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Steve Metz Chem 0330 John Kavouris October 29, 2012 Abstract In this lab exercise, column chromatography

was used to separate the components of spinach extract. The column was prepared using alumina, a very polar, solid stationary phase, and an elution gradient of a series of solvents for the mobile phase, beginning with nonpolar 9:1 hexane/acetone mixture. The components, then, of the spinach extracted were separated based on differences in polarity. This components were then analyzed along with the raw spinach extract via thin layer chromatography, in which the composition of the extract was established as well as the relative polarities of the different components by analyzing retention factors. Introduction Although distillation was once an important lab technique for separating mixtures, the separation and analysis of mixtures, today, is largely carried out using chromatography techniques. Chromatography, in a very basic sense, is the separation of a mixture into its components based on differences in physical and chemical properties. Though there are numerous specific types of chromatography, all types of chromatography are based on a stationary phase and a mobile phase. The idea being that differences in physical properties will cause the different components to adhere either to the stationary phase or the mobile phase, thus leading to separation. This idea is common through all types of chromatography. It is often differences in stationary phases that differentiate the different types of chromatography. For instance, in gas chromatography the stationary phase is a very thin layer of a non-volatile organic

compound, in its liquid phase, which is coating a solid column. The mobile phase, for gas chromatography, is an inert gas. The sample is vaporized when it is introduced to the gas chromatograph, the sample then equilibrates between its liquid and gas phases1. The components of the mixture that prefer the mobile gas phase will move through the column faster than the components of the mixture that prefer the stationary liquid phase. In general, compounds with lower boiling points prefer the mobile phase and elute faster2. Additionally, separation can be tailored by varying the liquid mobile phase, as different polarities, molecular weights, and functional groups will lead to separation. Further, gas chromatography is used to determine the relative amounts of components of a mixture via separation and test the purity of a substance. Column chromatography, on the other hand, differs from gas chromatography in that the stationary phase of column chromatography is solid and the mobile phase is liquid. Another major difference is the main physical property upon which the separation is based. For column chromatography, the separation is based mostly on polarity. The solid stationary phase used, generally silica gel or alumina, is very polar in nature. The size of the solid stationary phase is important. A particle that is too small will result in the solvent draining too slowly, while a particle too large will jeopardize the separation3. The liquid mobile phase, on the other hand, is typically a series of eluents of varying polarity. The elution gradient generally begins with very nonpolar eluents, such as hydrocarbons, and uses a series of increasingly polar eluents. These eluents are often mixtures of non-polar and polar solvents, allowing a wide and precise range of polarities. The theory being that the more polar fractions of the mixture will adhere to the stationary phase while the less polar fractions will be carried down the column by the less polar

Padias, Anne B. Making the Connections2: A How-To Guide for Organic Chemistry Lab Techniques, 2nd ed.; Hayden-McNeil: Plymouth, 2011. 2 Ibid. 3 Ibid.

eluents and collected. As these less polar fractions elute, eluents with increasing polarity will be used to carry the more polar fractions down the column. In this lab, column chromatography was utilized to separate the components of spinach extract. The setup for this lab utilized alumina powder as the stationary phase and the following eluents listed in order of increasing polarity: 9:1 hexane/acetone, 1:1 hexane/acetone, and methanol4. The least polar eluent, 9:1 hexane/acetone mixture would carry the non-polar components of the spinach extract down the column to be collected while the polar components would be left behind. As fractions are collected, the elution gradient will be followed until separation has been achieved and all fractions have been collected. As mentioned, the applications of column chromatography differ from the applications of gas chromatography. Column chromatography is typically used in conjunction with thin layer chromatography. The column chromatography separates the mixture into its components, these components are then analyzed using thin layer chromatography, which can check the effectiveness of column chromatography or be used preemptively to determine the appropriate solvent for column chromatography. Thin layer chromatography is also often used to monitor the progress of a reaction, and can also determine whether or not two compounds are identical5. Thin layer chromatography, while still relying on differences in physical properties to separate a mixture and having a solid stationary phase and liquid mobile phase like column chromatography differs in the number of solvents used. A single solvent is used in thin layer chromatography, and this solvent often must be determined experimentally. A desirable solvent will result in separation. The solid stationary phase for thin layer chromatography are very polar absorbents attached to the back of a thin layer chromatography

