UTPA STEM/CBI Courses/Organic Chemistry/Chromatography

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Course Title: Organic Chemistry

Lecture Topic: Chromatography

Instructor: Debasish Bandyopadhyay

Institution: The University of Texas-Pan American

Backwards Design[edit]

Course Objectives

  • Primary Objectives- By the next class period students will be able to:
    • Identify various colors
    • Know the name of various separation techniques
    • Recognize the difference between chromatographic separation with several other techniques
  • Sub Objectives- The objectives will require that students be able to:
    • Get an overall idea of some common organic solvents
    • Learn about capillary action

  • Difficulties- Students may have difficulty:
    • Get an overall idea of some common organic solvents
    • Learn about capillary action
  • Real-World Contexts- There are many ways that students can use this material in the real-world, such as:
    • Chromatography is used as a technique to separate the additives, vitamins, preservatives, proteins and amino acids. Some other chromatography uses are in the detection of drugs or medications in the urine and the separation of traces of chemicals in the case of fire in houses or buildings.
    • Chromatography has evolved to be one of the most widely used chemical techniques to separate particles and contaminates in chemical plants. For example, in the chemical industries, pesticides and insecticides like DDT in the groundwater and PCBs (Polychlorinated biphenyls) are removed by the process of chromatography. As a major testing tool, chromatography is used by government agencies to separate toxic materials from the drinking water and also to monitor air quality.

Model of Knowledge

  • Concept Map
    • Understanding of why separation is needed
    • Understanding of polarity of solvents
    • Understanding of polarity of organic compounds
    • Predicting various solvent systems
    • Capillary action

  • Content Priorities
    • Enduring Understanding
      • The governing principle of chromatography is that different chemicals in a mixture have different degrees of dissolving in a liquid or sticking to a solid surface.
      • Chromatography can identify a chemical and separate it from a dense mixture of other chemicals and show it on a surface. text
      • Various chemicals in a mixture have different sticking ability on a surface. By varying this process in many ways, the chromatography technique can be used to separate any amount of quantities ranging from micrograms (in laboratories) to tons (in chemical plants)
    • Important to Do and Know
      • In the chromatography processes, a stream of liquid that is called as mobile phase is made to flow through a tube known as column, and it is packed with porous solid material, called as the stationary phase.
      • The sample of the mixture that is to be analyzed is sent through the mobile phase and as the mixture proceeds in the tube, the compounds are separated. Chromatography is preferred over many other techniques as it doesn't cause any molecular changes in the composition of the chemicals involved.
      • In the field of organic chemistry and pharmacy, chiral compounds are very close to each other in terms of atomic or molecular weight, element composition, and the physical properties. However, they exist in two different forms, called as the enantiomers and optical isomers. Both these compounds though may appear to be same, have very different chemical properties. So, in pharmacy, chromatography becomes crucial to analyze the exact chiral compound so that correct medicines can be manufactured.
    • Worth Being Familiar with
      • Chromatography refers to a set of techniques used to separate different compounds. The word comes from the Greek chromatos (color) and graphein (to write). So, as one might guess, chromatography involves separating chemicals and identifying them by color.
      • It is commonly used in laboratories to isolate new compounds, analyze subtle differences between different environmental samples, and even in the sequencing of DNA.

Assessment of Learning

  • Formative Assessment
    • In Class (groups)
      • Different chromatographic procedure for undergraduate students
      • Thin layer chromatography
    • Homework (individual)
      • What is an everyday item that could be separated using paper chromatography?
      • Do you use chromatography to separate homogeneous mixtures?
  • Summative Assessment
    • Run thin layer chromatography (TLC) of a two component mixtures
    • Column packing for column chromatography

Legacy Cycle[edit]


By the next class period, students will be able to:

  • To separate pigments found in markers. (Rose Art works the best. Do not use Crayola- won't separate)
  • To determine the primary colors of pigments.
  • To calculate the Rf value of primary colors.

The objectives will require that students be able to:

  • Guess about the polarity of organic solvents
  • Measure solvent front and compound front i.e. in turn, the Rf value of the compound
  • Prepare the concentration of the solution to be used for separation


The unique chromatography experiment works extremely well and promotes insight and scientific inquiry of chromatography. The project is suitable for people of all ages.


Column chromatography: The stationary phase is a powdered adsorbent which is placed in a vertical glass column. The mixture to be analyzed is loaded on top of this column. The mobile phase is a solvent poured on top of the loaded column. The solvent flows down the column, causing the components of the mixture to distribute between the powdered adsorbent and the solvent, thus (hopefully) separating the components of the mixture so that as the solvent flows out of the bottom of the column, some components elute with early collections and other components elute with late fractions.

