ANALYTICAL CHEMISTRY
260 (IO). It can be seen that there is poor resolution of the spots, owing to the fact that the amino acids have small R, values in the tertamyl alcohol-2,4-Iutidine and consequently they do not travel far enough for resolution because of the limitation of the technique. Development stops as soon as the solvent reaches the top while by the method described here it keeps on going as long as-there is liquid in the dish above. LITERATURE CITED
(1) Block, R. J., ANAL.CHEM.,22, 1327 (1950). (2) Ibid., 23, 298 (1951).
(3)
Consden, R., Gordon, A. H., and Martin, 8. J. P., Biochem. J.. 38, 224 (1944).
(4) Datta, S. P., (1950).
Dent, C. E., and Harris, H., Science, 112,
621
22, 517 ( 5 ) Kowkabany, G. S . , and Cassidy, H. G., ANAL.CHEM., (1950).
(6) Ibid., 24, 643 (1952).
(7) llueller, J. H., Science, 112, 405 (1950). lluller, R. H., and Clegg, D. L., ANAL.CHEY.,23, 403 (1951). Toennies, G., and Kolb. J. J., Ibid., 23, 823, 1095 (1951). Williams, R. J., and Kirby, H.. Science, 107, 481 (1948). RECEIVED for review September 17, 1953. Accepted November 3, 19.53
Flow and Distribution of Solutions in Filter Paper Influence in Paper Chromatography SCOTT E. WOOD' and HAROLD H. STRAIN Argonne N a t i o n a l Laboratory, Lemont, 111. The flow and distribution of water and solutions in paper have been investigated in an eflort to place paper chromatography on a more quantitative basis. In upward flow in vertical strips of filter paper the rate of boundary migration between dry and moist paper decreases rapidly, and the amount of water per unit weight of paper decreases from the base of the strip to the advancing water boundarj ; the migration of the boundary between dry and moist paper for downward flow i n vertical strips of paper is nearly uniform. With downward flow in dry paper, the water distribution in the paper decreases slightly from the top of the strip to the advancing water boundary, but with downward flow in moist paper the water distribution is nearly uniform. The rate
I
N CHROMATOGRAPHY, the resolution of mixtures is effected by flow of the solution through porous, sorptive media. In electrochromatography, the separation of mixtures is effected by differential electrical migration in a solvent stabilized in porous media, but the accompanying electro-osmotic flow of the solvent may in turn affect the separations. For the description and the control of both separatory processes, the effect of the rate of flow of the solution and the effect of the distribution of the solution in the porous medium should be ascertained. Many publications have described the flow of liquids through porous media, and an extensive review of flow in paper has been prepared by Cassidy ( 2 ) . I n addition many studies ( 1 , 5 ,7 , 8 ,18) have been devoted to the effect of several variables, such as concentration, temperature, and rate of flow, on the migration of solutes relative to the migration of the solvents (the R values) in chromatographic systems. Krulla (6) and Takahashi ( I S ) have shown that there is a nonuniform distribution of liquid in vertical strips of moist filter paper. Schmidt (11) studied the flow of aqueous solutions of hydrogen chloride in vertical paper stripe and obtained approximately constant R values over a range of 20 cm. in the height of the water boundary. He incidentally demonstrated a nonuniform distribution of the solution in the paper. There have been few subsequent reports, however, concerning the effect of a nonuniform distribution of the solution in sorptive media on these R values. A thick Eaton-Dikeman filter paper has been employed extensively in electrochromatographic separations, but there is little information regarding its porosity and permeability. The rate of the movement of the water boundary in this papex has 1
On leave of absence from Illinois Institute of Technology, Chicago, I11
of boundary migration between dry and moist paper for radial flow in horizontal sheets of paper decreases rapidly, and the water distribution varies with the distance from the point where water or solution is added. The rate of migration of methyl orange dissolved in lactic acid solution, relative to the rate of migration of the solvent (R value) decreases slowly for upward flow b u t is constant, within experimental error, for downward and radial flow. The R value of fluorescein in radial flow decreases slightly. These results have been correlated with the quantity and the nonuniform distribution of t h e solution i n the paper. They have an important bearing upon the migration and the definition of zones i n practical chromatographic separations.
