Spray Technique for the Preparation of Thin layer Chromatography Plates lhor Bekersky, Department of Industrial Medicine, N e w York University Medical Center, N e w York 16, N. Y.
PI! plates for thin chromatography (8, 9) with a commercially available applicator, losses of adsorbent occur at Itimes due to the adsorbent thickening or hardening of tl slurry in the applicator. Because the physical properties adsorbents vary in physica ffrom v m m nna i ~it is often one h9tr-h batch tn to tho the nnext, difficult to reproduce the correct ratio of water to adsorbent in the preparation of plates. To overcome these difficulties, a new, simple and inexpensive method of preparing thin layer plates was developed. The applicator is an ordinary air spray bottle found in most lahoratones. A slurry of the adsorbent is sprayed on the plates and this gives a smooth and fairly uniform layer of adsorbent. In view of the ease of application by the spray technique, the inexpensive eaninment used. and the fact that waste of adsorbent i s ‘avoided, it seems desirable tobring thismethod to theattention of analytical chemists using thin layer chromatography. Furthermore, the method is very useful in tbe preparation of thin layers of adsorbent on microscope slides. This type of thin layer plates is often useful in rapid screening of mixtures, but the method of coating plates as described earlier ( 1 ) is time consuming and wasteful with respect to adsorbent. Using the spray technique, plates have been prepared with thicknesses from 250 microns to 1100 microns, depending upon the number of passes of the sprayer over the plate. The thickness of adsorbents on the plates were determined as described by Stab1 (8). Thicknesses at 40 uniformly d i e tlibuted positions on the plates were N THE THE PREPARATION OF
I layer
measured. The results are given in Table I. The sprayed plates showed a deviation of *40 microns from the average values shown in the table. The plates made with the applicator showed a deviation *20 microns from the average value. There is greater uniformity in thin layers made with the applicator; however, Stahl (8) has shown that for thicknesses greater than 100 microns, Rr values and distances of running are not much affected by thickness of adsorbent layer. The spray technique is expected to be useful in most applications of thin layer chromatography except those in which high uniformity is required, for example, in the measurement of spot intensities. The spray technique described here has been used in this laboratory with silica gel and alumina absorbents with varying proportions of binder and without binder and can undoubtedly be extended to other absorbents.
EXPERlMENTAl
Adsorbents used in this work are from Brinkmann Instrument Co., Great Neck, L. I. For aluminum oxide-G, 2OO-mesh, containing 5% calcium sulfate, 20 grams of adsorbent and 50 ml. of water were mixed and sprayed on three 8- X 8-inch borosilicate glass plates. Since soft glass plates sometimes cracked during activation, borosilicate glass was used. This, however, isnot aprerequisitefor thespray method. An air spray bottle (Fisher Scientific Co.) was used for spraying. This spray bottle is of the common type found in most chemical laboratories. The spray bottle consists of a 250-ml. Erlenmeyer flask with a ground glass joint to which i s fitted the spray nozzle. The plates were , i n horizontal position during snravine. An air nressure of 1.0 to I’.Z “PSI. was mosi satisfactory for spraying. For silica gel-G, 325-mesh, containing 13y0 calcium sulfate, the proportions used were 15 grams of adsorbent and 35 ml. of water for the preparation of three plates. Any num-
Table I. Thickness of Thin Layer Chromatographic Plates, Silica G e l Adsorbent Number of coatings Av. of adsorbent thickness
Spraved dates
1
2 3
250 600 1100
Applicator plate 1
250
Figure 1. Thin layer chromatogram of test mixture o n silica gel plate prepared b y spray technique VOL. 35, NO. 2, FEBRUARY 1963
261
grams of adsorbent on 8- x 8-inch plates with a thickness of 1000 to 1100 microns could he readily prepared by the spray method. In preparing thick plates three consecutive coatings are sprayed on the plate and each layer is allowed to dry for approximately 2 minutes before application of the next. Figure 1 shows a silicacoated plate prepared with the spray procedure, and Figure 2 shows one prepared with a commercial applicator. Both plates were activated at 130' C. for 11/*hours. A test mixture (Brinkmann Instrument Co., Great Neck, L. I.), consisting of 3 dyes (4-dimethylamino-azobenzene, indophenol, and Sudan Red G.), was applied to the plates and developed with benzene as solvent. Both plates required 40minutes to develop. Afourth spot at the origin of application is ascribed to an impurity in the test mixture. ACKNOWLEDGMENT
The author is indebted to B. L. Van Duuren for suggestions and encouragement in this work. LITERATURE CITED
Figure 2. Thin layer chrornotogram of test mixture on silica gel plate prepared b y applicator technique
ber of plates can be prepared by varying the amount of slurry. Also, the ratio of adsorbent to rater is not critical as i t is in the applicator technique. The spray bottle is held 7 to 9 inches from the snrface of the plate during
spraying. The nozzle should not be held too close to the surface of the plate; when this happens, irregularities or craters appear on the surface of the layer. Thick plates containing up to 20
Work was SuDDorted bv erant number C5946 from thk DepahGent of Health, Education and Welfare, U. S. Public
Health Sekoe, National Cancer Institute.
Diethylamine-Ninhydrin a s a Color Test Aid in Identification of Amino Acids on Paper Chromatograms Richard Circo and Bob A. Freeman, Department of Microbiology, University of Chicago, Chicago 37, 111.
different color tests have M been published for the identification of amino acids on paper chroANY
matograms. While there is at least one specific reagent for the color identification of each individual amino acid, relatively few universal reagents exist, which are capable of giving specific colors with a variety of amino acids and m
