Lab #2 – Paper Chromatography
Background
Chromatography is a very common and versatile separation technique that is most often used to separate small
samples. The components of the separated mixture can then be subjected to further analysis for identification.
Chromatography takes many forms, depending on the nature of the mobile phase and the stationary phase. You
will be performing paper chromatography; the stationary phase is paper, and the mobile phase is an acidified
butanol solution. The components of the mixture you will be separating differ in their solubility in the butanol
mobile phase, and in their ability to adhere to the paper stationary phase.
You will place small samples of several different metal cation solutions across the bottom of a rectangle of
chromatography paper. This paper will be placed in a beaker containing a small amount of the butanol solution
(aka, eluting solution). As the eluting solution is wicked up the paper by capillary action, the cation samples
will dissolve and rise with the solution. As the cations move up the paper, the solvent will gradually evaporate
and the cations will begin to adhere to the paper. A component that dissolves very well and adheres poorly will
travel a great distance. Conversely, a component that dissolves poorly and adheres to the paper well won’t get
very far.
All variations of chromatography operate on this basic premise, but each variation employs different mobile and
stationery phases. A paper chromatographer can choose from thousands of different eluting solutions to find
the one that affects the best separation, or resolution, for her particular mixture. Different types of paper are
also available. In thin layer chromatography (TLC), a thin sheet of plastic coated with one of hundreds of dried
gels replaces paper as the stationary phase, and the chromatographer again has the choice of thousands of
eluting solutions. In gas chromatography, an inert gas such as nitrogen replaces the eluting solution as the
mobile phase, and the sample is carried by the gas through a long coiled tube line with a dried gel. Hundreds of
different gel-lined coils are commercially available.
Chromatography is a qualitative procedure that only separates mixtures but doesn’t help directly in identifying
components. It turns out, however, that the distance the components travel in a particular stationary/mobile
phase combination is repeatable and quantifiable. By measuring the distance traveled by the component and
dividing by the total distance traveled by the solvent, we get what is called Rf. Since Rf is a function of the
component’s identity, it can be used as one piece of evidence, hopefully combined with other evidence, to
identify one or more components in the mixture.
Rf =
Distance component moves
Distance solvent moves
We will rely on various stains to help us identify the cations in our mixtures. Your chromatograph will have
two different unknown samples; these will be compared to known samples on the same chromatograph. As
each of the stains is applied, you will compare the results with the known spots to the colors and Rf’s that
appear in the unknowns.
Procedure
Use a pencil and straightedge to draw a straight line about 1 cm from one of the long edges of you
chromatography paper. Starting about 1.5 cm from one end, make a series of pencil mark notches that are 2 cm
apart. Use the pencil to prelabel the chromatograph so the bottom edge looks as shown below:
|
|
Ag+
|
Co+2
|
Cu+2
|
Fe+3
|
Hg+2
|
Known UK #
|
|
UK #
.
Place 1 or 2 drops of each of the six known samples (5 individual and 1 mixed) in a small test tube. Also get
two test tubes containing unknowns; you may have to share these. You will use very fine glass capillary tubes
to apply your sample to the line at the bottom of the chromatograph in the correspondingly labeled area. The
key is to apply sufficient sample to as small a circular area as possible. To accomplish this, you will apply a
small amount (which will spread in a small circular area), dry the spot with an incandescent light source, then
apply a 2nd and 3rd small application directly on top of the original spot, drying between applications.
Get 2 capillary tubes; you will each one for several samples, rinsing between samples. You’ll notice that when
the end of the capillary tube is submerged in liquid, the liquid is spontaneously drawn up the tube. Conversely,
when the tube is touched to paper, the liquid is wicked out of the tube. Practice making small spots on a piece
of circular filter paper using distilled water, then use the same technique to apply the 6 knowns and 2 unknowns
in the labeled area on the line at the bottom of your chromatography paper. To clean the tube between samples,
dip it into distilled water, touch it to paper towel to draw the distilled water out, then repeat. Important: since
the mixed known sample and the unknowns are more dilute, these samples should be spotted 4 or 5 times,
drying between applications.
Now that the paper has been spotted with all of your samples, it can be placed in the mobile phase for eluting.
Get 15 mL of eluting solution (acidified butanol) and place it in a 600 mL beaker. Form your chromatograph
into a cylinder and tape it near the top. Place the chromatograph cylinder into the 600 mL beaker with the
spotted bottom edge in the eluting solution. Cover the beaker with a large glass plate and set the beaker in the
fume hood. Your instructor will remove your chromatograph later in the day.
You will now prepare your standards, which will be used tomorrow to compare to your master chromatograph.
Get 3 pieces of circular filter paper and use a pencil to label the outer edge of each with the identities of the 5
ions (Ag+ , Co+2, Cu+2, Fe+3, Hg+2). Place a large, sloppy spot of each of the 5 ion solutions on each of the filter
papers. These will air dry overnight and be used in lab tomorrow. ***STOP***
You will collect 4 pieces of data to help you identify the components of your unknown solutions. First, observe
and record the color of any visible dry spots on your chromatograph. You will now apply 3 different stains to
your chromatograph. First expose your chromatograph to NH3 fumes by placing your chromatograph over a
petri dish containing a concentrated NH3 solution, which is in the fume hood. While there, also expose one of
your circular filter paper standards to the NH3. Observe and record the color of any NH3 complexes that form.
Comparing these colors to the known standards on your chromatograph and on your circular filter paper will
allow you to identify the components in your unknown solutions. After recording these observations, expose
your chromatograph and a second circular filter paper standard to H2S fumes, also found in a petri dish in the
fume hood. Observe and record the color of any sulfide compounds formed. Lastly, get the ferricyanide stain
spray bottle and, in the sink at your lab station, spray your chromatograph and the third circular filter paper with
it, being carefully to keep the spray in the sink. You needn’t drench the papers; slightly damp is good. Observe
and record the color of any ferricyanide complexes formed.
Rank each ion from 1st through 5th in terms of Rf, where 5 is the ion that traveled the greatest distance. Record.
Prelab summary
Procedural Tips: ***Be sure to record the Unknown numbers!!! Use one ODD #, one EVEN #
Always use pencil on chromatographs, not ink.
Spot mixtures more heavily as they are less concentrated.
Stain chromatographs in the correct order: NH3, H2S, ferricyanide spray
Safety: The eluting solution is a solution of approx. 4 M HCl and flammable butanol
Disposal: Chromatographs can be thrown in garbage. Excess samples can go in sink. Recycle eluting solution.
Data and Calculations:
Indicate which ions are present in your unknowns by writing the symbol for the ion
in the box containing the data that led you to that conclusion.
Sample
Ag+
Rf ranking
Color(s) – dry
Color(s) – NH3
Color(s) – H2S
Color(s) - spray
Co+2
Cu+2
Fe+3
Hg+2
Odd UK
#_______
Even UK
#_______
Prelab Questions
1. What difference in property(ies) is the basis for separating a mixture using paper chromatography?
2. What kind of component will travel the farthest in paper chromatography?
3. How would our results be different if we only soaked our chromatograph for 10 minutes, instead of the
recommended several hours?
4. How would our results be different if we soaked our chromatograph for 24 hours, instead of the
recommended several hours (assuming we used plenty of eluting solution)?