( 7 ) Iya, V. K., J . Rech. Centre .\-atl. Rech. Sci. Lab. Bellevue (Paris) 35, 91
LITERATURE CITED
(1) Faris, J . P., ANAL. CHEM.32, 520 (1960). ( 2 ) Faris, J. P., Warton, J. W.,Ibid., 34, 1077 (1962). (3) Fritz, J . S., Umbreit, G . R., Anal. Chim.Acta 19, 509 (1958). (4) Hamagushi, H., Kuroda, R., Aoki, K., Sueishita. R.. Onuma. X.. Talanta 10. 15s (1963). ’ ( 5 ) Hamagushi. H., Kuroda. R.. Onuma. N., Ibid, p. 120. ’ (6) Hamagushi, H., Kuroda, R., Shimizu, T., Anal. Chim.Acta 28, 61 (1963). I
,
I
,
’
(1956). (8) Iya, V . K., Loriers, J., Compt. Rend. 237, 1413 (1953). (9) James, D. B., Powell, J. E., Spedding, F. H., J . Inorg. Sucl. Chem. 19, 133 (I961 \ \ - - - - I
(10) Minczewski, J., Dybczynski, R., J . Chromatog. 7,568 (1961). (11) Radhakrishna, P., Anal. Chzm. Acta 8, 140 (1953). (12) Rezanka, I., Frana, J., Vobecky, M., Mastalka, A,, J . Znorg. .Vucl. Chem. 18,13 (1961). (13) Spedding, G. H., Powell, J. E.,
Daane, A . H., Hiller, M. A,, ildams, W . H., J . Electrochem. SOC. 105, 683 (1958) \..__,
(14) Strelow, F. W. E., AKAL. CHEM.32, 1185 (1960). (15) Strelow, F. W. E., unpublished data, Sational Chemical Research Laboratory, 1961-63. (16) x’ickery, R. C., J . Chem. Soc. 1955, p. 245. (17) Yoshimura, J., Takashima, Y., Waki, H., Xippon Kagaku Zawhi 79, 1169 (1958). RECEIVED for review Xovember 29, 1963. Accepted February 4, 1964.
Some Factors Affecting Mobility Measurements in Paper Electrophoresis R. H. HACKMAN and MARY GOLDBERG Division of Entomology, Commonwealth Scientific and Industrial Research Organization, Canberra, A.C.T., Australia
F The mobilities of serum albumin, glucose, and maltose, but not of glutamic acid and lysine, increased with the wetness of the paper. Both the applied potential gradient and the wetness of the paper influence the resolution of serum proteins in barbital buffer. In carefully controlled experiments it is possible to achieve a high degree of reproducibility as well as uniformity of conditions throughout a paper strip.
I
N PAPER ELECTROPHORESIS the dis-
tance travelled by a charged molecule depends on a number of factors and these have been discussed by McDonald (5) and Ribeiro, Mitidieri, and Affonso (7). Very little is known about the effects of variations in the ratio of water to paper-i.e., wetness-and wetness is certainly the one factor over which least control has been exercised. The apparatus described by Bailey and Hackman (1) has been modified and used to study the effects of paper wetness and, in the case of proteins, of potential gradient on mobility. The degree of wetness of the paper is controlled by the pressure applied to the paper. EXPERIMENTAL
The apparatus of Bailey and Hackman ( 1 ) was modified to give an increased flow rate of water and hence an increased rate of cooling. Half-inch inlet and outlet tubes were located on the center line of the lower surface of the bladder near each end and passed through the base of the apparatus. Experiment showed that with a flow rate of 4.5 liters per minute or less the pressure throughout the bladder was uniform. The flow rate was maintained a t 4.5 liters per minute by inserting a suitable choke in the outlet tube. 1220
ANALYTICAL CHEMISTRY
Water, thermostatically controlled a t 15’ f 0.1’ C., circulated through the overflow device and bladder. A strip of Whatman No. 3 filter paper (57 X 12.7 cm.) was wetted with buffer solution and the two ends (for a length of 7.5 cm.) pressed between filter paper. Surplus liquid was removed from the central 42 cm. by compressing the strip between two pieces of cotton cloth for 1 minute a t a pressure 4 mm. of mercury greater than that a t which the experiment was to be performed. The paper strip was removed and compounds ( 5 - p l . spots) to be investigated were placed along a line a t the center of the strip. Temperature rise in and evaporation from the exposed ends of the strip-i.e., those parts not under pressure-were prevented by the use of wicks made from Whatman No. 3 filter paper. Two pieces 14 X 12.7 cm. and two pieces 12.7 x 12.7 cm. were wetted with buffer solution and pressed between filter paper. A long and a short piece of the wick were placed above and below each end of the strip, the shorter pieces being outermost and the extreme ends of the strip and wick being in line. The wicks, including the ends of the strip, were completely enclosed in a polythene sleeve 14 cm. long. This arrangement gave a stepped effect to the wick, which, when the paper was put under pressure, minimized distortion of the bladder. Pressure was applied to the paper strip and the ends of the wicks were dipped into buffer solution in the electrode compartments. Each compartment contained 1 liter of buffer solution and the apparatus was levelled to prevent syphoning of liquid through the paper. The strip was allowed to come into equilibrium with regard to buffer content (and temperature) and experiments showed that the time required was 1.5 hours with a pressure of 24.3 mm. of mercury (0.47 p s i . ) and 1 hour at a pressure of 254.5 mm. of mercury (4.92 p.s.i.). A potential difference was applied between
