Circular Paper Chromatography Studies of Physical Factors That May Injiuence
Rf Values
.4BR.4EUM SAIFER AND IRWIN ORESICES Biochemistry Department, Division of Laboratories, Jewish Sanitarium and Hospitalfor Chronic Diseases, Brooklyn 3, N.Y . Circular paper chromatography has many advantages for use in the small laboratory, due to the simplicity of apparatus and techniques employed. The present paper deals with a systematic study of the physical factors which influence Rj for a simple amino acid system. Aside from pH and quality of paper used, which w-erekept constant, factors that influence R j significantly are temperature and length of exposed wick. Rate of solvent flow is best controlled by varying wick width. Application of this simple technique to qualitative and quantitative analysis of complex amino acid mixtures--e.p., protein hydrolyzates-is now under investigation.
R
UTTER (27, 28) has described a modified chromatographic technique employing circular filter paper in which components separate out in concentric rings instead of the usual spots. €€e lists the following as some of the advantages of his method: speed and sharpness of separations, simplicity and compactness of apparatus, reproducibility, removal of test samples during (or after) development, and control of rate of solvent flow. However, he does not give experimental data--e.g., R, values-for the circular chromatographic system. lfuller and Clegg (20-23) employed Rutter’s technique to study the kinetics of paper chromatogram development and the phenomenon involved in chromatographic separations in paper by semiautomatic methods. They found the circular technique both simple and extremely useful for their purpose. 4 series of papers dealing with the circular chromatographic technique and its application to the separation of amino arid mixtures has been published recently bv Giri and coworkers (6-10). These authors made a number of interesting innovations in Itutter’s original technique. They used a filtei paper n irk inserted in a slit and alternated unknown and known amino acids in n circle 4 cm. from the center (10). Using butanol-acetic acida a t e r as the developing solvent they w r e able to determine the flee amino acid composition of papala Iates (10) and of hydrolyzed casein (?). They also emplojetl an elution technique for the quantitative spectrophotometric determination of the amino wid-ninhydrin color ( 8 ) and in a recent publication (9) discuss some factors influencing the quantitative determination of amino atricls separated by circular paper chromatography. However, thcii work does not include a systematic study of the various p h j s i ~ dfactors which influence the circular chromatographic method. Itosebeek (26) has developed another techniqueof circular paper chromatography in which he employe a filter paper cone immersed in the elutant and just touching the center of the horizontally supported filter paper. While the components separate in bands with this system, the lack of an adjustable wick makes it impossible to control the rate of flow of the developing solvent. llarchal and Mittwer (17, 18) have described a modified circular chromatographic technique employing a paper wick which dips into the developing solvent and is attached to an enlarged section of filter paper suspended vertically. The substance being resolved-e.g., amino acids-spreads out vertically in a series of semicircular arcs. While this method appears to have no decided advantages over the circular horizontal method, these authors claim that their method can resolve into separate concentric arcs a mixture of hydroxyproline, arginine, and histidine which gave one large spot on a two-dimensional sheet chromatogram. If the circular paper chromatographic method is to compete surcessfully u-ith existing methods, a careful study of its reproduci-
bility and of the various physical factors that may influence the R f values is required. For this purpose the authors have chosen t o study, n-ith a modified Rutter technique, the chromatographic separation of a simple amino acid mixture composed of the three neutral amino acids, glycine, alanine, and valine, with watersaturated phenol as the developing solvent in a closed system, Factors which have been studied include effect of the presence of the inverse phase, of saturation of paper with solvent vapor, of variation of length and width of the paper wick, and of variation of temperature, time, concentration, and aging of the solvent. Experimental results substantiate the statements as t o the simplicity, reproducibility, and general usefulness of the method made by Rutter (27, 28). It is the authors’ conclusion that the Rutter method, when used in a closed system, has many advantages as a paper chromatographic procedure, particularly for the small laboratory where such techniques are used only occasionally. REAGENTS AND APPARATUS
