A liquid-liquid Microextractor for Solvents lighter than Water Use i n Phosphate Analysis PAUL L. KIRK AND MARYANN DANIELSON, U n k e r s i t y of California, Berkeley, Calif. LTHOUGH numerous liquid-liquid extractors for use with
A solvents lighter than water (1, S, 4, 5 ) have been described,
the use of all common designs of such special extractors with small volumes of liquid is difficult or impractical. The ordinary separatory funnel is most inconvenient when successive extractions must be made, because the aqueous phase must be withdrawn each time in order to remove the solvent phase. With small volumes, admixture of the two phases also occurs to some extent due to wetting of the stopcock and delivery tube with the first phase withdrawn, which then is collected in withdrawing the second phase. In making large numbers of multiple extractions with butyl alcohol in connection with phosphate analysis of tissue cultures and other biologi(sal materials by the Berenhlum and Chain (2) procedure, it was necessary to devise an apparatus capable of application to small volumes (down to 0.5 ml. of either phase), rapid in action, and giving reasonably rlean separation of the phases. The extractor described meets these requirements and is appliFigure 1. Liquid-Liquid cable t o most types of Extractor lighter than water solvents used to extract aqueous solutions. Presumably it ma\ b? used with any two immiscible solvents in which the lighter phase is thta ratracting solvent
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DESCRIPTION
The extractor (constructed by the Microchemical Specialties Company, Berkeley, Calif.) shown in Figure 1 consists of a conical chamber with a total volume of about 10 ml., attached as shown to a lower exit tube connected through a capillary stopcock to a side chamber with a capacity of about 5 ml. This is used for filling and admitting air t o the conical chamber. The top of the extraction chamber is attached to a three-way stopcock, one arm of which connects with a vacuum source, and the other is terminated with a simple rubber bulb. The top of the extraction chamber also carries a ground joint through which passes the stem of a small separatory funnel with a capacity of 2 to 3 ml. and a long capillary stem reaching to the bottom of the extraction chamber. The construction of the seal at the bottom and its relation to the tip of the small separatory funnel are important. The seal t o the capillary tubing should be made with a rather long taper, giving a conical bore into which the capillary tip is inserted so as nearly to fill it. OPERATIOIV
The liquid sample to be extracted is placed in the side chamber of the extractor with the stopcock open. The solvent is added to the same chamber, and the liquids are drawn by vacuum into the extraction chamber. If the vacuum is adjusted properly, a stream of air is drawn through the chamber a t a rate that gives good mixing of the phases without undue violence which may cause loss. After a sufficient mixing time, the three-way stopcock
is reversed; this stops the suction and allows the phases to separate. The chamber is then connected to the rubber bulb by further rotation of the stopcock. With the bulb the liquid is forced into the side chamber until the interface between the liquids is immediately below the orifice of the internal capillary stem, and the side stopcock is closed. The upper stopcock to the small separatory funnel is opened. If any of the aqueous phase has run into the stem, it is withdrawn by a gentle suction, after which the upper solvent phase is forced upward into the separatory funnel by pressure from the rubber bulb. The stopcock of the latter is closed to retain the solvent, after which another batch of solvent can be added t o the side chamber and the entire operation repeated for a second extraction. At the end of the final extraction, all of the solvent phase is confined in the upper separatory funnel which is lifted out of the apparatus and the solvent delivered into whatever container is desired. I n the case of phosphate analysis by the Berenblum and Chain method, this material is butyl or isobutyl alcohol extract of phosphomolybdic acid. I n this particular use, it is convenient to use the extraction chamber of the extractor for the reduction step of the analysis, which is carried out as follows: Instead of removing the separatory funnel from the extraction chamber after the final extraction, a tube connected t o a n evacuated waste bottle is inserted into the side chamber. The aqueous phase is blown with the rubber bulb into this chamber, where it is sucked into the waste bottle by the vacuum applied to the bottle. A water or acid rinse may be used to clean the extraction chamber, and is removed in the same manner. The extract is then allowed to flow down into the extraction chamber, where it is mixed with stannous chloride solution introduced through the upper chamber of the separatory funnel. After the reduction to molybdenum blue is complete, the phases are separated as described before; the alcohol solution of molybdenum blue is isolated in the separatory funnel, which is then removed and its contents are diluted t o volume and measured spectrophotometrically or otherwise. By this means the entire operation of extraction, washing (if desired), and reduction is performed in the same vessel; this prevents loss and conserves the time and trouble necessary for transfer to other containers. DISCUSSION
No extensive studies of the use of this extractor for general purposes have been made. That it may be used in a variety of ways is readily apparent. It is possible that air stirring would not be desirable with very volatile solvents such a$ ether. Unless the time of extraction was prolonged, however, it does not seem probable that serious loss of ether would occur. Immersion of the lower portion of the apparatus in ice water should be sufficient t o eliminate serious loss of the more volatile solvents. One analyst can readily operate trvo such extractors simultaneously. Because the time of manipulation is appreciably shorter 131th each one than the use of separatory funnels, the effective number of extractions in a given time is considerably more than twice that of a n operator using separatory funnels, and the results are more clean cut. The Berenblum and Chain method for determination of phosphate has been shown to be less subject to interference and to technical variations than other colorimetric phosphate methods. The development of molybdenum blue in the butyl alcohol phase serves to standardize the conditions of color formation and thus give exceptionally reproducible results, free of most of the shortcomings of phosphate analyses in which the color is developed in the aqueous phase with its variations in salt content, pH, reagents previously added, and other factors. The only significant limitation to its general use is the added inconvenience 1122
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V O L U M E 20, NO. 1 1 , N O V E M B E R 1 9 4 8 of the extraction step. The use of the extractor described effectively minimizes this difficulty and allows the full realization of all the advantages otherwise inherent in the Berenblum and Chain nncthod. LITER4TtiRE CITED
ANAL.ED.,13,
(3) Matchett, J. R., and Levine, J., ISD. ENG.CHEM., 264 (1941).
