Separating a Mixture of Biphenyl, Benzhydrol, and

Benzophenone by Thin-Layer Chromatography

Chem 3003/6 Laboratory
Abstract
Thin-Layer Chromatography (TLC) can be used to separate mixtures of Biphenyl,
Benzhydrol, and Benzophenone. The first procedure in this lab is to determine which solvent to
use because of the differing polarity each solvent by calculating the R​f​ differences. The solvent is
Dichloromethane. Then using the Dichloromethane, a standard R​f​ value for Biphenyl,
Benzhydrol, and Benzophenone is to be calculated. The standard R​f​ value for Biphenyl,
Benzhydrol and Benzophenone is respectively 1.00cm, 0.83cm, 0.89cm. Now, by comparing the
standard R​f​ to the observed R​f​ the identity of the unknowns can be determined. Unknown B is
Biphenyl, R​f​ is 1.00cm.
Introduction
The purpose of this experiment is to select the best suited solvent to separate a mixture of
Biphenyl, Benzhydrol, and Benzophenone using simple, inexpensive technique such as
Thin-Layer Chromatography. Then identify each compound using the calculated R​f​ value. TLC
is a technique that is used to quickly and efficiently separate quantities of compounds of less than
10 micrograms of the materials. It is a way to separate multiple solids from each other due to the
property of polarity. The Thin-Layer Chromatography will be used to investigate the polarity of
solvents on relative compounds in the mixture and also to identify unknown compounds
containing a combination of Biphenyl, Benzhydrol, and Benzophenone.This technique is in to 2
parts: the mobile phase and the stationary phase. The mobile phase carries the sample compound
through the stable medium. The stable medium is called the stationary phase.
In TLC, capillary action allows the liquid to gradually ascend a solid, stationary phase
coated with silica gel. Silica gel is the most commonly used stationary phase, SiO​2​ • nH​2​O . The
surface of Silica gel is polar which interferes with the polar compounds. A small sample of the
mixture is then applied to the bottom of the TLC glass plate at a specific small spot. This is a
preventative measure to be able to track each compound contained in the mixture. This plate is
then placed into a developing chamber. A developing chamber is a container with a shallow layer
of the mobile phase liquid (eluent). The eluent is also known as the solvent, it moves the
compounds over the silica gel surface. The distance the solvent travels is as a result of the
property of polarity. An important thing to note is to take the TLC out of the developing
chamber before the eluent reaches the top of the plate but also not take it out too early either. So
the TLC plate must be observed critically from time to time.
The stationary phase is the Silica gel, hydrated SiO​2​, it is very polar. This means that
other polar compounds will be attracted to it. The more polar the compound, the more attracted it
is to the stationary phase. The more attracted the compound is to the silica gel, the smaller the
distance it travels on the stationary phase. So a compound with very high polarity will not move
very far from the origin spot because polar molecules attract each other and a compound with
little polarity will move too far on the stationary phase. This is why an appropriate eluent is
needed. So an eluent with too high polarity will interact with the silica gel but it will cause the
compound to travel too far and not separate. An eluent with low polarity will not interact with
the silica gel, causing the compounds to barely move and not separate. After the compound have
been separated from each other, they can be viewed under UV light. Then the identification and
analysis of the results can occur using the R​f​. The calculated R​f​ values is then used to identify the
unknowns. The R​f​ is the retention factor which is the ratio of the distance that a compound
moves to the distance from the origin to the solvent front. Solvent front is the farthest point
reached by the solvent in chromatography.
Reaction schemes
Eluents:
Hexane Dichloromethane
C​6​H​14 CH​2​Cl​2
 Methanol
CH​3​OH

Sample compounds:
Biphenyl Benzhydrol
C​12​H​10 (C​6​H​5​)​2​CHOH

Benzophenone
C​13​H​10​O
Silica gel
SiO​2​ • nH​2​O

Results and Discussion

The experiment was divided into 3 parts.
Part 1
In the first part of this experiment, Thin Layer Chromatography is first performed using 3
different eluents to determine which eluent has the appropriate polarity for this procedure. So the
same mixture of Biphenyl, Benzhydrol, and Benzophenone is used in all 3 developing chambers.
On the TLC plates, one spot was made with 3 drops of the mixture slowly added to increase
visibility. This first spot marks the origin spot. Methanol, Hexane, and Dichloromethane were
each placed respectively into the 3 developing chambers. Then the TLC plate is placed into the
developing chamber and it is constantly watched. When the TLC plate is taken out, a solvent
front is marked then it is placed under UV light because the distances are not visible to the
human eye. The compound spots are marked and the distances from the spot to the origin are
measured with a measuring ruler.
Calculations for part 1
Methanol eluent:
R​f​= 3.00cm/4.00cm = 0.75cm
R​f​= 4.00cm/4.00cm = 1.00cm
Dichloromethane eluent
R​f​= 3.30cm/ 5.00cm= 0.66cm
R​f​= 4.20cm/ 5.00cm= 0.84cm
R​f​= 5.00cm/ 5.00cm= 1.00cm
Hexane eluent
R​f​= 0.10cm/ 3.90cm= 0.02cm
R​f​= 0.40cm/ 3.90cm= 0.10cm
R​f​= 2.10cm/ 3.90cm= 0..54cm
So in Methanol eluent, there were 2 visible spots which indicate that the mixture is not
completely separated. This is due to the high polarity of the eluent. If the eluent is too polar, then
all 3 spots should move closer to the solvent front and there should be no separation between the
mixture to be able to differentiate. This is what happened in the experiment. In the Hexane
eluent, the solvent is nonpolar so it does not react with the high polarity of the silica gel. The
mixture should not travel too far from the origin spot due to its movement being limited by the
polarity of the silica gel. The right solvent is the Dichloromethane solvent because it is polar
enough to interfere with the polarity of the silica gel but not too polar to remove the separation
between the mixture. Therefore, Dichloromethane is eluent with enough polarity but it is also
capable to separate and distinguish the mixture.
The data is summarized in the table below.

