chromatography experiment theory

• Random article
• Teaching guide
• Privacy & cookies

by Chris Woodford . Last updated: February 25, 2022.

Photo: High-performance liquid chromatography (HPLC) is a very fast and precise kind of liquid chromatography used in research labs. Here it's being used to analyze an antibiotic for controlling diseases in wheat. Photo by Jack Dykinga courtesy of US Department of Agriculture/Agricultural Research Service .

What is chromatography?

Photo: What makes ink blur on wet paper? What you're seeing here is chromatography in action. Different inks and papers produce very different effects. Try experimenting for yourself!

How does chromatography work?

Artwork: How chromatography works: here the mobile phase is a liquid (blue) and the stationary phase is a solid (gray). The green molecule spends most time in the liquid so moves fastest. The yellow molecule spends more time on the surface of the solid, so moves slower. The red molecule spends even more time on the solid surface, so moves slowest.

What are the different types of chromatography?

What is chromatography used for.

“ ... the pigments separate themselves from top to bottom in different colored zones.... such a preparation I call... the chromatographic method. ” Mikhail Tswett, 1906

Photo: What's your poison? A sample of vehicle exhaust is injected into a gas chromatography machine so the pollutants it contains can be analyzed. Photo by Warren Gretz courtesy of US DOE/NREL (US Department of Energy/National Renewable Energy Laboratory).

If you liked this article...

Don't want to read our articles try listening instead, find out more, on this website.

• Forensic science
• Mass spectrometers
• Paper Chromatography : A simple activity for young readers from the ever-excellent Science Buddies site.
• Candy Chromatography : Another Science Buddies activity. This one uses paper chromatography to investigate the colors in candy sweets.
• Leaf Chromatography : Using paper chromatography to investigate the chlorophyll pigments in green leaves.

For older readers

• Chromatography: Basic Principles, Sample Preparations and Related Methods by Elsa Lundanes et al. John Wiley & Sons, 2013. A broad, accessible introduction for undergraduate students and laboratory technicians. It begins with the general principles before discussing each type of chromatography in detail.
• Chromatography: A Science of Discovery by Robert L. Wixom and Charles W. Gehrke. John Wiley and Sons, 2010. A detailed history of the field that will be of most interest to professionals and chemists. Unlikely to interest general readers.
• Chromatography: Fundamentals and Techniques by Erich Heftmann (ed). Elsevier, 2004. Detailed coverage of the theory, types, and applications of chromatography.

For younger readers

• All About Chemistry by Robert Winston. DK, 2015. A bright, colorful, and fun introduction to chemical elements and how they combine to make up the matter in our world.
• Kitchen Science Lab for Kids: 52 Family Friendly Experiments from Around the House by Liz Lee Heinecke. Quarry/Quarto, 2014. Marker chromatography (p.58) is one of the experiments to try.
• Chemistry by Ann Newmark. DK, 2005. A colorful guide that takes us through the history of chemistry to the role of chemicals in everyday life.
• Chemistry for Every Kid: 101 Easy Experiments That Really Work by Janice VanCleave. Wiley, 1989/2020. A new reprint of an old-book—and you may be able to track it down secondhand. A great collection of simple, safe chemistry experiments for the curious young scientist.
• IBM Making Silicon to Sort Viruses and Other Nanoscale Biological Targets : by Dexter Johnson. IEEE Spectrum, 1 August 2016. What does the future hold for lab techniques like chromatography?
• Boston Marathon: Can Technology Do a Better Job of Finding Bombs? : by Douglas McCormick. IEEE Spectrum, 18 April 2013. Do techniques like gas chromatography and mass spectrometry have a place in bomb detection?
• Stationary phases move ahead by Jon Evans, Chemistry World. A look at some of the new materials being used in chromatography columns.
• To catch a cheating athlete by Rajendrani Mukhopadhyay, Chemistry World, 8 February 2010. Exploring the chemical tricks used to trap drug cheats.
• Hope for natural insect repellent : BBC News, 4 July 2006. How chemists are using gas chromatography to develop a new mosquito repellent.
• Michael Tswett by Trevor Robinson, Chymia, Vol. 6 (1960), pp. 146–161. Was Tswett really the inventor of chromatography? This article evaluates his life and work.
• Chromatography by William H. Stein and Stanford Moore, Scientific American, Vol. 184, No. 3 (March 1951), pp. 35–41. An old but still very helpful summary of the basic concepts.

