The Basis for Paper Chromatography

Transcription

1 APTER 4 Polarity The Basis for Paper hromatography bjectives The objectives of this laboratory are to: Use paper chromatography to determine the number of components in certain mixtures. lassify these components in terms of their relative molecular polarity. Predict the relative migration rates for a sample of known polarity with a number of solvents of different polarity. Define the terms molecular dipole and molecular polarity. Introduction Paper chromatography is a simple and extremely useful tool for separating covalently bonded compounds differing slightly in molecular polarity. Although widely used in the past in the biological, biochemical, and medical sciences for separation and identification of thousands of different compounds found in living systems, it has been largely replaced by more powerful and more convenient forms of chromatography. In this experiment we will use this tool to separate the components of a number of common substances such as inks and food colors, and we will make some educated guesses regarding the structure and molecular polarities of these materials by observation of their interaction with various solvents. Paper chromatography is based on the relative attraction of a compound for two different substances. ne of these substances (the paper) is moderately polar, and it is stationary. The other may be more or less polar, but it is a liquid and is mobile. Paper is composed of cellulose, which, in turn, is a polymer of glucose. A section of this polymer is illustrated in Figure 1. Recall that oxygen atoms have two bonding pairs of outer electrons and two pairs of unshared electrons. These unshared pairs of electrons give oxygen a lopsided charge distribution and produce a polar area at that region of the molecule. ellulose also contains a large carbon hydrogen fraction, which is, of course, nonpolar. 37

2 hemistry 143 Experimental hemistry Figure 1. ellulose is a linear polymer of glucose that is formed by beta glycosidic bonds. While portions of the molecule are nonpolar, the oxygen atoms provide sites of considerable polarity. Polar placed on the solid cellulose are attracted to these polar sites, and tend not to move with the moving solvent. The separation process in paper chromatography is accomplished by placing a small sample of the unknown mixture near the bottom of a strip of filter paper, then placing the filter paper in a small dish of solvent and allowing the solvent to migrate up the paper by capillary action. (The sample is placed above the solvent level so that the solvent does not cover the sample but migrates past it.) This is illustrated in Figure 2. As the solvent migrates up past the sample, the sample components have their choice of sticking to the relatively polar paper where they were placed or riding along in the ascending solvent. over (Saran wrap) movement Spot initially containing two or more constituents ayden-mcneil, LL Figure 2. Paper chromatography involves placing the unknown sample in a small spot on a strip of filter paper, which is then dipped in a shallow pool of solvent. As the solvent migrates up the paper, those sample components attracted more to the solvent than to the cellulose base of the paper migrate with the solvent, thus separating the sample into a number of bands of material. Each band moves at a rate related to the relative competitive attraction of the solvent and cellulose for the sample. The paper may be suspended from the top of the beaker, or it may be folded to stand alone in the solvent. The rate at which a compound will move up the paper strip is determined by the difference between its attraction for the stationary cellulose and the moving solvent. onsider, for example, the case of a mixture of a polar and nonpolar compound placed on the paper strip. If the paper is then placed in a nonpolar solvent, the nonpolar component 38

3 Polarity: The Basis for Paper hromatography hapter 4 will tend to move up the paper with the migrating solvent, while the polar component will tend to remain in place on the paper. n the other hand, if a very polar solvent were used, the polar compound would move up the paper with the rising solvent, while the nonpolar compound would remain in place. It is important never to allow the solvent to rise all the way to the top with the faster moving components because the components will group up at the top of the paper (Figure 3). Sample Paper Polar cellulose (stationary) Moving solvent Polar cellulose Paper Start ayden-mcneil, LL Figure 3. ere is a molecule s eye view of separation of by paper chromatography. The paper is made of with polar parts. Positive and negative parts of these protrude on the surface of the paper. The large dye to be separated also have some polarity and stick to the paper when a negative part of the dye molecule gets close to the positive part of one of the paper. They want to stay put. The solvent, which are moving up the paper by capillary action, rub past the dye molecule. If the solvent have polar parts, they will grab onto the dye molecule and try to carry it along with them. The dye molecule, however, is trying to hang onto the stationary polar paper. If there are a lot of polar solvent, they will slowly drag the dye molecule along with them. The chromatography system is said to have a stationary phase (the paper) and a mobile phase (the moving solvent). ere is the separation scheme: The rate at which a compound moves up the paper strip is determined by the difference between its attraction for the stationary cellulose and the moving solvent, A nonpolar solvent will carry nonpolar along as it migrates up the paper, while polar will tend to remain in place, A polar solvent will carry polar up the paper, while the nonpolar would tend to stay in place. ne may learn a lot about the polarity of the unknown molecule by observing its behavior with a number of solvents of different polarity. 39

