Chromatographic separation of spinach pigments.
Parts of this experiment adapted from Experience the Extraordinary Chemistry of Ordinary Things, B.C. Richardson and T.G. Casteen, Third edition, John Wiley & Sons, Inc., 1998.
IMPORTANT: BEFORE COMING TO LAB, set up an InterChemNet Account. Follow these instructions:
Your InterChemNet account is now ready for in-class data collection. At any time, you can go to the bookmarked page (www.interchemnet.com) to view and process data.
View this animation of thin-layer chromatography. Keep pressing "Play" until the topic changes to gas chromatography. Then close the animation window to return to this page.
Chromatography is a method of separating mixtures of substances into their pure components. There are many forms of chromatography, but in all forms, a mixture of substances is separated into pure components by passing the mixture over an insoluble material to which the substances stick to varying degrees. Substances that stick tightly to the insoluble material move very slowly, while those that stick loosely or do not stick at all move rapidly. The insoluble material over which the mixture passes is called the stationary phase (in the animation, the stationary phase is the surface of the TLC plate). The solvent (liquid or gas) carrying the solutes over the stationary phase is called the mobile phase (in the animation, the contents of the solvent bath).
Chromatography can be an analytical method, in which the investigator learns the number and nature of the components in a very small amount of a mixture, but does not actually isolate them. Or it can be a preparative method, in which the investigator uses a large quantity of the mixture to obtain useable amounts of each component.
A common analytical method is silica-gel thin-layer chromatography (TLC), in which the stationary phase is a thin coating of the very polar silica gel (oxides of silicon) on a glass or plastic plate, and the mobile phase is a solvent (or mixture of solvents) that is less polar than silica gel. Typical solvents are hydrocarbons like hexane (C6H14, very nonpolar), acetone (CH3COCH3, moderately polar), and ethanol (C2H5OH, very polar).
An common preparative method involving the same phases is silica-gel column chromatography (see figure above). A fine powder of silica gel in a tube (called a column) serves as the stationary phase, and mixtures of substances in appropriate solvents are passed through the column, more or less like filtration. The less-polar components of the mixture emerge from the bottom of the column first, followed by the more-polar components.
In the animation, recall in particular that, when the TLC plate is placed in the solvent bath, the solvent does not immerse the spot of the sample mixture. The solvent is below the spot, but then moves up across the spot, carrying the components of the sample mixture up the plate at different rates. If the spot is immersed in the solvent at the beginning, it will simply dissolve in the solvent and not rise up the plate.
Absorption spectroscopy is the measurement of absorption of electromagnetic radiation by a sample, which may be a pure substance or a mixtures. Absorption of electromagnetic radiation provides many kinds of information about the molecules of the sample. Infrared spectroscopy gives information about bond vibration in molecules. UV/visible spectroscopy reveals electronic energy levels in molecules. Microwave spectroscopy reveals motion and rotation of molecules. The result of this kind of measurement is an absorption spectrum, a plot of energy absorbed as a function of wavelength. Such a plot shows how much radiation is absorbed by the sample at each wavelength of radiation, as in this example:
This spectrum reveals that the sample absorbs UV radiation strongly at wavelengths below 240 nm, and absorbs visible light near 410 nm. A sample transmits light of the wavelengths that it does not absorb. It should not surprise you that this sample appears orange in color. The sample absorbs light at the blue end of the visible spectrum (roughly 400-700 nm), allowing light of higher wavelengths (yellows, oranges, reds) to pass through and reach the eye of the observer.
The following problems require calculations similar to those called for in the report on this experiment. Learn how to work these problems, showing your calculations with units. Similar questions may appear on your prelaboratory quiz. For guidance, read the remainder of this handout, and look at the Assignment/Report Form for this experiment.
Download and print the Procedure for this experiment. Study it carefully after reading this page.
Download and print the Report Form for this experiment. Bring it with you to lab. You will write data and observations on the form during lab, as you carry out the Procedure. After lab, you will complete the Report Form by carrying out calculations to give your final lab results. Completing your report will involve visiting the InterChemNet website make and print visible spectra of your pigments. If you need to use USM computers for this purpose, be sure to allow time to visit the USM computer lab for this work.
If it is not convenient for you to visit this page online, you might want to print it out as well.