Jeffrey A. Hurlbut
Metropolitan State College Denver, Colorado 80204
II
TLC Separation and Spectrophotometric ~ n c l l ~ s of is
0-
and p # i t r ~ h ~ i n e
A loborofory experiment
O u r chemistry department offers a second year, five-unit course entitled Advanced Quantitative Analysis in which we introduce the students to modern methods of analysis and separation. The lecture material is broken up into three sections, the first of which deals with spectrophotometric methods of analysis. Absorption, emission, and fluorescence spectrometry are covered (1,t). The second section is devoted to separations. Extraction, gas chromatography, column chromatography, and plane chromatography (1-5) as well as miscellaneous topics such as enzyme specific columns (4) and dry columns (5) are discussed. The third section deals with electroanalytical chemistry (B, 6). Then, if there is time, the students are introduced to nuclear magnetic resonance spectroscopy and mass spectrometry (1, 7). The laboratory work is designed to complement the lecture material, and the students are required to report their experimental results using a formal report procedure. This usually requires a fair amount of library work. In order to adequately cover the large amount of lecture material in the laboratory, some of the experiments have been combined into one. One such exercise involves the separation of a mixture of ortho and para-nitroaniline by thin-layer chromatography fol-
lowed by a determination of the amount of each isomer by spectrophotometric analysis. Ortho and para-nitroaniline are easily separated on a preparative thin layer plate using activated silica gel as the stationary phase. The yellow bands corresponding to the two isomers are readily visible, and a particular band can be identified using experimentally determined R, values. The two bands are scraped off, and each isomer is extracted into 95% ethyl alcohol. After determining ,A., and preparing calibration curves for the pure isomers, the student then dilutes the extracted isomers to the appropriate volumes and determines the absorbance of each. Finally, the concentration of each isomer is determined from the experimental data. This paper presents the details of the separation and of the quantitative analysis. Separation Using Thin-Layer Chromafography
Each student prepares three 8 X 8-in. thin-layer chromatographic plates. The plates are thoroughly washed with soap and water, rinsed with distilled water, and then rinsed with acetone. The dry plates are placed end to end on a flat surface, and runners consisting of four layers of masking tape are placed on two sides of the plates. A suspension of 36 g of silica gel (Mallinckrodt SilicAR, TLC-7) in 70 ml of water is
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prepared in an erlenmeyer flask and poured onto the plates. The silica gel is evenly distributed over the plates using a glass stirring rod which runs on top of the masking tape runners. The plates are smoothed over two or three times and are allowed to dry at room temperature for 2 hr. The masking tape is then removed, and the plates are dried a t llO°C overnight. Each student receives an unknown containing a p proximately 20 mg each of ortho and para-nitn~a~dine (CAIJTION: NlTR0.4NlLlSES AllKTOXIC A S D - CAN BE ABSORBED THROUGH THE SKIN). The sample is dissolved in ethyl acetate (CAUTION: ETHYL ACETATE IS FLAMMABLE AND TOXIC) and is diluted to exactly 10.0 ml in a 10-ml volumetric flask. The plate is then streaked one-half in. from the bottom with 1.00 ml of the solution. If available, an applicator similar to the one described by Alhadeff (8) can be used. At the same time the student should prepare another plate containing pure ortho and para-nitroaniline so that the R, values can be determined. The plates are developed in a closed container (a large desiccator) using 3 :1 hexane-ethyl acetate (V/V) as the solvent. After the two bands have completely separated hr), the position of the solvent front is marked with a scratch and the plates are removed from the developing chamber. The plates are allowed to dry a t room temperature, and the R, values are then determined. The R, value for ortho-nitroaniline is about 0.5, and the R , value for para-nitroaniline is about 0.2. The bands can be removed using an inexpensive zone extractor (9, l o ) , or they can be scraped off and quantitatively transferred to separate 30 ml, medium porosity filtering crucibles. The silica gel containing one isomer is then washed inside the crucible with three 20-ml portions of 95% ethyl alcohol, and the washings are caught in a 25 X 200 mm test tube which is placed inside a 1000-ml filtering flask. The other isomer is then extracted in a similar manner. The vacuum created by a water aspirator is adequate for the filtration. The ortho isomer is transferred to a 200-ml volumetric flask and diluted to the line with 95% ethyl alcohol. The para isomer is likewise transferred to a 500-ml volumetric flask and diluted to the line with 95% ethyl alcohol. These solutions are savedfor the quantitative analysis. \
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Quantitative Analysis Using a Spectraphotometer
The visual spectra of pure ortho and para-nitroaniline are obtained using 95% ethyl alcohol as a solvent, and the .A., is determined for each isomer. The A,, is 371 mfi for the para isomer and 401 mr for the ortho isomer.
