Unit 2 Assignment C
Chromatography
I was given the task of performing a variety of chromatographic techniques to separate and identify
different compounds within substances. I had to test the amino acids glycine, leucine, and lysine to
figure out an unknown compound labelled X using paper chromatography. I then used thin layer
chromatography to determine the compounds of extracted pigment from a geranium plant.
(P6) What is Chromatography?
Chromatography is a technique used to separate compounds from a mixture to allow for chemical
analysis to occur and to identify certain substances within the mixture. There are many different
forms of chromatography; gas chromatography and high-performance liquid chromatography are
often used within the industry as they are more sensitive and accurate when separating and
identifying compounds compared to paper and thin layer chromatography which is usually used in
schools and colleges. Chromatography is able to work as mobile phases separate out the compound
over a stationary phase. A mobile phase is a liquid that transports a mixture across an absorbent
solid material whereas the stationary phase is the solid material that absorbs the substances. In a
given space of time, different substances would travel different distances as some move faster than
others. This is because each chemical compound has a unique attraction to the mobile and
stationary phase.
Thin layer chromatography (TLC) is a more effective and accurate method than simple paper
chromatography as it can be used on a greater range of compounds and molecules. Paper
chromatography is a simple method that usually uses water as its mobile phase and paper as its
stationary phase. It is usually used in schools as it is simple to prepare and conduct and can easily
separate out any substances based on their solubility. It often gives clear visual results, or the results
can be easily viewed under an ultraviolet light. Even though it is a simple and fairly effective method
to use, it has a low range of possible uses as only a small number of substances can be used as they
need to be water soluble. On the other hand, TLC allows for a greater application of use as the plate
is coated in a thin layer of silica gel. This gel contains different hydroxyl groups which attract
substances differently based on their polarity. Molecules which are more polar move slowly up the
TLC plate as they have a great attraction to the silica gel therefore, they travel less distance, however
nonpolar molecules have a weaker attraction to the hydroxyl groups so are able to travel higher up
the plate at a greater speed. TLC often requires an aid to be able to view the results. Usually a UV
light, iodine, or a chemical spray such as ninhydrine are used in order for the different pigments to
be able to be seen. TLC is usually used in higher levels of education as it is not only quicker than
paper chromatography, but it also gives a more accurate indication of the progress of a reaction.
Both paper chromatography and thin layer chromatography can help to understand the purity of a
product or substance. In order to test the purity of a substance you can add a small sample of your
product, a completely pure version of your product and the reactants that were used to create the
product. Then using either form of chromatography, you can determine the products purity as a
pure substance should only have 1 spot appear up the paper/plate. If another spot appears then it is
clear to determine that the product is impure. Using the molecules RF value (Retention Factor) you
can determine the exact reactant(s) that are still present as each molecule will have its own specific
RF value. So, if any two spots from different samples have the same RF value, then you can
determine they both contain the same substance. The RF (Retention Factor) value of a molecule is
, calculated by dividing the distance travelled by the compound to the distance travelled by the
solvent, all the values should be less than 1.
Risk assessment-
Before completing my experiments, I conducted a risk assessment to identify any possible hazards
and know what to do in the event that they occurred. Firstly, I had to wear a lab coat and goggles to
protect my eyes and clothes from any potential splashes of fluid. I also tied back my long hair so that
I could clearly see what I was doing and to prevent any possible contamination of my hair onto the
chromatography paper and plate. There was also the risk of broken glass and receiving cuts as I was
working with a beaker, watch glass and capillary tube. Therefore, I had to handle my equipment with
care to prevent any breakages and accidents.
(P5) Paper Chromatography of Amino Acids-
The first experiment I conducted was identifying an unknown substance using paper
chromatography. I knew that the substance was a mixture of either glycine, leucine, or lysine. To find
out the two amino acids within my unknown sample, I performed this method of chromatography as
I knew it would be able to separate out the mixture. To conduct my experiment, I first took a piece
of chromatography paper and, about 1cm from the bottom, I drew a light pencil line. Then, I placed
4 small equally spaced dots on the line and labeled them so that I would be able to identify where I
placed my samples once the experiment was complete. Using a capillary tube, I placed a small dot of
each sample onto its allocated pencil dot and left it to air dry. I repeated this 3 times to ensure there
was enough of each of my samples to test. Then, I poured a small amount of my solvent into a
100cm3 beaker and, once my samples had dried, I placed the chromatography paper on the solvent,
ensuring it was not slanted and the solvent did not go beyond the pencil line as this could have all
impacted my results. I proceeded to cover the top of my beaker with a watch glass to reduce the
amount of solvent that was evaporated, and I left my chromatography paper in there until the
solvent stopped rising up the paper. Once I believed it had stopped, I carefully removed my
chromatography paper from the beaker and marked my solvent front with a light pencil line. Finally,
the technician sprayed my chromatography paper with ninhydrin, and I was able to clearly view the
components within my samples and calculate their RF values. Overall, I was left with multiple purple
and yellow large spots. I was able to use my RF values to determine that my unknown substance X
was a combination of leucine and glycine.
