Brooks C. Madsen
Florida Technological University Orlando, 32816
Thin-Layer Chromatogram Visualization A student experiment
As applications of thin-layer chromatography become more widespread in areas of chemistry, biology, toxicology, etc., exposure to the versatility associated with various visualization techniques becomes a critical part in the teaching of' thin-layer chromatography. Both selectivity and sensitivity associated with various chromatogram visualization techniques should be emphasized. The experiment described below is an amplification on one previously reported' with increased emphasis on visualization technique. It is not intended to represent another example where a complex separation problem is solved by tlc hut is intended to emphasize the importance of chromatogram visualization in a ~ i t u a t i o nwhere separation is less than perfect. The followin: techniques are considered: 254 nm uv. 365 nm uv. and selective sprav As each tech. . reaaents. nique is introduced, properties such as the destructive/ non-destructive character of each should be stressed. The tlc separation evaluated with the appropriate visualization techniques includes mixtures of common "over the counter" analgesic and antipyretic preparations and related compounds. The student unknown can be a commercial tablet or svnthetic unknown sunplied bv the. in.. stmctor. The following compounds can be investigated: acetaminophen (4'-hydroxyacetanilide), acetanilide, aminophenazone (4-aminoantipyrine), aminopyrine (4dimethylaminoantipyrine), antipyrine, aspirin, caffeine, phenacetin, salicylamide, and salicylic acid. Experimental
Preparation of Thin-Layer Plates Silica Gel G containing a 254 nm phosphor has been obtained from several sources. All give good results. Standard procedures are followed to coat 5 x 20-em glass plates with a 250-p layer of adsorbent. The layers are allowed to air dry for at least 24 hr prior to use. Activation of layers does not significantly affect results. Solutions containing approximately 0.5 g analgesic/100 ml of each methanol are used. One small drop, solution is spotted. Unknowns and "real" samples are prepared to
contain similar concentrations of individual analgesics. Chromatography and Chromatogram Visualization Careful spotting of a 5 x 20-cm thin-layer plate will allow up to six known compounds and one unknown mixture to be applied 6-7 mm apart. The experiment has been performed using the six compounds specifically indicated in the table. The actual purpose .of. the. experiment can, however, be accomplished with other combmat~ons. The plate is developed using 120:60:18:1 v/v benzene, diethylether, glacial acetic acid, and methanol in a two quart "mason jar" which has been previously equilibrated. Migration of the developing solvent to a distance 10-15 cm above the origin is adequate. The plate is removed from the chamber, solvent front marked, and the plate allowed to air dry. Visualization of the chromatogram is accomplished in sequence with 254 om uv, 365 nm uv, 2% w/v ferric chloride spray, and 1%w/v potassium ferricyanide spray. The 254 nm uv serves as the universal visualization technique for this particular application because all components can be located due to their quenching effect on the phosphor in the Silica Gel layer. Positive identification of all components cannot he 852 /Journal of Chemical Education
Visualization of Analgesics-Antipyretics U I ~ ~ ~ V ~ O I ~ ~
component
RI
Salicylic acid Aspirin0 Salicylamid@ Acetanilide Phenacotina Acetaminophen Caflein@ Anfipyrin@ Aminophene,on@ Aminopyrine
0.86 0.81 0.72 0.64 0.m 0.41 0.30 0.29 0.13
0.07
S .D ~. ~ Y e. ~eent~
PsCb KsFeloKsFeICNM d i n e ICNIeL mixture Vapor
254nm Quenchin$
365nm Fluoresconce
FaCb
Blue+
Paintblue
Violet
-
Blue
Blue
Violet
-
Blue
Blue-pray
-
-
Blue~gray -?
Blue+
Blue' Blue
Blue Blue
Bmwn Blue Blue
Bmwn Blue Blue
+ ++
--
++ ++ + ++ ++ ++
Rust Pink Violet
-
-
+ + +
+ ++ + ++ ++ +?
0 Components aeluallyused in laboratorycxperimcnt. easily detected, &Componentlocated as dark spot on fluorescent background; detectable. -undetectable. 'Color observed ir altcr nmavine kith ferric chloride and ootasium ferricvanide i n s . ~ . quence. 'Freshly prepared mixture. easily detected, +detectable. - undefeefable. Component l a s t e d an brown spot: ,Allow 5-10 min foreolar development.
