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B i o p h y s i c s D e mys tifie D
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molecules Molecules with more charge will experience a stronger force generated by the electric field
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Size Exclusion Chromatography
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Size exclusion chromatography (SEC) is another technique that relies on sedimentation SEC, however, uses gravity, or sometimes pressure, to sediment a solution through a gel The gels used in SEC, however, differ from those used in electrophoresis in the following way SEC gels are not one solid piece of gel, but rather a tightly packed suspension of gel beads or particles with spaces between them There are pores on the surface of the gel particles through which molecules pass to get into the gel matrix on the inside each particle or bead These pores, however, are very small, excluding larger molecules Smaller molecules enter these pores, but when they do it can take them some time to pass through or otherwise exit the gel bead This slows down the smaller molecules Larger molecules bump into the gel particles (as we saw with electrophoresis) but move around the spaces between the gel beads much faster than the smaller molecules that are temporarily trapped inside the gel beads The end result is similar to electrophoresis in that molecules are separated based on their size However, in SEC it is the larger molecules that pass through the gel faster and the smaller molecules that lag behind Using gels with different size pores can exclude different size molecules from the insides of the gel particles
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Spectroscopy
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As a general class of investigative techniques, spectroscopy typically involves sending some form of electromagnetic radiation into a sample and measuring various properties of the electromagnetic radiation that emerges from the sample For example, one can measure the intensity of the emergent radiation Other common properties include the direction of the emitted radiation and its polarization The measured property is then plotted as a function of the wavelength or frequency of the radiation; the resulting plot is called a spectrum (plural: spectra) Just as a reminder, all electromagnetic radiation can be characterized by wavelength or frequency The two are inversely proportional If we know one, we can calculate the other 5 c/ (3-1)
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chapter 3 B i o p h y s i c a l T e c h n i q u e s a n d a p p l i c aT i o n s
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where is the frequency, c is the speed of light (29979 3 108 m/s), and l is the wavelength So, plotting a spectrum by wavelength or frequency is more or less the same thing It certainly contains the same information The frequency, however, has the advantage that it is proportional to the energy; thus E 5 h where h is Planck s constant (6626068 3 10234 m2kg/s) Originally, the techniques of spectroscopy were developed using only the visible light portion of the electromagnetic spectrum [wavelengths of approximately 380 to 750 nanometer (nm)], and later grew to include ultraviolet, infrared, and a much broader range of wavelengths Over the years, additional techniques have been developed which do not necessarily involve electromagnetic radiation but which nonetheless produce a spectrum of sorts For example, electron spectroscopy measures the kinetic energy of electrons that emerge from the sample Mass spectrometry produces a spectrum as a function of mass By and large, however, most spectroscopic techniques involve electromagnetic radiation Therefore, when necessary in order to distinguish those forms of spectroscopy that use electromagnetic radiation from other uses of the word spectroscopy, we will specifically use the term EM spectroscopy There are dozens of spectroscopic techniques used in the biophysical sciences In the following sections we briefly describe a few of them Each type of spectroscopy teaches us something different about the biological sample, with some overlap in what we can learn from each technique Commonly, spectroscopic techniques provide information about the identity of biological molecules, about their structure, conformational transitions, binding, and kinetics The various spectroscopic techniques are classified according to the type of light (electromagnetic radiation) used and according to the properties of the emergent light measured Additionally, some spectroscopic techniques are further distinguished according to the conditions of the experiment that are controlled For example, if we measure the amount of light absorbed as we control and slowly vary the temperature of a sample, we would call this temperaturescanning absorbance spectroscopy A note on the use of the word light: Throughout this book we will often use the word light rather freely to mean electromagnetic radiation Strictly speaking, the word light is meant to distinguish those parts of the electromagnetic spectrum that are visible to living organisms In practice, light is something we are very familiar with, and use of the word light to mean electromagnetic (3-2)
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