Brother Joseph M. Walrh College of Santa Fe Santa Fe, New Mexico 87501
I
The Use of Talc as a TLC Adsorbent
A n investigation of certain flavonoids of the ornamental shrub, Poneinis trifoliata (Rafinesque), required a relatively inexpensive TLC adsorbent to be used in preparative thin-layer chromatography procedures. Talc was found to be quite suitable not only for the purification of samples but also for analytical or identification purposes. A search of thc literature using C A from 1948 through July, 1966, revealed that, whereas talc bad been used as a filtering agent and adsorbent for pharmaceuticals in column chromatography, very little had been reported on its usc as an adsorbent in TLC. An investigation of talc for column chromatography with certain flavonoids in 1948 showed that talc had no adsorptive capacity for quercetin, quercitrin, rhamnetin, xanthorhamnetin, homoeriodictyol, D-catechol, rutin, and naringin (I). I n 1951 talc was used by J. G. Iiirchner and his associates in one of the earliest applications of TLC in the investigation of essential oils from citrus fruits. Talc was reported as having good physical characteristics but slight resolution of tcrpenes (3). The recent texts have little to say about talc. Neither E. Stahl (3) nor E. V. Truter (4) mention it when discussing the different adsorbents for TLC, and J. M. Bnbbitt (5) has only a passing reference to the work of Kirchner and his associates previously mentioned. As will be shown later, talc has excellent physical characteristics for TLC applications, is easily applied, is inert to solvent systems in general, and is quite good for preparative work as well as showing good separatory capabilities with selected groups of compounds. In addition to its usefulness its economical price suggested this article. Talc costs about 50$ a pound (enough for fifty 8 X 8 in. plates) whereas most of the commercial TLC adsorbents range around $10.00 per pound and at least one variety sells for $19.00 per 100 g. The following applications are intended to be illustrative rather than exhaustive. Preparation and Characteristics of the Talc Plater
The talc used in the experiments reported in this paper was obtained from Matheson, Coleman, and Bell (#CB853, TX5, USP powder). The supplier stated that it had been acid-treated, water-levigated, and ground so that 98.5% was less than 77p (200 mesh), 85% less than 2 0 ~and , 55% less than lop. I t was used as such without further treatment. Slurries of talc with water produced plates with grainy surfaces that were rather hard, took a long time to dry, and gave streaky results. With methanol, ethanol, propanol, isopropanol, and butanol, the surface was much smoother and the development of the compounds spotted usually resulted in more compact spots. 294 / Journal of Chemical Education
Methanol was selected as the slurrying liquid, about S 3 . 5 ml of the alcohol usually being sufficient for 1g of talc. The slurrying was done by shaking talc and methanol in a glass-stoppered bottle for about one minute. A Desaga applicator was used with a setting of 20W250~for the analytical plates and about 3.30~for the preparative plates. Twenty-five grams of talc will easily coat three 20 X 20-cm plates (250~). Relatively good analytical results were also obtained when plates were made without the use of the applicator by simply pouring the slurry on the plate, then "jockeying" the plate about so that the slurry was spread out uniformly. Students introduced to TLC for the first time were usually successful on the second plate. A local glass cutter made plates ranging from 2 cm on up for use in :I variety of TLC chambers that included gas collecting bottles, tomato-sauce bottles, and peanut butter jars. The narrower plates were usually placed side by side on a small board for the hand-poured procedure. Activation of the plates at llO°C for 30 min, followed by storage in a desiccator, was found unnecessary in most of the applications, adding to the ease of preparation for beginners. At times the activation was actually less satisfactory than air drying; the data in Table 1 show that the separation of flavonoids is much better on the air-dried plates. (This may not be so applicable in other regions; the humidity of Santa 12e, New Mexico, is usually about 30'%, varying from 28% through 38%.) Consistent., cood results (Table 1) were usunllv obtained if the plates were developed within the period between 3 and 24 hr after being made. Plates developed up to tcn days after being made were still quite good, hut if developed after two weeks, streaking became pronounced. The last data refer to plates left in the open but away from solvent fumes. u
Table 1. Rr Values of Selected Flavonoids on Talc under Different Surface Conditions but in Same Solvent System, 15% . - CHaCOOH for 5 5 min
Flavonoids Naringin Neohesperidin Poncirin Rhoifolin Rutin Nobiletin Date of development (1966)
Condition of Talc Plate Activat,ed Aicdried at. 110°C dates. for t& weiks 1 hr* Air-dried. old .75 60 55 .63 0.00
.80 .77 .77 .52 .58 .58 .43 .43 .43 .26 .25 .28 .%O .43 .33 0.00 0.00 0.00
.XO .G8 .5O 37 .50 0.00
8/18
5/18
8/28
8/18
.70
6/7
These plates were spot,ted and developed when 1e.s than 24 hr old.
