Oct 18, 2019
Version 2
Chlorophyll Extraction and Spectral Analysis with Spectrometer Calibration V.2
- 1Independent Researcher
Protocol Citation: Victor vmr Rodriguez 2019. Chlorophyll Extraction and Spectral Analysis with Spectrometer Calibration. protocols.io https://dx.doi.org/10.17504/protocols.io.8eahtae
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2019
Last Modified: October 18, 2019
Protocol Integer ID: 28834
Keywords: chlorophyll, plant, extraction, spectroscopy
Abstract
This protocol is designed to be able to extract and analyze the concentration of chlorophyll within a sample of a given plant. The procedures of this protocol require using a spectroscope to determine the approximate level a chlorophyll within a given sample.
Guidelines
For proper extraction and spectroscopy of Chlorophyll concentration you must be able to measure and transfer liquids within a hundred micro-liters ensure that samples are separated and free of contaminants.
Materials
MATERIALS
Magnesium Sulfate Heptahydrate, ACS GradeGold BiotechnologyCatalog #M-020
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
STEP MATERIALS
Magnesium sulfate, heptahydrate, ACSBio Basic Inc.Catalog #MB0329.SIZE.2.5Kg
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
Magnesium sulfate, heptahydrate, ACSBio Basic Inc.Catalog #MB0329.SIZE.2.5Kg
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
AcetoneBio Basic Inc.Catalog #AC1200.SIZE.1L
Protocol materials
Magnesium sulfate, heptahydrate, ACSBio Basic Inc.Catalog #MB0329.SIZE.2.5Kg
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
AcetoneBio Basic Inc.Catalog #AC1200.SIZE.1L
Magnesium Sulfate Heptahydrate, ACS GradeGold BiotechnologyCatalog #M-020
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
Magnesium sulfate, heptahydrate, ACSBio Basic Inc.Catalog #MB0329.SIZE.2.5Kg
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
Magnesium sulfate, heptahydrate, ACSBio Basic Inc.Catalog #MB0329.SIZE.2.5Kg
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
Magnesium sulfate, heptahydrate, ACSBio Basic Inc.Catalog #MB0329.SIZE.2.5Kg
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
AcetoneBio Basic Inc.Catalog #AC1200.SIZE.1L
Safety warnings
This protocol requires the use of flammable solvents, and require the extraction of pigments that may stain clothing. Proper lab coat, eye protection, gloves in ventilation are required to conduct this chlorophyll extraction and concentration protocol. Also, care must be taken to ensure that all materials used are disposed properly, as many of the chemicals may be hazardous to health and environment.
Before start
In order to perform this chlorophyll extraction protocol you will need the following materials and chemicals:
the materials listed are based on one single sample, in must be multiplied based on the number of samples you would like to test
One 20 ml (minimum) test tube
40 ml beaker
Two 200 micro-liters PCR tubes ( although more may be needed based on the accuracy necessary for the procedures performed)
A 100 to 1000 micro-liter adjustable pipette
5 disposable 1000 micro-liter pipette tips (Number varies based on need and mistakes)
1200 g Centrifuge
One 20 micron filtration filter paper
Mortar and Pestle
Transfer Pipettes (as needed for contamination prevention)
50 ml Graduated Cylinder
Test tube stirrers
Approximate protocol time: 1 hours total
As needed Distilled water
15 ml Acetone
0.13g Magnesium sulfate
0.25g of sample
Spectrometer
Extraction of Chlorophyll
Extraction of Chlorophyll
Weigh out 0.25 g of sample subject and add it to a pestle.
1m
Weigh out 0.13 g of
Magnesium sulfate, heptahydrate, ACSBio Basic Inc.Catalog #MB0329.SIZE.2.5Kg
and add it to a pestle.
1m
Add 1.0 mL of
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
to the pestle.
1m
Grind the entire mixture until the sample is consistent and the
Magnesium sulfate, heptahydrate, ACSBio Basic Inc.Catalog #MB0329.SIZE.2.5Kg
is completely dissolved into the sample paste.
5m
Add the paste into a 40 ml beaker and wash out the pestle with 1.0 mL of
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
and empty it into the beaker.
1m
Add 13.0 mL of
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #34850
to the 40 ml beaker and mix the sample thoroughly, ensuring to press and remix the sample several times.
2m
Let the sample beaker stand for 15 minutes.
15m
Filter the sample into a 20 ml test tube using a 20 micron retention filter paper.
1m
Let the sample stand for a further 10 minutes.
10m
Sample Analyzation
Sample Analyzation
Add 200 µL of the sample to two PCR tubes and label them according to your needed labeling system. the two tubes will serve as double tests for verification.
1m
Centrifuge the two samples to 1200 g to settle out any particulates for 1 minute.
1m
From each PCR tube, transfer 100 µL of the sample from the top to another PCR Tube.
2m
Add 100 µL of
AcetoneBio Basic Inc.Catalog #AC1200.SIZE.1L
to a PCR tube and label it "base"
Use the "base" PCR tube to calibrate the spectrometer.
Place each tube in the spectrometer.
1m
Take to log base 10 of the absorbance percentage of the wavelength 647 nm and 664.5 nm and denote them as A647 and A664.
5m
To calculate the concentration...
CHLA=20.47A647 - 4.73A664
CHLB= 12.63A664 - 2.52A647
CHLTotal= CHLA + CHLB
Divide each concentration by 1000, multiply by the acetone used, then divide by the sample mass to get the mg/g of sample
CITATION
5m
Confirm readings match to within an acceptable margin of error.
5m
Citations
Step 17
Inskeep, William P., and Paul R. Bloom.. Extinction Coefficients of Chlorophyll a and b in N,N-Dimethylformamide and 80% Acetone.
doi:10.1104/pp.77.2.483