Analysis of the Color of Water Soluble Inks


If a dye separates into several dyes of different colors, then those individual dyes must have been responsible for the color of the original dye.  Using the color of the dyes, the values of the RF's for dyes in different solvents, and the visible spectra of the dyes, it is possible to determine if two food colors use a common dye to get their color.  Using this idea and the nature of the different subtractive primary colors, it is possible to identify the the origin of the color of a food coloring dye.  It is also possible to identify the specific dye that is used.  In this experiment the individual dyes from different foods and food colorings are independent variables and the colors of the chromatogram spots, RF's and the spectra are dependent variables.

Different solvents can change the effectiveness of a separation by changing the values of the RF'sThis can be investigated by chromatographing the dyes several times with different solvents.  In this part of the experiment the composition of the solvent is and independent variable and the values of the RF's are dependent variables.

There are three parts to the purpose of this experiment.  In general, they are to determine:

At the beginning of your report in your notebook, you must write the purpose of your experiment.  You can follow the format from above, but be specific.   For example, which foods are you studying?  Which solvents are you using?   Use the purpose section to ask the specific questions that you are trying to answer with your experiment.

Some Background About the Method

Paper chromatography is a method chemists use to separate compounds from one another, but not change them. In this section we will explore how this separation is made -- using different inks as mixtures. Molecules with similar arrangements of their atoms or molecular structures are attracted to each other. Water molecules have the structure shown below in which the two hydrogen atoms form a 104o angle with the oxygen at the vertex.

Because of this structure the oxygen end of the molecule has a small negative electrical charge and the hydrogen end has a small positive charge. Liquid water is held together by the attraction between the charges on different molecules. This is shown below for a small cluster of water molecules.

A molecule with these charged regions is called a polar molecule. Methanol (CH3OH) has a similar structure, and the methanol molecules are very soluble in water because of the mutual attraction between the two polar molecules.

A more complex, yet still similar molecule is cellulose, a molecule which is the basic component of paper. It is a very long molecule (a polymer) in which thousands of rings of six atoms each are linked together like beads. A portion of a cellulose molecule is shown below.

The polar -OH regions of these molecules are attracted to OH groups on adjacent cellulose chains helping to hold the fibers together in paper. Not surprisingly, water molecules, being polar, are also attracted to these regions and when paper is wet it loses strength because the water molecules get between the cellulose chains and weaken the attraction between them.

When the end of a piece of paper is dipped into water the water molecules keep finding new places (polar regions) to stick to and so the water molecules climb up the paper being replaced by new water molecules from below. Other molecules which might be dissolved in the water will also be carried along up the paper. This is applied to the separation of dyes in a technique known as paper chromatography.

A spot of dye is placed on the paper above the level of the water. As the water moves up, the dye molecules will move with it if they are more strongly attracted to the water molecules than to the paper molecules. If the dye molecules are more strongly attracted to the paper than to the water, they will move more slowly than the water or even not at all. What if the dye is a mixture? If two or more dyes have been mixed to form, each dye may move at a different rate as the water moves up the paper. If this happens, they will separate and we can identify them . This is shown in the sketches below for the separation of the dyes in a black ink..

After running the chromatogram, each separated "spot" can be assigned a Retention Factor (RF) which is characteristic of the specific dye(s) associated with it. The RF is a ratio of the distance the "spot" travels relative to the distance the solvent (water in this case) travels. The RF is calculated by dividing the "spot" distance by the solvent distance. This ratio should be a constant that is characteristic of the dye(s) in a particular spot under a particular set of chromatographic conditions (i.e., paper, water solvent, etc.)

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We can use this as evidence to help answer several questions about the inks.

Procedure for Obtaining a Chromatogram for a Dye
Separate the dyes in the inks of the Mr. Sketch pens assigned to you by your instructor using paper chromatography as outlined below.

  1. Cut pieces of filter paper into rectangular strips about 2 or 3 cm wide and at least 11 cm long. Trim one end as shown below. Use a pencil to draw a short line about 1 cm from the tapered end. Place a small spot of dye on the pencil line using a glass capillary tube, or better, a drawn-out glass disposable pipet with an open end.


  1. Fold about 1 mm of the filter paper strip along the dotted line (see above) and attach enough tape to it so it can be suspended across the bottle mouth as shown. Pour distilled water into the chromatography bottle until it is deep enough to just touch the bottom of a suspended strip of filter paper. Be sure the water does not touch the dye spot and the paper strip does not stick to the side of the glass jar. Place the lid over the jar.

