Thin Layer Chromatography and Column Chromatography Experiment

Thin Layer & Column Chromatography
By: Lisa Mickey

Introduction

                Thin layer chromatography (also known as TLC) is the physical separation of a mixture into its individual components by distributing the components between a stationary phase (the porous TLC plate) and a mobile phase (the solvent that moves through the stationary phase and carries the material that needs to be separated.  The driving force to separate components is capillary action.  This method can be used to determine how many different components (usually non-volatile) are in a sample.  Column chromatography is a method to physically separate all of the components (also usually non-volatile) of a mixture.  The driving force to separate components is gravity.  Both of these methods work based on polarity differences between components in a sample.

Procedures and Observations

                In order to perform thin layer chromatography, seven porous plates needed to be obtained that were in good condition with no chips or cracks.  These plates functioned as the stationary phase.  With pencil, two lines were drawn on each of the seven plates.  One line was drawn 3 mm from one end, and the other line was drawn 12 mm from the other end.  The line 3 mm from the end served as the “end” line, and the line 12 mm from the other end served as the “start” line.  2 mL of the orange solution for TLC analysis was obtained in a small beaker.  A very thin capillary was obtained and dipped into this solution.  The filled capillary was touched in the middle of the “start” line on each of the seven plates.  The capillary was not held to the plate, but rather just tapped, so the dot of the orange solution on the plate did not have a diameter of more than 3 mm.  At this point, seven different solvents needed to be prepared for each of the seven plates and put in a beaker.  Solvent 1 was 5 mL of petroleum ether.  Solvent 2 was a mixture of 5 mL of petroleum ether and 0.5 mL of ethyl acetate.  Solvent 3 was a mixture of 4 mL of petroleum ether and 1 mL of ethyl acetate.  Solvent 4 was a mixture of 3 mL of petroleum ether and 2 mL of ethyl acetate.  Solvent 5 was just 5 mL of ethyl acetate.  Solvent 6 was just 5 mL of diethyl ether.  Solvent 7 was just 5 mL of dichloromethane.  Each of the seven plates was placed in a different beaker with a different solvent.  The plates were labeled 1 through 7, based on the solvent in which they were placed.  Each of the seven beakers was covered in order to prevent evaporation while the solvents soaked up the plates.  Each of the plates was allowed to sit in the beaker until the solvent front soaked up to the “end” line drawn on each of the plates.  They were then removed and observed under the UV light.  Each observed spot under the UV light was circled in pencil.  Each plate, once again labeled 1 through 7 in order, appeared as shown:

In order to perform column chromatography, a mixture of 0.509 g of silica gel and 2 mL of unknown #2 was prepared and allowed to dry in a drawer for a week.  This left only a dry, orange powder for the experiment.  Because of these measurements and the information provided, it was known that 75 mg of azobenzene (melting point: 68 ˚C) and 75 mg of an unknown compound was present.  The unknown compound was either  ortho-, meta-, or para-nitroaniline (melting points were 71.5˚C, 112.5˚C, or 149˚C, respectively).   After a week passed the only the orange powder was left, the column chromatography apparatus was assembled.  A column containing a fritted disk and a stop cock was assembled with two clamps to stand vertically in a fume hood.  The column was assembled as to allowed for a 50 mL Erlenmeyer flask to fit under it for collection.  A funnel was also placed in the top of the column.  The three Erlenmeyer flasks to be used for collection of components of the sample were pre-weighed.  They all weighed in at 39.8 g.  The two mobile phases to be used to separate the components were prepared.  The first mobile phase, or eluent, was a mixture of 30 mL of petroleum ether and 1.5 mL of ethyl acetate.  The second mobile phase, or eluent, was just 20 mL of ethyl acetate.  2.04 g of silica gel was obtained and placed in a 10 mL Erlenmeyer flask and 5 mL of the first mobile phase (the mixture of petroleum ether and ethyl acetate) was added to the silica gel in the 10 mL Erlenmeyer flask.  This formed a white slurry.  1 mL of the first eluent was added to the column via pipet and the stop cock was closed.  The column was tilted and the slurry was added to the column.  1 mL of the same eluent was added to the Erlenmeyer flask with the residual silica and was swirled.  The remaining silica was poured into the column.  No air bubbles were present in the silica gel.  Some of the eluent was allowed to drain into a beaker since over 15 mm of eluent was over the silica gel.  The draining was stopped when about 4 mm of eluent was over the silica gel.  At this point, the orange powder was added to the column and the draining was allowed to begin again.  The first eluent was continuously being pipetted into the column in order to prevent the silica from drying.  As eluent was being added, gravity pushed the first band of orange down the column.  When the first band reached the fritted disk, the first Erlenmeyer flask for collection was placed under the column.  The first eluent continued to be added until all of the first band of the sample was collected in the first Erlenmeyer flask.  It took about 10 minutes for the first orange band to move down the column and be collected.  Once this first band was fully collected, a new Erlenmeyer flask was placed under the column for collection.  An orange band lighter in color than the previous band was collected in this flask.  A white band containing neither component of the sample appeared in the gel.  Below the white band was the lighter orange band being collected in the second Erlenmeyer flask.  Above it was another darker orange band containing the component of the sample to be collected in the third Erlenmeyer flask.  When the remainder of the orange band below the white band was collected, the eluent needed to be changed to the solvent containing just ethyl acetate.  This was what caused the final band to start moving down the column.  As the third orange band, approached the fritted disk, the third Erlenmeyer flask was placed under the column to collect the final component of the sample.   After all three samples were collected, three TLC plates were collected.  A thin capillary was dipped in each of the samples.  With pencil, two lines were drawn on each of the three plates.  One line was drawn 3 mm from one end, and the other line was drawn 12 mm from the other end.  The line 3 mm from the end served as the “end” line, and the line 12 mm from the other end served as the “start” line.  Each of the filled capillaries was touched in the middle of the “start” line on each of the three plates.  The capillary was not held to the plate, but rather just tapped, so the dot of the solution to be tested on the plate did not have a diameter of more than 3 mm.  The mobile phase, or solvent used for TLC on each of the three plates was a mixture of 4 mL of petroleum ether and 1 mL of ethyl acetate.  The solvent was allowed to soak each of the plates to the end line, and then the plates were removed and observed under the UV light.  Pencil was used to circles the marks that appeared on the plates under the UV light.  The plates were observed (the first fraction being on the left, the second fraction being in the middle, and the third fraction being on the right) as follows:

