Centrifugal Force in Paper Chroma tog ra p hy and Electrophoresis HUGH J. McDONALD, LUlZ
P. RIBEIRO,'
and LEONARD J. BANASZAK
Graduate School and Sfritch School o f Medicine, loyola University, Chicago 7 2, 111.
b A simple apparatus and techniques have been developed for centrifugally accelerated chromatographic separations, one- and two-dimensional, in circular, rotating paper sheets. The developed chromatograms resemble the classical radial patterns but development time is markedly reduced. For 16 amino acids, I?,values, using two developing solvents, were obtained b y the technique described and the conventional descending technique. Utilizing centrifugal force in combination with a high voltage direct current electrical field has made possible electrochromatographic separations, in horizontal rectangular paper sheets. Electrophoresis may b e discontinuous or continuous flow. Centrifugal and electrical forces may b e applied independently, in sequence, or simultaneously.
T
application of centrifugal force to paper chromatography and ionography provides a new dimension in these widely used techniques. A simple and effective apparatus is described for carrying out centrifugally accelerated separations in paper disks rotating in a horizontal plane. Use of centrifugal force in combination with a high voltage direct current electrical field has made possible relatively rapid electrochromatographic separations or planar electrophoresis, somewhat analogous to the type formerly achieved by the timeconsuming technique of hanging curtain electrophoresis. HE
CENTRIFUGALLY ACCELERATED PAPER CHROMATOGRAPHY
Some details of the technique, and earlier modifications of the apparatus, for carrying out centrifugally accelerated chromatographic separations in rotating circular paper sheets, have been described (2, 3, 6). A technique for conducting two-dimensional separations on a rotating paper disk has been published ( 5 ) . The developed chromatograms resemble those resulting from the classical stationary circular paper method, but 1 Permanent address, Biochemical Laboratory, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil.
the development time for an acceptable pattern 35 cm. in diameter is reduced from several hours to 5 to 20 minutes. The apparatus consists essentially of a carefully balanced motor-driven diskshaped head, rotating in a horizontal plane, and completely enclosing a circular sheet of filter paper held in position a t the center. While rotating, the paper sheet assumes a taut horizontal position. The solution to be fractionated isapplied, by micropipet, to the paper surface in the form of dots, or whole or partial rings. While the paper disk, 18 inches in diameter, is rotated a t from 300 to 1000 r.p.m., the developing solution is added a t a point away from its center as a fine, continuous stream. A schematic diagram of the current modification of the apparatus is shown in Figure 1. A constant-speed, 1/4-hp. motor is coupled with a variable-speed drive, completely mechanical in operation. This arrangement was designed to permit reproducible, constant speeds of the rotor from 250 to 1000 r.p.m. The unit is equipped with a brake to stop the rotor rapidly a t the end of a run, a desirable feature if diffusion and consequent enlargement of the colored zones occupied by individual components of a mixture are to be avoided. A tachometer and automatic timer are also provided. The rotor, made of Monel alloy, has an outside diameter of 50 cm., with a spacing of 2.5 em. between the lid and base. The device for feeding the developing liquid to the paper during a run consists of a screw-top glass pressure vessel containing the solution, which is fed through the capillary tip under 8 to 11 pounds nitrogen pressure. Rapid interchange of the capillary tips is provided for through use of a ball and socket joint between the tip and the main glass tubing. For proper development of the chromatogram, the liquid must be added as a fine continuous stream, a t about 0.8 to 1.5 ml. per minute. calibrated as water at 25' C., depending on the particular paper, rotor speed, etc. The rate of flow of liquid through the capillary tip is usually calibrated with water at 25' C. This makes i t unnecessary to restandardize the capillary for each solvent used. To initiate a sufficiently fine stream, rather than a series of drops, is sometimes difficult if a drop once forms on the tip of the capillary. This problem can be solved by
Figure 1 . Cross section of main f e a tures of apparatus for centrifugally accelerated chromatographic separations Lid of rotor Circular sheet of paper, 18 inches in diameter, on which chromatographic separations toke place-e.g., Whotman 3MM: it rests lightly on numerous support points c. liner, of heavy-stock paper-e.g., EatonDikeman No. 320, in close contact with bottom of rotor, d, so support points protrude through e. Removable glass capillary delivery tube f. Stopcock to regulate liquid flow to rotating paper disk g. Spring-loaded closure device, for opening in lid of rotor, equipped with Teflon bearing ring j. Drive shaft of rotor
a.
