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Lab CC: Separation of Unknown by Column Chromatography Purpose The purpose of this experiment is to acquaint yourself with some common techniques and glassware used by an organic chemistry to purify organic compounds. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.1 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC. Learning Objectives Correctly prepare solution of organic solvents Separate a mixture of solids based on them. difference of attraction for a solid (stationary) and/or liquid (mobile) phase . Calculate the retention factor (R ? ) of compounds. Confirm the identity of each compound based on their R. ? ) TLC analysis ( Laboratory Skills Perform a microscale chromatographic separation. Perform a TLC Calculate R ? Perform column chromatography. Equipment Small Pasteur pipet (5 3/4 inches ) Cotton Pipet bulb Paper towel 150 mL beaker Chemicals Alumina Hexanes Diethyl ether Acetone

Chromatography Background What is Chromatography? Chromatography is a technique that employs the partitioning of a solute between a stationary phase (solid, or sometimes liquid), and a mobile phase (liquid, or gas). Chromatography, in its various forms, is a purification and analytical technique that has the widest applicability in organic chemistry. Depending upon the system used, the purity of compounds can be from modest to very high (analytical) purity. There are many forms of chromatography, some of the common techniques are Gas Liquid Phase Chromatography (GPLC), Thin Layer Chromatography (TLC), and column chromatography. Gas Liquid Phase Chromatography (GLPC) In GLPC, the mobile phase is a gas (typically He or H 2 ) and the stationary phase is a viscous liquid absorbed in a thin layer on the surface of an inert material packed in a tube (packed columns) or on the walls of a long narrow capillary (WCOT – Wall Coated Open Tube) also referred to as Capillary GC. The columns are typically heated in an oven, and the sample is vaporized in a hot zone prior to injection into the column. We will not be doing this technique in sophomore level organic lab. Students interested in this technique should register for the Analytical Chemistry Lab course. Thin Layer Chromatography (TLC) TLC is a related form of chromatography in which a mobile phase (liquid) and a solid stationary phase (sorbent) is used. An example of a TLC chamber is shown in Figure CC.1. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.2 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC. References: 1. Horowitz, G. J. Chem. Educ. 2000 , 77 , 263. 2. Univ. of Colorado, Boulder, Chemistry & Biochemistry Department website. http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.html 3. Prof. R. Ronald ( Chromatography lecture notes )Department of Chemistry Washington State University, Pullman, WA

Chromatography a TLC chamber. The sorbent is spread in a thin layer, 0.25-2 mm thick, on an inert, rigid surface (glass, plastic, or aluminum plates) and the liquid phase is allowed to travel through the sorbent by capillary action from a solvent pool in which the bottom part of the plate is dipped. The samples to be analyzed are “spotted” near the bottom of the plate and travel upwards as they are carried along with the solvent. Egon Stahl invented the technique of TLC shortly after the end of WWII. Since that time, it has become one of the most useful and used analytical techniques in chemistry. TLC plates have a thin layer of silica gel dispersed evenly on an inert, rigid backing (glass, plastic, or aluminum). In the beginning, researchers had to make their own layers, but in the 1960’s commercially manufactured plates became readily available and the “art” of making TLC plates is mostly lost. For analytical TLC plates the layer is 0.25mm thick; and for preparative (separating compounds) TLC plates the layers can be up to 2 mm thick. The sorbent, typically silica gel 60 particles, is applied with an inert binder (usually a form of hydrated silica, or hydrated CaSO 4 , but sometimes an organic polymer) to make the layers stable and durable. Other sorbents such as cellulose, alumina (aluminum oxide) or reversed phase silica gel are also available. To visualize the separation of compounds, the plate can be held under a UV lamp after it is developed. The plate will fluoresce green, and compounds will appear as dark spots. If you have spotted the correct amount of sample, the major spots should be no more than 5-6 mm in diameter. You can only see spots under the UV lamp if the compound in the spot has a UV chromophore (absorbs UV light). Another method to visualize TLC plates is by placing the plate in a jar containing a small amount of iodine (I 2 ) – iodine from iodine vapor forms colored charge- transfer complexes with many compounds that appear as brown spots; The spots generated by iodine vapor are transitory and fade within a few minutes of removal from the I 2 chamber. Retention Factor (R ? ) is defined as the distance traveled by the compound divided by the distance traveled by the solvent. The R ? for a compound is a constant from one experiment to the next only if the chromatography conditions (such as solvents, adsorbent, concentration of spots, temperature) are kept the same. Since these Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.3 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC. Figure CC.1: An image of

