Introduction: (Initial Observation)
Chromatography is one of the most widely used methods for separating a mixture of chemicals. Separation is the first step of any chemical analysis. Chemists separate a mixture to it’s ingredients and then analyze and identify each ingredient. Paper chromatography is the most simple and the least expensive method of chromatography that we can do at home.
In this project I use chromatography to analyze the dyes used in the coating of M&M chocolate candies. I am wondering if these dyes are pure or they are a mixture of two or more other dyes. Chromatography should be able to separate and existing dye in the coatings and reveal hidden dyes.
Gather information about chromatography. Read books, magazines or ask professionals who might know in order to learn about different separation methods. Keep track of where you got your information from.
If you use the Internet!
If you use the Internet to gather information, search for Introduction to Chromatography.
You may find general information about chromatography or specific methods of chromatography.
Chromatography is the science of separation. The discovery of chromatography is generally credited to Tswett, who, in 1903 described his work on using a chalk column to separate the pigments in green leaves. The term “chromatography” was coined by Tswett to describe the colored zones that moved down the column.
Basically, chromatography involves the flow of a mobile (liquid) phase over a stationary phase (which may be a solid or a liquid). As the mobile phase moves past the stationary phase, repeated adsorption and desorption of the solute occurs at a rate determined chiefly by its ratio between the two phases. If the ratio is large enough, the components of the mixture will move at different rates, producing a series of bands (chromatographs).
CLICK HERE to see a demonstration of chromatography.
There are different methods of chromatography. You should focus on TLC (Thin Layer Chromatography) and PC (Paper Chromatography).
Paper Chromatography is a type of chromatography using filter or other special papers as the stationary phase. Spots of and reference materials are applied near one edge (or corner, for two-dimensional PC) of the paper. The edge of the paper is dipped in a solvent, which travels along it by capillarity, moving the components of the sample at rates depending on their relative solubility in the solvent. In two-dimensional PC, the paper is turned 90° and dipped it in a different solvent. The components of the sample mixture, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials. PC is especially useful for complex mixtures of amino acids, peptides, carbohydrates, steroids, and many other organic compounds and inorganic ions.
While gathering information, I learned that certain types of dyes are approved for use in Food, Drugs, and Cosmetics. These dyes are called FD&C dyes. In US, food and drug administration (FDA) must approve a dye before it can be used in food, drug or cosmetics.
What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.
The purpose of this experiment is to separate and identify the FD&C dyes from M&M’s or Skittles using paper chromatography.
This is a typical problem brought to analytical chemists. It may also be called a reverse engineering problem. For example a manufacturer of sugar coated chocolate may be wondering how his competitors such as M&M are able to produce products with such interesting colors. He has already searched and know that those colors are not standard food colors available in the market. He is wondering maybe such colors are produced by mixing a few other colors. Now he wants to test that. A chemist will use different separation methods to determine if the colors are a mixture of two or more colors.
The process used in such experiments is chromatography. A basic chromatography experiment may ask:
How do different food dyes vary in the velocity in which they migrate in a water based mobile phase?
For this question food color is the independent variable and the migration velocity is the dependent variable.
When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.
- The independent variable (also known as manipulated variable) is the color of M&M.
- The dependent variable also known as responding variable is the color of ingredients.
- Constants are experiment method, material and conditions.
- Controlled variable may be any environmental condition that may affect your experiment results (such as temperature, light, wind).
Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis. This is a sample hypothesis:
I think M&M dyes are pure. My hypothesis is based on my observation of the M&M colors and the fact that they don’t have different shades of each color.
Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”
Have you done chromatography before? If not, do some simple experiments before you start. Get some food dyes. Mix one drop of each dye in a plastic spoon. Use a toothpick to place a few dots of your mix on a white cotton fabric or filter paper about one inch from one end.
Wait until the dye dries. Place the end of paper or fabric which is close to the color spot in water or alcohol and keep the other end up.
While water or alcohol enter the fabric, they force the color spot to move up.
You can repeat this experiment using color markers. You may discover that each color is a mixture of two or more other colors.
