Introduction to Chromatography: Comparing Paper-, Column-, and Thin-layer Chromatography.
Scenario: A technical problem at a small packing and bottling plant resulted in the inadvertent mixing of several vats containing dyes resulting in an unwanted product. Lab teams are requested to determine the original components of the accidental mixture and suggestions for ways to separate them. The scenario and assignments are presented in the form of letters and memos (attached).
Safety, Handling, and Disposal: It is recommended that students read the M.S.D.S.'s of all chemicals used in this lab experiment. As always, the use of safety goggles are required, as is the compliance with standard laboratory safety rules.
Organic solvents like petroleum ether, acetone, ethanol, and isopropyl alcohol are highly flammable. These organic solvents should be kept away from spark sources and should be disposed of properly in marked solvent waste cans.
Materials: (per lab station)
-beakers and watch glasses
-Dyes (prepared by the instructor)
-tooth picks or capillaries
Column chromatography: (per lab station)
-Dyes (same as above)
-Several disposable Pasteur pipets, 14.5 x 0.5 cm
-Swab of glass wool
-Several grams of clean, white sand
-Several grams of alumina
-Several grams of silica gel
-Several grams of sodium bicarbonate
-Several grams of sucrose
-Mortar and pestle
-Small beakers or sample vials
Thin-layer chromatography (per lab station)
-dyes (same as above)
-Several sheets of TLC paper
-Pasteur pipets or capillaries for spotting
-UV light source
RE: Separation of dyes
Dear Gen.Chem team:
We operate a small bottling and packaging plant for household and commercial dyes. Yesterday, a major technical problem occurred during our night shift. Despite several fail-safe procedures, three vats of dyes were inadvertently mixed resulting in a brown dye for which we have currently no orders. To make matters worse, our chemist was involved in an accident and will not be available for several weeks. We hope that your team can analyze the enclosed sample of the mixed dyes and suggest ways of separating them into their original colors. Thank your for giving this matter your prompt attention and we are looking forward to your results.
TO: GEN. CHEM. LAB TEAMS
FROM: Peter Jeschofnig
RE: Lab Experiment: Dye Separation Project DATE: 7-18-1997
As you can see from the enclosed memo, you have been asked to solve a major dilemma. After receiving the letter from True Colors, Inc I called the manager and he thought that the colors involved are red, yellow and green, but he is not 100% sure.
Since we have already discussed several basic separation techniques including paper chromatography, column chromatography, and thin layer chromatography you already have the basic skills necessary to tackle this problem.
Each team is expected to use all three techniques, but may vary stationary and liquid phases as they wish.
I expect a detailed lab report with procedures used and results obtained as well as a short letter to the True Colors company summarizing your findings and recommendations.
Memo from True Colors, Inc.
Summary of Paper Chromatography
Summary of Column Chromatography
Summary of Thin-layer Chromatography
Paper chromatography is a simple, yet extremely effective method for determining the presence of a substance in a mixture or for separating components from a mixture. A strip of paper (special chromatography paper or simple filter paper) serves as the stationary phase. The mixture to be separated is placed in a small spot near one end of a strip and a solvent (or solvent mixture), called the mobile phase (or eluent), is passed over the spot. The solvent moves up the plate due to capillary action the same way that water will move up a paper towel and carries with it the various components in the spot.
The sample moves upward to the extent that it dissolves in the solvent more than it adheres to the adsorbent and is carried along by the solvent. Since every substance will have a slightly different solubility in a given solvent and will adhere to a solid adsorbent to a different degree, the different components in a mixture will be carried along by the solvent at different rates, and hence be separated. Differences in solubility and degree of adsorption are due to different sizes and shapes of molecules as well as difference in polarity. Thus if two compounds are started at the same place and the solvent passed over them (elution), one compound will move along the strip faster than the other.
After a period of time the flow of the mobile phase is stopped and the strip is dried. If the components of the mixture are colored the separated compounds can be observed directly. If they are colorless some method (usually chemical) must be used to show their presence. This process is called visualization.
Paper chromatography tells whether or not a sample is pure, and if it is not, how many different components are present. The positions of each spot can be compared with those from known compounds (standards) to ascertain the presence or absence of that substance. The location of the compound is indicated by a Rf value. For a given substance the distance it moves depends on the total time the experiment is carried out as well as on the physical and chemical properties of the system. However, the distance the compound moves relative to the distance the mobile phase moves is a characteristic of that compound and is known as the Rf value.