Huston et al. Chem 0330: Organic Chemistry I Laboratory Manual, Fall 2012-Summer 2013; Hayden-McNeil: Plymouth, 2013. 55 Padias, Anne B. Making the Connections2: A How-To Guide for Organic Chemistry Lab Techniques, 2nd ed.; Hayden-McNeil: Plymouth, 2011.

plate, silica gel and alumina, again, are preferred. The samples, often fractions produced via column chromatography, as was the case for this lab, are spotted onto the TLC plate, in small concentrated spots. The TLC plate is then developed using a solvent, as this solvent is drawn up the plate via capillary action, the less polar samples will prefer the mobile phase and move further up the plate than the more polar samples. The distance the sample moves is often described in a ratio against the total distance the solvent travels up the plate, this value is an Rf value. Thin layer chromatography was employed in this lab to analyze the components of spinach extract that had been separated via column chromatography and concentrated. Different types of chromatography differ in their processes and applications, but all rely on differences in physical properties to partition a mixtures components into a stationary and mobile phases in order to achieve separation. Reagent Table
Compound Hexane Acetone Structure M.W. (g/mol) 86.18 58.08 Amount 45 mL 25 mL 5 mL 25 mL Moles .34 .19 .07 .34 Density (g/mL) .6548 .791 m.p/b.p (C) -96/68 -95/56



50 mL






11 g




Ethyl Acetate




Experimental The three eluents were prepared for the column chromatographic separation. Each of the three eluents was mixed in clean 250 mL beakers. The 9:1 hexane/acetone eluent was prepared thoroughly mixing 45.1 mL of hexanes and 5.2 mL of acetone. 25.2 mL of hexanes and 25.0 mL of acetone were used to prepare the 1:1 hexane/acetone eluent. 50.2 mL of methanol was measured into another 250 mL beaker for use as the final eluent. Next, the alumina slurry was prepared by mixing 11.02 g of alumina with entirety of the 9:1 hexane/acetone eluent. Concurrently, the column was prepared by loosely placing a cotton ball in the bottom of the column that had been secured with two clamps, followed by approximately 1 cm of sand. The alumina batter was then poured into the column, the runoff was collected in a separate beaker and used to clean the sides of the beaker in which the alumina batter was mixed in order to insure all alumina was placed into the column. At this point, the stopcock was opened, and a separate 250 mL beaker was used to collect the runoff. Next, another 1 cm of sand was placed into the column. It is important to note that the column was kept moist by continually running eluent through it. Next, roughly 2 mL of spinach extract was added to the column via a pipet, followed by approximately 1 mL of he 9:1 hexane/acetone solvent, also added via pipet, slowly. Once the extract had migrated to the alumina, the 9:1 hexane/acetone eluent was continuously added. The extract was monitored as fractions formed, 9:1 hexane/acetone eluent was used until the first fraction was collected in a clean beaker. Following the collection of the first fraction, 1:1 hexane/acetone eluent was employed until the second fraction had eluted from the column. Methanol was then used as the final eluent until the third and fourth fractions were collected in a clean beaker. The four fractions were then boiled on the steam bath until thoroughly concentrated and reduced; boiling stones were used. Several drops of acetone were added to each

of the samples. As the samples boiled, a thin layer chromatography plate was prepared by tracing an origin line in pencil and marking spots for each of the four samples in addition to a spot for the whole spinach extract. The developing chamber was prepared by adding roughly 1 cm of 30% ethyl acetate in hexanes into a clean 400 mL beaker with a piece of filter paper folded in half, serving as a wick. The TLC plate was then spotted with each sample and the spinach extract using capillary tubes. The spotting was carried out by gently, albeit quickly, tapping a loaded capillary tube on the TLC plate in order to generate small, concentrated spots. The prepared TLC plate was then loaded into the developing chamber and covered. The TLC plate was allowed to develop until the solvent had migrated to about 3 cm from the top of the plate. The solvent front was marked with a pencil and the spots were circled for analysis later. Results The first fraction collected was very pale yellow. The second fraction collected was also very pale yellow and difficult to distinguish from the first fraction. The third fraction was very pale green and the third fraction was pale green. The figure below is a sketch of the developed TLC plate with each spot identified. The table accompanying the figure indicates the Rf values for each of the spots. Figure 2.1 Developed TLC Plate