b.
touching the drop on the tip of the capillary nith a clean dry paper tissue, while the liquid in the capillary is under gas pressure. The drop will be absorbed in the paper and a continuous stream of liquid 1%-illbegin to flow. It was found advisable to filter all developing solvents through Khatman N o . 1 paper to prevent clogging of the capillary tips during a run.
To have the rotating paper sheet completely enclosed and yet a t the same time add liquid from the capillary a t a point away from the center, a specially designed closure device is provided for the opening, 8 em. in diameter, in the center of the rotor lid. The closure device, shown in Figure 1, is essentially a spring-loaded flat cap, g, equipped with a thin circular Teflon bearing ring, 9.5 cm. in diameter, partially embedded in its surface, and held in position by snapping it just over center into a prepared groove. IT'hile the rotor is spinning, the cap remains stationary, the Teflon ring bearing down gently on the lid of the rotor to seal it completely. Although the Teflon ring may be used without any lubricant, a thin film of Dow-Corning stopcock grease, a silicone lubricant, decreases wear and improves the seal. The cap has a small VOL. 31, N O . 5, MAY 1959
825
hole through which the capillary feed tip, e, can be inserted during a run. I n the early phases of the development of the apparatus and technique, dye mixtures were often used because they yielded colored chromatographic patterns without further development, and required simple water solutions for their development, thus avoiding the problems involved in differential evaporation rates of individual solvents from multisolvent solutions. For the separation of such mixtures as bromophenol blue, methyl orange, and methyl red, Rhatman KO. 1 filter paper was used with an aqueous Verona1 buffer solution of ionic strength 0.05 and a p H of 8.7. For this system the R/ values, 0.86, 0.22, and 0.64, respectively, of the dyes were essentially unaffected by changes in rotational speed of the rotor from 300 to 925 r.p.m. (6). The flow of solvent !vas important in developing satisfactory chromatograms; 1.2 ml. per minute was satisfactory a t all rotational speeds above 250 r.p.m. The shape of the developed chromatogram was elliptical rather than circular in all types of paper utilized. For WhatmanNo. I and3MM, the major axis of the ellipse was parallel to the machine direction of the paper. The R f values for the dye components were essentially the same for Whatman KO.1, Eaton-Dikeman KO.613, and Cremer-Tiselius Munktells ( 6 ) ,and within the limits of experimental error, were the same whether the separation was carried out in a direction parallel to the long axis of the ellipse or at right angles to it. Amino acid mixtures were fractionated using Vhatman 3MkI paper and a solvent system of butanol-glacial acetic acid-water, in the volume ratio of 4 : 1: 1, respectively, or methyl ethyl ketone-propionic acid-water, in the
Table 1.
Figure 2. Relative positions and migration distances of 16 amino acids, and degree of separation of three mixtures of amino acids Elliptical broken line indicates perimeter of developing solvent at end of run. Arrow indicates long direction of Whatman 3MM sheets, 1 8 l / r X 2Z1/2 inches, from which circular sheet was cut. Dots on circular line, 7 cm. from center of disk, represent points of application of amino acids
volume ratio of 15: 5 :6, respectively (1). Solutions containing 10 mmoles per liter of each amino acid were prepared, using as solvent a n aqueous solution containing 10% isopropyl alcohol. I n the case of tyrosine and cystine, and in the mistures containing these amino acids, 6.V hydrochloric acid was added,
Rf Values of Amino Acids
(Whatman 3MM paper, 25’ C.)