You first dissolve the dye of candy coating in vinegar and use it to dye wool. Wash the wool so all the sugar will be removed. You will then use ammonia solution to separate dye from the wool. Let it evaporate and become concentrated. The resulting dyes are then separated using paper chromatography. If commercial food colors are used as standards, the separated dyes can be identified.
- Place a test tube containing a 10-15 cm length of white woolen yarn and 10-15 mL of household vinegar in a boiling water bath and heat for 4-5 minutes to remove any fluorescent dyes which could interfere with the separation of the dyes. After cooling, remove the yarn from the vinegar and let it drain.
- Place 5 or 6 M&M’s or Skittles in a test tube with enough household vinegar to cover the candies. Heat the tube in a boiling water bath until the colored coating dissolves. Avoid dissolving the interior of the candies.
- Carefully decant the solution which now contains the dyes, some sugar, etc. into another test tube. Avoid transferring the sediment.
- To extract the dyes, add the prepared length of woolen yarn and 3 mL of vinegar to the test tube containing the dye solution. Heat this tube in the boiling water bath for about 5 minutes with occasional stirring. Remove the yarn and rinse it with a little tap water.
- To release the extracted dyes, place the yarn and about 5 mL of clear household ammonia solution in a clean test tube. Mix with a stirring rod and then test the resulting solution with red litmus paper to make sure that it is basic. If not, add a bit more ammonia solution.
6. Heat the tube containing the yarn and ammonia in a boiling water bath for about 5 minutes with occasional stirring to release the dyes.
Picture in the right is from one of my trial tests. In this test I used only one yellow candy. That is why the color is so pale.
Remove the yarn and pour the solution containing the dyes into an evaporating dish. Heat the evaporating dish gently to concentrate the solution. Stop just short of dryness. If all the liquid evaporates, add a drop or two of distilled water and stir.
7. Cut two l0-cm × 20-cm pieces of chromatography paper. Draw a pencil line 1 cm from one long edge of each piece of paper. Mark six or seven equally spaced positions along the pencil line. Use toothpicks to place a small drop of the concentrated dye solution as possible on two of the positions on the pencil line on each piece of paper. Allow the spots to dry and spot them again in exactly the same positions to increase the amount of sample. Spot a third time and a fourth time if the color is not very intense. Use toothpicks to place one small drop of each food color sample on the remaining positions on each piece of paper.
8. Add 5 mL of vinegar to a 600-mL beaker. Carefully staple one piece of chromatography paper into a cylinder and place the paper in the beaker with the spots at the bottom of the cylinder. Be careful that the paper does not touch the sides of the beaker.
The cylinder is used to hold the paper vertically. You could hang the paper from a pencil or wood dowel instead.
Cover the beaker with aluminum foil and allow the chromatogram to develop until the developing solution has climbed to about l cm from the top of the paper. Remove the paper from the beaker, open it out and allow the paper to dry on a piece of paper towel.
9. Repeat step 8 using the second piece of chromatography paper, but develop with 5 mL of clear ammonia solution.
10. Use the following information to identify the specific dyes present in the candy coatings.
|Crown Colony Kit||Blue #1
|Crown Colony Singles||Red #40||Yellow #5
|Yellow #5||Blue #1
Crown, Durkee and McCormic are food color manufacturers. Using the results of your experiment and the above table, you may find out what brand color is used in each M&M candy.
11. Remaining interiors of candies may be disposed of with solid waste. Solutions that remain may be flushed down the drain.
Care should be exercised when using boiling water baths.
You may be wondering how do you come up with such a complex experiment procedures. Well, first you need to gather some information and do some tests. You need to know how can you separate dye from sugar and other ingredients.
In my test, first I tried to find the best way of separating dyes from candy. I started by 3 test tubes, containing water, ammonia and vinegar. I thought different pH solutions may have dissolve the dye and candy in different rates.
It seems that a vinegar solution gave the best result and water was my second choice.
I then needed to know what type of fabric can be dyed with the dyes used in candy coatings. Wool was proven to be the best.
Finally I needed to know what solution can extract the dye from wool. Ammonia seemed to be the best. Note that I used ammonia as a base and vinegar as an acid, because these two can easily evaporate and exit any solution.
