The use of Column Chromatography to separate two compounds from a solution The use of Column Chromatography to separate two compounds from a solution
Keithen Bailie Cast Keithen Bailie Cast
Introduction: Introduction:
Column chromatography is a method to separate or purify solutions t
Column chromatography is a method to separate or purify solutions t hrough a chromatographichrough a chromatographic column. The purpose of this lab is
column. The purpose of this lab is to understand the separation process of different compounds usingto understand the separation process of different compounds using column chromatography and organic properties. Column
column chromatography and organic properties. Column chromatography is defined as a chromatography is defined as a separationseparation method in which compounds migrate down a column at
method in which compounds migrate down a column at different rates. This process is used in bothdifferent rates. This process is used in both separation and purification of chemistry and biopharmaceuticals (2). Similar to thin layer
separation and purification of chemistry and biopharmaceuticals (2). Similar to thin layer chromatography, column chromatograph
chromatography, column chromatography has both a y has both a mobile and stationary phase in order to mobile and stationary phase in order to separateseparate out different compounds. This column is usually glass
out different compounds. This column is usually glass or plastic packed with a stationary phase thator plastic packed with a stationary phase that most often is silica or alumina.
most often is silica or alumina. On top of the column On top of the column there is a funnel where the mobile phase is there is a funnel where the mobile phase is addedadded and correspondingly at the bottom of the co
and correspondingly at the bottom of the column there is a valve to control outflow speed. lumn there is a valve to control outflow speed. In this labIn this lab the stationary phase was alumina. The stationary phase stays t
the stationary phase was alumina. The stationary phase stays t he same through the entire experiment,he same through the entire experiment, it is the constant.
it is the constant. The mobile phase is typically a solvent that allows The mobile phase is typically a solvent that allows certain substances to migrate. Thiscertain substances to migrate. This solvent is called an eluent (eluent buffer) this is
solvent is called an eluent (eluent buffer) this is used to separate compounds from each other orused to separate compounds from each other or ““eluteelute them
them” (1)” (1).. The column is created by a technique called the “slurry” method. This process is usingThe column is created by a technique called the “slurry” method. This process is using hexanes mixed with alumina to create and equ
hexanes mixed with alumina to create and equal packing layer for the column. al packing layer for the column. The mixture suspendsThe mixture suspends the alumina particles in the hexane liquid, which allows the
the alumina particles in the hexane liquid, which allows the particles to pack into the chromatographicparticles to pack into the chromatographic column equally (1). This method decreases the chance of alumina packing to
column equally (1). This method decreases the chance of alumina packing to tightly which can happentightly which can happen when directly pouring alumina into the column. As
when directly pouring alumina into the column. As in thin layer chromatography, the mobile phase is ain thin layer chromatography, the mobile phase is a solvent that has intermolecular forces that are favourable to one or more of the compounds allowing solvent that has intermolecular forces that are favourable to one or more of the compounds allowing migration through the stationary phase. There are two different mobile phases in this lab.
migration through the stationary phase. There are two different mobile phases in this lab. First, hexaneFirst, hexane is used to separate out one
is used to separate out one component of the solution and the second was 50/50 hexane/butyl methylcomponent of the solution and the second was 50/50 hexane/butyl methyl ether.
This thin layer chromatography is an extra step to ensure correct results. A sample of each compounds were placed on a TLC plate. The TLC plate has a stationary phase that is silica and the mobile phase was 30:1 toluene: ethanol. Both of the compounds were run through the TLC method twice since they w ere particularly dilute. Both compounds have color when they are concentrated, but due to the dilution each compound was not visible to the eye. Therefore in order to see migration distance UV light was used to track the compounds. The TLC plates contain f luorescent properties that appear when exposed to the 254nM of light (1). Using the retardation factor (Rf value) and an evaluation of each molecules intermolecular organic properties, the compounds will be i dentifies (equation 1). Organic properties for instance: Hydrogen bonding, polarity, dipole, and London forces all influence migration through the chromatographic column and the TLC plate (2). The compatibility of mixture compared to the mobile phases versus the stationary phase is highly impacted by polarity in this lab. Polarity form differing of electronegative charges in a compound resulting in a non neutral compound. This charge is from asymmetry and differing electronegativity of atoms inside the molecule. Highly polar solvents will
dissolve highly polar solutes, thus allowing more significant migration through the TLC and speed. This is an example of the rule “like dissolves like” (2). Other factors such as molecular orientation, size, and weight all impact the speed (column chromatography) and distance (TLC) of migration (3).
Rf= (migration distance of compound mM)/ (migration distance of solvent mM) Equation1
Results:
Subsection 1, Chemical Structures and Formulas: Figure 1, Structures of compounds separated
Name Formula Structure
Acetylferrocene [Fe(C5H4COCH3)(C5H5)]
Figure 2, Mobile and stationary phases
Name Formula Structure
Alumina (stationary column) Al2O3
Silica (stationary TLC) SiO2
Hexane (mobile Column) C6H14
Butyl Methyl Ether (Mobile) C5H12O
Toluene C7H8
Ethanol C2H6O
Subsection 2, Data Tables (3) Table 1, Retardation factor values
Yellow ( 1st through column) Orange (2nd)
TLC #1 Rf value 0.971 0.057
TLC #2 Rf value 0.947 0.078
In table 1, Rf values were calculated by equation1 and sample calculations shown in subsection 3. Both runs were done directly after the column chromatography to ensure accurate and proper separation was achieved.
