Raphael lkan and Eliezer Rapaporl
Hebrew University Jerusalem, Israel
II
Test Tube and G ~ S Rod S
chromatography, using thin layers of adsorbents on glass plates of various sizes has been applied qualitatively and quantitatively for the analysis of complex mixtures of natural products such as alkaloids, carbohydrates, lipids, steroids, terpenoids, and a wide variety of other organic and inorganic compounds. The preparation of chromatoplates (20 X 20 cm) requires the use of specially designed applicators, which are quite expensive. Furthermore, the development of chromatograms on the large glass plates requires from 30 min to several hours for satisfactory resolution of the components. These requirements are inconvenient when using chromatoplates for the routine analysis of large numbers of samples as well as for student laboratories and for lecture demonstrators. Several papers (1-5) were published recently describing the rapid preparation of microchromatoplates from microscope slides and from test tubes which were coated on their inner surface (6-7). These inexpensive methods for preparing thin layers do not give the highly uniform layers that may be obtained with the commercially available applicators, hut they are adequate for applications when the mixtures to be resolved are not highly complex. The present communication describes the use of test tubes, coated on their outer surface, as well as glass rods for thin-layer chromatography. This method eliminates the need for special applicators and glass plates of specific dimensions and the prolonged development periods. It also permits the quantitative recovery of the components and their spectroscopic determination. Apparatus and Techniques
Coating with Silica-Gel G: The most convenient sizes for routine use were test tubes 150 mm long with 14-15 mm od and glass rods of 5-10 rnrn diameter. The results presented here are based on these sizes. The test tubes and glass rods are thoroughly cleaned with a dichromate-sulfuric acid solution then with a dilute detergent solution, and finally are rinsed with water. A homogeneous suspension consisting of 1 part of Silica-Gel G and 2 parts of water is prepared in a test tube or a cylinder (20 cm long; 20-30 mm wide). The test tube or rod to he coated is dipped to a depth
TLC
of 12 cm into the slurry, taken out and held for afew seconds above the slurry until almost all draining has stopped, and then inverted and dried a t room temperature for 15 min. The coated test tubes or rods are then activated in an oven at 120-130°C for 45 min: cooled, and kept in a desiccator.
Coating with Silica-Gel G Imprepated with Silver Nitrate: The slurry is prepared as described above, except that a 10% solution of silver nitrate is used instead of water. The coated test tubes and rods are activated a t 120°C for 30 min. Spotting of Samples and Chromatography: One milligram of a substance in 1 ml of a suitable solvent is prepared and applied with a micropipet to the thinlayer 1cm above the closed end of the test tuhe or rod. The solvent is allowed to evaporate, and the test tube or Table 1.
Results of Separations on Silica-Gel G on Coated Test Tubes
Ergot Alkaloids* Ereobasine Ergocristinine Ergotaminine Amino,Acidsb Aspart~cAcid Glycine a-Alanine Isoleucine Tyrosine ~&bohgdrates" Glucose Fructose Sucrose A'Ieleaitose Dyesd p-Dimethylrtminoazohe~~ze~~e Sudan Red G Indo~henol
Color
RI
Violet Violet Violet Violet
0.44 0.50 0.70 0.76
Violet. Pink Pink Pink Pink
0.11 0.31 0.45 0.70 0.73
Blue Yellow Grey Grey
0.55 0.45 0.31 0.26
Yellow Red Blue
0.58 0.20 0.07
" Developing solvent: ethyl acetate-dimethylformrtmide-ethao ( 3 1.9:01). Developing time: 20 min. Detection: Ehrlich reagent (5% p-dimethylaminobenzddehyde in concentrated hydrochloric acid). Developing solvent: watecethznol-acetic aeid (1 :5:0.1),containing 10 mg ninhvdrin. Developing time: 45 mi". Detection: -The teit t&& are air dried a i d heated in sn oven a t llO°C, whereupon the colored spots appear. "Develo~inesolvent: n-butanol-acetic scid-ethvl ether-water (9:6:3:1).' ~evkvela~in~ time: 40 min. ~ e t e c t i o n :dlphenylamine (2%)-aniline (2%) in acetonp-phosphoric acid (5:5: 1). dDeveloping solvent: benzene. Developing time: 12 min.
Volume 44, Number 5, May 1967
/
297
Table 2.
Some Results with AgNOs Impregnated SilicaGel G.
Color
R, 0.45 0.50 0.60
0s.
Brawn Brown Brown Drown Brown Brown
Steroidsb Cholesterol Chaleitxnol Alloeholesterol SSitosterol p-Sitostmol
Grey Brown Blue Grey Brown
0.23 0.27 0.29 0.23 0.32
Olive oil : Triglycerider La
Lao LO? 0 2
03 or LSS
11.78
0.85 0.92
Number of double bonds 6 5 4 3 2 1 1
..