(4) Waprnan, M., and Wright, G. F., Ibid., 17,55 (1945). (5) Wollner, H. J., and Matchett, J. R., I b i d . , 10, 31 (1938).
RECEIVEDDecember 16, 1047. .\i3ed by grant3 f r o m t h e Research Board of t h e University of California, a n d t h e Committee on Growth, a c t i n s f o r t h e American Cancer Society.
(I’ Bairenscheen, H. K., .Wikr.ochim. Acta, 1, 319 (1937). ( 2 ) Berenblum, I., and Chain, E., Biochem. J . , 32, 281, 295 (1938).
Multiple Dropping Mercury Electrodes CLARK E. BKICKER AYD N. H. FURMAN Frick Chemical Laboratory, t’rinceton University, Princeton, .V. J .
HE iicc of several dropping mercury electrodes connected iu Tparalld was apparently first reported in the literature by Rlc(;llvery, Hawkings, and Thode (21. A further article on this subject by De Vries and Barnhart (1) causes the authors to submit this note on unfavorable experience with the use of similar multiple electrodes in 1942. The authors’ electrode was analogous to that described by De Vries and Barnhart, but had a fifth capillary dropping from the middle of the bulb as well as four capillaries connected at the sides of the bulb. This apparatus wa= constructed primarily for making electrolyses in order to recover small but identifiable amounts of reduction products E APPLIED that mere formed upon e l e c t r o l y s i s in t h e Figure 1. Use of Slultiple Electrodes neighborhood of the halfwave potential. These
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& volt
experiments will be described in a subsequent paper on the polarography of certain organic compounds. The authors also made experiments t,o determine whether the sensitivity could be increased or any other advantage could be derived from the use of multiple electrodes in polarographic determinations. The drop times of the various capillaries differed and irregular fluctuations in current were found a t the top of the wave, as indicated in Figure 1.
All curves of Figure 1 were taken with approximately molar cadmium chloride solution in 0.1 N potassium chloride. Curve 1 recorded with a single dropping electrode a t a sensitivity of 0.13 microampere per mm. Curve 2 recorded with the multipl? device with five capillaries dropping simultaneously, a t a sensitivity of 0.26 microampere per mm. Curve 3 recorded with a single dropping electrode at a sensitivity of 0.035 microampere per nini. The arrows point to 0.60 volt applied. Further irregular curves that were obtained are shown in Figure 2. The solution used for curves I and I1 of Figure 2 contained, per 100 ml., 2 ml. of 0.05 molar cadmium chloride and 2 1111. of 0.05 molar zinc chloride. The solution was 0.1 N in potassium chloride. Curve I is taken a t a sensitivity of 0.13 microampere per nim with a single dropping electrode and curve I1 a t a sensitivity of 0.38 microampere per nim. with the multiple electrode containing five capillaries. The solution for curves I11 and I V contained 0.002 mg. of cadmium per ml. and was 0.1 LVin potassium chloride. Curve 111 was taken a t a sensitivity of 0.013 microampere per mm. and curve IV with the multiple electrode was taken at a setting of 0.026 microampere per mm. The high sensitivity and the irregularities of the multiple electrode are such as to obscure almost entirely the position of the cadmium wave. At the high sensitivity of the galvanometer used in recording the polarogram from a solution containing only 2 micrograms of cadmium per ml. (curve IV, Figure 2), the actual cadmium wave was practically masked by the irrrgular fluctuations of the current. In view of these unfavorable findings, s o r k on the use of multiple electrode systems to increase sensitivity was discontinued. The authors have concluded that multiple dropping mercury electrodes may cause irregular fluctuations in current at the tops of polarographic waves, so that it is difficult or impossible to estimate the nave height accuratclv. LITER4TURE CITED
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(1) De Vries, T., and Barnhart, IT. S., Ar.41.. CHEM., 19, 934 (1947). ( 2 ) McGilvery, J., Hawkings, 1%. C., and Thode, H. G., Can. J . Research, 25B, 132 (1947).
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APPLIED
Figure 2. Irregular Curves
RECEIVED .January 29, 1948.