4 hemistry 143 Experimental hemistry If you have a group of dye that have only a small amount of polarity, you can still separate them by using solvents that have very little polarity. You can also use a square filter paper and use one solvent to separate the unknown mixture into several groups. Then rotate the paper 90 degrees and use a less polar solvent to separate the groups of similar polarity. Nonpolar dye will dissolve easily into nonpolar solvents and move along with the solvent front. In this experiment we will use four different solvents: water, methanol, acetone, and hexane. These solvents represent the spectrum from very polar (water), quite polar (methanol), polar with significant nonpolar properties (acetone), and nonpolar (hexane). We will try separating the components of a number of samples (ballpoint pen ink and four colors of food color) to determine the number of different components present in each of the mixtures, and the relative molecular polarities of each of these components (Figure 4). Water Methanol Increasing molecular polarity Acetone exane 40 Figure 4. Several suggested solvents are shown in this figure. These solvents differ markedly in their molecular polarity. Use of a number of different solvents permits one to make some educated guesses concerning the relative molecular polarity of an unknown substance.

5 Polarity: The Basis for Paper hromatography hapter 4 Safety Precautions bserve all normal laboratory precautions. Minimize your contact with organic compounds and inhalation of organic vapors. Materials Equipment 400 ml beaker Filter paper cut into squares Ruler Pasteur pipettes Reagents Deionized water Methanol Acetone exane Experimental Procedures 1. You will use five different solvents: DI water, 50% ethanol, 75% ethanol, 100% ethanol, and acetone. You will test four different food colors in each solvent. Then decide which solvent works best for each food coloring. 2. The solvent system in which the food color moves the fastest is not the best solvent; the best solvent is the one that separates the color best. 3. Place a small amount of solvent in the 400 ml beaker to cover the bottom. 4. Use Whatman filter paper as chromatography paper. You will draw a straight line with pencil (do not use pen, as the color from a pen will be carried with the moving phase) approximately 1 1½ cm from the bottom of the filter paper. 5. Squeeze a drop of each food coloring onto a well of the spot plate. Then use toothpick to spot the filter paper with food color. 6. It is important that the dots must be of the right size, not too big not too small; you should take the time to dry the food color dots first before putting the chromatography paper in the solvent. 7. You should blunt the end of a toothpick by snipping off a small portion of the tip (just snip off enough so the toothpick is no longer sharp). Dip the toothpick in one of the food colors and then spot the filter paper along the pencil line. Do this for each of the four colors, using separate toothpicks each time. (Give each spot equal spacing along the line.) Smaller is better than bigger with regard to spot size. Allow the spots to dry before starting the experiment. 41

6 hemistry 143 Experimental hemistry 8. Fold the filter paper in half lengthwise so that it will stand in the beaker. (Do not fold the paper on any of your spots.) Stand the filter paper up in the beaker, making sure that the solvent level is below the pencil line and sample dots. over the beaker with a plastic wrap and allow the solvent to draw up the paper until it is approximately 2 cm from the top. 9. TAs will make two different unknowns (Unknown A and B) for the students by mixing two different food colors for each unknown. The students will use the best solvent to identify the unknowns. 10. Students should provide the identification of unknown and a table with the R f value for all the known and unknown. Data Analysis ne of the methods used to analyze paper chromatography results is known as the retention factor. Simply put, the retention factor is the ratio of the distances moved of the component of interest and the solvent front (Figure 5). Paper hromatography front omponent B ayden-mcneil, LL A rigin or starting line R f = A B Figure 5. Retention factor. 42

7 Polarity: The Basis for Paper hromatography hapter 4 As part of the analysis for this experiment, you should calculate the retention factors of each component studied. Note that if a separation occurred, there may be more than one retention factor in each alley. Utilizing the retention factors as well as visual observations, analyze your chromatographic strips and determine which food colors make up your unknowns. Laboratory Report Please include this information in your report: a. What is the moving phase and what is the stationary phase in paper chromatography? What is the role of each one? b. Is food color polar or nonpolar? ow can you tell? c. ow does the solvent move through the paper? (apillary action and polarity) Which solvent (moving phase) was the best? ow did you decide? (The solvent that separates the color best.) d. Does the polarity of the solvent and the food color play any role? e. What is the retention factor (R f )? Why do you need to know that? f. Discuss a type of chromatography other than paper chromatography, for example: thin layer chromatography, gas chromatography, size exclusion chromatogram, ion exchange chromatography, affinity chromatography, or anything else. 43