Spectrophotometers and Extinction Coefficients Lecture Notes Absorbance The energy difference between the ground and excited states. Low Energy High Energy 750nm 350nm Red shift Blue shift Hypochromic hypsochromic Extinction Coefficients. Beer?s Law: A=?bc (A=absorbance, b=path length, c=concentration, e=extinction coefficient) To calculate an ?, you must know c and b, then measure A. Jablonski diagram Para-nitrophenol (PNP). PNP is often conjugated to enzymes to monitor their reaction with substrate because when linked to another molecule PNP has no major visible absorbance. When PNP is cleaved from the molecule both the acid and base forms are in equilibrium in solution. The basic form has an absorbance maximum at 405nm and hence can be easily used to monitor reactions (the acid form absorbs in the UV and often overlaps with the absorbance of biomolecules, so it is not used). In order to use PNP in this way, the rate of production of PNP at 405nm is measured versus time. This yields a dA/dt value. However, for the purpose of future calculations, a dC/dt value is more useful. You must know the ? of PNP at 405nm to make this conversion. If A=?bc, then dA/dt = ?b dC/dt Using the Spectrophotometer Page 64 of your manual has step-by-step instructions for using the spectrophotometer. You will use the spectrum, kinetic and photometric mode today. Results are most accurate when your absorbance max is below 1.5 AU. Which cuvette should I use? Deciding which cuvette you need to use to obtain a spectrum is the first step. There are two types of cuvettes that we will use in this class. Plastic cuvettes are suitable for measuring all visible absorbances (anything 350nm-800nm). Plastic cuvettes absorb light in the UV range (anything below about 350nm). For scan requiring this range, a quartz cuvette must be used. When you think you need a quartz cuvette, you must sign them out from your TA, then sign them back in at the end of class. They are very expensive so please be very careful with them. Running a blank. There are many factors that can contribute to your absorbance, solvent, cuvette. You must therefore run a blank (AKA baseline correction or reference) prior to running you sample. To do this, both cuvettes must contain the same thing and be of the same type (but may be of different sizes). Usually it is simply the buffer you are running your reaction in. Data Processing. ?nce you have your data, you need to process it in such a way that makes it useful to you. Usually the two most important pieces of info are??max and absorbance at that wavelength. See page 64 in your manual for more details on finding your peak. For certain scans, I will ask you to generate a curve of your data in excel. To do this you will have to write down the points in your notebook and manually put them in excel. See page 64. Experiment notes. You will have to dilute the PNP. Do only 1 scan of PNP in NaOH and HCl as long as your absorbance max is below 1.5. Dispose of all PNP waste in container in waste hood. When you are finished with lab work, please work on the post-lab outside of the lab.
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