Two Techniques for Paper Chromatography DONALD I. FRENCH and MELVIN R. GIBSON School o f Pharmacy, State College o f Washington, Pullman, Wash.
b Two techniques, which involve spotting crude extracts on paper strips and eluting separated alkaloids, provide accurate and simple methods for quantitative separation and preparation for alkaloidal assay of small quantities of solanaceous drugs. U M E R O U ~ workers
have reported satisfactory separation of solanaceous alkaloids by paper chroniatographic techniques, but have been concerned primarily with separations from solutions containing known alkaloids. The failure of paper chromatographic techniques to be adapted to quantitative procedures is often due to unsatisfactory separation of alkaloids from the paper. A method by which small amounts of crude drug can be extracted, the alkaloids of the extract separated on paper and eluted, and the eluates quantitatively assayed is described here. S o satisfactory way has been demonstrated for placing the large volume of plant extract on the paper prior to development. Brindle, Corless, and Koodhead (9)attempted to concentrate the extract by evaporation to a volume of v-orkable size, and Drey and Foster (4) used aliquot portions of a known volume. However, as these procedures introduce additional sources of error, a method was needed in which all intermediate steps between extraction of the crude drug and inoculation of the paper strip are eliminated. The process devised involves direct application of the extract to the paper strip.
the extractor and the two air outlets, as shown in Figure 1. These outlets are connected to a compressed air source. The stopcock of the extractor is adjusted to allow the extract from the solvent passing through the drug to drip on the paper a t a rate adjusted t o allow the paper t o dry between drops. I n this manner the entire extract is placed on a small spot of the paper strip. At least two extractions are run in this manner at one time. The paper strips are then developed in standard fashion with an appropriate solvent. Following development, the position of the separated alkaloids is determined by a control strip to which a color reagent has been added. The desired sections of the chromatograms are cut out and placed in the elution apparatus shown in Figure 2.
The sections to be eluted are placed between the plates of the paper holders, as shown in detail in Figure 3. These plates are held together with a small rubber band. The holders are
Figure 2.
ANALYTICAL CHEMISTRY
Elution apparatus
Bell jar Ring stand Solvent reservoir D. Clamp E. Capillary tube enclosed in rubber tubing F . Paper holder G. Evaporating dish H . Glass plate -1.
B. C.
Moore and Roylen (6) and Gregory (5) devised simple techniques for making transfers in paper chromatography. The theory of these techniques n-as adapted by the authors, and the extraction apparatus shown in Figure 1 was constructed.
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ELUTION
EXTRACTION A N D SPOTTING
A pledget of cotton is placed in the neck of the extractor and a small amount of crude drug is placed on the cotton (Figure 1). The extractor is attached to the solvent reservoir, R-hich, in turn, is filled with the extracting solvent. One end of a strip of chromatographic paper is placed between two glass plates with 1-inch centered holes. The plates (held together with rubber bands), with the paper secured between them, are placed horizontally below
attached t o the elution apparatus by inserting them into the rubber tubing which contains the capillary tube as shown in Figure 2. The solvent reservoirs are filled with eluting solvent, and the entire apparatus is covered with a bell jar to reduce evaporation. To obtain constant sloiv passage of the eluting solvent through the paper segment, the capillary tube a t E (Figure 2) is made from a 2-cm. section of thermometer. The eluate is collected in a small evaporating dish and, following the completion of elution, is evaporated to dryness under a stream of air. The residue is then ready to be assayed.
APPLICATION OF TECHNIQUES
Figure 1. Extraction apparatus
A. Inverted 50-cc. beaker B. Solvent reservoir C. Rubber stopper D. Extractor E . Crude drug powder F. Pledget of cotton G . Stopcock H. Air outlet plates with I . Glass centered 1-inch holes J. Chromatograph paper
This procedure was used for determining hyoscyamine and hyoscine in small quantities of Datura tatula L. root culture powder.
A small quantity (25 mg.) of the powder was weighed directly into the extractor and moistened with a 1 to 1 solution of 10% (w./v.) ammonia and 95% (w./v.) ethyl alcohol. The moistened drug was allowed to stand for 1 hour and \vas then extracted with 25
of chloroform. The extract was placed on a strip of Whatman KO. 1 c*hromatograph paper as described above. The extract-inoculated strips were developed for 10 hours with a sol1.ent composed of 15 ml. of concentrated hydrochloric acid and 100 ml. of 1-butanol saturated with distilled water. This solvent is similar to that proposed by Munier (7, 8). The position of the separated alkaloids was determined by comparing the strips to a control strip treated with Dragendorff’s reagent (1). The alkaloid sections (3 to 4 em.) of the chromatograms were cut out and placed in the elution apparatus. Each section was eluted for approximately 3.5 hours with 30 ml. of a solvent composed of 3% (w./v.) hydrochloric acid in 10% (v./v.) ethyl alcohol. Then the exhausted paper segments were removed, air-dried, and tested for complete removal of alkaloids by DragendoriT’s reagent. Elution was complete in all tests performed. The eluates were evaporated to dryness under a stream of air and assayed spectrophotometrically by the T’itali-Norin
LITERATURE CITED
1111.
