Two Techniques in Paper Chromatography Application to Ketosteroids DAVID M. TENNENT, JOHN B. WHITL.4, AND KLAUS FLOREY Merck Institute f o r Therapeutic Research and Research Laboratories, Merck & Go., Inc., Rahway, N . J. The first technique relieves the analyst of the necessity of finding color tests for many classes of compounds to be investigated on paper strips, and the second furnishes a simple and versatile apparatus for running strips of any desired length by ascending solvent flow. A direct photometric technique is presented for locating and semiquantitatively measuring on paper strips the amount of any substance which absorbs either visible light or ultraviolet, by means of an adapter which fits into the absorption cell compartment of the Beckman spectrophotometer, Model DU. The apparatus for ascending flow development of strips consists of lengths of glass tubing with inside diameter approximately equal to the width of the strip. The top of the tube is closed with a rubber stopper and the bottom is immersed in a small volume of solvent.
the slit width and the sensitivity knob. If the instrument cannot be brought to balance, the adjustment of the focusing mirrors in the spectrophotometer should be checked. When the zero setting has been made, the paper is advanced an arbitrary amount ( 5 to 10 mm., or 1/8 to turn of the indicator shaft) by turning the proper spindle, the spectrophotometer is brought to balance with the density knob, and the reading is recorded. Another movement of the paper is then made, the spectrophotometer is brought to balance again, and the second reading is recorded. This process is continued for the length of the strip.
T
WO techniques in paper strip chromatography (5) have been developed. The first is a means of locating and measuring the amounts of the different components on chromatograms without chemical treatment, provided these components absorb either visible light or ultraviolet; and the second is a simple and convenient method for running paper strips Fith ascending solvent flow (81). Zonesondeveloped paper chromatograms have been located and measured without chemical treatment by means of Geiger counters (19, go), with radioautographic techniques (3, 6, Y), by inspection in ultraviolet light (IO, 18, I?), and by ultraviolet photography (9, 15). Direct photometry of paper strips has been used to follow the development of colored substances ( I 6 ) , and to measure amino acid spots after reaction with ninhydrin ( I , 6 , 4 , 8)
Figure 1. Adapter for Direct Photometry of Paper Strip Chromatograms
I .2
adenine
APPARATUS 1.0
In the procedure reported the zones are located and semi-
1)
quantitatively measured with an adapter, illustrated in Figure 1, which fits into the absorption cell compartment of the Beckman spectrometer, Model DU.
0.8
The apparatus, made of metal, consists of a frame which eupports two split-shaft spindles on which the paper strip is wound. In assing from one spindle to the other the paper runs behind a guiie which holds it close t o a 9 X 7 mm. u indow in the supporting frame. A third shaft has near its lower end a rubber ring which is in contact with the aper. As the strip is wound from one spindle to the other the d i n shaft turns, indicating the distance which the strip has moved. bree rotation of the spindles is prevented by springs attached t o the frame. The paper strip to be evaluated is wound on one of the spindles, with the end to which the spot was applied innermost. The other end is run behind the guide to the other spindle and. the strip is wound from the first to the second spindle until the limit of total solvent traverse has nearly reached the window. At this point the paper in front of the window has not been touched by the solvent. This position is used for the’zero setting of the spectrophotometer. The apparatus is put into the spectrophotometer with the window on the side next t o the photocell. The wave length dial is set a t the absorption maximum of the compound being investigated. The density knob is set a t zero (100% transmittance) and the selector switch a t the 0.1 position. The dark current is adjusted to null in the usual manner, the shutter is opened. and the instrument is brought t o balance by adjusting
a2
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7
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n
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0 In
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U
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i
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RF Value
Figure 2. Absorbance Curve of Mixture of Uracil, Adenine, and Thymine Developed with water-saturated butanol i n presence of ammonia (29). Measured at 260 r n p
1748
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V O L U M E 2 3 , NO. 1 2 , D E C E M B E R 1 9 5 1 When the absorbance readings are plotted against the distance along the strip, measured in turns of the indicator shaft, the RF values of the different components can be calculated in the usual manner. A semiquantitative evaluation can be made by measuring the areas under the curve. The accuracy of such determinations was studied with single substances and with mixtures. The systems used were uracil, adenine, and thymine, as illustrated in Figure 2; and corticosterone and ll-desoxycorticcsterone (DOC), as illustrated in Figure 3. The absolute accuracy was 1 2 5 % when areas of the same substance on different strips were measured, and the relative accuracy was 2 ~ 5 %when the areas of different substances on the same strips were compared.
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Compound F
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Q)
u c
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I n the case of adrenal brei samples to which desosycorticosterone or Compound S had been added (11, 1 4 ) the acetone was distilled from the filtrate in vacuo and the ketosteroids extracted from the remaining aqueous phase with ethyl acetate. This was concentrated to dryness, the residue was taken up in petroleum ether, and the sdution was shaken repeatedly with 30% methanol. The corticoids were then extracted from the aqueous methanol solution with ethyl acetate, which was concentrated to small volume for paper strip analysis.
0
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a
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/DOC
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Figure 3.