Table 2.
Rr Values of Selected Flavonoids on Talc in Variovs Solvent Systems.
Flavonoids Naringin Neohesperidin Poncirin Rutin Rhoifolin Nohiletin
------Solvent 1" 25 .90 .i8 .65 .57 .52 0.00
.41 30 .24 .I3 .09 0.00
Syst,ems :P
.76 55 .42 .25 .I0 0.00
4 L 5 "
.31 .61
.42
54
.55
... .61
. 10
.86
.92
.4!J
.20
20% CHSOOH; 1'/* hr for 11 cm rise of solvent front. hr for 11.5 cm r i e of solvent front. " Methanol-wateliaeetic acid (5:15: 1 v/v); 11/2 hr. for 12 cm rise of solvent front. dTolnene-ethylacetate-melh~~~~l-acetie acid (27:4:9:4 v/v); lZIahr. for 15 cm rise of solvent front. Nitramethar~emethanol(7: 1 v/v); 40 min for 10 cm rise of solvent front,, a
a 12% CHGOOH; I:/*
Figure 1. Selected Rovonoids on talc in 15% CHaCOOH (1 hr 1 0 mid: 11) nobiletin, noringin, ond rhoifolin; (21 rhoifolin; I31 poncirin; (41 naringin; (51 nobiletin.
The talc forms a smooth, adhering surface that will not flake off. Some plates were developed three successive times in one solvent system, butanol-acctic acidmethanol (10: 1:4). Other plates were developed three times in 15% acetic acid. The surface remained smooth and adhering. The portion in the solvent had been eroded, but uniform flow of solvent was secured for succeeding developments by placing a thin slurry of the talc with a pipet to the base of the plate so that continuity of the adsorbent was conserved. Prepordive TLC on Talc
Plates, 20 X 20 cm, coated with talc to a thickness of 3.50p, were able to take as much as 3/n ml of a samplc containing G 8 mg of dissolved flavonoids. To describe one typical example, semi-purified naringin (Snnkist) was subjected to this preparative procedure, and five distinct bands were revealed under UV after a three-hour development in hutanol-acetic acid-carbon tetrachloride (10:1:4 v/v). A spot of authentic naringin in an adjacent strip on the same plate as the semi-purified material permitted identification of the main component. The bands averaged 1.5 cm in width a t the approximate R, values: 0.14; 0.31; 0.39; 0.65; (naringin) and 0.80. After the bands had been outlined under W, they were scraped off the plates and kept for the elution step. Bleached blotting paper cut into small rectangles madc excellent disposable scrapers, and the talc was caught on squares of foil-covered paper. Elution was carried out under vacuum on sintered glass funnels. About 10-15 ml of methanol completely eluted 1 g of talc. The eluate was evaporated under vacuum (20 in.) at 55°C by means of a Buchler "Flash-Evaporator". The main component was further re-chromatographed on talc using a different system, toluene-ethyl acetatemethanol-acetic acid (25 :3 :-5:3 v/v) for 11/, hr. Two contaminants, faint leading and trailing bands, separated, leaving a highly purified naringin which agreed in R, values with authentic naringin in five different solvent systems and manifested the same UV spectrum as the authentic naringin in the Bausch and Lomh Spectronic 600. I n this fashion about 200 mg of semipurified naringin were fractionated in a week on some thirty 20 X 20-cm plates and required somewhat less than lb. of talc.