now been determined for upward, downward, and radial How. For these conditions, the distribution of the water in the 11aper has been ascertained. In addition, the R values of methyl orang? and of fluorescein dissolved in 0.1M lactic acid solution have been determined under various conditions. For some conditions these R values vary slowly with the migration into the paper. This variation of R has been correlated with the quantity of liquid per unit quantity of paper, with the distribution of the liquid in the paper, and with the velocity of flow at the boundaries of the solute and of the solvent. The well-known narrowing of a wide zone of solute when radial flow is used is explained in terms of the different velocities of flow a t the leading and trailing boundaries of the zone. EXPERIMENTAL
Paper, which is manufactured from biological raw materials treated and handled in various ways, is not a uniform or easily characterized product. I n these experiments emphasis has, therefore, been placed upon the phenomenological aspects of the observations rather than upon a precise study of the o b v r r e i l effects.
A single kind of paper-Eaton-Dikeman, wood-cellulose paper, Grgde 301, 0.03 inch thick-was used in all experiments. This . paper was used either as received or was washed with 1.11 nitric acid and with water using downward percolation and subsequently dried a t room temperature. For the experiments using upward and downward flow, long strips, 5 cm. wide and usually 1 meter long, were emploJ-ed. For upward flow these strips were suspended with the lower end dipping into distilled water or into the solution. Both the paper and the beaker containing the liquid were enclosed in polyethylene sheet, shaped into a tube and sealed with Dow-Corning stopcock lubricant to prevent evaporation.
V O L U M E 2 6 , NO. 2, F E B R U A R Y 1 9 5 4
26 1
Table I. Migration of Boundaries of Water and of 0.1M .4cid Solution Saturated with Methyl Orange (Mo) and R Values for Methyl Orange Using Upward Flow Paper 1 (Unwashed) h, Tlinr, cm of nun. water
Paper 2 (Washed) h, Time, cni of min. water
19 80 97 127 169 215 265 30; 335 1 ,237 1,437 1.620 1.740 2.692
4 17 55 95 125 1064 1244 1429 1549 2499
13.7 17.0 28.9 32.2 36.1 40.1 43.5 46.0 47.5 72.0 74.8 77.5 79.1 88.3
11.6 16.1 25.4 31.3 34.3 72.3 75.7 78.9 80.8 89.5
Time, min. 5 10 20 35 50 65 80 95 110 125 185 200 230 260 320 380 440 1,376 1.460 1,640 1,760 1,880 2 815
Paper 4 (Unwashed) h, h’, cm. of cm. of solution Blo 11.7 13.6 16.7 10.7 22.6 25 3 27.4 29.2 31.1 32.5 38.0 39.4 41.3 43.6 47.2 50.2 53.2 74.6 75.4 77 6 79 6 80 6 88 7
7.0 7.9 9.7 11.7 12.7 15.3 15.8 17.1 18.3 18.9 22.4 22.7 23.8 25.3 27.3 28.6 29.7 39.4 39.8 41 0 41 8 42 3 46 9
R, h’/h 0.60 0.58 0.58 0.59 0.56 0.60 0.58 0.59 0.59 0.58 0.59 0.58 0.57 0.58 0.58 0.57 0.56 0.53 0.53 0 53 0 53 0 53 0 53
Table 11. Migration of Water Boundary Using Upward Flow on Paper 9 (Unwashed) Tiroe, AIin
h, Cm.
n
14.0 19.8 31.4 38.2 42.6 46.0 72.3 75.2 76.1 77.5 79.2 80.4 89.8 90.8 91.8 92.8 98.3 99.0
25 100 173 235 295 1,260 1.380 1,440 1.530 1.650 1.740 2.700 2.820 3 , no0 3,120 4,150
4,270
Rate, Mm./Min
...
2.32 1.55 0.91 0.73 0.57
...
0.24 0.15 0.16 0.14 0.13
...
0.08 0.06 0.08
...
