1872
ANALYTICAL CHEMISTRY
have little practical use, because the values of the variables depend upon the system, and the systems show considerable variations themselves. In addition, no method has been devised by which the opposing forces can be measured directly. ACKNOWLEDGMENT
(3) (4) (5)
Kunkel, H. G., and Tiselius, A., J . Gen. Physiol., 35, 89 (1951). Longsworth, L. G., J . Am. C h a . SOC.,69, 1288 (1947). Sato, T. R., Kisieleski, W. E., Norris, W. P., and Strain, H. H., ANAL.CHEW,25, 438 (1953).
( 6 ) Strain, H. H.,
J. Am. Chem. SOC.,61, 1292 (1939). (7) Strain, H. H., Sato, T. R., and Engelke, J. L., ANAL.C H E ~ I26, ., 90 (1954).
The radioactive dextran and sucrose, employed as flow indicators, were supplied by Norbert J. Scully of this laboratory. LITERATURE CITED
(1) Chen. Eng. News, 30,4244 (1952). (2) Engelke, J. L., Strain, H. H., and Wood, S. E., ANAL.CHEM.,26, 1864 (1954).
(8) Wood, S. E., and Strain, H. IT., Ibid.,26, 260 (1954). RECEIVED for review April 7, 1954. Accepted September 17, 1954. Pre. sented in part a t the syinposium on Electromigration in Stabilized Electrolytes, Division of Biological Chemistry and .4merican Association of Clinical Chemists a t the 124th Meeting of the AMERICAN CHEMICAL SOCIETY in Chicago, Ill., September 1953. Scott E Wood nas on leave of absence from Illinoiq Institute of T e r h n o l o ~ y .
Electrical Mobility of Phosphate Ions in Paper Electrochromatography JOHN L. ENGELKE1 and HAROLD H. STRAIN Argonne National Laboratory, Lemont,
111.
The electrical mobility of phosphate ions has been determined in phosphate solutions in paper with zones of radioactive phosphate as the migration indicator. This procedure, which eliminates solution boundaries, shows that the mobility of phosphate varies with the have nearly equal pH. The ions H 9 0 4 - and mobilities, whereas Pod--- migrates almost four times as fast. The electroosmotic flow of the phosphate solutions also varies with the pH. In strongly acid solutions, i t is toward the anode; in weakly acid and alkaline solutions, it is toward the cathode.
I
N WLUTIOSS in paper, M employed in electrochromtttography, there is no simple relationship between the mobilities of ions and their ionic charge. Depending upon the conditions, different ions with the same ionic charge may migrate a t the same or a t different rates. Likewise, the same element in different valence states may also migrate a t different rates (6-8). With many anions, the number of the ionic charges varies with the degree of the dissociation. There is, however, little information concerning the mobility of anions in paper moistened with solutions of different pH values. A succinct report by Sansoni and Klement ( 5 ) shows that the electrical mobility of phosphate ions in paper moistened with organic and inorganic buffers (other than phosphate) varies anomalously with the composition and pH of the solution. In these experimenta, the migration of the phosphate was determined from the movement of small zones of phosphate solutions which were located with molybdate reagent. This observed movement was corrected for the electroosmotic transport determined with separate zones of flow indicators. In this laboratory, the electrical mobility of phosphate ions in moist paper has also been determined aa a function of pH. Here, however, the measurements were made in phosphate solutions with zones of radioactive phosphate as the migration indicator. With this autodetection or self-detection method, there are no solution boundaries in the moist paper, and there is no variation in the kind of buffer (sodium and dissociated phosphoric acid) or in the concentration of total phosphate. With the use of secondary electrodes for the determination of voltage, the electrode reactions are far removed from the paper itself. The observed movement of the phosphate zones was corrected 1 Present address, Department of Chemistry, University of California, Berkeley 4, Calif.
for the electroosmotic transport, which was usually observed with zones of an easily detectable flow indicator-auch as hydrogen peroxide (8). But in the strongly alkaline solutions, owing to the dissociation of the peroxide, hydrazine was used as the flow indicator. In the strongly acid solutions, the electroosmotic flow was toward the anode, whereas in weakly acid and alkaline solutions, it was toward the cathode. In harmony with the earlier investigations (5), the mobility of the phosphate ions was a complex function of their ionic charge and of the p H of the solution. In addition, calculation of the proportions of the several iouic species present a t the different pII values indicates that particular mobilities may be assigned to each species. METHODS
The apparatus utilized for the investigation of electroomiosis in paper ( 2 ) was adapted to the determination of the electrical mobility of the phosphate ions. All the electrolytic solutions were prepared from phosphoric acid or from the sodium salts and their mixtures. All these solutions contained the same concentration (0.01M) of phosphate with variable proportions of sodium. These solutions were used to moisten the paper and to fill the electrode vessels h the same level ( 3 liters each). The pH of the solutions was determined with special calibrated glass electrodes. The temperature of the moist paper was 17' & 2" C. The paper was from a single roll of the Eaton-Dikeman product (Grade 301, 0.03 inch thick). It was different from that em loyed for the study of electroosmosis (2), and it eshibited d d r e n t electroosmotic properties. With this paper, the electroosmotic flow was toward the anode with 0.1M lactic acid as the electrolytic solvent. This paper was cut into strips 4 X 22 inches. These strips were soaked in the phosphate solution and placed between the water-cooled padded plates of the electrolysis cell. Two strips of paper (Eaton-Dikeman, Grade 301, 0.05 inch thick and 2 X 17.5 inches) were moistened and placed along the edges of the cell to help support the upper plate. After 20 minutes, the long paper strip was blotted gently a t the center where the indicators were to be added. The radioactive phosphate, dissolved in the buffer solution (10 pl.), was added to the center of the strip. The flow indicator (0.1M in the buffer solution) was added a s two separate zones (10 pl. each) at each side of the radioactive phosphate. The paper was covered with polyethylene, and voltage (140 f 1 volts, equivalent to 3 volts per cm.) was applied for 3 hours. The ends of the paper Mere then cut off, the cell was opened, and to locate the peroxide zones, the paper was sprayed with a solution of equal parts of sodium hydroxide (6M) and silver nitrate (0.1M) in ammonia (8%). This sprayed paper was dried uniformly on a flat surface, and the radioactive zone was located autographically. Both the phosphate and the peroxide zones were always symmetrical. Conventional chromatographic experiments show that spots
V O L U M E 2 6 , NO. 1 2 , D E C E M B E R 1 9 5 4 of radioactive phosphate were not sorbed by paper when washed with the solutions a t different hydrogen ion concentration. By contrast, the sodium was slowly sorbed by the paper, a phenomenon indicative of ion exchange properties. As a result of the mrption, a small pH gradient was formed. This gradient, with the more acid region at the leading boundary, as observed with indicator papers and with glasR electrodes.
-1.4. -I 3-I. 2 . -I I * '04
A - 1 1
CORRECTED?
,
/ I
1873 employed as a measure of the electroosmosis in the strongly alkaline solution. These observations show that mixtures of hydrogen peroxide and hydrazine may be separated electrochromatographically in alkaline solutions. They may also be separated i n arid solutions wherein the hydrazine forms a mobile cation. The movement of phosphatr corrected for the electroosmotic displacement BhoRs that the niobility is low in the strongly acid solutions and high in the strongly alkaline solutions. In the p l l range between 4 and 10, hov ever, the mobility values are largely within the range of 0.8 to 0.9 (cni./hour/volt/cm.). Qualitatively these results are in poor agreement with those reported b\ Sansoni and Klement (6) shoa ing a maximum near pH 8. For comparison of the mobilities R ith the Occurrence of particular ionic species, the proportions of the dlseociation products of phosphoric acid 11ere calculated from the dissociation constanti and plotted. The different ions present in the solutions miployed for the mobility determinations and the proportions oi these ions interpolated from the plota are reported in Table 1. From this table, the ionic nature of the phosphate changes from virtually 1 0 0 ~H2P04o at pII 4.5to abouC 100% HPOI-- a t pII 9 0. A e there is virtually no change in mobility over this range of pH, the H2P04- and HPO4-- ions must have about equal niobilities. Qualitatively, undissociated phosphoric aeid HaPo4 in the acid solutionsmust havea Ion mobility, and PO,--- in thealkaline solutions must have a high mobility. As the zones of the radioactive phosphate remained uniform, the several ionic species of phosphate must interchange rapidly relative to the rate of electrical migration.
Table I.
-2
PEROXl DE
Figure 1. Electrical Migration of Phosphate and Electroosmotic Displacement of Hydrogen Peroxide and Hydrazine as Function of pH in Paper Moistened with Phosphate Solutions (0.01M) Migration of phosphate is toward the anode (corre-, sponding to negative values). Displacement of hydrogen peroxide and hydrazine is toward the cathode in weakly acid and alkaline solutions (positive values) and is toward the anode in acid solutions (negative values)
The radioactive phosphate was prepared by neutron irradiation of crystalline sodium dihydrogen phos hate followed by prolonged hydrolysis with acid. Unless hyfrolyzed to orthophosphate, various comples radioactive phosphorus compounds migrated more slowly than the phosphate and yielded long diffuse indicator spots (6).
2.0 4.5 9.0 11.7
Ionic Species of Phosphoric Acid and Their Proportions at Various pH Values (Based on dissociation constants at 25' C.)
57
.. .. ..
43 100
2
...
.. 80
On the basis of two assumptions, the mobility of the nonionized HsPOa and the PO,--- may be calculated from the data in Figure 1 and Table I. First, the mobility of a particular ion is assumed to be the same a t different p H values; and second the mobility of the total phosphate is assumed to be the sum of the products of each ionic mobility times the proportion of that ion. The mobilities, calculated in this way from average values in Figure 1, are presented in Table 11. The values in parentheses are the mobilities assumed to be constant a t the different p H values.
RESULTS A N D DISCUSSION
Table 11. Mobilities of Ionic Species of Phosphoric Acid at Various pH Values
Mobilities. The observations on the electrical migration of phosphate ions are summarized in Figure 1. The observed migration of the phosphate zones, the observed electroosmotic displacement of hydrogen peroxide and hydrazine, and the corrected mobility of the phosphate are all presented as a function of PH. From Figure 1, the electroosmotic flow, indicated by hydrogen peroxide, is zero a t p H about 4.3. In the strongly alkaline range from pH 1 1 to 12, the movement of hydrogen peroxide is very small. Presumably the peroxide dissociates into HOn- ions (about 50% a t pH 12, based upon the dissociation constant). These negative ions undergo electrical migration to the anode thereby reducing the electroosmotic displacement toward the cathode. Because of this effect, the movement of hydrazine wa8
(l'itlues at p H 4.5 a n d 9.0 areaverages from Figure 1. Values in parentheses are assumed values. T h e remainder are calculated values) Ionic Mobilities, Cm./Hour/Volt/Cm. PTr HBPOI HIPOIHPOI-2.0 -0.05 (0.87) ... .. 4.5 ... 0.87 ... 9.0 ... ... 0.88 ... 11.7 ... ... (0.88) 3.2
Mobilities determined in moist paper are usually less than those determined in free solution and may vary with the amount of the solution in the paper (4). In the absence of sorption by the paper, the mobilities in paper should be indicative of the relative migration rates of the ions in free solution. Wherea in moist
1874
ANALYTICAL CHEMISTRY
paper the mobilities of the H,POI- and HPOI-- ions are about equal, the mobilities in free solution are proportional t o the charge ( I ) , both ions having the same ionic diameter ( 3 ) . The reason for this difference is not apparent, although it may be related, in part, t o variations of the migration system, as by the sorption of sodium ions. Electroosmosis. The dependence of the direction of the electroosmotic flow upon the pH as shown in Figure 1 points to a complex mechanism for the electroosmotic effect. The paperphosphate system acts as if it had amphoteric properties with a n isoelectric point a t about pH 4.3. The results virtually exclude the possibility that the electroosmosis is caused by the ionic transport of water. I n the acid region, most of the current is carried by hydrogen ions; hence, for ionic transport of water toward the anode, the hydration of the dihydrogen phosphate ion must be many times greater than that of the hydrogen ion,
a conclusion not supported by ionic mobilities or calculations of ionic diameters (3). LITERATURE CITED
Abbott, G. A . , and Bray, W.C.. J . Am. Chem. SOC.,31, 729, esp. 762 (1909).
Engelke, J. L., Strain, H. H., and Wood, S. E., Asrlz. CHEY., 26,1864 (1954).
