriculture over others of & similar nature not mentioned.
Constant Rate Flow Device for Electrolyte Eluents in Column Chromatography Raymond K. Main, Leonard J. Cole, Leroy M. Bryant, and Stanley
K.
Morris,
Biologicol and Medical Sciences Division, United States Nova1 Radiological Defense Laboratory, Son Fronscisco 24, Calif. URING
investigations concerned with
D gradient elution column chromatography (8), a device was needed to control accuratelv the flow of eluent (an electrolyte lolution) through the column. This had to he capable of delivering a fixed volume of electrolyte (within the range of 3 t o 5 ml.) per hour, a t a constant rate, for periods of unattended operation up to 2 weeks. 1558
ANALYTICAL CHEMISTRY
The column material did not afford sufficientresistance to flow in some cases to limit the rate to that desired. Because the concentration of electrolyte was constantly changing in this instance, control devices based upon the principle of capillary throttle brought abont either by the use of a Mariotte bottle and capillary bore tubing (3, 4, 61, a partially closed glass stopcock, a Teflon
needle valve, or tubing packed with sized glass powder ( I ) , failed to control the rate of flow satisfactorily over relatively long periods of time.
The device, shown in Figure meets cons.ts the foregoing of a cylindrical, flat-hottomed glass vessel, A , provided with a small outlet connected by suitable flexible tubing to a capillary bore glass tubing gooseneck-
shaped dropper, B. A is surrounded loosely by a stationary Lucite sleeve within which it may move freely in a vertical direction. A is annealed to and is supported by a glass rod. The lower end of this glass rod passes through a guide hole in a supporting Lucite plate and rests on top of the outer bearing surface (rise) of a constant speed cam, D. The cam is mounted on :L shaft which is connected to a constant speed electric motor, C, which turns tlie shaft a t a speed of 1 revolution per hour. As a coiibequence, the rod (and A ) is raised a t a eonstant speed tliroughout the cycle of the cam. The point at which liquid emerges from B ii on an exact level with the inside bottom of A when the latter is raised to the extreme upper limit by the cam and supporting rod. Two fixed platinum wire electrodes, X, X, extend through a cap, supported by the Lucite sleeve, down into A and barely touch the inside bottom of A when it is at the upper limit of the cam cycle. Liquid is led from a reservoir through a normally closed stainless steel solenoid valve (Skinner solenoid valve KO.V5hI 8050 CT, Skinner Electric Valve Division, New Britain, Conn., or equivalent. This normally closed valve operates on 110 volts 60 cycles alternating current and draws 0.23ampere starting current.) into A , by means of a stationary capillary bore supply tube. TVhen equilibrium is attained liquid fills the flexible tubing connecting A with B. As A rises a t a constant rate, liquid will drip from B in order to maintain a constant level of liquid in A . The rate at which liquid drips from B will be constant within the limits of precision of the constant speed cam. Thus, a constant flow of liquid is
achieved throughout tlie first cycle of the cam. The two platinum electrodes provide a n electrical switch arrangement to control a sensitive electronic relay, which in turn controls the solenoid operated valve in the supply tube to A. While the electrodes are immersed in the electrically conducting liquid (salt solution), an external current f l o w from one electrode to the other and through the electronic relay, nhich shuts off the current to the solenoid valve. Wlien the cani completes its cycle, the rod is a t the extreme upper limit. A:, the rod (and A ) drops suddenly by gravity from the upper t o the lower limit of the cam, the electrical circuit is broken a t tlie electrodes. This activates the relay, which in turn provides sufficient current to open the solenoid valve. Thus, salt solution is quickly supplied to A until the solution level rises to touch the electrodes again. At the instant that this occurs, electrical contact through the solution and electrodes is made and the supply is shut off by the solenoid valve. I n this manner A is filled at the beginning of each new cycle. As finally assembled, two such cams are mounted on a single shaft driven by one constant-speed motor (Cramer timing device synchronous motor, Type 112, 1 r.p.h., 115 volts, 60 cycles, 3 watts, manufactured by the R. W. Cramer Co., Centerbrook, Conn., and obtained from the Western Electro hlechanical Co. , Inc., Oakland, Calif.). The motor and cam bearings are attached to a Lucite base through which electrical leads are brought to the motor. This base also supports a Lucite surrounding cover (shown partially cut away in Figure I), which in
turn supports hoth the Lucite slcevw and (tlirough a rubber gronimct) tlic gooseneck droppers, B. To ensure proper operation, several precautions in construction are required. Each gooseneck dropper, B , must be constructed of 0.5- to 1.0-mm. bore t,ubing to ensure capillary attraction sufficient to fill the distal end of B automatically a t the beginning of each cycle. For the samc rcason tlie distal vertical pot,tiims oi B must IN lield to o. niiiiiniuni.