Time, hlin. 4,450 4,630 5,590 5,770 5.950 6,070 9,915 11,325 12,805 14,235 15,685 20.045 21,430 22,875 21,315 25,745 31,515
Rate, B I m /&fin.
h, Cm. Y9.7
100.5 104.9 105.8 106.8 107.1 115.7 118.2 120.9 122.2 123.6 128.1 129.7 130.9 131.2 131.6 134.9
0 04 0 04 0 05 006 0 03 0 02 0 02 0 01 0 01 0 01 0 01 0 01 0 01 0 002 0 003 0 006
0.06
For downward flow, the paper was draped over a small step ladder-shaped support made of rods of Pyrex glass No. 7740 and held in a shallow pan with the upper end of the paper dipping into the liquid contained in the pan. The ladder was fashioned in such a manner that the paper did not rest on any surface continuous in the direction of flow and the liquid could flow only within the paper itself. Again the paper was contained in a polyethylene tube, sealed a t the bottom; the pan and the top of the tube were covered with a sheet of polyethylene. Care was t:iken that the paper did not touch the polyethylene. For radial flow a sheet of unwashed paper was placed horizontally over a 6-inch crystallizing dish and covered with a glass plate. A narrow tab, formed by two parallel cuts to the center of the paper, dipped into the solution contained in the dish. Since serondary effects, such as the equilibration of the paper with the vapor of the solution or a nonuniformity in a given paper, were expected to alter only the observed values but not the basic phenomena, these effects were not studied. All houndaries were observed visually and all measurements
of distance were made to the nearest millimeter with a meter stick. The water boundary on the unwashed paper was observed rather easily because of the brown coloration that a p peared there. As no such coloration appeared on the acidwashed paper, the observation of this boundary was extremely difficult. The methyl orange boundary was diffuse and conseuently measurements on that boundary are not recise. For jetermination of the distribution of the solution in t i e paper, the paper strips used for vertical flow were cut into sections approsimately 5 cm. long. K i t h radial flow, a sector of the circle having an angle of approximately 90” was cut from the paper and this sector was then cut into four sections along circular arcs having radii of 1, 1.75, 2.25, and 2.75 inches. The sections were rapidly weighed, dried a t room temperature, and reweighed. The upward migration of water in dry paper, as indicated by the boundary between the dry and moist paper, is shown in the columns marked h in Table I for papers 1 (unwashed) and 2 (washed) and in Table I1 for paper 9 (unwashed, 2 meters long). The migration for upward flow of lactic acid solution containing methyl orange, again as indicated by the boundary between the dry and moist paper, is likewise given in the column designated h in Table I for paper 4 (unwashed). I n these tables, h is the distance of the boundary from the surface of the water in the beaker. The rate of migration averaged over each time interval in millimeters per minute for paper 9 is reported also in Table 11. The values have been omitted where the rate was changing rather rapidly with time and the time interval between successive measurements was necessarily large. The migration and consequently the rate of migration are essentially the same in all four papers except a t the beginning where the rate of migration is large and consequently the determination of zero time is difficult, if not arbitrary. The complete data have been presented as tables rather than graphs because of this agreement and also because the conditions and the duration of the experiments varied greatly. The migration of the boundary between dry and moist paper for downward flow is shown in the columns marked h in Table I11 for paper 5 (unwashed). The migration of lactic acid solutions of methyl orange using downward flow is similarly reported in Table I11 for papers 7 (washed) and 8 (unwashed). I n this table, h is again the distance of the boundary from the liquid surface. The position of the boundary was measured only after it had passed through that portion of the paper supported on the glass ladder and had entered the vertical portion of the paper. The choice of zero time is thus arbitrary. For this reason a comparison of the rates of migration is more significant than that of the actual distances migrated a t any particular time. Again the rates are almost the same in the three papers, although the rate in the washed paper (paper 7 ) is slightly greater than in the unwashed paper. The distribution of water in the paper in grams of water per gram of paper is given in Table IV both for upward and downward flow. The distribution for upward flow was determined in papers 1 (unwashed) and 2 (washed) a t the end of 48 hours and in paper 9 (unwashed) a t the end of 3 weeks. The distribution for downward flow was determined in paper 5 (unwashed) after 6.5 hours, a t which time the lower end was still dry. I n paper 6 (unwashed), water was allowed to flow through the strip until it dripped from the paper. (In this case the polyethylene tube was open a t the bottom.) This experiment was terminated after 24 hours. Paper 3 (unwashed) was thoroughly wet with water and hung in a sealed tube of polyethylene for 48 hours without a source of water. The sections of the various papers, given in the first column of the table, are numbered downward from the top of the moist paper. Small portions of the last section of apers 1 , 2 , and 9 and the first sections of papers 5 and 6 may havegeen actually dipping
Table 111. Migration of Boundaries of Water and of 0.1M Lactic Acid Solution Saturated with Methyl Orange (Mo) and R Values for Methyl Orange Using Downward Flow Paper 7 (Washed) h, om. of solution
Paper 5 (Unwashed) Time, nun. 0 30 60 150 170 190 210 230 250 280
Rate, mm./mia.
