Paper Chromatography 45526k
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Identification of Sugars by Paper Chromatography Section A4
WHAT IS CHROMATOGRAPHY? • a method of separating and identifying the components of a complex mixture • differential movement through a twophase system, in which the movement is affected by a flow of a liquid or a gas (mobile phase) which percolates through an adsorbent (stationary phase) or a second liquid phase
WHAT IS CHROMATOGRAPHY? • Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption • Basic Chromatographic Principles All chromatographic systems contain: A stationary phase A mobile phase Sample molecules
Type of Chromatography
Applications in the Real World
Why and What is it
Liquid Chromatography
test water samples to look for pollution,
Used to analyze metal ions and organic compounds in solutions. It uses liquids which may incorporate hydrophilic, insoluble molecules.
Gas Chromatography
detect bombs in airports, identify and quantify such drugs as alcohol, used in forensics to compare fibers found on a victim
Used to analyze volatile gases. Helium is used to move the gaseous mixture through a column of absorbent material.
Thin-Layer Chromatography
Paper Chromatography
Uses an absorbent material on detecting pesticide or insecticide residues in flat glass plates. This is a simple food, also used in forensics to analyze the and rapid method to check the dye composition of fibers purity of the organic compound.
separating amino acids and anions, RNA fingerprinting, separating and testing histamines, antibiotics
The most common type of chromatography. The paper is the stationary phase. This uses capillary action to pull the solutes up through the paper and separate the solutes.
PAPER CHROMATOGRAPHY • Paper Partition Chromatography • to emphasize the partitioning of solutes between a mobile phase and water adhering to the filter paper during chromatography
PAPER CHROMATOGRAPHY • a method for testing the purity of compounds and identifying substances • a useful technique because it is relatively quick and requires small quantities of material
PAPER CHROMATOGRAPHY Carried out by: • placing samples to be analyzed on sheets of filter paper • allowing an organic solvent to develop the chromatogram
PAPER CHROMATOGRAPHY • Mobile phase: – ORGANIC SOLVENT
• Stationary phase: – THE WATER WHICH IS HELD TO THE CELLULOSE FIBERS OF THE PAPER BY HYDROGEN BONDING
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• • • • • •
Prepare the Stationary Phase Spotting the Samples Developing the Chromatogram Spray the Chromatogram Heat the Chromatogram Identify Spots
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• Prepare the Stationary Phase • • • • •
Spotting the Samples Developing the Chromatogram Spray the Chromatogram Heat the Chromatogram Identify Spots
PREPARATION FOR THE STATIONARY PHASE 1. Draw a line, 2cm away from the edge of the filter paper using a pencil. 2. Divided the baseline into 6 spots. 3. Marked each spot and labelled according to the name of the standard sugars.
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• Prepare the Stationary Phase • Spotting the Samples • • • •
Developing the Chromatogram Spray the Chromatogram Heat the Chromatogram Identify Spots
SPOTTING THE SAMPLES 1. Using a capillary pipette, 4µl each of the given sugar solutions were applied. 2. Application is done 6 times for each spot (drying spot in each applications).
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• Prepare the Stationary Phase • Spotting the Samples • Developing the Chromatogram • Spray the Chromatogram • Heat the Chromatogram • Identify Spots
DEVELOPING THE CHROMATOGRAM • The paper was placed in the chamber containing the solvent. • The spots should not touch the solvent.
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• Prepare the Stationary Phase • Spotting the Samples • Developing the Chromatogram • Spray the Chromatogram – Aniline acid oxylate spray
• Heat the Chromatogram • Identify Spots
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• • • •
Prepare the Stationary Phase Spotting the Samples Developing the Chromatogram Spray the Chromatogram
• Heat the Chromatogram – Heat in a hot plate until spots become visible.