the electrodes in the buffer compartments so that the potential gradient along the central 25 cm. of the strip was 20 volts per cm. (or 5 volts per cm.) as determined in separate experiments by the use of platinum electrodes placed on the paper. At the conclusion of the experiment the paper strip was removed after the wicks had been torn off and discarded. The strip was dried and the positions of the spots were determined with an appropriate reagent. This procedure gave a linear relationship between migration distance and time and between migration distance and potential gradient for all the substances under investigation. No experiment was considered satisfactory unless the potential difference and current were constant throughout the entire experiment. At a constant potential difference, constancy of current is an indication of steady conditions both of temperature and of wetness of paper. The temperature of the paper, as measured in separate experiments by very small thermocouples enclosed in very thin glass tubing placed between the paper and the glass plate, was constant during an experiment, was uniform over the area of paper being used for mobility measurements, and was reproducible. The temperature recorded on top of the paper is not necessarily that of the paper itself, which is probably cooler. The ratio of water to paper for each buffer a t each pressure was measured by conducting experiments in which that part of the paper strip between the wicks was enclosed in Mylar film (0.0005 inch). Aipreviously marked area was cut from this central part (both film and paper), wrapped in Mylar film, weighed, and dried to constant weight a t 110’ C. Experimental conditions are given in the tables. Serum proteins were also separated under a potential gradient of 5 volts per em. for 8 hours. Amino acids were detected with ninhydrin, proteins with sugars with benzidine (8), bromphenol blue (9), and dextran with
alkaline permanganate (6) (only part of strip sprayed). The paper strips were scanned photometrically and the positions of maximum color intensity were taken to be the positions to which the compounds had migrated. Each experiment was repeated five times and there were four replicates on each strip of paper. ;is a comparison, the serum proteins were separated on 'Whatman Xo. 1 filter paper in an EEL paper electrophoresis apparatus using the horizontal open strip system. A potential gradient of 5 volts per cm. was applied for 16 hours. RESULlS
The results are given in Tables I, 11, and 111. Migration distances have been corrected for electro-Dsmotic flow and the mobilities have been calculated from these figures. Mobilities have not been corrected for obstructive effects of the filter paper fibers. The variability of migration distances is given as the standard error of the mean of 20 results and also as the standard deviations both within and between strips. The best resolution of serum proteins was obtained at a potential gradient of 5 volts per em. and :t pressure of 24.3 mm. of mercury; a t 20 volts per cm. the lower pressure gave the better resolution. The resoluticn given in the horizontal open strip system at a potential gradient of f volts per cm. was no better than that given in the apparatus of Bailey anc Hackman a t the same potential gradient and pressure of 24.3 mm. of mercury. DISCUSIION
The mobilities of glucose, maltose, and serum albumin, but not those of glutamic acid and lysine, were higher on the wetter papers (significantly different a t the 1% level). With the 7-globulin fraction of human swum the distance moved was too small for such a change to be detected. The ratio of the wetness of the paper for barbital buffer a t the t n o pressures did nct agree with the ratio of the currents, indicating that interaction had taken place between the paper and the buffer solutions. Hollinger and Lansing (4) reported that, when corrected for electro-osmotic flow, the mobilities of serum components in barbital buffer were independent of the wetness of the filter paper. Their conclusion is the opposite to that drawn from the results gixren in Table 111 above. Some comments may help to explain their results. Although they estimated the amount of buffer solution on the paper strip prior to placing the strip under pressure between glass plate>, the apparatus used allowed buffer solution to move into the strip from the electrode compartments. Since all their expei*iments were con-
Table I. Effect of Wetness of Paper on Mobilities of Amino Acids Acetate buffer, pH 5" a t 17.6"C. Potential gradient, 20 volts/cm. Time of migration, 60 minutes. Alanine used to measure electro-osmotic flow. M obi1ity Distanceb migrakd, cm.
Ratio of water to paper 1.58(254.5mm. Hg, 16.5 ma.)
