Column Chromatography lab report PDF

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Lab report Column Chromatography of a Reaction Mixture........................

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Desk: Chem 36.1 TA: 11/9/05 Experiment 8—Column Chromatography I. Introduction Column chromatography fins its advantage of simple TLC separations through its dual application to both a large or small scale setting. While TLC enables one to analyze the components and rate of completion of a specific reaction and its products, column chromatography allows for the collection as well as the isolation of different components in a certain reaction scheme. As was the case with TLC, alumina and silica are popular stationary phases for column chromatography due to their high polarity resulting in a higher retention of polar samples. This stationary phase will cause the less polar compounds to elute the fastest out of the column in order of polarity with the most polar compound eluting last. In selecting a stationary phase, the type and polarity of adsorbent as well as the size of the fractioning column all influence the rate of elution of a given sample. Manipulation of these factors will ultimately result in the most efficient separation of a mixture. A major difference from TLC in regards to column chromatography resides in the retention times. The longer retention times for column chromatography are associated with a more polar substance, while smaller Rf values in TLC are associated with more polar substances. This results from the variation in solvent flow in the different techniques, as in a TLC the solvent flows up and in column chromatography the solvent flows down, resulting in a different functional order of elution. Another factor which plays a major role in the separation of a mixture regards the solvent used as the mobile phase, which generally will be of a nonpolar or low polarity nature to enable compounds to adsorb to the stationary phase and slowly travel with the mobile phase if their

polarity warrants such movement. Nonpolar compounds will travel with the nonpolar mobile phase and thereby elute faster, while more polar components will reside in the column for longer times. As the elution continues, the solution will be collected in fractions to allow for complete separation of components. In this experiment, fluorenone is synthesized via the oxidation of fluorene. This reaction occurs in the presence of a phase-transfer catalyst, which in this experiment is Stark’s catalyst. This substance basically increases the rate of reaction by allowing the hydroxide present in the reaction to deprotonate acidic fluorene protons at a faster rate, which allows for faster formation of a carbanion. The increased reaction rate results from a decrease in the activation energy necessary to deprotonate the fluorene. By enabling the hydroxide to behave in such a way, the carbanion can then attack the oxygen in the air to avoid using chromium trioxide as an oxidizing agent. In avoiding use of chromium trioxide, a hazardous chemical waste of chromium is then eliminated from the process of formulating fluorenone, as the oxygen is gathered from the air instead of the chromium trioxide. This experiment involved the oxidation of fluorene to fluorenone under basic conditions with the addition of Stark’s catalyst for the reasons listed above. The mechanism, illustrated below, follows a general oxidation mechanism, beginning with a hydroxide ion deprotonating a fluorene proton under the influence of the catalyst. This forms a carbanion, which attacks atmospheric oxygen to ultimately form a ketone of fluorenone following removal of water. This experiment ran until the final mixture contained 50% fluorene and 50% fluorenone. The reaction progress was monitored through TLC, and the final product was separated by column chromatography with separate vials to collect fractions of solution as it eluted through the tube.

The final products were then analyzed for percent recovery, and the fluorenone underwent IR analysis. Figure 1: Oxidation Mechanism of Fluorene to Fluorenone

II. Procedure/Data Observations In order to oxidize fluorene to synthesize fluorenone, 5 milliliters of 10M NaOH and roughly 70 milligrams (72 milligrams in actuality) were added to a 25-milliliter Erlenmeyer flask with a ½ inch stirring bar over a magnetic stirrer. A volume of 5 milliliters of toluene was then added until all of the solid fluorene dissolved, and a yellow color formed, with a separation of organic and aqueous layers of the fluorene layer with toluene representing the organic layer and the layer containing the sodium hydroxide representing the aqueous layer. Two drops of Stark’s catalyst were added to the solution, and the solution was set to stir for up to 30 minutes, depending on the reaction progress, which was monitored through TLC. The TLC was run every five minutes and was developed in a solvent containing 20% dichloromethane in hexanes. A fluorene standard was spotted on the TLC plates to serve as a guideline towards how fast the reaction was progressing at every time interval. In spotting the plates, the reaction mixture was carefully dabbed with a capillary tube and transferred to the reaction plate. From here, the spots were examined for initial intensity with a UV lamp before being developed. After development, the spots were reexamined for intensity. The reaction was to be brought to a level where half of the fluorene had converted in fluorenone. This was

