CH 227
Chromatography(Adapted fromLaboratory Manual to AccompanyOrganic Chemistry: A Short Course, H. Hart, L. E. Craine, D. J. Hart, and T.K. Vinod 13th ed. Houghton-Mifflin, Boston, 2012.)
General Principles
Chromatography is a technique used to separate the components of a mixture and to purify substances. It was first developed in connection with the separation of colored compounds (hence the name, from the Greek chroma, color), but chromatography is now used to separate colorless substances as well.
There are two kinds of chromatographic separations. The first is called adsorption chromatography. This type depends on selective adsorption or adhering of the substance being purified on some highly porous surface known as an adsorbent. Different types of intermolecular interactions including H-bonding, dipole-dipole interactions, and Lewis acid-base interactions cause organic molecules to bind to the porous surface of the adsorbent. Adsorption chromatography may be carried out in vertical columns packed with an adsorbent (column chromatography). Alternatively, the adsorbent may be spread out in a thin layer on an inert surface as illustrated in this experiment (thin-layer chromatography).
The second general type of chromatographic separation is called partition chromatography. Here, separation depends on partitioning of the mixture’s components between two solvents. One of these solvents (called the stationary phase) is adsorbed on a solid support. The other solvent (called the mobile phase) passes through this support.
One kind of partition chromatography, illustrated in this experiment, is called gas-liquid chromatography or vapor-phase chromatography. In this technique, vapors of the mixture being purified (the mobile phase) are passed through a heated tube that contains a finely divided solid support on which a nonvolatile liquid (the stationary phase) is adsorbed. Separation depends on the different affinities of the mixture’s components for the nonvolatile liquid phase.
Thin-Layer Chromatography (TLC)
References:
Organic Laboratory Survival Manual, p. 223
SuperOrganicLab:
Then: click on “Lab Techniques,” then “Purification Procedures,” then double click on “Chromatography” and “Thin-Layer Chromatography”
MIT TLC Video:
Analysis of Analgesics (Pain Killers) by Thin Layer Chromatography
Your task is to identify the ingredients in a common pain-killer (analgesic).You will be given known samples of these compounds and you will need to compare them with the commercial samples, of which you will not know the composition. You will analyze the six known samples with the other members of your group.
Unfortunately, commercial pain-killers contain mixtures of compounds so you will not be able to use melting point as a comparison technique. Instead you will use thin layer chromatography, a method that allows you to visualize the number of ingredients in a mixture, and by comparison with known samples, to identify those materials.
Common ingredients in pain killers are:
Use a ruler with mm graduations to measure your plates.
Some things for you to consider during this experiment:
Use your 250-mL beaker as the developing chamber.
Place a piece of 9 cm filter paper, with one edge slightly folded down, in the developing chamber.
Use 10 mL of solvent in the developing chamber, poured over the filter paper in the beaker. Cover with a watch glass. Allow to equilibrate for 5 minutes before running a plate. A smaller version of the developing chamber you will use is pictured below (Figure 1).
The micropipets and TLC plates are on the side shelf.
To simplify your lives, we will have three solvent systems made up: 99% ethyl acetate/1% toluene; 90% ethyl acetate/10% toluene; and 97% dichloromethane/3% methanol. They will be in bottles in the hood. Experiment and see which system works better.
Figure 1. TLC Developing Chamber
with TLC Plate and Solvent Running Up
The plates are 1 inch x 3 inches. This is a perfect size for your notebooks. The grid on the pages is 4 lines to an inch. This makes it easy for you to draw pictures of your TLC plates without having to trace the plates. Just use your ruler as a straightedge.
For making the analyses solutions, weigh 10 mg (0.01 g) of compound and dissolve it, in a test tube, in 2 mL of dichloromethane (DCM). If it does not dissolve, add 2 mL of ethanol.
Draw a straight line ~1 cm from the bottom on the plate with a pencil. do not use a pen. Draw a tick mark across the line where you will spot each sample. It is important that the spots are not too close to the outside of the plate and not too close together. Three evenly spaced spots will give you optimal spacing. Use a 15 μL disposable applicator pipet to spot thedichloromethane solution of your sample to the thin-layer plate about 1 cm from the bottom and try to keep the spots at 1-2 mm in diameter.After the spots have dried, but before you place the plate into the developing chamber, look at the spots under the UV lamp light. If the spot is too light, you need to spot additional times. If the spot is too dark, you will not get good separation. Throw that plate away (regular trash can) and dilute your solution before spotting again. If you are not sure, ask your TA for guidance.
Place the plate in the developing chamber and cover with a watch glass.Do not let your TLC plate touch the filter paper! Allow the solvent to run to 1 cm from the top.
Visualize your developed plates with the UV lamp on the side shelf. Ask your TA to demonstrate. Mark each spot by drawing a circle around it. For example, see the figure below.
Each group has to do a TLC of all six ingredients. 3 Compounds per plate is optimal. DO NOT WASTE THE PLATES. We have to cut them up in the stockroom. It is labor intensive and time consuming. Also, use only one applicator pipet for each compound.
Each student must run a commercial pain killer on his/her own. You will need to co-spot some of the pure compounds on the same plate to compare Rf values. Report which analgesic compounds are contained in your commercial pain killer in your notebook and on the post-lab sheet.
