Experiment IV. Adsorption Chromatography Part A-Thin Layer Chromatography (TLC) Reading assignment: Techniques in Organic Chemistry 2nd ed pages 177-189, 208-233. 3rd ed pages 221-253 Introduction Chromatography is a technique used to separate mixtures of compounds for either analytical identification and quantification, or for preparative purification. A mixture of compounds is dissolved in liquid and the solution (mobile phase) is transported along an immobile non-soluble adsorbent (stationary phase). Along the way, the solute molecules interact with the stationary phase in repetitive adsorption/desorption steps. Various compounds have different degrees of interaction with the adsorbent and therefore have different mobilities. A compound that has stronger interactions with the stationary phase will move along slower than a compound that has weaker interaction with the adsorbent. This leads to separation due to difference in mobility. Thin Layer Chromatography. Thin Layer Chromatography (abbreviated as TLC) uses a thin layer of adsorbent material (stationary phase) that is bound to a solid support (glass, plastic or aluminum sheet). The most common stationary phase is silica gel, an amorphous form of silicon oxide (SiO2) that has hydrated surfaces. Silica gel is a porous material and therefore has a very large surface area, allowing for extensive interactions with molecules. TLC plates are prepared by mixing silica gel particles with compounds that act as an adhesive for binding the particles to the solid support. When a TLC plate is placed inside a container with solvent, the solvent will rise up the plate due to capillary forces. A capillary is a tube with a very small diameter (capillus is hair in Latin, therefore slender as hair). When a glass capillary is inserted into water, the water inside the capillary will rise above the level of the water outside, due to the interactions between the water molecules and the surface of the glass (Figure 1). Glass is mainly composed of silicon oxide which displays hydroxy groups on its surface. The hydroxyl groups form strong hydrogen bonds with water molecules and therefore the molecules will “climb” up the glass surface to gain binding energy. The adhesion of the molecules is countered by gravity forcing the water down. As the capillary becomes smaller, the weight of the water column inside of it decreases relative to the adhesion forces, and the water will climb higher. Do not underestimate the significance of capillary forces. Water transport in plants is due to capillary forces between water and cellulose. Water molecules “climb” inside microscopic capillaries (xylem) to incredible heights as in the case of the sequoia trees.

When silica gel particles are packed to a thin layer, microscopic gaps exists between the particles. The gaps create a network of capillaries, and therefore once a plate is inserted into a solvent, the solvent will rise up. Therefore, TLC relies on a thin layer of adsorbent as a stationary phase and the ascending solvent as a mobile phase. A drop of solution containing a sample for separation is placed at one end of the plate. This process is termed ‘spotting’ and the spot is the origin point for the TLC. The solvent from the sample will immediately evaporate leaving behind a spot of the compound(s) adsorbed to the stationary phase. The TLC plate is then transferred to a jar containing an appropriate volume of a suitable solvent (mobile phase). The mobile phase will ascend the TLC plate (layer of adsorbent) as a result of the capillary action and it will reach the sample. The components in the sample mixture will be dissolved and carried by the ascending solvent. This is a dynamic process in which the molecules are constantly adsorbed to the surface and dissolve back in the mobile solvent. Structural differences of the compounds are manifested in the differences in reversible "binding" between the solute(s) and the binding sites on the surface of the adsorbent. Each compound in the mixture will travel a specific distance along the TLC plate leading to a series of spots. When the solvent front is close to the top of the TLC plate, the plate is then removed from the jar and the position of the solvent front is marked with a pencil. The spots on the TLC plate are analyzed either directly if the compounds are colored or with the aid of an indicator (UV-light, or I2 if the compounds are colorless. Compounds or the components of a sample mixture differ in structure and/or in the presence, number and/or types of functional groups. Therefore, different compounds have different polarities and will vary in their capacity for interaction (intermolecular forces) with the adsorbent (stationary phase) that coats a TLC plate. As a result, different compounds will ascend a TLC plate at different rates (distances) relative to the solvent. In this way, components of a sample mixture are separated from one another, and often, one can identify the components of a mixture by comparing the distance of migration that an "unknown" traveled compare to the distance observed for "known substances" (standards). The distance that a compound travels along a TLC plate depends on few factors: the polarity of the compound (solute); the polarity of the solvent (mobile phase); and the polarity of the adsorbent (stationary phase). There is a reversible "binding" between the moving compound (solute) and the polar sites on the surface of a polar adsorbent. This "binding" is due to intermolecular forces. The same holds true for the interaction of solvent with the adsorbent. In total, there is a threefold competitive interaction between solute, solvent and adsorbent. The result of these interactions determines the relative rate (distance) at which a solute and the solvent front ascend the layer of adsorbent on the TLC plate. Thus, for a given mobile phase and adsorbent, the more polar the solute, the greater the interaction or affinity that the solute will have for the adsorbent and the less distance the solute will travel along the TLC plate. Alternatively, the less polar the solute, the weaker the interaction will be between the solute and