Filter Paper. Whatman S o . 1, 24 cm. in diameter. Untreated filter paper was used in all these runs. Developing Solvent. The solvent used in all these runs was made up of reagent grade phenol (70%), reagent isopropyl alcohol (5%), and distilled water (25%) by weight as recommended by Toennies and Kolb (29). All reagents \\-ere used as purchased without further purification. This solution was kept in an amber bottle and stored in a refrigerator when not in use. Amino Acid Solutions. o,L-Glycine, D,balanine, and D,L valine (Merck, 0.5 millimole each) were dissolved in 100 ml. of 10% isopropyl alcohol solution. Each of these amino acids was also prepared separately in the same concentration ( 5 millimoles per liter) in the same solvent. Ninhydrin Color Reagent. Ninhydrin (0.25 gram), reagent grade (Dougherty Chemical Co., Jamaica, X . Y.), was dissolved in 100 ml. of anhydrous acetone reagent. This reagent waa applied by dipping (instead of spraying) the chromatograms as suggested by Toennies and Kolb (29). This solution was kept in an amber bottle and stored in a refrigerator when not in use. Micropipets. Pipets, calibrated for 20 cu. mm. (usually employed for hemoglobin determination) were used to apply the amino acid solutions to the paper. A Gilmont ultramicroburet, lOO-cu.-mm. capacity, was also used to apply the amino acid solutions when a specified amount-e.g., 10 micrograms-was desired. Culture Dishes and Covers. Bottom dish (185 mm. in outside diameter and 70 mm. high), cover (190 mm. in inside diameter and 65 nim. high). These dishes are readily available for most laboratory supply companies. Petri Dishes with Covers. Bottom dish (90 mm. in outside diameter and 15 mm. high), cover (100 mm. in inside diameter and 10 mm. high) or 25-ml. beakers. Infrared heating lamp. Small electric fan. METHOD
The exact center of a circular sheet of Whatman No. 1 filter paper, 24 cm. in diameter, was found geometrically. This was
1539
ANALYTICAL CHEMISTRY
1540 then used as the master sheet to mark the center of all the other pieces of paper used in a run. A radius was drawn lightly in pencil from the center of the paper to the edge. A rectangular wick (4 X 50 mm.) was marked off along this radius. The heating lamp and fan were turned on, so that the temperature a t the surface of the paper was between 50' and 60' C. A 20-cumm. aliquot of the amino acid solution was drawn up in the micropipet with the aid of a piece of rubber tubing, and was then applied t o the paper in such amounts that the diameter of the wet spot was approximately 10 mm. This means that three to four applications were required for each pipetful until the entire 20 cu. mm. had been added. After the spot had dried, the wick was cut out carefully with a sharp razor blade and the wick was bent so that the crease went through the center point. The chromatogram WAS then ready to be placed in the culture dish for development. FILTER PAPER
24 CM DIAMETER
hours afterward. The colors began to fade after a few dags, but the chromatogram still remained usable for makiDu Rt measurements for several weeks after having been dipped. DETERMINATION OF Rf VALUES
Three diameters were drawn, so that the circle was divided into approximately 60" angles. On each radius the center of each colored band was marked off carefully and the R, calculated along each radius as follows :
R/ =
distance (in mm.) of center of circle to center of each band distance (in mm.) of center of circle to solvent front
The mean R f for each component is actually obtained by calculating the average of a t leafit six different measurements, one along each radius.
INITIAL SPOT
--
I-
111
RESULTS
I
I
II
Figure 1. Chromatographic Setup for Circular Chromatography Whatman No. 1 filter paper enclosed in culture dish
About 35 to 40 ml. of the phenol developing solvent were placed in the bottom half of a small Petri dish or in a small beaker. The paper chromatogram was then centered on the top of the culture dish with the paper wick suspended in the center of the developing solution. The level of the solvent was adjusted so that exactly 1 cm. of the wick was below the surface and the distance between the liquid surface and the horizontal plane of the paper was exactly 4 cm. This can best be done by placing a pencil mark 1 cm. from the edge of the wick and making certain that the wick is bent exactly perpendicular to the plane of the filter paper. The cover was then pressed down evenly over the overlapping paper, so as to form a tight seal of the entire system, which is illustrated in Figure 1. The dish was allowed to stand a t room temperature (24" to 28" in an air-conditioned room) or preferably in a constant temperature cabinet a t 25' for 24 hours. In this period the solvent front usually moves a radial distance of about 80 mm.
