Paper Chromatography of a Metal Cation Mixture

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CHEM 121L 
General Chemistry Laboratory 
Revision 3.0 
 
 
Paper Chromatography of a Metal Cation Mixture 
 
 
 To learn about the separation of substances. 
 To learn about the separation technique of chromatography. 
 To learn how to use properties of known substances to identify unknown substances. 
 
 
In this laboratory exercise we will separate and identify the cations in an aqueous mixture that 
possibly contains Fe
3+
, Ni
2+
 or Cu
2+
 salts. The separation will be achieved using Paper 
Chromatography. Identification of the cations will be affected by simultaneously running 
separate solutions, each containing a single salt of these ions, through the chromatographic 
system and comparing the results against those of the unknown solution. 
 
Separation of substances is a key component in most chemical processes. Reaction products 
need to be separated from associated by-products, complex natural systems need to be separated 
to obtain a desired component, etc. Thus, in some sense, the science of chemistry is really the 
science of separations. And, over the years, chemists have developed an enormous array of 
techniques for the separation of substances. 
 
In 1906 the Russian scientist Mikhail Semenovich Tswett reported separating the different 
colored pigments of plant leaves by passing a liquid extract of the leaves through a column of 
calcium carbonate (think powdered chalk). He coined the term chromatography, from the Greek 
words χρώμα (chroma or “color”) and γραφειν (graphein or “to write"), to describe this process. 
Presently, chromatography is the general name applied to a series of separation methods that 
employ a system with two phases of matter; a mobile phase and a stationary phase. The 
separation process occurs because the components of the mixture have different affinities for the 
two phases and thus move through the system at different rates. A component with a high 
affinity for the stationary phase moves more slowly, whereas one with a high affinity for the 
mobile phase moves more rapidly. 
 
Paper chromatography, probably the simplest chromatographic system, employs a strip of porous 
paper for the stationary phase. A drop of the mixture to be separated is placed on the paper, 
which is then dipped into a liquid, the mobile phase. The liquid travels up the paper as though it 
were a wick. The separation occurs as the liquid moves along the paper, carrying along with it, 
most rapidly, those components with a low affinity for the paper and leaving behind those with a 
high affinity for the paper. In this type of chromatography, the paper used is a highly purified 
cellulose with sufficient adsorbed Water such that the stationary phase is actually the liquid 
adsorbed to the solid cellulose fibers. 
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In a given chromatographic system, using the same type of paper, each substance can be 
characterized by a constant called the Retention Factor, Rf. By definition: 
 
 Rf = 
Distance from Origin Spot Travels
Distance from Origin Solvent Front Travels
 (Eq. 1) 
 
The Rf value is a characteristic property of each species in a mixture. The retention factor simply 
measures the fraction of the distance each species travels, relative to the distance the solvent 
travels. In order for this technique to be effective in resolving a mixture, the paper and eluting 
solvent must be chosen such that each compound in the mixture has a different Rf value. 
 
 
 
 
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It should be noted that a given compound’s Rf is not the only determinant of this method’s 
resolution. As each compound migrates, its spot broadens as the material diffuses away from the 
highly concentrated center of the spot. The reasons for this broadening are complex and will not 
be discussed here. Note only that the longer the system is allowed to develop, the broader will be 
the spots. And, this breadth also affects the resolution. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Thus, the breadth of each spot is another important parameter of a paper chromatographic 
system. And, the usual quandary encountered when establishing a chromatographic system is 
that the system should run long enough such that the distance each spot travels is different for 
each species, but not so long that broadening causes the spots to overlap. 
 
In general, Paper Chromatography is considered to be a low resolution technique. Other 
chromatographic techniques have been developed to significantly improve resolution. 
 
We will use this chromatographic technique to separate a mixture of salts (Cupric Nitrate, Nickel 
Nitrate and Ferric Nitrate) dissolved in water. The stationary phase will be a piece of Whatman 
Grade 1 Chr Cellulose Chromatography Paper. The eluting fluid, or mobile phase, will be 
primarily Acetone with a little Hydrochloric Acid added. The presence of each salt can be 
detected independently, once the separation has been completed. Ferric Nitrate, Fe(NO3)3, which 
contains iron, produces a rust color on wet paper. Cupric Nitrate, Cu(NO3)2, reacts with 
ammonia to produce a deep blue color. The Nickel Nitrate, Ni(NO3)2, reacts with the organic 
reagent Dimethylglyoxime (DMG) to produce a pink color. 
 
 
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Pre-Lab Safety Questions 
 
1. Do a short internet search for an NFPA 704 Fire Diamond for Acetone. What is the Degree 
of Hazard for Acetone's flammability? What does this rating mean? 
 
