CEfROM; 9339 Note zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA T hin-la ye r c hrom a t ogra phy ruf.TRKOVNJK, iM. KDLE$ of som e c oum a rin I. TABAKOYI~ de e iua t ile s and M. ZECEVIC - zyxwvutsrqponmlkjihgfedcbaZY Labor at or y of Or gani c Chemi st r y, Teeimological Fact& y, Uni ver si t y of Bmr ja Luk a, 78000 Banja Luk a ( Yugodavi a) (Fi r st received January2Oth, 1976; rev&d manuscriptreceived April 27th, 1976) Suitable methods for the separation and identification of 4hydroxycoumarin derivatives are desirable in view of the physiofogical activities of these compounds, and their usefulness in therapy. Paper and thin-layer chromato,gaphy (PC and TLC) have already been used in the examination of various anticoagulant dicoumarok’, of some coumarin derivatives with rodenticidalz*3, bactericidal and fungicidal activities and of several further types of coumarin derivatives of potential importancC*‘. We now report results obtained in experiments with a number of coumarin derivatives which were synthesized in our laboratory6-g. TLC on silica gel HF,, was employed and all of the compounds examined could be rapidly separated and identified. These results may be of practical advantage in preparative coumarin chemkry, enabling an efhcient follow-up of synthetic procedures. zyxwvutsrqponmlkjihgf TABLE I COLOURS OF CHROMATOGRAPHIC MARIN DERIVATIVES ON SILICA REAGE-NTS SPOTS PRODUCED BY REPRESENTATIVE COUGEL I-K& AFFER SPRAYING WITH VARIOUS For reagent composition see Experimenti section. Background colours were: (1) vy: (2) grey; (3) ly; (4) ly; (5) W; (6) yg. CoIours: w = white; br = browa; bl = blue; y = yellow; Iy = light yellow; ybl = yellowish blue; yg = yellowish green; ibr = light brown; vy = violetish yellow. R H CHO COCH3 NO -2 MpNHa& COaIz~ N=CBC=CH-CE=C I 0 Reagent I 2 3 4 5 6 W br br br lbr br bl bl Y bl bI Y bl y ly Y ly y 1~ y y bl ybl bl bl bl bl bl Y bl bl Iy 1Y 1Y W I W I& ; br ly ;; bf yg bl bl OF THE zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA SOLVENT zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC SY ST E_M S USED ON zyxwvutsrqponmlkjihgfedcbaZYXWVUT SILKi GEL HFa FOR THJ5 SEPAELAOF VdEOUS COUMkRIN DERIVATIVFS S-Y T&N KNFLUENCE OF SOLVENT COMPOSITION O;u & VAJXES FOR 3SUBSITiWTED 4ZWDROXYCOUMARLN DERIVATIVES IN TLC ON zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ SILICA GEL HF, R 1 2 3 4 5 6 7 Sofvenr system i ii ILL --- iv -CHO -CH=N-CC-NH2 -C(CHs)=NX%NHz -CH=N-NH-CO-NH2 -CH=N-NH-CS-NH2 -CH=N-NH-CO-NH-CeHs 0.25 0.35 0.50 0.32 0.14 - -C!i=N --N H -C=N 0.71 0.17 0.41 0.55 0.06 0.83 0.07 0.64 0.68 0.93 0.89 O-71 0.26 0.50 0.51 0.16 0.35 0.36 w30 0.57 0.64 0.96 zyxwvutsrqponmlkjihgfedcbaZYXWV 0.93 0.98 0.08 0.42 0.72 0.89 0.98 II S vi v co 'CH't 8 9 LO 11 12 13 -CO-CH, -CO-CH&X%CICzH5 -CO-CH=CH_csHs -CO-CH=CH-C&.