Versatile Technique of Paper Chromatography FILADELFO IRREVERRE and WILLIAM MARTIN National lnstitutes of Arthritis and M e t a b o l i c Diseases, National lnstitutes o f Health, Bethesda,
Md.
G is a filter paper cylinder. H and Z are, respectively, the glass cylinder and plate glass cover. Paper Cylinder. For the first dimension the paper is cut 15 x 16 inches. It is important that the edges be cut smoothly and evenly by means of a large paper cutter. A cross vvhere the spot is to be placed is marked on a corner 2 inches from both edges. Along the two longer edges holes are cut with a punch. In order that the holes on both edges of the paper will match, a multiple paper punch (Mutual Centamatic S o . 300) was used. The punched holes are approximately 6 mm. in diameter and they are 12 mm. from the edge. The paper is fashioned into a cylinder; the holes are matched and while they are held in place by a pair of glass rods inserted in the holes the paper is laced with a strip of the same kind of paper cut slightly less than 6 mm. wide. Care must be exercised to make the edges match so that the top of the cylinder is flat. The ends of the strip used for lacing should be very carefully tucked into the holes. S o metallic clip of any sort is needed. With a little patience and practice a perfectly round paper cylinder can be made with a minimum of handling and without leaving fingerprints on the pathway of the spots. The finished cylinder has a diameter of 116 mm. Wick. The simplest support for a wick is made by cutting two circular papers 121 mm in diameter and putting them on top of each other. At the center is inserted one strand of string (KnitCro-Sheen, J. & P. Coats, a very common brand of cotton thread with a diameter of approximately 1 mm.), which has been previously washed by descending chromatography with the same solvents to be used. A knot is tied at one end of each wick to prevent it from pulling out of the filter paper top. The following have also been used for wicks: 2, 3, and 4 strands inserted in the middle of the paper disks; 4 double strands placed equidistant from each other along a circle drawn, diameter equal to 1.0 cm. from center; 6 double strands, diameter equal to 1.5 em., 8 double strands, diameter equal to 2.0 cm.; 12 double strands, diameter equal to 3.0 em.; 16 double strands, diameter equal to 4.0 cm.; also 16 single or double strands, diameter equal to 6.8 cm. With wicks on circles of 3.0 cm. and above in diameter a center circle of smaller diameter is usually cut out in order to facilitate straightening the wicks a t the start of the development.
I n order to improve on the present methods of paper chromatography in which the solvent flow rate is fixed by solvent and paper, a versatile technique has been developed. The flow of solvent on the filter paper is varied and regulated by a system of wicks. The spots are made to travel any distance throughout the entire length of the paper, thereby increasing resolution. The chromatographic process can be made to go on even after the solvent front has reached the end of the paper or to stop by itself at any time. The system utilizes a relatively small all-glass apparatus for descending flow of solvent. The technique yields a more reproducible chromatogram and provides the investigator with a wider choice of working conditions and with a more bersatile tool in further studies on the mechanism of paper chromatography.
11I1: different methods in current use in paper chromatog1 raphy are essentially similar, in that the paper dips into the solvent and the latter rises or descends ( 3 , I O ) . These methods and their various modifications ( 1 , 2, 4,9 ) have one drawback in that the rate of flow of the solvent is fixed. As a result, a qolvent that works well for the separation of a mixture of solute on one type of paper often produces poor or very little separation on filter paper of different porosity. hliiller and Clegg (8) and Kowkabany and Cassidy ( 6 ) , using paper strips of a variety of shapes and an ascending technique, found that the rate of flow of solvent is affected by geometric shapes. Mueller ( 7 ) ,in an effort to overcome the poor resolution usuallj- observed with thick papers of high porosity, attached a thinner paper along the edge. This acts as a valve to slow down the flow. The present communication describes a versatile technique of paper chromatography. The main feature of this method is that the rate of flow of the solvent on the paper can be varied by means of a system of feeders or wicks, thus giving the chromatographer a wider choice of rates as well as more reproducible chromatograms. In addition, it utilizes a compact all-glass apparatus which is easy to clean and assemble, thus avoiding the danger of possible contamination from metals. A battery of a dozen jars occupies only a small space as compared to the usual aquarium tanks (9). In the authors’ opinion the technique i- adaptable to almost any need in paper chromatographic separat ions.
