Learning by Example
From TheLiquidPhase
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Undergraduate Chemistry Laboratory Experiment
==The Chemical Components of Ink: Exploring Paper, Column, and Countercurrent Chromatography.==
The Scenario
Congratulations, you have been chosen by the ACME ink company to be on a team of scientific researchers to develop new ink products. If you are reading this on a printed page, then you already are aware of the importance of ink in modern society. What you may not know, is that ink of any color is often a mixture of several compounds including pigments. Your first task, as part of the ACME ink company research team is to devise methods to separate the chemical components of ink. Since ink is not regulated by the Food and Drug Association, ink manufacturers do not have to divulge the ingredients of their ink formulations to the world. The ACME ink company would like to develop ways to separate and analyze the components of their competitor’s ink in order to see what they are up to.
Chromatography is the science of separation. Therefore, you will be test three chromatography techniques that have the potential to separate components of mixtures.
Paper Chromatography
The earliest form of chromatography to be developed was paper chromatography. Paper chromatography can be demonstrated by spilling a little water on a paper written with water-soluble ink. The ink dyes that were originally black or dark blue separate into a rainbow of colors. The reason the pigments in the ink separate is because they have different affinities for the paper versus the water. The paper acts as the solid support and attracts the different pigment molecules with different affinities. The paper also acts as a wick to draw the water through its fibers by capillary action. The water is the mobile phase and it solublizes the different pigment molecules in different ways.
Column Chromatography
Paper Chromatography is an example of adsorption chromatography that has proved to be a useful and versatile means of separating compounds to purify and identify them. The paper can be substituted by a plethora of different materials and the water can be substituted by a great many solvents and solvent combinations. Simple column chromatography works on the same principle of solid support and liquid mobile phase. In this case gravity causes the liquid phase to run downwards through the solid support made up of fine particles of silica or another substance. Some compounds stick to the solid support, others are carried along readily by the liquid. Most compounds, are somewhere in between, they stick a little to the solid support and are carried along a little with the liquid.
Liquid/Liquid Chromatography
Liquid-liquid chromatography differs from solid support chromatography by the fact that both phases are liquids. It is necessary to find a solvent combination that forms two distinct layers in order for liquid-liquid separations to work. Some compounds prefer the upper phase and will not go into the lower phase no matter how much the two layers are shaken together. Other compounds prefer the lower phase and stay there. Most of the compounds will be distributed between the upper and lower phases. It takes several mixing and settling operations to separate them from the compounds that prefer either the upper or the lower phase. Liquid-liquid chromatography is a simple form of countercurrent chromatography (CCC) that separates compounds through the continuous interaction between two immiscible liquid phases. Although the concept may seem straightforward, getting the two phases to mix well and then separate has led to the development of sophisticated instrumentation such as high-speed CCC (HSCCC). Yoichiro Ito and colleagues made the first CCC instrument in Japan in 1966. In 1971, a version was developed with no rotary seals, which led to the development of coil planet centrifuge CCC instruments. Coil planet centrifuges are devices in which the column, which is a coil of inert tubing, is rotated about a center point like a planet rotates while it revolves around the sun. The centrifugal force can be exploited to hold one of the liquid phases in the column while the other is pumped through. This allows for the continuous mixing and settling of the immiscible liquids throughout the length of the column rather like an arrangement of hundreds of separatory
Procedure
Paper Chromatography
This lab is best done in groups of two or three.
1. Obtain 2 strips of filter paper. 2. With a pencil, label the strips at the top with the letters of your first names. 3. Draw a line with the pencil about 1.5 centimeters from the bottom of the strips. 4. Spot the ink & methanol solution in the middle of the pencil line. 5. Carefully pour 20 mL of Lower Phase into a 250 mL beaker. 6. Carefully pout 20 ml of Upper Phase into a second 250 mL beaker. 7. Fasten a paper clip on the labeled end of each paper strip. Suspend the strip on a wooden stick so that the end hangs down into the beaker into the solution. Do not let the solution cover the ink spot. 8. Let the paper develop (the water will crawl up the paper). Do not jostle the paper or the jar while it is developing. This will take awhile. 9. Take the strip out before the solvent front has reached the letter – about 2 centimeters from the end. Make a mark with a pencil where the solvent front has reached. 10. Let the paper dry by hanging it in a dry beaker. 11. Describe the separation pattern of your strip the in the report section.
Column Chromatography
Part II. Column Chromatography.