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Chromatography factors are difficult to keep constant from experiment to experiment, relative R ? values are generally considered. This means that the values are reported relative to a standard, or it means that you compare the Rf values of compounds run on the same plate at the same time. The larger an R ? of a compound, the larger the distance it travels on the TLC plate. When comparing two different compounds the compound with the larger R ? is less polar because it interacts less strongly with the polar adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a mixture, you can predict that a compound of low polarity will have a larger R ? value than a polar compound run on the same plate. The retention factor can be measured by taking the distance traveled by the compound and dividing it by the distance traveled by the solvent. For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the R ? is equal to 0.75 (or 2.1/2.8). See Figure CC.2 for an example of how to calculate R ? values. Figure CC.2: How to calculate the retention factor for a TLC plate. Using a mm scale, measure the distance traveled by developing solvent from the line, where you spotted the fractions (this is known as solvent front or SF). Measure the distance traveled by each spot from the original line, where you spotted the fractions. Divide the distance traveled by the compound by distance traveled by the solvent. Repeat for all spots on TLC plate. Column Chromatography Column chromatography is another form of chromatography in which a mobile phase (liquid) and a solid stationary phase (sorbent) is used.In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (or metal) column and the mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique to isolate desired compounds from a mixture. The mixture to be analyzed by column chromatography is applied to the top of the column. The liquid solvent (the eluent) is passed through the column. Equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Because the different Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.4 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

Chromatography components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or eluents, are collected as the solvent drips from the bottom of the column. Column chromatography is separated into two categories, depending on how the solvent flows down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography. If the solvent is forced down the column by positive air pressure, depending upon the amount of pressure used it is classified either as Low Pressure Liquid Chromatography (LPLC), Medium Pressure Liquid Chromatography (MPLC), and High Pressure (sometimes called High Performance) Liquid Chromatography (HPLC). These are all related forms of chromatography in which the mobile phase is a liquid and the stationary phase is a porous solid. LPLC,MPLC and HPLC differ mainly in the type of columns and in the pressure applied to the mobile phase (eluent) due to the different particle sizes used to pack the columns. LPLC (Low Pressure Liquid Chromatography – or Flash Chromatography): The term ”flash chromatography” was coined by Professor W. Clark Still because it can be done in a “flash. This is a ”state of the art” method commonly used in organic chemistry laboratories. The sorbent is packed into columns (usually glass or plastic) and the mobile phase is applied to the top of the column and allowed to percolate through the sorbent under a slight positive pressure (2-10 psi) of air or nitrogen. The samples are applied to the top of the column and then eluted out the bottom. MPLC (Medium Pressure Liquid Chromatography): A preparative technique and employs glass, or plastic columns limited to between 50-200 psi. HPLC(HighPressure(sometimescalledHighPerformance): This technique employs pumps and columns capable of delivering and withstanding pressures up to 8000 psi. To resist these high pressures, HPLC columns and fittings are made from stainless steel; the pumps have sapphire pistons, ruby check valves and sapphire valve seats. Typically, HPLC is an analytical tool and uses various detection methods to achieve quantitative information on the separation of the analyte. The Experiment Summary of the Procedure The purpose/object of this experiment is to apply column chromatography to separate two compounds and to analyze their purity by TLC analysis. Chromatography is the most important separation technique in organic chemistry. This experiment will allow you to practice and investigate this technique. A mixture of two organic compounds benzyl, benzoin, and alumina has been prepared. See Figure CC.3 for the chemical structures of benzyl and benzoin. You will separate the two organic compounds benzyl and benzoin from each other (alumina is not soluble in your mobile phase and hence will remain on the column) Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.5 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