h, om. of water
1.9 1.8 1.7 2.1 1.7 1.8 1.9 1.6
39.0 45.4 51.2 67.2 70.5 74.6 77.9 81.5 85.3 90.1
... 2.1
Time, mln. 0
15 30 45 60 75 184 210 240 270
Rate, mm./min.
...
3.4 2.9 2.5 2.5 2.5 2.4 2.0 2.3 2.2
26.3 31.4 35.8 39.6 43.4 47.1 73.3 78.5 85.4 91.9
h’, om. of
Mo
15.8 19.8 22.9 24.4 27.9 29.7 47.2 50.2 54.0 59.0
R,
h’/h 0.60 0.63 0.64 0.62 0.64 0.63 0.64 0.64 0.63 0.64
Time. mln. 0
20 40 60 80 100 120 260 280 300 325
Paper 8 (Unwashed) h, cm. of solution
Rate, mm./min.
...
2.7 2.3 2.3 2.0 2.5 1.8 2.0 2.0 1.8 1.6
28.1 33.5 38.0 42.6 46.6 51.5 55.1 81.5 85.5 89.1 93.2
h’. cm. of 210 17.1 20.8 23.8 26.4 29.8 32.3 34.8 51.8 54.1 56.8 58.8
R
h’jh 0.61 0.62 0.63 0.62 0.64 0.63 0.63 0.64 0.63 0.64 0.63
ANALYTICAL CHEMISTRY
262
Table I\’.
Paper
Section 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Uppermost
95 minutes of observation. The migration of the water boundary in unwashed papers 1, 4, and 9 is almost identical, 6 9 while that in washed paper 2 is slightly greater (Table I). The rate of downDownward Upward ward flow in washed paper is also slightly 24 3 weeks, faster than with unwashed paper as unwashed unwashed shown by comparison of papers 7 and 8 0.38 2.68 (Table 111). 0.47 2.68 2.66 0.57 The movement of the rrater boundary 2.61 0.62 2.68 0.74 in paper 4 follows the equation, d h / d t = 0.72 2.61 ( a / h ) - 6, given by Peek and McLean 0.81 2.64 0.90 2.66 ( I O ) . The values of a and 6 for this 0.87 2.58 1 .oo 2.58 paper are determined to be 5.25 sq. cm. 2.60 1 .05 per minute for a and 0.056 cm. per minute 1.17 z.61 1.28 -. 67 for 6. .\wording to this equation the 1.39 2.63 1.51 2.66 rate becomes zero when h reaches a maxi1.65 2.72 mum equal to a/6 which, for these values Dripping 1.81 1-..I 42 of a and h, would be 94 cni. The meas2.07 2.21 urements with paper 9 were made to 2.23 determine 15-hether or not a maximum 2.40 2.50 could be obtained in reasonable time. 2.62 2.71 S o niasinium was reached after 3 weeks. 2.76 The constants of the Peek and 2.93 McLean equation for this paper using the data for the first 72 hours are 4.80 for a and 0.046 for b and give rates of flow in reasonable agreement with those observed over this time interval. However, aftrr i 2 hours the calculated rates steadily deviate from the observed. The maximum height using these values is 104 cm. When all the data are used, a equals 4.58 and b equals 0.036 giving a maximum of 127 cm. These latter values, however, give rates in good agreement with those observed only a t the start of the experiment and after the boundary has moved 100 cm. Peek and McLean considered the forces due to capillarity and gravity, but it now appears that still an additional force is required to explain the results. This force might be that due to a diffusion potential in or along the fibers of the paper. Since the first section of paper 9 contained 0.38 gram of water per gram of paper while the paper in contact with water vapor contained approximately 0.12 gram of water per gram of paper, there was still a discontinuity a t the water boundary. The integral of Peek and McImn’s equation setting h equal to zero when t equals zero is
D i s t r i b u t i o n of W a t e r in G r a m s per G r a m of Paper 1
2
Upward
Upward
3
__
Flow - ..