Thin Layer Chromatography: The stationary phase is a powdered adsorbent which is fixed to a aluminum, glass, or plastic plate. The mixture to be analyzed is loaded near the bottom of the plate. The plate is placed in a reservoir of solvent so that only the bottom of the plate is submerged. This solvent is the mobile phase; it moves up the plate causing the components of the mixture to distribute between the adsorbent on the plate and the moving solvent, thus separating the components of the mixture so that the components are separated into separate "spots" appearing from the bottom to the top of the plate.

Gas Chromatography: The stationary phase is a high-boiling liquid. (Think of it as a viscous oil, or waxy substance.) This high-boiling liquid is packed into a long, narrow glass or metal column. The mixture to be analyzed is loaded by syringe into the beginning of this column. The mobile phase is an inert gas which continuously flows through the column. The components of the mixture distribute between the stationary high-boiling liquid (these components are either condensed or absorbed on the high-boiling liquid) and mobile gas (vapor) phase moving through the column. The gaseous mixture flows through a detector at the end of the column and if it has been successfully separated, the components show as different 'blips' or peaks on a recorder.


In Gas Chromatography, the determining factor in how fast a component travels is usually (but not always) the boiling point of the compound. (If a polar high-boiling liquid adsorbent is used in the GC column, the polarity of the components determines the elution order.) In Column and Thin Layer chromatography, the stationary phase (the adsorbent: silica gel or alumina) is polar, and the polarities of both the component of the mixture and the solvent used as the mobile phase are the determining factors in how fast the compound travels.


Column chromatography is used to separate and purify components of a mixture. TLC and GC are usually (but not always!) used only to analyze mixtures: to determine the number of components and to see if a desired component is present. TLC is often used to determine the "ideal system" for a column chromatography procedure (as explained in the following paragraphs).


Determining solvent systems for TLC and Column Chromatography


Chromatography is a method of separating out materials from a mixture. Ink is a mixture of several dyes and therefore we can separate those colors from one another using chromatography. When ink is exposed to certain solvents the colors dissolve and can be separated out. When we expose a piece of paper with ink on it to a solvent, the ink spreads across the paper when the ink dissolves. Some inks are water-soluble, so you can use water as the solvent. Inks which are not water soluble are often alcohol-soluble and you can use Isopropyl alcohol as the solvent to create your chromatograph. Different ink pens use different types of ink and this is obvious when you expose the ink to a solvent. A banding pattern of the components of the ink mixture is called a chromatograph. Follow the instructions below to discover which pen was used to write a note.

Pre-Lesson Quiz[edit]

  1. What is chromatography?
  2. How the term “chromatography” has been derived?
  3. Who invented chromatography?
  4. What is retention factor (Rf)?
  5. What is polarity of solvent?
  6. Which one is more polar- methanol or hexanes?
  7. Which one is less polar- ethyl acetate or diethyl ether?
  8. How TLC spots are typically visualized?
  9. How Rf is calculated?
  10. What is a stationary phase?
  11. What is slurry?
  12. What is eluent?
  13. What is absorbent?
  14. What is a mobile phase?
  15. What is flow-rate?
  16. What is a “gravity column”?
  17. Mention the name of a “mixed chromatography” technique.
  18. What is the “bed” of a column?
  19. Mention the name of two commonly used packing materials.
  20. What should be the ration of bed: slurry for an ideal column?

Test Your Mettle Quiz[edit]

  1. What is the significance of Rf ?
  2. How Rf can be measured?
  3. Give the name of a common stationary phase in column chromatography?
  4. Give the name of a common mobile phase in gas chromatography?
  5. Why is it important to avoid air bubbles in the column during chromatography?
  6. What property does paper chromatography make use of to separate its components?
  7. What is Van Deemter equation?
  8. What kind of packing material you will select to separate a basic mixture by column chromatography?
  9. What kind of packing material you will select to separate a acidic mixture by column chromatography?
  10. What is dielectric constant?
  11. How dielectric constant influences the polarity of a solvent?
  12. What is “Ion-exchange chromatography”?
  13. On which principle “Ion-exchange chromatography” works?
  14. To separate amino acid mixture what chromatographic technique should be followed?
  15. What chromatographic technique should be followed to separate proteins?
  16. What are the uses of chromatography in science, medicine and law enforcement?
  17. What chromatographic technique should be followed for the determination of the components a plant contains?
  18. What is “preparative TLC?”
  19. How does column chromatography works?
  20. Is it feasible to use column chromatography for quantitative analysis? Why or why not?