INTRODUCTION
One of the common problems a practical chemist faces today is the determination of whether a particular substance is present in a sample. The chemical discipline which deals with this problem is analytical chemistry, and more specifically, qualitative analysis. Examples include the presence of cocaine in urine, alcohol in blood, PCBs in fish, lead in water, and carcinogens such as benzopyrene in the air we breathe.
Such questions are particularly important since only a minute quantity of some contaminants can affect the health of humans or pollute a large body of water. The analysis of very small quantities of analyte (the substance to be analyzed) is often referred to as trace analysis and quantities analyzed for are commonly present to the extent of one microgram or less.
Paper chromatography is a simple, yet extremely effective method for determining the presence of such small quantities of analyte. A strip of paper serves as the stationary phase. The mixture to be separated is placed in a small spot near one end of a strip and a solvent (or solvent mixture), called the mobile phase (or eluent), is passed over the spot. The solvent moves up the plate due to capillary action the same way that water will move up a paper towel and carries with it the various components in the spot.
The sample moves upward to the extent that it dissolves in the solvent more than it adheres to the adsorbent and is carried along by the solvent. Since every substance will have a slightly different solubility in a given solvent and will adhere to a solid adsorbent to a different degree, the different components in a mixture will be carried along by the solvent at different rates, and hence be separated. Differences in solubility and degree of adsorption are due to different sizes and shapes of molecules as well as difference in polarity. Thus if two compounds are started at the same place and the solvent passed over them (elution), one compound will move along the strip faster than the other.
After a period of time the flow of the mobile phase is stopped and the strip is dried. If the components of the mixture are colored the separated compounds can be observed directly. If they are colorless some method (usually chemical) must be used to show their presence. This process is called visualization.
Paper chromatography tells whether or not the sample is pure, and if it is not, how many different components are present. The positions of each spot can be compared with those from known compounds (standards) to ascertain the presence or absence of that substance. The location of the compound is indicated by an Rf value. For a given substance the distance it moves depends on the total time the experiment is carried out as well as on the physical and chemical properties of the system. However, the distance the compound moves relative to the distance the mobile phase moves is a characteristic of that compound and is known as the Rf value.
The choice of solvent in paper chromatography is crucial. The solvent must dissolve the various components in the mixture and there must be at least slight differences in solubility of each component. Therefore, there will be no separation (or as is often stated, no resolution). Also if the components are insoluble they will remain at the origin, unmoved. If all of the components are very soluble in the solvent system then each component of the mixture may travel along with the leading edge of the solvent ("the solvent front") unseparated. In practice it is usually found that one pure solvent will not give resolution and a mixture of solvents must be found for each sample to be analyzed.
Although paper chromatography is simple by not requiring elaborate equipment or complex procedures it still requires careful attention to detail and good technique to obtain satisfactory results.
In this experiment you will determine the colors used by Mars Inc. in M and M's, the world famous candy and moneymaker for clubs and organizations. You will be given four "standard" food colors: blue, yellow, red, and green. The standards themselves may be a mixture of two or more colors. These standards will be run along with the color extract from 6 different M and M's: tan, brown, green, yellow, red, and orange. You will determine whether each standard color consists of one color or a mixture. In addition you will determine if any of the standard colors are used by Mars Inc. in M and M's.
PROCEDURE
1. Obtain one M and M of each color: tan, brown, green, yellow, red, and orange. Moisten a toothpick and scrape the color off of each M and M using a separate toothpick for each M and M color.
2. Obtain four strips of chromatography paper that are approximately 3 x 10 cm. Draw a thin line with a pencil (do not use a pen!!!!) across the paper 10 mm (1 cm) from the bottom. Draw the line just hard enough to be visible. As many as three different samples can be applied to each piece of paper.
3. Wipe the colored material on the end of one toothpick onto the paper about ¼ the way across. The toothpick may be discarded. Be sure to label the sample identity. Continue adding each color to the paper in the same manner. You should be able to fit three different colors on each piece of paper.
4. Place one drop of each standard onto a watch glass. One drop will be enough for an a number of lab groups. Use the toothpick as in step 4 to apply the standard to the chromatography paper. Only one drop will be required to get a dark enough standard spot. Be sure to label the sample identity. Wait overnight before proceeding with step 5.
5. After applying all of the samples you should have four pieces of paper with spots on them: two pieces with three spots and two pieces with two spots for a total of ten samples. After the spots have dried overnight carefully place one of the paper strips in one of the elution chambers, and place one of the other strips in the other elution chamber. Prepare two elution chambers by pouring the eluting solvent (35 % ethyl alcohol in water by volume) into the jars to a depth of about 3 mm (just enough to cover the bottom of the chamber). Put the jar cover on the prevent evaporation of the solvent. Place the lids on the chambers. The spots must be at the bottom of the chambers and above the solvent level. The solvent front will travel up the paper rapidly at first and then will slow down. The solvent front is allowed to rise to within 2 cm of the top of the paper strip if time permits. The paper should not touch the side of the jar. When complete the paper is removed from the chamber, the top of the solvent front is marked with a pencil, and the paper strip is allowed to dry. If there is enough time the other two sample strips can be run, if not wait until the next day. Pour the remaining solvent into the designated waste container.
6. Measure the distance from the origin (initial pencil line) to the center of each spot on the paper strips. If the spots are large or irregular in shape it is necessary to estimate where the center is. Also measure the distance from the original line to the solvent front. Make all measurements to the nearest millimeter. Record the measurement on the Data Sheet and note the color for each spot.
7. In the space provided on the Data Sheet, calculate the Rf value for each spot:
Rf = component distance
solvent distance
Attach a sample paper strip to the report sheet.
DATA SHEET
I. Distance measurements and colors of spots.
Blue standard:
Yellow standard:
Red standard:
Green standard:
Red M+M:
Tan M+M:
Brown M+M:
Orange M+M:
Yellow M+M:
Green M+M:
II. Rf calculations:
Blue standard:
Yellow standard:
Red standard:
Green standard:
Red M+M:
Tan M+M:
Brown M+M:
Orange M+M:
Yellow M+M:
Green M+M:
QUESTIONS
1. From a comparison of the Rf values, what conclusions can be made about the similarities or differences in the dyes used in each of the candies?
2. From a comparison of the Rf values, what conclusions can be made about the number of components contained within each standard food color?
3. For each candy color indicate which dyes may be present by comparison to your standard.