2. Is Acetone considered a significant health hazard? 
 
3. Do a short internet search for a Safety Data Sheet (SDS) for Ammonia. What First Aid 
measures should be taken if someone inhales significant Ammonia? 
 
 
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Procedure 
 
1. Prepare about 25 mL of eluting solution (mobile phase). This is a mixture of 19 mL of 
acetone and 6 mL of 8 M HCl. Pour this into a 1000 mL beaker; which will act as the 
chromatography tank. Cover the beaker with a piece of aluminum foil. This allows the 
atmosphere within the beaker to become saturated with solvent vapor and helps to give a 
better chromatographic separation. 
 
2. Obtain a piece of chromatography paper. Handle this from the side edges or with gloves 
on as the oils from hand can alter the properties of the paper surface sufficiently to 
affect the running of the chromatogram. Draw a pencil line about 2 cm from the long 
edge of the paper. This line will indicate the origin. 
 
3. Draw out 3 capillary tubes, using the micro Bunsen burner, to use as a "spotter". (Your 
instructor will show you how to do this. Make sure your eluting fluid and the Acetone 
stock are a safe distance from the burner's flame as Acetone is highly flammable.) 
Using a different "spotter" tube for each solution, transfer a drop of each solution listed 
below to the penciled line to give a spot about 0.5 cm in diameter. Each spot should be 
about 3 cm apart. Allow the spot to dry momentarily, then transfer a second drop of each 
solution to the appropriate spot. With a pencil, identify each spot on the paper. The 
solutions are: 
 
 1) Ferric Nitrate, Fe(NO3)3 
 2) Nickel Nitrate, Ni(NO3)2 
 3) Cupric Nitrate, Cu(NO3)2 
 4) An unknown mixture of the above salts. 
 
4. Form the paper into a cylinder without overlapping the edges. Fasten the paper at the top 
and bottom with staples. 
 
5. Carefully place the paper cylinder into the chromatography tank and replace the cover. 
Wait as the solvent front moves up the paper. Do not move the beaker. If the movement of 
the solvent front appears to stall, burp the fumes in the chromatography tank by briefly 
opening the coverand blowing into the beaker. Do this lightly , so as not to blow the 
fumes back into your face. If the solvent front remains stalled, consult with your 
instructor concerning what to do next. 
 
6. When the solvent has risen to within 1 cm of the top of the paper, remove the paper from 
the tank and quickly mark the solvent front with a pencil. Remove the staples and dry the 
paper with a hot air dryer. If no hot air dryer is available, dry the paper over a luke warm 
Hot Plate. 
 
7. Working in the fume hood, hold the paper just over the top of a large evaporating dish 
containing about 100 milliliters of concentrated ammonia solution. A deep blue color will 
indicate the presence of cupric nitrate. Any white "smoke" which appears is really 
Ammonium Chloride due to a reaction between Hydrochloric Acid in remaining eluting 
solution and the Ammonia: 
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 Hydrochloric Acid + Ammonia Ammonium Chloride 
 
 or 
 
 HCl(aq) + NH3(aq) NH4Cl(s) 
 
8. While the paper is still moist with ammonia, spray the paper with the DMG solution. A 
pink color will indicate the presence of nickel nitrate. If no pink color appears for the 
"Known" nickel salt, again hold the paper over the ammonia dish. This should help “bring-
out” the nickel spots. 
 
9. For each spot, measure the distance the spot traveled and the breadth of each spot. 
 
 
 
 
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Data Analysis 
 
1. Calculate the Rf value for each known salt. 
 
2. Calculate the Rf value for each spot produced by the unknown. 
 
3. Identify the cation salts in the unknown mixture. 
 
 
 
 
 
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Post Lab Questions 
 
1. If you had not been told there were only three possible compounds in your unknown 
mixture, would you be able to determine the number of compounds in your mixture based 
on your chromatographic results? Explain. (Hint: Consider what would happen if two compounds 
have the same Retention Factor.) This is a problem with any separation technique. 
 
2. Two extreme values for Rf are 1 and 0. Explain what each value means in terms of the 
compound's affinity for the paper versus the eluting solution. 
 
3. Why do you mark the solvent front immediately upon removal of the filter paper? 
 
4. Amino acids, the building blocks of proteins, can easily be separated via paper 
chromatography. In fact, Insulin, the first protein whose amino acid composition was 
determined, had its composition determined by paper chromatography. Consider a mixture 
containing three amino acids that has been separated via paper chromatography. It is found 
the retention factor for each component is as follows: 
 
 Component Rf 
 Glycine 0.37 
 Proline 0.65 
 Leucine 0.76 
 
 Sketch a diagram of a chromatogram. Assume the piece of paper is 20cm high and the 
solvent front runs 17cm from the original spot, dotted 1cm above the bottom of the paper. 
Be as quantitative as possible.