Experiment 1:

The fact that different substances have different solubilities in a given solvent can be used in several ways to affect a separation of substances from mixtures in which they are present. You will see in an upcoming experiment how fractional crystallization allows you to obtain pure substances by relatively simple procedures based on solubility properties. Another widely used resolution technique, which also depends on solubility differences, is chromatography.

In a chromatographic experiment, a mixture is deposited on some solid adsorbing substance, which might consist of a strip of filter paper, a thin layer of silica gel on a piece of glass, some finely divided charcoal packed loosely in a glass tube, or even some microscopic glass beads coated thinly with a suitable adsorbing substance and contained in a piece of copper tubing.

The components of a mixture are adsorbed on the solid to varying degrees, depending on the nature of the component, the nature of the adsorbent, and the temperature. A solvent is then caused to flow through the adsorbent solid under applied or gravitational pressure or by the capillary effect. As the solvent passes the deposited sample, the various components tend, to varying extents, to be dissolved and swept along the solid. The rate at which a component will move along the solid depends on its relative tendency to be dissolved in the solvent and adsorbed on the solid. The net effect is that, as the solvent passes slowly through the solid, the components separate from each other and move along as rather diffuse zones. With the proper choice of solvent and adsorbent, it is possible to resolve many complex mixtures by this procedure. If necessary, a given component can usually be recovered by identifying the position of the zone containing the component, removing that part of the solid from the system, and eluting the desired component with a suitable good solvent.

The name given to a particular kind of chromatography depends upon the manner in which the experiment is conducted. Thus, there is column, thin-layer, paper, and gas chromatography, all in very common use. Chromatography in its many possible variations offers the chemist one of the best methods, if not the best method, for resolving a mixture into pure substances, regardless of whether that mixture consists of a gas, a volatile liquid, or a group of nonvolatile, relatively unstable, complex organic compounds.

In this experiment, you will use paper chromatography to separate a mixture of metallic ions in solution. A sample containing a few micrograms of ions is applied as a spot near one edge of a piece of filter paper. That edge is immersed in a solvent, with the paper held vertically. As the solvent rises up the paper by capillary action, it will carry the metallic ions along with it to a degree that depends upon the relative tendency of each ion to dissolve in the solvent and adsorb on the paper. Because the ions differ in their properties, they move at different rates and become separated on the paper. The position of each ion during the experiment can be recognized if the ion is colored, as some of them are. At the end of the experiment their positions are established more clearly by treating the paper with a staining reagent which reacts with each ion to produce a colored product. By observing the position and color of the spot produced by each ion, and the positions of the spots produced by an unknown containing some of those ions, you can readily determine the ions present in the unknown.

*Adapted from Slowinski, E. J., Wolsey, W. C. Chemical Principles in the Laboratory 9th ed.

CHEM 403 Exp 1

It is possible to describe the position of spots such as those you will be observing in terms of a quantity called the Rf value. In the experiment, the solvent rises a certain distance, say L centimeters. At the same time a given component will usually rise a smaller distance, say D centimeters. The ratio of D/L is called the Rf value for that component:

= = distance component moves distance solvent moves

The Rf value is a characteristic property of a given component in a chromatography experiment conducted under particular conditions. It does not depend upon concentration or upon the other components present. Hence it can be reported in the literature and used by other researchers doing similar analyses. In the experiment you will be doing, you will be asked to calculate the Rf values for each of the cations studied.


From the instructor obtain an unknown and a piece of filter paper about 19 cm long and 11 cm wide. Along the 19-cm edge, draw a pencil line about 1 cm from that edge. Starting 1.5 cm from the end of the line, mark the line at 3-cm intervals. Label the segments of the line as shown in Figure 1, with the formulas of the ions to be studied and the known and unknown mixtures.

Figure 1: Paper Chromatography Preparation

You will be spotting five known cations and an unknown solution that may contain up to three of the cations. The known cations are prepared as 0.1 M solutions of the following compounds:

AgNO3 Co(NO3)2 Cu(NO3)2 Fe(NO3)3 Ni(NO3)2

In solution these substances exist as ions. The metallic cations are Ag+, Co2+, Cu2+, Fe3+, and Ni2+, respectively. One drop of each solution contains about 50 micrograms of cation.

CHEM 403 Exp 1

Your instructor will furnish you with a fine capillary tube, which will serve as an applicator. Practice the application procedure by dipping the applicator into one of the colored solutions and touching it momentarily to a paper towel or spare piece of filter paper. The liquid from the applicator should form a spot no larger than 8 mm in diameter. Practice making spots until you can reproduce the spot size each time.

To prevent cross-contamination, it is important to clean your applicator between each different sample. Clean the applicator by dipping it into distilled water and then touching it to a paper towel to remove the liquid. Continue contact until all the liquid in the tube is gone. Repeat the cleaning procedure one more time.

When you are ready to prepare your filter paper, dip the applicator into one of the cation solutions and put a spot on the line on the filter paper in the region labeled for that cation. Clean the applicator twice, and repeat the procedure with another solution. Continue this approach until you have put a spot for each of the five cations and the unknown on the paper, cleaning the applicator between solutions. Dry the paper by moving it in the air. If a spot is not visible, apply that solution again to the same spot; this procedure will increase the amount of each ion in the spots, which will make it easier to find the spot after it elutes up the filter paper. Make sure that you dry the spots between applications, since otherwise they will get larger.

Form the paper into a cylinder in such a way that the edges are parallel but do not overlap. The pencil line at the bottom of the cylinder should form a circle, approximately anyway, and the two edges of the paper should not quite touch. Carefully staple the top corners together. Do not staple the lower edges of the paper together.

Place the cylinder in the eluting solution that has been provided in a 600-mL beaker, with the sample spots down near the liquid surface. The paper should not touch the wall of the beaker. Cover the beaker with a watch glass. The solvent, which is a mixture of hydrochloric acid, ethanol and butanol, will gradually rise by capillary action up the filter paper, carrying along the cations at different rates. After the process has gone on for a few minutes, you should be able to see colored spots on the paper, showing the positions of some of the cations. When the eluting solution has risen to within about 2 cm of the top of the filter paper (it may take about 75 minutes), remove the cylinder from the beaker and carefully take out the staple. Draw a pencil line along the solvent front. Dry the paper under a heat lamp until it is quite dry, and all cation spots are visible.

Measure the distance from the straight line on which you applied the spots to the solvent front, which is distance L in your Rf equation. Then measure the distance from the pencil line to the center of the spot made by each of the cations, when pure and in the known; this is distance D. If your solvent front is uneven, your value of L may vary for your calculations. Calculate the Rf value for each cation. Then calculate Rf values for the cations in the unknown. How do the Rf values compare? Note the colors of the spots in your unknown as a second way to confirm which cations are present.

Then, with your paper on a paper towel, spray it with a staining reagent. Note any color changes of your cations, and if the cations you identified in your unknown stain in the same way as the known cations.


Prelab Assignment

1. Explain, in your own words, how samples can be separated into their components using chromatography.

2. If you have two samples in this experiment that produce spots with the same Rf value, can you assume they contain the same cation? Why or why not? What other observations could you use to determine if the two spots are the same?

3. The solvent moves 3 cm in about 5 minutes. Why shouldn’t the experiment be stopped at this point instead of waiting the 75 minutes for the solvent to travel the entire 10 cm?

4. In this experiment it takes about 10 microliters of solution to produce a spot 1 cm in diameter. If the Cu(NO3)2 solution contains about 6 g Cu2+ per liter, how many micrograms of Cu2+ ion are there in one spot of that size?

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