Text copyright © Chris Woodford 2009, 2020. All rights reserved. Full copyright notice and terms of use .

Rate this page

Tell your friends, cite this page, more to explore on our website....

• Get the book
• Send feedback

Chemistry Learner

It's all about chemistry.

• Chemical Bonds
• Chemical Reactions
• Materials Chemistry
• Organic Chemistry
• Periodic Trends
• Periodic Table Groups
• How to Read Periodic Table
• Naming Covalent Compounds Worksheets
• Net Ionic Equation Worksheets
• Types of Chemical Reactions Worksheets
• Word Equations Worksheets
• Valence Electrons Worksheets
• Graphing Periodic Trends Worksheets
• Periodic Trends Ionization Energy Worksheets
• Atomic Structure And Isotopes Worksheets

Paper Chromatography

How does paper chromatography work, stationary and mobile phases, paper chromatography experiment, applications.

Paper chromatography is a simple and cost-effective separation technique that separates and identifies different components in a mixture. [1-4]

In paper chromatography, a specialized paper acts as the stationary phase, while a liquid solvent is the mobile phase. The mixture to be analyzed is applied to the paper. As the solvent moves up through capillary action, it carries along the individual components of the mixture at different rates based on their solubility and affinity for the stationary phase.

The principle behind paper chromatography lies in the differential partitioning of compounds between the stationary and mobile phases. The stationary phase typically consists of cellulose fibers embedded in filter paper or thin-layer chromatography plates. These fibers provide an adsorbent surface for compounds to interact with.

Understanding the mechanism behind paper chromatography requires knowledge of several key processes. [1-4]

The first process is capillary action, which refers to the ability of liquids to flow through narrow spaces against gravity. In paper chromatography, capillary action allows the solvent to move up the paper strip due to its attraction to the fibers in the paper. As the solvent moves up, it carries the solutes in the analyzed mixture. This migration of solutes is driven by two main mechanisms: adsorption and partitioning.

Adsorption occurs when solute molecules adhere to the fibers or other surfaces within the paper. It can be influenced by polarity and molecular size, with more polar or larger molecules having stronger interactions with the stationary phase.

Conversely, partitioning involves solute molecules distributing themselves between two immiscible phases – in this case, between the stationary phase (paper) and mobile phase (solvent). The extent of partitioning depends on factors such as solute polarity and affinity for either phase.

As solutes migrate up through capillary action, they may experience different degrees of adsorption and partitioning along their journey. This results in their separation based on their characteristics. By analyzing how far each component migrates on a chromatogram – a visual representation of separated components – scientists can determine properties such as retention factor (R f ) values and identify unknown substances based on known reference compounds.

Stationary and mobile phases play crucial roles in separating components of a mixture. [1-4]

The stationary phase refers to the absorbent material fixed on the chromatography paper. It can be made of cellulose or other materials with high absorbency. The stationary phase acts as a substrate for the sample mixture to interact with during separation.

On the other hand, the mobile phase is the solvent or liquid that moves through the stationary phase, carrying the sample components. The mobile phase must have good solubility with the components of interest. It should be able to flow easily through the paper.

As the mobile phase moves through the stationary phase, it interacts differently with each mixture component based on their solubility and affinity for both phases. This differential interaction leads to separation as different components travel at different rates along the paper.

Choosing an appropriate combination of stationary and mobile phases is important for effective separation in paper chromatography. Factors such as polarity, viscosity, and compatibility between phases must be considered to achieve optimal results.

Performing a paper chromatography experiment involves several essential steps to ensure accurate results. The process begins with preparing samples for paper chromatography, then spotting the sample on the paper strip, and finally, developing the chromatogram. [1-4]

Preparing the samples is crucial in obtaining reliable data. It involves selecting appropriate substances to analyze and ensuring they are suitable for chromatography. Samples can be liquid or solid and must be dissolved or crushed into a solution before application.

Next, spotting the sample on the paper strip is done carefully to ensure accurate separation. A small spot of the prepared sample is placed near one end of a designated area on the filter paper strip. It is essential to use a capillary tube or micropipette for precise and consistent application.

