Chlorophyll analysis - spectrophoptometry

Chlorophyll Analysis

This guide is primarily for the lab researcher who will spectrophotometrically measure the chlorophyll content of laboratory cultures of phytoplankton.   Those wishing to determine chlorophyll in natural seawater samples or in samples containing significant amounts of phaeophytin should consult fluorometric and other techniques. Some variation in this protocol maybe required for species that are particularly resistant to extraction.  This may include overnight extractions in a freezer, boiling methanol extraction, and sonication. 

1. Sample preparation
1. Filter a known volume of culture onto a 25 mm glass fiber filter.

-         Whatman GF/F filters are better suited for small cells;  Gelman A/E  are adequate for cells greater than 4 um is size.

-         Swirl your culture before sampling to ensure homogeneous sampling.

-         Keep vacuum level low (< 10 in Hg) to prevent cell breakage and low filtration efficiency.

2. Rinse funnel walls with small amount of filtered seawater.
3. Remove the funnel.  Fold the filter in quarters and place in a grinding vessel. 

-         Try not to touch the filter with your “oily, acidic” fingers.  Use forceps and the tip of the grinding vessel mouth as extensions of your hands.

-         The grinding vessel is pretty tough; you can tap the vessel bottom on the bench to get the filter to the bottom of the vessel.

4. Place enough 90% acetone in the grinding vessel such that the filter is covered (about 1-2 mls).

-         Keep in mind that you want to keep the total acetone extract volume for the entire analysis to less than 10 ml.

-         For “tough” cells, use instead 80% Methanol instead of acetone and boil the sample for 5 minutes at 75-80 degrees C. 

5. Homogenize the filter using the motorized Teflon pestle.

-         Use Safety glasses!

-         Hold tightly to the vessel, insert the pestle into the vessel and turn on the motor (use REVERSE position and about setting 3 on the dial). Use an up & down motion while keeping the pestle immersed in the acetone.  When filter is suitably macerated, turn off the motor while pulling the pestle from the narrow part of the vessel at the same time.  This keeps the pestle from becoming stuck when it stops. 

-         Rinse the pestle into the vessel with a small amount of 90% acetone.  The acetone volume should now be about ½ to ¾ of the narrow portion of the vessel. 

6. Place the vessel in a dark box and repeat the above steps for other samples.

2. Clarify the sample preparations.

Samples can be clarified using filtration or centrifugation.   The filtration method is best suited for situations in which you have many samples to analyze (see Kevin for help on this method).  Centrifugation is more often used in our lab.   It is more economical (but less time efficient). 

1. Remove the sample from the dark box and carefully pour sample into a 12 ml conical glass centrifuge tube.

-         Gently vortex the sample prior to pouring into the centrifuge tube.  This keeps the ground-up filter from “sticking” to the vessel when you try to pour it out. 

-         To make a vortex, hold the top of the vessel between your thumb and index finger.  Flick the bottom of the vessel with the fingers of you other hand.

2. Rinse the vessel 2 or 3 times with small amounts of 90% acetone.  Pour the rinses into the centrifuge tube.  Once again, try to keep your total acetone extract low. 
3. Vortex the centrifuge tube gently to ensure that the extract is homogeneous. 
4. Balance your sample against a blank tube or another sample and use the Dynac clinical centrifuge.

-         Spin at top speed for 5 minutes.

5. Remove the tubes from the centrifuge and note sample extract volumes.
6. For best results, your extract should have some color to it, but not be too colored.  Absorbencies beyond 1.5 result in peak flattening and inaccuracies. Experience will help you make good estimates of this using just your eyes. 

3. Spectrophotometric determination

                       In most circumstances, you will want to make your measurements using the Aminco DW-2000 or DW2 instruments.   Please refer to the guides on using this instrument should you not be familiar with it.  If needed, you can also use the HP 8451 Diode Array spectrophotometer.  However, this is abit less desirable as it has a 2 nm bandpass and does not directly measure the absorbance at 647 nm required for chlorophyll b containing organisms. 