J
EXPERIMENTAL
Apparatus. A borosilicate glass cylinder 6 X 18 inches with X 6.5 X 6.5 inches was used. Both the top a plate glass cover
of the cylinder and the glass plate are ground smooth and fine, using for the final grinding a 3 F Grade Carborundum. This eliminates the use of grease seals. -4s shown in Figure 1, two funnels are used for an adjustable platform. A is a 115-mm. funnel; A’ is an SO-mm. funnel with the stem cut off and replaced by glass tubing approximately 130 mm. long and 10 mm. in inside diameter. This should slide freely on funnel A . C is a Teflon ring with a setscrew of the same material. (Teflon is used as it is impervious to most of the organic solvents used in chromatography.) I t slides on stem A and holds the funnel A‘ a t any height. B is a crystallizing dish (Corning No. 3140) 100 mm. in diameter, with the height cut to 33 mm. D is a round, flat glass plate ‘/a inch thick and 5 inches in diameter. It has been found useful to cut a hole 40 mm. in diameter into the center of this glass plate to facilitate the straightening of some types of wicks. E is a round filter paper top with its one-strand wick, F .
Figure 1. Apparatus A . 115-mm. funnel A ’ . 80-mm. funnel B. C.
Crystallizing dish Teflonring
F. G. H. I. J.
top One-strand wick Filter paper cylinder Glass cylinder Plate glass cover Additional weight on top of plate glass
D. Glass plate E. Round filter paper
n
257
When several strands are placed equidistant from each other on a circle, the solvent flow upward and from each strand an everwidening circle of solvent emanates, with the result that the solvent front moves as though originating from one circle. By this technique it is possible to regulate or vary the rate of flow of the solvents by varying the number of strands and their distance from the center of the circular filter paper top and/or varying the effective length of the wickthat is, the distance between the liquid surface and the filter paper top. Typical Two-Dimensional Run. A quantity of the solution to be chromatographed is measured and placed on a silicon-treated glass slide or on a piece of Teflon. After the droplet of liquid has evaporated to a very small sphere, it is transferred to the filter paper cylinder made as described by just touching it to the cross mark. This method permits the application of small discrete spots to the Daper. Next the platforms A , A’ adjusted to the desired height by
A N A L Y T I C A L CHEMISTRY
258 means of the Teflon ring are placed in the glass jar. This adjustable ring can be replaced by a length of glass tubing. Different sizes will provide changes in the height of the wick. Dish B containing the solvent is next placed on A' and the paper cylinder is gently lowered into position with the end next to the spot a t the top. The circular paper top with wick must be centered. I n the case of a 16-strand top with a center circle cut out the strands should be straightened as they are immersed in the liquid. This is done with the aid of a curved glass rod. Then the round glass plate is very gently lowered on the circular filter paper top t o keep the latter in firm contact with the cylinder. If the circular glass plate has no hole in the middle, it is necessary to lower it and to lift it a t the end of the run by means of tongs fashioned out of a stainless steel wire. For additional weight on top of the plate glass (J in Figure 1) cylindrical sections 1.5 inches wide cut from a 1-liter beaker and weighing approximately 50 grams were used. When weak and thin filter papers are used such as Whatman S o . 1 or 4 the plate glass is sufficient.
I
I
I I LEUCINE
I
I
I
I
1
I
N E -V A IL
-1
8 . 4 1
v -
0
1
I
1
2
Figure 3.
I
1
I
I
I
I
~
3 4 5 6 7 \/TIME TO REACH 4 0 CM. ( H O U R S )
8
9
Relation between Rate of Flow of Developer and Rf Value
rn Nor L e u c i n e a-Amino Butyric Acid
AGIycine
w
m 2 0
1
2
3 4 5 6 L E N G T H OF W I C K ( C M . )
7
8
Figure 2. Relation between Rate of Flow of Developer and Length of Wick
Y
0
3 1
$1 P O
To measure the height of the wick a t the beginning of the run a cathetometer was used. Other types of measuring devices may also be used. The paper cylinder is put in the jar and the top sighted. S e x t the top level of the liquid is measured and the difference between the two readings represents the vertical length of the wick on which the liquid travels. After development in the first solvent, the chromatogram is dried in the hood a t room temperature for several hours or overnight. Then, the paper is trimmed to a new size, such as 14.5 X 15 inches, and laced into a cylinder 14.5 inches tall. For thin and weak filter papers such as Whatman No. 1 or 4, it map be necessary in the second run t o trim off both punched edges from the previous run. The same procedure is followed for setting the second dimension as the first. At the end of the run the chromatogram is dried as above and then processed in the usual way. When it is desired to let a run stop by itself, the amount of solvent needed to effect the desired resolution of the compounds must be determined. This is done by putting a measured volume 100 ml.-on the dish and noting the amount of solvent-e.g., left after a trial run. The difference between the two volumes represents the amount taken u p by the filter paper. This amount plus 2 ml. is the exact volume needed to stop a chromatographic run by itself. DISCUSSION
I n Figure 2 the rate of travel is plotted against the length of wick. The solvent used was tert-amyl alcohol-2,4-lutidine (1 to 1j saturated with' water ( 1 , 2 ) and the paper was S & S 598. It can be seen that given a certain type of wick-Le., 8 double strands on a circle 2.0 cm. in diameter-the square root of time varies directly with the length. By keeping the length of wick constant the rate of travel can also be varied by increasing the number of double strands. For instance, 4 double strands placed equidistant from one another on a circle 1.0 cm. in diameter will give a slower rate than 6-double strand wicks on a 1.5-cm. diameter circle, which in turn will be slower than 8 double strands on a 2.0-cm. diameter circle, etc. The effective length of the wick
0
I
I
I
I
[
,
1
2
3
4
5
6
\/TIME
Figure 4.