Amino Acid Cystine Lysine Histidine .4rginine -4spartic
Serine Glycine Threonine Alanine Proline Tyrosine
Methionine Valine Phenylalanine Isoleucine Leucine Development time 826
Butanol-Glacial Acetic AcidH.O. - , Vol. Ratio. 4:1: 1 Conventional, kentrifugally accelerated descending
Methyl Ethyl KetonePropionic Acid-H20, Vol. Ratio. 15:5:6 Conventional, ’ Centrifugally accelerated descending
0.06 0.12 0.14 0.16 0.18 0.19 0.20 0.24 0.31 0.34 0.39 0.48 0.53 0.55 0.62 0.65
0.08 0.17 0.18 0.22 0.23 0.24 0.26 0.31 0.38 0.41 0.45 0.55 0.59 0.63 0.68 0.70
0.04 0.11 0.13 0.16 0.13 0.14 0.16 0.18 0.23 0.27 0.41 0.43 0 41 0.54 0.52 0.55
0.15 0.32 0.35 0.40 0.32 0.33 0.35 0.40 0.44 0.48 0.57 0.57 0.56 0.63 0.63 0.65
7 hr.
15 min.
4 hr.
17 min
ANALYTICAL CHEMISTRY
drop by drop, with stirring, until solution was complete. I n almost all experiments, 2 4 aliquots of the amino acid solutions were spotted on the paper sheet 7 cm. from the center. When the solution was applied to the paper in the form of a n arc, 10- to 20pl. samples were used. After development, a11 chromatograms were dried in a current of warm air, sprayed xith a solution containing 0.2% ninhydrin in 95% butanol-5% acetic acid (2.49. Color development of the chromatograms was carried out by placing the paper sheets in a current of warm air. Table I shows R , values for 16 individual amino acids, as obtained by centrifugally accelerated paper chromatography and by the conventional descending technique, under similar conditions (paper, migrationdistance, dereloping solvent, temperature, etc.). The R f values represent the average of a t least three separate determinations. I n the centrifugally accelerated esperiments, the average calculated standard deviation for the butanol-acetic acid-n ater solvent was 0.011, and for the methyl ethyl ketone-propionic acid-water solr e n t was 0.021. I n the esperiments using the descending technique, the average calculated standard deviation for both developing solvents was 0.006. The R , ralues of the amino acids
C -c
1 Figure 3. Apparatus electrochrornatography
for
centrifugally
A.
accelerated
Top view of rotor 6 . Underneath side of rotor C. Side view of apparatus, with drive mechanism, brake, tachometer, automatic timer, deleted d. O v a l feeder cup e. Collector troughs, swing out when heat rotates f. Platinum-plated electrodes g. Brass collector rings h. Spring-loaded graphite brushes Capillary feed for developing liquid j. k. Capillary feed for solution to be fractionated m. Drive shaft of rotor n. Silicone gasket, above and below paper sheet p. Spring-loaded closure device, for opening in top section, r, of rotor
studied, obtained on the rotating paper disk, were in all cases slightly higher than those obtained by the conventional descending technique.