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Journal of Chemical Education
The dilutions should be made on the assumption that the molar absorptivities are about 5,400 and 15,000 for the ortho and para isomers respectively. A plot of absorbance versus concentration for the two compounds can be prepared by the student a t the experimentally determined , A for each isomer. Ninety-five percent ethyl alcohol is used as the solvent, and at least four different points should be determined for each calibration curve. The Beer-Lambert law is obeyed between 1.0 X 10-=M and 6.0 X 10-SM for the para isomer, and between 0.50 X 10-'M and 1.8 X 10-'M for the ortho isomer. The absorbance of each solution obtained from the separation experiment is now determined, and the concentration of each isomer is calculated with the aid of the calibration curve. The number of milligrams of each isomer given to the student can now be calculated. Results and Discussion
If the students pair up, then they can easily complete the experiment in two 3-hr laboratory periods, and students using proper technique can obtain better than =t10% accuracy. The preceding experiment gave students the o p portuuity to experience and evaluate both thin-layer chromatography and spectrophotometric, quantitative analysis. Also, the students obtained experience in preparing thin-layer chromatography plates, and they obtained experience in making standard solutions. Finally, they became familiar with the operational procedure for a spectrophotometer. The separation experiment can also be easily performed using a 2 X 40 cm column packed with 60 g of activated'8Ck200 mesh silica gel. Benzene was found to be a good solvent, and excellent separations can be obtained if the flow rate is held between 4 to 5 ml per minute. Acknowledgment
The author wishes to thank Professor B. R. Baker for the use of laboratory space and equipment. Literature Cited (1) PECBOK, R. L.. AND SHIELDB, D. L. '(Modem Methods of Chemical Analysis," John Wiiev & Sons. Ino..New York. 1968. (2) Farrz. J. 8.. A N D Scxeax. JR..G. H.. "Quantitative Analytical Chemiatry:' 2nd d.. Allynand Baeon. Ino.. Boston, 1969. (3) Gnucmn, G. M.. J. Cn~r. Enuc.,46,729 (1969). P., W u ~ c ~ e M., a , *ND ANPINSEN,C. B., B i ~ ~ h ~ l l l i d l ~ , (4) C*u~nec*S*s, 61.636 (1968). (5) LOET. B.,GOOOMIN. M. M.. ~ h ~ ~ . 2026 ~ ~ d(DW. . . a 1967). ofs Analytical Chem(6) Smoa. D. A,. A N D WEBT,M. W., " F ~ n d ~ m e n t d istry," 2nd 4..Holt, Rinehnrt and Winston. Inc., N e w Yark, 1969. m nM.. , A N D BABSI.EB. G. C., "Speotrometdo Identificsr (7) S ~ r v ~ n s ~ R. tion of Oresnie Camoounds." 2nd d.,John Wile, & Sons, Ino.. .. . ....., .... . (8) ALMADEFP, E.S., J. Cner. Eouo., 46, 249 (1969). M.. W.. J. C x e r . Eonc., 44, 110 (1967). (9) R u c ~ e ~ r m (lo) Knx, G.,AND Swmcaos, V., J. Cxeu. Eorrc., 16, 763 (1969).
