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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

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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