The choice of solvent in paper chromatography is crucial. The solvent must dissolve the various components in the mixture and there must be at least slight differences in solubility of each component. Therefore, there will be no separation (or as is often stated, no resolution). Also if the components are insoluble they will remain at the origin, unmoved. If all of the components are very soluble in the solvent system then each component of the mixture may travel along with the leading edge of the solvent ("the solvent front") unseparated. In practice it is usually found that one pure solvent will not give resolution and a mixture of solvents must be found for each sample to be analyzed.
As more of a substance is present, it will spread over a greater area, and potentially overlap with other components on a chromatogram. This is called band broadening. Although the largest dot produces the most vivid colors, the smallest dot shows the most distinct break between colors.
1. Obtain a strip of filter paper that is approximately 20 x 15 cm. Draw a thin line with a pencil (do not use a pen !!) across the paper 10 mm (1 cm) from the bottom. Draw the line just hard enough to be visible.
2. Obtain a set of food colors, or any other colors available.
3. Using a toothpick or capillary tube place a dot of a color sample onto the pencil line. Place additional dots of other color samples on the same line about 2 cm apart.
4. After the spots have dried bring the ends of the paper together and staple them to form a roll (the ends should not overlap). The pencil line with the sample dots should be at the bottom of the rolled up paper
5. Prepare an elution chambers by pouring the eluting solvent (water) into the jars to a depth of about 3 - 5 mm just enough to cover the bottom of the chamber). Place the rolled up paper into the chamber. The spots must be at the bottom of the chambers and above the solvent level. The solvent front will travel up the paper rapidly at first and then will slow down. The solvent front is allowed to rise to within 2 cm of the top of the paper strip if time permits. The paper should not touch the side of the jar.
6. When complete, the paper is removed from the chamber, the top of the solvent front is marked with a pencil, and the paper strip is allowed to dry.
7. Measure the distance from the origin (initial pencil line) to the center of each spot on the paper strips. If the spots are large or irregular in shape it is necessary to estimate where the center is. Also measure the distance from the original line to the solvent front. Make all measurements to the nearest millimeter and record them.
8. Calculate the Rf value for each spot:
Rf = component distance/solvent distance
9. Using fresh filter paper, repeat the procedure using different solvent, such as 25% ethanol - 75% water; 75% ethanol - 25% water; 100 % ethanol; methanol-water mixtures; isopropyl-water mixtures. When using volatile solvents such as alcohol, acetone, etc. place a watch glass over the top of the beaker to prevent the solvent from evaporating.
10. After completing these steps you should be able to identify the optimum solvent for your sample and be able to separate a mixture of components analyzed so far.
Thin-layer chromatography consists of a stationary phase immobilized on a glass or plastic plate and a solvent. The sample, either liquid or dissolved in a volatile solvent, is deposited in spots onto the stationary phase. Unknowns can be identified by simultaneously running standards with the unknown. One edge of the plate is then placed in a solvent reservoir and the solvent moves up the plate by capillary action. The different components in the mixture move up the plate at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase.
Although it is possible to make one's own TLC plates,
a variety of different TLC plates are available commercially and their
use saves considerable time in the laboratory.
1. Cut a 3cm x 8cm strip of TLC material and draw a thin pencil line 1 cm from the end (parallel to the short dimension).
2. Place 1 or 2 sample dots on the pencil line (evenly spaced). If the sample is not very concentrated, repeated spotting might be useful. When the first dot is dry, place another dot of the sample on top of the first (dried) spot. This may be repeated several times.
3. The chromatogram is developed by placing it into a developing chamber (beaker with watch glass lid) containing 5 mm of appropriate solvent. The sample spots must be dry and above the solvent level. Make sure to replace the lid. Frequently a piece of solvent saturated filter paper is placed inside the jar (it must not touch the TLC plate) to aid in developing efficiency.
4. The TLC plate is removed from the chamber when the solvent front nears the top of the plate.
5. Visualization of colorless separated compounds
is achieved by placing the dried TLC plate under an UV lamp in a viewing
box or darkened area. Colorless compounds could also be visualized by placing
the plate in an iodine vapor chamber for a few seconds. The compound spots
need to be marked by pencil, as the spots will fade rapidly. The elution
characteristics should be reported as Rf values. The Rf value is the measure
of the travel of a substance up the place (from the pencil line) divided
by the distance traveled by the solvent from (from the pencil line). When
measuring the distance of the compound spot it is important to measure
the estimated center of the spot and not its tip.
All contents copyrighted (c) 1998
Peter Jeschofnig, Ph.D., Professor of Science, Colorado Mountain College
All Rights reserved
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