TITLE: Calibrating 10-AU fluorometer to quantify extracted chl-a
AUTHORS: Shelby Whitehead, Samantha Coy, Steven Wilhelm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method is for use for calibrating the 10-AU flurometer using chl-a extracts. The method also provides guidelines for extracting chl-a and calculating absorbance using a spectrophotometer, making dilutions of extracts that can be used to create differetn chl-a concentrations</div></div>

[STEPS]
?. [Chlorophyll Extraction]
Extract chlorophyll from   of gound spinach with   of 90% acetone at  overnight.
1 g
5 ml
-20 °C
?. [Spectrophotometry]
Use the Biomate 5 spectrophotometer to measure the adsorption of extracted chlorophyll by adding   to a quartz cuvette. Make sure the cuvette is in the correct position in the machine.Zero the machine by blanking with   of 90% acetone. Measure "multi λ" with  λ1 = 664 nm  and  λ2 = 647 nm.  (Jeffrey 1975)Zero the machine by blanking with  of 90% acetone.
1 ml
1 ml
?. [Fluorometry]
Calculate the concentration of diluted chlorophyll extract using the absorbance values from the spectrophotometer and the equation:  concentration of chlorophyll a = 11.93(λ1) - 1.93(λ2)(Jeffrey 1975)
If using an organism that has more pigments, will have to subtract those from total absorbance (i.e. chlorophyll a - chlorophyll c). The spinach we used did not require subtraction of chlorophyll c.Example:   11.93(0.736) - 1.93(0.479) = 7.85601 ug/ml
?. [Calibrate Fluorometer]
Convert answer from  to ug/L, because this is the readout for the fluorometer.
?. [Calibrate Fluorometer]
Turn on the 10-AU Fluorometer and allow the instrument to warm up for at least 30 minutes.
?. [Spectrophotometry]
Based on the absorbance value from  :  dilute the chlorophyll extract so that the absorbance is in optimum range for the fluorometer. Optimum rangle is Abs λ1 and λ2= 0.3-0.85. Example:Undiluted Abs λ1= 1.88  Abs λ2= 1.32420-fold dilution: Abs λ1= 0.736 Abs λ2= 0.479
?. [Calibrate Fluorometer]
Create solutions of varying concentrations to test the fluorometer readings. Use C1V1=C2V2 to make solutions at different concentrations based on expected field concentrations, for example: 20, 10, 5, 1 and 0.5 ug/L. Also, create an acetone blank (with no chlorophyll).
For example:7,856 ug/L (C1)For a 5 ug/L concentration (C2) in 7 mls acetone (V2)(7,856 ug/L)(V1)=(5 ug/L)(7 ml)V1= 4.45519 ug
?. [Calibrate Fluorometer]
Use the concentration most expected in the field to set as the solid standard.
?. [Calibrate Fluorometer]
Clear past calibrations on screen #2.6.
?. [Calibrate Fluorometer]
Access screen #2.43 and set range control to "manual" and range to the appropriate level for the standard chosen. For example, we set range control to "Man" and "Med."
?. [Calibrate Fluorometer]
Access screen #3.2. Loosen the sensitivity locking screw using an allen wrench. (The sensitivity lock is the small screw to the left of the number pad.)
?. [Calibrate Fluorometer]
Insert and cover your selected standard concentration. Use a coin or tool to slowly turn the sensitivity knob (located to the right of the number pad, under the power button) to adjust the %FS to the value appropriate for the standard concentration according to Table 1 in . A deviation of 5% less than or greater than the table is acceptable. When %FS reaches the designated value, tighten the sensitivity lock.At this point, sensitivity is set.
Example:For 5 ug/L.       FS = 21%
?. [Calibrate Fluorometer]
Access screen #2.43 again, and set range control to "auto."
?. [Calibrate Fluorometer]
Access screen #2.1. Insert and cover the acetone blank created in  . Press "1" to Run Blank. Wait for the reading to stabilize for about 8 seconds, and then press "0."  The instrument will take 15 seconds to complete blanking. At this point, blank is set.
?. [Calibrate Fluorometer]
Calibrate with your standard:
?. [Calibrate Fluorometer]
Access screen #2.2 and input the actual concentration of your standard. Return to screen #2.0
?. [Calibrate Fluorometer]
Insert and cover the standard.
?. [Calibrate Fluorometer]
Run standard by pressing "3." Wait about 8 seconds for reading to stabilize.
?. [Calibrate Fluorometer]
Press the "*" to set the calibration point. After about 15 seconds, a message with appear that says "Finished".
?. [Calibrate Fluorometer]
Test the 10-AU fluorometer using the concetrations created in   to assure appropriate readout.
Example: Expected       Observed5 ug/L            5.01 ug/L1 ug/L            1.11 ug/LBlank              0.0 ug/L