  1. Allow the water to rise on the paper until it is about 1 cm from the top. Remove, label, and allow the paper to air dry. Record results in your notebook (color, measured distances,solvent, observations). Be sure to put your dried chromatograms in the results section of your note book. 

Procedure for Obtaining a Visible Spectrum of a Dye Solution

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  1. Be sure the spectrophotometer is turned on.
  2. Set the wavelength knob to 600 nm.
  3. Using the zero adjust knob on the left side, set the needle to read 0% transmittance (%T) on the top of the meter.  (Nothing should be in the sample compartment)
  4. Fill a cuvette with deionized water and insert it in the sample compartment with the line facing the front.  Close the top.
  5. Use the 100% adjust knob on the right side to set the needle to 100% with the water-containing cuvette in the holder.  Remove the cuvette and set it aside without emptying it.
  6. Fill the other cuvette with your solution.
  7. Insert it in the instrument and close the cover.  Read the absorbance from the bottom scale on the meter.  Record, in the results section of your notebook, the wavelength and corresponding absorbance reading.
  8. Remove the cuvette, close the top, and change the wavelength to a setting which is 20 nm lower.
  9. Reset the 0%T if it has changed (empty sample compartment).
  10. Insert the cuvette of deionized water and reset the 100%T.
  11. Replace the water cuvette with your sample-containing cuvette and read and record the absorbance again.
  12. Repeat steps 8 through 11 until you reach 360 nm.
  13. Make a graph of your spectrum in your notebook.  Place the absorbance values on the y-axis and the wavelengths on the x-axis.  Use a full page for this graph and be absolutely certain that the distances on each page are constant.  Ask your instructor to check your graph if you are not sure about it.

A More Specific Procedure for this Experiment

  1. Using pure water as a solvent, obtain chromatograms of the three authentic food dyes which are provided (FD&C Red #40, Blue #1 and Yellow#5).  Let them dry and measure (in mm) the distances traveled traveled by the solvent and the leading edge of each spot.  Record your data.  Tape the dry chromatograms in your notebook with the data.
  2. Repeat Step 1 using saturated sodium chloride (table salt) solution as a solvent.
  3. Repeat Steps 1 and 2 using the commercial food colorings as samples.
  4. Obtain a visible spectrum for one of the dyes found in either the blue, green or red food colorings.  Prepare a solution for this analysis by obtaining a chromatogram of the dye, with an intense spot, in an appropriate solvent.  Cut off the section with the color of interest and put this paper in a test tube with deionized water to extract the pure dye.  You will need about 4 mL of this solution so you may need to make more than one chromatogram to get a solution with enough dye to see the color.   If the filter paper falls apart in you solution, it will be necessary to filter it to remove the floaties.  Your instructor will show you how to make a micro-filter.   Also make a solution of the corresponding FD&C reference dye.
  5. Obtain a spectrum of the FD&C dye, and also of the dye that you extracted from your chromatogram, using the spectrophotometer.  If two dyes have the same wavelength for maximum absorbance then they are probably the same dyes.
  6. Prepare a concentrated solution of the colored coating from a single color of candy (M&M's or Skittles).  This is best accomplished by placing 11 drops of water into a very small beaker.  Then add one candy and swirl until the color is removed and you begin to see the white inner layer.  Use tweezers to remove the candy.  Repeat with three more of the candies (one at a time) in the same beaker of solution.  Obtain a chromatogram of this solution to identify the color(s) of the dyes used.  Often manufacturers will use the lake form of the dyes.  These are the same dyes as above but are rendered insoluble in water by reaction with aluminum ions.   When a dye in the lake form is chromatographed there will be a spot of the color which does not move and the chromatogram will show a long stripe of the color.
  7. Prepare a concentrated solution of one flavor of a powdered drink mix by dissolving about one gram in a very small amount of pure water.  Obtain chromatograms of this solution in the water and saturated salt solutions to determine the dyes that are used.  Extract a dye and determine its visible spectrum.


The Conclusion section which you include at the end of your report in your notebook is very closely related to the Purpose section.  In the Conclusion section, answer each of the questions you wrote in your Purpose.  Base your answers on the data you reported in the Results section.

For example:


Can you suggest any extensions to this experiment?  Think about changing the independent variables and the dependent variables that you could measure or observe.   This may suggest new experiments  that you could propose.  One product that you might study is the ink in various kinds of pens.