Chromatography Figure CC.3: Structures of benzoin and benzyl. After you have separated the components, you will identify them by performing a TLC experiment and measuring the R ? of each compound and comparing it to the R ? of the reference compound (benzyl or benzoin). Safety Precautions Students working in the Chemistry lab should observe all PPE and safety requirements enforced by your Lab Instructor: full-sleeved shirts, full pants, and shoes covering all areas of the foot. Wear splash-proof goggles always. Wear gloves when handling chemicals. Remove soiled gloves and wash hands. No eating or drinking in the lab. All chemicals including alumina should be managed in the hood to avoid inhalation. All liquid chemical waste should be discarded in the containers located in the dispensing hoods. Avoid excessive evaporation of organic solvents. Used glass pipes, alumina, and TLC plates should be disposed of in solid waste containers. No flames or hot plates should be used in the laboratory when hexane and diethyl ether are used. Review the SDS for alumina, benzyl, and benzoin before lab. Procedure Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.6 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

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Chromatography Figure CC.4: Images showing how to pack a pipet column. Images from Prof. Lisa Nichols at Butte College. 1. Plug a Pasteur pipet (small 5 3/4 inches length) with a small amount of cotton. Caution: too much cotton will ruin the separation 2. Add dry alumina (2-4 grams) to a depth of 5 cm. Gently tap the pipet to pack the alumina. This is your chromatography column. See Figure CC.4 for images showing how to pack the pipet column. 3. Pre-elute the column with Hexanes; keep the column wet with hexanes as you prepare the product for loading. 4. Weigh out approximately 150 mg (0.15 g) of a mixture of benzil:benzoin:alumina. Transfer the mixture to the column. 5. Add more hexanes to the top of the column and force the solvent through the column with a pipet bulb: place the pipet bulb on top of the column, squeeze the bulb ( ask a TA for help ) and then remove the bulb while it is still squeezed. You must be careful not to allow the pipet bulb to expand before you remove it from the column, or you will draw solvent and silica into the bulb. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.7 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC. Part I: Column Chromatography

Chromatography 6. Prepare 15 mL each of eluent A, eluent B, and eluent C in Erlenmeyer flasks. You can use a syringe to measure the ether. Label all the containers and remember to keep them covered with either a watch glass, a cork or Parafilm. This will prevent the solvent from evaporating and hence changing the concentration of your solutions. a. Eluent A: 13.5 mL of hexanes + 1.5 mL of diethyl ether b. Eluent B: 12 mL of hexanes + 3 mL of diethyl ether c. Eluent C: 7.5 mL of hexanes + 7.5 mL of acetone 7. Run a total of 10 mL of eluent A (9:1 hexane: diethyl ether) through the column, until the first component (yellow) is off the column. Do not use more than 15 mL of eluent A unless directed by your TA. Collect and save the eluent in a pre-weighed beaker or an Erlenmeyer flask. (fraction 1) 8. When the solvent eluting from the column is clear, change the eluting solvent to eluent B (8:2 hexanes: diethyl ether). Run a total of 10 mL of eluent B through the column. Collect in a separate pre-weighed beaker/flask. (fraction 2) 9. Change the receiving flask and continue to elute the column with eluent C (10-15 ml 1:1 acetone: hexanes). (fraction 3) 10. Obtain a TLC of all fractions (Part II). Part II: Thin Layer Chromatography 1. Prepare a developing chamber by placing a 2.5 x 6 cm piece of paper towel in a 150 mL beaker. Add developing solvent (8:2 Hexanes:Diethyl Ether) to the chamber to a depth of no more than 1/2 cm. Cover the beaker with a watch glass. The reason for covering the beaker is to make sure that the atmosphere in the beaker is saturated with solvent vapor. To help this, the beaker is often lined with some filter paper soaked in solvent. Saturating the atmosphere in the beaker with vapor stops the solvent from evaporating as it rises up the plate. 2. Obtain a silica coated TLC plate, avoid touching the coating on the plate handle only by its edge. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.8 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

