AIDS FOR THE ANALYST Volume Collector for Chromatographic Column
(not shown in diagram) of the drop counting funnel arm support is lengthened to elevate the drop counting funnel arm. Electrical Circuit. The negative electrode is connected to ground (phototube housing). A short length of platinum wire dips into the mercury a t the top of the positive electrode and is connected by a piece of shielded wire to the cathode lug of the phototube.
B. Hamilton, Alfred I. d u Pont Institute of t h e Nernourr Foundation, Wilrnington 99, D e l . Paul
devices for the direct measurement of fraction volVumes collected from chromatographic columns have been deARIOUS
vised in conjunction with model types of collectors (1-4, 6). Direct measurement appears preferable because volumes collected by time flow will increase appreciably as resistance to f l o ~ increases through gradual packing of the column with use.
DROP COUNTING FUNNEL
*
PHOTOTUBE
-_ Figure 1.
Siphon 4ssemblj
Volumes measured hy drop counting n ill alter 11hen the concentration of developing buffer solution is changed-e.g., changing from 0 . l M citrate buffer a t p H 5, to 0.5X citrate buffer a t p H 5 results in a 25 to 305 reduction in volume because of the smaller drops of the more dense solution. The volume of fractions collected by neighing will also vary inversely as the density of developing solution: there are also the added labor and difficulty of selecting a large number of tubes of uniform weight and absorbance. To obviate these difficulties the assembly described below was designed for use with the Technicon drop counting fraction collector. It can be easily installed with essentially no change in the apparatus or its electrical circuits,
OPERATIOK
The light intensity knob on the drop counting box of the Technicon fraction collector is turned to 0, the sensitivity knob set a t 50, and t,he niicrofles counter set a t 1. The elect,rical control circuit follons a path from the positivr dipping electrode t’hrough the fluid in the siphon to the sealed-in negative electrode to ground. When the siphon empties, current’ is interrupt’ed inti1 sufficient fluid drainage from the wall plus effluent collects n the bottom of the siphon and re-establishes fluid contact bexeen the electrodes. Since t,he photocell circuit in this apparatus is reversed, “break” followed by “make” gives the poeitive impulse necessary t o energize the mechankm bringing the next tube under the discharge funnel. The height of the positive electrode is adjusted t o give a t,ime interval betrveen break and make of about 45 spconds, ample for complete drainage of the siphon. This control circuit carries i pa. and 35 volts direct current. Chromatograms obtained with an ultrasensitive relay (0.001 pa.) interposed betiveen the siphon electrodes and the phototube circuit were similar in all respects. I t was therefore concluded that electrolytic effects such as the conversion of arginine to ornithine as noted by Stein and Moore ( 7 ) for larger current density (0.2 to 1 ampere) ’vere probably absent with the i-pa. current. This method of collection has proved extremely reliable during months of continuous (24-hour) use and the volume delivered remains constant, regardless of changes of temperature, or of buffer or ion concentration in the effluent. However, for 1-ml. siphons operation is most sat’isfactory when the developing solutions contain a detergent as described by Moore and Stein ( 5 ) . Alcohols and acetone have sufficient conductivity to be used as controlling fluid, but chloroform, petroleum ether, ligroine, carbon disulfide, benzene, or toluene will not carry enough current to activate eit’her the 7-pa. or the 0.001-pa. circuits. If necessary or desirable, evaporation error from the siphon presumably could be minimized as descrihed by &der and Mader
(4). LITERATURE CITED
APPARkTUS
Siphon. A siphon of borosilicate glass (6) of the dimensions indicated in Figure 1 delivers approximately 1 ml. Melting the glass near the bottom of the siphon and gently blowing out or sucking in enable it to be adjusted exactly to 1 ml. Siphons fashioned in this laboratory delivered I ml. i lY0; the calibration was the same when rechecked 4 months later. A 2-cm. length of No. 24 platinum wire is sealed in the bottom of the siphon and is the negative electrode. The positive electrode is a 6-cm. length of No. 22 platinum wire, sealed through the lower end of a 15-cm. length of borosilicate glass tubing ( 5 mm. in outside diameter) filled with clean mercury. The electrode is adjusted and locked securely in the correct position by tightening the setscrew in the half-split collar of the Lucite holder. The holder is screwed to a split ring of Lucite mounted around the top of the siphon. The assembly is mounted securely in any convenient manner to the phototube housing of the fraction colIector. Discharge of the siphon takes place into a funnel in the position normalIy occupied by the dropping cup; the lower end of the funnel is beveled, fire polished, and provided with a solid 1-cm. drainage tip similar t o that shown on the siphon. The tip of the funnel just clears the apparatus frame when the phototube housing is moved to the off position. The adjustable post
(1) Dutton, H. J., and Castle, F. J., ANAL.CHEM.,25, 1427 (1953). (2) Hickson, J. L., and Whistler, R. L., Ibid., 25, 1425 (1953). (3) Mader, C., and Mader, G., Ibid., 25, 1423 (1953). (4) Ibid., p. 1556. (5) AMoore, S., and Stein, W. H., J. Bid. Chem., 192, 663 (1951). (6) Schram, E.,and Bigwood, E. J., ANAL.CHEM.,25, 1424 (1953). (7) Stein, W-.H., and Moore, S., J . Bid. Chem., 190, 103 (1951).
Fraction Collector for Chromatography-Addendum On present models of the recently described apparatus [Schram,
E., and Bigaood, E. J., ANAL.CHEM.,25, 1424 (1983)], the grid of the triode is now connected through a 1-megohm resistance in order to prevent the electrodes from becoming passive, as has been observed with high conductivity eluents after long periods (6 months) of continuous operation. Simultaneously the battery voltage has been lowered to 4.5 volts and the 20-megohm resistance increased to 60 megohms. Current between the electrodes is then in the IO-’ A. range.
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