I also tried to remove dies from candies using a few drops of cold water in a spoon. It worked well. I just had to use my finger to rub the candies and practically wash the dyes off the candy coatings.
You may modify the experiment procedures and equipment based on what you can have access to. I did not have chromatography paper, so I used filter paper.
Regular paper will not work because it also contains white pigments, starch and many other chemicals. White cotton fabric may work as well.
Need a control experiment?
The control experiment may be another paper chromatography experiment you perform at the same time with a mixture of known food colorings.
Need a graph?
Chromatography is a natural way of making graph. It works because each substance moves at a different speed when placed in the path of a mobile phase (solvent). If you want to make your own graph, make a graph to show how much does each color travel in certain period of time (speed of stationary phase).
Make a bar graph and use one bar for each of the colors you found in your sample. For example if you test a green color and discover that it is separating to blue and yellow colors, measure the distance each of the two final colors travel in one hour. For example if the yellow color travels 4 inches and the blue color travels 2.5 inches, you must make a yellow bar that is 4 inches tall and a blue bar that is 2.5 inches tall.
If your experiment lasted only for 30 minutes, you must double the amounts to know the travel distance in one hour.
After you know the travel distance per hour for 2 or more colors, you may make a bar graph with one bar for each color.
Materials and Equipment:
This is a sample list of equipment. You may use other material and equipment, so you must make your own list.
M&M’s and/or Skittles
white household vinegar
clear, colorless household ammonia solution
food colors (used in practicing chromatography)
red litmus paper
Bunsen burner, alcohol burner or hotplate
600-mL beakers 25-cm × 150-cm test tubes
l0-mL graduated cylinder
test tube holder
chromatography paper (MiniScience# P610 works fine)
boiling chips (Boiling chips are small glass balls or beads. They simply prevent violent boiling and make the bubbles smaller.)
Results of Experiment (Observation):
Paper chromatography is an important separation technique that depends upon differences in how strongly the dyes are adsorbed onto the paper (stationary phase) and how soluble the dyes are in the developing solvent (mobile phase). In paper chromatography, a small amount of the mixture to be separated is placed close to the edge of a piece of paper. The edge of the paper is then immersed in a developing solution. As the developing solution ascends up the paper by capillary action, the. components of the sample are carried along at different rates. To prevent evaporation of the developing solution, this process is carried out in a closed container.
Each component of the mixture will move a definite distance on the paper in proportion to the distance that the solvent moves. This ratio, H f = distance component moves/distance solution moves, can be calculated for each component to aid In identification. Hf values are dependent upon the paper, the developing solution, and the amount of sample used.
Candies, such as Mix’s or Skittles, contain FD&C dyes, sugar, and other organic and Inorganic substances In their coatings. If the coating is dissolved in an acidic solution, the FD&C dyes can be adsorbed from the solution by wool. The dyes can be released from the wool in an alkaline environment. Thus the dyes can be separated from other substances in the coatings. This simplified equation represents the equilibrium involved:
Wool + Dye + H+ <====> Dyed Wool+
A solution of ammonia can be used to supply sufficient OH- to reduce the concentration of the H+ and shift the equilibrium to the left releasing the dyes.
Keep your developed papers and use them as a part of your display.
No calculation is required.
Summary of Results:
Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.
It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.
Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.
Related Questions & Answers:
What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.
- The yarn used In this experiment must be 100% wool.
- Two different developing solvents are used to illustrate the significant dependence of this technique on the solvent used and because the vinegar separates the yellow and blue dyes better while the ammonia separates the reds and yellows better.
- Since it Is difficult to apply comparable quantities of the extracted dyes and the food colors to the chromatogram, It is likely that the Hf values will not be very reproducible. The separations are sufficiently distinct that you can identify the components qualitatively.
Jenkins, C., Science and Children, April 1986, p. 25. — This article describes a chromatography experiment for young children using Kool-Aid and lists the FD&C dyes In various brands of food colorings.
McDuffie, T.E. Jr. and Anderson, J., Chemistry Experiments from Daily Life, J. Weston Walch, Publisher, 1980, p. 77. — This work describes a similar experiment which uses non-consumer solutions for the extraction and for developing the chromatogram.
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