Mean of Standard 20 error of results mean
Amino acid Glutamic acid 4-5.53 0.072 Lysine -5.96 0.139 1.99(24.3mm. Glutamic Hg, 20.7ma.) acid i-5.69 0.072 Lysine -6.41 0.139 a Walpole (IO)half strength. * +, movement toward anode; -, movement toward
Standard deviation Within Between strip strips
em2
sec. volt -1 x 10-5
0.066 0.108
0.352 0.453
7.68 8.28
0.060
0.291 0.754
7.90 8.90
0.156
cathode.
Table II. Effect of Wetness of Paper on Mobilities of Carbohydrates Borate buffer, pH loa at 18.0' C. Potential gradient, 20 volts/cm. Time of migration, 80 minutes. 2,3,4,6-Tetramethyl-glucose used to measure electro-osmotic flow. Mobility Distanceb migrated, em. cm. Standard Standard deviation Pee. -1 Mean Ratio of water of 20 error of Within Between volt-' mean strip strips X 10-5 to paper Carbohydrate results 1.51 (254.5mm. Glucose +9.32 0.122 0.114 0.315 9.71 Hg,21.7ma.) Maltose +3.37 0.051 0.065 0.201 3..51 2.04 (24.3mm. Glucose +10.42 0.122 0,174 0.704 10.89 Hg, 30.0ma.) Maltose +3.87 0.051 0.075 0.254 4.03 Ref. (3). * movement toward anode. 5
+,
Effect of Wetness of Paper on Mobilities of Human Serum Proteins Barbital buffer, pH 8.W at 16.7"C. Potential gradient 20,volts/cm. Time of migration, 120 minutes. Dextran used to measure electro-osmotic flow. Mobility Distance* migrated in cm. cm. Mean Standard Standard deviation sec. --I volt-' Ratio of water Serum of 20 error of Within Between x 10-5 to paper fraction results mean strip strips 1.54(254.5mm. Albumin +7,l5 0.058 0.127 0.247 4 96 Hg, 10.2 ma.) ?-Globulin fl.58 0.038 0.091 0.146 1 10 5 54 1.98(24.3mm. Albumin +7.98 0.058 0.147 0.274 1.10 Hg, 15.6ma.) ?-Globulin $1.59 0.038 0.166 0.191 a Ref. ( 2 ) * movement toward anode
Table 111.
+,
ducted a t the same pressure the effect would be for all strips, irrespective of their initial wetness, to acquire eventually the same ratio of buffer solution to paper. Their observations on the displacement of dextran and their need to make adjustments to the voltage during experiments indicates that the system as a whole was not in equilibrium. They reported that pressures below 1 p.s.i. gave poorer resolution, which is in contrast to the results obtained with the apparatus of Bailey and Hackman and suggests that they may not have had a uniform pressure over the paper strip. Better resolution of the protein components of human blood serum was obtained on wetter papers. The differences were concerned with tailing of
the components rather than spreading a t right angles to the direction of travel. It is possible that adsorption of the undernatured protein on the paper may influence mobility as t,he wetness of the paper decreases. I n carefully controlled experiments, as described in this paper, t'he reproducibility of mobility measurements is very good. The low standard deviation between replicat,es run on the same strip indicates the uniformity of conditions throughout, the strip. The low standard deviation between the results obtained on different strips indicates that experimental conditions are reproducible. With compounds such as amino acids and simple sugars good separat'ions can be obtained by paper electrophoresis in this enclosed strip-type of apparatus VOL. 36, NO. 7, JUNE 1964
1221
Over a wide range of pressures. Other compounds, such as serum proteins, give better resolution at lower pressures. LITERATURE CITED
( 1 ) Bailey,
S. W.,Hackman, R. H., J .
Chromatog. 8, 52 (1962). ( 2 ) Flynn, F. V., De Mayo, P., Lancet 261, 235 (1951).
( 3 ) Foster, A. B., Newton-Hesrn, P. A,, 1956, p. 30. Stacey, ( 4 ) Hollinger, N. F., Lansing, R. K., J. Chromatog. 5 , 38 (1961). ( 5 ) McDonald, H. J., “Ionography,” Year Book Publ., Inc., Chicago, 1955. ( 6 ) Pacsu, E., Mora, T. P., Kent, P. W., Science 110, 446 (1949). ( 7 ) Ribeiro, 1,. P., Mitidieri, E., hffonso, 0.R., “Paper Electrophoresis,” Elsevier, Amsterdam, 1961. M.j
(8) Smith, I., “Chromatographic and Electrophoretic Techniques,” T’ol. 1 , 2nd ed., p. 250, William Heinemann, London, 1960. ( 9 ) Ibid., Vol. 2, p. 11, William Heinemann, London, 1960. (10) Walpole, G. S.,J . Chem. SOC. 105, 2501 (1914).
RECEIVED for review November 22, 1963. Accepted January 27, 1964.
