Organic Chemistry Lab Thin Layer Chromatography Spring 2019-2020 PDF

Organic Chemistry Lab Thin Layer Chromatography Spring 2019-2020 TLC TLC Thin Chromatography Layer TLC is one of the most common techniques used in organic chemistry. It is a separation...

Organic Chemistry Lab Thin Layer Chromatography Spring 2019-2020 TLC TLC Thin Chromatography Layer TLC is one of the most common techniques used in organic chemistry. It is a separation technique that allows the separation of the components of a sample based on the difference in their partitioning between a stationary phase and a mobile phase. Why do we use TLC in the Laboratory ? Know the number of compounds in a mixture Test the purity of a sample Determine an unknown compound, by comparing its behavior on TLC to that of a known reference Follow the course of the reaction 2 Concept What are the components of TLC? We partition a solute between 2 phases 2. sample Solute 3. Mobile phase 1. Stationary (Eluent) phase On what basis does the solute partition itself between both stationary phase and eluent? 3 Concept This phase is stationary : It doesn’t MOVE! Stationary phase Silica Stationary Phase is more polar than alumina Most common TLC’s contain silica stationary phases Adsorbent: Fine powder: Silica Plate Alumina Fig.1: TLC plates 4 Concept NOTE: Polar compounds are compounds which have polar bonds and no symmetry. A 2. Solute polar bond is a bond that contains 2 atoms with different electronegativities. In organic chemistry polar bonds are: C-O; C-N, C- Can be either a solid or a liquid halogens solubilized in a solvent Non polar Can consist of one or more compounds Can be either polar or non-polar Increasing elution order Due to the difference in polarity of the compounds, each have different affinities to the eluent and to the polar stationary phase! Polar compounds have higher affinity to Fig.2: The elution order of different types polar compounds and lower affinity to of organic compounds non-polar ones Non-polar compounds have higher affinity to non-polar compounds and 5 lower affinity to polar ones Concept 3. Eluent It carries the constituents of the solute on the stationary phase Solvent: Can be one solvent or a mixture of solvents Not all solutes have the same Polarity!!! So the eluent will carry different solutes at different rates on the stationary phase allowing for separation Fig.3: The polarities of common organic solvents 6 Experimental Details Step 1: Draw a baseline using a PENCIL on the TLC plate Baseline 1 cm Fig.4: a schematic representation of a TLC plate 1. We can’t use a Pen because the pen contains organic compounds which might solubilize in the solvent 2. Make sure to draw the line smoothly to avoid removing the silica 7 Experimental Details Step 2: Place your solute using a capillary tube on Baseline Fig.6: The tip of the capillary tube (figure showing 5 tubers) A B 1 cm A B 1 cm AB 1 cm Fig.5: loading the solute on the TLC plate 1. Don’t forget to label the solute 2. You should use different capillaries to spot different compounds 3. The spots shouldn’t be very concentrated otherwise they will form a tail Fig.7: Tailing 4. The spots shouldn’t be very diluted otherwise they will not be spoted clearly 5. The spots should be distnat from each other so that the spots will not mix 6. The spots should be far away from the side of the TLC plate to see clearly the migration of the spots 8 Experimental Details Step 3: Place the TLC plate in a closed beaker that contains the eluent. Fig.8: TLC plate being eluted inside a beaker 1. The beaker should be closed to avoid evaporation of the solvent. Evaporation of the solvent will lead to changing of the polarity of the eluent especially in the case of mixtures. 2. The level of the eluent should be lower than the base line drawn in step 1 or else you will solubilize your solute in the eluent. 9 Experimental Details Step 4: Allow to migrate. Fig.8: TLC plate being eluted inside a beaker 1. You should remove the TLC plate before the eluent reaches the top otherwise all the spots will mix. 2. You should mark the position the eluent reached (solvent or eluent front). This is essential for analysis. 3. If your spots don’t migrate with the chosen eluent make sure to use a more polar solvent.(see slide 15) 4. Allow the TLC to dry prior to analysis 10 Experimental Details Step 5: Detect the spots. Fig.10: visualizing the TLC plate 1. See by eye 2. Use UV light 3. Stain with iodine or other dyes 11  Analysis of Results Calculate Retention factor (that is from the baseline to the 𝑐𝑒𝑡𝑛𝑒𝑟 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑝𝑜𝑡) Distance traveled by solute 𝑅𝑓 = Distance traveled by solvent (𝑡ℎ𝑎𝑡 𝑖𝑠 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒 𝑡𝑜 0 < Rf < 1 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡 ) Baseline Fig.11: Rf calculation If my stationary phase is silica (polar): Polar compounds migrate slower so they have smaller Rf values Non-polar compound migrate faster so they have larger Rf values 12 Analysis of Results Every spot corresponds to one compound if you are using the correct conditions The number of spots indicate the number of compounds One spot, then the sample A has one compound (it is pure) 1 Two spots, then the sample is 1 a mixture (not pure). B The acid (C)has a lower Rf so it is more polar. The aldehyde (B) has higher 2 2 Rf so it is less polar C 13 Fig.12: TLC of pure sample (1) vs, mixtures (2) Analysis of the Results If two compounds have the same Rf values under the same conditions, This means that they might be the same. Elution A B X A B X Fig.13: Using TLC to identify an unknown 14 Analysis of Results Effect of solvent polarity on Rf If the compounds are not moving well on the silica (so you cannot see a good separation to calculate the Rf) then increase the polarity of the solvent (use a A B C D A B C D Fig.14 more polar eluent) If the compounds are moving too much on the silica (so you cannot see a good separation to calculate the Rf) then decrease the polarity of the solvent (use a less polar eluent) A B C D A B C D Fig.15 Using the good eluent A B C D A B C D Fig.16 15  Analysis of Results We can take different TLC measurements will performing a reaction B B A A After 5 min After 30 min Fig.17: using TLC to monitor the progress of a chemical reaction After 5 min the Reaction mixture (RXN) shows 2 spots. Spot A has the same Rf of SM so they are the same and spot B have the same Rf value of P so they are the same. This shows that the reaction started to take place After 30 minutes, Spot B looks more intense will spot A tends to disappear showing that the reaction ended. 16 Experiment o-nitroaniline p-nitroaniline 17 Experiment Hydrogen bonding H-bonding Nitro p-nitroaniline o-nitroaniline O-nitroaniline can’t form H-bonds with silica P-nitroaniline forms H-bond with the silica P-nitroaniline will move slower on TLC, will have a smaller Rf This is why polar compounds have small Rf value 18