SOLENOID VALVE COI L
A
i i
2021w
I Figure 2.
1
4’
I
13
Circuit for electronic relay
Secondly, the tips of the platinum electrodes must be adjusted to a level with the distal tip of B. If these tips were placed higher than this level, the solenoid valve would remain open sufficiently long to allow electrolyte to fill A to a level higher than that desired. Liquid would then siphon out of B (and thus from A ) until electric contact was broken a t the electrodes in A . Such an event would cause opening of the solenoid valve and allow entry of fluid into A sufficient to cause a closing of the circuit again across the electrodes. This would cause a n undesired intermittent siphon action iradependent of the control of the action of the cam. Thirdly, the fall surface of the cam should be undercut sufficiently to allow full instantaneous drop of the rod at the end of each cam cycle. Ideal operating conditions are easily attained by reasonable attention to the above details. &lore elegant control of adjustment of the height of B could be obtained by a suitable screw arrangement.
A circuit for the sensitive electronic relay to control the solenoid valve is shown in Figure 2 . The gas tetrode relay tube will pass 0.50 ampere and is controlled across X and X by a current of approximately 10 pa. If the relay does not operate properly as constructed, the leads to the secondary of the transformer should be reversed.
Figure 1.
Cutaway diagram of constant rate flow device For essential measurements, see text
I n the authors’ laboratory the device was constructed to deliver 4.5 ml. of electrolyte per hour. By inserting a suitable close-fitting glass sleeve around the inner walls of A , the effective volume delivered from A was reduced to 3.1 ml. per hour. As used in column chromatography, the electrolyte solution dripping from B was conducted downward through a vertical thistle tube directly to the top of the column. A small layer of liquid over the column was maintained automatically by the VOL. 29,
NO. 10, OCTOBER 1957
1559
arrangement described by Tonipkins, Khym, and Cohn ( 5 ) . During the chromatography of certain macromolecular substances, changes in resistance of the column to flow w r e observed. T o compensate for these changes, the thistle tube, connected to the top of the column through a ground-glass joint, provided a source of hydrostatic head which automatically compensated for such changes. The fill-up time of A was 15 seconds, a negligible 0.4'% of the delivery hour.
face of the cam was 2.5 cni., so that the device delivered a volume of 4.5 ml. during one cycle. To contain possible splash, 1 cm. more was allowed in the inner length of cylinder A, making thr inside length 3.5 em. Obviously, thr rate of delivery of eluent may be liniitetl to any value desired by varying one or both of these variables.
Two such double devices have given satisfactory service for 1 year. The essential dimensions of the device are those of the cylindrical, flat-bottomed glass vessel, A , and the length of the fall surface of the constant-speed cmn. I n the device as used by the authors, the inside diameter of A was 1.6 cm., and the length of the fall sur-
The authors are indebted to Walter Gora, Shops Branch, Engineering Division, USNRDL, for these constant speed cams, made from Lucite by hand, and for the machine work on this device. The circuit for the electronic relay was designed by David F. Covell, Nuclear and Physical Chemistry Branch, Chemical Technology Division.
ACKNOWLEDGMENT
LITERATURE CITED
Gevantman, L. H., XIain, R. IC., Bryant, L. €I,,;In-a~.CHEX 29, 170 (1957).
\rain, R. K., C O ~ P1,. , ,J., :Irch. Biochem.nnd Riophp.68, 18ii (1Mi). ['age, F. i r , , 77,.a7L,7. i ~ ~ l , ~ n r sOC. i n i / 49, 1033-8 (1953).
Stead, Brenda, Page, F. XI., Ilrnbigh, K. G.. D ~ S C U S S ~Fnradaii O ~ L S SOC. 1947, 90.2, 263-TO. ( 5 ) Tompkins, E. K., Khym, J. S., Cohn, IT. E., J . .lin. Cheni. SOC.69, 2769-77 (1947). ( G ) T'ogt, TTalter, Cheiii. Ing.-Yeck. 23, 580-1 (1931).
