Unit 2C applied science Complete
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Najah Bendriss
Unit 2C: Practical Scientific Procedures and Techniques.
When a particular substance needs to be analysed or used for different applications, we use
chromatography to distinguish it from other substances in its mixture. Chromatography is
the process of identifying substances and the purity of a chemical substance through the
separation of the mixture. There are four main types of chromatography, liquid
chromatography, gas chromatography, thin-layer chromatography and paper
chromatography. I carried out experiments on paper and thin-layer chromatography.
P6, The use of chromatographic techniques to separate mixtures:
In forensic science chromatography can be used to determine the cause of death at a crime
scene as it can detect the presence of alcohol, drugs or poison in a person body. If a person
is caught drunk-driving chromatography can be used to discover the alcohol content present
in the body. Specific types of explosives can be distinguished from debris if an explosion has
taken place, this allows a more thorough investigation of the crime, body fragments,
including blood, nails or hair can also be used to identify the victims.
Factors which affect the separation of compounds:
Polarity of the solvents: The higher polarity of the solvent the better it competes
with the stationary phase and results in a higher Rf value. Aspartic acid and lysine are
both polar amino acids this means that they both should have a higher Rf value than
leucine which is non-polar, however, my results show that leucine has the highest Rf
value at 0.169 cm this could be because I did not transfer an equal amount of each
amino acid to my chromatography paper.
Effect of molecule size on its mobility: the size of the molecule in stationary phase
will affect it in mobile phase as smaller molecules travel further on the
chromatogram whereas larger molecules will only travel a short distance or will
remain in their original position. Lysine has the heaviest molecular weight at 146.19
g/mol, aspartic acid is next at 133.11 g/mol and the lightest of the three is leucine
which has a molecular weight of 131.17 g/mol. From my chromatogram you can see
that leucine has travelled the furthest from the base line. Aspartic acid has travelled
slightly further than lysine which correctly displays their slight difference in
molecular weight.
Separation of amino acids by paper chromatography:
Aim: To identify the amino acids present in the mixture by measuring the Rf values of spots
on a paper chromatogram and comparing values of known standards.
Chemicals used:
Ninhydrin spray (2% of ninhydrin spray in ethanol)
DL – Aspartic acid, 0.01 M in 10% propan-2-01
DL – Leucine acid, 0.01 M in 10% propan-2-01
DL – Lysine acid, 0.01 M in 10% propan-2-01
Amino acid mixture
1
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Najah Bendriss
Solvent: Butanol: Acetic acid: Water (12:3:5)
Apparatus:
Capillary tube
Plastic gloves
Pencil and ruler
Chromatography paper (12cm × 22cm)
250cm3 beaker
Oven
Introduction:
A mixture of unknown amino acids can be separated and identified by means of paper
chromatography. The positions of the amino acids in the chromatogram can be detected
by spraying with ninhydrin, which reacts with amino acids to yield highly coloured
products.
Method:
On a clean sheet of chromatography paper (12cm × 22cm), I marked a light pencil
line parallel to the bottom and around 2cm up.
Along the line I marked 4 light crosses, at intervals of 1.4cm apart.
I labelled each cross as Asp, Leu, Lys, U. (U stands for unknown mixture).
Using capillary tubes, I placed a small amount of each appropriate solution on its
positions along the line of chromatography paper, ensuring the spot was no
larger than 3mm in diameter.
I allowed the paper to dry in the air for a few minutes.
I added a second portion of the unknown to make sure that there were sufficient
quantities of each component of the unknown will be present for good visual
observation when the paper is developed.
Rf = distance travelled by the amino acids from the spot/ distance travelled by solvent.
Results:
Amino acids Distance travelled Distance travelled Rf value = A/B
by the amino acids by the solvent (B), (3 decimal places)
(A), (cm) (cm)
Asp 2.5 10.5 0.238
Leu 6.5 10.5 0.619
Lys 2.2 10.5 0.210
Unknown amino (cm)
acids
U1 2.2 10.5 0.210
U2 6.5 10.5 0.619
2
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Assignment
written by
najbo123
www.stuvia.com
Downloaded by: najbo123 | Want to earn £756
Distribution of this document is illegal extra per year?