A liner of Eaton-Dikeman, KO.320, a heavy stock paper approximately 2.5 mm. thick and 18 inches in diameter, was laid down in close contact with the bottom of the lower section of the rotor. Holes were cut in the paper sheet, so that the stainless steel support points protruded through. One hundred milliliters of developing solvent mas spread over its surface, from a pipet, and a second sheet of Whatman 3MM paper was mounted above it, resting lightly over the stainless steel support points. The lid was placed on the rotor, the Teflon-ringed closer device lowered over the opening in the center of the lid, and the rotor run a t 900 r.p.m. for 5 minutes. I n this manner, the atmosphere within the rotor was equilibrated with respect to solvent vapor. After 5 minutes, the flow of developing solvent was started. I n the case of the butanolacetic acid-water solvent, the true flow rate of solvent was 1.0 ml. per minute, and it was continued for 15 minutes. For the methyl ethyl ketonepropionic acid-water solvent, the true flow rate was 1.1 ml. per minute, and it mas run for 17 minutes. Whatman 3MM paper has also been used as the liner in the rotor but does not appear
C
Figure 4. Elliptical feeder cup attached to drive shaft of the rotor by circular hub a and two spokes, b Inner circular broken line represents path traced on paper sheet by stream of liquid from a glass capillary tip, represented b y arrow e, used to develop the electrochromatogram. Outer circular broken line indicates path of stream of liquid containing solution to be fractionated as it is fed into the feeder cup, from a second capillary tip, d. for continuous operation of unit. The two stainless steel capillaries, c, one a t each end of bottom of oval cup, allow solution to be fractionated to leak into paper sheet a t two points symmetrically positioned a w a y from center of sheet, and along medion line parallel to its longer sides
to be as satisfactory as the much heavier stock paper, Eaton-Dikeman No. 320. With a liner of Whatman 3MM, 25 ml. of solvent was used to wet it initially, and before each subsequent run. The former paper stays flat in the bottom of the rotor and has less tendency to lose the solvent from its perimeter when rotated a t high speed. The same liner may be utilized for several runs. Each time a new sheet of Whatman 3MM paper is placed in the rotor 25 ml. of developing solvent is added to the Eaton-Dikeman No. 320 liner. The RI values appeared, within the limits of experimental error, to be the same regardless of the angle of their path of migration to the major axis of the ellipse representing the perimeter of the solvent front. A chromatogram, developed with methyl ethyl ketone-propionic acidwater, showing the relative positions reached by 16 individual amino acids and the degree of separation of three mixtures of amino acids, is shown schematically in Figure 2. The arrow was drawn parallel to the long direction of the Whatman 3MM sheets, 181/a x 22’/* inches, from which the circular sheet was cut. The arrow is parallel to the major axis of the ellipse marking
the perimeter of the solvent front. The experimental conditions were as described above for this solvent. CENTRIFUGALLY ACCELERATED ELECTROCHROMATOGRAPHY
In the usual curtain electrophoresis, the solution to be fractionated is applied to the top of a vertical sheet of paper, the edges of which are fitted with electrodes. I n the n e w r planar electrophoresis described here, the paper is horizontal a i d is rotated so that the material undergoing fractionation moves across the paper under the stimulus of both electrical and centrifugal, instead of electricsl and gravitational, forces. Some details of the technique and earl). modifications of the apparatus for carr\.ing out the centrifugall?. accelerated electruchromatographic separations have been puhlished ( 2 , 4 , 7 ) . -1schematic diagrsnl of thc current apparatus is illustrated in Figure 3. Thc drive mechanism and auxili:q. components, such as brake. tachometer, and automatic timer, are similar to those oi the centrifugslly accelerated paper chro1r.atogrsphy apparatus. The rotatsble head consists of a lower (Bakelite) and an upper (Lucitej shallow trsy (each section measuring 56 X 23 cni.) which c m be clmiped together to yield a chamber. l h e rectangular sheet of filter paper is moistened, blotted wit11 JYhatman 31111 paper, VOL. 31, NO. 5, MAY 1959
827