WORKsupported, in part, by funds provided by the Bureau of Medicine and Surgery, U. S. Navy Department. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official, or reflecting the views of the Department of Uefense.
Ferricyanide and a Modified Periodate Chromatographic Spray for Reducing and Nonreducing Sugars Dwight F. Mowery, Jr.,l Ripon College, Ripon, Wis. DDITION
of tert-butyl alcohol to an
14 aqueous sodium periodate solution
reduces the spreading of spots when the solution is used as a spray for detection of reducing and/or nonreducing carbohydrates on paper chromatograms. This solution may be stored in the spray bottle and replenished from a large stock bottle as required. A slightly acidic benzidine spray is used in conjunction with the periodate spray and reveals carbohydrates as white spots on a blue background. A useful additional spray is an alkaline ferricyanide qolution which, when followed by the hame benzidine spray, may be used for dctection of reducing sugars only. All threc sprays are stable for 6 months without appreciable loss of effectiveness. Recently considerable energy has heen directed toward the development of sensitive spray reagents for the detection of nonreducing carbohydrates on paper chromatograms. Two main types have emerged as being more sensitive than the others-the silver-ammonia reagent ( 1 , 3, 7 ) and the periodate reagent (1-5, 7, 8, 10, 11). The objective of the work rcported here \vas the development of a spray reagent of high sensitivity for nonreducing carbohydrates which would contain enough nonaqueous solvent to reduce spot spreading and still be stable enough for storage in a spray bottle refilled from a large supply bottle as needed. The possibility of such a spray based upon the silver1 Present address, Xew Bedford Institute of Technology, X e w Bedford, Mass.
1560
ANALYTICAL CHEMISTRY
ammonia type was ruled out a t once, owing to the danger of formation of silver fulminate upon long storage. I n modifying the aqueous sodium periodate spray only one author ( 5 ) has reported addition of an organic solvent to the periodate solution to reduce spot spreading. I n this case acetone was added, but the final solution was stable for only about 3 hours. As part of the present investigation, a large number of reagent grade solvents were tested and tert-butyl alcohol (Eastman Kodak 820) was finally found to produce a highly stable solution with sodium periodate and at the same time reduce the spreading of spots. Th? utility of this solvent depends on the great difficulty of oxidation of a tertiary alcohol and the high degree of purity available a t reasonable cost. It was found that 25% by volume was about the maximum that could be added without precipitating sodium periodate; this addition slowed the reaction rate of the periodate m-ith glycols so that the time had to be increased from about 5 to 30 minutes. The most satisfactory spray for visualizing the carbohydrate spots was a slightly acid benzidine solution, a constant acidity being produced by addition of ammonium nitrate rather than free mineral acid as in previous formulations of this reagent. Another solution for use in conjunction nith this benzidine spray, found useful in detecting reducing sugars, consists of potassium ferricyanide in alkaline aqueous tert-butyl alcohol. All three of these solutions are stable for 6 months and the spots
produced are also usually visible for several months. COMPOSITION A N D M O D E OF APPLICATION OF SPRAYS
Ferricyanide Spray for Reducing Sugars. Potassium ferricyanide (0.008 mole) and trisodium phosphate (0.002 mole) are dissolved in a mixture of 750 ml. of water and 250 ml. of tert-butyl alcohol. The solution (pH about 13.0) is sprayed lightly but evenly on both sides of the chromatogram using about 25 ml. per square foot. The sprayed chromatogram is then heated in an oven a t 80' to 90' C. for 5 minutes and is finally sprayed with the benzidine reagent to produce white reducing sugar spots on a blue background. Only slight deterioration of the ferricyanide solution was observed in 6 months and it could easily be restored by addition of a small amount of potassium ferricyanide. Periodate Spray for Reducing and Nonreducing Sugars. Sodium metaperiodate (0.03 mole) is dissolved in 750 ml. of water and 250 ml. of tertbutyl alcohol are added. The solution ( p H about 6.2) is applied in the same way as the ferricyanide solution and the chromatogram is then allowed t o dry for 30 minutes a t room temperature and finally sprayed with the benzidine reagent to produce white or yellow spots on a blue background. The spots change slowly to orange on a brown background but are still visible after several months. Benzidine Spray for Use after Each of the Preceding Sprays. Benzidine (0.03 mole of Eastman Kodak X-33) is dissolved in 500 ml. of tertbutyl alcohol and this solution is mixed with a solution of ammonium