• Identify Spots
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• • • • •
Prepare the Stationary Phase Spotting the Samples Developing the Chromatogram Spray the Chromatogram Heat the Chromatogram
• Identify Spots
CHROMATOGRAM (1A4-1)
CHROMATOGRAM (1A4-2)
RESULTS 1A4-2
1A4-1 Sugar
Distance traveled by sugar
Sugar
Distance traveled by sugar
Galactose
2.6 cm
Galactose
3.6 cm
Glucose
3.3 cm
Glucose
3.9 cm
Fructose
5.1 cm
Fructose
5.5 cm
Maltose
1.0 cm
Maltose
1.4 cm
Sucrose
1.7 cm
Sucrose
2.2 cm
Unknown
1.5 cm
Unknown
2.1cm
Distance traveled by farthest sugar = 5.1 cm
Distance traveled by farthest sugar = 5.5 cm
COMPUTING THE Rf VALUE Rf
distance traveled by unknown distance traveled by solvent front
• i.e. Galactose (1A4-1) – Distance traveled by the sugar = 2.6 cm – Distance traveled by farthest sugar = 5.1 cm 2.6 cm Rf 5.1 cm
Rf 0.51
1A4-1
RESULTS 1A4-2
Sugar
Rf Value
Sugar
Rf Value
Galactose
0.51
Galactose
0.65
Glucose
0.65
Glucose
0.71
Fructose
1.00
Fructose
1.00
Maltose
0.20
Maltose
0.25
Sucrose
0.33
Sucrose
0.40
Unknown
0.29
Unknown
0.38
DISCUSSION • Mobile phase: Organic solvent system – Butanol: Ethanol: Water (52:32:10) solvent system
• Stationary phase: Water molecules embedded in Whatman filter paper • Color visualization: Reaction of reducing sugars with aniline acid oxalate and the addition of heat – Dehydration which produced fulfural from hydroxymethyl-fulfural
DISCUSSION • Problem: Solvent ran off the paper distance traveled by unknown Rf distance traveled by solvent front
• Solution: Change the denominator to the distance traveled by the farthest sugar • Unknown: Sucrose
SOURCES: • Jack, R. (1995). Basic Biochemical Laboratory Procedures and Sampling. New York: Oxford University Press, pages 84-85. • Furfural. Accessed on 07/08/09 at http://en.wikipedia.org/wiki/Furfural • HMF. Accessed on 07/08/09 at http:// en.wikipedia.org/wiki/Hydroxymethylfurfural
WHAT IS CHROMATOGRAPHY? • a method of separating and identifying the components of a complex mixture • differential movement through a twophase system, in which the movement is affected by a flow of a liquid or a gas (mobile phase) which percolates through an adsorbent (stationary phase) or a second liquid phase
WHAT IS CHROMATOGRAPHY? • Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption • Basic Chromatographic Principles All chromatographic systems contain: A stationary phase A mobile phase Sample molecules
Type of Chromatography
Applications in the Real World
Why and What is it
Liquid Chromatography
test water samples to look for pollution,
Used to analyze metal ions and organic compounds in solutions. It uses liquids which may incorporate hydrophilic, insoluble molecules.
Gas Chromatography
detect bombs in airports, identify and quantify such drugs as alcohol, used in forensics to compare fibers found on a victim
Used to analyze volatile gases. Helium is used to move the gaseous mixture through a column of absorbent material.
Thin-Layer Chromatography
Paper Chromatography
Uses an absorbent material on detecting pesticide or insecticide residues in flat glass plates. This is a simple food, also used in forensics to analyze the and rapid method to check the dye composition of fibers purity of the organic compound.
separating amino acids and anions, RNA fingerprinting, separating and testing histamines, antibiotics
The most common type of chromatography. The paper is the stationary phase. This uses capillary action to pull the solutes up through the paper and separate the solutes.
PAPER CHROMATOGRAPHY • Paper Partition Chromatography • to emphasize the partitioning of solutes between a mobile phase and water adhering to the filter paper during chromatography
PAPER CHROMATOGRAPHY • a method for testing the purity of compounds and identifying substances • a useful technique because it is relatively quick and requires small quantities of material
PAPER CHROMATOGRAPHY Carried out by: • placing samples to be analyzed on sheets of filter paper • allowing an organic solvent to develop the chromatogram
PAPER CHROMATOGRAPHY • Mobile phase: – ORGANIC SOLVENT
• Stationary phase: – THE WATER WHICH IS HELD TO THE CELLULOSE FIBERS OF THE PAPER BY HYDROGEN BONDING
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• • • • • •
Prepare the Stationary Phase Spotting the Samples Developing the Chromatogram Spray the Chromatogram Heat the Chromatogram Identify Spots
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• Prepare the Stationary Phase • • • • •
Spotting the Samples Developing the Chromatogram Spray the Chromatogram Heat the Chromatogram Identify Spots
PREPARATION FOR THE STATIONARY PHASE 1. Draw a line, 2cm away from the edge of the filter paper using a pencil. 2. Divided the baseline into 6 spots. 3. Marked each spot and labelled according to the name of the standard sugars.