The following studies were undertaken in order to investigate the various physical factors which may influence the reprotlucibility and accuracy of circular paper chromatography. Reproducibility of Results (Ry Values). Twenty cubir millimeters of the amino acid mixture (glycine, alanine, and valine) were applied to each filter paper circle in quadruplicate and chromatogramed, and the colors were developed exactly as described above, This run was repeated a t approximately weekly intervals for a total of 32 individual chromatograms. hlthough fresh solvent was used for each chromatogram, several different batches of solvent were employed in the course of these erperiments. Similar runs were performed for each of the three amino acids separately, employing the same technique, in order to determine whether the R, values obtained for the individual amino acids were different from those obtained when all three were run together. +4statistical summary of the results obtained in thefie runs is given in Table I, together with a typical set of results used to calculate a single R/ value for each amino acid. Effect of Presence of Inverse Phase on R/ Values. The literature states that the presence of the "inverse" phase--i.e.,
I
L
5
/
GLYCINE
moB 40b
ALANINE
B
O
VALINE
50 PO
2
3' GLYCINE
I
i
1 a
56
62
The top cover wa8 removed together with the chromatogram. Care should be taken that the liquid adhering to the wick does not splash on to the filter paper. The solvent front was sketched in lightly and accurately with a sharp-pointed lead pencil. The chromato ram was removed and dried in front of a fan for 3 to 4 hours or %y standing overnight in a well ventilated room. A small amount of ninhydrin solution in acetone was poured into a shallow enamel tray and the chromatogram was dipped in the solution in such a manner as t o wet the encircled area thoroughly. The paper was allowed to dry in air and was usually dry within 5 t o 10 minutes. The colored rings began to appear in about 0.5 hour but the colors continued to intensify for several
76
B
86
1 b
RADIUS OF SOLVENT FRONT-MM
Figure 3.
Table I. Amino Acids Glycine5 Alsninea Valine" G1y cin e Alanine Valine
98
Effect of Width of Wick on R/ Values
TIME OF STANDING IN PRESENCE OF SOLVENT-HRS
Figure 2. Effect of Saturation of Paper with Solvent Prior to Running Chromatogram on RJ Values
i
80
--- -- -- - - - WIDTH - _ - _OF _WICK-KM-
Reproducibility of R, Values No. of
y Detns. Range of Values Mesn 32 0.46-0.53 0.49 7.5 32 0.58-0.62 0.60 8.9 0.76 11.7 32 0.74-0.77 16 0.45-0.49 0.48 7.5 16 0.59-0.62 0.61 8.9 0.78 15 0.76-0.79 11.7 Typical Set of Results for Calculation of R / Values R/ Values (Measured along Radii)
Glycinea 7.5 0.51,0.47,0.50,0.49,0.51,0.51 Alenine" 8.9 0.61 0.56 0.59 0 . 6 2 0.59 0.61 Valine5 11.7 0.78: 0.73: 0.75: 0.76: 0.75: 0.76 a All three amino acids chromatogramed together
Standard Devistion 10.023 10.014 10.017 iO.011 f0.007 rtO.009
R / Av
Calcd:' 0.50
0.60 0.76
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3 Effect of Presence of Inverse Phase on RJ Values
Table 11.
_ _ - ~ _ _R I Values .4nlino
Acidsa Glycine
10
.4lanine
10
Valine
10
a
y
One-phaxe (phenol saturated with water)
Two-phase (phenol saturated with water plus water saturated with phenol!
0 . 4 6 , 0 . 4 6 , 0 48. 0 . 4 7 , 0.47, 0.48 Av. 0.47 0 60, 0 . 6 0 , 0 . 6 3 , 0 . 6 2 . 0.61, 0.63 A v . 0 . 62 0 75, 0 . 7 6 . 0 . 7 7 , 0 76. 0 7 7 , o 79 .4v. 0 . 7 7
0 . 4 3 , 0 . 4 3 , 0 . 4 7 , 0 47, 0 45, 0 . 4 8 0.46 0 3 5 , 0 . 5 4 , 0 GO, 0 . 5 9 , 0.58.0.62 0 58 0 72, 0 . 7 0 . 0 . 7 3 . 0 . 7 5 , 0 7.5 0.73
A11 three nniino acid.; chro:narr~grsiiiedtogether.
__--
--
'or-
__-____-___
LENGTH OF EXPOSED WICK-CM 55 15 20 25 TIME OF DEVELOPMENT -HRS
Figure 4. Effect of Distance from Developing Surface to Paper on Rf Values W h e n solvent front travels a fixed radial distance of 80 mm.
water saturated with phenol -is required for reproducible results and compact spots ( 4 , 16, SO). In order to determine the efrcct of the presence of this v-ater-rich phase in the enclosed system on the R/ values, about 30 ml. of water saturated with phenol was placed in the bottom of each culture dish prior to running the chromatograms. The procedure, except for the addition of the mter-rich phase, was performed exactly as described above. The results obtained in these runs are given in Table 11. As a comparison there are inrluded runs performed under identical conditions in which only the single phase--i.e., phcnol saturated 4 ith water-was present in the system.