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R, Value Superimposed Absorbance Curves
Developed with water-saturated x j lene.
applied near the bottom of a strip, which was then developed by the procedure of Hotchkiss ( I S ) . One drop of concentrated ammonia was put on the upper end of the strip just before the stopper was inserted. The absorbances were measured a t 260 mp. The symmetry of the peaks indicates that this system approaches the ideal in partition chromatography, and that the separation may be based on distribution. These methods were applied to the analysis of mixtures of C B I ketosteroids. Figure 3 presents the superimposed results from individual strips on rshich pure ketosteroids xere developed with water-saturated xylene. The absorbances were measured at 245 mp. Although the zones are more diffuse than those obtained by the elegant procedure of Zaffaroni, Burton, and Keutmann (62), who used different solvent systems, xylene offers advantages in that the RF values of the components can be determined, and an overnight period of development is sufficient. The shape of these curves indicates that this system is operating b y adsorption rather than by distribution. When this procedure n-as applied to samples that contained biological material, a preliminary purification was required to remove interfering substances.
Measured a t 245 rng
ACKNOWLEDGMENT
The authors wish to express their appreciation to Michael Kniazuk and Omer Lucier for assistance in the development and construction of the adapter. They are greatly indebted to Gregory Pincus, The Worcester Foundation for Experimental Biology, Shrewsbury, Xlasa., for the corticosterone used. LITERATURE CITED
For chromatograms, Whatman XO.I paper, purchased in rolls 0.5 inch (1.25 em.) wide, is drawn through 4foot lengths of glass tubing 15 mm. in outside diameter and cut off so that the strips rxtend a few inches a t each end. One end of the paper is cut t o a point about 1.5 inches long, and the material to be analyzed is applied almut 0.5 inch above this. The strip is then drawn into the tubing until about 0.5 inch of the point is left extending out. The other end of the tubing is closed ITith a tightly fitting rubber stopper after removal of the excess paper. The tube is then placed, open end down, in the developing solvent. This may be contained in test tubes for individual samples, or in a beaker if a number of strips are being run with the same solvent. The purpose of cutting the lower end of the strip to a point is to prevent rise of t'he solvent from the reservoir along the line of contact of the paper strip with the glass. JVhen this occurs, a converging solvent front. results, causing trailing of the zones. I n this technique the glass tubing serves as a support for the strip, isolating it from other strips, and as a sealed unit which11 contains t,he solvent vapors. It has not been found necessary to use an additional solvent reservoir to maintain sat,uration inside the tube. The length of the tubing can be varied to w i t the immediate purpose. A similar technique, using test tubes, has heen reported by Rockland and Dunn (18). Small scale isolation work can he carried out by running as many as five replicat'e strips in one tube. The preparation is applied to each in t.he usual manner, they are t,hen stapled together above and below the point of application, and only the middle strip extends down into the solvent,.
(1) Block, R. .J., S c i e m e , 108, 608-9 (1018). (2) Bull, H. B., Hahn, J . W., and Baptist, V. H . , J . Am. Chem. Soc., 71, 550-3 (1949). (3) Calvin, M., and Benson, -4.-4., Science, 109, 140-2 (1949). (4) Clegg, D. L., ANAL.CHEM.,22, 48-59 (1950). (5) Consden, R., Gordon, A. H., and Martin, A . J. P., Biochem. J . , 38, 224-32 (1944). (6) Fink, R. hI., Dent, C. E., and Fink, K.. N a t u r e , 160, 801--3 (1947). (7) Fink, R. XI., and Fink, K., Science, 107, 253-4 (1948). (8) Fosdick, L. S., and Blackwell, R. Q., Ibid., 109, 314-15 (1949). (9) Goeller, J. P., and Sherry, S., Proc. Sor. Ezptl. B i d . M c d . , 74,381-2 (1950). 9, 130-1 (10) Haines, IT. J., and Drake, N. A , , Federation PPOC., (1950). (1 1) Hayano, bl., Dorfman, R. I., and Prins, D. A . , Proc. ,Sot. E.c.pt2. Bid. .Wed., 72, 700-1 (1950). (12) Holiday, E. R., and Johnson, E. A , , Nature, 163, 216-17 (1949). (13) Hotchkiss, R. D., J . B i d . C'hem., 175, 315-32 (1948). (14) hIcGinty, D. A., Smith, G. N., Wilson, M. L., and Worrel, C . P., Science, 112, 506 (195u). (15) Markham, R . , and Smith, J. D., .Vatwe, 163,250-1 (1949). (16) MCiller, R . H., and Clegg, D. L., A N . ~ L .CHEM., 21, 1123-5 (1949). (17) Pereira, A . , and Serra, J.A4., Science. 113, 387-8 (1951). (18) Rockland, L. B., and Dunn, 11.S., Ibid., 109,53940 (1949). (19) Taurog, A., Tong, W., and Chaikoff, I. L., N a t u r e , 164, 181-2 (1949). (20) Tomarelli, R. hZ.,and E'lorey, K., Science, 107,630-1 (1948). (21j Williams, R. J., and Kirby, H., Ihid., 107,481-3 (1948).
( 2 2 ) Zaffaroni, A , , Burton, R. B., and Keutmann, E. H., Ihid., APPLICATIONS
d n exaniple o f the application of t,hese techniques is presented in Figure 2. Ai mixture of uracil, :tdenine, and thymine was
111,6-8 (1950).
RECEIVED March 3, 1951. Presented a t the Rleeting-in-Jliniature
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SOCIETY. I. January 8. 1951. Xorth Jersey Section, AMERICAXC H E ~ I I C A
the
