STPM-Biological Molecules -Analytical Technique

STPM-Biological Molecules -Analytical Technique 

Paper Chromatography 

This guide summarizes the principles and applications of paper chromatography, focusing on pigment separation. 

I. What is Paper Chromatography? 

Paper chromatography is a separation technique used to analyze mixtures of similar chemicals, such as photosynthetic pigments, proteins, amino acids, nucleic acids, nucleotides, fatty acids, monosaccharides, and disaccharides. It works by exploiting the differential movement of these chemicals through a porous medium (paper) using a common solvent. 

II. Principles of Pigment Separation: 

The Medium: A piece of porous, absorbent paper (cellulose fibers) acts as the stationary phase. 

The Process: A concentrated sample (e.g., leaf extract) is spotted onto the paper. The paper is then placed in a solvent (e.g., petroleum ether), which acts as the mobile phase. Capillary action draws the solvent up the paper, carrying the pigments with it. Pigments separate based on their differing interactions with the stationary and mobile phases. 

Factors Affecting Separation: The rate at which a pigment moves depends on: 

Solubility: More soluble pigments move faster. 

Molecular Size: Smaller pigments move faster. 

Charge: Pigments with similar charges to the paper move faster; opposite charges lead to slower movement due to attraction. 

Retention Factor (Rf): The Rf value is used to identify pigments. It's a constant for a given pigment and solvent: 

Rf = (Distance travelled by the pigment) / (Distance travelled by the solvent) 

Comparing the Rf value of an unknown pigment to known standards helps identify the unknown. 

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III. Advantages of Paper Chromatography: 

Simplicity: Easy to perform, requiring minimal equipment (paper, dropper, boiling tube). 

Uniqueness: Offers separation where other techniques may fail. 

Speed: Quick results (e.g., leaf pigment separation in under 30 minutes). 

IV. Limitations of Paper Chromatography: 

Sample Size: Only small amounts of material can be separated at once. 

Resolution: Similar pigments (e.g., chlorophyll a and b) may overlap, hindering complete separation. 

V. Study Questions: 

Define chromatography and explain its underlying principle. 

Describe the role of the stationary and mobile phases in paper chromatography. 

List three factors influencing pigment separation in paper chromatography and explain how each factor affects the pigment's movement. 

What is the Rf value, and how is it calculated? Why is it important? 

What are the advantages and limitations of using paper chromatography? 

Give examples of mixtures that can be separated using paper chromatography. 

VI. Practice Problems: 

If a pigment travels 4 cm and the solvent travels 6 cm, what is the Rf value? 

Why might two pigments with very similar structures be difficult to separate using paper chromatography? 

Definition & Principle: 

Electrophoresis is a laboratory technique used to separate charged molecules (like amino acids, proteins, and DNA fragments) based on their differing migration rates in an electric field. This differential movement occurs because charged molecules are attracted to the oppositely charged electrode. 

II. Methodology: 

Medium: The separation takes place within a gel matrix (typically agarose gel, but polyacrylamide gel or even paper can be used). This gel acts as a sieve, slowing down the movement of larger molecules more than smaller ones. 

1. Sample Preparation: The sample (e.g., a mixture of DNA fragments) is mixed with a buffer solution (often acidic) to ensure the molecules carry a net charge. A tracking dye is added to visually monitor the progress of the electrophoresis. The sample is then loaded into wells at one end of the gel. 

1. Electrophoresis Setup: The gel is placed in a chamber filled with buffer solution. Electrodes are positioned at opposite ends of the chamber. Applying an electric current causes the charged molecules to migrate through the gel towards the electrode with the opposite charge. Smaller, more highly charged molecules move faster and further. 

1. Visualization: After electrophoresis, the separated molecules are visualized. This might involve staining the gel with a dye that binds to the molecules of interest or using fluorescent labels incorporated during sample preparation. ​
   
2. III. Applications: 
3. Protein Separation: Electrophoresis is ideal for separating proteins due to its gentleness; separated enzymes often remain active. ​
4. Disease Diagnosis: Analyzing blood plasma proteins can reveal the presence of antibodies produced in response to pathogens. Comparison with standard antibodies aids in disease identification.Forensic Science (DNA Fingerprinting): DNA is cut into fragments of varying lengths, and these fragments are separated to create a unique banding pattern for each individual. 
   
   DNA Sequencing: Electrophoresis is used to separate DNA fragments of different lengths, allowing scientists to determine the order of nucleotides in a DNA sequence. 
   
   IV. Limitations: 
   
   Sample Size: Electrophoresis is only suitable for separating small quantities of material. 
   
   Charge Limitations: Uncharged molecules or molecules with very similar charges will not separate effectively using this technique. 
   
   V. Key Terms: 
   
   Anode: The positive electrode. 
   
   Cathode: The negative electrode. 
   
   Agarose gel: A common gel matrix used in electrophoresis. 
   
   Buffer solution: Maintains pH and provides ions for conductivity. 
   
   Tracking dye: A dye added to the sample to monitor its progress. 
   
   Kilobase pairs (kb): A unit of measurement for DNA fragment length. 
5. VI. Study Questions: 
6. Explain the principle behind electrophoresis. Why do molecules move in an electric field? 
7. Describe the components of an electrophoresis apparatus. 
8. What are the advantages of using electrophoresis for protein separation? 
9. How is electrophoresis used in forensic science and disease diagnosis? 
10. What are the limitations of electrophoresis, and why do these limitations exist? 
11. What factors influence the rate of migration of a molecule during electrophoresis? (Consider size, charge, and gel matrix).