then clamped in position. -4ttached along the full length of each side of the bottom tray are platinum-plated electrodes. f, connected to a high voltage direct current source, through two brass collector rings, g, and spring-loaded graphite brushes, h (Figure 3, C). The upper tray, with the paper sheet held in position, is placed over the lower tray, so that the platinum electrodes on both trays make good contact with the long sides of the paper sheet, and is then clamped in position. The paper sheet when positioned properly does not touch either the upper or lower plate but is suspended in a horizontal plane approximately midway between them. A collection trough, e. divided into nine individual compartments, is attached by a hinge arrangement to each end of the bottom tray in such a way that the troughs srring out into the same horizontal plane as the plane of rotation of the rotor, when the latter is set in motion. A series of V-shaped drip points is cut into each end of the rectangular paper sheet, to correspond with the number of drip collector compartments or cups in each of the two collector troughs. For continuous operation, the solution to be fractionated is added continuously from a glass capillary tip to the oval, shallon-. stainless steel cup, d (Figure 3). It is designed as an annular trough (outside dimensions, major axis, 9.5 em.; minor axis, 9 em.) attached to the drive shaft of the rotor by two slender spokes, b (Figure 4). The cup rotates with the paper sheet. The major axis of the oval or elliptical cup is arranged parallel to the long dimension of the rotor and the spokes holding the cup to the axle are a t right angles t o this, or congruent with the minor axis of the ellipse. TWOstainless steel capillary tips, one in each end of the bottom of the oval cup, are provided so that the solution to be fractionated can leak into the paper sheet a t tIvo points symmetrically positioned an-ay from the central point of the rectangular sheet and along the median line parallel to the long sides of the paper sheet. The centrifugal force tends to propel the solution in a thin straight line from the center, while the electrical field, acting a t an angle to the centrifugal field, causes a drift of positively or negatively charged components to the electrode of opposite electrical sign. The result is that the components of the migrant solution assume distinct paths of migration (Figure 3, A ) . The design of the oval feeder cup (Figure 4) is essential to the proper operation of the unit as a whole.
As the liquid to be fractionated is added to the cup from the glass capillary tip, represented by the arrow, d, held firmly in place above its annular opening under continuous-operation conditions, it describes a circle within the annular opening of the cup as the paper sheet turns through 360 degrees. The placement of the capillary tip must be adjusted closely so that the circle reniains within the annular opening of the 828
ANALYTICAL CHEMISTRY
Figure 5.
Electrochromatogram from spot application of 5-pI. aliquot
Solution containing 0.01 mole per liter, each, of arginine, histidine, proline, tyrosine, and aspartic acid. Experimental conditions. Phosphate buffer; pH, 6.4; ionic strength, 0.008; potential gradient, 17 volts per cm.; current, 1 6 mm.; rotor speed, 31 6 r.p.m.; temperature, 2 5 " C.; paper, Whatman 3MM, 2 3 X 5 6 cm.; flow rate of developing solution, 0.8 ml. per minute. Time, 15 minutes. Displacement of amino acids, cm., from point of origin to approximate center of spot: arginine, 8.2; histidine, 8.1 ; proline, 8.6; tyrosine, 6.6; aspartic acid, 9.0
cup through a full turn of the paper sheet; otherwise the liquid to be fractionated would spill over the edge of the cup and onto the paper sheet. For batch-type operation, a small sample of the mixture can be added to the cup, and no further additions made. For micro quantities of sample, the paper sheet may simply be "spotted" with the liquid to be analyzed, a t two points away from the center, as shown by the arrows in Figure 5, and the development carried through in the usual way. As the paper sheet rotates, provision must also be made for introduction of more liquid near its center, to prevent drying out. The liquid for this purpose is added to the rotating paper sheet in a thin, continuous jetlike stream from a second glass capillary tip, represented in Figure 4 by the arrow, e. The developing liquid in this way is added directly t o the paper as a ring (rather than a t two point sources, c, as is the solution to be fractionated), which, under the influence of the centrifugal field, spreads out uniformly in a widening circular path so as to saturate the paper sheet thoroughly from the center on out t o its perimeter. The capillary tip is positioned carefully over the paper, so that the diameter of the initial ring of developing liquid is somewhat less than the shorter diameter of the inside wall of the elliptical cup. To cause the electrophoretic separations, a direct current potential is applied t o the strip electrodes, to yield a potential gradient of from 8 to 40 volts per em. across the dampened paper sheet. TKO Heathkit units, Model PS-3 (Heath Co., Benton Harbor, Mich.), connected in series niay be used as the source of electrical power, and the ionic strength of the buffer solution saturating the paper can be adjusted to maintain the current carried by the sheet within the rated capacity of the power supply.
