Unit 2C Chromatography Report:
I have just began working at a forensic lab known as ‘CrimeSolve’ as a technical assistant. In order for me to
reach my goals of progressing further in this job, I will need to be able to prove that I am competent and
talented in various practical skills and practices. For me to be able to do this, I will be assisting in identifying
substances that have been found in crime scenes. For this, I will need to show that I can conduct a variety
of chromatographic techniques which will separate and help me to identify different components in
mixtures. I will show that I have managed to carry out these techniques in my report, which consists of me
explaining the techniques to show that I understand it, as well as analysing and evaluating my results that I
have gotten. I will also make sure to suggest future improvements that I will make to my experiments, so as
I can ensure I get the most reliable and accurate results as I can. So, I have carried out various different
methods of chromatography throughout this report, including thin layer chromatography, paper
chromatography using amino acids, as well as paper chromatography of plant photosynthetic pigments.
This was done so as I could ultimately find which method of chromatography is the most effective, as
shown when I compared them in my report below.
What is chromatography?
Chromatography is a method used in many scenarios and contexts to separate components from a mixture.
In chromatography, there is a thing known as the stationary phase, and the mobile phase. The stationary
phase is the thing that does not move the sample, so for example, in paper chromatography that I have
carried out, the paper is the stationary phase. As well as this, the stationary phase in thin layer
chromatography is the silica gel on the TLC plate. The mobile phase is the thing that does move with the
sample, so again, in paper chromatography that is the solvent, as well as in TLC, the solvent is the mobile
phase.
When analysing a chromatogram, the pigments are separated and have travelled different distances from
each other, and this is because of the components all have different amounts of solubility. The pigment that
has travelled farthest up the stationary phase and is closest to the solvent front is the most soluble,
whereas the colour closest to the origin line, and has travelled the least is less soluble.
As well as this, attraction to the stationary phase is also a factor that effects how far the pigments travel.
So, the pigments that are more attracted to the mobile phase, so in our case the solvent, the faster it will
travel. While pigments that are more attracted to the stationary phase will travel more slowly. Polarity is a
huge factor as to why attraction effect how fast the pigments travel. Molecules will have a positively
charged side and a negatively charged side, and due to opposites attracting, the more positively charged
side will be attracted to the negatively charged side of another molecule, and the bigger the difference, the
more polar the molecule will be. So, the more polar the solvent is, the quicker the sample will travel up.
,Thin layer chromatography-
Preparing my pigment solution-
1. Firstly, collect your leaves from the crime scene and finely chop them up into a mortar containing
some sand. Sand is required for this part as when it is being mixed with the leaves, it will aid in
tearing and uncovering the inner leaf.
2. Next, add around 2cm3 of propanone into the mortar.
3. Grind all of this together in the mortar until there is a thick green slurry. Throughout mixing it, make
sure to keep adding little amounts of propanone when it is needed. Not much liquid is needed in
the end result, as only a few drops are needed to put onto the TLC plate.
Preparing the chromatography paper-
1. When picking up the TLC plate, ensure that only the sides are being held, and avoid touching the
silica gel on the surface. This is to make sure that finger oils are not transferred to the TLC plate,
which will consequently make the sample move around it or stop it from dissolving straight
upwards, as it will not dissolve through it. This would make the results inaccurate when measuring
the distances.
2. Next, draw a pencil line 1cm above the bottom of the TLC plate on the silica gel, on the non-shiny
side, so the line needs to be just above the solvent, or the sample on the line will dissolve into the
solvent, rather than up the TLC plate. Using pencils are necessary as this will not dissolve up the TLC
plate in the mobile phase, whereas with pen, the ink would dissolve.
3. Then transfer the chloroplast pigment into a capillary tube, ensuring that no plant debris goes in
with it (as this will hinder the chromatogram), and put a small spot in the middle of the drawn
pencil line, which is the baseline of the chromatogram. Do this step carefully to make sure that the
TLC plate surface does not break, and that the spot put on are not too big.
4. Quickly dry the spot in order to prevent it from spreading. Repeat this.
Risk assessment-
Hazard Risk Precaution
Glassware. Could shatter and cut oneself. Do not keep on the edge of your workbench.
Wear goggles in case it shatters.
Propanone. Highly flammable. Can cause Wear goggles. Remember to close the lid on the
eye irritation. jar it is contained in to prevent spillages. If
spilled, clean immediately.
Cyclohexane. Highly flammable, can cause Wash hands thoroughly if in contact, wear
harm if contact to skin, can be goggles to prevent it getting in your eyes, and a
fatal is swallowed. lab coat to prevent contact with your skin.
Petroleum ether. Can cause dizziness if inhaled, if Wear goggles and a lab coat. If spilled, remove
ingested may cause nausea and all ignition sources form the area and clean
vomiting. immediately.