1 1
...
a S = snt.urated acid, e.g., stearie; 0 = munoenoic acid, e.g., aleic; L = dienoic acid e.g., linoleic. Developing solvent: ehlorofmm. 1)eveloping iime: 35 min. Detect,ion: spraying with 507; sulfuric acid followed by heating in an oven at 150°C far 10 min. Developing solvent: chloroform. Developing time: 30 min. Detertirm: Spraying with 50y0 sulfuric arid followed by charring at 150°C for 10 min.
1
Journal of Chemical Educafian
5
~~
.-,
~~~
7 -
~
The coat,ing is accomplished by a simple and reproducible method. It is possible to use test h b e s and glass rods of varyingsises. Thin layers containing ailver nitrate or possibly other specific resgents can be wed. The application of the samples to be analyzed is simple. The detection of compounds wit,h chi-omogens is easy, much easier than d h test tubes, coated an the inside. The compounds separated can easily he scrapped of? for spectroseopie measurements and for preparative plrposes.
The following simple experiments (Tables 1and 2) are recommended for use in lectures and student laboratories. When thin-layers of Silica-Gel G were applied to the separation of closely related colnpounds such as the const.it,uent,sof natural fatty oils, no efficient separation could he achieved (8). However, impregnation of silica gel thin layers with silver nitrate permits a t least the separation of the unsaturated from the saturated constituents. The former give complexes with the silver ions, and these complexes migrate much more slowly than the saturated components that do not give complexes. This principle has been applied by Dutton, Scholfield, and Jones (9) to mixtures of isomeric fatty acids esters, by de Vries (10) and by Cubero and Mangold (If) t.o lipids, by Ilcan and Cudzinovski (1.8) to cholesteryl esters, by IBan (IS) to triterpenes, and by Gupta and Dev (14) to sesquiterpene hydrocarbons. Table 2 illustrates the use of test tubes coated with AgN03 impregnated Silica-Gel for the separation of olive oil and steroids. The efficiency of impregnated Silica-Gel is illustrated in the figure. The sterols and the corresponding stanols have practically the same R, values (0.23) on unimpregnated
/
4
thin layers of silica; the superiority of the impregnated gel is obvious. I n conclusion, the following advantages of the coated test tubes and rods might be emphasized:
Laborafory and Lecture Experiments
298
3
. -
rod is loaered carefully into a larger test tube containing a few ml of developing solvent. The roated test tube or rod is held in the "developing tube" with the aid of the two halves of hollowed cork stopper. The solvent is allowed to rise to a height of 7-8 cm above the point at which the sample has been applied. The test tube is then removed from the container, airdried, and sprayed lightly with an appropriate chromogen. The distance traveled by each component is measured and the R, calculated.
+
2
Examples of test tube TLC. I1 I Triglycerider of olive oil on Silica-Gel G impregnated with silver nitrate. ( 2 ) Chole.tero1, cholestonol, and allocholerterol on Sillco-Gel G imnremated with silver nitrate. 131 ~ r ~ o r t i r mid, glycine, a-olonine, ond isolewine on Plaster of Paris. 14) Arportic acid, glycine, a-olanine, and iholeucine on Silica-Gel G. (51 p-Dimethylaminoozobenrene, Sudan Red G , and indophenol on Silica-Gel G.
Finally it may be mentioned that equally good results were achieved with test tubes and glass rods coated with plaster of Paris. Literature Cited
(1) ANWAR, M. IT., J. CHEM. EDUC., 40, 29 (1963). (2) HANSBURY, E., OlT, D. G., A N D PERRINGS, J. D., J. CHEM. EDU~., 40, 31 (1963). C., J. CHEM.EDUC., 40, 32 (1963). (3) ROIAINS, J. J., Mikroehim. A d a , 529 (1962). (4) PEIFER, S., J. CHEM. EDUC., 43, 145 (1966). (5) SAMUEIS, (6) LIE, K. B., A N D NYC,J. F., J . Chromatoy., 8 , 75 (1962). (7) BEMILLER, J. N., J. CHEM.EDUC., 41, 608 (1964). ( 8 ) MALINS, D. C., A N D WEKELL,J. C., J. CHEM.EUUC., 40, 531 (1963). , J., SCWOLFIELD, C. H., AND JONES, E. P., Chem. (9) D u r m ~11. Ind. (London), 1874 (1961). B., Chem. Ind. (London), 1049 (1962). (10) DEVRIES, J. M., AND MANGOI~D, 11. K., Microcham. J., 9, (11) CWBERO, 227 (1965). R., AND CUDZINOVSKI, M., J . Chromalog., 18, 422 (12) IKAN, (IQRT,\. ~- - - ,
(13) IKAN, R., J . Chromalog., 17, 5Q1(1965). (14) GUITA,A. S., AND DEV, S., J. Chromalog., 12, 189 (1963).
+
+
