Chromatography Notes and Labs

Target Audience 9 - 12
Student Handout Version

Background:

Organic analysis of unknown substances includes analytical techniques such as Chromatography, Spectophotometry and Mass Spectrometry. Chromatography is a means of separating and identifying organic components. It is especially useful to separate mixtures i.e. many illicit drugs contain many different materials to dilute the drug of interest. Gas Chromatography separates molecules using high temperature system to vaporize all the components and subsequently separated on a column.

Theory of chromatography uses the different polarities of the stationary phase, mobile phase and the liquid mixture of different compound to be separated. The compounds that have the most similar polarity to the mobile phase i.e. solvent or eluant will move the fastest and be closer to the solvent line than the other types of molecules which have less similar polarity to the solvent. The compounds more attracted to the stationary phase i.e. paper or thin layer plate will also move more slowly. Thus, molecules can be separated from each other based on their different polarities.

Gas Chromatography (GC) separates mixtures based on their distribution between a stationary liquid phase and a mobile gas phase. This is a more accurate technique. Paint chip s, fibers and plastics can be tested in a GC. A chromatogram with a specific pattern is produced and can be used to identify materials found at a crime scene as belonging to a suspect.

HPLC (High Performance Liquid Chromatography) uses a stationary phase that is a thin film with a mobile gas phase. Again a pattern specific to particular molecules is produced and can be matched to standard references or to a suspect.

TLC (Thin Layer Chromatography) can be an inexpensive screening test which is often used prior to more expensive analytical tests. This chromatography is done on thin layer plates of glass or plastic coated with silica (SiO2) or alumina (Al2O3). These compounds are run in an appropriate solvent and visualized with ultraviolet light.

Notes: This kit is very user friendly. It contains five organic dye molecules with different polarities; the one that matches the solvent polarity the most moves furthest with the solvent. Silica (SiO2) is coated on plastic. Handle with gloves. The plates are spotted with the different dyes. Then, the spotted plates are placed in jars with lids and the solvent (eluant) which was acetone and water (50:50 ). After the plates are run and the dyes have moved, calculate the Rf values= distance dye moves/ distance solvent moves.

This lab can be done in one or two days depending on the amount of lecture notes given. The plates take about 15-20 min to run in the solvent and 5-10 minutes to load the dyes. One drop of dye is enough for the entire class, so I set out 8 plates (one per dye and one for each unknown mixture)

LAB on TLC Lab: Thin Layer Chromatography Flinn Scientific catalog # AP4504

Purpose: To learn to use a separation technique of chromatography using known organic dye molecules and three unknown dye mixtures.

Equipment:

  1. TLC plates (sheets) (in kit)
  2. Capillary tubes (in kit)
  3. Jars with lids or 250 nml beakers with cover (watch glass) to hold a 10 cm TLC plastic strip (not in kit)
  4. Rulers
  5. Pencils
  6. Graduated cylinders, 10 ml
  7. Scissors
  8. Bunsen burner
Reagents:
  1. 5 dyes (in kit)
  2. Acetone and water chromatography solvent (in kit)
Procedure:
  1. Make spotters by heating the middle of a capillary tube and pulling apart and breaking it in the middle to produce two spotters. Need one spotter for each dye and the unknowns.
  2. Label plates with pencil, one cm from bottom draw line to put the dyes onto (3 spots per plate). Label the top of the plate 1,2,3, or 4,5, unknown.
  3. Spot with a very small amount of dye using a clean capillary spotter for each substance.
  4. After applying the dyes, set into chromatography chamber and leave undisturbed till solvent reaches top of plate.
  5. Calculate Rf values= distance dye moves/ distance solvent moves and observe dyes under fluorescent light.
Conclusion or Data Analysis:
  1. Match Rf values with the unknown.
  2. Match color and fluorescence with the unknowns.
  3. Describe chromatography process.
  4. Describe other types of chromatography and their purposes.
References and Resources:
Flinn Scientific catalog # AP4504
"Criminalistics: An Introduction to Forensic Science" seventh edition By Richard Safestein Ed. Prentice Hall

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