Once all samples are spotted on the filter paper strip, it is time for the development of the chromatogram. This step involves placing one end of the strip into a solvent traveling up through capillary action. The choice of solvent depends on factors such as solubility and desired separation distance.

As the solvent moves up through the filter paper strip, it carries different components in each sample. These components separate based on their affinity for stationary (filter paper) and mobile (solvent) phases. The separation occurs due to differences in molecular size, polarity, or other physical properties.

Throughout this process, it is important to maintain controlled conditions such as temperature and humidity to ensure reproducibility. Further analysis can be conducted once an optimal separation has been achieved, which can take several minutes or hours depending on various factors, including solvent choice and sample composition.

The diverse applications of paper chromatography across various fields are listed below. [1-4]

• It plays a crucial role in forensic analysis by separating and identifying different components in complex mixtures, such as blood or ink samples.
• Aids in the analysis of crime scene evidence, allowing forensic scientists to determine the presence of specific substances and identify potential suspects based on chromatographic patterns
• Enables the separation of different dyes used in food coloring, helping to ensure compliance with regulatory standards and quality control measures
• Determines the authenticity and safety of food products by identifying and quantifying specific components present in complex food matrices
• Separate and identify active ingredients, impurities, and by-products in pharmaceutical formulations.
• Chem.libretexts.org
• Swe.mit.edu
• Chemlab.truman.edu

Trending Topics

© 2024 ( Chemistry Learner )

Chemistry Dictionary

A complete A-Z dictionary of chemistry terms.

Are you a chemistry student? Visit A-Level Chemistry to download comprehensive revision materials - for UK or international students!

Paper chromatography

Introduction to paper chromatography.

Paper chromatography is a chromatography technique used to separate mixture of chemical substances into its individual compounds. Paper chromatography is used to teach TLC or other chromatography as it is very similar to TLC.

Principles of paper chromatography

All chromatography follow the same principle. Paper Chromatography consists of two phases: one mobile phase and one contiguous stationery phase. The stationery phase a paper and the mobile gas is solvent. The compound mixture moves along with the mobile phase through stationery phase and separates depending on the different degree of adhesion (on the paper) of each component in the sample or the compound mixture.

Explanation

The stationery phase.

The paper chromatography is very similar to Thin layer chromatography. Difference is, instead of using a thin layer of silica on metal, it uses a special type of chromatography paper as stationery phase. This paper is made of cellulose. Cellulose is a polymer of simple sugar, glucose.

Cellulose contains -OH group similar to the silica or alumina on the TLC plate. The surface of cellulose is thus very polar. So the compounds can form hydrogen bond or can interact by van der waals dispersion forces and dipole dipole forces.

Paper chromatography works in few steps:

Step 1:  A horizontal line is drawn near one end (about 1.5 cm from the bottom edge) of the paper. In figure below 6 is the horizontal line.

Step 2:  The sample needs to be separated is placed as a small drop or line on to the paper using capillary tube. Labelling the drop by a pencil with an alphabet or number help to identify the compound later. In figure above 3 and 4 are the drops labelled. The drops are then soaked on the paper and dried.

Step 3:  The paper is then placed into a sealed container with a swallow layer of  suitable solvent. The solvent level must be lower than the pencil line or drop on it. The container need to be covered to stop the solvent to evaporate.

Step 4:  The solvent rises up the paper chromatography taking each component of the sample with it. The components travel with the solvent depends on three things:

• The polarity of the sample molecule. The non polar components travel faster than the polar component.
• The attraction between the sample molecule and the solvent or solvent mixture.
• The attraction between the sample and the silica.

Suppose any sample compound mixture contains three colored molecules  green, blue and red. According to their polarity, the order of these compounds is green<blue<red. Thus the most non polar green will travel first along with the mobile phase. Then blue and at last most polar compound the red one.

Step 5: When the solvent rises near the end of the paper then the paper should be taken out from sealed container and air dried. The paper with separated bands of components are then observed under UV-light.

The compounds in the sample travels along with solvent to give separate bands on the paper. The distance travelled by same compound with respect to the solvent is always constant. Thus the ratio of the distance that the compound travelled and the distance that the solvent travelled is denoted as R f . And mathematically expressed as:

• Paper chromatography is an chromatography technique used to separate mixture of chemical substances into its individual compounds.
• Paper chromatography consists of two phases: one mobile phase and one contiguous stationery phase.
• Paper used in paper chromatography is made of cellulose.
• A suitable solvent (mobile phase) is moved along with a compound mixture through the paper according to the polarity and the degree of adhesion of each component on the stationery phase.
• The ratio of the distance that the compound travelled and the distance that the solvent travelled is denoted as R f .