1. Obtain a pre-packed silica gel column with an isopropanol slurry. 2. Put 10 mL of isopropanol in a 10 mL graduated cylinder. 3. Put 10 mL of ethanol in a second 10 mL graduated cylinder. 4. Put 10 mL of methanol in a third 10 mL graduated cylinder. 5. Put an Erlenmeyer flask under the column outlet. 6. Open the pinch clamp and let the solvent run down to the silica gel bed. Practice stopping the flow with the pinch clamp before it reaches the bed. 7. When the top of the solvent touches the silica gel bed, stop the flow. Do not let the level of the solvent “disappear” into the silica gel at any time during the experiment. 8. Carefully add 5 drops of ink solution to top of the bed with a plastic pipette. 9. Open the pinch clamp and let the ink flow until it is level with the top of the bed. 10. Close the pinch clamp and add 10 drops of isopropanol to the top of the silica gel bed. 11. Open the pinch clamp and let the solvent flow until it is level with the top of the bed. 12. Close the pinch clamp and add another 10 drops of isopropanol to the top of the silica gel bed. 13. Open the pinch clamp and let the solvent flow until it is level with the top of the bed. 14. Close the pinch clamp and add the rest of the isopropanol to the top of the silica gel bed. 15. Open the pinch clamp and let the isopropanol flow until it is level with the top of the bed. Write your observations in the report section of this handout. Try to catch the different bands of color eluting from the column in separate beakers 16. When the isopropanol reaches the silica gel bed, close the pinch clamp and add the ethanol to the column. You may have to let it run a bit so it doesn’t overflow. 17. Open the pinch clamp and let the ethanol flow until it is level with the top of the bed. Write your observations in the report section of this handout. 18. When the ethanol reaches the silica gel bed, close the pinch clamp and add the methanol to the column. You may have to let it run a bit so it doesn’t overflow. 19. Open the pinch clamp and let the methanol flow. Write your observations in the report section of this handout.
Liquid/Liquid Chromatography
1. Add 25mL of Upper Phase (UP) to each (8 of them) separatory funnels 2. Add 25mL of Lower Phase (LP) to the separatory funnel on the left (#1). 3. Add 6 drops of ink to separatory funner #1.. 4. Put the stopper in place and shake separatory funnel #1 vigorously. 5. Let the contents settle and remove the glass stopper. 6. Drain the lower layer (LP1) into Erlenmeyer #1. 7. Pour Erlenmeyer #1 into separatory funnel #2. 8. Add 25mL of Lower Phase to separatory funnel #1. 9. Shake the separatory funnels #1 and #2 vigorously. 10. Let the contents settle and remove the glass stoppers. 11. Drain the lower layer from separatory funnel #1 into Erlenmeyer #1. 12. Drain the lower layer from separatory funnel #2 into Erlenmeyer #2. 13. Pour Erlenmeyer #1 into separatory funnel #2. 14. Pour Erlenmeyer #2 into separatory funnel #3. 15. Add 25mL of Lower Phase to separatory funnel #1. 16. Shake the separatory funnels #1, #2, & #3 vigorously. 17. Let the contents settle and remove the glass stoppers. 18. Drain the lower layer from separatory funnel #1 into Erlenmeyer #1. 19. Drain the lower layer from separatory funnel #2 into Erlenmeyer #2. 20. Drain the lower layer from separatory funnel #3 into Erlenmeyer #3 21. Repeat this operation until you have all 8 separatory funnels with two distinct layers! 22. When all six have two distinct layers drain each one into a bottle. 23. Line up the bottles and write your observation in the report section of this handout.
Prelab
(Answer on a separate sheet of paper and hand in at the beginning of the lab period.)
1. Give a short definition of chromatography that makes sense in the context of this experiment.
2. In addition to the example given in the first paragraph of this handout, give another example where is it important to be able to separate complex mixtures.
3. Safety: You inadvertently knock over one of the beakers you are working with and it falls to the floor and breaks. What two things should you do? What 2 things could you have done to prevent this awful misfortune from happening?
Report
(Due one week after the experiment has been completed.)
Part I.
Describe the color separation on your paper strips.
Describe two ways that paper chromatography is similar to TLC (labs 1 and 3).
Describe two ways that paper chromatography is different from TLC.
Part II.
Describe the color separation you observed on the column.
Describe two ways the paper and column chromatography were similar.
Describe two ways the paper and column chromatography were different.
Part III.
Describe the color separation you observed in the separatory funnels.
Parts I, II, & III:
According to these three methods, how many colored compounds make up black ink?
Why do you think they use a mixture of compounds instead of just one black substance?
How could you identify these pigments once you have separated them?
Instructor's Notes
From the Bricker & Sloop article: Use Sheaffer’s Skrip blue-black soluble ink (ordered on-line from Office Max) 1-butanol / 0.5 M aqueous HCl
Paper Chromatography: Resolution is not great. I decided to use PC because we had already done 2 labs with TLC. TLC would definitely work better.
Column Chromatography: CC is an art difficult to describe on a protocol. I pre-packed the columns just to save the headache. The sequence works pretty well as far as resolution. I used “Kontes Disposaflex Glass Columns” 8x150 mm, 6 mL capacity ordered from Fisher K420166-1001
Countercurrent Distribution: You need a lot of separatory funnels! I found a box full of 125 mL sep funnels that worked great. The number of sep funnels can be varied, 6 would be the minimum, I would think.