Chromatography What is it?

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1 Chromatography Most things that are colored are mixtures of different substances of various colors. In a mixture you have several different kinds of chemicals that are all next to each other but not reacting. Since it is just a mixture and not a compound, the different chemicals can be separated. Since each pen manufacturer, or juice manufacturer, or indeed each type of grass uses a somewhat different formula to produce its colors, each possesses a unique, identifiable character. As a matter of fact, the same basic technique is used for identifying all sorts of things including DNA. A technique known as chromatography, which is a term taken from the Greek language & means written in color, will be used to separate the substances into their various colors. This is exactly the same technique that a real crime lab would use to determine the manufacturer of the ink used in a note found at the crime scene or later sent in as a ransom note. It would also be used on any stains or juices found at the scene, etc. To separate the substance into it s components a piece of filter paper, that has a similar composition to that used in most coffee filters is used. Sometimes the paper is a wide strip of paper, sometimes it is a long strip. You may do it either way. If you have a lot of different substances to test, the wide one is often most convenient. If you only need to test a couple, it is often just as easy to use the long ones. Of course it depends on what is available in the lab. To give you some knowledge on how to handle both kinds, we will try both. Regardless of the type of paper used, the idea is to put a fairly small dot of the substance to be tested about 1.5 cm up from one edge of the paper. The bottom of the paper is then dipped into a liquid of some sort, such that the liquid level is below the level of the dot. The liquid is chosen such that it will dissolve the substances that make up the color. The liquid will travel up the paper, dissolve the colors, and drag them along as the liquid continues to travel up the paper. Since the molecules making up the colors are of different sizes and hence different weights and different dissolving properties, they will migrate up the paper at different rates. This will then separate them into their various components. These signature chromatograms can then be compared against knowns to determine what the unknown is. We will only be trying a couple kinds of chromatograms that use water as the liquid to dissolve the substances and separate them into their component parts. We will leave some of the more exotic separations for you to experience later. PenChromatograms 1.Take a wide rectangular piece of filter paper and use a pencil to draw a line about 1.5 cm from the bottom edge of 1 long side. 1.5

2 2. Place small dots of each of the known materials you are to test spaced about 1 cm apart along the line you just drew. Be sure to label what the dots are on the paper ABOVE the dot. 3. Roll the paper up like a cylinder & staple the ends together. staples 4. Put about 1 cm of water in a Petrie dish. 5. Set your chromatography paper in the dish with the dot side down. Be sure the water level in the Petrie dish does not touch any of the dots on the line you made. 6. While that chromatogram is developing do the same thing over for the knowns of another set of materials. 7. Take 2 strips of chromatogram paper and make a line about 1.5 cm from the bottom with a pencil on each Bring your prepared pieces of chromatogram paper to the instructor who will place a dot on the papers as your unknowns. 9. Hang the papers in a beaker by placing a wooden stick through the hole in the top & resting the wooden stick on the lip of the beaker. 10. Use your wash bottle to put enough water in the beaker so that the bottom of the chromatograph paper just is in the water, but not so much that the dots are in the water. Be sure that you do not get any water on the chromatography paper as you are putting it in. 11. The chromatograms are done when the water level gets up to about 1.5 cm from the top. 12. Take the chromatograms out and spread them on a paper towel to dry. 13. Which of the knowns do your crime scene chromatograms match up to?

3 Juice Chromatograms Juice Chromatograms are done very much like pen chromatograms except for the method used to put the material on the paper. In pen chromatograms you can use the pen to place a dot on the paper directly, but in juice chromatograms it is necessary to use an instrument to place the juice on the chromatography paper. A toothpick works well for this. Dip the toothpick in the juice and touch it to the chromatography paper on the line. Do not let too much liquid get on the paper. The dot should be about the size you got when you did the pen. Use a different toothpick for each of your known juices. You do not want the juices contaminated. With juices, it is usually necessary to let the liquid from the first dot soak in a bit and then go back and put a second dot right in the same place as the first dot. You should let the liquid soak in for seconds between dots. It is usually best to apply 3-4 dots of juice in the same place to get enough pigment to make a visible chromatogram. Each juice then should have a different place on the paper.

