Written on 1-26-98 by John Scott Surfus
Revised on 01-04-01 by Stan Houston
Procedure for leaf pigment analysis on the H.P.L.C.
Solvent Mixtures: from Thayer and Björkman, 1990
Solvent A-> 85:15, Acetonitrile: Methanol
Solvent B-> 68:32, Methanol: Ethyl Acetate
Materials needed:
leaf punch of a known area
rubber stopper
forceps, pencil or other tool to remove leaf disks from the punch
light meter with quantum sensor
liquid nitrogen in dewar
sample canes
nunc vials for leaf disks
notebook and writing instrument
Theory:
An HPLC (High Performance Liquid Chromatograph) system, like all chromatography
systems, separates substances according to their relative adhesion to a "stationary phase"
(typically a solid substance) and a "mobile phase" (typically a liquid or gas flowing past
the stationary phase). The heart of an HPLC is the column, which consists of tightly
packed plastic beads (the stationary phase). Through this column is pumped the pigment,
(or other substance), sample dissolved in an appropriate solvent (the mobile phase). As
the mobile phase passes through the column, different photosynthetic pigments are
retarded to different degrees according to their hydrophobicity and hydrophilicity. A
detector measures the pigments as they come off the column according to their
absorbance or optical density (OD) at a pre-set wavelength (or wavelengths). If
everything is running properly, the different pigments emerge from the column one at a
time rather than all at once, resulting in separate peaks on the output. The detector
automatically measures the area of each peak; this area can then be related to pigment
concentration through a series of separate calibration steps.
Procedure:
Prior to sampling a leaf it is important to make a note of what you are sampling. For
example, note the species, growth conditions (sun/shade), (high/low nutrient levels), and
incident and horizontal light levels.
It is also important to note how many disks to collect for your sample. Each species has a
different concentration of pigments. This might require experimentation with various
numbers of disks. For example, if I was to extract pigments from a clover sample I would
use 2 disks of area .5877 cm2 in 5ml of solvent.
Collect leaf samples by punching leaf disks using a rubber stopper and a leaf punch. Leaf
disks must then be quick-frozen in liquid nitrogen. Noting the area of these disks is
critical; the accuracy of your pigment extraction depends on it.
Note: Liquid nitrogen can be very dangerous if improperly handled. Be sure you are
fully aware of the safety procedures before handling this substance!
Keep the time between punching and freezing as short as possible by having all sample
holders ready and labeled ahead of time. Try to minimize changes in light intensity
during the sampling process (i.e. do not transfer samples from sun to shade or visa versa
before freezing) as this can cause changes in xanthophyll pigment concentration.
Once frozen in liquid nitrogen, the leaf sample is stable indefinitely (unless you run out
of nitrogen!). For long-term storage, samples can be transferred to a -60 to -80 degree
freezer.
Instrument set up
1. Plug in the refrigerated centrifuge and press the start button, (it takes approximately 25
minutes to cool down to -1 degrees centigrade). Make sure the centrifuge program reads
3 minutes at around 10,000 rpm and from -1 to -5 degrees centigrade.
2. Turn on all components of the HPLC, making sure that the injection port is in the up
position and that the pump on the LC_10AS liquid chromatograph is ON,
3. also turn on the red over head lamp. Fill the black and the blue ice containers with ice
from the basement or the third floor cell biology lab and get all pigment extraction
materials.
************************************************************************
************************************************
Note: Sometimes when I have turned the machines on, they automatically start to run!
This means you will not be able to load the parameters from the IC card and the setup
will basically look screwy if you try to run a sample. How to decide if this is the case:
1. The chromatogram is running, that is you see the pen moving and a line being plotted.
2. The "run" light on the LC_10AS is lit.
What to do:
· Hitting the start/stop button on the chromatograph will ONLY stop the plotting! You
must hit the "run" button on the LC_10AS to stop the run (at least according to the
manual).
· Letting the program run will not solve the problem, as it will run for over 10 hours!
************************************************************************
************************************************
Loading the HPLC Program
1. Load the parameters from the I.C. card on to the Chromatopac by pressing (load) then
type ("para1") and press (return),
2. then press (time prg.) (return), then (Y) (return) (esc).
3. To list these parameters on the chromatogram press (llist) followed by the (para), then
(enter). (llist) and (para) are buttons on the keyboard.
Materials needed for extraction:
mortar and pestle
TRIS solution which is (85% acetone: 15% distilled water (v:v), with a 0.1 Molar tris
solution adjusted to pH 8 with HCL)
100% acetone
brown microfuge tubes (B tubes)
Two or more ice containers (We have black and blue)
disposable syringe with filter
disposable pipettes
15ml calibrated, conical centrifuge tubes (C tubes)
5ml and 2ml nunc vials
liquid nitrogen in a storage container and a 4L dewar
· Write at the top of the notebook page and on the HPLC paper the identifying notes of
this run (date, species, treatment, disk area) and leave space for the final volume (i.e. the
final leaf pigment volume you have in the C tube.
