Chromatography
Aim:
To identify the components in a mixture.
Calculate the Rf values.
Introduction:
Within this assignment I will need to demonstrate skills in a range of practical procedures and
techniques. Chromatography is a technique for separating and identifying different compounds in
a mixture. There are numerous forms of chromatography, each with a similar goal but different
mobile and stationary phases. The stationary phase in paper chromatography is paper. The
stationary phase in thin layer chromatography is a thin layer of adsorptive material placed on a
sheet of glass, plastic, or aluminium foil. The chemicals will go up the paper faster if you use more
polar solvents. This is because the attraction of polar molecules to polar phases increases as
polarity increases. For example, because the stationary phase is usually polar, polar compounds
will cling to it more and appear lower in the chromatogram. In chromatography, the mobile phase
must be considered. For instance, using water (a polar substance) as a mobile phase will give
different results than using a less polar mobile phase. This is because polar substances dissolve in
polar water molecules. They mix readily because both types of molecules engage in hydrogen
bonding.
Chromatography is used in several industries such as forensics for DNA (deoxyribonucleic acid)
fingerprinting and
bioinformatics. Chromatography is also used in the food industry for separating and identifying
additives, vitamins, preservatives, proteins, and amino acids
I am aiming to ensure that the experiment is reliable and valid enough to compare to known
values or data from experiments that have already been carried out. This will enable me to test
the repeatability and reproducibility of the experiment.
Method:
Paper chromatography:
1. On a piece of paper, a pencil line was drawn.
2. A mixture of salt, small amount of alcohol and grass was grinded with a pestle and mortar
until the pigment was extracted in liquid form.
3. A capillary tube was used to spot the pigment onto the centre of the line.
4. The spot was pigmented but small by not applying too much pressure and re applying the
spot by spotting and drying with a hair dryer.
5. The spot process was repeated 3-5 times.
6. A gas jar was filled with cyclohexane, propane and petroleum in the ratio of 3:2:1 till the
baseline.
1
, 7. The paper was placed inside, and the top of the paper was folded and wedged into place
by placing a watch glass on top to hold in place.
8. The paper was removed once the solute had reached the top.
9. A pencil line was used to mark where the line reached. (Solvent front)
10. The pigment of where it travelled was circled
11. The Rf value was calculated
TLC:
1. On a TLC (Thin Layer Chromatography) plate a pencil line was drawn on silica side of the
TLC plate.
2. The pigment mixture was spotted onto the TLC line to make a concentrated spot.
3. Once it was done the TLC plate was transferred to a beaker which was filled to the baseline
with cyclohexane, propane and petroleum in the ratio of 3:2:1 .
4. The TLC plate was balanced against the beaker and a watch glass was placed on top.
5. Once it reaches near top the TLC plate was removed and the solvent front was marked
with pencil.
6. The spots were circled.
7. The Rf values were calculated.
Amino acids:
1. The workspace was wiped down to make the surface clean.
2. A3 paper was placed down on the table.
3. One person put a glove on to touch the paper.
4. Another paper was placed onto the A3 paper which the amino acids are spotted on.
5. A baseline was drawn with pencil.
6. 5 spaces equally spaced out for the 4 known amino acids and the unknown sample,
7. The abbreviations were written under each spot.
8. For each spot it was concentrated by using a capillary tube to spot it then drying it with
hair dryer until the spot was concentrated. This was repeated for each amino acid and the
unknown one.
9. The paper was left on the side for the preparation of the Shandon tank.
10. The Shandon tank was filled till the baseline of the paper.
11. The paper was turned into a cylinder shape without making it overlap and secured with
clips to hold.
12. The paper was placed into the Shandon tank.
13. Once the solute reached the top, it was removed, and solvent front was marked with
pencil.
14. The paper was placed in the oven for 30-60 seconds then removed.
15. It was then sprayed with ninhydrin dye and placed back into oven for 30 seconds.
16. The spots were circled and Rf values calculated.
17. The unknown sample was identified by comparing the spots in the unknown and the
known.