, Stuvia.co.uk - The Marketplace for Revision Notes & Study Guides
Najah Bendriss
Unit 2C: Practical Scientific Procedures and Techniques.
When a particular substance needs to be analysed or used for different applications, we use
chromatography to distinguish it from other substances in its mixture. Chromatography is
the process of identifying substances and the purity of a chemical substance through the
separation of the mixture. There are four main types of chromatography, liquid
chromatography, gas chromatography, thin-layer chromatography and paper
chromatography. I carried out experiments on paper and thin-layer chromatography.
P6, The use of chromatographic techniques to separate mixtures:
In forensic science chromatography can be used to determine the cause of death at a crime
scene as it can detect the presence of alcohol, drugs or poison in a person body. If a person
is caught drunk-driving chromatography can be used to discover the alcohol content present
in the body. Specific types of explosives can be distinguished from debris if an explosion has
taken place, this allows a more thorough investigation of the crime, body fragments,
including blood, nails or hair can also be used to identify the victims.
Factors which affect the separation of compounds:
Polarity of the solvents: The higher polarity of the solvent the better it competes
with the stationary phase and results in a higher Rf value. Aspartic acid and lysine are
both polar amino acids this means that they both should have a higher Rf value than
leucine which is non-polar, however, my results show that leucine has the highest Rf
value at 0.169 cm this could be because I did not transfer an equal amount of each
amino acid to my chromatography paper.
Effect of molecule size on its mobility: the size of the molecule in stationary phase
will affect it in mobile phase as smaller molecules travel further on the
chromatogram whereas larger molecules will only travel a short distance or will
remain in their original position. Lysine has the heaviest molecular weight at 146.19
g/mol, aspartic acid is next at 133.11 g/mol and the lightest of the three is leucine
which has a molecular weight of 131.17 g/mol. From my chromatogram you can see
that leucine has travelled the furthest from the base line. Aspartic acid has travelled
slightly further than lysine which correctly displays their slight difference in
molecular weight.
Separation of amino acids by paper chromatography:
Aim: To identify the amino acids present in the mixture by measuring the Rf values of spots
on a paper chromatogram and comparing values of known standards.
Chemicals used:
Ninhydrin spray (2% of ninhydrin spray in ethanol)
DL – Aspartic acid, 0.01 M in 10% propan-2-01
DL – Leucine acid, 0.01 M in 10% propan-2-01
DL – Lysine acid, 0.01 M in 10% propan-2-01
Amino acid mixture
1
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Najah Bendriss
Solvent: Butanol: Acetic acid: Water (12:3:5)
Apparatus:
Capillary tube
Plastic gloves
Pencil and ruler
Chromatography paper (12cm × 22cm)
250cm3 beaker
Oven
Introduction:
A mixture of unknown amino acids can be separated and identified by means of paper
chromatography. The positions of the amino acids in the chromatogram can be detected
by spraying with ninhydrin, which reacts with amino acids to yield highly coloured
products.
Method:
On a clean sheet of chromatography paper (12cm × 22cm), I marked a light pencil
line parallel to the bottom and around 2cm up.
Along the line I marked 4 light crosses, at intervals of 1.4cm apart.
I labelled each cross as Asp, Leu, Lys, U. (U stands for unknown mixture).
Using capillary tubes, I placed a small amount of each appropriate solution on its
positions along the line of chromatography paper, ensuring the spot was no
larger than 3mm in diameter.
I allowed the paper to dry in the air for a few minutes.
I added a second portion of the unknown to make sure that there were sufficient
quantities of each component of the unknown will be present for good visual
observation when the paper is developed.
Rf = distance travelled by the amino acids from the spot/ distance travelled by solvent.
Results:
Amino acids Distance travelled Distance travelled Rf value = A/B
by the amino acids by the solvent (B), (3 decimal places)
(A), (cm) (cm)
Asp 2.5 10.5 0.238
Leu 6.5 10.5 0.619
Lys 2.2 10.5 0.210
Unknown amino (cm)
acids
U1 2.2 10.5 0.210
U2 6.5 10.5 0.619
2
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