Who Wrote the Note?

Analysis of Ink by Thin Layer Chromatography

Introduction:

Chemists use the technique of chromatography to separate compounds for purification and identification. Many types of chromatography have been developed, but utilization of a moving phase and a stationary phase is common to this category of techniques. In thin layer chromatography, the method used in this experiment, the moving phase is the solvent, and the stationary phase, or adsorbant, is a thin layer of adsorbant particles attached to a solid plate.

Chromatography works because of differences in polarity between the stationary phase, the solvent, and the components of the mixture being studied. When using a solvent with low polarity, the majority of the components of the mixture, if not all of them, will be held more strongly to the adsorbant than the solvent molecules, so the solvent will not displace them and carry them along with it. If a more polar solvent is chosen, it will displace the majority of the molecules in the sample, but depending on the polarity of each of the components, some may travel farther with the solvent than others, resulting in separation of each of the components. The distances traveled with the solvent are characteristic of different molecules and make identification possible (see Figure 1).

A solvent much more polar than the components of the mixture will displace all of the molecules and carry them all very easily on the solvent front, yielding no separation that would be useful in identification. Finding a solvent suitable for separation of a mixture or identification of a sample usually requires trying several solvents of different polarity.

Purpose:

The purpose of this lab is to use thin layer chromatography to identify an unknown ink sample.

Equipment:

Chromatography jar / Tweezers
Capillary tubes or microdroppers / Methanol
Scissors / Developing solvent: mixture of n-butyl alcohol, ethyl alcohol, water, acetic acid:
60/10/20/0.5
TLC plates / Ruler
Suspect pens / Pencil
Evidence note / Pasteur pipette or disposable pipette
Scintillation vial

Safety:

  • Always wear safety goggles.
  • Wear gloves and wash thoroughly with soap and water if you get solvent on you.

Procedure:

  1. Prepare a developing chamber by adding about 0.5 cm or 2mL of developing solvent to the chromatography jar, and closing the lid until needed.
  1. Prepare the TLC plate:
  2. Obtain a TLC plate. Handle the plate by the edges only, making sure to avoid touching the white silica layer (the side you are using).
  1. With a pencil (not a pen), use the ruler to gently draw a line about 1.0-1.5 cm (the width of your pinky finger) from the bottom of the plate. Be careful not to chip off the silica layer.
  1. As shown in the diagram (Data section), draw a cross hair (+) for each sample.
  1. Write your initials in pencil at the very top or bottom of the plate.
  1. Prepare the evidence:
  2. Cut a piece of the evidence note that contains approximately 2-3 cm of ink.
  1. Place the piece of evidence note into a scintillation vial, and using a pipette, add methanol one drop at a time until the ink is extracted (removed) from the paper. The solution should be fairly dark. Try to use as little methanol as possible so the solution is fairly concentrated.
  1. Spot the plate with evidence ink:
  2. Immerse the capillary tube into the evidence ink solution until some liquid is drawn into the capillary. If using a microdropper, make sure the end of the capillary tube that is in the dropper is below the surface of the liquid. This may take a few tries.
  1. Very gently press the end of the capillary tube at the crosshair on the TLC plate. Keep the spot small by touching the capillary quickly to the plate and then removing it immediately. Allow the spot to dry and repeat spotting directly over your original spot.
  1. Record the sample identity on diagram below (Data section).
  1. Spot the plate with suspect ink:
  2. Using each suspect pen, gently make an ink dot at each crosshair on the same TLC plate you used to spot the evidence ink. Make sure the ink dots do not touch one another.
  1. Record the sample identities on the diagram.
  1. Develop the sample:
  2. Before placing the TLC plate in the developing chamber, place the TLC plate alongside the jar containing the solvent. The solvent level must be below the spots and the sides of the plate must never touch the jar.
  1. Place the plate vertically in the developing jar with the top end leaning against the jar.
  1. Reseal the jar and allow the solvent to rise until it is about 2-3 cm from the top of the plate. If the solvent does not rise far enough before the end of the lab period, your teacher will remove the plate from the jar and mark the solvent front.
  1. Allow the TLC plate to dry.
  1. Mark the center of each spot with a pencil and then measure the distance traveled by each component.
  1. Using the TLC plate diagram, draw what your plate looks like. In the table on the following page, record the distances from the original location of the spot to the solvent front and the distance from the origin to the center of each spot.
  1. Clean up:
  2. Dispose of all liquids as instructed by your teacher.
  3. Wash your hands with soap and water.

Data:

Analysis and Calculations:

  1. Calculate the Rf for each spot by dividing the distance the spot traveled by the distance the solvent traveled. The Rf value is characteristic of each substance and may be used for identification of the substance.

# / Sample / Distance traveled by solvent front (cm) / Distance traveled by sample (cm) / Rf
1
2
3
4
5
6
7
  1. Compare the results of the suspect ink samples to the results of the evidence note ink. What do the results indicate about the evidence note?

3.Were any of the inks inseparable using this solvent? Why?