1. Make a wavelength scan of absorbance from 375 to 750 nm.

-         Be sure to store the data on the computer

2. From the UTILITIES menu, choose to transfer you data to a DOS text file.
3. EXIT the DW2000 software and change directory to GWBASIC

-         DOS command is:  cd gwbasic

4. Start the chlorophyll calculation program.

-         Type:  gwbasic dw2000

5. Use the up/down arrow keys to select READ FROM DISK and press ENTER

-         to see files, enter 2

-         your file from step 2 should have a PRN extension

-         enter the file name (without extension) that you want to read

-         if successful, you should see the spectra displayed on the screen

6. Select SETUP using the F2 function key

-         then use the up/down arrow keys to choose DATA TYPE and enter 1 for CHLOROPHYLL

-         use arrow keys to choose SPECIES TYPE and choose the appropriate chlorophyll content (chl b or c containing organisms) and solvent (acetone or methanol)

-         use arrow keys to select FINISHED

-         Enter volume filtered, volume extract, and cell count if known.  If cell count is unknown, then enter a value of 1e6.

7. Select RECALC. by pressing the F6 function key

-         the chlorophyll calculations should be displayed and you should write down the appropriate numbers.  The calculations are based upon the equations of Jeffrey and Humphrey, 1975.

8. Use the ESC key to exit the program
9. If you have a problem with the program, use CTRL + Break to halt the program and type SYSTEM to return to the DOS operating system.  Start over from Step 4.

4. Calculations and Reference

Multiply values by volume of acetone extract and divide by the volume filtered (all in milliliters).  The results are in milligrams per liter.  Note that each OD value is corrected for the absorbance at 750 nm  (e.g. OD₆₆₄ = Abs₆₆₄ – Abs₇₅₀).

For Chlorophyll c containing organisms in 90% Acetone:

Chl a = (11.47 * OD₆₆₄) – (0.4 * OD₆₃₀)

Chl c = (24.36 * OD₆₃₀) – (3.73 * OD₆₆₄)

And in 80% Methanol:

Chl a = (12.66 * OD₆₆₅) – (0.5 * OD₆₃₅)

Chl c = (31.25 * OD₆₃₅) – (5.79 * OD₆₆₅)

                       

                       

For Chlorophyll b containing organisms in 90% Acetone:

                                    Chl a = (11.93 * OD₆₆₄) – (1.93 * OD₆₄₇)

                                    Chl b = (20.36 * OD₆₄₇) – (5.5 * OD₆₆₄)

                        And in 80% Methanol:

                                    Chl a = (16.5 * OD₆₆₅) – (8.3 * OD₆₅₀)

                                    Chl b = (33.8 * OD₆₅₀) – (12.5 * OD₆₆₅)

           

There is a trichromatic calculation (90% Acetone) for determining chlorophylls in a mixed phytoplankton assemblage in which both chlorophyll b and c containing organisms are present.

                                    Chl a = (11.85 * OD₆₆₄) – (1.54 * OD₆₄₇) – 0.08 * OD₆₃₀)

                                    Chl b = (-5.43 * OD₆₆₄) + (21.03 * OD₆₄₇) – (2.66 * OD₆₃₀)

                                    Chl c = (-1.67 * OD₆₆₄) – (7.60 * OD₆₄₇) + (24.52 * OD₆₃₀)

                        *** Chl c calculation may lead to results which are 24% too low

Reference:

Jeffrey, S.W. and Humphrey, G. F. 1975.  New Spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae, and natural phytoplankton.  Biochem. Physiol. Pflanz. 167: 191-194.

Jeffrey, S.W. and Welschmeyer, N.A.  Spectrophotometric and fluorometric equations in common use in oceanography. In  Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods.   Appendix F; 597-615

More recently, the following equations have been determined for 100% Methanol:

            Chl a = (16.29 * OD₆₆₅) – (8.54 * OD₆₅₂)

            Chl b = (30.66 * OD₆₅₂) – (13.58 * OD₆₆₅)

Reference:

Porra, R. J., Thompson, W.A., and Kriedemann, P.E. 1989.  Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents:  verification of the concentration of chlorophyll standards by atomic absorption spectroscopy.  Biochimica et Biophysica Acta 975:  384-394.

For cyanobacteria extracted in boiling methanol, we have typically used:

            Chl a = 13.42 * OD₆₆₅

Reference:

Mackinney, G. 1941.  Absorption of light by chlorophyll solutions.  J. Biol. Chem. 140:  315-322.

