|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
Different solvents can change the effectiveness of a separation by changing the
values of the RF's. This 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:
- the origin of the color of food dyes in commercial food coloring, by separating
these dyes using paper chromatography and by considering how mixtures of primary
colored dyes filter white light.
- the identity of dyes used to color several foods and food colors, by comparison
of chromatograms, RF's, and visible spectra to those found for
FD&C reference dyes.
- how the separation of the dyes is affected by the solvent by studying the effect
of solvent composition on the retention of dyes on the paper.
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
|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
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.
- 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.
- 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.
- 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
- Be sure the spectrophotometer is turned on.
- Set the wavelength knob to 600 nm.
- 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
- Fill a cuvette with deionized water and insert it in the sample compartment with
the line facing the front. Close the top.
- 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
- Fill the other cuvette with your solution.
- 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.
- Remove the cuvette, close the top, and change the wavelength to a setting which
is 20 nm lower.
- Reset the 0%T if it has changed (empty sample compartment).
- Insert the cuvette of deionized water and reset the 100%T.
- Replace the water cuvette with your sample-containing cuvette and read and record
the absorbance again.
- Repeat steps 8 through 11 until you reach 360 nm.
- 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
- 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.
- Repeat Step 1 using saturated sodium chloride (table salt) solution as a solvent.
- Repeat Steps 1 and 2 using the commercial food colorings as samples.
- 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.
- 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.
- 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.
- 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.
- Use your spectra to prove whether your extracted dye is one of the FD&C
reference dyes and, if so, which one.
- Give a specific explanation of the origin of the color observed for each food
dye. In your explanation refer to the subtractive primary color(s) which are used in
the dyes. How do these primary colors result in the color of the dye? is
a description of how these dyes work.
- Are there any dyes which are used in more than one food? How do you know
this? Use the colors and the RF's as evidence to convince the reader that
your conclusion is supported by evidence.
- How does the change in solvent affect the separation? Refer to specific
values of the RF's to illustrate your conclusion. Does this tell us
anything about how the dye prefers the paper or the moving solvent? What solvent
gives the best separation?
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.