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Solvent/Solution Mol. Wt. (g/mol) Acetic Acid Ethyl Acetate Hexane Methanol Toluene 60.05 88.10 86.17 32.04 92.14 Safety Data Irritating to eyes and skin. Harmful if swallowed. Irritating to eyes. Highly flammable. Highly flammable. Irritating to eyes. Harmful by inhalation. Highly Flammable. Toxic by inhalation, in contact with skin and if swallowed. Highly Flammable. Harmful by inhalation. 1. Preparation of the Developing Chamber Using the chromatography jar provided, place 3-4 mL of the solvent mixture (which is also provided to you) in the chromatography jar, cover tightly and allow to stand 5 - 10 minutes before using. CHROMATOGRAPHY JARS SHOULD BE FILLED IN THE FUMEHOOD AND STAY IN THERE UNTIL DISPOSAL TIME (Note: Dispose of solvent in the “Organic Waste” container). The developing solvent system that was provided is a mixture of: 74 mL ethyl acetate 25 mL hexane 1 mL acetic acid *When finished with the lab empty the jar into the waste, DO NOT WASH JAR with water, simply leave upside down where you obtained it 2. Preparation of the Sample a) Obtain an unknown sample from your TA b) Write down the number of your unknown sample, then remove the label from your vial and stick it onto your 10 mL erlenmeyer flask. c) Dissolve your unknown in 4 mL of methanol-toluene (1:1) in your 10 mL Erlenmeyer flask. [NOTE: some insoluble material will not dissolve. This is normal.] FYI: The reference standards have been prepared at a strength of 25 mg/mL. 3. Application of the Samples a) Obtain 2 of the commercially prepared fluorescent Silica Gel TLC plates provided. b) To know where your spots started, draw LIGHTLY in pencil a horizontal line 1 cm from the bottom of the TLC plate. If you push too hard, you will crack the silica. c) You will have five solutions (4 reference compounds and 1 unknown) to examine. They should be spotted on the coated side (not smooth) on the line 1 cm from one end of the sheet, equally spaced apart, with the outer two spots about 0.75 cm from the edge of the sheet. The unknown should be placed in the centre, with one reference compound on each side. You should use two TLC plates. You should use one plate to run your unknown plus two of the reference samples, and the other plate to run the unknown plus the remaining two reference samples. To apply a sample, touch one end of an applicator capillary tube to the solution, and then gently and quickly touch the Silica Gel plate at the proper spot. It is important to touch the plate very lightly and not to gouge a hole in the absorbent on the plate. It is also important to touch the plate very briefly so that the entire contents of the capillary tube is not transfer to the plate. Use the
other end of the capillary tube or a fresh capillary tube for each sample. The sample spots should not be larger than 2 mm diameter. d) BEFORE elution, look at the TLC plates under the ultraviolet lamp (see step 5) to make sure some compound is present. Reference Unknown Reference 1cm Solvent Front TLC Before Elution TLC After Elution (example) 4. Development of the Chromatogram When each plate has been prepared, place the plate, spotted end down, in the developing jar. Make sure that the solvent pool begins below the spots. To develop both plates at once, have them sitting back to back (shiny sides without sample) in the developing jar. Cover tightly, do not disturb (do not touch the developing jar until elution is complete, you will be able to see the solvent rise up on the silica), and allow about 5 - 10 minutes for the solvent to rise to within about 1 cm from the top of the plate - do not allow the solvent to run all the way to the top of the TLC plate! Remove the TLC plate(s) and immediately mark the solvent front lightly in pencil (a single horizontal line across). Allow the TLC plates to dry (IN THE FUMEHOOD!). 5. Visualization The colourless compounds are visualized by illumination of the plate with an ultraviolet lamp. Many substances, particularly aromatic compounds, will show a bright fluorescence, which may have a characteristic colour. The thin layer plates used contain a trace of fluorescent dye. Compounds which are fluorescent show up as bright spots on a light background; any others appear as a dark spot since they quench the fluorescence of the background dye. Circle the spots lightly in pencil, and note any distinctive colours. CAUTION: DO NOT LOOK DIRECTLY AT THE UV LAMP! 6. Comparison of the Unknown with Reference Standards (a) Calculate the Rf values of the reference compounds and the components of the unknown. Only once do you need to show a full set of calculations. (b) Draw the chromatogram (developed TLC plate) to scale in your lab notebook; identify and
Page 1 and 2: Experiment 1. Thin in Layer Chromat
Page 3 and 4: power Diethyl Ether Ethyl Acetate A
Page 5: Part B: The Separation of Fluorene
Page 9 and 10: 1. Preparing the Column a) You will

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