Figure 1: Capilary forces inside capillary tube (left) and bwtween silica gel particles (right).

Figure 2: TLC plate.

the adsorbent, and the greater the distance that the solute will travel along the TLC plate. The following series in the Table 4.1 below is arranged roughly in the order of decreasing polarity of certain functional groups. The functional group at the top is the most polar and has the greatest affinity to a polar adsorbent such as silica gel. The functional group at the bottom has the least attraction to a polar adsorbent such as silica gel.

Table 4.1 : Relative Chromatographic Adsorbabilities of Some Functional Groups

The polarity of a solvent also influences the rate (distance) at which a solute ascends a TLC plate for a given solute and adsorbent. An increase in the polarity of a solvent increases the interaction of the solvent with the polar sites of the adsorbent. The increase in solvent-adsorbent interactions in turn reduces the solute-adsorbent interactions. Therefore, the solute will spend less time interacting with the adsorbent and travel further along the TLC plate. An approximate order (the "elutropic series") in terms of decreasing polarity of common elution solvents is as follows, with the most polar solvents at the top of Table 4.2.

Table 4.2: The Elutropic Series. The two most common adsorbents used in TLC are silica gel and alumina. The activity of the adsorbent influences the rate or distance a solute will migrate. The most active adsorbents "bind" the solute molecules more tightly. The activities of the adsorbents are greatly affected by their water content and the most active samples are those that contain the least amount of moisture.

Figure 3: Definition of retardation factor (Rf).

carboxylic acidsalcohols, amines and thiolsaldehydes, ketones and estershalogenated hydrocarbonsunsaturated hydrocarbonssaturated hydrocarbons

Watermethanol, ethanolacetoneEthyl acetatediethyl etherdichloromethanetoluenehexanes

The retardation factor (Rf value) of a solute is defined as the ratio of the distance traveled by the compound to that of the solvent front in a given time (Figure 3). Thus, Rf values will vary from a minimum of 0.0 to a maximum of 1.0. The Rf values for a given compound will vary widely with changes of adsorbents and/or solvent. Also, the Rf value can greatly change with the content of moist in the adsorbent. I: TLC analysis of ortho-hydroxyacetophenone and para-hydroxyacetophenone.

Equipment and supplies You need a 100 mL beaker containing a wick (cut filter paper) and a watch glass as a cover. Silica gel TLC plates and capillary tubes will be supplied by your TA. Iodine visualization jars and UV lamps are located in the laboratory. Preparation and development of TLC plates Note: Spotting solutions on a TLC plate require some TLC (tender loving care). As the contact time between the capillary and the plate is shorter, less solution is sucked out of the capillary and the spots are smaller. Smaller spotting area will lead to more defined spots and better separation. Use a pencil to gently mark the origins of three "lanes" on the coated side of a silica gel TLC plate, with each mark 1 cm from the bottom of the TLC plate. Dip a capillary tube into a 1% TBME solution of o-hydroxyacetophenone. The solution will be drawn into the tube by capillary action. Very briefly and gently touch the end of the capillary tube to the pencil mark in lane 1 on the TLC plate. In the process, transfer the smallest drop of solution possible to the TLC plate. Then do the same for lane 2. Repeat the process with 1% TBME solution of p-hydroxyacetophenone in lanes 2 and 3. As a consequence, lanes 1 and 3 will contain pure o-hydroxyacetophenone and p-hydroxyacetophenone, respectively, and lane 2 will be co-spotted with both components. While working in the fume hood, pour ~1 mL of 30:70 ethyl acetate:petroleum ether into the development jar (the 100 mL beaker), and allow the solvent to soak the wick. The solvent-wet wick saturates the air in the jar with solvent and enhances chromatographic separations on the TLC plate by approximating equilibrium conditions. Hold a TLC plate by its edges so that no finger oils contaminate the adsorbent and cause confusing artifacts. Then carefully lower the TLC plate into the beaker so that the main body of the solvent does not touch the spotted portion