However, more recent work has suggested that Mackinney’s extinction coefficients are too low.  Hence, we should re-examine which equation to use for cyanobacteria.

Chlorophyll analysis

Australian National Algae Culture Collection - Methods
Chlorophyll (spectrophotometric analysis)

Method adapted from Lesley Clementson CSIRO (April, 2002)
Sampling

Water samples collected for chlorophyll analysis from the field and from cultures are filtered through 47 mm and 25mm GF/F filters respectively. For field samples, if filtering directly from the Niskin bottle, the filtrate must be collected so the volume of water filtered can be measured. The pressure used during filtering should be low (@ 5 mm Hg) to prevent cell breakage. Filtering and then handling of filters should be performed under dimmed lighting.




Volume to filter


Filter size

Field Sample


no less than 2 litres and preferably 4 or more litres


47mm GF/F

Culture Sample


Typical coloured culture may only need 25 mL


25mm GF/F



After filtering, remove the filter from its holder (with the vacuum still applied, if possible, to remove as much moisture as possible from the filter) and place in a cryo-tube. Label each tube with cruise number, station number, depth and pigment number. (When filtering directly from the Niskin bottle under pressure rather than vacuum, the excess moisture in the filters must be removed before freezing. This can be done by using a second pump that can apply a vacuum to the filter holder or by folding the filter in half and blotting dry with white paper towel.

Analysis

All glassware is cleaned in dilute Extran solution, rinsed three times with MQ water and once with acetone (AR). Scissor blades are wiped clean on tissues between samples.



Frozen filters are cut into halves and placed in a clean 10 ml centrifuge tube. 3 ml of 100% acetone (see note on solvents below) is added to the tube. Cover tube with parafilm and vortex for 30 seconds before placing the tube in an ice-water bath whilst the filter and acetone are sonicated for 15 minutes. The filter and acetone are then stored for at least 18 hours at 4°C. After this time, 0.2 ml MQ water is added to each tube (solvent » 90:10 acetone : water) and the filter and solvent sonicated for another 15 minutes. Solvent and filter are then transferred quantitatively to a Biorad column (see figure x) containing a small GF/F filter acting as a plug. The sample tubes are rinsed with 2 x 0.5 ml of acetone/water (90:10) which is quantitatively added to the Biorad column. Each Biorad column is fitted into a centriguge tube and centrifuged for 5 minutes at 5000 rpm. The filtrate is stored in the cool and dark (small foam esky) just prior to analysis. The absorbance of the filtrate is measured using a U.V./visible spectrophotometer with 10 mm path length optical glass cells (40 mm cells can be used if the colour of the extracts is very pale). Absorbance is read at wavelengths of 750, 664, 647 and 630 nm. The absorbance at 750 nm is subtracted from the absorbance at each of the other three wavelengths and substituted into the following equations:



[chl. a]extract = 11.85A664/l - 1.54A647/l - 0.08A630/l

[chl. b]extract = 21.03A647/l - 5.43A664/l - 2.66A630/l

[chl. c]extract = 24.52A630/l - 1.67A664/l - 7.60A647/l

A = corrected absorbance.

l = path length in cm.

The concentration of each chorophyll in the sample in µg/L is obtained by the following equation:

[chl.x]sample = [chl.x]extract * (v/V)

v = volume of extract in ml.

V = volume of seawater filtered in litres.



The total concentration of chorophyll in the sample in µg/L is obtained by the following equation:

[chl.]total = [chl.a]sample + [chl.b]sample + [chl.c]sample



Solvents

Solvents used for phytoplankton pigment extraction are many and varied. Dimethyl formamide has the highest extraction efficiency but is particularly toxic and therefore difficult to handle. Methanol is another highly efficient extractor, particularly for hard to extract species such as some cyanobacteria. Unfortunately accurate equations to determine chlorophyll a, b and c are not available for methanol pigments. Acetone (either 90 or 100%) is therefore regarded as the most suitable solvent for a broad range of marine phytoplankton species and is widely used for both field samples and cultures. If in doubt, protocols in Jeffrey et al, 1997 detail the suitability of various solvents for a range of different classes and species of microalgae (full ref below).



“Phytoplankton pigments in oceanography: Guidelines to modern methods. 1997. Ed. S. W. Jeffrey, R. F. C. Mantoura, S. W. Wright. Unesco Publishing”