No great problem is introduced by the fact that the electrodes run along the
full length of each side of the paper sheet. As the developing liquid is introduced near the center of the sheet, it flows out a t a uniform rate to form a circle of steadily increasing diameter. L4sthe front of this circular wave reaches the sides of the sheet uhere the electrodes are attached, it divides evenly and flows along the electrodes to the four outside corner collection cups, impelled by centrifugal force. In this way, any electrode products which collect in the edges of the paper sheet in contact with the electrodes are continually washed aa ay. The hole in the center of the top plate of the rotor, which accommodates the elliptical cup, and through whjch both the solution to be fractionated and the developing liquid are added, is sealed as in the simpler chromatographic unit. The spring-loaded closure device and Teflon bearing ring are represented in Figure 3, C, by p . ,4s an additional measure, to reduce evaporation of solvent from the paper and maintain equilibrium conditions as regards solvent vapor within the rotating head, soft silicone rubber gaskets, 1 X 20 X 0.3 cm., make intimate contact with the paper sheet a t each end of the rectangular head. The gaskets (n,Figure 3, C), one above and one below the paper sheet a t each end of the rotor, are positioned adjacent to the serrated ends of the paper sheet, but allow the nine paper points to be exposed within the compartments of the collection troughs. I n general, heavier stock paperse.g., Vhatman 3?IIhI--were preferable for planar electrophoresis. For separations of dye mixtures, such as aniline red, bromophenol blue, and methyl orange, generally satisfactory conditions were: Verona1 buffer, pH 8.6, ionic strength 0.01; rotor speed, 300 to 400 r.p.m.; flow rate of developing solvent, 0.6 to 0.8 nil. per minute. The paper vias prenetted, mounted in the
head of the instrument, and rotated for 5 minutes to achieve an even wetness. It was then stopped and 5-pl. aliquots of the mixture to be separated were applied. The rotor was now set a t 400 r.p.m., the developing solution added as a fine stream, and a dire-t current potential of 18 volts per cm. applied. After 20 minutes, a bromophenol blue spot had traveled outward 7 . 5 em. (chromatographic displacement) ; and 2 em. in the direction of the anode [ionographic migration ('7) 1. For separation of amino acids, representative experimental conditions might be illustrated by the fractionation of a solution containing glycine, lysine, and glutamic acid. The conditions were: sample, 0.01M solution of glycine, lysine, and glutamic acid; rotor speed, 400 r.p.m.; flow of rate of developing solvent, 0.6 nil. per minute; buffer and other conditions as above. After 15 minutes, the amino acids had all moved 7.5 cm. from the point of application and mere 1.9 em. apart ( 7 ) . The electrocliroinatogram shown in Figure 5 is a further illustration of the type of separations achieved. It was ohtained from a spot application a t the tn o points indicated by the arrows.
-1 5-pI. aliquot of a solution contain-
ing 0.01 mole per liter each of arginine, histidine, proline, tyrosine, and aspartic acid was applied. The experimental conditions were: phosphate buffer, pH 6.4; ionic strength, 0.008; potential gradicnt, 17 volts per cm.; current, 16 ma.; rotor speed, 316 r.p.m.; temperature, 25" C.; paper, Khatman 3MM, 23 X 46 cm.; flow rate of developing solution, 0.8 ml. per minute; time, 15 minutes. The displacement of the amino acids, in centimeters, from the point of origin to the approximate center of each zone, was: arginine, 8.2; histidine, 8.1; proline, 8.6; tyrosine, 6.6; aspartic acid, 9.0. The distance of separation betn-een the arginine and aspartic acid zones mas 4.5 cm. As the pattern on one half of the electrochromatogram is practically a mirror iniage of that on the other, an authentic sample of one amino acid can be applied to the paper on one side and a mixture on the other. \Then development has been completed, the paper sheet can be folded in half, and the presence or absence of the knon n amino acid in the mixture determined by observing congruence of developed zones. The centrifugal and electrical forces niay be applied independently, in scquence, or simultaneously. It is thus
possible to carry out simple chroniatographic or ionographic as well as electrochromatographic separations. LITERATURE CITED
(1) Clayton, R. A., Strong, F. M., A \ . ~ L . CHEM.26, 1362 (1954).
