TITLE: Determining Chlorophyll Concentration using CuSO4  Magnesium-Copper Exchange Titration
AUTHORS: Victor Rodriguez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed to be able to extract  and analyze the concentration of chlorophyll within a sample on a molecular level. The procedures of this protocol require using copper II sulfate and hydrochloric acid  as a means of trituration to determine the approximate level a chlorophyll  within a given sample.</div></div>

[STEPS]
?. [Preparation of Sample and Extraction]
Add of sample plant matter or other test article to a mortar.
2 g
?. [Preparation of Sample and Extraction]
Add  of  crystals to the mortar.
1 g
?. [Preparation of Sample and Extraction]
Grind the sample item and  together until the leaves are ground up, the magnesium sulfate is completely dissolved  into the plant liquid and a liquid starts to form.
?. [Preparation of Sample and Extraction]
Transfer as much of the solid and liquid slurry to a 20ml test tube.
?. [Preparation of Sample and Extraction]
Add  to the 20ml test tube. This is the raw mix.
8 ml
?. [Preparation of Sample and Extraction]
Add  of  to the mortar to wash up and collect as much of the sample as possible into the 20ml test tube.
1 ml
?. [Preparation of Sample and Extraction]
Use a glass stirrer to stir the raw mix. Take care to press the plant slurry down to the base of the test tube, then agitate the slurry again to mix. Repeat this many times for  minutes. Ensure that the acetone is fully incorporated into the slurry.
?. [Preparation of Sample and Extraction]
Let the raw mix stand for  minutes.
?. [Preparation of Sample and Extraction]
Use a glass stirrer to stir the raw mix again for  minute.
?. [Preparation of Sample and Extraction]
Using a 20 micron retention filtration paper to filter the raw mix into another test tube. Ensure to use a new filter paper for each separate sample. This is the refined mix.
?. [Dilution Phase  ]
Determine the resolution needed for the concentration.  There resolution is the interval of dilution. For example, a 10% resolution will result in a dilution interval of 100%, 90%, 80% etc. The smaller the resolution the narrower the margin of error is. For this protocol, the resolution used will be 10%.
?. [Dilution Phase  ]
Label ten 5ml vials in descending order from 100% to 10%.
?. [Dilution Phase  ]
Add Acid Activated mix to each 5ml vial at volume based on the formula Vol = 1000ul * Per with Per representing the percentage written on each vial. Use the percentage or decimal, not whole number. Ex. 10% = .10
?. [Copper Transfer Phase]
Prepare a 60mgCuSO4 /mlH2O concentration solution of to be referred to as the copper (II) sulfate Solution.
?. [Dilution Phase  ]
Add to the refined mix  and mix with clean glass stirrer  for  minute to create the Acid Activated Mix.
1 ml
?. [Dilution Phase  ]
Let the Acid Activated Mix stand for   minutes.
?. [Dilution Phase  ]
Add  to each 5ml vial at volume based on the formula Vol = 1000ul * (1-Per) with Per representing the percentage written on each vial. Use the percentage or decimal, not whole number. Ex. 10% = .10
?. [Dilution Phase  ]
Gently stir each vial by rotating them in a circular motion for  seconds each.
?. [Dilution Phase  ]
Using an adjustable pipette, dispense  of Acid Activated Mix from each vial to the corresponding PCR Tube. Ensure to change the pipette tip for every transfer in order to ensure contamination prevention.
100 µl
?. [Dilution Phase  ]
Label The proper amount of PCR tubes corresponding to the percentages listed on the vials.
?. [Copper Transfer Phase]
Add  copper (II) sulfate Solution to each PCR tube.
100 µl
?. [Test Phase]
Close all PCR tubes and Centrifuge all tubes at  for 2 minutes.
Centrifuge: 1200 34
?. [Test Phase]
In a PCR rack, let the samples stand for 6 hours
?. [Test Phase]
Centrifuge all tubes at  for 2 minutes.
Centrifuge: 1200 34
?. [Results Phase]
Observe which PCR is the lowest percentage concentration not to have any trace of precipitated Copper (II) Sulfate. (A blue crystalline precipitate at the bottom. Take note of the percentage number to be used as the variable P-.
?. [Results Phase]
Assign the highest percentage value vial to have visible copper (II) Sulfate precipitate the variable P+.
?. [Results Phase]
Use this formula to calculate the mean value for the concentration in g/ml. (6*10-4 * 893.51)/(P- * 159.609)+(((6*10-4 * 893.51)/(P+ * 159.609) - (6*10-4 * 893.51)/(P- * 159.609))/2)= Con
?. [Results Phase]
Use this formula to calculate the margin of error of the concentration.((6*10-4 * 893.51)/(P+ * 159.609) - (6*10-4 * 893.51)/(P- * 159.609))/2= Moe
?. [Results Phase]
Write the final results as Con ± Moe