Subsection 3, Sample calculations
Retardation factor for each TLC was as shown:
Rf= (migration distance of compound)/ (solvent front migration) Rf=36.0mM/38.0mM
Rf=0.947 (1)
Discussion:
The purpose of this lab was to understand the mechanism of column chromatography and how/why molecules migrate at different speeds when the same force of gravity is acting upon them. Part one of this lab was column chromatography. Once the column was set up correctly the TA put six droplets of the solution, containing Acetylferrocene and Ferrocene, into the column. The chromatography began by
first adding hexanes to separate Ferrocene out. The migration of Ferrocene was easy to visualize due to its yellow color. The movement of Ferrocene was directly related to the solvent used. The solvent, hexane, has a non polar affinity “like dissolves like” therefore moved the Ferrocene through the
stationary phase while the polar Acetylferrocene did not migrate. The stationary phase, alumina, is very polar and has no affinity for non polar Ferrocene. Ferrocene has a very symmetrical structure, as seen in figure 1; this permits efficient movement through the both phases resulting in quicker travel down the column. Acetylferrocene did not travel with the hexane mobile phase. This is because Acetylferrocene is polar due to the functional group off of one of the five carbon rings. This polar group is a ketone and because of the Oxygen atom ketone has a substantial electronegativity gap between C and O. Also hybridization affects the polarity due to sp3 trigonal planer geometry. This geometry has 120 degrees bond angles creating an asymmetrical charge distribution. This negatively charged functional group creates a net dipole toward the Oxygen in Acetylferrocene. Furthermore, the functional group creates asymmetry on the Acetylferrocene molecule (resulting in polarity). The second part of column
chromatography was to elute Acetylferrocene down through the column. In this step a solvent that was 50/50% hexane and butyl methyl ether. This solvent moved Acetylferrocene through the column
because of the 50% butyl methyl ether’s polarity (figure 2). The compound did eventually move through the column, but at a more gradual pace compared to Ferrocene. This observation suggested that the stationary phase, concentration of the mobile phase, and structural composition all effected movement down the column. The highly polar stationary phase, alumina, has an affinity for Acetylferrocene. This “competition” for the polar side of the molecule is one reason for the difference in migration time between the compounds. The mobile phase used to elute Acetylferrocene was 50/50 concentration (50% butyl methyl ether is the “important solvent”), compared to 100% hexane concentration to move Ferrocene. Consequently this solvent: solute relationship lessened interaction between Acetylferrocene and butyl methyl ether (approximately 50% slower). The last rationalization is the structure of
passage down the column more fractious in comparison to a more linear compound. After both
compounds were collected each one was submitted for further analysis by a TLC test. In the thin layer chromatography test, a drop of each compound was placed on a TLC plate until the concentration was potent enough for viewing under UV light. The TLC plate was coated in silica, a highly polar stationary phase. The mobile phase, 30:1 toluene: ethanol mixture (figure 2), was used to test the migration of each substance based on their individual organic properties. Toluene, as seen in figure 2, is a non polar solvent mixed with ethanol a compound that has H bonding capabilities. This mixture of solvents is beneficial for the migration of Ferrocene. This highly concentrated solvent (30:1) interacts well with Ferrocene, because toluene is a non polar molecule. Ferrocene moves easily through the stationary silica (very polar) on the TLC plate as a result of the molecules symmetrical, non polar nature. Ethanol, miscible in toluene, has a polar molecular structure as seen in figure 2. Along with polarity the ethanol contains a hydroxyl group (OH), thus giving H bonding capabilities. T he intermolecular properties of ethanol have little to no effect on the movement of Ferrocene. However, ethanol is directly linked to the migration of Acetylferrocene. Acetylferrocene has an oxygen atom on the ketone functional group which facilitates hydrogen bonding with hydroxyl group from ethanol. Consequently, from the H
bonding and polarity similarities, Acetylferrocene does migrate through the silica stationary phase. Acetylferrocene migrates a fraction of the distance that Ferrocene travels (table 1). This suggests three probable explanations for the migration gap between the two compounds. First, the mobile phase was 30:1 toluene: ethanol, this low concentration of ethanol is nearly insignificant compared to the highly abundant toluene. Second, the highly polar stationary phase has an affinity for polar Acetylferrocene. This means Acetylferrocene spent a greater amount of time in the stationary phase than Ferrocene. The third rationale is because of the structure of Acetylferrocene. Compared to Ferrocene, the structure of Acetylferrocene is more bulky, reducing movement speed through the s tationary phase.
Error analysis of this lab involved both random and systematic error. Systematic error results from instruments that measure inaccurately. Possibilities of systematic error could be any of the
following: measuring stick (TLC data), column apparatus, amount of alumina measured, the
concentration of the solution (Ferrocene/Acetylferrocene). The results in this lab do not suggest that systematic error occurred. Random error is the irreproducibility of making measurements, thus
effecting precision of collected data. Potential random errors are: thickness of silica coat on TLC plate, equal dispersion of alumina in the chromatographic column, fluorescent indicators in TLC plate. Errors that are random are usually eradicated by averaging data values and/ or removing outliers.
Consequently, in lab it is important to have multiple trials to ensure results are accurate and precise as possible.
Reference:
1. Haynes, W M; Lide, David. Handbook of Chemistry and Physics. Hbcpnetbase [Online] 2011, 8, 1-3. http://www.hbcpnetbase.com.proxyau.wrlc.org/
2. Williamson, K; Milnard, D; Masters, K.Column Chromatography. Macroscale and Microscale Organic Experiments. Houghton Mifflin Company: Boston, NY, 2007; 184-200
3. Xiong Cariao; Zhou Xiayo, et al. Characteristics of column packing materials in high
performance liquid chromatography by charge detection. Analytical Chemistry 2011 [online]. 83, 13, 5400-5406. http://web.ebscohost.com/ehost.
