BIOL 1406

PreLab 7.1

What is chromatography?

Cells are composed of thousands of different molecules. To study these molecules, scientists must be able to isolate them in pure form. To isolate just one type of biomolecule from the thousands of others present in a cell can be a daunting task, like looking for a needle in haystack. Fortunately, powerful separation techniques developed over the past century make it possible to isolate virtually every type of molecule known to occur in living cells. Two of the most powerful techniques for purifying biomolecules are chromatography and electrophoresis. In this lab you will use chromatography to separate a mixture of lipid-soluble pigments extracted from spinach leaf cells. In labs 8 and 9 you will use electrophoresis to separate mixtures of proteins and nucleic acids.

Use the interactive exercise below to learn more about chromatography.

The pigments present in spinach leaf cells are used to absorb light energy needed for photosynthesis. The color of each pigment depends on which wavelengths of visible light it reflects and which wavelengths it absorbs. Green pigments, for example, reflect most of the wavelengths in the green region while they strongly absorb light in the red and blue regions. A scanning spectrophotometer is an instrument that can automatically measure how much light at each wavelength is absorbed by a given pigment, over an entire range of wavelengths. This is referred to as the pigment’s absorption spectrum. Because each pigment’s absorption spectrum is unique, like a fingerprint, it can be used to identify the pigment.

During lab, you will use TLC to separate a mixture of pigments that have been extracted from spinach leaf cells. After you have separated the pigments, you will use a scanning spectrophotometer to plot the absorption spectra for 3 of the pigments and then use this information to identify the 3 pigments.

Your Turn
In cell and molecular biology, what are electrophoresis and chromatography used for? Check your answer.
During chromatography, a moving substance called flows past a stationary substance called . Check your answer.
In this experiment, what does TLC stand for? Check your answer.
In this experiment, what will TLC be used for? Check your answer.
What is an absorption spectrum? Check your answer.
How will you identify the different pigments that will be isolated during your experiment? Check your answer.


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