Please rate these notes

• History & Society
• Science & Tech
• Biographies
• Animals & Nature
• Geography & Travel
• Arts & Culture
• Games & Quizzes
• On This Day
• One Good Fact
• New Articles
• Lifestyles & Social Issues
• Philosophy & Religion
• Politics, Law & Government
• World History
• Health & Medicine
• Browse Biographies
• Birds, Reptiles & Other Vertebrates
• Bugs, Mollusks & Other Invertebrates
• Environment
• Fossils & Geologic Time
• Entertainment & Pop Culture
• Sports & Recreation
• Visual Arts
• Demystified
• Image Galleries
• Infographics
• Top Questions
• Britannica Kids
• Saving Earth
• Space Next 50
• Student Center

paper chromatography

Our editors will review what you’ve submitted and determine whether to revise the article.

• Open Library Publishing Platform - DRAFT – Organic and Biochemistry Supplement to Enhanced Introductory College Chemistry - Thin Layer (TLC) And Paper Chromatography (PC)
• Chemistry LibreTexts Library - Paper Chromatography - Separation and Identification of Five Metal Cations
• Oregon State University - College of Engineering - Paper chromatography
• Academia - Paper Chromatography
• Journal of Emerging Technologies and Innovative Research - Paper Chromatography: A Review

paper chromatography , in analytical chemistry , technique for separating dissolved chemical substances by taking advantage of their different rates of migration across sheets of paper. It is an inexpensive but powerful analytical tool that requires very small quantities of material.

The method consists of applying the test solution or sample as a spot near one corner of a sheet of filter paper. The paper is initially impregnated with some suitable solvent to create a stationary liquid phase . An edge of the paper close to the test spot is then immersed in another solvent in which the components of the mixture are soluble in varying degrees. The solvent penetrates the paper by capillary action and, in passing over the sample spot, carries along with it the various components of the sample. The components move with the flowing solvent at velocities that are dependent on their solubilities in the stationary and flowing solvents. Separation of the components is brought about if there are differences in their relative solubilities in the two solvents. Before the flowing solvent reaches the farther edge of the paper, both solvents are evaporated , and the location of the separated components is identified, usually by application of reagents that form coloured compounds with the separated substances. The separated components appear as individual spots on the path of the solvent. If the solvent flowing in one direction is not able to separate all the components satisfactorily, the paper may be turned 90° and the process repeated using another solvent.

Paper chromatography has become standard practice for the separation of complex mixtures of amino acids , peptides , carbohydrates , steroids , purines , and a long list of simple organic compounds . Inorganic ions can also readily be separated on paper. Compare thin-layer chromatography .

• Skip to primary navigation
• Skip to main content
• Skip to primary sidebar

• FREE Experiments
• Kitchen Science
• Climate Change
• Egg Experiments
• Fairy Tale Science
• Edible Science
• Human Health
• Inspirational Women
• Forces and Motion
• Science Fair Projects
• STEM Challenges
• Science Sparks Books
• Contact Science Sparks
• Science Resources for Home and School

Paper Chromatography Experiment

March 17, 2021 By Emma Vanstone Leave a Comment

This simple felt tip pen paper chromatography experiment is a great way to learn about this particular method of separating mixtures .

WHAT IS CHROMATOGRAPHY?

Chromatography   is a technique used to separate mixtures. Information from a chromatography investigation can also be used to identify different substances.

In chromatography, the mixture is passed through another substance, in this case, filter paper. The different-coloured ink particles travel at different speeds through the filter paper, allowing the constituent colours of the pen ink to be seen.

All types of chromatography have two phases: a mobile phase where the molecules can move and a stationary phase where they can’t move. In the case of paper chromatography, the stationary phase is the filter paper, and the mobile phase is the solvent ( water ).

The more soluble the ink molecules, the further they are carried up the paper.

The video below shows chromatography in action.

You’ll need:

Filter paper or paper towel

Felt tip pens – not washable or permanent

A container – glass, jar or plate

Instructions

Pour a small amount of water onto a plate or into the bottom of a jar.