Table 2.1 Rf Values Accompanying Figure 1.1. Spot 1 Rf Values


Spot 2 .23

Spot 3 .51

Spot 4 .92

Spot 5 .92

Spot 6 .51

Spot 7 .098

Spot 8 .51

Spot 9 .15


Rf=distance spot traveled/distance of solvent front = .6 cm/6.1cm = .098

Discussion The goal of this lab was to separate spinach extract into its components using column chromatography and analyze this components using thin layer chromatography techniques. The eluents, in increasing polarity, used for the column chromatography were a 9:1 hexane/acetone solvent, a 1:1 hexane/acetone solvent, and methanol. A 9:1 hexane/acetone mixture, that is 90 percent hexanes and 10 percent acetone, is a very nonpolar solvent. This contrasted heavily with the very polar solid alumina mobile phase. Thus, any components of the spinach extract that were not very polar in nature would have preferred the mobile phase and eluted through the column. The solvents used increased in polarity in order to elute increasingly polar components of the extract. The acetone was added to the concentrated fractions in order to have enough of each sample to spot the TLC plates with. Analysis of the TLC plate reveals that the raw spinach extract contained four components. The Rf values of these components were .098, .23, .51, and .92. Spot 5 from fraction 1 had an Rf value of .98, thus, it is likely that spot 5 was the same component as spot 4 from the raw spinach extract. Spot 6 from fraction 2 had an Rf value of .51, matching spot 3 from the raw spinach extract, a similar conclusion can thus be made that the component isolated in fraction 2 is the same compound as spot 3 from the raw spinach extract. The separation achieved using column chromatography was not ideal, however. The third

fraction, when spotted, resulted in two spots on the developed TLC plate. Thus, it can be concluded that fraction three actually contained two components of differing polarity, leading to the two spots on the developed TLC plate, spots 7 and 8, and two separate Rf values, .098 and .51 respectively. Thus, fraction 3 was not perfect separation; it contained two different components. Further, these spots matched spots 1 and 3 from the raw spinach extract, which had identical Rf values of 0.98 and .51. This matching makes sense, as fraction 3 was isolated from the raw spinach extract. The addition of the first eluent too soon after the addition of the extract could account for this error. Additionally, fraction 4 contained a spot with an Rf value of .15, which is close, though not exactly matching the Rf value of .23 from spot 2. It is difficult to conclude, then, that these compounds are the same. A possible source of error for this inconsistency is the over or under reduction of fraction 4, leading to an inconsistent reaction with the very polar alumina backed TLC plate. It is also noteworthy that fraction 1 contained the component of the mixture that traveled the furthest, fraction 2 contained the component that traveled the second furthest, fraction 3 contained two components, one of which matched the component isolated in fraction 2 and another component, that traveled the shortest distance. Fraction 4, finally, contained the component that traveled the third furthest, though it is notable that the component isolated by fraction 4 and one of the components from fraction 3 were very close in terms of distance traveled. Given that the solid stationary phase, alumina backing, for thin layer chromatography is very polar, and the liquid mobile phase was relatively nonpolar, the components that were less polar in character would travel further, and thus have higher Rf values. It is unsurprising then, that fraction 1, the fraction that eluted first during column chromatography in which it preferred the least polar solvent, traveled the furthest, confirming its relatively nonpolar nature. Similar conclusions follow for fractions 2 and 3. However, the

component isolated by fraction 4 traveled slightly further than component represented by spot 7 in fraction 3. Thus, the conclusion would be that the component isolated by fraction 4 is less polar than the component represented by spot 7, even though fraction 3 before fraction 4 during column chromatography. This error could possibly be explained by the use of methanol, a very polar solvent, to elute both the third and fourth fractions during column chromatography.