Complete this worksheet in conjunction with your lab book Exercise 8 "Spectrophotometry" This means you have to read/do the lab book procedures using this worksheet as a guide. For example, if the lab worksheet says to do a particular procedure in your lab book, go to the lab book to see and execute the directions. This worksheet is where you will record your data, answer questions, etc. There often are parts in the lab book exercise that we don't do so that is why you use the lab worksheet to guide you to what you need to do. The completed worksheet is what you submit (the lab book pages are not checked nor collected by your instructor). AGENDA FOR LAB #4 Spectrophotometry: Remote lab students Parts A & C only, then submit worksheet via Blackboard *****PART A ALL STUDENTS Review lab book Figures 8.1 & 8.2 + Search & View the two Youtube videos listed in order below + Read the passage below. Then answer the posed questions. "How does a spectrophotometer work?" ByBioNetwork (58sec, Nov 2011) https://www.youtube.com/watch?v=pxC6F7bK8CU This video describes the inner workings of the spectrophotometry instrument (as also shown in the figure below). Solution Wavelength Selector Detector White Light Incident Light Transmitted Light The difference between the incident and transmitted light indicates the absorbance [PDF] The Use of the Spectrophotometer and Beer's Law www.apsu.edu/files/...of_the_Spectrophotometer_and_Beer's_Law_F08.pdf A linear relationship exists between absorbance and concentration known as Beer's Law (A = + bc) where is the length of the path traveled by Ight through the sample, c is the concentration and is a molar absorptivity constant that depends on both wavelength and substance. This inear relationship between concentration and absorbance allows scientists to use spectroscopy for quantitative measurements of unknown samples. Using a calibration curve prepared from standard solutions (solutions of known concentration), the concentration of an unknown solution can be interpolated by linear regression "What is a Standard Curve?" By The Penguin Prof (6:52, July 2014) https://www.youtube.com/watch?v=OluczWOOQ This video details how a spectrophotometer is used to create a graph of absorbance readings at known protein concentrations #1) Explain the difference between the transmittance & absorbance readings on a spectrophotometer. #2) For what purpose is a spectrophotometer used in the lab? #3) Would a sample solution that has a high absorbance reading, have a LOW or a HIGH concentration? #4) When a standard curve is constructed, what label/units are on the X axis of the graph? What label/units are on the Y axis of the graph? (Note that Absorbance values are unitless while wavelength units are in nanometers.) #5) What is the purpose of a standard curve? You are to read lab book Procedure 8.1 Absorption Spectrum of Cobalt Chloride" then search & view the Youtube video: "Spectrophotometer demo & practice experiment byBioNetwork (6:26, Nov 2011) https://www.youtube.com/watch?v=xHQM4BbR040 This video shows a lab technician using a spectrophotometer to conduct a procedure similar to that in your lab book procedure 8.1, but instead of using cobalt chloride, they use red food coloring as their sample solution. They also use MilliQ water as their blank (MilliQ is highly purified water). They record absorbance of the sample at a range of wavelength in order to determine the wavelength at which the sample shows maximum absorbance. Watch the whole video then complete Table 8.2 below and answer the questions posed. LAB BOOK PROCEDURE 8.1 DETERMINE THE ABSORPTION SPECTRUM Table 8.2 Absorbance Spectrum: Use graph @ end of video to complete absorbance data points in this table Absorbance Absorbance Wavelength 380 nm 405 nm 430 nm 455 nm Wavelength 480 nm 505 nm 530 nm 555 nm #6)What wavelength is the peak absorbance of the red food coloring sample? #7)Would you expect an absorption spectrum graph of chlorophyll (green plant pigment involved in photosynthesis) to be the same or not of that for the red food coloring as done in this experiment? Why? #8)What's the purpose of an absorption spectrum?