ultimately determined when the intensity of the spots of the reaction mixture on the TLC plate were of a similar magnitude. The organic layer was separated from the aqueous layer and washed in a separatory funnel three times with 5 milliliters of 5% HCl followed by 5 milliliters of saturated NaCl. The aqueous layer was removed from the separatory funnel after each wash, in order to fully isolate the organic components. The remaining toluene mixture was then dried over anhydrous sodium sulfate in a 25-milliliter Erlenmeyer flask in order to remove as much water from the solution as possible. After roughly ten minutes of drying, the toluene solution was decanted into a tared 100milliliter beaker. The remaining sodium sulfate was washed with 3 milliliters of toluene to ensure complete product transfer and similarly decanted into the 100-milliliter beaker. The remaining toluene was left to evaporate until the following lab period under the hood. In preparation of the actual column chromatography, the column first had to be assembled. A microscale column was used, to account for the small amount of sample that would be tested in the experiment. A stopcock with a fritted filter disk was firmly attached at the bottom of the column to control the movement of the solvent in the column. After the valve was closed and a funnel was on top of the column, a solvent of hexanes was placed in the column up to the bottom of the funnel. Then roughly 4.5 grams (4.54 grams in the case of this experiment) of dry alumina was slowly sprinkled into the column while gentle tapping with a pen to ensure effective packing of the stationary phase. After the alumina leveled off, the stopcock was opened to allow sufficient solvent out until the solvent is flat just above the alumina. In preparing the fluorene and fluorenone for the chromatography, ten drops of both dichloromethane and toluene were added to the solution to dissolve the compounds. This was then added to the surface of the alumina, and then the solvent was drained until the dichloromethane/toluene mixture was just

covering the alumina. Drops of hexanes were then added to the column and drained until a narrow band of color was seen within the column. This was then covered with a small 4 millimeter layer of sand in order to stabilize the column’s flatness and was filled with solvent. After the column is prepared, 3 milliliter fractions of solution were eluted into separate containers, which ended up totaling 16 fractions in this experiment. After collecting each fraction, a TLC was developed in 20% dichloromethane in hexanes for each fraction with roughly 5 fractions on a slide. A UV lamp was used to verify spotting before and after the development. The Rf values were determined by dividing the movement of the spots for each molecule by the distance the solvent front traveled. The results of the TLC are summarized in the table below: Table 1: Rf Values for Fluorene-Fluorenone Fractions Lane Number 1* 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Rf value 0.357 0.339 0.357 0.339 0 0.054 0.054 0.054 0.054 0.037 0.046 0 0 0 0 0 0

*Lane 1 was used to plot the fluorene standard.

From this like fractions were combined into tared beakers in order to determine the final masses of the fluorene and fluorenone. After the like fractions were combined, a stream of nitrogen gas was blown over to evaporate the solvent, concentrating the solution. If no product or both products were present in a fraction, they were discarded. After the dichloromethane fully evaporated, dry masses were taken of the two beakers containing fluorene and fluorenone, as

well as an IR spectroscopy of the final fluorenone product. The mass measurements are tabulated below: Table 2: Mass of Fluorene and Fluorenone Compound Fluorene Fluorenone

Mass of Beaker (g) 29.61 21.71

Mass of Beaker with Compound (g) 29.640 21.745

Product Mass (g) 0.030 0.035

III. Results/Discussion/Conclusions The TLC plates were used in the first half of the experiment in order to determine the rate of reaction of the fluorene to fluorenone. When the intensity on the two substances was equal in size, the reaction was halted, which was at roughly 25 minutes. The calculations of percent recovery displayed relatively successful results in ending the reaction at the halfway point, losing a small fraction of the fluorene most likely in some of the discarded fractions, which showed minimal elution. The TLC however showed relatively small spots in regard to the intensity of the products, but this was most likely a result of insufficient sample placed on the origin for elution. The overall yield of the reaction is simply calculated by adding the total masses of the product and dividing by the original reactant mass. Each separate percent recovery can be calculated by using the separate masses of the products and dividing them by the original reactant mass. The percent recoveries of the reaction are calculated below: Yield Calculation: Total and product yields of fluorene and fluorenone Total Percent Yield: [(0.030 g + 0.035 g)/0.072 g] x 100 = 90.3% Fluorenone Percent Yield: (0.035 g/0.072 g) x 100 = 48.6% Fluorene Percent Yield: (0.030 g/0.072 g) x 100 = 41.7%

For the most part, this data coincides with the findings of the TLC plate, which argued the reaction was roughly at halfway to completion. As stated above, the lower yield of fluorene most likely was lost in discarded fractions, which also probably accounted for the lower overall yield of the final products.

The TLC plates in the second half of the experiment served to determine the location of the final products of the reaction. The Rf values were calculated as described above and summarized in the table. Listed below is a sample calculation of an Rf calculation of lane 3 as observed in a 20% dichloromethane in hexanes mobile phase: Sample Calculation: Rf value for lane 3 in 20% dichloromethane in hexanes Rf = distance spot traveled/distance solvent front traveled = 2.0 cm/5.6 cm = 0.357