TLC identification of Fiovonoids on Toic
Representative solvent systems that proved useful in identifying selected flavonoids are shown in Table 2. The amount of flavonoids applied to the spots on the plate shown in Figure 1 ranged from a low of 0.2 rg for nobiletin to a high of 2 pg for naringin, though identification could be made routinely on half those amounts. The flavonoids were dissolved in methanol in roncentrations that required 3-5 p1 for adequate spotting. "Microcaps," 5-p1 pipets, obtained from Drummond Scientific Co., 500 Parkway S., Broomall, Pa., were used in the quantitative work for the results reported in this article. Students can readily make their own pipets by drawing out glass tubing and run an approximate calibration by correlating spot size and number of spots per pipet with one of the "microcaps". For application of larger quantities, the B-D Disposable Pipettes (20 p1, No. 2720-P) equipped with convenient plastic handles can be obtained from Beeton, Dickinson, and Company, Rutherford, New Jersey. Ultraviolet light (long wavelength) was used to bring out some of the flavonoids which show charwteristic fluorescent colors (6). Spraying the plates with aluminum chloride (1% in methanol) enhances the fluorescence of some flavonoids while masking the distinctive colors of others. Figure 1 is a photograph and diagram of one plate taken under W. The photograph~x-as made with a camera fitted with an f-6.3 Wollensak lens at 6.3 shutter opening and exposed for 15 sec under UV light (both long and short wavelengths) from a "Mineralight" W-SL-13 lamp. The W light came from the same side as the thin layer so that the talc background fluorescence would be lessened and thus permit better contrast. Sugars identified on Talc
Selected sugars were separated to a reasonable extent on talc as shown in Table 3 and Figure 2. The sugan were obtained from Sigma Chemical Co. They were dissolved in methanol, from 20-30 mg in 4 or 5 ml of alcohol (so that 1-2 pl would be sufficient for most pnrposes). The alcohol permitted more rapid drying and prevented contamination by microorganisms. Galactose, cellohiose, and lactose formed saturated solutions nnder these conditions although methanol-water mixtures (4: 1 or 3: 2 v/v) produced a single phase system. The sugars were brought out, after development and Volume 44, Number 5, May 1967
/
295
Table 3.
Colors with Aniline Oxdie Acid Spray
Sugars (- ) Arabinose a 14) Xvlose ~ , ,-"-~ (+)Galactose ore (+)Glucom L (+)Rhamnose D
~
D-Cellobime -Lactose
Table 4. Rt Values of Selected Amino Acids on Talc.
Rr Valuer of Selected Sugars on Talc.
pink oink brown brown olive brown brown
Amino Acids -Solvent
system*-
1
2b
3'
4d
.33 .47 .26 .li .53
.70 .85
.49 .56 .43 .34 .ti6
.45 .65
...
.ll
...
.58 .93 .38
...
.. .
.33
. .. .45 .77
...
...
a Butanol-water-formic acid (10: 10: 1 v/v); organic layer, 21/z hr for 10.5 em rise of solvent front. Nitromethsne-methanol (4: 1 v/v); 32 min. for 11 cm rise of
solvent front. Butanol-water-formic acid (10: 10: 2 v/v); single phase; Z1/* hr for 11 em rise of solvent front. * Butsnol-acetic acid-toluene (9:1:6 v/v); 11/1 hr for 11 cm rise of solvent. fmnt.