, My chromatogram analysis:
Now that I had looked at the methods and why certain things needed
to be done, I then went on to do this practical myself. There were
four different chromatograms that were done between my group,
one with 6, 12, 18 and 24 spots. For my chromatogram I had used 12
spots. The sample that I had put onto my TLC plate was a mix of the
leaves and propanone. I had put my sample onto my drawn pencil
line 1cm up from the bottom and put it into a small bottle containing
the solvent, and this is known as the mobile phase. Once the solvent
had moved up to 5mm below the top of the TLC plate, I took it out of
the bottle and drew the solvent front at the top and put dots in the
middle of each colour separation I had gotten, which was three.
Though I did get some colour separations, it wasn’t the best
chromatogram as the colours were a little bit smeared, which made it
hard to know where the middle of each colour was to draw my dots
on. This was a little successful though as I had managed to get it so as
my colours did not run up at an angle, which means I didn’t get any
finger oils or dust on the silica gel, and I hadn’t moved the bottle
containing the TLC plate when the mobile phase was in progress. This
is good as I can be more accurate with measuring the distances of each pigment. Throughout this practical I
did make some mistakes, for example, at the beginning of the experiment I had dropped my TLC plate onto
the floor, which resulted in the silica gel getting dust on it, as well as my finger oils when I was picking it
back up. This is a problem as this causes the sample to move around these areas, making it go up at an
angle, making my measurements less accurate, though I was lucky and didn’t end up with this problem. As
well as this, I believe that I had left an insufficient amount of time to let the spots dry before adding
another one onto the TLC plate, which makes the sample spread and make the spot bigger, effecting the
results of my chromatogram. I also believe that I had gotten some plant debris onto my TLC plate, which is
problematic as this causes my sample to run up a lot slower if at all and left my colours really flat and
splotchy. I had realised that this was the problem and why I had gotten bad results thanks to the feedback
given to me by my teacher, and so I had decided to change the method the next time I carry out this
practical, to avoid plant debris from affecting the quality of my chromatogram again.
Now that I have had a look at my chromatogram, I had decided to go
and compare mine with my classmate, who had all used different
amounts of spots to me, I looked at one with 6, 18, and 24 spots. The
chromatogram that had used 24 spots of the sample had no colour
separations as it didn’t move up at all. This could be due to the fact
that way too much of the sample was used, consequently getting
some plant debris onto the TLC plate like shown in the picture. So,
this tells me that if I am going to do another chromatogram, I should
not use up to 24 as this is way too much. You can also see the outline
of the sample is pretty big, showing that they may have not left
enough time for each spot to dry when applying another one. This
could be due to the fact that there was so much of the sample on it
that it took a really long time to dry, which gives it too much time to
spread.
I have just began working at a forensic lab known as ‘CrimeSolve’ as a technical assistant. In order for me to
reach my goals of progressing further in this job, I will need to be able to prove that I am competent and
talented in various practical skills and practices. For me to be able to do this, I will be assisting in identifying
substances that have been found in crime scenes. For this, I will need to show that I can conduct a variety
of chromatographic techniques which will separate and help me to identify different components in
mixtures. I will show that I have managed to carry out these techniques in my report, which consists of me
explaining the techniques to show that I understand it, as well as analysing and evaluating my results that I
have gotten. I will also make sure to suggest future improvements that I will make to my experiments, so as
I can ensure I get the most reliable and accurate results as I can. So, I have carried out various different
methods of chromatography throughout this report, including thin layer chromatography, paper
chromatography using amino acids, as well as paper chromatography of plant photosynthetic pigments.
This was done so as I could ultimately find which method of chromatography is the most effective, as
shown when I compared them in my report below.
What is chromatography?
Chromatography is a method used in many scenarios and contexts to separate components from a mixture.
In chromatography, there is a thing known as the stationary phase, and the mobile phase. The stationary
phase is the thing that does not move the sample, so for example, in paper chromatography that I have
carried out, the paper is the stationary phase. As well as this, the stationary phase in thin layer
chromatography is the silica gel on the TLC plate. The mobile phase is the thing that does move with the
sample, so again, in paper chromatography that is the solvent, as well as in TLC, the solvent is the mobile
phase.
When analysing a chromatogram, the pigments are separated and have travelled different distances from
each other, and this is because of the components all have different amounts of solubility. The pigment that
has travelled farthest up the stationary phase and is closest to the solvent front is the most soluble,
whereas the colour closest to the origin line, and has travelled the least is less soluble.
As well as this, attraction to the stationary phase is also a factor that effects how far the pigments travel.
So, the pigments that are more attracted to the mobile phase, so in our case the solvent, the faster it will
travel. While pigments that are more attracted to the stationary phase will travel more slowly. Polarity is a
huge factor as to why attraction effect how fast the pigments travel. Molecules will have a positively
charged side and a negatively charged side, and due to opposites attracting, the more positively charged
side will be attracted to the negatively charged side of another molecule, and the bigger the difference, the
more polar the molecule will be. So, the more polar the solvent is, the quicker the sample will travel up.