· Make sure the wavelength is 445 on the SPD-10AV, if not, type "445" and press enter,
and make sure that solvent A will flow through the system first. If not press {func} on the
LC-10AS until this display {1.20 sv (0)} is showing, the last number will be blinking and
0 is for solvent A and 1 is for solvent B. To change, type the appropriate # and press
(enter) then press (ce) which will bring the display back to where you started.
· When solvent A pressure reads about 58 (anything is almost possible, experience is
helpful here) take up 50 ul of 100% acetone and inject into the HPLC injection port. Turn
the handle down, this will start the run. Run this acetone blank until the HPLC column is
clean and fully filled with solvent. You can tell when the system is ready if there are no
peaks on the paper readout.
1. Place 1 C tube, 2-6 B tubes, 1 5ml nunc vial and 1 syringe with filter into the black ice
bucket and cover with the black top.
2. Put all other materials in the blue ice bucket except for the mortar and pestle and the
Hamilton syringe.
3. Fill the 4L liquid nitrogen dewar with liquid nitrogen (if not already full).
Pigment extraction
Turn off the overhead lights while keeping the red lamp on, (Note: keep the lights off
until you have injected the sample into the HPLC and you have protected your 5ml
extract from excess light). Pour some liquid nitrogen into the mortar (about one fourth
full) with the 4L dewar, then get your frozen leaf disks from the large storage container,
(be careful not to get frostbite while handling the cane and nunc vials, use the blue cryogloves). Put the leaf disks into the liquid nitrogen that is in the mortar. When the liquid
nitrogen is almost all boiled off start to grind the disks, (annual plants will break apart
easily while evergreens and deciduous plants will take more work). After grinding for
about 1 minute or until all of the big leaf particles are ground up, put 1 or 2 drops of the
TRIS solution onto the leaf particles in the mortar and grind again, (repeat this until all
signs of leaf integrity are gone). Now pour this green soup into one of the B tubes in the
ice, then wash the insides of the mortar and pestle with more TRIS solution and grind so
as to get the remaining pigment and pour into the B tube, (repeat this until both B tubes
are full, 1.5ml each). Then place both full B tubes into the black ice bucket and put the lid
on. If the centrifuge temperature readout is between -1 and -5 centigrade place the B
tubes filled with your extract into the centrifuge opposite each other, close the lid and
press the start button, (if the temperature is not at -1 to -5 wait until it is).
Note: Open and close the centrifuge lid quickly so as to keep the internal temperature
below 0 degrees centigrade. Make sure the centrifuge is properly balanced and the rotor
is fully attached. A loose or unbalanced centrifuge rotor becomes a flying missile that can
harm you and damage the centrifuge.
Now wash the mortar and pestle with soap and water, then with distilled water and with a
dry & clean towel. When the centrifuge stops spinning, quickly open the lid and take out
both B tubes and replace the centrifuge lid. Pour the supernatant of both B tubes into the
C tube and note the volume (it should be around 3ml but 2.9 to 3.1 is acceptable). Resuspend the pellets in the B tubes with 100% acetone so that the final volume in the C
tube will be 5ml. Break up the pellet with a metal spatula and replace the B tubes into the
centrifuge and press start again. Replace the lid on the black ice bucket and when the
centrifuge stops spinning again, take out the B tubes, close the centrifuge lid and pour the
supernatant in to the c tube and note the final volume and write this down in your notes
and on the HPLC paper.
Note: Centrifuging for 3 minutes at 10,000 rpm may not be sufficient to remove all the
leaf particles. At this point you might have to pour your 5ml of extract into 4 clean B
tubes and centrifuge again.
Pour the final supernatant into a plastic syringe with its filter and filter this into the 5 ml
nunc vial that is in the black ice bucket. Repeat until all of your extract is in the 5ml nunc
vial. (Try to use only one syringe filter per sample as they are very expensive). Mix up
the extract in the 5 ml nunc vial and replace into the black ice bucket to keep cool.
Pigment separation with an HPLC
Procedure:
Look at the display window on the LC-10AS and note the pressure of solvent A, it should
be around (57-61), if not something is wrong. Typically this is correct only if the filters in
the column are new. Don't be surprised if the pressure is about 70-80.
If so then take your full nunc vial and stir up the extract. Do the next 3 steps very
quickly!
1. With the Hamilton syringe take up 50 ul of pigment from your extract and inject the
sample into the sample port.
2. Turn the sample port clockwise until it stops then
3. press the (zero) button on the top left of the SPD-10AV,
4. then take the syringe out of the sample port. Put your 5ml nunc vial with the pigment
extract back in the black ice bucket put the lid on, now you can turn on the lights. Now
clean your Hamilton syringe with 100% acetone, and make sure you have identified your
sample run on the HPLC paper and in the notebook. If you have another sample to extract
get your supplies ready and repeat the above steps.