( 2 ) McDonald, H. J., Bermes, E. IT-., Shepherd, H. G., Chromatog. Methods 2, No. 1, 1 (1957).
(3) McDonald, H. J., Bermes, E. K., Shepherd, H. G., Nuturzc~zssenschafte,L 44, 9 (1957). (1) McDonald, H. J., Bermes, E. IT-., Shepherd, H. G., Proc. Chenl. SOC. (London), 1957, 23. (5) McDonald, H. J., MeKendell, L. V., Naturwissenschajten 44, 616 (1958'1. (6) McDonald, H. J., McKendell, L. V.. Bermes. E. W.,J. Chronzatog. 1, 259 (1958).' ( i )McDonald, H. J., Ribeiro, I,. P., Federation Proc. 17,272 (1958). RECEIVED for review November 17, 1'358. Accepted March 4, 1959. Project snpported in part by a grant-in-aid from the Chicago Heart Association. Leonard J. Banaszak is holder of the New Horizons Fellowship, for 1958-59, supported b>Labline, Inc., Chicago. Luiz P. Ribeiro, postgraduate research fellow 1957-58, acknowledges receipt of a travel grant from the National Research Council Of Brazil. The apparatus described in thls article is manufactured by and avdable from Labline, Inc., Chicago 22, Ill.
Diffusion Analysis of Ions in Gels MARVIN ANTELMAN Hampshire Chemical Corp., Nashua, N. H. SISTER DENISE EBY Saint Joseph College, Emmifsburg, Md. GEORGE 8. KAUFFMAN Fresno State College, Fresno, Calif.
b Methods were needed for qualitative separation of ions and their quantitative estimation by radial diffusion in gels. Mixtures of two and three cations were easily identified after resolution in gelatin and poly(viny1 alcohol) gels. Diffusion distances for cations and anions were noted and compared in gelatin and sodium alginate gels. Individual cations and anions diffuse in accordance with Fick's law, and can be determined b y plotting the variation of radial distance against ionic concentration. Common cations and anions may be quantitatively determined in concentrations between 0.1N and lO.ON, with an This accuracy to approximately 1 technique i s valid for both the cationic and anionic species in a given compound, as each constituent diffuses independently.
yo.
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objective of this work was to develop further qualitative and quantitative tests for ions by utilizing the property of diffusion in gelatinous media in contradistinction t o solvent flow capillary action and absorption vs. gravity, the properties utilized in paper chromatographic separations. Whereas analytical separations have been accomplished by diffusion alone in gels (f-8),no method has been previously developed for utilizing the property of diffusion (independent of current or other influence) for determining ions quantitatively. This paper describes the partial resolution and detection of some common cations and anions by radial differential diffusion taking place in concentrated gels under the influence of chemical potential gradients, and the behavior of i n d i ~ ~ d u a l i o r i s ~respect ~ i t h to the variHE
ables of time, concentration, and distance when undergoing diffusion, n-hich enables their deteimination. GEL PREPARATION
Thick concentrated gels of gclatin, poly(ving.1 alcohol), and sodium alginate were prepared. Gelatin plates were prepared by softening 16 grams of food grade gelatin in 50 ml. of ccld distilled water, then adding 150 ml. of boiling distilled water instantly to the mass. The hot solution nas poured into Petri dishes to a depth cf 1.5 cm. (watch glasses may also be used). Sodium alginate plates were prepared from Kelcosol (Kelco Co.) in the same manner by dissolving 3 grams of alginate in 100 ml. of hot distilled water. Poly(vinyl alcohol) plates were prepared from Elvanol 72-60 (Du Pont). In this case it was necessary to heat the alcohol (40 grams per 200 ml. of distilled VOL 31, N O . 5, MAY 1 9 5 9
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