5
Hung Downward Total Time. Hours
48
48
48
6.5
unwashed
washed
unwashed
unwashed
0.7fi 0.93 1.07 1.17 1.32 1.41 1.44 1.61 1.71 1.89 2.03 2.23 2.30 2.38 2.56 2.58 2.62 2.71 3.12
1.96 2.01 2.07 2.13 2.17 2.27 2.42 2.42 2.46 2.50 2.55 2.68 2.67 2.70 2 78 2.81 2.83 2.85 2.82 2.78
2.56 2.41 2.43 2.33 2.30 2.27 2.23 2.25 2.17 2.14 2.05 2.15 2.15 2.06 2.01 1.04 1.75 1.39 Dry
0.67 0.95 1.06 1.23 1.30 1.49 1.54 1.72 1.82 2.00 2.19 2.26 2.36 2.49 2.57 2.65 2.79 3.04
Lowest
in the water because of the difficulty of cutting the paper exactly a t the surface of the water. I n the discussion it is assumed that the lactic acid solution would be distributed like water in the paper because the density of the dilute lactic acid solution is very close to that of water. Also the concentration of the lactic acid in the solution is expected to be constant in the paper except in the region of the lactic acid front (the R value of lactic acid on the washed paper is 1.00 and on the unwashed paper is 0.83). The slight adsorption of lactic acid on the unwashed paper would not materially change the basic phenomena. The R values of methyl orange in 0.1M lactic acid solution are given in Table I for upward flow (paper 4, unwashed) and in Table I11 for downward flow (papers 7, washed, and 8, unwashed). I n these tables, h’ is the distance in centimeters of the methyl orange boundary from the surface of the solution. The consistency of the R values reported in the tables show a possible error of 2 to 5% in these determinations, varying with the position of the boundaries in the paper. The distribution of the lactic acid solution in the paper using radial flow (paper 11, unwashed) was 1.21, 1.08, 0.91, and 0.59 grams of solution per gram of paper in the four sections used, moving from the center outward. The R value of methyl orange in a saturated solution using radial flow was 0.68 (paper 10, unwashed) and was constant, while that of fluorescein varied from 0.40 to 0.36 (paper 12, unwashed) as the boundary progressed outward. For these last determinations, small portions of the saturated dye solution were added a t the juncture of the tab and the horizontal paper before the lactic acid solution was allowed to flow into the paper.
When the logarithm is expanded in series for small values of h, keeping only the first significant term, this equation becomes
h2 = cut DISCUSSION OF RESULTS
Comparison of the migration and the rate of the migration in Tables I and I1 with those in Table I11 shows that the overall rate of upward flow is much slower than that of downward flow, an effect which may be attributed to the force of gravity. The initial rates for both upward and downward flow are of the same order of magnitude but that for the upward flow decreases rapidly and attains a value of about 0.1 mm. per minute in about 24 hours. At the end of about 48 hours, the boundary is still moving with a velocity of about 0.09 mm. per minute and a t the end of 3 weeks the velocity is less than 0.01 mm. per minute. I t appears from papers 7 and 8 (Table 111) that the rate of migration with downward flow decreases slightly. Paper 5 (Table 111)gives some evidence of a slight decrease also, but in this paper the rate is essentially constant within the accuracy of measurement. The rate of migration of the water boundary in radial flow decreased from 0.50 to 0.28 mm. per minute in
(2)
This last equation is given by Fujita (5),but this analysis shows that his equation is applicable only to small values of h. The empirical equation h2 = at p (3)
+
given by Muller and Clegg (9) is obtained on integrating the equation of Peek and McLean, setting h equal to a constant when t equals zero and also expanding the logarithm keeping on]:- the first significant term. The results given in Table IT’ show that the distribution of water with upward flow, papers 1, 2, and 9, is far from uniform while that with downward flow, paper 5 , is much more uniform. There is little difference between unwashed paper (paper 1) and washed paper (paper 2). The results reported for radial flow (paper 11) show that the aeight of water per unit weight of paper decreases much more rapidly with radial flow than with upward flow. The same decrease takes place in 7 cm. in radial flow as in approximately 20 cm. in paper 1 and 50 cm. in paper 9.