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• Prepare the Stationary Phase • Spotting the Samples • • • •
Developing the Chromatogram Spray the Chromatogram Heat the Chromatogram Identify Spots
SPOTTING THE SAMPLES 1. Using a capillary pipette, 4µl each of the given sugar solutions were applied. 2. Application is done 6 times for each spot (drying spot in each applications).
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• Prepare the Stationary Phase • Spotting the Samples • Developing the Chromatogram • Spray the Chromatogram • Heat the Chromatogram • Identify Spots
DEVELOPING THE CHROMATOGRAM • The paper was placed in the chamber containing the solvent. • The spots should not touch the solvent.
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• Prepare the Stationary Phase • Spotting the Samples • Developing the Chromatogram • Spray the Chromatogram – Aniline acid oxylate spray
• Heat the Chromatogram • Identify Spots
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• • • •
Prepare the Stationary Phase Spotting the Samples Developing the Chromatogram Spray the Chromatogram
• Heat the Chromatogram – Heat in a hot plate until spots become visible.
• Identify Spots
PROCEDURE • Prepare the developing chamber – Organic solvent: Butanol:Ethanol:Water (52:32:10)
• • • • •
Prepare the Stationary Phase Spotting the Samples Developing the Chromatogram Spray the Chromatogram Heat the Chromatogram
• Identify Spots
CHROMATOGRAM (1A4-1)
CHROMATOGRAM (1A4-2)
RESULTS 1A4-2
1A4-1 Sugar
Distance traveled by sugar
Sugar
Distance traveled by sugar
Galactose
2.6 cm
Galactose
3.6 cm
Glucose
3.3 cm
Glucose
3.9 cm
Fructose
5.1 cm
Fructose
5.5 cm
Maltose
1.0 cm
Maltose
1.4 cm
Sucrose
1.7 cm
Sucrose
2.2 cm
Unknown
1.5 cm
Unknown
2.1cm
Distance traveled by farthest sugar = 5.1 cm
Distance traveled by farthest sugar = 5.5 cm
COMPUTING THE Rf VALUE Rf
distance traveled by unknown distance traveled by solvent front
• i.e. Galactose (1A4-1) – Distance traveled by the sugar = 2.6 cm – Distance traveled by farthest sugar = 5.1 cm 2.6 cm Rf 5.1 cm
Rf 0.51
1A4-1
RESULTS 1A4-2
Sugar
Rf Value
Sugar
Rf Value
Galactose
0.51
Galactose
0.65
Glucose
0.65
Glucose
0.71
Fructose
1.00
Fructose
1.00
Maltose
0.20
Maltose
0.25
Sucrose
0.33
Sucrose
0.40
Unknown
0.29
Unknown
0.38
DISCUSSION • Mobile phase: Organic solvent system – Butanol: Ethanol: Water (52:32:10) solvent system
• Stationary phase: Water molecules embedded in Whatman filter paper • Color visualization: Reaction of reducing sugars with aniline acid oxalate and the addition of heat – Dehydration which produced fulfural from hydroxymethyl-fulfural
DISCUSSION • Problem: Solvent ran off the paper distance traveled by unknown Rf distance traveled by solvent front
• Solution: Change the denominator to the distance traveled by the farthest sugar • Unknown: Sucrose
SOURCES: • Jack, R. (1995). Basic Biochemical Laboratory Procedures and Sampling. New York: Oxford University Press, pages 84-85. • Furfural. Accessed on 07/08/09 at http://en.wikipedia.org/wiki/Furfural • HMF. Accessed on 07/08/09 at http:// en.wikipedia.org/wiki/Hydroxymethylfurfural
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