How to Use Spectrophotometry to Analyze Chlorophyll Concentrations Extract Filtered Chlorophyll Samples Wear gloves any time you work with methanol. It is toxic and can absorb through the skin. 1. Remove your filters from the freezer. Label a test tube with your name, section number, sampling period (e.g. Pre-salt or Post-salt), and the [Cl]. 2. Using tweezers, unfold the filter. 3. Cut the filter into small pieces using scissors and put all the pieces into your labeled test tube. Use a clean glass stir rod to push the filter down toward the bottom of the tube. 4. Add 6 mL methanol to the test tube. 5. Mash the filter gently with the glass stir rod ~5-10 times. 6. Cover the test tube with parafilm, making sure you get a good seal. 7. Gently shake the test tube until all filter bits are submerged, keeping your gloved thumb over top the parafilm to prevent any leaks. 8. Place your labeled test tube into your lab section's test tube rack in the fridge. These samples will be left at (4?) to extract for the next 48 hours.
Use a Spectrophotometer to Measure Absorbance Wear gloves any time you work with methanol. It is toxic and can absorb through the skin. 1 2 A Blank B C D E ... Mesocosm Mesocosm A Mesocosm B A Mesocosm B 3 4 ... Schematic of 96-well plate. Orient with A1 in upper left corner It does not matter whether the pre-salt or post-salt samples goes into column 1 or 2 1. Remove your test tubes from the fridge. 2. Carefully unwrap the parafilm from the top. Avoid shaking the sample - this could release fibers from the filter paper into the solution which could interfere with the absorbance reading. 3. Use the micro-pipette to transfer 100 uL of the solution into a well in the 96- well plate provided. Make sure you record which sample ([Cl], and pre- or post- salt) you put in each well (see diagram above). Don't forget to change tips between samples. 4. Add 100 uL of pure methanol to well A1. This will act as the blank. 5. Carefully place the plate onto the spectrophotometer tray. NEVER PUSH THE TRAY CLOSED. Always use the button! 6. The protocol "BIOL212 Module 4 - Chl a and b" has been programed to read the absorbance of your samples at 664 nm and 647 nm. These are the wavelengths of highest absorbance for Chl a and b respectively. 7. Run the protocol. 8. The program will produce an Excel file containing the absorbance values. Record the absorbance values (corrected for the blank) at both wavelengths for each of your pre- and post-salt samples in your lab notebook. You will use these values to calculate the concentration of Chl a and b in your mesocosm.
Calculate Chlorophyll Concentrations Use your measured absorbance values to calculate the chlorophyll concentration of your mesocosm water. To calculate the chlorophyll concentration (ug/L) of your methanol extraction, use the