Find a way to suspend the filter paper over the water so that just the very bottom touches the water. If you do the experiment in a jar, the easiest way to do this is to wrap the top of the filter paper around a pencil, clip it in place, and suspend it over the top of the jar.

Our LEGO holder worked well, too!

Use the felt tip pens to draw a small circle about 1cm from the bottom of the filter paper with each colour pen you want to test.

Suspend the filter paper in the water and watch as the ink moves up the filter paper.

You should end up with something like this! The end result is called a chromatogram.

What happens if you use washable pens?

If the inks are washable, they tend to contain just one type of ink, so there is no separation of colour.

Below, only a couple of the inks have separated compared to the non-washable pens above.

Why does chromatography work?

When the filter paper containing the ink spots is placed in the solvent ( in this case, water ), the dyes travel through the paper.

Different dyes in ink travel through the chromatography filter paper at different speeds. The most soluble colours dissolve and travel further and faster than less soluble dyes, which stick to the paper more.

I’ve created a free instruction sheet and chromatography experiment write up to make the activity even easier.

Extension task

Experiment with different types and colours of pens. Depending on the type of ink used, some will work better than others.

Try chromatography with sweets .

Steamstational also has a great leaf chromatography investigation.

More separation experiments

Clean up water by making your own filter .

Separate water and sand by evaporation .

Make colourful salt crystals by separating salt and water.

Separate liquid mixtures with a bicycle centrifuge .

Last Updated on May 20, 2024 by Emma Vanstone

Safety Notice

Science Sparks ( Wild Sparks Enterprises Ltd ) are not liable for the actions of activity of any person who uses the information in this resource or in any of the suggested further resources. Science Sparks assume no liability with regard to injuries or damage to property that may occur as a result of using the information and carrying out the practical activities contained in this resource or in any of the suggested further resources.

These activities are designed to be carried out by children working with a parent, guardian or other appropriate adult. The adult involved is fully responsible for ensuring that the activities are carried out safely.

Reader Interactions

Leave a reply cancel reply.

Your email address will not be published. Required fields are marked *

Microbe Notes

Thin Layer Chromatography: Principle, Parts, Steps, Uses

Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.

In this physical method of separation, the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Depending upon the stationary phase and mobile phase chosen, they can be of different types.

Table of Contents

Interesting Science Videos

What is Thin Layer Chromatography (TLC)?

Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent solid / (liquid) spread over a plate and liquid as a mobile phase.

Principle of Thin Layer Chromatography (TLC)

• Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase.
• After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.
• It is thus based on the principle of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent, its treatment and nature of solvents employed. The components with more affinity towards stationary phase travels slower. Components with less affinity towards stationary phase travels faster.
• Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character is identified by means of suitable detection techniques.

Components of Thin Layer Chromatography (TLC)

TLC system components consists of:

• TLC plates,  preferably ready made with a stationary phase: These are stable and chemically inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The stationary phase on the plates is of uniform thickness and is in a fine particle size.
• TLC chamber-  This is used for the development of TLC plate. The chamber maintains a uniform environment inside for proper development of spots. It also prevents the evaporation of solvents, and keeps the process dust free.
• Mobile phase-  This comprises of a solvent or solvent mixture The mobile phase used should be particulate-free and of the highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, a stationary phase.
• A filter paper-  This is moistened in the mobile phase, to be placed inside the chamber. This helps develop a uniform rise in a mobile phase over the length of the stationary phase.

Procedure of Thin Layer Chromatography (TLC)

The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize. These days, however, ready-made plates are more commonly used.

• With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots.
• Then, samples solutions are applied on the spots marked on the line in equal distances.
• The mobile phase is poured into the TLC chamber to a leveled few centimeters above the chamber bottom.
• A moistened filter paper in mobile phase is placed on the inner wall of the chamber to maintain equal humidity (and also thereby avoids edge effect).
• Now, the plate prepared with sample spotting is placed in TLC chamber so that the side of the plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid.
• The plate is then immersed, such that the sample spots are well above the level of mobile phase (but not immersed in the solvent) for development.
• Sufficient time is given for the development of spots.
• The plates are then removed and allowed to dry.
• The sample spots are then seen in a suitable UV light chamber, or any other methods as recommended for the given sample.