1541
prior to performing the chromatogram is essential for good results ( 1 , I I ) . To check the effect of this factor on the R, values, the filter paper was placed within the enclosed chromatographic chamber, prepared in the usual manner, except that the wick was not inserted in the liquid until after the time interval noted in Figure 2. After the stated time interval had elapsed, the wick wa8 inserted in the developing solvent in the prescribed manner and the chromatogram run exactly as described above. The results obtained in duplicate runs for the three amino acids chromatogramed together are shown in Figure 2. Effect of Width of Wick on R, Values. I n these runs the width of the wick was varied from 2 to 8 mm., the length of the rectangular wick being kept constant at 50 mm., 10 mm. of which is immersed in the developing solvent. Each wick size was run in triplicate for the combined three amino acids and the chromatograms were performed exactly as previously described. The average R, values obtained in these runs for the three amino acids and the average radius of the solvent front for each wick width are given in Figure 3. Effect of Distance from Surface of Developing Solvent to Plane of Paper on R / Values. The mixture of the three amino acids was chromatogramed in the prescribed manner except for the variation in the length of the wick, the width being kept constant at 4 mm. For each wick length shown in Figure 4, 1 em. was added and the level of the developing solvent so adjusted that exactly 1 cm. of the wick lay beneath the surface of the liquid. The results obtained in these runs, performed in quadruplicate, are shown in Figure 4. Effect of Variation of Temperature on R , Values. The relatively small dimensions of the culture dishes and their flat surfaces permit them t o be conveniently stacked in refrigerators or constant-temperature cabinets (27, 28). After the chromatograms of the three standard amino acids had been set up in the usual manner, the culture dishes were placed in either a refrigerator or constant-temperature cabinet a t i o 2,4 O , and 37" C. for 24 hours. A4tleast eight separate determinations were performed a t each temperature. The results obtained in these runs are shown in Figure 5 ,
Figure 6. Effect of Variation of Time of Development on Rj Values
70-
-
'ALANINE
v)
3 EO z U
50
40!J
Ib
I5
I
I
20
25
30
35
a0
- C' Effect of Temperature Variation on RJ Values TEMPERATURE
Figure 5.
Effect of Saturation of Paper with Solvent Vapor Prior to Chromatograming. A number of investigators have held that saturation of the paper with the vapors of the developing solvent
Effect of Variation of Time of Running Chromatogram on R , Values. The mixture of the three amino acids (2O-cu.-mm. volumc) was chromatogramed exactly as described under Methods except that the filter paper was removed, dried, and developed at the time interval stated in Figure 6. Four separate determinations were run for each of the five time intervals. The results obtained for these runs and the average Rj values for the three amino acids at the various time intervals are given in Figure 6. Effect of Variation of Amino Acid Concentrations on R, Values. I n order t o determine the effect of the variation of concentration of the three amino acids on their R, values, two sets of experiments were performed. In the first set the concentrations of all three amino acids were varied simultaneously from 10 to 2 5 7 as shown in Figure 7 . I n another set, the concentration of each amino acid was varied separately over the same range, the
ANALYTICAL CHEMISTRY
1542 others being kept constant a t 107. The various amounts of the individual amino acids were added with a microburet. The results obtained in these runs are shown in Figure 8. Effect of Use of Fresh us. Stored Phenol on Rl Values. Because the use of freshly prepared or redistilled phenol is continuously being recommended in the literature ( 5 ) ,it was decided to investigate this factor for circular paper chromatography, as it would be advantageous not t o have to prepare this reagent for each day’s run. A batch of phenol solution was prepared exactly as described under Reagents and stored in a refrigerator. Both the stored phenol, and a phenol solution freshly prepared on the date the determinations were performed, were used as the developing solvents to run the chromatograms simultaneously over a period of several months. Although the data obtained in these runs are not included in this paper, it can be stated that no significant differences in Rj values were obtained for the amino acid mixture, whether “stored” or freshly prepared solvent was used. DISCUSSION O F RESULTS
The original method of Rutter (47) was modified for the ultimate purpose of separating and identifying the larger number of amino acids present in protein hydrolyzates. These modifications consisted of the use of filter paper of larger diameter (24 cm.), a culture dish and tightly fitting cover to provide a sealed system, and the cutting of the wick without removing a complete segment of the circular filter paper.