++
+
++
made based strictly on migration behavior because similar R, values are obtained far salicylic acid, aspirin, and salicylamide, for acetanilide and phenacetin, and for caffeine and antipyrine. Only salicylic acid and salicylamide are observed under 365 nm uv thereby demonstrating the selectivity of this visualization procedure. The significance of employing a definite sequence in application of visua1izatib.n techniques can he further emphasized if the plate is then sprayed with ferric chloride solution, allowing a few minutes far color development, then spraying with potassium ferricyanide solution. The initial spray will allow certain components to be visualized as colored spots while others do not appear until the second spray is applied. Caffeine and acetanilide are not detected with these spray reagents. The tahle summarizes visualization results. Discussion
This experiment has advantages which many student orientated tlc experiments lack. The analysis of a "real" sample is involved and students are required to think about what each visualization technique can offer to solution of the problem of comnonent identification. Correct answers depend on successful evaluation of the visualization techniques and interpretation of results as well as diIeCtR f for the and components. The six components used in this experiment have been selected in such a fashion that the presence or absence of two, phenacetin and aminophenazone, can he confirmed by simple comparison of migration behavior. Visualization under 254 nm uv is adequate for these two components and little interpretation is required. Caffeine and antipyrine, khich similar R, values in the sol. vent systemused, require interpretation of additional information before the presence of one or both is confirmed. In actual practice the student unknowns are selected to contain only one of these two components. The determination of which component is present is based on the color reaction of antipyrine with the ferric chloride and potassium ferricyanide spray reagents. Caffeine is not detected with these sprav . . r e a-~ e n t s .Positive response with 254 nm uv and positive response with spray reagents confirm the presence of antipyrine while positive response with 254 nm uv and negative response with the reagents con'Lieu, V. T., J. CHEM. EDUC., 48,418 (1971).
firm the presence of caffeine. Chloramine-T spray reagentZ can also be used as a selective reagent for caffeine. If both antipyrine and caffeine are suspected to be present, then two separate chromatograms can he sprayed, one for antipyrine and one for caffeine. Careful evaluation of visualization observations is important when samples contain aspirin and/or salicylamide. Both have similar Ri values. Based on visualization data presented in the table, uv and spray reagent information should he sufficient to establish the presence of either or both. The blue fluorescence of salicylamide is easily detected. If either aspirin or salicylamide but not both are present in the student sample, 254 nm and 365 nm data will confirm the presence of one or the other. In general student unknowns do not contain both. Unless the solutions containing aspirin are used shortly after preparation, the visualization and chromatographic behavior of the aspirin-containing samples change due to decomposition. Formation of salicylic acid as a result of this decomposition will result in an additional spot on the chromatogram. Salicylic acid migrates only slightly faster than aspirin and the "aspirin" sample gives two spots. The visualization behavior of salicylic acid under uv light and 2Stahl, Egon, "Thin Layer Chromatography," 2nd ed., Springer-Verlag,New York, 1969, p. 457. =Ref. 2, p. 527. R e f . 2, p. 525.
upon exposure to the ferric chloride and potassium ferricyanide spray reagents is nearly identical to that of that of salicylamide. Unless the separation is performed carefully and unless the presence of two distinct spots is observed, confirmation of the presence of partially decomposed aspirin can be misinterpreted to indicate the presence of salicvlamide. Samules susnected to contain both aspirin and salicylic acid should be dissolved and chromatographed immediately so complications associated with this decomposition can he avoided. The sway reagents, ferric chloride solution and wtassium ferricy&ide-solution, are more commonly uskd as a mixture3 hut as such do not illustrate the increased selectivity which can sometimes result when a series of spray reagents is used. The experiment described above does not include application of all types of visualization techniques and reagents but is representative of'those most commonIv used. Iodine vavor can be used to visualize most of the ~ompounds.The table includes results that are obtained when the ferric chloride-potassium ferricyanide mixture or iodine vapor is used for vi&alization. Students are encouraged to evaluate the effect that other solvent systems4 have in achieving reasonable separations and to evaluate other visualization reagenk3 Many variations on this experiment are possible if combinations of the ten analgesic-antipyretic compounds are incorporated into the basic scheme.
Volume 50, Number 12, December 1973 / 853