of the plate. Place the 70 mm watch glass over the beaker and observe the passage of the solvent up the surface of the TLC plate. When the solvent front is about 5 mm from the top of the TLC plate, remove the plate from the beaker and immediately mark the position of the solvent front on the TLC plate with a pencil. Allow the solvent to evaporate from the TLC plate in the fume hood. Visualizing the chromatogram Observe the developed TLC plate under short-wave (254 nm) ultraviolet (UV) light. Be careful not to look into the UV light. Use a pencil to draw a circle around any spots that appear dark against the bright green background. Each time you finish using the UV light, turn the UV light off. Also, visualize the TLC plate with the iodine by placing your developed TLC plate in a jar that contains I2 crystals. Dark spots will form on the TLC plate where iodine adheres to o- or p-hydroxyacetophenone. Remove the plate after approximately one minute, close the jar and draw a cross through any spots where iodine has adsorbed to the TLC plate. It is necessary to make these marks promptly because the spots will disappear as the iodine deadsorbs and vaporizes. Measure the distances from the level of the original sample spot to the level of the solvent front and also to the level of the respective centers of the o- and p-hydroxyacetophenone spots. Then calculate the respective Rf values for o- and p-hydroxyacetophenone. Attache your developed TLC plates to your finished report. Which compound belongs to which spot? Explain why these two compounds have significantly different Rf values even though both compounds have the same functional groups.

o-Hydroxyacetophenone has a melting point of 4-6 °C. p-Hydroxyacetophenone has a melting point of 109-111 °C! Explain the basis for the sizable difference in melting points of these two compounds A II: TLC analysis of analgesic components and an unknown mixture.

The active ingredients of five common OTC analgesic tablets are shown in the Table 4.3 below. Note that if one could separate and visualize each component by TLC, one could clearly distinguish among these five preparations since each preparation would give a unique chromatogram. In this part of the experiment you will determine the component(s) present in an unknown mixture described below. Your unknown may contain one or any combination of two or three of the following components: Acetylsalicylic acid, acetaminophen, caffeine or ibuprofen.

Table 4.3: Analgesics Components (mg/tablet) Brand name Acetylsalicylic

acid Acetaminophen Caffeine Ibuprofen

Tylenol - 325 - - Anacin 400 - 32 - Bayer Aspirin 325 - - - Excedrin 250 250 65 - Advil - - - 200

Procedure Analyzing the unknown: Place a small sample of the unknown (crushed tablet) inside a test tube and add about 1 ml of ethyl acetate. . Mix the solid with the solvent using a spatula. Not all the solid will be dissolve since the tablet contains insoluble structural materials. Use the top solution and spot it on a TLC plate together with known standards. Analyzing standards: Spot a silica gel TLC plate with a drop of your unknown and two standards (acetaminophen, ibuprofen) next to it. Use another plate and spot the unknown and the other two standards (caffeine and acetylsalicylic acid). Develop the TLC plate using ethyl acetate as solvent. (Note: This is not the same solvent system that was used in the hydroxyacetophenone analysis.) As before use short-wave UV light and iodine to visualize the TLC plate. Use a pencil to mark the spots on your TLC plates. If you not sure that one of the spots in your unknown is identical to a standard, co-spot the unknown with the standard and run the TLC. If the spots are not identical, they will appear as two distinctive spots. Remember to work in a fume hood when evaporating the solvent. Save all your developed TLC plates and submit these along with your report.