This calculation was reused to solve for the Rf values of the other fractions as well. An examination of the TLC plates enabled one to isolate the fractions according to similar spotting. It also testified to the strength of column chromatography in separating components that enter a system, as there were distinct spots for each presence of fluorene as well as fluorenone. The first lane was used to establish a fluorene standard in order to verify the products that were being spotted on the TLC plates. The TLC plates showed like fluorene fractions at an average Rf value of 0.345, which generally dominated the start of the reaction in lanes 2 through 4. This would make sense as fluorene is less polar than fluorenone, due to the presence of the carbonyl group on the fluorenone, which would make fluorene elute faster in the nonpolar mobile phase of column chromatography setup. This value also generally correlated to the Rf value of the fluorene standard from the first lane. These spots followed by nothing showing up on the fifth lane, which would most likely indicate the reaction was occurring at this time and was in an intermediate phase. Lanes 6 through 9 as well as 10 and 11 showed signs of fluorenone, which fell under an average Rf value of 0.050. The final six lanes showed no significant spotting off of the origin, which most likely indicated they consisted of pure solvent. Those lanes were collected to ensure the entire product was collected in the reaction.

The IR analysis further reified the presence and purity of the fluorenone product in a few critical ways. First, the peak at 1708 cm-1 corresponded to the presence of a carbonyl group, which was critical in identifying the fluorenone and verifying it was not actually fluorene. The peak at 3060 cm-1 corresponds to the presence of some form of an aromatic ring structure, which would also hint towards the fluorenone structure, but was also present in the fluorene. The peak is critical in defining whether or not an aromatic structure exists within a compound, as without its presence there is generally no aromaticity. Similarly, the peak at 1450 cm-1 verified the presence of a benzene ring structure within the substance. The peak at 3392 cm-1 is most likely a result of water intrusion on the substance, as that peak generally correlates to some form of a hydroxide group, which would not really be present in either unless in some form of the transition stage. The high atmospheric absorption of potassium bromide is most likely the reason for the inclusion of such a peak, as any prolonged exposure of the material to the air would almost definitely result in the visibility of a hydroxide peak. While other peaks are displayed on the spectrum, the majority of them exist within the fingerprint region, and the ones in the functional group region do not seem to correlate with any actual functional groups. It is possible they might simply be contaminants from the beaker the fluorenone was collected in, but the size of the peaks aren’t significant enough to deem them major contaminants in the fluorenone structure. All of the above results support the strength of column chromatography as a separation technique from the percent yields to the TLC plates to the IR analysis. The relative success of the oxidation reaction is mostly shown through the high percent yield of the reaction in conjunction with the strong IR spectrum of the fluorenone product, which showed minimal impurities and definitive peaks at critical points. The TLC plates also clearly identified when the oxidation

reaction had come to its halfway point, as well as the fractions which contained fluorene versus fluorenone. These all combined to illustrate a successful oxidation reaction coupled with an efficient isolation and separation through column chromatography. IV. Post-lab Exercise 1. Write a detailed mechanism for the formation of fluorenone from fluorene. Explain the purpose of the phase transfer catalyst (PTC). Refer to your organic lecture text. The mechanism for the formation of fluorenone from fluorene is shown below:

The phase-transfer catalyst, which in this case is the Stark’s catalyst, accelerates the reaction rate of the hydroxide group on the sodium hydroxide in deprotonating the fluorene. It does this by forcing the inorganic hydroxide group out of the aqueous phase into the organic phase through the positively attached component on the salt of the catalyst, which thereby attracts a negatively charged ion which it finds in the hydroxide group. This allows the hydroxide ion to then complete the oxidation reaction as drawn above. 4. You notice that a colored band remains on the column after collecting many fractions. You believe that this band is the desired product. How can you accelerate the rate at which the compound elutes? The elution of a compound can be accelerated through either a more polar mobile phase or through flash chromatography. By using a more polar mobile phase, polar compounds

will have a greater interaction with the mobile phase, thereby traveling out of the column at a faster rate. In this experiment, it would be possible to do this by adding more dichloromethane to the solvent to counterbalance the nonpolarity of the hexanes. If the polarity is changed dramatically, it may result in cracks in the column due to the rapid change in the mobile phase, ultimately resulting in flawed separation. In using flash chromatography, nitrogen gas inserted throw the top of the column works to push the solvent throw the system at a faster pace, moving the elution at a faster rate with its decreasing volume. However, increasing the rate of elution may risk poorer separation because of the increased motion of the mobile phase. 6. A student begins to run a column at 4:00 pm. By 4:25 pm, the labs close, and the student is not nearly finished with the separation. The student decides to cap up the column in order to finish it the next day. Why is this a bad idea? By simply capping the column, the student leaves the compound within the column to filter throw until the bottom and simply accumulate, as opposed to fractioning out the column over intervals of time. By allowing the product to elute like this will result in the collection of a large fraction the next day, which would most likely result in the separation of no materials. This would require repeating the chromatography, which already is a very time-consuming process. Additionally, by capping off the column, there is a good potential the solvent may simply dry out and the student would be left with a dry column with cracks which will guarantee a poor separation. V. References Minard B.; Masters, K.; Halmi, T.O.; Williamson, K.L.; Lab Guide for Chemistry 35 & Chemistry 36, 2005-2006 Edition, p. 193-204....