263
V O L U M E 2 6 , N O . 2, F E B R U A R Y 1 9 5 4 Paper 3 shows that the distribution becomes nonuniform when a paper saturated with water is hung in a gravitational field. In this case, however, much more u ater is held on the paper than is taken up by the paper with upward flow. K i t h downward flow, continued until the mater actually drips from the paper, the distribution is uniform as shown in paper 6. The curves obtained when the weight ot nater per unit weight of paper are plotted as a function of the section number are very similar to those of Takahashi (13) given by Fujita ( 3 ) . The R values of methyl orange for upward f l o ~(Table I ) are distinctly different from those for downward flow (Table 111) and still different from those for radial flow. Also the values obtained with downward and radial flow, papers 7 and 8 (Table 111) and 10. are essentially constant, but those with upward flow definitely decreasc n hen the migration of the boundar) becomes fairly large (paper 4, Table I). The R values for fluorescein show a small but definite decrease. A correlation should exist between the R values and the quantity of solution and the distribution of the solution in the paper. From phenomenological considerations, R maj- be hhown to depend on the concentration, the rate of floa, the ndsorDtion isotherms of both solute and solvent, and the distilbution of the solution in the paper. For the present treatment the solution is assumed to be dilute and to contain a single solute in a single solvent. The distance between the boundar) of solute in the paper and the surface of the liquid in which the paper is dipping or from the center in radial flow is designated as y and that between the solvent boundary in the paper and the surface of the liquid or the centcr in radial flow is designated as 2 . so that y will alaays be equal to or smaller than z . I t is furthei assumed that when adsorption occurs on the paper equilibrium is attained instantaneously, that the (*oncentration of the solute in the solvent is constant and equal to that in the bulk of the solution, and that the boundaries of the solute and of the solvent me sharp. The value of R for the solute is here defined as the ratio of g/: and the change of R in respect to z is given by the relation
where du/dt and dt/dt arc the velocities of the migration of the solute and solvent boundaries. These velocities can be expressed in terms of the velocity of the solution a t y and that of the solvent at z by means of mass balance equations. Thus if p u represents the grams of the solution per unit volume of paper just at the y boundary, pa the density of the bulk solution, c the concentration of the solute in terms of grams or moles per unit volume of solution, and f ( r ) the adsorption isotherm of the solute in terms of grams or moles of adsorbed solute per unit volume of paper, the mass balance equation for the solute at y is
(5) where c y is the velocity of the solution a t the y boundary. this equation
From
+
The quantity p y c / [ p , c p o f ( c ) ] is the fraction of the total quantity of solute per unit volume of paper which is in solution and not adsorbed. Similarly for the solvent a t the z boundary where
where pL is the grams of solvent per unit volume of paper and is the adsorption isotherm of the solvent on the paper, assumed to he a function of pz. Then combining Equations 4, 6 , and 7 .
