Basic Guide to a Spectrophotmeter
Procedure for measuring the absorption spectrum of betacyanin

Measuring Absorbance of Betacyanin
Each group has an extract of beet betacyanin that was prepared in distilled water (dH2O), two small test tubes (cuvettes) which fit into the spectrophotometer, two graduated bulb pipettes (3-4mL), an empty flask, a squeeze bottle filled with dH2O, and a large 1L beaker for rinsing.  

Step 1.  Turn on the Spectronic 20 to allow it to warm up (10-15 minutes)

Step 2.  In one cuvette, place 5mL of beet betacyanin

Step 3.  In the second cuvette, place an appropriate blanking solution. 

• What is the appropriate blank for this investigation?  

Hint: All compounds have light absorbing characteristics, which may at one or more wavelengths.  Blanking eliminates those characteristic wavelengths from detection. What compounds are involved in this assay?  Which one needs to be eliminated from detection?

Step 4.  Prepare a data collection table with two columns using graph paper.  Label one for wavelength and another for Absorbance.  The range of wavelengths will be from 500nm to 600nm in 10nm increments.  Each student will collect data and graph it (see Lab Assignment below).

Step 5.  Adjust the Spectronic 20 to total Absorbance or zero transmission using the zeroing knob (on/off) on the left.  Turn it until the needle aligns on top of the  ¥ line, just to the left of 2 on the Absorbance scale.

Step 6.  Select the first wavelength.  Wipe the bottom half of the blanking cuvette with a tissue wipe and insert the cuvette with the blanking solution into the sample holder of the Spectronic 20 and adjust to 0 Absorbance (on the right-side of the scale) using the blanking knob. Remove the blanking cuvette. 

• Be sure to use proper blanks and to blank the spectrophotometer at each wavelength for each sample.  Failure to do so will in all likelihood produce inaccurate readings.  

Step 7.  Place the cuvette with betacyanin into the Spectronic 20 after wiping it as in Step 4.  Record the absorbance.  Remove the cuvette.

Step 8.  Select the next wavelength and repeat steps 6 and 7.  Periodically check for total Absorbance/zero transmission (see Step 5)

Lab Assignment
Determining lmax (the wavelength of maximum absorbance
Construct a graph by plotting the absorbance of the solution as a function of wavelength.  

• Would wavelength be on the x or y axis of your graph?  Does it matter?

From your graph determine lmax for betacyanin dissolved in water.  See lmax in introduction to Spectrophotometry