Chromatography (Chapter 7)

​PAPER CHROMATOGRAPHY

By Group 13

• Paper chromatography is a liquid partition chromatography

• The end of the paper is dipped in solvent mixture consisting of aqueous and organic components

• The solvent soaks in paper by capillary action because of fibrous nature of paper

• The aqueous component of the solvent binds to the cellulose paper and thereby forms stationary phase with it

• The organic component of the solvent binds continues migrating, thus forming the mobile phase

BASIC CONCEPT

• Mobile Phase rises up by capillary action

• Testing sample is concentrated as a minute spot at the bottom of the filter paper

• Sample mixture gradually rises up with the mobile phase which is liquid

• Compounds in the mixture will be separated according to their ability of solubility

• More Polar substances will move slower and less polar substances will travel faster.

SEPARATION PRINCIPLE

​A specialized, highly uniform and absorbent filter paper (cellulose)

​​Chromatography Paper (Stationary Phase)

A liquid (often water, ethanol, or a mixture of organic solvents) placed in the container

Solvent (Mobile Phase)

​A container (like a beaker or jar) that holds the mobile phase and the paper. It is usually covered to ensure the atmosphere is saturated with solvent vapor, which prevents the solvent from evaporating as it moves up the paper

Chromatographic Chamber

​MAIN COMPONENTS OF THE EQUIPMENT

The mixture (e.g., ink, dye, plant extract) applied as a small, concentrated spot near the bottom of the paper

​​Sample (Baseline)

Used to draw the baseline and apply the sample spots

Pencil/Capillary Tube

​Used to check the progress of a chemical reaction over time by monitoring the presence of reactants and the appearance of products

​​Reaction Monitoring

​Used extensively for the separation and detection of free amino acids, sugars and related compounds in biochemical analysis

​​Amino acids, Carbohydrates & Peptides

​APPLICATIONS IN LABORATORY OR INDUSTRY

​Used for the qualitative analysis of drug purity, identifying active compounds and potential impurities in drug development and manufacturing control

​​Pharmaceutical Industry

​Essential for analyzing and identifying both natural and synthetic food colours, additives and dyes in products(e.g. soft drinks, jams, sweets) to ensure compliance with regulatory standards

​​Food & Beverage Industry

​Used to test for and analyze pollutants and contaminants in environmental samples, such as detecting pesticides or heavy metals in water and soil

​Environmental Monitoring

• Simplicity & Ease of Use : The setup and procedure are very straightforward, requiring minimal training to perform

• Cost : Inexpensive method, require only basic, readily available equipment (filter paper, solvent and container)

• Minimal Sample Requirement : Only small amount of the sample mixture for analysis

• Non-Destructive : The separated components can be recovered from the paper after analysis if needed further testing

• Versatility : It can be adapted to separate both inorganic and organic compounds with varying polarities

• Excellence : Effective for determining what components are present in mixture (e.g. separating plant pigments or food dyes)

​ADVANTAGES OF PAPER CHROMATOGRAPHY

LIMITATIONS OF PAPER CHROMATOGRAPHY

• Low Resolution : It often fails to adequately separate compounds that have similar properties, resulting in overlapping spots and making precise identification difficult

• Limited Sample Capacity : Not suitable for separating or purifying large amounts of a sample

• Slow Process : While often rapid, the seperation process can sometimes take a long time(hours) depending on the solvent and the complexity of the mixture

• Inability : For mixtures with many components, the limited resolution makes the chromatogram difficult to interpret

• Not Suitable for Volatile Substances : Compounds that evaporate easily may be lost before the separation is complete, leading to inaccurate results

• Difficulty in Reproducing Results : Results can be highly dependent on environmental conditions (temperature & chamber saturation), the quality of paper and manual technique, making it hard to achieve consistent, reproducible results

Multiple Choice

What is the main purpose of paper chromatography?

• To determine the pH of a solution

• To separate components in a mixture

• To measure the concentration of a solution

• To determine the melting point of a substance

Multiple Choice

What is the stationary phase in paper chromatography?

• Solvent

• Chromatography paper

• Sample

• Air

Multiple Choice

A food safety laboratory is testing a batch of imported candy to ensure no illegal, non-permitted colorants have been added. Which application of paper chromatography is most directly relevant to this quality control task?

• Separation of free amino acids for clinical diagnostics

• DNA and RNA fingerprinting

• Qualitative analysis of food dyes and additives

• Monitoring of polymerization reactions in chemical manufacturing

Multiple Choice

A student successfully uses paper chromatography to separate a dye mixture into four distinct coloured spots. What is the primary limitation of this technique that prevents the student from precisely determining the mass or concentration of each individual separated dye?

• The technique is inherently a qualitative method and lacks the necessary instrumentation and precision for accurate quantitative analysis

• The high cost of the specialized cellulose paper makes quantitative analysis prohibitively expensive

• It can only be used with inorganic compounds, and dye mixtures are organic

• The resolution is too high, causing the spots to overlap and making measurement impossible

​THANK
YOU