Lab 1-1 Separation of Mixtures : Chromatography
Skill Set
How to separate mixtures via differential rates of migration
Objective:
Separating mixtures is a key process for chemical analysis and in fact is one of the most important processes in chemistry today. The particular branch of chemistry called analytical chemistry is concerned with the separation of mixtures. Part I studies the resolution of inks by paper chromatography. Part II deals with the resolution of a mixture of colored indicator dyes by thin-layer chromatography.
General Introduction
to Chromatography:
The word chromatography means color writing. The name was chosen at the beginning of this century when the method was first used to separate colored components from plant leaves. Chromatography in its various forms is perhaps the most important known method of chemical analysis of mixtures.
Paper and thin-layer chromatography are simple techniques that can be used to separate mixtures into the individual components of the mixture. The methods are very similar in operation and principle, differing primarily in the medium or material used in the separation for the analysis.
Paper chromatography uses ordinary filter paper, which consists primarily of the polymeric carbohydrate cellulose, as the medium on which the mixture to be separated is applied. Thin layer chromatography (universally abbreviated as TLC) uses a thin coating of aluminum oxide (alumina) or silicagel on a glass microscope slide or plastic sheet to which the mixture to be resolved is applied.
A single drop or spot of the unknown mixture to be analyzed is applied about half an inch from the end of a strip of filter paper or a TLC slide. The filter paper or TLC slide is then placed in a shallow layer of solvent or solvent mixture in a jar or beaker. Since filter paper or the coating of the TLC slide is permeable to liquids, the solvent begins rising by capillary action.
As the solvent rises to the level at which the spot of mixture was applied, various effects can occur, depending on the constituents of the spot.
The components of the spot that are completely soluble in the solvent will be swept along with the solvent front as it continues to rise.
The components that are not at all soluble in the solvent will be left behind in the original location of the spot. Most other components of the unknown spot mixture will be found between. Components in the spot that are somewhat soluble in the solvent will be swept along in the solvent front, but to different extents, reflecting their different solubilities. The original spot of mixture is then spread out into a series of spots or brands, with each spot or brand representing one single component if the single mixture.
The separation of a mixture by chromatography is not solely a function of the solubility of the components in the solvent used, however. The filter paper or TLC slide coating used in chromatography is not inert, but consists of molecules that may interact with the molecules of the components of the mixture being separated. Each component of the mixture is likely to have a different extent of interaction with the filter paper or slide coating. This differing extent of interaction between the components of a mixture and the molecules of the support forms an equally important basis for the separation. Filter paper or the TLC slide coating absorbs molecules on its surface to differing extents, depending on the structure and properties of the molecules involved.
To place a paper chromatography or TLC separation on a quantitative basis, a mathematical function called the retention factor, Rf, is defined:
Rf = Distance traveled by spot/distance traveled by solvent front
The retention factor depends on what solvent is used for the separation, and on the specific composition of the filter paper or slide coating used for a particular analysis. Because the retention factors for particular components of a mixture may vary if an analysis is repeated under different conditions, a known sample is generally analyzed at the same time as an unknown mixture on the same sheet of filter paper or slide. If the unknown mixture produces spot having the same Rf values as spots from the unknown sample, then an identification of the unknown components has been achieved.
Paper chromatography and TLC are only twp examples of many different chromatographic methods. Mixtures of volatile liquids are commonly separated by a method called gas chromatography. In this method, a mixture of liquids is vaporized and passed through a long tube (column) of solid adsorbent material coated with an appropriate liquid, by the action of a carrier gas (usually helium). As the paper chromatography, the components of the mixture will have different solubilities in the liquid coating and different attractions for the solid absorbent material. Separation of the components of the mixture thus occurs as the mixture process through the tube. The individual components of the mixture exit the tube one by one and are usually detected by electronic means. A final very important chromatographic technique is called high performance liquid chromatography (HPLC). In HPLC, liquid mixtures to be analyzed are blown through a column of absorbent material under high pressure from a pump, resulting in a very quick passage through the column. HPLC is routinely used in medical and forensic labs to analyze biological samples. For example, blood samples can be analyzed for the presence of alcohol or illicit drugs in just a few minuets using HPLC.
Introduction
Although paper chromatography is a very simple technique, it is still used frequently for analysis of mixtures of colored substances (or for substances which can be made colored by treatment with an appropriate reagent). For example, biologists often use paper chromatography can also be used for simple analyses of protein extracts (amino acids).