4 Chromatography What is it? Some materials appear homogenous, but are actually a combination of substances. For example, green plants contain a mixture of different pigments. In addition, the black ink in the pens that are used in this experiment is a mixture of different colored materials. In many instances, we can separate these materials by dissolving them in an appropriate liquid and allowing them to move through an absorbent matrix, like paper. Chromatography is a method used by scientists for separating organic and inorganic compounds so that they can be analyzed and studied. By analyzing a compound, a scientist can figure out what makes up that compound. Chromatography is a great physical method for observing mixtures and solvents. The word chromatography means "color writing" which is a way that a chemist can test liquid mixtures. While studying the coloring materials in plant life, a Russian botanist invented chromatography in His name was M.S. Tswett. Chromatography is such an important technique that two nobel prizes have been awarded to chromatographers. Over 60% of chemical analysis worldwide is currently done with chromatography or a variation thereon. Chromatography is used in many different ways. Some people use chromatography to find out what is in a solid or a liquid. It is also used to determine what unknown substances are. The Police, F.B.I., and other detectives use chromatography when trying to solve a crime. It is also used to determine the presence of cocaine in urine, alcohol in blood, PCB's in fish, and lead in water. Chromatography is used by many different people in many different ways. Chromatography is based on differential migration. The solutes in a mobile phase go through a stationary phase. Solutes with a greater affinity for the mobile phase will spend more time in this

5 phase than the solutes that prefer the stationary phase. As the solutes move through the stationary phase they separate. This is called chromatographic development. How it works In all chromatography there is a mobile phase and a stationary phase. The stationary phase is the phase that doesn't move and the mobile phase is the phase that does move. The mobile phase moves through the stationary phase picking up the compounds to be tested. As the mobile phase continues to travel through the stationary phase it takes the compounds with it. At different points in the stationary phase the different components of the compound are going to be absorbed and are going to stop moving with the mobile phase. This is how the results of any chromatography are gotten, from the point at which the different components of the compound stop moving and separate from the other components. In paper and thin-layer chromatography the mobile phase is the solvent. The stationary phase in paper chromatography is the strip or piece of paper that is placed in the solvent. In thin-layer chromatography the stationary phase is the thin-layer cell. Both these kinds of chromatography use capillary action to move the solvent through the stationary phase. What is the Retention Factor, Rf? The retention factor, Rf, is a quantitative indication of how far a particular compound travels in a particular solvent. The Rf value is a good indicator of whether an unknown compound and a known compound are similar, if not identical. If the Rf value for the unknown compound is close or the same as the Rf value for the known compound then the two compounds are most likely similar or identical. The retention factor, Rf, is defined as

6 Rf = distance the solute (D1) moves divided by the distance traveled by the solvent front (D2) Rf = D1 / D2 where D1 = distance that color traveled, measured from center of the band of color to the point where the food color was applied D2 = total distance that solvent traveled The Different Types of Chromatography

7 There are four main types of chromatography. These are Liquid Chromatography, Gas Chromatography, Thin-Layer Chromatography and Paper Chromatography. Liquid Chromatography is used in the world to test water samples to look for pollution in lakes and rivers. It is used to analyze metal ions and organic compounds in solutions. Liquid chromatography uses liquids which may incorporate hydrophilic, insoluble molecules. Gas Chromatography is used in airports to detect bombs and is used is forensics in many different ways. It is used to analyze fibers on a persons body and also analyze blood found at a crime scene. In gas chromatography helium is used to move a gaseous mixture through a column of absorbent material. Thin-layer Chromatography uses an absorbent material on flat glass or plastic plates. This is a simple and rapid method to check the purity of an organic compound. It is used to detect pesticide or insecticide residuesin food. Thin-layer chromatography is also used in forensics to analyze the dye composition of fibers. Paper Chromatography is one of the most common types of chromatography. It uses a strip of paper as the stationary phase. Capillary action is used to pull the solvents up through the paper and separate the solutes. The table below summarizes the information from above. Type of Applications in Why and What is it

8 Chromatography the Real World Liquid test water samples Chromatography to look for pollution, Gas Chromatography detect bombs in airports, identify and quantify such drugs as alcohol, used in forensics to compare fibers found on a victim Used to analyze metal ions and organic compounds in solutions. It uses liquids which may incorporate hydrophilic, insoluble molecules. Used to analyze volatile gases. Helium is used to move the gaseous mixture through a column of absorbent material. Thin-Layer Chromatography Paper Chromatography detecting pesticide or insecticide residues in food, also used in forensics to analyze the dye composition of fibers separating amino acids and anions, RNA fingerprinting, separating and testing histamines, antibiotics Uses an absorbent material on flat glass plates. This is a simple and rapid method to check the purity of the organic compound. The most common type of chromatography. The paper is the stationary phase. This uses capillary action to pull the solutes up through the paper and separate the solutes.