TO
REACH 4 0
I 7
, 8
1 9
CM. (HOURS)
Effect of Rate of Flow of Solvent on Diameter of Spots
affects the rate more than the number of wicks. Different papers and solvents should give similar results though the values would be different. Using a fairly fast paper (S &. S 598) and tert-butyl alcoholformic acid-water (70 to 15 to 15) (1, 2 ) a mixture of arginine, alanine, valine, and leucine shows a definite though small increase of the RI values when the average solvent speed is increased from 0.68 to 4.58 cm. per hour as shown in Figure 3. With a slow solvent, such as tert-amyl alcohol-2,4-lutidine, the R, values likewise show definite but smaller increases. Kowkabany and Cassidy (6) using filter paper strips and ascending technique showed that under their experimental condition the R, values of glycine and valine are not affected by the flow rates they employed. However, it had been observed in the examination of many different filter papers ( 5 ) that the faster papers often showed higher R f values. S o w with this versatile technique such effects can be demonstrated in the same filter paper. Figure 4 shows the effect of solvent rate on the size of the spots for glycine, a-aminobutyric acid, and norleucine. The faster the speed, the larger are the spots. I n Figure 5,A and B are 2 two-dimensional chromatograms of a mixture of 18 amino acids in tert-amyl alcohol-2,4-lutidine as the first solvent and tert-butyl alcohol-formic acid as the second solvent a t two different speeds. The amino acids spots a t the slower speed are smaller and more discrete as compared with those of the higher speed. The spots in B developed a t a faster rate than those in 4 , which, although more diffuse, are still identifiable.
V O L U M E 26, NO. 2, F E B R U A R Y 1 9 5 4 I n the tert-butyl alcohol-formic acid solvent the slower speed took 24 hours whereas the faster speed took only 7 hours. Figure 5,C is a two-dimensional chromatogram of the same mixture of amino acid? in the same pair of solvents. I t Tvas developed longer to shoiv thatrmore resolution could be achieved
259 for the slower moving amino acids. The rest of the spots have gone off the paper. For comparison Figure 5 , D is included showing a two-dimensional chromatogram of the same mixture of 18 amino acids in the same solvents and paper hut obtained h r the ascending technique
u
Msth
0
z n t 3
J I
t NI
J
0
r
0 0
il
a
! l o
U c
1 - B U T Y L A L C O H O L - F O R M I C ACID
1 - B U T Y L A L C O H O L - F O R M I C ACID
(A)
(B)
Hist
WA
z
0
S A n
I2
-I-
J I
:-1
0 I 0
u J a I
3 J I.
6
4 cu I
J
0 I 0 0
J
J
> I
f
a
J
> 5 a
Cy5
1-BUTYL ALCOHOL
c -FORMIC ACID
(C) Figure 5.
W
1-BUTYL
.-. ALCOHOL-FORMIC ACID (D)
Two-Dimensional Chromatograms of Mixture of Amino Acids Using the Same Grade of Filter Papers and the Same Pair of Solvents A. B.
R u n a t a slow rate R u n a t a fast rate
D.
R u n by ascending method of Williams and Kirby (10)for comparison
C.
Developed longer t o give more resolution t o slower moving spots
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., Dent, C. E., and Harris, H., Science, 112, 621 (1950). 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 paper 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.
.
