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Use of the Spectrophotometer
E x e r c i s e 3A
Use of the Spectrophotometer Introduction The spectrophotometer is utilized by molecular biologists for accurate preparation and analysis of many types of samples. This exercise is designed to familiarize students with this instrument. Spectrophotometers have varied applications in the qualitative analysis of sample purity, DNA and protein quantitation, cell density measurements, and assays involving enzyme-catalyzed reactions. Spectrophotometry is based on the simple premise that various compounds will differentially absorb specific wavelengths of light in either the ultraviolet (UV, 200-400 nm), visible (VIS, 400-700 nm), or near-infrared (near-IR, 700-900 nm) range. Photometric assays may directly measure sample absorbance at a given wavelength or indirectly measure an enzymatic reaction product or related binding substance that absorbs light in amounts directly proportional to the absorbance of the target compound (such as assays for protein concentration). Nevertheless, all spectrophotometers employ the same basic structural components designed to detect these variations between absorption wavelengths and densities. The general components common to most spectrophotometer systems include a light source, wavelength selector, fixed or adjustable slit, cuvette, photocell, and analog or digital readout (Figure A-1.3A). The light source (specific for either UV or visible ranges) will emit light that is passed through the wavelength selector, usually a prism, diffraction grating, or set of screening filters, where a specific wavelength of "monochromatic" light is selectively generated (defined by its maximum emission at this wavelength). This light is then directed toward a thin slit (usually adjustable) to regulate its relative intensity before it passes through a cuvette containing the sample of interest. Cuvettes differ with respect to their absorbance characteristics and 174
Alternative Protocols and Experiments
175
SLIT LIGHT SOURCE
PHOTOCELL READOUT I LENGTH / LSELECTORJ
0.24
CUVETTE
Figure A-1.3A. Diagram of components of spectrophotometers.
one must be careful and consistent when making measurements. Finally, absorbance is detected by a photocell in which electrons in the refracted light generate an electrical current that can be amplified and measured to yield an absorbance value, or optical density (OD), for the given sample. The OD reading is corrected against a blank, which contains all of the reagents in the experimental sample except the test compound, to obtain a true measure of the optical density of the sample. The data obtained from spectrophotometric analyses are important for determining experimental parameters and analyzing results, especially in conjunction with data from other analytical techniques.
Reagents/Supplies Bromphenol blue (1.25%, w/v) Cuvettes (alternatively, colorimeter tubes if needed) Micropipettors and tips Pipets, 10 ml Test tubes, 18 x 150 mm
Equipment Spectrophotometer (if not available, colorimeter can be used) Vortex
176
Appendix 1
Instructor's Note It will be important to demonstrate to the students the proper use of instrumentation used in your laboratory for this class. This exercise can be done using a variety of instruments to measure optidal density. You may wish to have the students compare values obtained from different instruments.
Procedure 1. Warm up (about 20 minutes) the spectrophotometer set at 540 nm or a Klett colorimeter (green filter, wavelength range of 500 to 570 nm) before use. The Klett reading can be converted to optical density (OD) by multiplying the Klett reading by 0.002. 2. Place 10 ml of distilled water in each of eight test tubes. 3. Use micropipettors to add to each successive tube the following amounts of bromphenol blue (1.25%, w/v): 0.5, 1, 2, 4, 10, 20, 50, and 100/~1. Manufacturer instructions for use of micropipettors should be followed scrupulously (see Appendix 5, Use of Micropipettors). 4. Vortex each tube until the dye is in solution. 5. Set the spectrophotometer/colorimeter to zero with distilled water. 6. Transfer the above dye solutions from least concentrated to most concentrated into the same cuvette or Klett tube from reading to reading. 7. Record the readings and graph the results.
References Brown, S. B. (1980). "An Introduction to Spectroscopy for Biochemists." Academic Press, New York.
Alternative Protocols and Experiments
177
Harris, D. A., and Bashford, C. L. (1987). "Spectrophotometry and Spectrofluorimetry: A Practical Approach." IRL Press, Washington, D.C.
Questions 1. What may account for the difference in OD values obtained using a Klett colorimeter with a green filter as compared to a spectrophotometer set at 540 nm? 2. Consistency of micropipettor usage depends on strict attention to what operational procedures? 3. Explain the relationship among absorbance value, optical density, and percent transmittance.