VALINE
70
p 0 5
401 0
GLYCINE
I
I
I
1
5 IO I5 20 AMINO ACID CONCENTRATION J l G
25
Figure 7 . Effect of Variation of Amino Acid Concentration on Rj Values When concentrations of all three amino acids are varied simultaneously
Increased diameter of the filter paper circles permitted a greater distance of travel of the solvent front with corresponding better resolution of the individual amino acids and increased accuracy of Rj measurements. The use of culture dishes as a sealed system also resulted in increased reproducibility of Rl values by preventing evaporation of the developing solvent. Such dishes can easily be cleaned, transported, and stacked so that they can be placed in already available refrigerators or constant-temperature cabinets. Cutting of the wick without removing a segment of the paper permits the edges of these dishes to provide horizontal support for the filter paper circles without sagging or requiring any other supporting device. The tolerance between the top and bottom halves of these culture dishes are usually sufficiently close so that the thickness of the filter paper is all that is required to make an air-tight sealed system for running the chromatograms. In the original runs adhesive tape or Parafilm was used t o seal the system, but no appreciable difference was found in results when these were omitted from the technique. Preliminary studi:s with various grades of filter paper indicated that Whatman Nos. 1 and 4 and Stancien No. 333 are all suitable for amino acid separations. Whatman No, 1 was chosen mainly because it gave very satisfactory results and is the paper most widely used for chromatographic analysis. The data in Table I show that this modified Rutter method gives a high order of reproducibility of R/ values both for the three
individual amino acids and also when they are chromatogramrd as a group--e.g., S.D. = =k 0.02 Rf unit. One reason for the increased precision of the method is the means by which the R f for a component is calculated. As was shown experimentally by Muller and Clegg (%2), filter paper has a preferred orientation which causes the solvent to flow a t different rates along the paper. Therefore, the spots formed in paper sheet chromatography are seldom circular, or compact, making the matter of choice of the center a rather arbitrary one and the Rj calculation dependent on a single measurement. In contrast, circular chromatography permits a large number of measurements to be made from the initial spot to the circumference of the circle. This measurement is made to the center of the circular band rather than to either edge, as there is some increase in band width with increasing concentration. The R f values reported in this paper are therefore the mean of six different measurements, as is illustrated in Table I. The results in this table also show that there is often greater variability in the R, values obtained along different radii on a single chromatogram than between duplicate circular chromatograms run under identical conditions. This technique, therefore, serves to eliminate any need for pretreatment of paper as recommended by some authors (24). A number of other investigators, besides those already tited (SO-SS), have made careful studies of some of the physical factors that influence paper chromatography. Bate-Smith (1) has studied the conditions necessary to obtain accurate Rj values. Similar detailed studies for paper strip chromatography were made by Kowkabany and Cassidy (14). LIcFarren (f5,1 6 ) and others (11, 15’) have shown the importance of pH as a factor in the paper chromatography of amino acids. In the authors’ studies reagents and developing solutions were maintained at constant pH’s throughout these runs. The three neutral amino acids-glycine, alanine, and valinewith widely different RI values were selected for this study. As these could be determined by the modified Rutter technique with considerable accuracy, the changes in RJ values, if any, produced by variation of the physical factors, could then be readily determined. Several investigators (4, 15, 30) have stated that the presence of the “inverse” phase-i.e., water saturated with phenol-is necessary for reproducible results, particularly where sheets are contained in large enclosed chambers. Others ( 2 ) employing smaller enclosed containers have not found the presence of this “inverse” phase necessary for good results. The authors results shon- that, for the culture dishes used in the circular chromatographic system, the presence of the inverse phase causes an increased rate of travel of the solvent front, which results in diffusion and irregularity of the circular bands. This phenomenon is reflected in the greater variability of the R f values obtained in the two-phase system as compared to the one-phase system, although the average values are in fair agreement. The presence of the inverse phase in a small enclosed system, therefore, serves to complicate the procedure and gives poorer results than n-hen the developing solvent is used alone. A number of investigators (1, 12) have recommended that the paper be equilibrated with the atmosphere in the chamber prior to developing the chromatogram. While this may be important where paper sheets are enclosed in large chambers, the necessity for performing this step for smaller vessels has been questioned by Block (2). The authors’ results, as given in Figure 2, indicate a drop in R, with an increase in the time during R-hich the paper remained in contact with the vapor in the enclosed system. For the most part these differences are small, being within *0.04 Rj unit or twice the standard deviation. While the distance which the solvent travels remains about the same, there is a tendency for more diffuse bands with greater time of saturation. It is a t least evident that presaturation introduces an additional time-consuming step which adds nothing to the accuracy of the procedure and is therefore not desirable.
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3 I