Equipment list:
2
Aim:
To identify the components in a mixture.
Calculate the Rf values.
Introduction:
Within this assignment I will need to demonstrate skills in a range of practical procedures and
techniques. Chromatography is a technique for separating and identifying different compounds in
a mixture. There are numerous forms of chromatography, each with a similar goal but different
mobile and stationary phases. The stationary phase in paper chromatography is paper. The
stationary phase in thin layer chromatography is a thin layer of adsorptive material placed on a
sheet of glass, plastic, or aluminium foil. The chemicals will go up the paper faster if you use more
polar solvents. This is because the attraction of polar molecules to polar phases increases as
polarity increases. For example, because the stationary phase is usually polar, polar compounds
will cling to it more and appear lower in the chromatogram. In chromatography, the mobile phase
must be considered. For instance, using water (a polar substance) as a mobile phase will give
different results than using a less polar mobile phase. This is because polar substances dissolve in
polar water molecules. They mix readily because both types of molecules engage in hydrogen
bonding.
Chromatography is used in several industries such as forensics for DNA (deoxyribonucleic acid)
fingerprinting and
bioinformatics. Chromatography is also used in the food industry for separating and identifying
additives, vitamins, preservatives, proteins, and amino acids
I am aiming to ensure that the experiment is reliable and valid enough to compare to known
values or data from experiments that have already been carried out. This will enable me to test
the repeatability and reproducibility of the experiment.
Method:
Paper chromatography:
1. On a piece of paper, a pencil line was drawn.
2. A mixture of salt, small amount of alcohol and grass was grinded with a pestle and mortar
until the pigment was extracted in liquid form.
3. A capillary tube was used to spot the pigment onto the centre of the line.
4. The spot was pigmented but small by not applying too much pressure and re applying the
spot by spotting and drying with a hair dryer.
5. The spot process was repeated 3-5 times.
6. A gas jar was filled with cyclohexane, propane and petroleum in the ratio of 3:2:1 till the
baseline.
1
, 7. The paper was placed inside, and the top of the paper was folded and wedged into place
by placing a watch glass on top to hold in place.
8. The paper was removed once the solute had reached the top.
9. A pencil line was used to mark where the line reached. (Solvent front)
10. The pigment of where it travelled was circled
11. The Rf value was calculated
TLC:
1. On a TLC (Thin Layer Chromatography) plate a pencil line was drawn on silica side of the
TLC plate.
2. The pigment mixture was spotted onto the TLC line to make a concentrated spot.
3. Once it was done the TLC plate was transferred to a beaker which was filled to the baseline
with cyclohexane, propane and petroleum in the ratio of 3:2:1 .
4. The TLC plate was balanced against the beaker and a watch glass was placed on top.
5. Once it reaches near top the TLC plate was removed and the solvent front was marked
with pencil.
6. The spots were circled.
7. The Rf values were calculated.
Amino acids:
1. The workspace was wiped down to make the surface clean.
2. A3 paper was placed down on the table.
3. One person put a glove on to touch the paper.
4. Another paper was placed onto the A3 paper which the amino acids are spotted on.
5. A baseline was drawn with pencil.
6. 5 spaces equally spaced out for the 4 known amino acids and the unknown sample,
7. The abbreviations were written under each spot.
8. For each spot it was concentrated by using a capillary tube to spot it then drying it with
hair dryer until the spot was concentrated. This was repeated for each amino acid and the
unknown one.
9. The paper was left on the side for the preparation of the Shandon tank.
10. The Shandon tank was filled till the baseline of the paper.
11. The paper was turned into a cylinder shape without making it overlap and secured with
clips to hold.
12. The paper was placed into the Shandon tank.
13. Once the solute reached the top, it was removed, and solvent front was marked with
pencil.
14. The paper was placed in the oven for 30-60 seconds then removed.
15. It was then sprayed with ninhydrin dye and placed back into oven for 30 seconds.
16. The spots were circled and Rf values calculated.
17. The unknown sample was identified by comparing the spots in the unknown and the
known.
Equipment list:
2