,Thin layer chromatography-
Preparing my pigment solution-
1. Firstly, collect your leaves from the crime scene and finely chop them up into a mortar containing
some sand. Sand is required for this part as when it is being mixed with the leaves, it will aid in
tearing and uncovering the inner leaf.
2. Next, add around 2cm3 of propanone into the mortar.
3. Grind all of this together in the mortar until there is a thick green slurry. Throughout mixing it, make
sure to keep adding little amounts of propanone when it is needed. Not much liquid is needed in
the end result, as only a few drops are needed to put onto the TLC plate.
Preparing the chromatography paper-
1. When picking up the TLC plate, ensure that only the sides are being held, and avoid touching the
silica gel on the surface. This is to make sure that finger oils are not transferred to the TLC plate,
which will consequently make the sample move around it or stop it from dissolving straight
upwards, as it will not dissolve through it. This would make the results inaccurate when measuring
the distances.
2. Next, draw a pencil line 1cm above the bottom of the TLC plate on the silica gel, on the non-shiny
side, so the line needs to be just above the solvent, or the sample on the line will dissolve into the
solvent, rather than up the TLC plate. Using pencils are necessary as this will not dissolve up the TLC
plate in the mobile phase, whereas with pen, the ink would dissolve.
3. Then transfer the chloroplast pigment into a capillary tube, ensuring that no plant debris goes in
with it (as this will hinder the chromatogram), and put a small spot in the middle of the drawn
pencil line, which is the baseline of the chromatogram. Do this step carefully to make sure that the
TLC plate surface does not break, and that the spot put on are not too big.
4. Quickly dry the spot in order to prevent it from spreading. Repeat this.
Risk assessment-
Hazard Risk Precaution
Glassware. Could shatter and cut oneself. Do not keep on the edge of your workbench.
Wear goggles in case it shatters.
Propanone. Highly flammable. Can cause Wear goggles. Remember to close the lid on the
eye irritation. jar it is contained in to prevent spillages. If
spilled, clean immediately.
Cyclohexane. Highly flammable, can cause Wash hands thoroughly if in contact, wear
harm if contact to skin, can be goggles to prevent it getting in your eyes, and a
fatal is swallowed. lab coat to prevent contact with your skin.
Petroleum ether. Can cause dizziness if inhaled, if Wear goggles and a lab coat. If spilled, remove
ingested may cause nausea and all ignition sources form the area and clean
vomiting. immediately.
, My chromatogram analysis:
Now that I had looked at the methods and why certain things needed
to be done, I then went on to do this practical myself. There were
four different chromatograms that were done between my group,
one with 6, 12, 18 and 24 spots. For my chromatogram I had used 12
spots. The sample that I had put onto my TLC plate was a mix of the
leaves and propanone. I had put my sample onto my drawn pencil
line 1cm up from the bottom and put it into a small bottle containing
the solvent, and this is known as the mobile phase. Once the solvent
had moved up to 5mm below the top of the TLC plate, I took it out of
the bottle and drew the solvent front at the top and put dots in the
middle of each colour separation I had gotten, which was three.
Though I did get some colour separations, it wasn’t the best
chromatogram as the colours were a little bit smeared, which made it
hard to know where the middle of each colour was to draw my dots
on. This was a little successful though as I had managed to get it so as
my colours did not run up at an angle, which means I didn’t get any
finger oils or dust on the silica gel, and I hadn’t moved the bottle
containing the TLC plate when the mobile phase was in progress. This
is good as I can be more accurate with measuring the distances of each pigment. Throughout this practical I
did make some mistakes, for example, at the beginning of the experiment I had dropped my TLC plate onto
the floor, which resulted in the silica gel getting dust on it, as well as my finger oils when I was picking it
back up. This is a problem as this causes the sample to move around these areas, making it go up at an
angle, making my measurements less accurate, though I was lucky and didn’t end up with this problem. As
well as this, I believe that I had left an insufficient amount of time to let the spots dry before adding
another one onto the TLC plate, which makes the sample spread and make the spot bigger, effecting the
results of my chromatogram. I also believe that I had gotten some plant debris onto my TLC plate, which is
problematic as this causes my sample to run up a lot slower if at all and left my colours really flat and
splotchy. I had realised that this was the problem and why I had gotten bad results thanks to the feedback
given to me by my teacher, and so I had decided to change the method the next time I carry out this
practical, to avoid plant debris from affecting the quality of my chromatogram again.
Now that I have had a look at my chromatogram, I had decided to go
and compare mine with my classmate, who had all used different
amounts of spots to me, I looked at one with 6, 18, and 24 spots. The
chromatogram that had used 24 spots of the sample had no colour
separations as it didn’t move up at all. This could be due to the fact
that way too much of the sample was used, consequently getting
some plant debris onto the TLC plate like shown in the picture. So,
this tells me that if I am going to do another chromatogram, I should
not use up to 24 as this is way too much. You can also see the outline
of the sample is pretty big, showing that they may have not left
enough time for each spot to dry when applying another one. This
could be due to the fact that there was so much of the sample on it
that it took a really long time to dry, which gives it too much time to
spread.