Some common techniques for visualizing the results of a TLC plate include

• Iodine Staining: is very useful in detecting carbohydrates since it turns black on contact with Iodine
• KMnO4 stain (organic molecules)
• Ninhydrin Reagent: often used to detect amino acids and proteins

Retention Factor (R f ) Value

• The behaviour of a compound on a TLC is usually described in terms of its relative mobility or Rf value.
• Rf or Retention factor is a unique value for each compound under the same conditions.
• The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant:
• solvent system
• thickness of the adsorbent
• amount of material spotted
• temperature
• Since these factors are difficult to keep constant from experiment to experiment, relative Rf values are generally considered.
• Relative Rf” means that the values are reported relative to a standard.
• The Rf value is calculated using the following equation:

Applications of Thin Layer Chromatography (TLC)

• In monitoring the progress of reactions
• Identify compounds present in a given mixture
• Determine the purity of a substance.
• Analyzing ceramides and fatty acids
• Detection of pesticides or insecticides in food and water
• Analyzing the dye composition of fibers in forensics
• Assaying the radiochemical purity of radiopharmaceuticals
• Identification of medicinal plants and their constituents 

Advantages of Thin Layer Chromatography (TLC)

• It is a simple process with a short development time.
• It helps with the visualization of separated compound spots easily.
• It helps in isolating of most of the compounds.
• The separation process is faster and the selectivity for compounds is higher (even small differences in chemistry is enough for clear separation).
• The purity standards of the given sample can be assessed easily.
• It is a cheaper chromatographic technique.

Limitations of Thin Layer Chromatography (TLC)

• It cannot tell the difference between enantiomers and some isomers.
• In order to identify specific compounds, the Rf values for the compounds of interest must be known beforehand.
• TLC plates do not have long stationary phases. Therefore, the length of separation is limited compared to other chromatographic techniques.
• https://owlcation.com/stem/tlc-thin-layer-chromatography-Principle-Procedure
• https://www.utsc.utoronto.ca/webapps/chemistryonline/production/tlc.php
• https://en.wikipedia.org/wiki/Thin-layer_chromatography
• https://chem.libretexts.org/Demos%2C_Techniques%2C_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography
• https://www.slideshare.net/LavakusaBanavatu/thin-layer-chromatography-43293607

About Author

Sagar Aryal

1 thought on “Thin Layer Chromatography: Principle, Parts, Steps, Uses”

Leave a comment cancel reply.

Save my name, email, and website in this browser for the next time I comment.

• Analytical Chemistry

Column Chromatography

What is column chromatography.

In chemistry, Column chromatography is a technique which is used to separate a single chemical compound from a mixture dissolved in a fluid.

Column chromatography separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates which allows them to get separated in fractions. This technique can be used on a small scale as well as large scale to purify materials that can be used in future experiments. This method is a type of adsorption chromatography technique.

Table of Content

• Applications
• Frequently Asked Questions – FAQs

Column Chromatography Principle

When the mobile phase along with the mixture that needs to be separated is introduced from the top of the column, the movement of the individual components of the mixture is at different rates. The components with lower adsorption and affinity to the stationary phase travel faster when compared to the greater adsorption and affinity with the stationary phase. The components that move fast are removed first whereas the components that move slowly are eluted out last.

The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of the components is expressed as:

R f = the distance travelled by solute/ the distance travelled by the solvent

R f is the retardation factor.

Column Chromatography Diagram

Elution is a chemical process that involves removing a material’s ions by ion exchange with another material. The chromatographic technique of extracting an adsorbed substance from a solid adsorbing media using a solvent. The eluent is the solvent or mobile phase that passes through the column. When the polarity of the eluent matches the polarity of the molecules in the sample, the molecules desorb from the adsorbent and dissolve in the eluent.

The fraction of the mobile phase that transports the sample components is known as eluent. The mixture of solute and solvent that exits the column is known as an eluate. The eluate is made up of the mobile phase and analytes. A substance that separates and moves constituents of a mixture through the column of a chromatograph. The eluent in liquid chromatography is a liquid solvent whereas in gas chromatography is a carrier gas.

Column Chromatography Procedure

Before starting with the Column Chromatography Experiment let us understand the different phases involved.