Chromatography 3. Lightly draw a pencil line approximately 1 cm from the end of the plate. You may mark each lane with a number or symbol so you can keep track of what you spotted. With a pencil, lightly mark 1, 2, 3, and 4. See Figure CC.5 for how your spotted TLC plate should look. Figure CC.5: How to calculate the retention factor for a TLC plate. 4. Draw up some of the solution 1 with a micropipette. Gently touch the tip of the micropipette onto the surface of the silica-coated plate. If the spot (approx. 1 mm) is not visible, apply some more of the solution on top of the original spot. The spot should be small and concentrated. Repeat for 2 and 3. 5. Obtain a solution of pure benzoin and spot on the TLC plate as 4. 6. Place “spotted” TLC plate in developing chamber being sure that pencil line with spots is above the level of the solvent. The level of solvent in the solvent chamber should be no more than 5-6 mm and there should be at least 5 mm from the top of the solvent to where the solvent front encounters the sample. It is important that the solvent level is below the line with the spot on it. 7. Allow developing solvent to migrate approximately 3/4 of the way up the length of the TLC plate. Remove plate from chamber and immediately mark the position to which the solvent rose. 8. Allow the plate to air dry. 9. Observe the plate under a UV lamp. Mark all the spots visible in UV light with a pencil. Draw a picture of TLC plate (scale picture to the correct size of plate) in your report sheet and notebook. 10. Stain TLC with iodine in an iodine chamber and draw a picture of TLC plate in your report sheet and notebook. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.9 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

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Chromatography 11. Calculate the R ? for each spot. Show TLC plate to your TA. 12. Discard TLC plate in waste container marked “Alumina Waste”. 13. Identify the compound in each fraction. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.10 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

Data Tables for Lab 5: Separation of Unknown by Column Chromatography Report Table CC.1: Part I: Column Chromatography Compound Total Amount of Material Used Chromatography mixture (1:1:3 benzil:benzoin:alumina) Report Table CC.2: Part II: Thin Layer Chromatography Compound Spot # Distance UV S/W/I* travelled (mm) R ? Fraction 1 (9:1 1 hex: ether) Fraction 2 (8:2 2 hex: ether) Fraction 3 (1:1 3 acetone: hex) Benzyl 4 *S = strong absorption (dark); W = weak absorption; I = invisible on UV Draw an image of your TLC plate below. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.1 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC. Report Sheet: Chromatography Name: Section: Date: Alumina 9:1 hexanes: ether 8:2 hexanes: ether 1:1 hexanes: acetone Benzoin 5

Chromatography Report Sheet Lab Report Your Lab Report (6 points) should be neatly handwritten or typed. All chemical structures should be hand drawn. Lab report is due within 7 days of lab day - before Experiment 6 lab recitation for your section. You will receive a 0.5 pt penalty for each hour after the due date and time. Introduction (1.25 pts) : Answer the following questions. 1. Briefly describe the purpose of the experiment. (0.5 pts) 2. Mention the most significant differences or similarities between the following: a. Stationary phase in column chromatography and mobile phase in column chromatography (0.25 pts) b. Gravity column chromatography and flash column chromatography (0.25 pts) c. Thin layer chromatography and column chromatography (0.25 pts) Safety (0.5 pts) Describe any safety measures taken for this experiment. Mention some of the dangers posed by the chemicals that were used. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.2 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

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Chromatography Report Sheet Experimental (0.5 pts) Describe in a short paragraph (4-6 sentences) what you did in these two experiments. Mention the techniques and chemicals that were used. Results: Data and Calculations (1.5 pts) Complete the data tables below. Report Table CC.3: Data Table Compounds Amounts Used Color Solvent front in mm = Report Table CC.4: Data Table Compound Spot # Distance traveled Intensity under (mm) UV (strong/weak) R ? Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.3 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

Chromatography Report Sheet Discussion (2 pts) Answer the following questions as part of your discussion. 1. Discuss the theory involved in this lab. If your experimental results were not as predicted, discuss reasons for this. (0.5 pts) Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.4 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC. Fraction 1 Fraction 2 Fraction 3 Benzyl Benzoin

Chromatography Report Sheet 2. Imagine you had a mixture of three compounds: phenacetin, acetaminophen, and aspirin (see Report Figure CC.1 below). A TLC plate developed in 1:9 ethanol: hexane showed three spots of R ? 0.1, 0.2, and 0.3. Which compound would have the highest, middle, and lowest spots? Provide reasoning for your answer. (1.5 pts) Report Figure CC.1: Structures of phenacetin, acetaminophen, and aspirin. Conclusion (0.25 pts) Draw a picture of your TLC plate (scale picture to the correct size of the plate) and identify each spot visible under UV light as benzyl or benzoin. Catalyst Education OER Labs are licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 license. CC.5 To access our full list of experiments, visit https://links.labflow.com/OER. Lab Version CC.

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