Part B: Column chromatography. Reading assignment: Techniques in Organic Chemistry pages 206-226. Column chromatography is a preparative method for separating and isolating compounds from mixtures. The method is used for obtaining compounds from natural sources or for purifying products from reaction mixtures. The underlying physical principles of column chromatography are identical to those we learned for TLC. Essentially column chromatography is an upside-down version of TLC (Figure 4). Instead of having a thin layer of adsorbent attached to a solid support, a column is filed with a larger amount of adsorbent and the mixture is loaded on top of it. While TLC relies on capillary forces for moving the solvent, in column chromatography an eluent is allowed to percolate through the column by gravity. As the eluent is moving down the column it carries the soluble compounds with it. Compounds having strong interactions with the adsorbent move more slowly than compounds having weaker interactions with the adsorbent. Under the right conditions the compounds will separate in distinctive bands and each band will come out of the column individually. The bands are collected and the solvent is evaporated to give a clean compound. Column packing: Column chromatography starts with packing a column with an adsorbent material. The most common adsorbent is silica gel. Silica gel (SiO2) is commercially available in various particle sizes and has the appearance of a white powder. Other common adsorbents are alumina (Al2O3) and reverse phase silica1. The particle size has a tremendous effect on separation. As the particle size decreases, the relative surface area per unit weight increases. More surfaces are available for interaction with the solute and therefore better separation per unit weight is achieved. While smaller particles enhance separation, they also slow the solvent’s flow rate through the column. Small particles are packed well together and the resistance to solvent flow increases. Pressure can be applied to facilitate the flow. The column is usually made of glass and is clamped vertically onto a stand. Good packing is crucial for achieving a good separation. The material needs to be evenly distributed along the column and the surface needs to be flat. This will ensure that the compounds will be moving in parallel bands and that it will decrease mixing between bands. The first step in packing a column is the creation of mechanical barrier that will support the adsorbent (Figure 5). The barrier needs to allow for solvent to flow but at the same time keeps the adsorbent inside the column. A cotton or glass wool plug is inserted into the bottom of the column and a layer of sand is added on top.

1 Reverse phase silica is chemically modified silica gel that its surface was covered with long alkyl chains (typically 8 or 18 carbons). This modification transverse the silica gel surface from being hydrophilic to being hydrophobic. Reverse phase silica is used for separation of highly polar compounds.

Figure 4: Separation of mixture of compounds by column chromatography.

The sand has a dual function: it acts as a filter to prevent leaking of small adsorbent particles out, and it creates a flat surface to supporting layering the adsorbent. The adsorbent is added either ‘wet’ or ‘dry’. In wet packing the adsorbent is suspended in solvent and the slurry is transferred into the column. In dry loading the adsorbent is added directly to the column.

Figure 5: Assembly and dry loading of micro column. Column loading: Once a column is packed, the mixture intended for separation is loaded on top of the column. Two main methods are used to load the column: wet and dry loading. In wet loading the mixture is dissolved in a minimal amount of solvent and the solution is transferred to the top of the column. It is important to apply the solution on the walls of the column to minimize disruption of the column filling. In dry loading the mixture is first dissolved in a minimal amount of solvent and some of the adsorbent material is added. After the solution is mixed with the solid, the solvent is evaporated to dryness. This process results in adsorption of the mixture onto the surface of the adsorbent. The solid powder is then added on top of the column. Dry loading is used mainly when the mixture is only soluble in solvents that are more polar then the eluent of choice for the column. If excessively polar solvent is used for wet loading, it will stay inside the column and increase the local polarity. This can jeopardy the separation on the column as effectively the compound will experience the polarity of the solvent that was used for loading. Dry loading allows for separation in time and space between the processes of dissolving and loading. This is achieved by first dissolving the mixture in a polar solvent, but loading the column only after the polar solvent was evaporated and the compound is adsorbed to the surface of the adsorbent. Therefore this process eliminates applying the polar solvent onto the column. Eluting the column: Once the mixture is loaded on the column, eluent (a solvent or a mixture of solvent) is added and is allowed to percolate through the column. The eluent seep down the

column due to gravity (“gravity column”) or alternatively is forced through the column by applying air pressure (“flash column’’). The solvent is collected at the bottom of the column in several fractions. The optimal solvents system for eluting the column is determined by finding good separation conditions on TLC plate prior to running the column. The eluent can have the same polarity throughout the separation process (isocratic) or the polarity can be progressively increased (applying a gradient). A typical gradient starts with a non polar solvent (i.e. hexane) and as the column is progressing, an increasing amount of a polar solvent (i.e. ethyl acetate) is added. Following the progress of the column: If a compound is colored, it is easy to follow the progress of the compound in the column and collect the relevant solution. Yet, most compounds are not colored and therefore the eluent is collected in fractions (usually in test tubes). Each fraction is monitored for its contents. The most common way is to run a TLC of each fraction. A drop from each fraction is placed on a TLC plate (next to a drop from the crude mixture) and the TLC is developed). Figure 6 describes a TLC of a typical separation. Based on the TLC, fractions containing similar spots are combined and the solvent is evaporated to give the desired product. Fractions that have impurities are collected separately and can be purified again. In this example there is a good separation between the two top spots. Fractions 2-4 and 6-8 will each give a pure compound. The separation between the second and third spot was less efficient and therefore fraction 9 has a mixture of both. Fractions 10-11 have the third pure compound.