f(p,)
dK = dt
1
-
2
(z- R )
(8)
where
The equation is applicable to the three types of flow considered. Thus R is a function of the quantity of solution and its distribution in the paper, as represented by p,, and pz as well as the concentration and adsorption isotherms. I t should be emphasized that vy, u,, pu, and p n are all functions of z and change with the position on the paper. Also and u1 both depend upon the rate of flow of the solution into the paper. This is particularly important in radial flow since the rate of flow of solution into the paper will depend upon the dimensions of the tab. The complexity of the problem of the flow ( I O ) and distribution (3) of a liquid in paper makes further development of Equation 8 exceedingly difficult. I t is possible, however, to discuss the behavior of R using Equation 8. If R has the definition given ahove and is constant, then R must equal 2. I t is equal to the fiaction of the total solute which is in solution and not adsorbed only under the conditions that f ( p 2 ) is zero and that v y equals vz. This latter condition implies that the distribution of the solution in the paper must be uniform for flow in strips of paper of uniform cross section. However, R may still be constant and equal to Z \\hen f ( p , ) is positive and finite, that is, when the solvent is adsorbed. The condition that the distribution of the solution in the paper must be uniform would still have to be Eatisfied. In such a cape
for flow in strips of paper of uniform cross section. This expression for R is exactly the same as obtained on calculating R on the basis of a mass balance for the solute assuming uniform distribution. The R value for radial flow, calculated on the same basis, equals the square root of the expression in Equation 10. -4s seen from Equation 10, the value of R, for both flows, is a function of p , even though the distribution is uniform. The observed value of R for methyl orange with downward flow, for which the distribution is essentially uniform, is 0.63. The observed value for radial flow is 0.68 in comparison to the square root of 0.63 which is 0.79. The quantity of solution per unit volume of paper is quite different in the two cases and the distribution of the solution is not uniform for radial flow. I t is then impracticable to make comparisons between these different values or to attribute the difference either to the quantity of the solution or to the distribution of the solution alone. Further considerations are possible: Z might be constant but unequal to R rather than equal to R as just discussed, and also Z might be variable. When Z is constant, the integral of equation 8 is
R =
z - ( 2 - Ro)z/zo
(11)
when the integration constant is evaluated by making R equal to Ro when z equals zo. If R is less than Z , dR/& is positive and R will increase with z approaching 2 in the limit. Conversely, if R is greater than 2, R will decrease, again approaching 2 in the limit. When Z is not constant but a function of z , R will either increase or decrease depending upon the relative values of R and this term. There seems to be no a priori reason why this first term should be equal to R or should be constant, and it is expected that R should vary. Thus for radial flow, R, when defined in terms of squares of radii as done by Holmgren ( 4 ) ,can be strictly constant only when the distribution is uniform. Comparison between the R value for radial flow and that for flow in strips of paper can be made
264
ANALYTICAL CHEMISTRY
only when the distribution is uniform and when the quantity of solution per unit quantity of paper is the same in the two papers. .4lso comparison of R values of the same solute between different experiments can be made only when the quantity of solution per unit quantity of paper is the same in the different experiments. This is particularly important for radial flow. If R is not a constant, then its rate of change with t becomes important. From Equation 8, it is seen that the absolute value of dR/dz must decrease with increase of z unless the absolute value of Z - R increases rapidly with t . For further insight into this question, Equation 8 may be written as
For flow in a strip of paper of uniform cross section, L,P, and are proportional to the quantity of solution crossing the boundary a t y and z , respectively, per second. Consequently u,p, will be equal to or greater than v,p,. For radial flow v,p,y and z ~ ~ p ,are z proportional to the quantity of solution crossing the respective boundaries and again v Y p Y is equal to or greater than z',p,. On the other hand pz f(p,) will be less than p u f(c)(pole) and consequently the whole term could have values less than unity but not necessarily so. If i t does have values less than unity as R must have, then the difference between this term and R may be small so that R does not change rapidly vith 2. For the systems examined in this paper, R does not appear to be a sensitive function o f t . With downward flow, for which the distribution is rather uniform, R for methyl orange in lactic acid solution is a constant within experimental error. With upward flow, for which the distribution decreases with z , R decreases slowly. With radial flow for which the distribution markedly drcreasc~swith z , R for methyl orange appeared to be constant.