Eluent Solvent front distance Distance travelled by R​f​ Calculated

from origin spot mixture

Methanol 4.00cm 3.00cm 0.70cm

4.00cm 1.00cm

Dichlorome 5.00cm 3.30cm 0.66cm

thane 4.20cm 0.84cm
5.00cm 1.00cm

Hexane 3.90cm 0.10cm 0.02cm

0.40cm 0.10cm
2.10cm 0.54cm

Part 2
Now that the appropriate eluent has been solved, the order of elution has to be proved.
There will be 3 different developing chambers filled slightly with Dichloromethane eluent. There
will be 2 origin spots: one spot for the stock solution and the other spot is for the compound is
determined. A stock solution spot is added to the original spot. Each TLC plate is also spotted
with Biphenyl, Benzhydrol, and Benzophenone respectively. So that the distance traveled by the
compound can be compared to the stock solution to confirm the identity of the compound. The
values from this part of the experiment will be used to determine the identity of the unknown in
the next procedure.
Calculations for part 2
Biphenyl:
R​f​= 4.70cm/4.70cm= 1.00cm
Benzhydrol:
R​f​= 3.90cm/4.70cm= 0.83cm
Benzophenone:
R​f​= 4.20cm/4.70cm= 0.89cm
The data can also be found below:

Eluent Compound Distance Solvent Front R​f

travelled by Distance
compound

Dichloromethane Biphenyl 4.70cm 4.70cm 1.00cm

Dichloromethane Benzhydrol 3.90cm 4.70cm 0.83cm

Dichloromethane Benzophenone 4.20cm 4.70cm 0.89cm

Part 3: Determining the unknown

Thus far, Dichloromethane was determined to be the best-fitted eluent for this experiment. The
order of elution has been calculated. The same concept is then applied to the last part of the
experiment. The unknown selected was unknown B. For this procedure, the unknown is spotted
on the TLC plate which is then placed in a developed chamber containing Dichloromethane.
When the solvent climbs up close to the end of the TLC plate, it is taken out of the developing
chamber. The eluent front is marked with a pencil and the TLC plate is placed under the UV
light. There the distance the compound traveled will be measured and compared to the values in
part 2. The Rf is then calculated. Then it was determined that its values were similar to Biphenyl.
The identity of unknown B is Biphenyl.
Calculations for Part 3
Unknown B
R​f​= 5.30cm/5.30cm= 1.00cm
The data is summarized below

Eluent Test for: Solvent front Distance travelled by R​f

Distance the unknown

Dichloromethan Unknown B 5.30cm 5.30cm 1.00cm

e

Conclusion
The experiment was divided into 3 stages, the stages are interconnected and have to go in
the right order. The first stage was determining the eluent to use in the experiment. The choice of
eluent affects the experiment in a way that choosing the wrong eluent will completely ruin the
experiment. The second stage was used to discover the order of elution of the compounds. The
information determined in this stage is then used in the next stage to deduce the identity of the
unknown.
Post Lab questions
1. (a) Hexane, Toluene, Dichloromethane, Ethyl Acetate, Methanol
(c) The more polar compounds have higher R​f​ compared to the compounds with low
polarity. This is due to the attraction of the compound to the eluent used. The stronger the
attraction of the compound to the eluent, the longer the distance travelled. The weaker the
attraction, the shorter the distance travelled by the compound.
2. Dichloromethane was chosen as the best eluent because it caused separation between the
compounds in the mixture and each mixture can be clearly distinguished. Hexane wasn’t
chosen because its polarity is too low hence there was no separation of the mixture. The
compounds barely moved from the origin spot due to how nonpolar Hexane is. The
polarity of the silica gel severely limited the movement of the compounds. Methanol
wasn’t chosen because its polarity is too high. The high polarity of Methanol caused the
polarity of the silica gel to be less effective because polar molecules attract each other. So
the compounds ascended so high on the TLC plate that it became blown out. 2 of the
compounds did not separate, this is evidence of an inappropriate eluent for this
experiment.