Kielland, J., J . Am. Chem. Soc., 59, 1675 (1937). Kunkel, H. G., and Tiselius, 4.,J . Gen. Physiol., 35, 89 (1951). Sansoni, B. V., and Klement, R., Angew. Chem., 6 5 , 4 2 2 (1953). Sato, T. R., Kisieleski, W. E., Norris, W.P., and Strain, H. H., ANAL.CHEW,25, 438 (1953). S a t o , T. R., Sori-is, W. P., and Strain, H. H., Ibid., 26, 267 (1954).
Strain, H. H., Sato, T. R., and Engelke, J. L., Ibid.,26, 90 (1954). RECEITED for review .4pril 8, 1954. Accepted September 17, 192. John
L. Engelke was Resident Student Associate, Michigan College of Mining and Technology.
Gradient and Rate Aspects in Paper Chromatography BETTY-JEAN ACKERMAN and HAROLD G. CASSIDY Sterling Chemistry Laboratory, Y a l e University, N e w Haven,
This work was initiated to provide information about the thickness and properties of the film of developer in paper chromatography, and the rate of flow of fluids in paper. It w-as found that there is a mass gradient of liquid along the strip of paper up which liquid has risen, such that the thickness of the film decreases from the surface of the developer liquid to the front of the mobing liquid. Data showing the thickness of this film are presented for 10 present-day papers, together with information about thickness of the papers, weight per unit area of the dry papers, and rate of rise of water at 6-cm. height of rise. Correlations between structure of molecules and rate of rise of the liquid in paper are summarized from a study of 141 liquids. This information should be useful to chromatographers through its comparison of rates of flow of liquid and information on how much liquid is carried by the paper. The existence of the gradient also supports conclusions about the way the Rl’s of the zones behave.
A
N AXALYSIS of paper chroniatographic processes s1ion.s
that as the developer liquid rises along the paper there may be expected in it the mass gradient reported by Krulla (7’) and, if the developer is a mixed one, one or more concentration gradients due to frontal analysis ( 5 ) . The first part of this paper provides further information about such gradients. The second part summarizes a study of rates of rise of liquids in paper. MASS GRADIENT IN MOBILE PHASE
The materials and methods were the same as used in previous work ( 5 ) . Temperature control was maintained, and the chromatography chamber was lined with damp paper (except for two narrow vertical windows) so as to facilitate vapor saturation (18). Papers were conditioned (“equilibrated”) for 18 hours, as it has been found that only slight changes in weight occurred after this period. Table I gives some properties of the papers used. It has been shown by Krulla (7‘) and others (15, 1 9 ) that, as a liquid rises along a strip of filter paper, the weight of liquid per unit area of the paper decreases upward to the front of the rising
Conn.
liquid. Thus, there is a gradient in the mass of liquid along the paper, decreasing to the front. I n the terms used in paper chromatography, the sum of the cross-sectional areas of the mobile phase ( A L ) and the nonmobile phase (A,) [in the definition of R,:Rp = A L / ( A L CYAS)]decreases to the front of the developer. This was noticed by Consden, Gordon, and Alartin (51in their definitive paper. Fujita ( 4 ) has given a quantitative description of the shapes of the curves found.
+
Table I.
Some Properties of Papers AV.
Papera
Av. Thickness, Mni.
W t , Dry Paper/ Area Dry Paper bIg./CA.
dh/dt for Dist. Water a t 6 Cm., C ni./hlin. (Machine Direction)
Time for Water t o Rise 18 Cni., RIin.
0.17 8.3 0.1G 173 E.-D. 950 0.19 8.5 0 19 147 952 a W. = Whatman S 9. = Schleicher and Schuell, E.-D. = Eaton-Dikeman. Thickness wasrnkasured with a n Ames gage, No. 25. One-, two-, and three-sheet thicknesses were measured for each paper a t four places and calculated t o average single-sheet thickness. The rates of rise were determined a t 25.1”C., except W.1 done a t 24O C., and represent the averages.
The mass gradient was confirmed for 10 filter papers in presentday use and for a number of different conditions (Figures 1 t o 3). It is evident that the mass gradient is found in downward as well as upward development, and takes the same form in both. The only other data available for modern paper are the very precise measurements of Wood and Strain ( 1 9 ) with another paper and solvent system. When a binary developer of aqueous phenol was employed a mass gradient of the same general S-shape was found also. Concentration gradients show up, in many cases, as multiple fronts. These phenonena have been observed in various connections by others. Thus, for example, Lederer (8) found two fronts