Since this could possibly be a fortuitous case, the experiment using fluorescein was made in which R was found to decrease. These considerations throw new light on the behavior of chromatographic zones in circular paper chromatography. For radial flow we have pointed out that v,p, > u,p, and therefore vy/v. 2 zp,/ypY. It appears from this equation that o y is probably greater than vI. Accordingly the migration of the solution at any given boundary with a certain radius would be greater than that at any other boundary with a greater radius. If then a rather wide zone of a solute is present on the paper, the velocity of the solution a t the forward edge would be less than the velocity at the trailing edge. I n such cases the zone would become narrower and would appear better defined as i t moves outward but the concentration of the solute in solution in the zone would tend to remain constant.
ilpI
+
LITERATURE CITED (1) Austin. C. R., and Shipton, J.. J . Council Sci. Ind. Research, 17,
+
I
115 (1944). (2) Cassidy, H . G., A x ~ L CHEM., . 24,1415 (1952). (3) Fujita, H., J . P h y s . Chern,, 56, 625 (1952). (4) Holmgren, A. I., Biochem. Z., 14, 181 (1908); Kolloid-Z., 4, 219 (1908). ( 5 ) Kowkabany, G. N.. and Cassidy, H. G., ANAL.CHEM.,24, 643 (1952). (6) Krulla, R., 2. p h y s i k . Chem., 66,307 (1909). (7) Le Rosen. A. L., J . A m . Chem. Soc.. 69,87 (1947). (8) Le Rosen, .4.L.. and Rivet, C. A . , Jr., AN.AL.CHEM.,20, 1093 (1 948). (9) lluller, R. H., and Clegg, D. L., Ibid., 23, 403, 408 (1951). (IO) Peek, R. L., Jr., and McLean, D. A., IND.EXG.CHEM.,ANAL. ED.,6 , 8 5 (1934). (11) Schmidt, H., Kolloid-Z., 24, 49 (1919). (12) Strain, H. H., A N . ~ L CHEM., . 22,41 (1950). (13) Takahashi, A,, Kagaku, 20,41 (1950). RECEIVED for review August 20,
1953.
4ccepted Sovember 21. 1953.
Chromatographic Separation of Sugars with Hydrocellulose 1. D. GEERDES, BERTHA A. LEWIS, REX MONTGOMERY, and FRED SMITH Division
o f Agricultural
Biochemistry, University of Minnesota, St. Paul, M i n n .
Although cellulose and certain types of modified cellulose have been used successfully for separating various mixtures of sugars and their methyl ethers, there is need of an adsorbent with a better resolving power and a greater capacity. The process of dissolving cellulose powder in phosphoric acid, followed by precipitation with water, produced a desirable hydrocellulose for use in the chromatographic separation of methylated sugars. Both the resolving power and capacity of this hydrocellulose were superior to other cellulose adsorbents. However this
P
-1RTITION chromatographic analysis (6) has become a standard technique for the separation, identification, and quantitative determination of sugars (5, 8, 11 ) and cellulose has been extensively used as the adsorbent (f2). For preliminary identification purposes and the separation of small quantities of compounds, cellulose in the form of filter paper is generally satisfactory. I n order t o obtain larger quantities for complete characterization, the paper has been replaced by columns of cellulose powder (3, 8). Usually, for column Peparation a single solvent mixture, is preferred ( 2 ) , since the romplete separation of a mixture of sugars can be effected by means of an automatic fraction collector without attention once the process has been started, but in some instances it appears to be an advantage to use R series of solvents (4).
improvement did not extend to the separation of free sugars. It is believed that part of the success of the modified column is due to the top of the adsorbent being kept in place by a glass-enclosed metal weight. The anomeric forms of ethyl-crhamnofuranoside have been separated. The increased capacity and improved resolution obtained with the modified columns make it possible to effect separationof practical amounts of methylated sugars without being involved in the slow development rates of larger columns. Although cellulose and certain hydrocelluloses have been used successfully for separating various mixtures of sugars and their methyl derivatives ( 2 , 8 ) , there is need of an adsorbent with a better resolving power and a greater capacity. On the assumption that there is considerable association of cellulose molecules through hydrogen bonding (IO), a considerable portion of the surface of the cellulose plays little or no part in chromatographic separations and hence the capacity of unmodified cellulose is low. Some support for this view is forthcoming from the observation that modification of the cellulose by acid (9) and by oxidizing agents ( 1 ) acting in heterogeneous systems has been shown t o improve its chromatographic properties for certain purposes. I n view of this it seemed that if the orderly arrangement of the cellulose molecules was disrupted completely by dissolving the