Use of the Spectrophotometer Introduction The spectrophotometer is utilized by molecular biologists for accurate preparation and analysis of many types of samples. This exercise is designed to familiarize students with this instrument. Spectrophotometers have varied applications in the qualitative analysis of sample purity, DNA and protein quantitation, cell density measurements, and assays involving enzyme-catalyzed reactions. Spectrophotometry is based on the simple premise that various compounds will differentially absorb specific wavelengths of light in either the ultraviolet (UV, 200-400 nm), visible (VIS, 400-700 nm), or near-infrared (near-IR, 700-900 nm) range. Photometric assays may directly measure sample absorbance at a given wavelength or indirectly measure an enzymatic reaction product or related binding substance that absorbs light in amounts directly proportional to the absorbance of the target compound (such as assays for protein concentration). Nevertheless, all spectrophotometers employ the same basic structural components designed to detect these variations between absorption wavelengths and densities. The general components common to most spectrophotometer systems include a light source, wavelength selector, fixed or adjustable slit, cuvette, photocell, and analog or digital readout (Figure A-1.3A). The light source (specific for either UV or visible ranges) will emit light that is passed through the wavelength selector, usually a prism, diffraction grating, or set of screening filters, where a specific wavelength of "monochromatic" light is selectively generated (defined by its maximum emission at this wavelength). This light is then directed toward a thin slit (usually adjustable) to regulate its relative intensity before it passes through a cuvette containing the sample of interest. Cuvettes differ with respect to their absorbance characteristics and 174
Alternative Protocols and Experiments
175
SLIT LIGHT SOURCE
PHOTOCELL READOUT I LENGTH / LSELECTORJ
0.24
CUVETTE
Figure A-1.3A. Diagram of components of spectrophotometers.
one must be careful and consistent when making measurements. Finally, absorbance is detected by a photocell in which electrons in the refracted light generate an electrical current that can be amplified and measured to yield an absorbance value, or optical density (OD), for the given sample. The OD reading is corrected against a blank, which contains all of the reagents in the experimental sample except the test compound, to obtain a true measure of the optical density of the sample. The data obtained from spectrophotometric analyses are important for determining experimental parameters and analyzing results, especially in conjunction with data from other analytical techniques.
Reagents/Supplies Bromphenol blue (1.25%, w/v) Cuvettes (alternatively, colorimeter tubes if needed) Micropipettors and tips Pipets, 10 ml Test tubes, 18 x 150 mm
Equipment Spectrophotometer (if not available, colorimeter can be used) Vortex
176
Appendix 1
Instructor's Note It will be important to demonstrate to the students the proper use of instrumentation used in your laboratory for this class. This exercise can be done using a variety of instruments to measure optidal density. You may wish to have the students compare values obtained from different instruments.
Procedure 1. Warm up (about 20 minutes) the spectrophotometer set at 540 nm or a Klett colorimeter (green filter, wavelength range of 500 to 570 nm) before use. The Klett reading can be converted to optical density (OD) by multiplying the Klett reading by 0.002. 2. Place 10 ml of distilled water in each of eight test tubes. 3. Use micropipettors to add to each successive tube the following amounts of bromphenol blue (1.25%, w/v): 0.5, 1, 2, 4, 10, 20, 50, and 100/~1. Manufacturer instructions for use of micropipettors should be followed scrupulously (see Appendix 5, Use of Micropipettors). 4. Vortex each tube until the dye is in solution. 5. Set the spectrophotometer/colorimeter to zero with distilled water. 6. Transfer the above dye solutions from least concentrated to most concentrated into the same cuvette or Klett tube from reading to reading. 7. Record the readings and graph the results.
References Brown, S. B. (1980). "An Introduction to Spectroscopy for Biochemists." Academic Press, New York.
Alternative Protocols and Experiments
177
Harris, D. A., and Bashford, C. L. (1987). "Spectrophotometry and Spectrofluorimetry: A Practical Approach." IRL Press, Washington, D.C.
Questions 1. What may account for the difference in OD values obtained using a Klett colorimeter with a green filter as compared to a spectrophotometer set at 540 nm? 2. Consistency of micropipettor usage depends on strict attention to what operational procedures? 3. Explain the relationship among absorbance value, optical density, and percent transmittance.