Determine and record the Rf value of each component. (The distance traveled by each spot is measured from the origin to the center of the spot.
Rf = distance compound has traveled from origin
distance developing solvent has traveled from origin
Since we are using DCM this week, we will have special bottles in the hood labeled "Halogenated Organic Waste." We will always use these bottles when disposing of halogenated organics.
Waste Disposal
Discard the developing solvents containing DCM into a halogenated organic liquids waste bottle. The ethyl acetate/toluene developing solvents should go into the nonhalogenated organic liquids waste bottle.
Gas-Liquid Chromatography (GLC)
Gas-liquid chromatography (GLC)* is an accurate and rapid process for separating and analyzing the components of a volatile mixture. The apparatus required for this type of chromatography consists of the following essential parts:
1.Injection block. A heated chamber for introducing and vaporizing the sample.
2.Packed column. Usually a length of metal tubing packed with a porous solid that is thinly coated with a high-boiling liquid. The tube is located in an oven that can be heated at a controlled temperature. Most modern gas-liquid chromatographs use capillary columns (0.5 mm – 1.0 mm in diameter) that are considerably longer instead of the packed columns.
3.Carrier gas. The gas (usually helium or nitrogen) that carries the sample through the heated column.
4.Detector. A device used to detect each component of the sample as it appears at the exit of the packed column. The intensity or area of the signal indicates the quantity of the eluted component.
*Gas-liquid chromatography is also called vapor-phase chromatography (VPC).
The instrument works as follows: The injection block, column oven, and detector are heated to the desired temperature, and the inert carrier gas is passed through the apparatus. Actually, the carrier-gas stream is split. Part of it passes through the entire apparatus, and part of it goes directly to the detector. One common type of detector, called a thermal conductivity detector, consists of two heated wire filaments, one exposed only to the reference carrier-gas stream and the other exposed to the effluent gas from the column. This is the detector used in the chromatographs we will use.
First, a small sample of the material to be analyzed is injected. The sample vaporizes in the injection block and is carried through the column, through the detector, and eventually out of the apparatus. As the sample is eluted from the column and passes through the detector, one of the detector wires is exposed to a mixture of carrier gas plus sample, and the other wire is exposed only to the reference stream of carrier gas. The difference between the two gas streams causes a difference in the electrical conductivity of the wires, and this difference is automatically recorded. In this way a gas-liquid chromatogram is obtained.
procedure
Separation of a Two-Component Mixture
Inject a small sample, approximately 1 μL of the second, fifth, and last fractions from the fractional distillation of the hexane-toluene mixture (Experiment 3). Assume that the area under each curve is approximately proportional to the amount of material present, and calculate the ratio of the two substances in each fraction.Record the conditions used for your chromatographic separationin your notebook. The ratios as well as the GC chromatography conditions are found in the print out you will get from your chromatograph.
Paper Chromatography
General Principles
Paper chromatography is a form of partition chromatography that is especially appropriate for separating ultra-small quantities of materials. The cellulose of the filter paper contains adsorbed water, which is the stationary phase. The sample is applied as a spot to the filter paper, and the second solvent is allowed to pass through the filter paper by capillary action. As it does so, the components of the sample move through the paper at different rates. These rates are proportional to the partition coefficients between the adsorbed water and the particular solvent used for elution.
In this experiment, paper chromatography will be used to separate some synthetic food dyes into their components. The apparatus is a Petri dish, covered, containing solvent and containing a sheet of filter paper punctured at the center, and a rolled filter-paper wick is placed through the hole.
Chromatograms of Yellow and Blue Food Dyes
Partially fill the large halves of two 100 x 15 mm Petri dishes with n-propyl alcohol and water 2:1 by volume (about 30 mL of solvent per dish). Using capillary tubes open at both ends as pipets, place a drop of yellow food coloring at the center of one sheet of paper and a drop of blue food coloring at the center of a second sheet. The dye spot should be about the size of a dime (about 1.5 cm wide). When the spots are dry, insert the wicks so that they extend about 2 mm on one side and 8 mm on the other. Rest the sheets of filter paper on the rims of the Petri dishes with the longer part of each wick extending into the solvent. Cover the papers with the smaller halves of the Petri dishes, and allow the chromatograms to develop for 10 min.
Carefully remove the wicks and allow the chromatograms to dry. You should note the following observations in your lab notebook.
- What distances did the dyes move?
- Did the dyes move equal distances?
- Did the yellow dye appear to be homogeneous?
- Did the blue dye appear to be homogeneous?
- If either was nonhomogeneous (a mixture), how many separate dyes were present and what were their colors?
- Which dye moved the farthest?
Testing a Green Dye
We will now use this technique to determine whether a commercial green food color is (1) homogeneous, (2) made by mixing the yellow and blue dyes studied above, or (3) compounded in some other way.
On one sheet of filter paper, place a spot of green food dye. On another sheet, place a spot of green dye made by mixing equal amounts of the yellow and the blue dyes used above. (Mix these dyes well on a watch glass before applying the spot.) As before, insert the wicks and develop the chromatograms for 10 min.
Answer the following questions in your lab notebook.
- Is the green food coloring a single dye?
- Is it made by mixing yellow and blue dyes?
- Are the dyes the same as the yellow and blue food colorings?
Waste Disposal
The aqueous food dye solutions may be washed down the sink.
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