Based on the observations made under the UV light, the second fraction collected actually contained the same component as the first fraction.  The second and first fractions were combined into one Erlenmeyer flask.  The two Erlenmeyer flasks containing the two different components of the sample were left in a drawer for a week to dry.  The following week the melting points were tested for each of the two dried components.  0.015g of the red-orange component was obtained and the observed melting point of this component was 66˚C to 69˚C.  0.022g of the yellow-orange component was obtained and the observed melting point of this compound was 108˚C to 113˚C.

Discussion

                The results show that polarity of the eluent is the deciding factor as to how far a component of a sample will travel on a TLC plate. If the mobile phase is very non-polar, the non-polar components of the sample will travel farther up the TLC plate than the polar components.  If the mobile phase is very polar, the polar components will travel farther up the TLC plate than the non-polar components.  Petroleum ether is very non-polar and ethyl acetate is polar.  Diethyl ether is polar and dichloromethane is also polar.  None of the seven plates had all four spots resolve under the UV light.  When the mobile phase was strictly petroleum ether, the spot did not move at all on the TLC plate.  As ethyl acetate was added in higher concentration to petroleum ether as the mobile phase, in general, more movement was observed in the spots.  Also, a lot of movement was observed when diethyl ether and dichloromethane were used as the mobile phases.  In general, this shows that the components making up the sample were more polar than non-polar, but each had a different polarity from the other.  The eluent that worked best was solvent 3.  This was a mixture of 4 mL of petroleum ether and 1 mL of ethyl acetate.  The eluent was polar enough for separation, but not too polar like for plates 4 through 7 where only one spot could be observed.  Only plates 2 and 3 showed more than one spot, but they still only showed three spots instead of four.  This could be because of the eluents not being properly covered, so they evaporated during the experiment.  Also, some plates could have been left in the mobile phase for too long and not taken out at the right time.  The Rf values for the spots on plate 2 were 0.044, 0.162, and 0.471 respectively.  The Rf values for the spots on plate 3 were 0.044, 0.103, and 0.191 respectively.  Since none of the seven plates had four resolved spots, the TLC spots could not be assigned a substance.

The results show that polarity of the eluent is the deciding factor as to how quickly a component of a sample will travel down a column for a collection in column chromatography.  If an eluent traveling through a silica gel is less polar, the less polar component will travel down the gel quickly for collection, while the polar component stays stationary in the gel.  A more polar eluent was necessary to start the more polar component of the sample to travel down the gel for collection.  The first and middle fractions collected were azobenzene.  This is because the melting point of this sample was observed to be 66˚C to 69˚C, and the literature melting point of azobenzene given in class was 68˚C. Since there was originally 75 mg of azobenzene present in the sample and only 0.015 g was obtained, a recovery rate of 20% was calculated.  The other fraction collected via the more polar eluent was meta-nitroaniline because the observed melting point was 108˚C to 113˚C and the literature melting point given in class of this compound was 112.5˚C.  Since there was originally 75 mg of meta-nitroaniline present in the sample and only 0.022 g was obtained, a recovery rate of 29.3% was calculated.

Calculations and Figures

Recovery rate:

Azobenzene- 0.015 g = 15 mg

(15 mg obtained/ 75 mg originally in sample) x 100% = 20. % recovery rate

Meta-nitroaniline- 0.022 g = 22 mg

(22 mg obtained/ 75 mg originally in sample) x 100% = 29% recovery rate

Rf Values for plates with more than one resolved spot: