Setting Up Prepare a developing chamber by placing a folded filter paper length- wise in a wide-mouth bottle (Fig. 6.3). As directed by your instructor, prepare three or four mixtures of eluants that contain different ratios of varying pairs of the follow- ing solvents: petroleum ether, bp 60-80 °C (760 torr), chloroform, acetone, and ethanol. For example, prepare 10 mL of a 90:10 mixture of petroleum ether and chloroform to use as one eluant. Add an amount of the eluant to the developing chamber so that it forms a 1-cm layer on the bottom of the container. Screw the cap onto the bottle tightly, and shake the container well to saturate the atmosphere of the chamber with vapors of the solvent. Preparing and Developing a Plate Obtain three or four 3-cm x 10-cm strips of silica gel chromatogram sheets without a fluorescent indicator. Handle the strip only by the sides to avoid contaminating the plate with oils from your hands. Place one pencil dot about 1 cm from the left side and about 1 cm from one end of one sheet and another about 1 cm from the right side the same distance from the bottom as the first. Using a capillary pipet, carefully apply a small spot of a 10% solution of commercial azoben- zene in toluene, which you should obtain from your instructor, over one of the pencil dots. Do not allow the spot to diffuse to a diameter of more than 1-2 mm during ap- plication of the sample. Repeat this process for each strip. Allow the spots to dry and then expose the plates to sunlight for one to two hours (or a sunlamp for about 20 min). When the irradiation is complete, apply another spot of the original solution on the plate over the second pencil dot in the same manner as just described and allow each strip to dry. Place a strip in the developing chamber, being careful not to splash solvent onto the plate. Both spots must be above the solvent level. Allow the solvent to move to within approximately 2-3 mm of the top of the strip and then remove the strip. Repeat this process for each additional strip using a different eluting solvent as directed by your instructor. Mark the position of the solvent front with a pencil, and allow the plate to air-dry. Analysis Note the number of spots arising from each of the two original spots. Pay particular attention to the relative intensities of the two spots nearest the starting point in each of the samples; these are syn-azobenzenes. Calculate the R-values of each of the spots on your developed plate. In your notebook, include a picture of the developed plate drawn to scale as a permanent record. Identify the solvent mixture that gave the best separation of syn- and anti-azobenzene.