-CH~p -N=CH-C+Hs -N=CH+H,-NO,-p 0.91 0.68 0.78 0.92 0.04 0.05 0.87 0.50 0.81 0.87 0.06 0.09 O-o”6 0.47 0.75 0.84 - 0.85 0.75 0.86 0.97 0.88 0.84 0.85 o-i4 0.96 0.98 0.82 0.80 0.93 0.92 0.88 14 --N=cn 0.75 0.12 - 0.65 0.66 0.76 EXPERIMENTAL The vohme of each sample appikd ~2s IQp!; the concentr2tion of each compound was 0.1% (dissolved in ethanol or a c e t ic acid), Merck Type 60 cokercial pl2tes (ZOO x 200 mm) coated with a 5G&m layer of ii.@=_ gel EfFw (Stahl) were used for TLC. Cbrozxatograms were develdped i;r a solvent-vapour-satuated atmasphere at 20”; the relative air humidity was 55-&I”/& Chkomatogq.@ic spots anti vimaGed either by_W iliumin2tion at 360 and 254pm, respectively, or by spraylqg - & \ N / zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA N zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0.72 0.60 0.37 0.31 0.50 - 0.11 0.07 O-08 0.39 0.81 0.75 0.62 0.55 0.77 =f% 0 CGH c (N C& -O.P CH 3 with the following reagents: (1) I o/0 KMnO, in $1 N l&SC?,;- (2) Q.7 0AAg_NO, in a&one containing 6.5 7: of water and 1 “i, ofmethyl cellosolve; (3) a mixture of equal volumes of 1% K,Fe(CN& in- 50% ethanol (a) and 1% FeCl, in50% ethanol (b); (4) 1 ok aqueous FeCI, containing 0.5% of I&O,; (5) 5 ‘A zyxwvutsrqponmlkjihgfedcbaZYX me&anoIic KOH; (6) a mixture of one volume of 3a and two volumes of 3b. zyxwvutsrqponmlkjihgfedcbaZYXW RESULTS AND DISCUSSION Table I shows results obtained when reagents i-6 (ref. IO) were used to visualize the spots formed by representative compounds separated by use of the solvent system benzene-acetic acid-acetone (17 : 1:2). Each compound could also be located by means of its fluorescence on irradiation with light from a WI lamp. Using reascents l-6 for visualization, differentiy coloured spots were obtained with various &hydroxycoumarin derivatives.FeSu~tingeither from COmplexform~tion(Feagents 3, 4 and 6), or from salt formation (reagents 2 and 5). In experiments with other solvent systems (summarized in Table II), various newiy synthesized coumarin derivatives were separated on O-5-mm plates of silica gel I-IF,, giving the Rf values shown in Tables III and IV. The RF values presented are the averages from three experiments. REFERENCES 1 2 3 4 5 6 7 8 9 IO S. RamiC, J. GrujibVasit and M. Trkovnik. Gh. Nenz. Tehzoi. BosneKerc&govbre,15 (1967) 105. J. L. Ramant and A. Benoit, J. Pharm. Beig., 21 (1966)293. H. A. Rues&, Z. Anal. Chem., 250 (1970) 125. C. F. van Sumere, G. Wolf, H. Teuchy and J. Kint, J. Chronmogr., 20 (1965) 48. P. Daenens and M. Van Boven, J. Chramatogr., 57 (1971) 319. M. Trkovnik, M. La&n, Z. StuniCand D. Nahmijas, Urg. Prep. Pruced. int., 7 (1975) 47. M. Trkovnik, M:Kuld, M. La&n and B. BobareviC, Z. Naturfursch. B, 29 (1974) 580. M. Trkohk, M. KuIeS and K. KruniC. Org. Prep. Proced. Int., 7 (1975)26. M. Trkovnik, M. Kekie, M. KuleS and A. Gorenc, Z. iVarmforsch. B, 30 (1975) 237_ E. S’Cihl,Di7nuschicht-Chrontatagraphie, 2nd cd., Springer, Berlin, Heidelberg, New York, 1967, p. 813.