drying, by spraying with aniline-oxalic acid mixture (7). Solutions of aniline and oxalic acid, both 0.02 M in water, are kept in the cold until just before use, then equal volumes of each are mixed together and sprayed on the plates. After the plates are dried a t room temperature they are developed at llO°C for 10 min or left a t room temperature overnight. This brings out the distinctive colors of the sugars (Table 3), which persist for several hours and then become uniformly brown. With this spray, unlike the aniline-diphenylamine spray, the talc background remains white. Though these sugars can he identified in the 2-3 pg range, G 8 pg were the amounts usually spotted. Separation of Amino Acids
Representatives of aromatic, heterocyclic, straight and branched chain aliphatic, and snlfur-containing amino acids n-ere selected for separation on talc. Table 4 and Figure 3 indicate the type of separation obtained. The amino acids were dissolved in methanol, 25-30 pg in 5 ml of alcohol. Tyrosine required a 1: 1 mixture of methanol and benzene to produce a satisfactory solution. The usual spotting sample was in the 810 pg range though quantities in the 2-3 pg range could be brought out hy the ninhydrin spray (0.3y0 in 95% ethanol). The plates are sprayed with ninhydrin and
Figure 2. Selected sugars on talc in butanol-waterformis acid (10:lO:l v/vl 2 I : (11 glucose; (21 rhomnore; (31 glvcore and rhomnose; (41 ambinore; (51 xyloss; (61 arabinose and xylore; (71 galactore; (81 lactose.
296
/
Journal of Chemical Education
~bAlanine bcysteine Glycine histi ti dine. HC1 ~~rkoleucine &Proline >Tryptophan >Tyrosine
4 o l v e n t Systems----1'
3'
.33 .34 .23
origin to 0.11 .79 .38 .82
...
.39 .38 .40 .21 .83 .57 .90 .73
.20-.40d .15-.55 .10-.30 .0&.08 .70 .20-.57 .ti5 .25
"Butanol-water-formic acid (1O:lO: v/v) organic layer; 2'/2 hr for 11 om rise of solvent front. Butanol-water-scetio acid (4: 1 :6 v/v) organic layer; 3 hr for 10.5 cm rise of solvent front. =Ethyl acetate-formic soid (90%) (11:2 v/v); 45 min for 10 cm m e of solvent front.
*
The double R, values indicate the lower and upper end of the spot as a measure of spot size. Width was less than 0.5 cm.
air-dried for about 5 min then developed at llO°C for 10 min. The first two systems of Table 4 gave rather compact spots, as a rule, whereas in the third system some of the amino acids were elongated; only isoleucine, tryptophan, and tyrosine gave spots that ranged from 0.5-1.0 cm. The amino acids were obtained from Nutritional Biochemicals Corporation. Acknowledgments
The author is grateful to Dr. S. H. Wender of the University of Oklahoma for initiation into TLC and flavonoid research and for supplying most of the flavonoid standards reported in this article. The work of Bro. Emil Guyot of the College of Santa Fe in preparing the photograph is also gratefully acknowledged. This work was an outgrowth of an investigation supported by NSF Grant GE-6892 in the AYE program originating a t the University of Oklahoma. Lilemlure Cited (1) GAGE,T. B., GALLEMORE, C., AND WENDER,S. H., Proc. Okla. Acad. Sci., 29, 71 (1948). (2) KIRCHNER, J. G., MILLER, J. M., AND KELLER, G. J., Anal. Chern., 23, 420 (1951). E., "Thin-Layer Chromatngrrtphy," Springer-Trerlag, (3) STAHL, Berlin, 1965 pp. 29-34. (4) TRUTER, E. V., '*ThinFilm Chromatography," Interscience Ltd., London, 1963, pp. 14-19.
Figure 3. Selected amino acids on talc in butanolwater-formicacid (10:10: 1 V/V) (2L/lhrl: ( I I hirtidinei (21 cysteine; (31 tryptophan> (41 histidine, cysteine, ond (51 proline; tryptophan; (61 ikoleucine; (71 olanine; (81 glycine.
(5) BOB BIT^, J. M., "Thin-Layer Chromatography," Reinhold Publ. Corp., New York, 1963, pp. 14-29. (6) GEISSMAN,T. A. (Editor), "The Chemistry of Flavonoid
Compounds," The Macmillm Co., New York, 1962, p. 51. (7) WILLIAMS, R. T., IND SYNGE,R. L. M. (Edibm), "Partition Chromatography," Biochemical Society Symposia No. 3, Cambridge Univ. Press, 1950, p. 57.
Volume 44, Number 5, May 1967
/
297