Solvent front = 4cm
Spot Distance moved from Calculation Rf value
baseline (cm)
Glycine 3.1 3.1/4 0.78
Lysine 2.3 2.3/4 0.58
Leucine 3.5 3.5/4 0.88
1X 3.5 3.5/4 0.88
2X 3.1 3.1/4 0.78
Chromatography
I was given the task of performing a variety of chromatographic techniques to separate and identify
different compounds within substances. I had to test the amino acids glycine, leucine, and lysine to
figure out an unknown compound labelled X using paper chromatography. I then used thin layer
chromatography to determine the compounds of extracted pigment from a geranium plant.
(P6) What is Chromatography?
Chromatography is a technique used to separate compounds from a mixture to allow for chemical
analysis to occur and to identify certain substances within the mixture. There are many different
forms of chromatography; gas chromatography and high-performance liquid chromatography are
often used within the industry as they are more sensitive and accurate when separating and
identifying compounds compared to paper and thin layer chromatography which is usually used in
schools and colleges. Chromatography is able to work as mobile phases separate out the compound
over a stationary phase. A mobile phase is a liquid that transports a mixture across an absorbent
solid material whereas the stationary phase is the solid material that absorbs the substances. In a
given space of time, different substances would travel different distances as some move faster than
others. This is because each chemical compound has a unique attraction to the mobile and
stationary phase.
Thin layer chromatography (TLC) is a more effective and accurate method than simple paper
chromatography as it can be used on a greater range of compounds and molecules. Paper
chromatography is a simple method that usually uses water as its mobile phase and paper as its
stationary phase. It is usually used in schools as it is simple to prepare and conduct and can easily
separate out any substances based on their solubility. It often gives clear visual results, or the results
can be easily viewed under an ultraviolet light. Even though it is a simple and fairly effective method
to use, it has a low range of possible uses as only a small number of substances can be used as they
need to be water soluble. On the other hand, TLC allows for a greater application of use as the plate
is coated in a thin layer of silica gel. This gel contains different hydroxyl groups which attract
substances differently based on their polarity. Molecules which are more polar move slowly up the
TLC plate as they have a great attraction to the silica gel therefore, they travel less distance, however
nonpolar molecules have a weaker attraction to the hydroxyl groups so are able to travel higher up
the plate at a greater speed. TLC often requires an aid to be able to view the results. Usually a UV
light, iodine, or a chemical spray such as ninhydrine are used in order for the different pigments to
be able to be seen. TLC is usually used in higher levels of education as it is not only quicker than
paper chromatography, but it also gives a more accurate indication of the progress of a reaction.
Both paper chromatography and thin layer chromatography can help to understand the purity of a
product or substance. In order to test the purity of a substance you can add a small sample of your
product, a completely pure version of your product and the reactants that were used to create the
product. Then using either form of chromatography, you can determine the products purity as a
pure substance should only have 1 spot appear up the paper/plate. If another spot appears then it is
clear to determine that the product is impure. Using the molecules RF value (Retention Factor) you
can determine the exact reactant(s) that are still present as each molecule will have its own specific
RF value. So, if any two spots from different samples have the same RF value, then you can
determine they both contain the same substance. The RF (Retention Factor) value of a molecule is
, calculated by dividing the distance travelled by the compound to the distance travelled by the
solvent, all the values should be less than 1.
Risk assessment-
Before completing my experiments, I conducted a risk assessment to identify any possible hazards
and know what to do in the event that they occurred. Firstly, I had to wear a lab coat and goggles to
protect my eyes and clothes from any potential splashes of fluid. I also tied back my long hair so that
I could clearly see what I was doing and to prevent any possible contamination of my hair onto the
chromatography paper and plate. There was also the risk of broken glass and receiving cuts as I was
working with a beaker, watch glass and capillary tube. Therefore, I had to handle my equipment with
care to prevent any breakages and accidents.
(P5) Paper Chromatography of Amino Acids-
The first experiment I conducted was identifying an unknown substance using paper
chromatography. I knew that the substance was a mixture of either glycine, leucine, or lysine. To find
out the two amino acids within my unknown sample, I performed this method of chromatography as
I knew it would be able to separate out the mixture. To conduct my experiment, I first took a piece
of chromatography paper and, about 1cm from the bottom, I drew a light pencil line. Then, I placed
4 small equally spaced dots on the line and labeled them so that I would be able to identify where I
placed my samples once the experiment was complete. Using a capillary tube, I placed a small dot of
each sample onto its allocated pencil dot and left it to air dry. I repeated this 3 times to ensure there
was enough of each of my samples to test. Then, I poured a small amount of my solvent into a
100cm3 beaker and, once my samples had dried, I placed the chromatography paper on the solvent,
ensuring it was not slanted and the solvent did not go beyond the pencil line as this could have all
impacted my results. I proceeded to cover the top of my beaker with a watch glass to reduce the
amount of solvent that was evaporated, and I left my chromatography paper in there until the
solvent stopped rising up the paper. Once I believed it had stopped, I carefully removed my
chromatography paper from the beaker and marked my solvent front with a light pencil line. Finally,
the technician sprayed my chromatography paper with ninhydrin, and I was able to clearly view the
components within my samples and calculate their RF values. Overall, I was left with multiple purple
and yellow large spots. I was able to use my RF values to determine that my unknown substance X
was a combination of leucine and glycine.
Solvent front = 4cm
Spot Distance moved from Calculation Rf value
baseline (cm)
Glycine 3.1 3.1/4 0.78
Lysine 2.3 2.3/4 0.58
Leucine 3.5 3.5/4 0.88
1X 3.5 3.5/4 0.88
2X 3.1 3.1/4 0.78