In this process, you will do some very simple paper chromatography analyses of some ink mixtures. As you know, inks for pens come in many different, bright colors- particularly in pen sets used by small children for working on their coloring books. Such brightly colored inks, however, are often mixtures of primary color inks. For example, a felt tip pen having what appears to be bright purple ink may actually contain a mixture of blue and red inks. Similarly, what appears to be orange ink may be a mixture of red and yellow inks. Although this Part I is very simple, you will clearly see the basis for chromatographic analyses, and will perhaps gain some insight into the great importance of the various chromatographic methods.
Materials and
Chemicals Needed
Filter paper for chromatography (5 x 10 cm), latex surgical gloves, ruler, pencil, acetone water mixture (50-50 mix), felt tip pens (water-soluble and permanent)
Safety Precautions
Wear safety glasses at all times while in the laboratory.
Acetone is highly flammable. No open flames are permitted in the laboratory. Acetone may be toxic if inhaled or absorbed through the skin.
Procedure
It is OK to record all observations on this sheet of paper.
Because the skin contains oil which can interfere with the chromatogram (or paper strip), latex surgical gloves should be worn from this point onward in the procedure to prevent contamination of the chromatogram. A pencil must be used in the following procedure for marking the chromatogram, since ink from a ball-point pen would also undergo chromatography.
Obtain a sheet of paper (chromatography paper cut to a specific, consistent length…minimum 10 cm) prepared for the chromatographic analysis. Draw a light pencil line across the paper about 1 cm from each end. On the lower pencil line, lightly mark a small circle. These circles are where the inks will be applied to the paper.
From your instructor, obtain several ink samples containing water-soluble inks. Apply a small single spot of a different ink to each of the small circles you drew on the paper. Allow the spots to dry completely. Record on your notebook the original color of each ink applied to the filter paper.
When the spots are completely dry, apply a second spot of each ink to its respective place on the filter paper. Applying a second spot of the same dye builds up a larger sample of dye on the filter paper.
Clean a beaker (or special chromatography jar) for use in developing each of the chromatograms.
Add distilled water to the beaker to the depth of approximately one-quarter of an inch.
Make certain that the ink spots on the filter paper are completely dry. If the spots are dry, fold the filter paper in half lengthwise.
Carefully suspend and lower the filter paper (with the spots at the bottom) into the water in the beaker. Make certain not to wet the spots as you lower the filter paper, and do not move the beaker to avoid splashing water onto the spots.
Position the chromatogram to allow the water to rise in the filter paper until it reaches the bottom of the paper and below the lower pencil line. Allow to set until the liquid has moved up the paper until it reaches the upper line or stabilizes position. Then carefully remove the paper and set it on a clean paper towel. Quickly dry the filter paper using a heat gun or hair dryer if one is available.
Determine the retention factor values (Rf) for each of the ink spots (if a combo color Rf of each color) and record.
Obtain from your instructor several permanent ink markers (which are not water soluble). Repeat the chromatographic procedure using a new strip of filter paper and a 50% acetone-water mixture as the solvent (caution). Determine and record the Rf values for the permanent inks.
Save your chromatograms for submitting with your lab report.
Questions (part 1)
1.Use a chemical dictionary or
encyclopedia to find a specific definition of chromatography and report your findings in your own words
here.
2. Use a chemical encyclopedia to report the sorts of samples which might be analyzed by paper chromatography (as opposed to some other method).
3. On a paper chromatogram, a spot produced by a red ink traveled 5.2 cm, whereas the front of solvent used for the chromatographic separation traveled 8.9 cm during the experiment. Calculate Rf for the red ink.
4. In preparing a TLC slide or filter paper for chromatography, a baseline is drawn for positioning the spots in pencil. Why is ink never used for drawing the baseline?
Results and Observations
Water
Distance traveled by color(s)
A.
1.
2.
3.
B.
1.
2.
3.
C.
1.
2.
3.
D.
1.
2.
3.
Distance traveled by solvent front-
Spot color Rf
A.
1.
2.
3
B.
1.
2.
3
C.
1.
2.
3
D.
1.
2.
3
Water/Acetone Mix
Distance traveled by color(s)
A.
1.
2.
3
B.
1.
2.
3
C.
1.
2.
3
D.
1.
2.
3
Distance traveled by solvent front-
Spot color Rf
A.
1.
2.
3
B.
1.
2.
3
C.
1.
2.
3
D.
1.
2.
3
Questions part 2
7. Why is a mixture of acetone and water used to separate the second set of inks (non water soluble)?
8. Why was it necessary for you to wear plastic surgical gloves while preparing and developing your chromatogram?
9. List those samples which are single-component inks.
10. List those samples which are multi-component inks, identifying the colors of the components used to give the overall color of the ink.