Mobile phase – This phase is made up of solvents and it performs the following functions:

• It acts as a solvent-sample mixture that can be introduced in the column.
• It acts as a developing agent – helps in the separation of components in the sample to form bands.
• It acts as an eluting agent – the components that are separated during the experiment are removed from the column
• Some examples of solvents used as mobile phases based on their polarity are – ethanol, acetone, water, acetic acid , pyridine, etc.

Stationary phase – It is a solid material which should have good adsorption properties and meet the conditions given below:

• Shape and size of particle: Particles should have a uniform shape and size in the range of 60 – 200μ in diameter.
• Stability and inertness of particles: high mechanical stability and chemically inert. Also, no reaction with acids or bases or any other solvents was used during the experiment.
• It should be colourless, inexpensive and readily available.
• Should allow free flow of mobile phase
• It should be suitable for the separation of mixtures of various compounds.

Column Chromatography Experiment

• The stationary phase is made wet with the help of solvent as the upper level of the mobile phase and the stationary phase should match. The mobile phase or eluent is either solvent or a mixture of solvents. In the first step the compound mixture that needs to be separated, is added from the top of the column without disturbing the top level. The tap is turned on and the adsorption process on the surface of silica begins.
• Without disturbing the stationary phase solvent mixture is added slowly by touching the sides of the glass column. The solvent is added throughout the experiment as per the requirement.
• The tap is turned on to initiate the movement of compounds in the mixture. The movement is based on the polarity of molecules in the sample. The non-polar components move at a greater speed when compared to the polar components.
• For example, a compound mixture consists of three different compounds viz red, blue, green then their order based on polarity will be as follows blue>red>green
• As the polarity of the green compound is less, it will move first. When it arrives at the end of the column it is collected in a clean test tube. After this, the red compound is collected and at last blue compound is collected. All these are collected in separate test tubes.

Column Chromatography Applications

• Column Chromatography is used to isolate active ingredients.
• It is very helpful in separating compound mixtures.
• It is used to determine drug estimation from drug formulations.
• It is used to remove impurities.
• Used to isolate metabolites from biological fluids.

Types of Column Chromatography:

1. Adsorption column chromatography – Adsorption chromatography is a technique of separation, in which the components of the mixture are adsorbed on the surface of the adsorbent.

2. Partition column chromatography – The stationary phase, as well as mobile phase, are liquid in partition chromatography .

3. Gel column chromatography – In this method of chromatography, the separation takes place through a column packed with gel. The stationary phase is a solvent held in the gap of a solvent.

Frequently Asked Questions – FAQs

What is the principle involved in column chromatography.

The basic principle involved in column chromatography is to adsorb solutes of the solution with the help of a stationary phase and further separate the mixture into discrete components.

What is column chromatography?

It is a precursory technique used in the purification of compounds based on their hydrophobicity or polarity. In this chromatography process, the molecule mixture is separated depending on its differentials partitioning between a stationary phase and a mobile phase.

What is the main advantage of column chromatography?

The main advantage of this chromatography technique is that the stationary phase is less expensive and can be easily disposed of as it undergoes recycling.

How are the compounds separated in this technique?

The separation is similar to that of TLC where the compound mixture is carried by a mobile phase via a stationary phase.

Which compounds elute out first in the column chromatography technique?

Non-polar compounds. The polar compounds will strongly commune with the silica when compared to the non-polar compounds.

What is elution in column chromatography?

What are the limitations of column chromatography, what are the different types of column chromatography, what is an adsorption column chromatography, what is gel column chromatography.

To learn more about the different types of Column Chromatography from the experts, register to BYJU’S now!

Other important links:

Put your understanding of this concept to test by answering a few MCQs. Click ‘Start Quiz’ to begin!

Select the correct answer and click on the “Finish” button Check your score and answers at the end of the quiz

Visit BYJU’S for all Chemistry related queries and study materials

Your result is as below

Request OTP on Voice Call

Leave a Comment Cancel reply

Your Mobile number and Email id will not be published. Required fields are marked *

Post My Comment

What is developing agent?

Chromatographic procedure generally involves introducing at the top of the column the mixture of the components to be separated, developing the mixture with a suitable agent, and collecting the components in separate effluent fractions. The best developing agent in thin-layer chromatography was Petroleum ether: ethyl acetate.

Register with BYJU'S & Download Free PDFs

Register with byju's & watch live videos.