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EXPERIMENT IX: Separation of Two Dyes-- Column Chromatography Chromatography is a general and widely used method of separating mixtures of compounds....

question 4 of experiment 9
63 EXPERIMENT IX: Separation of Two Dyes-- Column Chromatography Chromatography is a general and widely used method of separating mixtures of compounds. On a large scale it is used to prepare pure samples from a mixture and on a very small scale it is used to analyze the components of a mixture. In column chromatography, a solution of the mixture to be separated is introduced at the top of a vertical column packed with finely divided solid already wet with solvent. Fresh solvent (called the eluent) is then added, and as it passes down the column carrying the components of the mixture with it, they are selectively adsorbed and desorbed. Different compounds will have different affinities for the two phases. In the course of the "development" of the chromatogram, each compound is adsorbed by and extracted from the stationary phase many times. Thus, compounds with higher preference for the solvent than the stationary support will be eluted more rapidly, and will be collected in the early fractions. Compounds with lower preference for solvent than the stationary support will be eluted in the later fractions. The extent to which adsorption occurs is a function of the molecular structures of the different compounds to be separated. Usually polar compounds are most firmly adsorbed and stay on the column longer. Sometimes several solvents or mixtures of solvents may be required to elute substances from the column. It is best to start with a relatively nonpolar eluent and progress gradually to more polar eluents. In this experiment, two dyes, malachite green and crystal violet, will be separated by column chromatography using basic alumina, A1 2 O 3 , as the solid adsorbent and water or dilute aqueous HCl as the solvent. Solvent (mobile phase) Filter paper Basic Alumina (stationary phase) Glass wool Stopcock
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64 Equipment and Chemicals: 25 ml buret with tapered end, fitted with a 1" piece of silicone rubber tubing and stopcock 9" plastic tube (use as a plunger) 1 cm diameter filter paper 5g basic alumina (Al 2 O 3 ) dye mixture (0.1 g crystal violet and 0.1 g malachite green in 200 ml warm water) 0.5M HC1 9" plastic disposable pipets 3 dram glass vials glass wool plastic powder funnel fitted with a short piece of Tygon tubing Caution: crystal violet: cancer suspect agent, toxic malachite green: toxic Special container for waste: alumina/glass wool waste Setting Up The Column 1. Using the 9" plastic tube as a plunger, push enough glass wool to the bottom of the buret to form approximately a 1/4" plug. 2. With the plastic tube resting on top of the glass wool and the stopcock closed, add 2-3 ml deionized water and gently poke the glass wool to remove any trapped air. Remove the plastic tube. 3. Fill the column to within about 1" from the top with deionized water. 4. Attach the plastic powder funnel to the top of the buret using the short piece of Tygon tubing. Slowly sprinkle 5 g of basic alumina (aluminum oxide) into the powder funnel. The alumina will pass through the water and settle on the glass wool in the lower part of the buret. Use an extra long plastic disposable pipet to loosen any alumina that sticks to the funnel or to the top of the buret. 5. When most of the alumina has settled, remove water from the top of the column with the extra long plastic disposal pipet until the water level is about 1/4" above the alumina. Place a 1cm diameter filter paper on the top of the buret and gently push it down onto the top of the alumina using the 9" plastic tube. This will help to keep the column from being disturbed by the addition of eluents/dye mixture to the column.
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