(1) Block, R. J., LeStrange, R., Zweig, G., “Paper Chromatography,
a Laboratory hianual,” p. 136, Academic Press, Sew York, 1952. ( 2 ) Brindle, H., Corless, J. E., Woodhead, H. B., J . Pharni. and Pharniacol. 3,793 (1951). (3) Colby, A. R., Beal, J. S.,J . A m . Pharin. Assoc., Sci. Ed. 41, 351 (1952). (4) Drey, 12. E. h.,Foster, G. E., J . Pharni. and Ph,arrnacol. 5 , 839 (1953). (5) Grpgory, G. F., Science 121, lti9 Figure 3. Paper holder (detail of Figure 2, F)
reaction as reported by Colby and Beal (3). The elution apparatus was found to be 99.6 =k 0.4% efficient in eluting known concentrations (ranging from 0.05 to 0.2 irg.) of hyoscyamine.
(1955). ( 6 ) Moore, A. hl., Boylen, J. B., Zbid., 118, 19 (1953). ( 7 ) Munier, R., Bull. SOC. chim. biol. 33, 857 (1951). ( 8 ) Zbid., p. 862.
RECEIVED for review October 22, 1!)56. ilccepted February 5,1957. Investigation supported in part by funds provided for biological and medical research by the State of IVashington Initiative Measure NO. 171.
Determination of Phenolic Glycosides and Aglycones on Paper Chromatograms JOHN B. PRIDHAM’ The Institute o f Paper Chemistry, Appleton, Wis
b The method was developed in order that small amounts of phenols and phenolic glycosides, present in enzymic digests, could b e accurately determined. The technique has been used for the determination of catechol and arbutin over a range of 0 to 100 y , and saligenin from 0 to 70 y. The errors in all cases were within *4%. (p-Hydroxyphenyl)-P-gentiobioside has also been determined in enzymic digests after separation of the components of the digests by paper partition chromatography. Quantitative analysis of mixtures of phenols and phenolic glycosides may b e rapidly carried out by the simple procedure described. Only a few microliters of the solutions to b e analyzed are required, and application of these solutions to the paper as single spots ensures good chromatographic separations of the components. The completeness of separations may be readily observed in view of the fact that the colorimetric reactions are carried out on the chromatograms.
T
methods available for the determination of phenols and their derivatives have recently been reviewed by Clarke and Nord ( 5 ) ,Trim (IS), and Bray and Thorpe ( 3 ) . The analytical principles involved are many and varied, but the majority of techniques have been devisrd for specific phenolic compounds and little attention has been paid to the quantitative separation and determination of mixtures of phenols. One of the few exceptions, however, is Stone and Blundell’s method, where the phenolic aldehydes obtained by alkaline nitrobenzene oxidation of lignin are separated by paper partition chromatography and then determined spectrophotometrically after elution from the paper with ethyl alcohol (9). This method has recently been utilized for the determination of phenolic carboxylic acids ( I ) . A good chromatographic spray reagent for phenols is diazotized p-nitroaniline ( d ) , which has also been used for the determination of p-hydroxybenzoic acid and p-hydroxyberizaniitle in the ether extracts of urine ( 2 ) . HE
In the procedure described herein the author has utilized this reagent, in conjunction with paper partition chromatography, to determine phenolic coinpounds in mixtures. The technique used is similar t o that described for determination of sugars with p-anisidine hydrochloride (6). The solution containing the phenols is applied to the chromatographic paper ns small discrete spots, and, after development with a suitable solvent, the rhroniatogram is dried and sprayed with n solution of diazotized p-nitroaniline buffered with sodium acetate. The excess moisture is then allowed to evaporate, the spots are cut from the chromnbogram, and the color is eluted from the paper with a solution of potassium hydroxide in aqueous methanol. Finall>-3 the absorbance of the resulting solution is nieaaured in a spectrophotometer. The intensity of color bears a linear rcllationship t o the weight of phenolic coniPresent address, Chemistry Dcpartnieiit, West Mains Road, The I-niversity, Edinburgh 9, Scotland. VOL 2 9 .
NO. 8. AUGUST 1957
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