Separation of Various Cations by Reversed-Phase Partition Chromatography Using Neutral Organophosphorus Compounds JEROME W. O’LAUGHLIN and CHARLES V. BANKS lnstitufe for Atomic Research and Department o f Chemistry, Iowa State University, Ames, Iowa
b The use of the neutral organophosphorus compoun ds tri-n-butylphosphate (TBP), tri-n-octylphosphine oxide (TOPO), and bis(di-n-hexylphosphiny1)methane (HDPM) as stationary phases in the reversed-phase partition chromatography of various metal chlorides, nitrates, and perchlorates was investigated. RI and R, values are reported as a function of the acid concentration of the mobile phase for the movement of a large number of cations on paper impregnated with TBP, TOPO, or HDPM. Elution curves are also given for several alkali metal and alkaline earth perchlorates on columns packed with Kel-F treated with these three extractants.
R
PARTITION chromatography is a very useful technique for the separation of various cations. This technique is closely related to liquid-liquid extraction except that the water-insoluble extractant is immobilized on some stationary] inert support. The reversed-phase description arises because of the convention, especially in paper chromatography, of regarding the organic phase as the mobile phase. Fidelis and Siekierski (’7) reported the separation of the lighter rare earths on columns of kieselguhr impregnated with TBP, using 15.1 to 15.8X nitric acid as the mobile phase. I n a later paper (8) these authors extended this work to the heavier rare earths which are normally more difficult to separate. They obtained fair separations using 11.5M, 12.3J1, 13.0X nitric and concentrated hydrochloric acid as the mobile phases. Gw6idi and Siekierski (11) reported the separation of various oxidation states of plutonium using this technique and Siekierski and Sochaka (27) reported the separation of calcium and scandium EVERSED-PHASE
1222
ANALYTICAL CHEMISTRY
on T B P columns using 6M hydrochloric acid as the mobile phase. Mikulski and Stroliski (60) gave a separation of zinc, manganese, and cobalt from iron on a T B P column and in another paper (21) describe the separation of tin(I1) and tin(1V) and of tin, tellurium] and antimony. Small (28) incorporated T B P into a cross-linked copolymer of styrene and divinylbenzene to give a material having useful properties as a column packing and he called this technique “gel liquid extraction” or GLX. He presented elution curves showing the separation of uranium and thorium, yttrium and thorium, and iron and yttrium nitrates, and in a later paper (69) showed the elution curves for rare earth nitrate mixtures. Pierce and Peck (24, 26) investigated the separation of the rare earths on columns packed with a polyvinyl chloride-polyvinyl acetate copolymer impregnated with di(2-ethylhexyl)orthophosphoric acid (HDEHP) using gradient elution with perchloric acid. Cerrai and others (6] 4, 6) also studied the separation of the rare earths on cellulose columns and paper treated with H D E H P using hydrochloric acid as the mobile phase. Testa (SO) reported the separation of a number of cations on paper treated with tri-n-octylamine. He reported the separation of zirconium and hafnium among other interesting separations. Cerrai and Testa also reported a number of separations using cellulose (1) and Kel-F (3) impregnated with TOPO as the column packing. Several different authors (9, 12, 25) have recently reported the use of a Kel-F column impregnated with T B P for the separation of uranium from a variety of other elements. Dietrich (6) described the use of a column packed with glass beads coated
with T O P 0 to remove uranium from urine. Winchester (52) used an alumina column impregnated with H D E H P to separate rare-earth mixtures. Fritz and Hedrick (10) described the separation of iron(II1) on columns packed with Haloport-F impregnated with 2-oc tanone. I n this report the recently synthesized extractant, bis(di-n-hexylphosphiny1)methane or H D P M (M), is compared with T B P and TOPO as the stationary phase in reversed-phase partition chromatography. Data on the movement of a number of metal chlorides, nitrates] and perchlorates as a function of the acid concentration of the mobile phase are presented using both paper and column chromatography. EXPERIMENTAL
Apparatus and Reagents. Schleicher and Schuell, KO.589 paper was used throughout this investigation. Disk chromatograms were run on Blue or Red Ribbon paper and sheet chromatograms on Blue or Orange Ribbon paper (58 X 58 cm.). The sheet chromatograms were developed in a “Chromatocab” (Research Specialties Co., Richmond] Calif.). Disk chromatograms were developed in Petri dishes. Kel-F, a fluorocarbon polymer, was obtained from the Minnesota hlining and Manufacturing Co. (hlolding Powder Grade 3010 or 300). This resin was a mixture of particle sizes and the 60-80 mesh fraction was used in this work. A typical column equipped with a floa-type conductivity cell for monitoring the effluent (when water was used as the mobile phase) is illustrated in Figure 1. The cell was used in conjunction with a Wheatstone bridge using a Leeds & Northrup KO. 1553 Ratio Box and associated equipment recommended in its bulletin DB-1199. Any unbalance signal from the bridge
