Introduction CHM 130 Paper Chromatography Chromatography is one of many techniques to separate the compounds in a mixture and to identify unknown substances. It is widely used in chemistry and biology. All types of chromatography involve two different immiscible phases in contact with each other. The mobile phase is moving and the other phase, stationary phase, is not moving. In paper chromatography, a solvent moves from one end of a paper to the other end, as the paper absorbs it. Since it is moving, the solvent is the mobile phase. The paper is the stationary phase. (More specifically, the cellulose molecules in the paper contain many polar OH (hydroxyl) groups that serve as the stationary phase). Chromatography works by capillary action. To separate a mixture of chemicals, a small amount of the mixture is first placed near the edge of an absorbent paper. That same edge is wetted with solvent. The solvent travels up the paper by capillary action, carrying the mixture with it. The various chemicals in the mixture travel different distances. The distance a chemical travels is determined by its interaction with the solvent (mobile phase) and with the hydroxyl groups attached to the paper. Substances that dissolve more readily in the solvent will move farther than the substances that have a higher attraction for the paper. When the solvent has moved the entire length of the paper, the paper is removed from the solvent and dried. The different chemicals in the mixture will be located at different positions on the chromatogram. At this point, we can calculate the R f (ratio of fronts) factor for each substance. For a particular substance the R f factor is defined as R f = Retention factors are useful in comparing the results of one chromatogram to the results of another. If the conditions in which the chromatogram are run remain unchanged (same mobile and stationary phases), the retention factor for a given material should remain constant. This allows unknowns to be compared to known materials. If the retention factor of an unknown does not match that of a known material, they are not the same compound. The various chemicals visible on the chromatogram can often be identified by their positions or their colors or both. If the mixture contains colored compounds, each different compound will appear on the chromatogram as a colored spot or a streak in a particular place. The color and location of each compound can be used as a basis for identification. The color and location of unknown compounds separated under specific chromatographic conditions can be matched with the color and location of known compounds subjected to the same conditions. Chromatograms with uncolored compounds can sometimes be treated with chemicals to color them for identification. Materials / Equipment chromatography paper pencil solvent (0.1% NaCl) ruler 600 ml beaker one set of colored marking pens (crayola or cra-z-art) plastic wrap or watch glass stapler hair dryer 1
Procedure 1. Mark the chromatography paper as shown below. Use only black pencil to write on the paper. 19.0cm 9.0cm pencil line 1.0cm from the bottom pencil marks numbered and 2.0cm apart 1 2 3 4 5 6 7 8 9 10 first and last numbers 0.5cm from edges 1. Obtain a set of marking pens and draw a small dot of ink from a red pen on the first pencil mark on the piece of chromatography paper. Continue placing colored dots on the pencil marks in the same order as the visible spectrum: red, orange, yellow, green, blue, and violet. Place other colors such as black, brown, and pink at the end. (If you cannot find every color, that is OK, try to get a range of colors.) Record the spot number, ink color, and brand of the pen in the data table. 2. Staple the paper into a cylinder, dot side out, being careful not to allow the edges to touch one another (Figure B). 3. Pour around 20 ml of the solvent into a large 600mL beaker. Place the paper cylinder into the beaker with the dots at the bottom. Initially, the colored dots should be above the solvent as shown in Figure C. Be very careful that the solvent does not touch the colored dots. Cover the beaker with plastic wrap or a watch glass. 4. Wait for the solvent to move up the paper. It will take several minutes. 5. Remove the paper cylinder when the solvent reaches ~2.0 cm from the top of the chromatography paper. Carefully remove the staples and set the chromatography paper on a paper towel. Draw a line along the highest point the solvent reached with a pencil and quickly blow dry the paper with a hair dryer. 6. Measure the distance traveled from the pencil line at the bottom of the paper to the solvent line at the top of the paper. Record this as Solvent Distance on the data table. 7. Measure the distance traveled from the pencil line at the bottom to the approximate center point of each colored splotch. Record each distance as Spot Distance on the data table. If the original ink spot has separated into multiple colors, then each new color must be measured and recorded separately. 2
Name Section Lab Partner Paper Chromatography Lab Questions Answer each of the following questions. Base your answers (conclusions) on reasonable interpretations of your data and provide justification. 1. Why is it important to keep the spots applied to the chromatography paper small? The spots spread out, so if they are too close, the ink will run together 2. Why did some ink dyes travel to the top of the paper and other ink dyes remained near the bottom of the paper? Explain in terms of the attractions of the particles for either the mobile liquid phase or the solid paper stationary phase. Ink that is attracted to the solvent will move towards the top of the paper. Ink that is attracted to the paper will stay near the bottom. Attractions are most commonly due to polarity of the molecules: Polar inks are attracted to polar solvent, nonpolar inks are attracted to nonpolar paper. 3. If a spot travels 2.3 cm during paper chromatography while the mobile phase travels 6.6 cm, what is the R f value of the substance in the spot? 4. What is the stationary and mobile phase used in this lab? Stationary phase is the chromatography paper, because it does not move during the experiment Mobile phase is the salt solution, because it moves up the paper 5. In preparing the chromatography paper, a baseline is drawn in pencil for the positioning the spots. Why is ink never used for drawing the baseline? The ink in the pen may also move with the solvent. This will create a mixed spot with the ink that is being tested. It also eliminates the reference line that is needed to measure the distance the ink and solvent travelled for the R f equation. 3
DATA TABLE You may need to divide some of the boxes into multiple rows if the ink separated into more than one color. Spot Number Ink Color Solvent Distance (cm) Spot Distance (cm) R f value 1 2 3 4 5 6 7 8 9 10 4
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