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Unformatted text preview: e’s parfocality. This can result in damage to the objective lens or slide. The microscope could be accidentally bumped against objects (or your lab mates) and damaged by this collision. The whole eyepiece tube could fall off the microscope and be badly damaged. This can result in damage to the mechanical stage assembly. This results in a loss of the friction that holds the stage at a selected focus. In this condition the microscope will not stay focused. Time will be wasted in finding the plane of the focus of the next specimen. This will make finding a desired object much more difficult and time consuming. Less light will be available for viewing the specimen. The usefulness condenser diaphragm for adjusting specimen contrast will be lowered. 13. Attempting to view low contrast specimens with the light too bright and the condenser diaphragm The specimen will probably not be visible. too widely open. It will be difficult if not impossible to gain a three 14. Improper adjustment of the inter-ocular distance. dimensional image of the specimen. 15. Attempting to view specimen with eyes too close The specimen will be difficult if not impossible to to the oculars. see. Biology 05LA – Fall Quarter 2012 Lab 2 – page 1 LAB 2: SPECTROPHOTOMETRY AND QUANTITATIVE DATA ANALYSIS This exercise will introduce the use of a spectrophotometer (or colorimeter) to measure a particular chemical property of many biological (and non-biological) materials. We will also learn to quantify spectrophotometric data, learn several "wet lab" techniques, and learn how to properly prepare scientific graphs. Be advised that you will be using all of these newly acquired skills in the labs of the weeks to come, so come to lab prepared to master these skills – your potential success in these future labs will be greatly enhanced if you do so. Absorption Spectra All solutions that are colored to the human eye absorb electromagnetic radiation in the visible portion of the electromagnetic spectrum (Fig. 1). The color that we perceive represents those wavelengths of light that are not absorbed by the substance, or, conversely, represents those wavelengths that are transmitted. For example, if a solution is green, it absorbs in the blue and red portions of the spectrum. If it is red, we would expect it to absorb in the green and blue portions of the spectrum. Another way of saying this is that a solution transmits light of a color complementary to that which it absorbs (Fig. 1, color wheel). The absorption of certain wavelengths of electromagnetic radiation is as characteristic of a compound as is its molecular weight, solubility properties, melting point or any other intrinsic property. Thus, the absorption spectrum of a compound may be used to identify it. The fact that a compound, solution or substance is not colored does not mean that it does not absorb electromagnetic radiation. It merely means that it does not absorb visible light. However, it may absorb strongly in other regions of the spectrum. Water, for example, is colorless, but it absorbs far infrared light, a band of wavelengths to which the human eye is insensitive. The radiation that is absorbed by water is converted to heat, and the water becomes warm. Many other compounds of biological importance absorb in non-visible regions of the electromagnetic spectrum. Absorbance and transmittance are measured with either a colorimeter or a spectrophotometer. The instruments used in Biology 05LA are technically called colorimeters. A colorimeter is an instrument that selectively projects narrow bands of visible light upon a solution (see Fig. 2). A spectrophotometer does the same thing but also can utilize wavelengths in non-visible regions of the near ultraviolet and infrared. Both colorimeters and spectrophotometers function in the same manner. Light from a source Comparable Wavelength Lengths: (in cm) UCR to Rubidoux 106 Size of blue whale. 104 Common Name McDonald ’s hamburger. 102 Hertzian waves; radio, TV, and microwaves. 100 Size of an amoeba 10-2 Infra red. Red blood cell. 10-4 Visible spectrum. Ultra violet. AIDS virus 10-6 H atom. 10-8 Violet (400nm) Gamma ray. 10-12 10-14 Orange (600nm) Yellow (550nm) Blue Green (450nm) (500nm) X-ray. 10-10 Electron Red (650nm) Cosmic ray. Figure 1: The Electro magnetic Spectrum. The range of wavelengths of the EMS is shown. The visible spectrum represents a narrow band of the EMS. The approximate wavelengths of the colors in the visible spectrum are given in the color wheel. Colors that are complementary fall on opposite sides of the wheel. Biology 05LA – Fall Quarter 2012 Lab 2 – page 2 that emits light at the required wavelength is focused upon a monochromator by a lens-like collimator. The monochromator, either a prism or diffraction grating, separates the light into its component wavelengths. Once the light has been separated, the wavelength required for the analysis is selected from all the available wavelengths. This selection occurs when the operator adjusts a control on the front of the instrument which moves a slit ( selector) that allows onl...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.

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