Figure 6: Following the progress of column chromatography by TLC.

Separating a three-component mixture by column chromatography Overview: In the next experiment you will be provided with an unknown containing three components: ferrocene, acethyl ferrocene and 9-fluorenone (Fig 7). You will have to separate the compounds using column chromatography and find the amount of each. First you will pack a column with silica gel and load the unknown on it. Then you will elute the compounds from the column by using a series of solvents with progressive increases in polarity. .The degree of separation will be followed by TLC.

The chemical structure of ferrocene, acethyl ferrocene and 9-fluorenone

Procedure Dry column packing: (Refer back to Figure 5) Take a 6 inch micro column and remove the plastic tip of the column. Create a support for the stationary phase (silica gel) by placing a small wad of cotton inside the tip. Make sure that the cotton is not squeezed too tightly. Clamp the column onto a stand and make sure that the column is vertical. Using a small plastic filter funnel add sand to give a thin layer of sand (3-5 mm) above the wad of cotton. Gently tap the column so that the sand will form a flat surface parallel to the bench. Follow this by addition of silica gel until the column is filled to little less then half way. Tap the column again so that the contents of the column will settle and become well packed. Dry column loading: Weigh about 100 mg of unknown mixture (record the exact weight) and transfer the sample into a small conical vial. Add to the vial about 100 mg of silica gel and wet the mixture with 2-3 drops of tBME. Stir the contents of the vial with a spatula until the solvent evaporates and the solid behaves like a dry powder. Transfer the solid mixture into the packed micro column. Cover the colored mixture with an additional layer of sand (~5 mm). Eluting the column: Before you begin the chromatography, obtain a rack of test tubes (8 test tubes, 13x100 mm). You will collect fractions of about 5 ml in each tube. Place the first tube under the column. To begin the chromatography, measure 10 ml of petroleum ether in a graduated cylinder. Using a plastic pipette, carefully add the mobile phase directly to the top of the column without disrupting the top layer of sand. The mobile phase will percolate through the column. Continue adding more mobile phase until the column is almost full. Use air pressure to facilitate the solvent flow through the column, but be sure not to let the level of the mobile phase drop below the upper layer of sand. You will observe an orange band at the top of the silica gel traverse down the column. After the first 5 ml are eluted from the column, switch to a new test tube. Elute the column with the rest of the petroleum ether. At this point, switch to a more polar solvent system (25 ml of 8%tBME/ petroleum ether). Continue eluting the column with this solvent system, changing tube every 5 ml. Once all the tBME/ petroleum ether is used, switch to pure tBME (about 5 ml) until the red band has been collected.

Using air pressure Fold-back about ½ inch of one end of a latex tube (Figure 8). This will provide a flat end (especially for a worn tube) and resistance to a griping pressure. Connect the other end to an air outlet. Gently open the valve and adjust the airflow. It is better to start with low flow and gradually increasing it, then to have an excessive pressure. Hold the folded end between your fingers and establish a contact between the tube and the top of the column. Don’t try to attach the tube, just secure a tight contact. This will result in enhanced flow of solvent. Pull back the tube when the solvents get about ¾ of an inch above the sand level. Be careful not to dry the column. Your TA will demonstrate the method.

Figure 8. Applying air pressure to a microcolumn.

Caution! Never attach the tube to the column. This can result in over pressure and burst shattering of the glass.

TLC analysis the fractions and product isolation: Obtain silica gel TLC plates and analyze the individual fractions to determine the extent of separation using a TLC solvent system 15% tBME /85% petroleum ether. Transfer the appropriate fractions to a pre-weighed small Erlenmeyer flask with boiling chips and gently evaporate the contents of the flask using a sand bath (60 °C). Weigh the compounds and measure their melting point. Report the mass and percent of each compound relative to the amount of the mixture along with respective melting points Questions:

1. Why is tBME more polar then petroleum ether? 2. Why evaporate at 60 °C, why not carry out the evaporation more quickly by heating the solvent to 100 °C?