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Let's become acquainted with chromatographic techniques as a method of separation (purification) and identification of substances.
You'll need the following equipment: 600-mL beakers (3), capillary pipets, glass stirring rods (4), metric ruler, 4-inch watch glasses (3), paper chromatography strips, evaporating dishes
You'll need the following materials: 0.5 M Cu(NO3)2, 0.5 M Ni(NO3)2, 15 M NH3, 1% dimethylglyoxime in ethanol, 0.5 M Fe(NO3)3, solvent (90% acetone, 10% 6 M HCl, freshly prepared), isopropyl alcohol, unknown solution (vial #3)
A little background: Chromatography is a technique often used by chemists to separate components of a mixture. The first chromatography occurred by allowing a solution of color pigments to flow down a column packed with an insoluble material such as starch, alumina (Al2O3), or silica (SiO2). Because different color bands appeared along the column, the process was called chromatography. Because of the simplicity and efficiency of this method, this technique has wide applicability for separating and identifying compounds.
The basis of chromatography is the partitioning of compounds between a stationary phase and a moving phase. Stationary phases have enormous surface areas. The molecules or ions of the substances to be separated are continuously being adsorbed and then released (desorbed) into the solvent flowing over the surface of the stationary phase. This brings about a separation of the components.
In this experiment, Fe3+, Cu2+, and Ni2+ will be separated with a solvent that consists of a mixture of acetone, water, and hydrochloric acid. Other solutions being used include a 2:1 ratio of isopropyl alcohol to water, used to separate components of ink. Also, a 1% solution of dimethylglyoxime in ethanol will be used to develop nickel ions. A 15 M solution of ammonium will be used to develop copper ions. In this experiment, the term “front” refers to the farthest line that an ion or solvent reached. See figure 10.1 in the Observations section for an example.
In this experiment, Rf values will be calculated. An Rf value refers to the ratio of the distance traveled by a compound to the distance traveled by the solvent. Those distances can be easily measured with a ruler directly from the chromatogram.
In this experiment, some ions will have to be developed. Ions will develop because of a reaction between the ion and a developing reagent. For example, when dimethylglyoxime reacts with Ni2+, the following reaction occurs, producing a strawberry red color:
Developing ions gives color to otherwise colorless ions.
There is an alternate method for paper chromatography than the one outlined in this experiment. It involves a circular filter paper and a wick. Figure 10.2 shows how the filter paper should be set up:
In order for elution to occur, the solvent must pass over the wick. The process of chromatography when done in this fashion looks like this:
In this experiment, the ascending strip technique will be used to determine Rf values and to separate and identify unknown ions from solution.
Procedure:
1. Four strips of chromatography paper about 15 cm long were obtained. Each strip was marked with a pencil dot. The strips were prepared as such:
2. Three 600-mL beakers were obtained. Some development solvent was poured into two beakers to a depth of 10 to 12 millimeters. The strips of paper were labeled (one each of unknown, known, trace Cu2+, and ink). The labeled ends of the strips were attached to a 6 inch glass rod by folding the ends over the rod and clipping the paper together using paper clips.
3. Some of each appropriate solution was added to the chromatography paper, depending on its label, using capillary pipets. The ink strip was spotted using a felt-tip pen.
4. One rod with two non-ink strips clipped to it was placed into the first beaker, containing an acetone/hydrochloric acid solution. Another rod with one non-ink strip clipped to it was placed into a second beaker containing the same solution. A third rod with the ink strip clipped to it was placed into the third beaker containing isopropyl alcohol. The chromatograms were not allowed to touch one another or the walls of the beakers. Each beaker was covered with a watch glass. Each beaker looked like this:
5. When the solvent had nearly reached the union of the folded part of the paper, the watch glass was carefully removed and the glass rod holding the strips was removed. The solvent fronts were marked with a pencil.
6. About 5 mL of 15 M ammonium hydroxide was poured into a clean, shallow dish and the chromatogram containing the knowns was placed on top of the dish. The paper was not permitted to dip into the solution. Any changes in color were noted.
7. Another piece of chromatography paper was dipped into dimethylglyoxime. This paper was used as a brush to paint the original chromatograms. Any change in color was noted and recorded.
Observations:
Color of solutions: Cu2+ clear light blue; Fe3+ clear pale yellow; Ni2+ clear green
Ion requiring no development: Fe3+
Are the solvent front and the Fe3+ front the same? Yes.
Color of ion developed by ammonia: Blue
Ion developed by ammonia: Cu2+
Color of ion developed by dimethylglyoxime: Red
Ion developed by dimethylglyoxime: Ni2+
Ring front distances for knowns: Solvent: 78.0 ± .5 mm; Fe3+: 75.0 ± .5 mm; Cu2+: 64.0 ± .5 mm; Ni2+: 25.0 mm
Ions present in unknown: Cu2+, Ni2+
Ring front distances for unknowns: Solvent: 79.0 ± .5 mm; Ni2+: 10.0 ± .5 mm; Cu2+: 67.0 ± .5 mm
Attached to the end of this report are two chromatograms. These are graphic illustrations of the products of the procedure and they show how qualitative and quantitative observations were made. A sample chromatogram might look like this:
Results:
Rf values for knowns (distance traveled by ions divided by distance traveled by solvent): Fe3+: 75.0 ± .5 mm / 78.0 ± .5 mm =
75.0 ± .67% mm / 78.0 ± .64% mm =
.96154 ± 1.31% =
.96 ± .01
Cu2+: 64.0 ± .5 mm / 78.0 ± .5 mm =
64.0 ± .78% mm / 78.0 ± .64% mm =
.82051 ± 1.42% =
.82 ± .01
Ni2+: 25.0 ± .5 mm / 78.0 ± .5 mm =
25.0 ± 2.00% mm / 78.0 ± .64% mm =
.32051 ± 2.64% =
.32 ± .01
Rf values for unknowns (distance traveled by ions divided by distance traveled by solvent):
Ni2+: 10.0 ± .5 mm / 79.0 ± .5 mm =
10.0 ± 5.00% mm / 79.0 ± .63% mm =
.12658 ± 5.63% =
.13 ± .01
Cu2+: 67.0 ± .5 mm / 79.0 ± .5 mm =
67.0 ± .75% mm / 79.0 ± .63% mm =
.84810 ± 1.38% =
.85 ± .01
Relative amounts: The Rf values for Cu2+ are close; the Rf values for Ni2+ are not close.
Discussion: The percent difference between Rf values can help determine the precision of an experiment. The first Rf values are those of copper, and the second are those of nickel.
(.85-.82)/.82 * 100 = 3.66% difference between Cu2+ Rf values
(.32-.13)/.13 * 100 = 146.15% difference between Ni2+ Rf values
The copper Rf values were close, but the nickel values were not close. This could be due to a number of sources of error. For example, when applying the solutions through the capillary tubes, some of the solution might have spilled or otherwise been applied wrong. If the painting with dimethylglyoxime did not fully coat the original chromatogram, then the distance from the pencil dot would not be accurate and the Rf value would be thrown off accordingly.
Furthermore, errors in placement of the chromatograms inside the beakers could lead to contamination errors. If the chromatograms touched other chromatograms, or the sides of the beakers, then foreign substances might have interfered with the development of the desired ions.
The theory associated with this experiment is that ions in solution can be separated and identified through the chromatographic process, regardless of the mixture. Furthermore, the Rf value is an intensive property, so that value can be used to identify substances if the Rf value is known. This process can be used to separate cations from solution and to separate dyes into their components.
There are several ramifications for this experiment. For example, if an unknown aqueous substance had to be identified, the cation could be determined by chromatographic process. Furthermore, any aqueous substance that had to be separated could be separated by this process. There are very few applications of chromatography beyond separation and identification.
Conclusion: The goal of the experiment was achieved. Chromatographic techniques were learned as a method of separation and identification of substances. Rf values were calculated successfully.